Synthesis of a carbocyclic sialic acid analogue for the inhibition of influenza virus neuraminidase

Article abstract of DOI:10.1016/S0008-6215(01)00079-9

The influenza virus neuraminidase (NA) is essential for viral infection and offers a potential target for antiviral drug development. We prepared a carbocyclic sialic acid analogue, potentially able to inhibit NA. Its structure is an analogue of the trans

Full text of DOI:10.1016/S0008-6215(01)00079-9

www.elsevier.nl/locate/carres
Carbohydrate Research 332 (2001) 23–31
Synthesis of a carbocyclic sialic acid analogue for the
inhibition of influenza virus neuraminidase
Armandodoriano Bianco,a,* Mario Brufani,b Fedele Manna,c Cristiana Melchionia
aDipartimento di Chimica, Uni6ersita` ‘La Sapienza’, Piazzale Aldo Moro n. 5, I-00185 Rome, Italy
bDipartimento di Scienze Biochimiche, Uni6ersita` ‘La Sapienza’, Rome, Italy
cDipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Atti6e, Uni6ersita` ‘La Sapienza’,
Rome, Italy
Received 5 June 2000; received in revised form 22 February 2001; accepted 8 March 2001
Abstract
The influenza virus neuraminidase (NA) is essential for viral infection and offers a potential target for antiviral
drug development. We prepared a carbocyclic sialic acid analogue, potentially able to inhibit NA. Its structure is an
analogue of the transition-state of the reaction catalysed by NA. As starting material, quinic acid was selected owing
to its ready availability and its stereochemical feature suitable for the target structure. The quinic acid was first
converted in the shikimic acid; then two of the three hydroxyl functions of this product were selectively functionalised
to obtain the target molecule (3R,4S,5R)-4-acetamido-3-guanidino-5-hydroxycyclohex-1-ene-1-carboxylic acid.
© 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Antiviral agents; Sialic acid; Shikimic acid
1. Introduction
acid and its adjacent carbohydrate moiety on
a variety of glycoconjugates. The inhibition of
NA activity is lethal to the virus which cannot
be released by the cell membrane; this limits
the mobility of the virus itself and so it pre-
vents the progress of the viral infection. It has
been proposed a mechanism of influenza virus
NA reaction in which the driving force comes
solely from the induction and stabilisation of
the oxocarbonium intermediate A3,4 (see
Scheme 1).
On the basis of this mechanism, it is possi-
ble to inhibit the NA enzyme by structurally
analogue molecules to the oxonium cation
transition state. Several NA inhibitor ana-
logues have been synthesised;5–7 in particular,
Kim et al. reported the synthesis of compound
GS4071 (Scheme 2) which is a potent NA
inhibitor with IC50 value of 1 nM.8 Subse-
quently, GS4104 (the ethyl ester prodrug of
The first step of infection by influenza
viruses subtypes A and B is mediated by the
interaction of sialic acid with two major sur-
face viral glycoproteins, haemagglutinin (HA)
and neuraminidase (NA). These proteins are
both essential for infection and offer potential
targets for antiviral drug development. The
HA of influenza viruses A and B is responsible
for viral attachment to host cells by binding to
terminal sialic acid residues of surface glyco-
conjugates.1,2 The NA (EC 3.2.1.18) catalyses
the hydrolysis of the a-(2“3) and a-(2“6)
glycosidic linkage between a terminal sialic
* Corresponding author. Fax: +39-06-490631.
E-mail address: armandodoriano.bianco@uniroma1.it (A.
Bianco).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00079-9

24
A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
Scheme 1.
Scheme 2.
GS4071) was found to be highly orally
bioavailable in several animal species and effi-
cacious in both the mouse and ferret models
of influenza infection by oral administra-
tion.9,10 Zanamivir (GG167, see Scheme 2)11–13
is another ‘transition state analogue’ of A
which exhibits the potent NA inhibitory activ-
ity. Based on the results previously obtained,
we aimed our research at the synthesis of new
compounds by using a carbocyclic template
instead of the dihydropyran ring of the
DANA system (DANA is the 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid).
material quinic acid 1. Quinic acid 1 was con-
verted into methyl shikimate 5 as shown in
Scheme 3.14 Quinic acid 1 was transformed
into methyl ester 2 which was then acetylated,
by controlling the reaction conditions, to
obtain the triacetyl derivative 3 in which the
tertiary hydroxyl remains free.
The two hydroxyls at C-3 and at C-4, in cis
position to each other, were protected by for-
mation of the O-isopropylidene derivative 6
(Scheme 4) and then the free hydroxyl was
protected by acetylation to afford compound
7.
To explain this regioselectivity, there is not
an obvious reason; probably the hydroxyl at
C-1 in quinic acid could take an axial orienta-
tion to undergo the elimination with the anti
proton and the substituents at C-3 and C-5
may have an important role in the dehydra-
2. Results and discussion
Considering the oxocarbonium ion (Scheme
1) as the transition-state, we prepared a cyclo-
hexene analogue 17 (Scheme 2) which has the
necessary conditions to imitate the transi-
tion state. These consist on acidic, acetamido
functions and the planar carbon correspond-
ing to C-2 of the sialic acid. Also the guani-
dino group has been introduced on the allylic
position; that should be right to occupy the
region of the NA active site that is normally
filled to glyceric chain of sialic acid.
1
tion process. In fact, on the basis of the H
NMR coupling constants observed for H-4 in
3 (J4,5 4, J4,3 12 Hz), the acetoxy group at C-3
adopts an equatorial position while that at
C-5 adopts an axial orientation. Obviously the
transition state, which corresponds to the de-
hydration that produces 4, is more energeti-
cally favoured than that which would produce
the other isomer.
The carbocyclic analogue 17 was prepared
according to the synthetic strategy depicted
in Schemes 3 and 5 using as chiral starting
The synthetic strategy for the preparation
of 17 continues, as described in Scheme 5,

A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
25
Scheme 3.
Scheme 4.
starting from methyl shikimate 5. While this
work was in progress, as a part of a PhD15
thesis project on inhibitors of NA and HA,
Kim and co-workers8 described the synthesis
of compound GS4071 (see Scheme 2) with a
strategy that is similar to that described in
Scheme 5.
group at 2090 cm−1 in the IR spectrum. Com-
pound 10 was treated with methanesulfonyl
chloride to provide the mesyl derivative 11, in
which the three hydroxyl functions at C-3, -4
and -5 are protected selectively with three
different groups. The 1H NMR spectrum of 11
showed the presence of a multiplet at l 4.77
due to the proton at C-4, now deshielded by
the esterification of OH-4 with methanesul-
fonic acid, and an additional singlet at l 3.12
due to the three protons of the mesyl group.
Conversion of the azido group in 11 to the
acetamido derivative 12 was efficiently accom-
plished in 50% yield via a two-step sequence:
reduction of the azide functionality with
triphenylphosphine in tetrahydrofuran and
acetylation of the amine with acetyl chloride.
In the 1H NMR spectrum of 12, it is noted the
presence of a singlet at l 2.09 due to the acetyl
group on nitrogen. Product 12 was treated
with sodium hydride in tetrahydrofuran to
provide N-acetylaziridine 13 by means of a
SN2 type intramolecular reaction.
Methyl shikimate 5 was esterified with thio-
nyl chloride to the cyclic sulfite 8 as a stable
1
intermediate in 97% yield. In the H NMR
spectrum of 8, there were two multiplets at l
4.87 and at l 5.58, ascribable to the protons at
C-4 and at C-3, deshielded owing to esterifica-
tion of the attached hydroxyls. Then the hy-
droxyl at C-5 of 8 was protected by means of
acetylation, affording product 9. The sulfite
ring opening of 9 has been carried out with
sodium azide in dimethylformamide; this reac-
tion allows the introduction of the azido
group in the allylic position and releases the
hydroxylic function at C-4, providing deriva-
tive 10. This complete regiospecific ring open-
ing is a consequence of a favoured azide ion
attack at the allylic C-3 position of 9. The
structure 10 was verified by the presence of the
characteristic absorption band of the azido
1
The H NMR spectrum of 13 shows the
shielding of two protons at C-3 and at C-4 (by
about 1 ppm) that give a multiplet at about l

26
A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
3.17. Treatment of 13 with sodium azide in
dimethylformamide in the presence of ammo-
nium chloride allowed ring opening of the
acetylated aziridine and formation of 14. The
ring-opening reaction was completely regio-
specific, giving azide 14 as the only pro-
duct. In the 1H NMR spectrum of 14, one could
notice the presence of two multiplets at l 4.08
and at l 4.35 due to protons at C-3 and at
C-4, respectively, which were deshielded.
the presence of the Lindlar catalyst resulting
in the selective reduction of the azide in the
amino group without interference with the
CꢀC double bond. This reaction afforded
derivative 15.
Treatment of 15 with N,N%-bis(tert-butoxy-
carbonyl)thiourea,16 provided bis-(BOC) pro-
tected guanidine 16. Finally, the hydrolysis of
the methyl ester and the acetyl group at C-5
followed by the removal of the BOC groups,
furnished the final product 17. Biological tests
on the sialic acid analog 17 are in progress.
The azido group in allylic position was then
subject to catalytic reduction with hydrogen in
Scheme 5.

A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
27
tetraacetylated product. To the reaction mix-
ture was added MeOH and the volatile mater-
ials were removed under diminished pressure.
The residue was purified by chromatography
(9:1 CHCl3–EtOAc) to give 3 (380 mg, 47%)
3. Experimental
General methods.—1H NMR spectra were
measured with a Varian Gemini 200 MHz
spectrometer and chemical shifts are expressed
in ppm from TMS. Infrared spectra were reg-
istered with a Shimadzu IR-470 spectrometer.
The starting material, quinic acid 1, was ob-
tained from Aldrich Chemical Co. Product
purification was obtained by solid–liquid
column chromatography on E. Merck 0.063–
0.20 nm Silica Gel; the elution mixtures were
determined case by case. E. Merck TLC plates
coated with Kiesel-Gel 60 F254 were employed
to monitor the reactions using 2 N H2SO4 and
heating at 120 °C.
Methyl quinate (2).—Quinic acid 1 (1 g)
was dissolved in MeOH; concd H2SO4 was
added in catalytic quantity. The reaction mix-
ture was allowed to stand at rt overnight.
When the reaction was complete as evidenced
by TLC (3:2 CHCl3–EtOAc), the solution was
neutralised with Dowex-21K (Cl−) previously
regenerated with NaOH. The mixture was
filtered on gooch and the filtrate was evapo-
rated under pressure to give methyl ester 2
(1.070 g, quantitative yield) as a white solid
which crystallised from MeOH, mp 118 °C
(Lit. 120 °C17). Compound 2 did not give a
correct carbon analysis but was pure enough
to be utilized in the next step. Quinic acid 1,
1H NMR (300 MHz, D2O): l 1.78 (m, 1 H,
H-2ax), 1.90 (m, 1 H, H-6ax), 1.96 (m, 1 H,
H-2eq), 2.07 (m, 1 H, H-6eq), 3.41 (dd, 1 H, J
3.3, 9.3 Hz, H-4ax), 3.90 (m, 1 H, H-3ax), 4.02
1
as a colourless amorphous solid. H NMR
(200 MHz, CDCl3): l 1.84 (s, 3 H, AcO), 1.90
(s, 3 H, AcO), 1.96 (s, 3 H, AcO), 2.05 (m, 4
H, H-2ax, H-2eq, H-6ax, H-6eq), 3.71 (s, 3 H,
CO2CH3), 4.98 (dd, J 4, 12 Hz, 1 H, H-4),
5.35 (m, 2 H, H-3, H-5). Anal. Calcd for
C14H20O9: C, 50.60; H, 6.07. Found: C, 50.48;
H, 6.19.
Methyl 3,4,5-tri-O-acetylshikimate (4).—
Triacetyl derivative 3 (500 mg) was dissolved
in pyridine; dropwise a solution of thionyl
chloride (0.2 mL) in pyridine (0.3 mL) was
added. The reaction mixture was allowed to
stand at rt. After 2 h a brown syrup was
formed; it was diluted with water and the
reaction product 4 was extracted with EtOAc;
the organic phase was washed first with 2 N
HCl and then with water. The organic extract
was dried on anhyd Na2SO4 and filtered; the
solvent was evaporated and the residue was
purified by chromatography (9:1 benzene–
EtOAc) to give 4 (285 mg, 61%) as a colour-
less solid. 1H NMR (200 MHz, CDCl3): l 2.00
(s, 3 H, AcO), 2.02 (s, 3 H, AcO), 2.03 (s, 3 H,
AcO), 2.41 (m, 1 H, H-6ax), 2.91 (m, 1 H,
H-6eq), 3.72 (s, 3 H, CO2CH3), 5.25 (m, 2 H,
H-4, H-5), 5.71 (m, 1 H, H-3), 6.85 (m, 1 H,
H-2). [h]D −129° (EtOH, c 1.88); Lit. −168°
(MeOH)14d. Anal. Calcd for C14H18O8: C,
53.50; H, 5.77. Found: C, 53.37; H, 5.86.
Methyl shikimate (5).—Triacetyl derivative
4 (500 mg) was dissolved in MeOH (10 mL);
an alcoholic solution of concd H2SO4 (0.5 mL
in 5 mL of MeOH) was added. The reaction
mixture was allowed to stand at rt overnight.
The reaction mixture was neutralised with a
methanolic suspension of barium carbonate;
the solution was filtered through Celite and
the solvent was evaporated under diminished
pressure. The residue was purified by chro-
matography (9:1 EtOAc–CHCl3) to give 5
(275 mg, 92%) as a colourless solid which
crystallised from AcOH–pet. ether, mp 112–
1
(m, 1 H, H-5eq). Methyl ester 2, H NMR
(300 MHz, D2O): l 2.01 (m, 4 H, H-2ax,
H-2eq, H-6ax, H-6eq), 3.49 (dd, 1 H, H-4ax),
3.69 (s, 3 H, CO2CH3), 4.05 (m, 2 H, H-3ax,
H-5eq). Anal. Calcd for C8H14O6: C, 46.60; H,
6.84. Found: C, 50.48; H, 6.19.
Methyl 3,4,5-tri-O-acetylquinate (3).—
Methyl ester 2 (500 mg) was dissolved in
pyridine (0.5 mL) and Ac2O (1 mL) was
added. The reaction mixture was allowed to
stand at rt. After 2 h, compound 3 was par-
tially formed as evidenced by TLC (9:1
CHCl3–EtOAc); after this time there is a part
of substrate that has not yet reacted; neverthe-
less the reaction was interrupted because
longer reaction times afforded principally the
1
113 °C (Lit. 113–114 °C14a). H NMR (200
MHz, D2O): l 2.19 (dd, 1 H, H-6ax), 2.67 (dd,
1 H, H-6eq), 3.68 (m, 1 H, H-5), 3.73 (s, 3 H,

28
A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
lowed by dropwise addition of thionyl chlo-
ride (0.4 mL). The reaction mixture was
warmed to rt and stirred for 2 h. After this
time, the reaction was complete (TLC 7:3
CHCl3–EtOAc). The mixture was diluted with
CHCl3 and was washed with water and brine
and dried on anhyd Na2SO4. The solvents
were evaporated under diminished pressure,
and the residue was purified by chromatogra-
phy (9:1 CHCl3–EtOAc) to give 8 (480 mg,
CO2CH3), 3.97 (m, 1 H, H-4), 4.36 (m, 1 H,
H-3), 6.77 (m, 1 H, H-2). Anal. Calcd for
C8H12O5: C, 51.06; H, 6.43. Found: C, 50.84;
H, 6.56.
Methyl 3,4-O-isopropylideneshikimate (6).—
Methyl shikimate 5 (500 mg) was dissolved in
anhyd acetone (5 mL); 2,2-dimethoxypropane
(5 mL) and Dowex-50 (H+) (catalytic quan-
tity) were added. The reaction mixture was
stirred at rt for 3 h. The mixture was filtered
on gooch and the filtrate was evaporated un-
der pressure to give 6 (596 mg, quantitative
yield). For its characterisation, a crude sample
(100 mg) was purified by chromatography (3:2
EtOAc–CHCl3) to give 6 (92 mg) as a colour-
less solid. 1H NMR (200 MHz, CDCl3): l 1.36
(s, 3 H, CCH3), 1.41 (s, 3 H, CCH3), 1.69 (bs,
1 H, OH), 2.23 (m, 1 H, H-6ax), 2.76 (dd, 1
H, H-6eq), 3.73 (s, 3 H, CO2CH3), 3.85 (m, 1
H, H-5), 4.05 (t, 1 H, H-4), 4.71 (m, 1 H,
H-3), 6.89 (m, 1 H, H-2). Anal. Calcd for
C11H16O5: C, 57.89; H, 7.07. Found: C, 57.73;
H, 7.18.
Methyl 5 - O - acetyl - 3,4 - O - isopropylide-
neshikimate (7).—Isopropylidene derivative 6
(100 mg) was dissolved in pyridine (0.5 mL);
Ac2O (1 mL) was added. The reaction mixture
was allowed to stand at rt for 1 h. After this
time, MeOH was added to the reaction mix-
ture and the solvents were concentrated under
diminished pressure. The residue was diluted
with EtOAc; to the organic phase 2 N HCl
acid was added; then the solution was washed
with water until neutralisation. The organic
extract was dried on anhyd Na2SO4, filtered
and evaporated under diminished pressure to
afford derivative 7 (118 mg, quantitative
yield). For its characterisation, a crude sample
(20 mg) was purified by chromatography (7:3
CHCl3–EtOAc) to give 7 (17 mg) as a colour-
less solid. 1H NMR (200 MHz, CDCl3): l 1.38
(s, 3 H, CCH3), 1.40 (s, 3 H, CCH3), 2.08 (s, 3
H, AcO), 2.25–2.38 (dd, 1 H, H-6ax), 2.72–
2.83 (dd, 1 H, H-6eq), 3.78 (s, 3 H, CO2CH3),
4.20 (t, 1 H, H-4), 4.71 (m, 1 H, H-3), 5.15 (m,
1 H, H-5), 6.89 (m, 1 H, H-2). Anal. Calcd for
C13H18O6: C, 57.77; H, 6.71. Found: C, 57.60;
H, 6.84.
1
97%) as a colourless oil. H NMR (200 MHz,
CDCl3): l 2.26–2.42 (m, 1 H, H-6a), 2.85 (m,
1 H, H-6b), 3.81 (s, 3 H, CO2CH3), 3.95 (m, 1
H, H-5), 4.87 (m, 1 H, H-4), 5.58 (m, 1 H,
H-3), 6.91 (m, 1 H, H-2). Anal. Calcd for
C8H10O6S: C, 41.02; H, 4.30; S, 13.69. Found:
C, 40.83; H, 4.42; S, 13.74.
Methyl 5-O-acetylshikimate 3,4-cyclic sulfite
(9).—Compound 8 (300 mg) was dissolved in
pyridine (0.5 mL); Ac2O (1 mL) was added.
The reaction mixture was allowed to stand at
rt for 1 h. After this time MeOH was added to
the reaction mixture and the solvents were
concentrated under diminished pressure. The
residue was diluted with EtOAc. To the or-
ganic phase 2 N HCl was added; then the
solution was washed with water until neutrali-
sation. The organic extract was dried on an-
hyd Na2SO4, filtered and evaporated under
pressure to give 9 (320 mg, 91%). Compound
9 is pure enough to be used in the following
synthetic step. For its characterisation, a
crude sample (20 mg) was purified by chro-
matography (9:1 CHCl3–EtOAc) to give 9 (17
1
mg) as a colourless oil. H NMR (200 MHz,
CDCl3): l 2.10 (s, 3 H, AcO), 2.38 (m, 1 H,
H-6ax), 2.89 (dd, 1 H, H-6eq), 3.79 (s, 3 H,
CO2CH3), 4.91 (m, 1 H, H-4), 5.11 (m, 1 H,
H-5), 5.54 (m, 1 H, H-3), 6.91 (m, 1 H, H-2).
Anal. Calcd for C10H12O7S: C, 43.48; H, 4.38;
S, 11.60. Found: C, 40.33; H, 4.49; S, 11.52.
Methyl (3S,4R,5R)-5-acetoxy-3-azido-4-
hydroxycyclohex-1-ene-1-carboxylate (10).—
Compound 9 (200 mg) was dissolved in anhyd
DMF (4 mL); sodium azide (200 mg) was
added. The mixture was stirred at rt for 20 h.
The reaction mixture was then diluted with
EtOAc, washed with saturated ammonium
chloride, water, and brine, and dried on anhyd
Na2SO4. The solvents were evaporated and
the residue was purified by chromatography
Methyl shikimate 3,4-cyclic sulfite (8).—To
a solution of 5 (400 mg) in THF (8 mL) at
0 °C was added triethylamine (0.8 mL), fol-

A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
29
SO2CH3), 3.77 (s, 3 H, CO2CH3), 4.31 (m, 1
H, H-3), 4.77 (m, 1 H, H-4), 5.20 (m, 1 H,
H-5), 5.61 (d, 1 H, NH), 7.19 (m, 1 H, H-2).
Anal. Calcd for C13H19NO8S: C, 44.69; H,
5.48; N, 4.01; S, 9.18. Found: C, 44.31; H,
5.62; N, 4.15; S, 9.32.
(4:1 CHCl3–EtOAc) to give 10 (168 mg, 92%)
1
as an oil. H NMR (200 MHz, CDCl3): l 2.08
(s, 3 H, AcO), 2.13–2.32 (m, 1 H, H-6ax),
2.79–2.98 (m, 1 H, H-6eq), 3.70 (s, 3 H,
CO2CH3), 3.79 (m, 1 H, H-4), 4.11 (m, 1 H,
H-3), 4.98 (m, 1 H, H-5), 6.62 (m, 1 H, H-2).
IR: 2090 cm−1 (N3). Anal. Calcd for
C10H13N3O5: C, 47.06; H, 5.13; N, 16.46.
Found: C, 46.82; H, 5.21; N, 16.33.
Methyl (3S,4S,5R)-5-acetoxy-3,4-(acetyle-
pimino)cyclohex-1-ene-1-carboxylate (13).—
Compound 12 (100 mg) was dissolved in THF
(10 mL); NaH (25 mg) was added. The reac-
tion mixture was allowed to stand at rt for 4
h. The excess NaH was neutralised with CO2;
then the volatile material was evaporated, the
crude residue was purified by chromatography
(1:1 CHCl3–EtOAc) to give 13 (48 mg, 65%)
Methyl (3S,4R,5R)-5-acetoxy-3-azido-4-
(methylsulfonyloxy)cyclohex - 1 - ene - 1 - car-
boxylate (11).—Compound 10 (200 mg) was
dissolved in CH2Cl2 (10 mL) and to this solu-
tion, at 0 °C, was added triethylamine (0.4
mL) followed by dropwise addition of
methanesulfonyl chloride (0.2 mL). The reac-
tion mixture was stirred at 0 °C for 1 h. Then
it was diluted with CH2Cl2. The organic phase
was washed with water and brine, dried on
anhyd Na2SO4, and the volatile materials were
removed under diminished pressure. The
residue was purified by chromatography (9:1
CHCl3–EtOAc) to give 11 (186 mg, 72%) as
1
as an oil. H NMR (200 MHz, CDCl3): l 2.10
(s, 3 H, NAc), 2.16 (s, 3 H, AcO), 2.35 (m, 1
H, H-6ax), 2.85 (m, 1 H, H-6eq), 3.14 (m, 2
H, H-3, H-4), 3.77 (s, 3 H, CO2CH3), 5.58 (m,
1 H, H-5), 7.19 (m, 1 H, H-2). Anal. Calcd for
C11H15NO5: C, 56.91; H, 5.97; N, 5.53. Found:
C, 56.70; H, 6.07; N, 5.47.
Methyl (3R,4S,5R)-4-acetamido-5-acetoxy-
3-azidocyclohex-1-ene-1-carboxylate (14).—A
mixture of 13 (70 mg), sodium azide (70 mg),
and ammonium chloride (35 mg) in DMF (4
mL) was stirred at rt for 20 h. The reaction
mixture was diluted with EtOAc, washed with
water and brine, and dried on anhyd Na2SO4.
The solvent was evaporated under diminished
pressure, and the residue was purified by chro-
matography (1:1 CHCl3–EtOAc) to give a
mixture of products which was dissolved in
Ac2O (1 mL) and stirred for 2 h. Excess Ac2O
was evaporated to afford 14 (52 mg, 61%) as
1
an oil. H NMR (200 MHz, CDCl3): l 2.12 (s,
3 H, AcO), 2.32–2.51 (m, 1 H, H-6ax), 2.91–
3.09 (m, 1 H, H-6eq), 3.12 (s, 3 H, SO2CH3),
3.79 (s, 3 H, CO2CH3), 4.30 (m, 1 H, H-3),
4.77 (m, 1 H, H-4), 5.19 (m, 1 H, H-5), 6.72
(m, 1 H, H-2). Anal. Calcd for C11H15N3O7S:
C, 39.64; H, 4.54; N, 12.61; S, 9.62. Found: C,
39.48; H, 4.61; N, 12.53; S, 9.48.
Methyl (3S,4R,5R)-3-acetamido-5-acetoxy-
4 - (methylsulfonyloxy)cyclohex - 1 - ene - 1 - car-
boxylate (12).—To a solution of 11 (200 mg)
in THF (15 mL) was added triphenylphos-
phine (140 mg) and the solution was stirred at
rt for 3 h. The solvent was evaporated, the
crude residue was dissolved in CH2Cl2 (15
mL) and cooled at 0 °C, and pyridine (0.5
mL) was added followed by dropwise addition
of acetyl chloride (0.25 mL). The reaction
mixture was stirred at 0 °C for 1 h. Then it
was diluted with EtOAc. The organic phase
was washed with water and brine, dried on
anhyd Na2SO4, and the volatile materials were
removed under diminished pressure. The
residue was purified by chromatography (3:2
EtOAc–CHCl3) to give 12 (106 mg, 50%) as
1
an oil. H NMR (200 MHz, CDCl3): l 2.08 (s,
3 H, NHAc), 2.12 (s, 3 H, AcO), 2.51 (m, 1 H,
H-6ax), 2.91 (m, 1 H, H-6eq), 3.78 (s, 3 H,
CO2CH3), 4.08 (m, 1 H, H-4), 4.31 (m, 1 H,
H-3), 5.05 (m, 1 H, H-5), 5.53 (d, 1 H, NH),
6.76 (m, 1 H, H-2). IR: 2090 cm−1 (N3). Anal.
Calcd for C12H16N4O5: C, 48.65; H, 5.44; N,
18.91. Found: C, 48.33; H, 5.62; N, 18.70.
Methyl (3R,4S,5R)-4-acetamido-5-acetoxy-
3-aminocyclohex-1-ene-1-carboxylate (15).—
Compound 14 (50 mg) was dissolved in EtOH
(2 mL) and treated with the Lindlar catalyst
(10 mg) and stirred at rt under 101 kPa of
hydrogen for 12 h. The reaction mixture was
filtered through Celite, and the catalyst was
washed with 1:1 hot MeOH–water. The
1
an oil. H NMR (200 MHz, CDCl3): l 2.08 (s,
3 H, NHAc), 2.15 (s, 3 H, AcO), 2.34 (m, 1 H,
H-6ax), 2.90 (m, 1 H, H-6eq), 3.12 (s, 3 H,

30
A. Bianco et al. / Carbohydrate Research 332 (2001) 23–31
(40 mg, 84%), as a colourless amorphous
filtrate was evaporated on a rotatory evapora-
tor. The residue was purified by chromatogra-
phy (1:1 MeOH–CHCl3) to give 15 (32 mg,
70%) as a colourless amorphous solid. The
compound did not give a correct nitrogen
analysis, but was pure enough for further use.
1H NMR (200 MHz, CDCl3): l 2.06 (s, 3 H,
NHAc), 2.10 (s, 3 H, AcO), 2.35 (m, 1 H,
H-6ax), 2.83 (dd, 1 H, H-6eq), 3.75 (s, 3 H,
CO2CH3), 3.92–4.08 (m, 2 H, H-3, H-4), 5.01
(m, 1 H, H-5), 5.56 (d, 1 H, NH), 6.26 (m, 1
H, H-2). Anal. Calcd for C12H18N2O5: C,
53.33; H, 6.71; N, 10.36. Found: C, 53.28; H,
6.82; N, 9.51.
1
solid. H NMR (200 MHz, D2O): l 1.98 (s, 3
H, NHAc), 2.19 (m, 1 H, H-6a), 2.67 (m, 1 H,
H-6b), 3.68 (m, 1 H, H-5), 3.99 (m, 1 H, H-4),
4.24 (m, 1 H, H-3), 6.66 (m, 1 H, H-2). Anal.
Calcd for C10H16N4O4: C, 46.87; H, 6.29; N,
21.86. Found: C, 46.68; H, 6.13; N, 21.62.
Acknowledgements
This work was supported by Istituto Pas-
teur-Fondazione Cenci Bolognetti, Universita`
di Roma ‘La Sapienza’.
Methyl (3R,4S,5R)-4-acetamido-5-acetoxy-
3-[2,3-bis(tert-butoxycarbonyl)guanidino]cyclo-
hex-1-ene-1-carboxylate (16).—To a solution
of amine 15 (100 mg), N,N%-bis(tert-butoxy-
carbonyl)thiourea (110 mg) and triethylamine
(15 mL) in dry DMF (5 mL) cooled to 0 °C
was added HgCl2 (100 mg). The heteroge-
neous reaction mixture was stirred for 45 min
at 0 °C and then at rt for 15 min, after which
the reaction was diluted with EtOAc and
filtered through a pad of Celite. The volatile
materials were removed under diminished
pressure. The residue was chromatographed
(9:1 EtOAc–hexane) to give 16 (156 mg, 84%)
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1
as a pale oil. H NMR (200 MHz, CDCl3): l
1.45 (s, 18 H, t-Boc), 1.95 (s, 3 H, NHAc),
2.01 (s, 3 H, OAc), 2.33–2.45 (m, 1 H, H-6a),
2.83 (m, 1 H, H-6b), 3.77 (s, 3 H, CO2CH3),
4.06–4.13 (m, 1 H, H-4), 4.32–4.43 (m, 1 H,
H-3), 4.98 (m, 1 H, H-5), 6.46 (d, 1 H, NH),
6.89 (m, 1 H, H-2). Anal. Calcd for
C23H36N4O9: C, 53.91; H, 7.03; N, 10.93.
Found: C, 53.77; H, 7.18; N, 10.85.
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hydroxycyclohex-1-ene-1-carboxylic acid (17).
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solution). The reaction mixture was stirred at
rt for 17 h, cooled at 0 °C, neutralised by
bubbling CO2, and acidified with Dowex-50
(H+). The mixture was stirred at rt for 4 h.
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volatile materials were removed under dimin-
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reverse-phase chromatography on Lichroprep
RP-8 E. Merck eluting with water to give 17
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31
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.
.