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European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2014	Eurjchem	Publishing	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.5.4.601‐606.1096	

	
	

	

	

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

	 	 	

Synthesis	and	biological	evaluation	of	shikimic	acid	derivatives	

Van	Hung	Nguyen	a,	Van	Cuong	Pham	a,*,	Huong	Doan	Thi	Mai	a,	Thanh	Nguyen	Le	a,	
Thi	Minh	Hang	Nguyen	a,	Van	Nam	Vu	a,	Huu	Giap	Tran	a,	Thi	Thao	Do	b,		
Wim	Dehaen	c,	and	Van	Minh	Chau	a	

a	Institute	of	Marine	Biochemistry,	Vietnam	Academy	of	Science	and	Technology,	18	Hoang	Quoc	Viet	Road,	Cau	Giay,	Hanoi,	Vietnam	
b	Institute	of	Biotechnology,	Vietnam	Academy	of	Science	and	Technology,	18	Hoang	Quoc	Viet	Road,	Cau	Giay,	Hanoi,	Vietnam	
c	Department	of	Chemistry,	Cathonic	University	of	Leuven,	Celestijnenlaan	200F	3001,	Leuven,	Belgium	

*Corresponding	author	at:	Institute	of	Marine	Biochemistry,	Vietnam	Academy	of	Science	and	Technology,	18	Hoang	Quoc	Viet	Road,	Cau	Giay,	Hanoi,	Vietnam.		
Tel.:	+84.4.37917049.	Fax:	+84.4.37917054.	E‐mail	address:	phamvc@imbc.vast.vn	(V.C.	Pham).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT

	
DOI:	10.5155/eurjchem.5.4.601‐606.1096	

Received:	25	May	2014	
Received	in	revised	form:	14	July	2014	
Accepted:	17	July	2014	
Online:	31	December	2014	

KEYWORDS	

	 From	shikimic	acid,	new	series	of	oseltamivir	analogues	containing	either	ether	sulfur	or	ether
oxygen	at	C‐3	position	were	prepared	and	evaluated	for	their	biological	activities.	This	is	the
first	 report	 on	 synthesis	 of	 oseltamivir	 analogues	 with	 ether	 sulfur	 at	 C‐3.	 Except	 for
compounds	4d,	4f,	7c	and	7d,	the	remaining	compounds	were	found	to	be	active	against	MCF‐
7,	LU‐1	and	KB	cell	lines.	As	oseltamivir	had	no	cytotoxicity	against	different	cell	lines	such	as
MCF‐7,	LU‐1,	KB,	MDCK,	MRC‐5,	VERO,	MK	and	293,	the	modification	of	alkyl	groups	at	C‐3	of
the	oseltamivir	 ring	 framework	 could	 significantly	 increase	 the	 cytotoxicity	 for	 this	 class	 of
compounds.	 Loss	 of	 neuraminidase	 inhibition	 activity	 of	 these	 synthetic	 oseltamivir
analogues	 suggested	 that	 the	 alkyl	 ether	 groups	 at	 C‐3	 should	 be	 critical	 for	 their	 anti‐
neuraminidase	activity.	

Sulfur	
Azides	
Amines	
Cytotoxicity	
Asymmetric	synthesis	
Nucleophilic	substitution	

	
1.	Introduction	
	

Shikimic	 acid	 is	 well	 known	 as	 starting	 material	 for	
production	 of	 oseltamivir	 phosphate	 (Tamiflu),	 an	 active	
neuraminidase	 inhibitor	 [1‐5].	 Although	 of	 some	 reports	 on	
total	 oseltamivir	 synthesis	 from	 other	 sources,	 shikimic	 acid	
remains	the	principal	starting	material	for	making	oseltamivir.	
Shikimic	 acid	 is	 a	 key	 precursor	 for	 an	 enormous	 number	 of	
aromatic	 compounds,	 e.g.	 aromatic	 amino	 acids,	 vitamins	 and	
host	of	secondary	metabolites	[6‐9],	and	is	the	ideal	precursor	
for	tracer	studies	because	it	is	generally	permeable	to	cells	and	
is	 chemically	 stable.	 As	 shikimate	 pathway	 does	 not	 occur	 in	
mammals,	much	 effort	 has	 been	made	 to	 synthesize	potential	
enzyme	 inhibitors	 in	 order	 to	 develop	 antimicrobial	 reagents.	
Many	 of	 these	 compounds,	 by	 virtue	 of	 their	 structural	
similarity	 to	 shikimic	 acid,	 have	 been	 targeted	 as	 likely	
inhibitor	 of	 enzyme	 on	 the	 shikimic	 acid	 pathway	 and	 are	 of	
biological	 interest	 as	 potential	 antifugal	 [10,11],	 antibacterial	
[11],	 antitumor	 [12,13],	 and	 antiparasitic	 agents	 [14].	Besides	
its	 bio‐significance,	 shikimic	 acid,	 which	 is	 a	 highly	 function‐
nalized	 carbocycle,	 is	 also	 an	 industrially	 useful	 chiral	
compound	[15].	In	our	search	of	bioactive	compounds	based	on	
shikimic	 acid,	 we	 reported	 here	 the	 synthesis	 and	 biological	

activity	of	oseltamivir	analogues	containing	either	ether	sulfur	
or	 ether	 oxygen	 at	 C‐3	 position.	 To	 our	 knowledge,	 the	
synthesis	of	oseltamivir	analogues	with	ether	sulfur	at	C‐3	has	
not	 been	 reported	 yet.	 Also,	 there	 are	 a	 few	 reports	 on	 the	
cytotoxicity	 of	 compounds	 based	 on	 shikimic	 acid,	 whereas	
several	 shikimic	 acid’s	 derivatives	 have	 been	 found	 to	 be	
potential	 antitumor	 agents	 such	 as	 pericosines	 [12,16].	
Furthermore,	 the	 plant	 star	 anise	 (Illicium	 verum),	 widely	
distributing	 in	 the	 tropical	 and	 subtropical	 zones	 of	 Asia	 is	
considered	as	a	rich	source	 for	extracting	shikimic	acid	which	
should	be	used	as	starting	material	for	different	purposes	[17].	
	

2.	Experimental	
	
2.1.	Instrumentation	

	
Chemical	 reagents	were	purchased	 from	Aldrich	Chemical	

Co.	 and	 used	 without	 further	 purification.	 Optical	 rotations	
were	recorded	on	a	Jasco	P‐2000	polarimeter	in	EtOH.	Melting	
points	 were	 recorded	 on	 a	 MELTEMP3.0	 instrument,	 and	 IR	
spectra	were	measured	on	a	Nicolet	Impact‐410	FT‐IR	spectro‐
meter.	ESI‐MS	were	recorded	on	an	Agilent	1100	LC‐MSD	Trap	
spectrometer.	



602	 Nguyen	et	al.	/	European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	
	

	

 
	

Scheme	1
	

	
The	NMR	spectra	were	recorded	on	a	Bruker	spectrometer	

operating	at	500.13	MHz.	1H	chemical	shifts	were	referenced	to	
CHCl3	at	7.27,	while	the	13C	chemical	shifts	were	referenced	to	
the	central	peak	of	CDCl3	at	77.0.	Column	chromatography	was	
performed	on	Merck	silica	gel	60	(70‐230	mesh).	

	
2.2.	Synthesis	

	
2.2.1.	General	procedure	for	compounds	2a‐e	

	
To	 a	 solution	 of	 N‐acetyl	 aziridine	 1	 (1.0	 mmol)	 in	

dichloromethane	 (5	mL)	 was	 added	 InCl3	 (20	mg,	 0.1	mmol)	
and	 thiols	 (2.0	 mmol).	 The	 reaction	 mixture	 was	 stirred	 at	
room	 temperature	 for	 12	 h.	 The	 solids	 were	 filtered	 off	 and	
washed	 with	 dichloromethane.	 The	 filtrate	 was	 evaporated	
under	vacuum	to	give	crude	products	2a‐e	which	were	used	for	
the	next	step	without	purification	(Scheme	1).	

	
2.2.2.	General	procedure	for	compounds	3a‐e	

	
To	a	solution	of	compounds	2a‐e	(0.5	mmol)	in	a	mixture	of	

ethanol	(2	mL)	and	water	(0.3	mL)	was	added	sodium	azide	(10	
eq).	 The	 reaction	mixture	was	heated	 at	 80	 °C	 for	 6	h.	Water	
(20	mL)	was	added	and	the	mixture	was	extracted	with	EtOAc	
(3	 ×	 20	 mL).	 The	 organic	 layers	 were	 combined,	 dried	 over	
sodium	 sulfate	 and	 concentrated	 under	 diminished	 pressure.	
The	 residue	 was	 purified	 over	 a	 silica	 gel	 column	 chroma‐
tography	(n‐hexane/EtOAc	gradient)	yielding	compounds	3a‐e	
(Scheme	1).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(butylthio)cyclohex‐
1‐ene‐1‐carboxylate	(3a):	Color:	Yellow,	amorphous	solid.	Yield:	
46.2%.	1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.90	(t,	J	=	7.2	Hz,	3H,	
CH3‐4′′),	 1.30	 (t,	 J	=	7.2	Hz,	3H,	CH3‐2′),	 1.38	 (m,	 2H,	CH2‐3′′),	
1.54	(m,	2H,	CH2‐2′′),	2.07	(s,	3H,	CH3‐CO),	2.25	(m,	1H,	Ha‐6),	
2.52	(m,	2H,	CH2‐1′′),	2.90	(dd,	J	=	5.5,	17.5	Hz,	1H,	Hb‐6),	3.58	
(q,	 J	=	9.0	Hz,	 1H,	H‐4),	 3.76	 (br.	 d,	 J	=	9.0	Hz,	 1H,	H‐3),	 4.04	
(ddd,	J	=	5.5,	9.0,	10.0	Hz,	1H,	H‐5),	4.22	(q,	J	=	7.2	Hz,	2H,	CH2‐
1′),	5.72	(d,	 J	=	9.0	Hz,	1H,	NH),	6.88	(br.	s,	1H,	H‐2).	13C	NMR	
(125	MHz,	CDCl3,	δ,	ppm):	13.5	(1C,	CH3),	14.1	 (1C,	CH3),	21.9	
(1C,	 CH3),	 23.4,	 29.7,	 29.8,	 31.6,	 45.6,	 55.4,	 58.9,	 61.0,	 127.9,	
139.1,	165.5,	171.1.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(phenylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (3b):	 Color:	 Yellow,	 amorphous	 solid.	
Yield:	42%.	 1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.27	(t,	 J	=	7.3	
Hz,	3H,	CH3‐2′),	1.98	(s,	3H,	Ac),	2.01	(m,	1H,	Ha‐6),	2.80	(dd,	J	=	
5.0,	17.5	Hz,	1H,	Hb‐6),	3.68	(q,	J	=	8.7	Hz,	1H,	H‐4),	3.93	(m,	1H,	
H‐5),	4.12	(br.	d,	J	=	8.7	Hz,	1H,	H‐3),	4.21	(m,	2H,	CH2‐1′),	6.43	
(d,	 J	=	8.7	Hz,	1H,	NH),	6.90	(br.	 s,	1H,	H‐2),	7.26‐7.32	(m,	3H,	
Ar‐H‐2′′,	H‐6′′	 and	H‐4′′),	 7.46	 (m,	2H,	Ar‐H‐3′′	 and	H‐5′′).	 13C	
NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.1,	23.3,	29.7,	49.2,	55.6,	58.9,	
61.0,	128.2,	128.4,	129.1,	131.8,	134.0,	138.1,	165.4,	171.1.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(o‐tolylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (3c):	 Color:	 Yellow,	 amorphous	 solid.	
Yield:	40%.	 1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.27	(t,	 J	=	7.2	
Hz,	3H,	CH3‐2′),	1.93	(s,	3H,	Ac),	2.16	(m,	1H,	Ha‐6),	2.44	(s,	3H,	
CH3‐7′′),	2.85	(dd,	J	=	4.5,	17.5	Hz,	1H,	Hb‐6),	3.67	(q,	J	=	9.0	Hz,	
1H,	H‐4),	4.04	(ddd,	 J	=	5.0,	9.0,	9.0	Hz,	1H,	H‐5),	4.18	(m,	2H,	
CH2‐1′),	4.19	(m,	1H,	H‐3),	6.16	(br.	s,	1H,	NH),	6.84	(br.	s,	1H,	
H‐2),	7.12‐7.25	(m,	3H,	Ar‐H‐4′′,	H‐5′′	and	H‐6′′),	7.45	(d,	J	=	7.5	
Hz,	1H,	Ar‐H‐3′′).	13C	NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.1,	20.9,	
23.3,	 29.8,	 48.4,	 56.5,	 58.7,	 61.0,	 126.6,	 127.7,	 128.3,	 130.7,	
132.3,	133.6,	137.5,	140.7,	165.5,	171.1.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(ethylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (3d):	 Color:	 White,	 amorphous	 solid.	
Yield:	38%.	 1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.22	(t,	 J	=	7.2	
Hz,	3H,	CH3‐2′′),	1.29	(t,	 J	=	7.2	Hz,	3H,	H‐2′),	2.06	(s,	3H,	Ac),	
2.23	(m,	1H,	Ha‐6),	2.53	(m,	2H,	CH2‐1′′),	2.87	(dd,	J	=	5.0,17.5	
Hz,	1H,	Hb‐6),	3.67	(m,	1H,	H‐4),	3.69	(m,	1H,	H‐3),	3.95	(ddd,	J	=	
5.0,	9.5,	 9.5	Hz,	1H,	H‐5),	4.20	 (q,	 J	=	7.2	Hz,	2H,	CH2‐1′),	 6.16	
(br.	s,	1H,	NH),	6.88	(br.	s,	1H,	H‐2).	13C	NMR	(125	MHz,	CDCl3,	
δ,	ppm):	14.1,	14.6,	23.4,	24.1,	29.8,	45.5,	55.2,	58.9,	61.0,	127.9,	
139.1,	165.5,	171.1.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(benzylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (3e):	 Color:	 Yellow,	 amorphous	 solid.	
Yield:	39%.	 1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.29	(t,	 J	=	7.2	
Hz,	3H,	CH3‐2′),	2.00	(s,	3H,	Ac),	2.20	(m,	1H,	Ha‐6),	2.83	(dd,	J	=	
5.0,	 17.5	 Hz,	 1H,	 Hb‐6),	 3.62	 (m,	 1H,	 H‐3),	 3.68	 (m,	 1H,	 H‐4),	
3.74	(d,	J	=	13.0	Hz,	1H,	Ha‐1′′),	3.80	(d,	J	=	13.0	Hz,	1H,	Hb‐1′′),	
3.91	(ddd,	J	=	4.5,	10.0,	10.0	Hz,	1H,	H‐5),	4.17	(q,	J	=	7.2	Hz,	2H,	
CH2‐1′),	5.56	(d,	J	=	7.0	Hz,	1H,	NH),	6.78	(br.	s,	1H,	H‐2),	7.22‐
7.31	 (m,	 5H,	 Ar‐H).	 13C	 NMR	 (125	MHz,	 CDCl3,	 δ,	 ppm):	 14.1,	
23.5,	 29.7,	 34.9,	 45.4,	 54.9,	 58.7,	 61.0,	 127.4,	 127.8,	 128.6,	
129.0,	137.6,	138.6,	165.4,	170.8.	

	
2.2.3.	General	procedure	for	the	synthesis	of	compound	4a‐e	
	

To	a	mixture	of	compounds	3a‐e	(0.1	mmol)	in	methanol	(2	
mL)	was	added	Lindlar’s	catalyst	(10	mg).	The	reaction	mixture	
was	 stirred	 under	 hydrogen	 atmosphere	 for	 5	 h.	 The	 solids	
were	 filtered	 off	 and	washed	with	methanol.	 The	 filtrate	was	
concentrated	to	dryness.	The	residue	was	purified	by	silica	gel	
column	chromatography	eluted	with	CH2Cl2:MeOH	(20:1,	v:v)	to	
provide	compounds	4a‐e	(Scheme	1).	

(3R,4R,5S)‐ethyl	 4‐acetamido‐5‐amino‐3‐(butylthio)cyclo	
hex‐1‐enecarboxylate	 (4a):	 Color:	 White,	 amorphous	 solid.	
Yield:	 82%.	 [α]D25:	 ‐30.8	 (c,	 0.15,	 CHCl3).	 1H	 NMR	 (500	 MHz,	
CDCl3,	δ,	ppm):	0.88	(t,	J	=	7.5	Hz,	3H,	CH3‐4″),	1.28	(t,	J	=	7.0	Hz,	
3H,	CH3‐2′),	1.38	(m,	2H,	CH2‐3″),	1.52	(m,	2H,	CH2‐2″),	2.08	(s,	
3H,	Ac),	2.24	(m,	1H,	Ha‐6),	2.46	(m,	1H,	Ha‐1″),	2.54	(m,	1H,	Hb‐
1″),	2.88	(m,	1H,	Hb‐6),	3.20	(m,	1H,	H‐5),	3.47	(br.	d,	J	=	9.7	Hz,	
1H,	H‐3),	3.81	 (q,	 J	=	9.7	Hz,	1H,	H‐4),	 4.17	 (q,	 J	=	7.0	Hz,	2H,	
CH2‐1′),	6.53	(br.	s,	1H,	NH),	6.86	(br.	s,	1H,	H‐2).	

	



Nguyen	et	al.	/	European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	 603	
 

	

 
	

Scheme	2
	
	

13C	NMR	 (125	MHz,	 CDCl3,	 δ,	 ppm):	 13.6,	 14.2,	 21.9,	 23.6,	
29.4,	 31.5,	 31.9,	 46.6,	 50.8,	 55.2,	 60.9,	 128.9,	 139.3,	 165.9,	
171.9.	
MS	(ESI,	m/z):	315	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(phenylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (4b):	 Color:	 White,	 amorphous	 solid.	
Yield:	 85%.	 [α]D25:	 ‐9.2	 (c	 0.17,	 CHCl3).	 1H	 NMR	 (500	 MHz,	
CDCl3,	δ,	ppm):	1.26	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	2.00	(s,	3H,	Ac),	
2.03	 (m,	 1H,	 Ha‐6);	 2.80	 (dd,	 J	=	 4.5,	 17.5	 Hz,	 1H,	 Hb‐6),	 3.17	
(ddd,	J	=	4.5,	10.2,	10.2	Hz,	1H,	H‐5),	3.75	(q,	J	=	10.2	Hz,	1H,	H‐
4),	3.90	(br.	d,	J	=	10.5	Hz,	1H,	H‐3),	4.16	(m,	2H,	CH2‐1′),	6.27	
(d,	 J	 =	 10.2	 Hz,	 1H,	 NH),	 6.89	 (br.	 s,	 1H,	 H‐2),	 7.27‐7.33	 (m,	
3ArH,	H‐3′′,	H‐4′′	and	H‐5′′),	7.46	(dd,	J	=	1.5,	8.5	Hz,	2ArH,	H‐2′′	
and	H‐6′′).	13C	NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.2,	23.6,	32.1,	
50.2,	50.9,	 56.4,	60.9,	128.3,	129.0,	129.3,	132.0,	134.0,	138.1,	
165.8,	171.6.	MS	(ESI,	m/z):	335	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(o‐tolylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (4c):	 Color:	 Yellow,	 amorphous	 solid.	
Yield:	 83%.	 [α]D25:	 ‐42.9	 (c	 0.14,	 CHCl3).	 1H	 NMR	 (500	 MHz,	
CDCl3,	δ,	ppm):	1.26	(t,	J	=	7.1	Hz,	3H,	CH3‐2′),	1.96	(s,	3H,	Ac),	
2.15	 (m,	 1H,	Ha‐6),	 2.43	 (s,	 3H,	 CH3‐7′′),	 2.82	 (dd,	 J	=	4.5	 and	
17.5	Hz,	1H,	Hb‐6),	3.14	(ddd,	J	=	4.5,	9.5	and	9.5	Hz,	1H,	H‐5),	
3.80	(q,	J	=	9.5	Hz,	1H,	H‐4),	3.93	(br.	d,	J	=	9.5	Hz,	1H,	H‐3),	4.15	
(m,	2H,	CH2‐1′),	6.07	(br.	s,	1H,	NH),	6.82	(br.	s,	1H,	H‐2),	7.15	
(dt,	J	=	1.5,	8.0	Hz,	1H,	Ar‐H‐4′′),	7.18	(dt,	J	=	1.5,	8.0	Hz,	1H,	Ar‐
H‐5′′),	7.21	(dd,	 J	=	1.5,	8.0	Hz,	1H,	Ar‐H‐3′′),	7.45	(dd,	 J	=	1.5,	
8.0	Hz,	1H,	Ar‐H‐6′′).	13C	NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.1,	
20.9,	 23.5,	 32.5,	 49.6,	 50.9,	 57.4,	 60.9,	 126.6,	 128.0,	 129.0,	
130.7,	 132.7,	133.2,	137.4,	140.6,	166.0,	 171.4.	MS	 (ESI,	m/z):	
349	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(ethylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (4d):	 Color:	 Yellow,	 amorphous	 solid.	
Yield:	 82%.	 [α]D25:	 ‐52.4	 (c	 0.55,	 CHCl3).	 1H	 NMR	 (500	 MHz,	
CDCl3,	δ,	ppm):	1.22	(t,	J	=	7.5	Hz,	3H,	CH3‐2′′),	1.29	(t,	J	=	7.0	Hz,	
3H,	CH3‐2′),	2.09	(s,	3H,	Ac),	2.24	(m,	1H,	Ha‐6),	2.50	(m,	1H,	Ha‐
1′′),	2.58	(m,	1H,	Hb‐1′′),	2.85	(m,	1H,	Hb‐6),	3.19	(ddd,	J	=	5.0,	
10.0,	10.0	Hz,	1H,	H‐5),	3.47	(br.	s,	J	=	10.0	Hz,	1H,	H‐3),	3.83	(q,	
J	=	10.0	Hz,	1H,	H‐4),	4.20	(q,	J	=	7.0	Hz,	2H,	CH2‐1′),	6.40	(d,	J	=	
10.0	 Hz,	 1H,	 NH),	 6.88	 (br.	 s,	 1H,	 H‐2).	 13C	 NMR	 (125	 MHz,	
CDCl3,	δ,	ppm):	14.2,	14.6,	23.6,	23.7,	32.0,	46.6,	50.9,	55.3,	60.9,	
128.9,	139.2,	165.9,	171.9.	MS	(ESI,	m/z):	287	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(benzylthio)cyclo	
hex‐1‐ene‐1‐carboxylate	 (4e):	 Color:	 White,	 amorphous	 solid.	
Yield:	 83%.	 [α]D25:	 ‐113.1	 (c	 0.53,	 CHCl3).	 1H	 NMR	 (500	MHz,	
CDCl3,	δ,	ppm):	1.27	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	2.05	(s,	3H,	Ac),	
2.21	 (m,	 1H,	 Ha‐6),	 2.84	 (dd,	 J	=	 4.5,	 17.5	 Hz,	 1H,	Hb‐6),	 3.11	
(ddd,	J	=	4.5,10.0,	10.0	Hz,	1H,	H‐5),	3.36	(br.	d,	J	=	10.0	Hz,	1H,	
H‐3),	3.72	(d,	J	=	13.0	Hz,	1H,	Ha‐1′′),	3.78	(d,	J	=	13.0	Hz,	1H,	Hb‐
1′′),	3.90	(q,	J	=	10.0	Hz,	1H,	H‐4),	4.14	(m,	2H,	CH2‐1′),	6.10	(br.	
s,	 1H,	 NH),	 6.76	 (br.	 s,	 1H,	 H‐2),	 7.21‐7.31	 (m,	 5H,	 Ar‐H).	 13C	
NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.1,	23.6,	32.0,	34.5,	46.7,	51.0,	

54.9,	60.9,	127.2,	128.5,	128.7,	129.1,	137.7,	138.8,	165.8,	171.8.	
MS	(ESI,	m/z):	349	[M+H]+.	
	
2.2.4.	General	procedure	for	compounds	5a‐f	
	

To	 a	 solution	 of	 compound	1	 (0.6	mmol)	 and	 alcohols	 (4	
eq.)	 in	anhydrous	CH2Cl2	(0.5	mL)	was	added	borontrifluoride	
diethyl	 etherate	 (1.5	 eq)	 in	 CH2Cl2	 (0.5	 mL)	 at	 ‐8	 °C.	 The	
reaction	 mixture	 was	 stirred	 for	 1	 h.	 Aqueous	 solution	 of	
potassium	carbonate	5%	(15	mL)	was	added	and	 the	mixture	
was	extracted	with	CH2Cl2	(3	×	15	mL).	The	organic	layers	were	
combined,	washed	with	water	(5	mL)	and	concentrated	under	
reduced	 pressure.	 The	 residue	 was	 chromatographed	 (n‐
hexane:EtOAc	gradient)	to	obtain	compounds	5a‐f	(Scheme	2).		

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐(cyclohexylmethoxy)‐5‐
((methylsulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5a):	 Color:	
White,	amorphous	solid.	Yield:	55%.	1H	NMR	(500	MHz,	CDCl3,	
δ,	 ppm):	 0.91‐0.98	 (m,	 2H,	 cyclohexane),	 1.15‐1.30	 (m,	 3H,	
cyclohexane),	1.33	(t,	J	=	7.1	Hz,	3H,	CH3‐2′),	1.54‐1.79	(m,	6H,	
cyclohexane),	2.05	(s,	3H,	Ac),	2.75‐2.87	(m,	2H,	CH2‐6),	3.06	(s,	
3H,	mesyl),	3.26	(dd,	J	=	6.5,	8.5	Hz,	1H,	Ha‐1′′),	3.47	(dd,	J	=	6.5,	
8.5	Hz,	1H,	Hb‐2′′),	4.11	(dd,	J	=	1.5,	9.0	Hz,	1H,	H‐3),	4.23	(q,	J	=	
7.1	Hz,	2H,	CH2‐1′),	4.33	(ddd,	J	=	2.0,	8.0,	9.0	Hz,	1H,	H‐4),	5.29	
(m,	1H,	H‐5),	5.85	(d,	J	=	8.0	Hz,	1H,	NH),	6.96	(br.	s,	1H,	H‐2).		

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐(2‐cyclohexylethoxy)‐5‐
((methylsulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5b):	 Color:	
White,	amorphous	solid.	Yield:	48%.	1H	NMR	(500	MHz,	CDCl3,	
δ,	 ppm):	 0.86‐0.93	 (m,	 2H,	 cyclohexane),	 1.12‐1.29	 (m,	 3H	
cyclohexane),	1.31	(t,	J	=	7.1	Hz,	3H,	CH3‐2′),	1.36‐1.69	(m,	8H,	
cyclohexane	 and	 CH2‐2′′),	 2.03	 (s,	 3H,	 Ac),	 2.73‐2.84	 (m,	 2H,	
CH2‐6),	3.03	(s,	3H,	mesyl),	3.48	(m,	1H,	Ha‐1′′),	3.66	(m,	1H,	Hb‐
1′′),	4.11	 (dd,	 J	=	1.8,	9.0	Hz,	1H,	H‐3),	4.22	 (q,	 J	=	7.1	Hz,	2H,	
CH2‐1′),	4.30	(ddd,	J	=	2.0,	7.5,	9.0	Hz,	1H,	H‐4),	5.28	(m,	1H,	H‐
5),	5.84	(d,	J	=	7.5	Hz,	1H,	NH),	6.94	(s,	1H,	H‐2).		

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐(cycloheptyloxy)‐5‐((methyl	
sulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5c):	 	 Color:	 White,	
Amorphous	 solid.	 Yield:	 52%.	 1H	 NMR	 (500	 MHz,	 CDCl3,	 δ,	
ppm):	 1.30	 (t,	 J	 =	 7.1	 Hz,	 3H,	 CH3‐2′),	 1.32‐1.39	 (m,	 2H,	
cycloheptane),	 1.49‐1.56	 (m,	 4H,	 cycloheptane),	 1.58‐1.65	 (m,	
6H,	 cycloheptane),	 2.02	 (s,	 3H,	 Ac),	 2.69‐2.74	 (m,	 1H,	 Ha‐6),	
2.80‐2.85	(m,	1H,	Hb‐6),	3.04	(s,	3H,	mesyl),	3.65	(m,	1H,	H‐1′′),	
4.07	(dd,	J	=	1.5,	8.5	Hz,	1H,	H‐3),	4.21	(q,	J	=	7.1	Hz,	2H,	CH2‐1′),	
4.26	(m,	1H,	H‐4),	5.22	(m,	1H,	H‐5),	5.89	(br.	s,	1H,	NH),	6.96	
(br.	s,	1H,	H‐2).		

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐isobutoxy‐5‐((methyl	
sulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5d):	 Color:	 White,	
amorphous	 solid.	 Yield:	 60%.	 1H	 NMR	 (500	 MHz,	 CDCl3,	 δ,	
ppm):	0.90	(t,	J	=	6.5	Hz,	6H,	CH3‐3′′	and	CH3‐4′′),	1.31	(t,	J	=	7.1	
Hz,	3H,	CH3‐2′),	1.83	(m,	1H,	H‐2′′),	2.03	(s,	3H,	Ac),	2.73‐2.85	
(m,	2H,	CH2‐6),	3.04	(s,	3H,	mesyl),	3.19	(dd,	J	=	6.5,	8.5	Hz,	1H,	
Ha‐1′′),	3.43	(dd,	J	=	6.5,	8.5	Hz,	1H,	Hb‐1′′),	4.11	(dd,	J	=	2.0,	9.5	
Hz,	1H,	H‐3),	4.23	(q,	J	=	7.1	Hz,	2H,	CH2‐1′),	4.32	(m,	1H,	H‐4),	



604	 Nguyen	et	al.	/	European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	
	
5.27	(m,	1H,	H‐5),	5.85	(d,	J	=	8.0	Hz,	1H,	NH),	6.94	(br.	s,	1H,	H‐
2).	

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐(4‐fluorophenoxy)‐5‐
((methylsulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5e):	 Color:	
White,	amorphous	solid.	Yield:	75%.	1H	NMR	(500	MHz,	CDCl3,	
δ,	ppm):	1.31	(t,	 J	=	7.0	Hz,	3H,	CH3‐2′),	1.99	(s,	3H,	Ac),	2.78‐
2.83	(m,	2H,	CH2‐6),	3.02	(s,	3H,	mesyl),	4.19‐4.25	(m,	3H,	CH2‐
1′	and	H‐3),	4.38	(ddd,	J	=	2.0,	8.5,	10.5	Hz,	1H,	H‐4),	4.52	(d,	J	=	
11.5	Hz,	1H,	Ha‐1′′),	4.67	(d,	J	=	11.5	Hz,	1H,	Hb‐1′′),	5.25	(m,	1H,	
H‐5),	5.80	(d,	J	=	8.5	Hz,	1H,	NH),	6.95	(br.	s,	1H,	H‐2),	7.04	(t,	J	=	
8.5	Hz,	2H,	Ar‐H‐4′′	and	H‐6′′),	7.32	(dd,	J	=	5.5,	8.5	Hz,	2H,	Ar‐
H‐3′′	and	H‐7′′).	

Ethyl	 (3R,4S,5R)‐4‐acetamido‐3‐(3,4‐dichlorophenoxy)‐5‐
((methylsulfonyl)oxy)cyclohex‐1‐ene‐1‐carboxylate	 (5f):	 Color:	
White,	amorphous	solid.	Yield:	45%.	1H	NMR	(500	MHz,	CDCl3,	
δ,	ppm):	1.31	(t,	 J	=	7.0	Hz,	3H,	CH3‐2′),	2.02	(s,	3H,	Ac),	2.76‐
2.83	(m,	2H,	CH2‐6),	3.04	(s,	3H,	mesyl),	4.21‐4.25	(m,	3H,	CH2‐
1′	and	H‐3),	4.42	(ddd,	J	=	2.5,	8.0,	10.0	Hz,	1H,	H‐4),	4.50	(d,	J	=	
12.0	Hz,	1H,	Ha‐1′′),	4.65	(d,	J	=	12.0	Hz,	1H,	Hb‐1′),	5.23	(m,	1H,	
H‐5),	5.99	(d,	J	=	8.0	Hz,	1H,	NH),	6.93	(br.	s,	1H,	H‐2),	7.15	(dd,	J	
=	 2.0,	 8.5	 Hz,	 1H,	 Ar‐H‐7′′),	 7.41	 (d,	 J	=	 8.5	 Hz,	 1H,	 Ar‐H‐6′′),	
7.43	(d,	J	=	2.0	Hz,	1H,	Ar‐H‐3′′).	
	
2.2.5.	General	procedure	for	compounds	6a‐f	
	

To	a	solution	of	compounds	5a‐f	(0.4	mmol)	in	a	mixture	of	
ethanol:H2O	 (5:1,	 v:v,	 5	mL)	was	 added	 sodium	 azide	 (3	 eq.).	
The	reaction	solution	was	stirred	for	8	h	at	reflux.	The	mixture	
was	cooled	to	room	temperature	and	water	(15	mL)	was	added,	
then	extracted	with	EtOAc	(3	×	15	mL).	The	EtOAc	solution	was	
dried	 over	 sodium	 sulphate,	 concentrated	 under	 reduced	
pressure	 and	 the	 residue	 was	 purified	 by	 silica	 gel	 column	
chromatography	(n‐hexane:EtOAc	gradient)	to	give	compounds	
6a‐f	(Scheme	2).	

Ethyl	(3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(cyclohexylmethoxy)	
cyclohex‐1‐ene‐1‐carboxylate	 (6a):	 Color:	 White,	 amorphous	
solid.	Yield:	71%.	1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.90‐0.97	
(m,	2H,	cyclohexane),	1.13‐1.25	(m,	3H,	cyclohexane),	1.31	(t,	J	
=	7.1	Hz,	 3H,	 CH3‐2′),	 1.54‐1.76	 (m,	 6H,	 cyclohexane),	 2.04	 (s,	
3H,	Ac),	2.26	(m,	1H,	Ha‐6),	2.86	(dd,	J	=	5.0,	17.0	Hz,	1H,	Hb‐6),	
3.24	(dd,	J	=	6.5,	8.5	Hz,	1H,	Ha‐1′′),	3.45	(dd,	J	=	6.5,	8.5	Hz,	1H,	
Hb‐1′′),	3.54	(m,	1H,	H‐3),	4.12	(m,	1H,	H‐4),	4.22	(q,	J	=	7.1	Hz,	
2H,	CH2‐1′),	4.43	(m,	1H,	H‐5),	5.61	(d,	J	=	8.0	Hz,	1H,	NH),	6.84	
(s,	1H,	H‐2).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(2‐cyclohexyl	
ethoxy)cyclohex‐1‐ene‐1‐carboxylate	 (6b):	 Color:	 Yellow,	
amorphous	 solid.	 Yield:	 88%.	 1H	 NMR	 (500	 MHz,	 CDCl3,	 δ,	
ppm):	 0.86‐0.93	 (m,	 2H,	 cyclohexane),	 1.12‐1.27	 (m,	 3H,	
cyclohexane),	1.30	(t,	J	=	7.1	Hz,	3H,	CH3‐2′),	1.34‐1.69	(m,	8H,	
cyclohexane	 and	CH2‐2′′),	 2.05	 (s,	 3H,	Ac),	 2.26	 (m,	 1H,	Ha‐6),	
2.86	(dd,	J	=	5.5,	17.5	Hz,	1H,	Hb‐6),	3.46‐3.54	(m,	2H,	CH2‐1′′),	
3.67	(m,	1H,	H‐3),	4.14	(m,	1H,	H‐4),	4.21	(q,	J	=	7.1	Hz,	2H,	CH2‐
1′),	4.45	(m,	1H,	H‐5),	5.67	(d,	 J	=	8.0	Hz,	1H,	NH),	6.84	(br.	 s,	
1H,	H‐2).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(cycloheptyloxy)	
cyclohex‐1‐ene‐1‐carboxylate	 (6c):	 Color:	 White,	 amorphous	
solid.	Yield:	53%.	1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.30	(t,	J	=	
7.1	Hz,	3H,	CH3‐2′),	1.31‐1.41	(m,	2H,	cycloheptane),	1.49‐1.66	
(m,	8H,	cycloheptane),	1.79‐1.90	(m,	2H,	cycloheptane),	2.03	(s,	
3H,	Ac),	2.22	(m,	1H,	Ha‐6),	2.85	(dd,	J	=	5.7,	17.5	Hz,	1H,	Hb‐6),	
3.26	 (m,	1H,	H‐3),	 3.62	 (m,	 1H,	H‐1′′),	 4.21	 (q,	 J	=	7.1	Hz,	2H,	
CH2‐1′),	4.32	(m,	1H,	H‐4),	4.56	(m,	1H,	H‐5),	5.71	(d,	J	=	7.0	Hz,	
1H,	NH),	6.77	(s,	1H,	H‐2).	13C	NMR	(125	MHz,	CDCl3,	δ,	ppm):	
14.1,	 22.5,	 23.2,	 23.9,	 28.2,	 29.8,	 34.2,	 34.9,	 38.5,	 51.6,	 61.1,	
72.2,	78.0,	80.0,	127.4,	137.0,	165.7,	170.6.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐isobutoxycyclohex‐
1‐ene‐1‐carboxylate	(6d):	Color:	Yellow,	amorphous	solid.	Yield:	
57%.	1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.88	(d,	J	=	6.5	Hz,	3H,	
CH3‐3′′),	0,89	(d,	J	=	6.5	Hz,	3H,	CH3‐4′′),	1.29	(t,	J	=	7.1	Hz,	3H,	
CH3‐2′),	1.79	(m,	1H,	H‐2′′),	2.03	(s,	3H,	Ac),	2.25	(m,	1H,	Ha‐6),	
2.85	(dd,	J	=	5.5,	17.5	Hz,	1H,	Hb‐6),	3.17	(dd,	J	=	6.5,	9.0	Hz,	1H,	

Ha‐1′′),	3.42	(dd,	 J	=	6.5,	9.0	Hz,	1H,	Hb‐1′′),	3.60	(m,	1H,	H‐3),	
4.05	(m,	1H,	H‐4),	4.20	(q,	J	=	7.1	Hz,	2H,	CH2‐1′),	4.39	(m,	1H,	
H‐5),	5.93	(d,	J	=	8.0	Hz,	1H,	NH),	6.83	(t,	J	=	2.0	Hz,	1H,	H‐2	).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(4‐fluorophenoxy)	
cyclohex‐1‐ene‐1‐carboxylate	 (6e):	 Color:	 Yellow,	 amorphous	
solid.	Yield:	56%.	1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	1.30	(t,	J	=	
7.0	Hz,	3H,	CH3‐2′),	1.99	(s,	3H,	Ac),	2.28	(m,	1H,	Ha‐6),	2.88	(dd,	
J	=	5.5,	17.5	Hz,	1H,	Hb‐6),	3.62	(m,	1H,	H‐3),	4.08	(m,	1H,	H‐4),	
4.22	(q,	J	=	7.0	Hz,	2H,	CH2‐1′),	4.49	(d,	J	=	11.5	Hz,	1H,	Ha‐1′′),	
4.54	(m,	1H,	H‐5),	4.67	(d,	J	=	11.5	Hz,	1H,	Hb‐1′′),	5.60	(d,	J	=	7.5	
Hz,	1H,	NH),	6.86	(d,	J	=	2.0	Hz,	1H,	H‐2),	7.02	(t,	J	=	8.5	Hz,	2H,	
Ar‐H‐4′′	and	H‐6′′),	7.29	(dd,	J	=	5.5,	8.5	Hz,	2H,	Ar‐H‐3′′	and	H‐
7′′).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐azido‐3‐(3,4‐dichloro	
phenoxy)cyclohex‐1‐ene‐1‐carboxylate	 (6f):	 Color:	 Yellow,	
amorphous	 solid.	 Yield:	 47%.	 1H	 NMR	 (500	 MHz,	 CDCl3,	 δ,	
ppm):	1.31	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	2.02	(s,	3H,	Ac),	2.30	(m,	
1H,	Ha‐6),	2.90	(dd,	J	=	5.5,	17.5	Hz,	1H,	Hb‐6),	3.62	(m,	1H,	H‐3),	
4.10	 (m,	1H,	H‐4),	 4.22	 (q,	 J	=	 7.0	Hz,	2H,	CH2‐1′),	4.48	 (d,	 J	=	
12.0	Hz,	1H,	Ha‐1′′),	4.56	(m,	1H,	H‐5),	4.67	(d,	J	=	12.0	Hz,	1H,	
Hb‐1′′),	5.60	(d,	J	=	8.0	Hz,	1H,	NH),	6.86	(t,	J	=	2.0	Hz,	1H,	H‐2),	
7.15	(dd,	J	=	1.7,	8.2	Hz,	1H,	Ar‐H‐7′′),	7.42	(d,	J	=	8.2	Hz,	1H,	Ar‐
H‐6′′),	7.44	(d,	J	=	1.7	Hz,	1H,	Ar‐H‐3′′).	

	
2.2.6.	General	procedure	for	synthesis	of	compound	7a‐f	
	

A	mixture	of	compounds	6a‐f	(0.1	mmol)	and	Ph3P	(2	eq.)	
in	THF:H2O	(10:1,	v:v)	was	heated	at	50	°C	for	16	h.	The	mixture	
solution	 was	 concentrated	 to	 dryness	 and	 the	 residue	 was	
purified	by	column	chromatography	on	silica	gel,	eluted	with	n‐
hexane:EtOAc	gradient,	affording	compounds	7a‐f	(Scheme	2).	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(cyclohexyl	
methoxy)cyclohex‐1‐ene‐1‐carboxylate	 (7a):	 Color:	 Yellow,	
amorphous	 solid.	 Yield:	 89%.	 [α]D25:	 ‐74.8	 (c	 1.4,	 CHCl3).	 1H	
NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.78‐0.85	(m,	2H,	cyclohexane),	
1.03‐1.14	(m,	3H,	cyclohexane),	1.20	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	
1.39‐1.45	(m,	1H,	cyclohexane),	1.66	(m,	5H,	cyclohexane),	1.93	
(s,	3H,	Ac),	2.05	(m,	1H,	Ha‐6),	2.67	(dd,	J	=	5.0,	17.5	Hz,	1H,	Hb‐
6),	2.83	(m,	1H,	H‐5),	3.09	(dd,	J	=	6.5,	8.5	Hz,	1H,	Ha‐1″),	3.33	
(dd,	J	=	6.5,	8.5	Hz,	1H,	Hb‐1″),	3.67	(m,	1H,	H‐4),	3.95	(m,	1H,	H‐
3),	4.11	(q,	J	=	7.0	Hz,	2H,	CH2‐1′),	6.23	(br.	s,	1H,	NH),	6.71	(br.	
s,	1H,	H‐2).	MS	(ESI,	m/z):	339	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(2‐cyclohexyl	
ethoxy)cyclohex‐1‐ene‐1‐carboxylate	 (7b):	 Color:	 Yellow,	
amorphous	 solid.	 Yield:	 92%.	 [α]D25:	 ‐26.4	 (c	 0.83,	 CHCl3).	 1H	
NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.80‐0.87	(m,	2H,	cyclohexane),	
1.07‐1.20	(m,	3H,	cyclohexane),	1.35	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	
1.30‐1.36	(m,	2H,	CH2‐2″),	1.37	(m,	1H,	cyclohexane),	1.57‐1.64	
(m,	5H,	cyclohexane),	1.99	(s,	3H,	Ac),	2.09	(m,	1H,	Ha‐6),	2.21	
(br.	s,	2H,	NH2),	2.74	(dd,	J	=	5.0,	17.5	Hz,	1H,	Hb‐6),	2.99	(m,	1H,	
H‐5),	3.39	(m,	1H,	Ha‐1″),	3.58	(m,	1H,	Hb‐1″),	3.70	(m,	1H,	H‐4),	
4.05	(m,	1H,	H‐3),	4.15	(q,	J	=	7.0	Hz,	2H,	CH2‐1′),	6.20	(br.	s,	1H,	
NH),	6.76	 (br.	 s,	1H,	H‐2).	 13C	NMR	(125	MHz,	CDCl3,	 δ,	ppm):	
13.9,	 22.7,	 25.6,	 25.6,	 26.3,	 29.6,	 29.7,	 33.0,	 38.0,	 49.2,	 55.8,	
60.9,	74.5,	77.1,	129.7,	137.1,	166.1,	172.2.	MS	(ESI,	m/z):	353	
[M+H]+.	

Ethyl	(3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(cycloheptyloxy)	
cyclohex‐1‐ene‐1‐carboxylate	 (7c):	 Color:	 Yellow,	 amorphous	
solid.	 	 Yield:	 93%.	 [α]D25:	 ‐88.8	 (c	 1.25,	 CHCl3).	 1H	 NMR	 (500	
MHz,	CDCl3,	δ,	ppm):	1.26	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	1.32	(m,	2H,	
cycloheptane),	 1.49‐1.59	 (m,	 8H,	 cycloheptane),	 1.76‐1.92	 (m,	
2H,	cycloheptane),	2.00	(s,	3H,	Ac),	2.10	(m,	1H,	Ha‐6),	2.71	(dd,	
J	=	5.0,	17.5	Hz,	1H,	Hb‐6),	3.16	(m,	1H,	H‐1″),	3.48	(m,	1H,	H‐5),	
3.58	(m,	1H,	H‐3),	4.17‐4.19	(m,	3H,	H‐4	and	CH2‐1′),	6.02	(br.	s,	
1H,	 NH),	 6.72	 (br.	 s,	 1H,	 H‐2).	 13C	 NMR	 (125	 MHz,	 CDCl3,	 δ,	
ppm):	 14.1,	 22.6,	 22.6,	 23.5,	 28.3,	 28.3,	 33.4,	 33.9,	 34.9,	 35.0,	
49.0,	60.8,	74.6,	79.4,	79.6,	129.4,	137.6,	166.2,	172.3.	MS	(ESI,	
m/z):	339	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐isobutoxycyclohex‐
1‐ene‐1‐carboxylate	(7d):	Color:	Yellow,	amorphous	solid.	Yield:	
89%.	[α]D25:	‐4.8	(c	0.67,	CHCl3).	



Nguyen	et	al.	/	European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	 605	
 

	
Table	1.	Cytotoxic	activity	of	compounds	4a‐f	and	7a‐f.	
Compounds	 IC50	(μg/mL)	 	 Compounds	 IC50	(μg/mL)	

KB	 MCF‐7	 LU‐1 KB MCF‐7	 LU‐1
4a	 72.0	 38.2	 40.1	 	 7c	 98.7	 97.6	 99.8	
4b	 9.6	 8.0	 8.8	 	 7d	 >100	 >100	 >100	
4c	 39.7	 42.9	 45.0 7e 84.6 72.7	 74.1
4d	 >100	 >100	 >100 7f 15.7 15.0	 15.3
4e	 79.7	 62.5	 82.9 Ellipticine 1.1 0.9	 0.9
7a	 84.9	 80.0	 79.7 Oseltamivir	phosphate >100 >100	 >100
7b	 27.6	 24.1	 25.8 	

	
	

1H	NMR	(500	MHz,	CDCl3,	δ,	ppm):	0.84	(d,	 J	=	6.5	Hz,	3H,	
CH3‐3″),	0.88	(d,	J	=	6.5	Hz,	3H,	CH3‐4″),	1.27	(t,	J	=	7.0	Hz,	3H,	
CH3‐2′),	1.77	(m,	1H,	H‐2″),	2.01	(s,	3H,	Ac),	2.12	(m,	1H,	Ha‐6),	
2.75	(dd,	J	=	5.0,	17.5	Hz,	1H,	Hb‐6),	2.99	(m,	1H,	H‐5),	3.10	(dd,	J	
=	8.0,	15.0	Hz,	1H,	Ha‐1″),	3.36	(dd,	J	=	8.0,	15.0	Hz,	1H,	Hb‐1″),	
3.74	(q,	 J	=	9.0	Hz,	1H,	H‐4),	4.06	(m,	1H,	H‐3),	4.17	(q,	J	=	6.5	
Hz,	 2H,	 CH2‐1′),	 6.03	 (br.	 s,	1H,	NH),	 6.79	 (br.	 s,	 1H,	H‐2).	 13C	
NMR	(125	MHz,	CDCl3,	δ,	ppm):	14.0,	19.0,	19.2,	23.0,	28.6,	33.3,	
49.2,	55.9,	60.9,	75.2,	80.8,	129.9,	137.1,	166.1,	172.3.	MS	(ESI,	
m/z):	298	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(4‐fluorophenoxy)	
cyclohex‐1‐ene‐1‐carboxylate	 (7e):	 Color:	 Yellow,	 amorphous	
solid.	 Yield:	 88%.	 [α]D25:	 ‐38.75	 (c	 0.5,	 CHCl3).	 1H	 NMR	 (500	
MHz,	CDCl3,	δ,	ppm):	1.16	(t,	J	=	7.0	Hz,	3H,	CH3‐2′),	1.86	(s,	3H,	
Ac),	2.03	(m,	1H,	Ha‐6),	2.65	(dd,	J	=	5.0,	17.5	Hz,	1H,	Hb‐6),	2.75	
(m,	 1H,	H‐5),	 3.71	 (q,	 J	=	9.0	Hz,	 1H,	H‐4),	 4.03	 (m,	 1H,	H‐3),	
4.08	(q,	J	=	7.0	Hz,	2H,	CH2‐1′),	4.34	(d,	J	=	11.5	Hz,	1H,	Ha‐1″),	
4.50	(d,	J	=	11.5	Hz,	1H,	Hb‐1″),	6.63	(br.	s,	1H,	NH),	6.72	(br.	s,	
1H,	H‐2),	6.89	(t,	J	=	8.5	Hz,	2H,	Ar‐H‐4″	and	H‐6″),	7.17	(dd,	J	=	
5.5,	8.5	Hz,	2H,	Ar‐H‐3″	and	H‐7″).	13C	NMR	(125	MHz,	CDCl3,	δ,	
ppm):	14.1,	23.5,	33.8,	49.8,	56.7,	60.9,	69.4,	76.4,	115.2,	115.3,	
129.7,	129.8,	130.9,	133.6,	136.1,	161.4	and	163.3	(C‐F),	165.9,	
171.0.	MS	(ESI,	m/z):	351.0	[M+H]+.	

Ethyl	 (3R,4R,5S)‐4‐acetamido‐5‐amino‐3‐(3,4‐dichloro	
phenoxy)cyclohex‐1‐ene‐1‐carboxylate	 (7f):	 Color:	 White,	
amorphous	 solid.	 Yield:	 68%.	 [α]D25:	 ‐50.8	 (c	 1.0,	 CHCl3).	 1H	
NMR	 (500	MHz,	 CDCl3,	 δ,	 ppm):	 1.30	 (t,	 J	=	7	Hz,	 3H,	 CH3‐2′),	
2.04	(s,	3H,	Ac),	2.18	(m,	1H,	Ha‐6),	2.81	(dd,	J	=	5.0,	17.5	Hz,	1H,	
Hb‐6),	3.04	(m,	1H,	H‐5),	3.85	(q,	 J	=	9.0	Hz,	1H,	H‐4),	4.22	(m,	
3H,	CH2‐1′	and	H‐3),	4.44	(d,	J	=	12.0	Hz,	1H,	Ha‐1′′),	4.65	(d,	J	=	
12.0	Hz,	1H,	Hb‐1′′),	5.41	(d,	J	=	9.0	Hz,	1H,	NH),	6.85	(br.	s,	1H,	
H‐2),	7.15	(dd,	J	=	1.5,	8.0	Hz,	1H,	Ar‐H‐7′′),	7.41	(d,	J	=	8.0	Hz,	
1H,	Ar‐H‐6′′),	 7.45	 (d,	 J	=	1.5	Hz,	 1H,	Ar‐H‐3′′).	 13C	NMR	 (125	
MHz,	CDCl3,	δ,	ppm):	14.1,	23.5,	33.9,	49.7,	56.3,	61.0,	68.5,	76.8,	
126.9,	 129.6,	 130.3,	 131.2,	 131.7,	 132.5,	 135.6,	 138.2,	 165.9,	
171.0.	MS	(ESI,	m/z):	401	[M+H]+.	

	
2.3.	Cytotoxicity	assay	
	

The	cancer	cells	(KB,	MCF‐7	and	LU‐1)	were	maintained	in	
Dulbecco's	D‐MEM	medium,	supplemented	with	10%	fetal	calf	
serum,	 L‐glutamine	 (2	mM),	 penicillin	 G	 (100	UI/mL),	
streptomycin	 (100	μg/mL)	 and	 gentamicin	 (10	μg/mL).	 Stock	
solutions	 of	 compounds	 were	 prepared	 in	 DMSO/H2O	 (1/9),	
and	cytotoxicity	assays	were	carried	out	 in	96‐well	microtiter	
plates	 against	 KB,	 MCF‐7	 and	 LU‐1	 cancer	 cell	 lines	 (3	 ×	
103	cells/mL)	 using	 a	 modification	 of	 the	 published	 method	
[18].	 After	 72	h	 incubation	 at	 37	 °C	 in	 air/CO2	 (95:5)	with	 or	
without	 test	 compounds,	 cell	 growth	 was	 estimated	 by	
colorimetric	measurement	of	stained	living	cells	by	neutral	red.	
Optical	 density	 was	 determined	 at	 540	nm	 with	 a	 Titertek	
Multiscan	 photometer.	 The	 IC50	 value	 was	 defined	 as	 the	
concentration	of	sample	necessary	to	inhibit	the	cell	growth	to	
50%	 of	 the	 control.	 Ellipticine	 was	 used	 as	 a	 reference	
compound.	

	
3.	Results	and	discussion		
	

The	 synthesis	 of	 oseltamivir	 was	 previously	 described	
starting	from	shikimic	acid,	in	which	the	synthesis	via	aziridine	

intermediate	was	considered	to	be	an	effective	method	[19,20].	
The	 approach	 in	 this	 work	 is	 preparation	 of	 ether	 sulfur	 or	
ether	oxygen	at	C‐3	position	of	oseltamivir	analogues	based	on	
ring	 opening	 of	 aziridine	 compound	 by	 different	 thiols	 or	
alcohols.	 Accordingly,	 aziridine	1	was	 obtained	 from	 shikimic	
acid	 through	 five	 steps	 in	high	yields	using	previous	 reported	
methods	[19,20].		

Aziridines	 are	 highly	 strained,	 three‐membered,	 nitrogen	
containing	 heterocycles	 that	 is	 easily	 opened	 by	 nucleo‐
philes	 in	 order	 to	 relieve	 the	 strain.	Yadav	and	co‐workers	
demonstrated	that	the	InCl3	could	promote	the	ring	opening	of	
aziridines	by	nucleophiles	including	thiols	under	mild	reaction	
conditions	 with	 high	 regioselectivity	 [21].	 Consequently,	
aziridine	 1	 was	 successfully	 reacted	 with	 different	 thiols	
(ethylthiol,	 n‐butylthiol,	 benzylthiol,	 thiophenol	 and	 o‐methyl	
thiophenol)	 in	 the	 presence	 of	 InCl3	 at	 room	 temperature	 to	
afford	 compounds	 2a‐e	 in	 high	 yields	 (63‐91%)	 with	 high	
stereoselectivity	 and	 regioselectivity.	 The	 next	 step	 was	
nucleophilic	 replacement	of	OMs	group	at	 the	C‐5	position	by	
the	 N3	 group.	 Treatment	 of	 compounds	 2a‐e	 with	 NaN3	
afforded	the	intermediates	3a‐e.	The	inversion	of	configuration	
at	 C‐5	was	 observed	 for	 compounds	3a‐e	 as	 indicated	 by	 the	
coupling	constants	of	H‐5	which,	had	two	anti	 (J	=	8.7‐9.0	Hz)	
and	a	gauche	(J	=	0.0‐1.0	Hz)	coupling	constants.	Thus,	a	trans	
relationship	between	H‐4	and	H‐5	was	revealed	for	compounds	
3a‐e.	Finally,	compounds	3a‐e	were	reduced	by	hydrogenation	
in	 the	 presence	 of	 Lindlar’s	 catalyst	 to	 provide	 4a‐e,	
respectively	(Scheme	1)	[19].	

In	 the	 second	 approach,	 the	 analogues	 containing	 ether	
oxygen	at	C‐3	were	also	prepared	starting	from	1	by	aziridine	
ring	opening	with	alcohols	in	the	presence	of	BF3.Et2O	to	yield	
compounds	 5a‐f	 which	 were	 then	 converted	 into	 the	
corresponding	azide	derivatives	6a‐f.	The	series	of	alkyl	or	aryl	
ethers	 at	 C‐3	 7a‐f	 was	 obtained	 from	 the	 corresponding	
intermediates	6a‐f	by	reduction	with	Ph3P	(Scheme	2)	[22].	

The	 synthetic	 compounds	 were	 evaluated	 for	 their	
cytotoxicity	 against	 three	 human	 cancer	 cell	 lines,	 KB	 (mouth	
epidermal	carcinoma	cells),	LU‐1	(human	lung	cancer	cells)	and	
MCF‐7	 (human	breast	 cancer	 cells).	Except	 for	4d,	7c	 and	7d,	
the	 remaining	 compounds	 were	 found	 to	 be	 active	 against	
three	 tested	 cancer	 cell	 lines	 (Table	 1).	 It	 was	 found	 that	
oseltamivir	has	no	cytotoxicity	against	different	cell	 lines	such	
as	 MDCK,	 MRC‐5,	 VERO,	 MK	 and	 293	 cells	 [23].	 Also,	 when	
tested	 against	 KB,	 LU‐1	 and	 MCF‐7	 cell	 lines,	 oseltamivir	
phosphate	had	no	 inhibition	even	at	 the	 concentration	of	100	
µg/mL.	 Thus,	 the	 modification	 of	 alkyl	 groups	 at	 C‐3	 of	 the	
cyclohexene	ring	significantly	increased	the	cytotoxicity	for	this	
class	 of	 compounds.	 In	 order	 to	 compare	 the	 neuraminidase	
inhibition	 activity	 with	 oseltamivir	 phosphate,	 the	 synthetic	
compounds	4a‐e	and	7a‐f	were	also	 evaluated	 for	 their	 in	
vitro	 influenza	 A	 neuraminidase	 inhibitory	 activity	 by	 an	
enzymatic	 assay.	However,	these	compounds	had	no	inhibition	
activity	 even	 at	 the	 concentration	 of	 50	 µM.	 This	 observation	
suggested	that	the	3‐o‐pentyl	group	at	C‐3	of	oseltamivir	played	
an	important	role	for	the	neuraminidase	inhibitory	activity	of	
oseltamivir.	This	 is	 in	agreement	with	the	previous	reports	on	
several	 oseltamivir	 analogues	 such	 as	 compounds	8‐11	which	
are	less	active	than	oseltamivir	(Figure	1)	[24].	
	



606	 Nguyen	et	al.	/	European	Journal	of	Chemistry	5	(4)	(2014)	601‐606	
	

 
	

Figure	1.	Compounds	taken	from	the	ref.	[24].	
	
4.	Conclusion	

	
We	have	described	the	synthesis	and	biological	evaluation	

of	oseltamivir	analogues	containing	either	ether	sulfur	or	ether	
oxygen	 at	 C‐3	 position	 for	 cytotoxic	 activity.	 Many	 of	 the	
synthetic	 derivatives	 exhibited	 cytotoxicity	 against	 the	 three	
tumor	 cell	 lines	 (KB,	 LU‐1	 and	 MCF‐7).	 This	 is	 the	 first	
synthesis	of	oseltamivir	analogues	with	ether	sulfur	at	C‐3.	
	
Acknowledgements	
	

This	 work	 was	 supported	 by	 the	 Vietnam	 National	
Foundation	 for	 Science	 and	 Technology	 Development	
(NAFOSTED)	 and	 the	 Research	 Foundation	 Flanders	 (FWO)	
(Project	No:	04/2010).	
	
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