2-Deoxy-2,3-didehydro-N-acetylneuraminic acid analogs structurally modified by thiocarbamoylalkyl groups at the C-4 position: Synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1

Article abstract of DOI:10.1016/j.bmc.2008.05.055

4-O-Thiocarbamoylmethyl-Neu5Ac2en 3 has strong inhibitory activity toward human parainfluenza virus type 1 (hPIV-1) sialidase compared with the parent Neu5Ac2en 2. We synthesized analogs having thiocarbamoylethyl- 4 and thiocarbamoylpropyl group 5 at the C-4 position of 2. The inhibition degrees of 4 and 5 were weaker than that of thiocarbamoylmethyl analog 3, indicating a remarkable effect of the carbon chain length in thiocarbamoylalkyl groups at the C-4 position on inhibitory activities against hPIV-1 sialidase.

Full text of DOI:10.1016/j.bmc.2008.05.055

Bioorganic & Medicinal Chemistry 16 (2008) 6783–6788
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Deoxy-2,3-didehydro-N-acetylneuraminic acid analogs structurally
modified by thiocarbamoylalkyl groups at the C-4 position: Synthesis
and biological evaluation as inhibitors of human parainfluenza virus type 1
a
a
b
Kiyoshi Ikeda a, , Kazuki Sato , Reiko Nishino , Shinya Aoyama , Takashi Suzuki b,c, Masayuki Sato a
*
a Department of Organic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
b Department of Biochemistry, School of Pharmaceutical Sciences and Global COE Program for Innovation in Human Health Sciences,
University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
c CREST, Japan Science and Technology Agency, Saitama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
4-O-Thiocarbamoylmethyl-Neu5Ac2en 3 has strong inhibitory activity toward human parainfluenza
virus type 1 (hPIV-1) sialidase compared with the parent Neu5Ac2en 2. We synthesized analogs having
thiocarbamoylethyl- 4 and thiocarbamoylpropyl group 5 at the C-4 position of 2. The inhibition degrees
of 4 and 5 were weaker than that of thiocarbamoylmethyl analog 3, indicating a remarkable effect of the
carbon chain length in thiocarbamoylalkyl groups at the C-4 position on inhibitory activities against
hPIV-1 sialidase.
Received 13 March 2008
Revised 22 May 2008
Accepted 28 May 2008
Available online 12 June 2008
Keywords:
Sialic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Human parainfluenza virus type 1 sialidase
inhibition
4-O-Thiocarbamoylethyl-Neu5Ac derivative
4-O-Thiocarbamoylpropyl Neu5Ac
derivative
1. Introduction
xyl group at C-4 position. As part of a program aimed at new sial-
idase inhibitors against hPIV-1, we describe herein the synthesis of
Human parainfluenza virus type 1 (hPIV-1), which belongs to
genus Respirovirus, family Paramyxovididae, is a serious human
pathogen causing upper and lower respiratory disease and is
known to be a cause of laryngotracheobronchitis (croup) in infants
and young children1; however, there is no known potential inhib-
itor of hPIV-1 infection. N-Acetylneuraminic acid (1, Neu5Ac) and
various related derivatives, sialic acids, play an important role in
various biochemical and biological processes.2 Influenza sialidase,
a key enzyme responsible for propagation of the influenza virus,
is a target of drug design. A variety of 2-deoxy-2,3-dehydro-N-
acetylneuraminic acid (Neu5Ac2en) analogs (2) have been synthe-
sized as competitive sialidase inhibitors.3 Neu5Ac2en derivatives
with structural modifications at the C-4 position are particular
candidates for the design of potent inhibitors against anti-para-
myxovirus agents.4 We found that 4-O-thiocarbamoylmethyl-
Neu5Ac2en (3)5 has strong inhibitory activity toward hPIV-1
sialidase compared with 2 (Fig. 1).
analogs having thiocarbamoylethyl- 4 and thiocarbamoylpropyl
group 5 at the C-4 position of Neu5Ac2en 2 and their inhibitory
activities against hPIV-1 sialidase.
2. Results and discussion
2.1. Chemical synthesis
As outlined in Scheme 1, the synthesis of compounds 4 and 5
beganwithcompounds7and8asthekeyintermediate,respectively.
For the synthesis of 4-O-cyanoalkylated analogs of Neu5Ac2en 7
and 8, methyl 5-acetamido-8,9-O-isopropylidene-2,3,5-tri-deoxy-
D
-glycero-D
-galacto-non-2-enopyranoso-nate 66 was chosen as the
starting material. First, for the synthesis of 7, the reaction of 6 with
3-bromopropionitrile in the presence of sodium hydride in dimeth-
ylformamide (DMF) at room temperature resulted in the recovery
of 6 by b-elimination of 4-O-thiocarbamoylethyl moiety of 7, due
to retro-Michael reaction under strongly basic conditions (Table
1, entry 1). Selective 4-O-cyanoethylation of 6 with 3-bromopropi-
onitrile in the presence of silver oxide and a catalytic amount
of tetra-n-butylammonium iodide (TBAI) in DMF at 50 °C success-
fully gave 4-O-cyanoethyl derivative 7 in 58% yield (entry 2).
We were interested in comparing inhibitory activity against
hPIV-1 sialidase of a few thiocarbamoylalkyl groups in the hydro-
* Corresponding author. Tel./fax: +81 54 264 5108.
E-mail address: ikeda@ys2.u-shizuoka-ken.ac.jp (K. Ikeda).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.055

6784
K. Ikeda et al. / Bioorg. Med. Chem. 16 (2008) 6783–6788
Figure 1.
Scheme 1. Synthesis of 4 and 5.
Table 1
Synthesis of 7 and 8
Entry
Conditions
Time (h)
1
12
12
1
Products
Yieldsa (%)
1
2
3
4
5
BrCH2CH2CN, NaH, DMF, rt
BrCH2CH2CN Ag2O, TBAI, DMF, 50 °C
CH2@CHCN, DBU, CH3CN, 0 °C
BrCH2CH2CH2CN, NaH, DMF, rt
BrCH2CH2CH2CN, Ag2O, TBAI, DMF, rt
7
7
7
8
8
N.R.b
58
70
50
70
12
a
Isolated yields alter purification.
No reaction.
b
Interestingly, the Michael addition reaction of 6 with 10 molar
equivalent of acrylonitrile in the presence of 1.0 molar equivalent
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrile at
0 °C smoothly proceeded to give 7 in 70% yield (entry 3). For the
preparation of 8, 4-O-cyanopropylation of 6 with 4-bromobutyro-
nitrile in the presence of sodium hydride in DMF at room temper-
ature successfully afforded 4-O-cyanopropyl derivative 8 in 50%
yield (entry 4). The reaction of 10 molar equivalent of 4-bromobu-
tyronitrile and 5.0 molar equivalent of silver oxide in DMF gave
compound 8 in 70% yield (entry 5).
Thiocarbamoylalkylation of the C-4 position of 7 and 8 by a pre-
viously reported method7 with AcSH–pyridine did not proceed.
Therefore, the isopropylidene group of 7 and 8 was removed with
80% AcOH at 80 °C for 1 h and the resulting alcohols were subse-
quently acetylated to give cyanoalkyl compounds 9 and 10 in
92% and 99% yields over two steps, respectively (Scheme 2).
For the conversion of cyanoalkyl groups of 9 and 10 to the thi-
ocarbamoyl group, we examined the thiocarbamoylation of 9 and
10 using AcSH–pyridine in CH2Cl2; however, the reaction did not
proceed (Table 2, entries 1 and 5). When using BF3ꢀOEt2 instead
of pyridine, the reaction gave the expected compound 12 in 63%
yield, in contrast to the result of 9 (entries 2 and 6). The thiolysis
of nitrile group of 10 with AcSH and BF3ꢀOEt2 involves the interme-
diacy of the acylthioimidate.8 Treatment of 9 with AcSH–trimeth-
ylsilyl chloride in CH2Cl2 resulted in the recovery of 9 (entry 3).
Interestingly, the thiocarbamoylation of 9 with AcSH–BnNH2 gave
11 in 33% yield (entry 4).
Treatment of 12 with 0.1 M KOH–MeOH (1:1) for 12 h at room
temperature afforded the expected compound 5 in 70% yield; how-
ever, hydrolysis of 11 with 0.1 M KOH–MeOH (1:1) for 12 h at
room temperature led to b-elimination of 4-O-thiocarbamoylethyl
moiety to give 2 under strong basic conditions. Therefore, we fo-
cused on the search for an efficient method in mild conditions
for the hydrolysis of methyl ester residue of 11 without b-elimina-
tion of 4-O-thiocarbamoylethyl moiety.
Pig liver esterase (PLE)9 has been successfully used in the
smooth hydrolysis of b-substituted methyl esters, specifically in
substrates susceptible to b-elimination under strong basic condi-
tions.10 We examined the enzymatic hydrolysis of methyl ester
group of 13 by PLE in neutral aqueous conditions. Thus, deprotec-
tion of acetyl groups of 11 with trimethylsilyl chloride in MeOH
was performed to afford 13 in 76% yield. Hydrolysis of 13 with

K. Ikeda et al. / Bioorg. Med. Chem. 16 (2008) 6783–6788
6785
Scheme 2. Synthesis of 9 and 10.
Table 2
Synthesis of 11 and 12
Entry
Substrate
Conditions
Product
Yielda (%)
1
2
3
4
5
6
9
9
9
9
10
10
AcSH, pyridine, CH2Cl2, 12 h
AcSH, BF3OEt2, CH2Cl2, 12 h
AcSH, TMSCI, CH2Cl2, 12 h
AcSH, BnNH2, 48 h
AcSH, pyridine, CH2Cl2, 12 h
AcSH, BF3OEt2, CH2Cl2, 12 h
11
11
11
11
12
12
N.R.b
N.R.
N.R.
33
N.R.
63
a
Isolated yields after purification.
No reaction.
b
PLE (Sigma) in 0.01 M potassium phosphate buffer, pH 7.0, at
35 °C for 20 h successfully afforded 4 in quantitative yield, after
purification by chromatography on silica gel and then desalting
by Bio-Gel P-2, followed by lyophilization from a H2O suspension
(Scheme 3). This is the first example of enzymatic hydrolysis of
methyl ester of sialic acids using PLE in neutral aqueous
conditions.
microsphere that interacts with the C-4 position of Neu5Ac2en in
the cavity of the catalytic pocket in HN glycoprotein. These find-
ings should provide useful information for the development of
anti-human parainfluenza virus compounds.
3. Experimental
All melting points are uncorrected. Optical rotations were mea-
sured with a JASCO P-1030 (Japan) digital polarimeter. IR spectra
were recorded on a SHIMADZU IRPrestige-21 (Japan) spectrome-
2.2. Biological evaluation
ter. 1H NMR spectra were recorded with
a JEOL ECA-500
The behavior of compounds 4 and 5 toward hPIV-1 sialidase was
(500 MHz) (Japan) instrument. 13C NMR spectra were recorded
with a JEOL ECA-500 (126 MHz) (Japan) instrument. Chemical
shifts are expressed in ppm relative to Me4Si (d = 0) in CDCl3
and in D2O referenced to HOD (4.85 ppm) as internal standards.
Fast-atom-bombardment (FAB) mass spectra were obtained with
a JEOL JMS-700 (Japan) mass spectrometer in the positive-ion
mode using an NBA. High resolution mass spectra (HR-MS) were
recorded on a JEOL JMS-700 (Japan) instrument under Fab condi-
tions. Column chromatography was performed on silica gel 60
(70–230 mesh, Merck). Desalting was carried out with an ASAHI
CHEMICAL Micro Acylizer G1. All reactions were monitored using
TLC (silica gel 60F254, E. Merck, Germany) by charring after spray-
ing 5% H2SO4 in MeOH and then heating.
tested by our previously reported method.5b As can be seen in Table
3, 4-O-thiocarbamoylethyl-
Neu5Ac 5 had inhibitory activities IC50 68
respectively. However, the degree of inhibition was lower toward
hPIV-1 sialidase than 4-O-thiocarbamoylethyl-3 (IC50 9 M).
In conclusion, 4-O-thiocarbamoylethyl-4 and 4-O-thiocarba-
moylpropyl-Neu5Ac 5 were synthesized via the key compound 6.
Compounds 4 and 5 exhibited decreased sialidase inhibition com-
pared with 3. The reason for the lower inhibitory activity of com-
pounds 4 and 5 is unclear in this study, but it was found that the
difference in the carbon chain length of the substituent in the hy-
droxyl group at the C-4 position of 2 might affect inhibitory activ-
ities against hPIV-1 sialidase. It is possible that hPIV-1 has a
4
and 4-O-thiocarbamoylpropyl-
l
M and IC50 102 M,
l
l

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K. Ikeda et al. / Bioorg. Med. Chem. 16 (2008) 6783–6788
Scheme 3. Synthesis of 4 and 5.
For the preparation of 7 using acrylonitrile11 and DBU, to a solu-
Table 3
Inhibitory activities of 4 and 5
tion of 6 (111 mg, 0.32 mmol) and acrylonitrile (170 mg, 3.2 mmol)
in CH3CN (2 mL) was added DBU (49 mg, 0.32 mmol) at 0 °C, and
the mixture was allowed to stir for 12 h at the same temperature
under Ar. After the addition of saturated aqueous NH4Cl solution
to the reaction mixture, the mixture was extracted with chloro-
form and the organic layer was washed with saturated aqueous
NaCl solution, dried over anhydrous MgSO4, and concentrated to
dryness. The residue was chromatographed by silica gel with
CHCl3–MeOH (50:1) to give 7 (89 mg, 70%).
a
Entry
Compound
IC50 (lM)
1
2
3
4
2
3
4
5
300
9
68
102
a
Inhibitory activities were determined by the method according to Ref. 5b.
3.1. Methyl 5-acetamido-2,6-anhydro-4-O-(2-cyanoethyl)-3,
5-dideoxy-8,9-O-isopropylidene-D-glycero-D-galacto-non-2-
enonate (7)
3.1.1. Methyl 5-acetamido-2,6-anhydro-4-O-(3-cyanopropyl)-3,
5-dideoxy-8,9-O-isopropylidene-D-glycero-D-galacto-non-2-
enonate (8)
Under argon, sodium hydride (22 mg, 0.92 mmol) at 0 °C was
added to a solution of 6 (245 mg, 0.71 mmol), propargyl bromide
(172 mg, 1.42 mmol) in anhydrous DMF (5 mL) was added, and
the mixture was stirred for 1 h at the same temperature. After
the addition of MeOH (1 mL), the solvent was concentrated to dry-
ness. The residue was chromatographed by silica gel with 50:1
CHCl3–MeOH to give 8 (142 mg, 52%). IR (KBr): 3275, 2254, 1724,
Compound 6 (90 mg, 0.26 mmol) was dissolved in anhydrous
DMF (5 mL) and stirred for 1 h with freshly activated MS 4 Å
(0.30 g). To the mixture were added 3-bromopropionitrile
(55 mg, 0.39 mmol), Ag2O (302 mg, 1.3 mmol), and TBAI (48 mg,
0.13 mmol), and the mixture was allowed to stir for 48 h in the
dark under an argon atmosphere. Insoluble materials were filtered
through a Celite 545 and the filtrate was concentrated to dryness.
The resulting residue was chromatographed by silica gel with
1645 cmꢂ1 1H NMR (500 MHz, CDCl3) d:1.33, 1.38 (s, each 3H),
.
1.83–1.96 (m, 2H), 2.08 (s, 3H), 2.39–2.54 (m, 2H), 3.52–3.56 (m,
2H), 3.73–3.81 (m, 1H), 3.76 (s, 3H), 4.02 (m, 1H), 4.07 (dd, 1H,
J = 5.2, 9.2 Hz), 4.12–4.18 (m, 2H), 4.23 (dd, 1H, J = 2.3, 8.6 Hz),
4.32 (ddd, J = 6.3, 5.2, 2.9 Hz), 4.58 (br, 1H), 6.01 (d, 1H,
J = 2.3 Hz), 6.09 (d, 1H, J = 7.5 Hz). 13C NMR (126 MHz, CDCl3)
d:13.8, 14.1, 23.1, 25.2, 25.3, 27.1, 48.4, 52.5, 65.6, 67.2, 70.1,
74.3, 74.3, 107.2, 109.2, 119.6, 145.7, 162.3, 173.0. Positive-ion
FABMS (NBA): m/z 413 [M+H]+, 435 [M+Na]+. Positive-ion HR-FAB-
MS (NBA) (m/z) Calcd for C19H29N2O8 [M+H]+: 413.1924, Found
413.1942.
CHCl3–MeOH (50:1) to give 7 (52 mg, 56%). ½a D24
ꢁ
+48.4 (c 0.33,
.
CHCl3). IR (KBr): 3286, 2243, 1724, 1645 cmꢂ1
1H NMR
(500 MHz, CDCl3) d: 1.35, 1.39 (s, each 3H), 2.09 (s, 3H), 2.55–
2.68 (m, 2H), 3.56 (dd, 1H, J = 4.6, 7.5 Hz), 3.66 (ddd, 1H, J = 4.6,
5.2, 9.8 Hz), 3.79 (s, 3H), 3.89 (ddd, 1H, J = 4.6, 5.2, 9.8 Hz), 4.07
(d, 1H, J = 10.9 Hz), 4.09 (dd, 1H, J = 4.6, 8.6 Hz), 4.14–4.20 (m,
2H), 4.45 (m, 1H), 4.37 (dd, 1H, J = 2.3, 8.6 Hz), 4.52 (d, 1H), 5.73
(d, 1H), 6.00 (d, 1H,J = 2.3 Hz). 13C NMR (126 MHz, CDCl3) d: 19.4,
23.1, 25.2, 27.0, 48.1, 52.4, 62.3, 67.2, 70.0, 74.1, 75.0, 77.3, 106.4,
109.2, 118.2, 146.1, 162.1, 173.1. Positive-ion FABMS (NBA): m/z
399 [M+H]+, 421 [M+Na]+. Positive-ion HR-FABMS (NBA) (m/z)
Calcd for C18H27N2O8 [M+H]+: 399.1767, Found 399.1744.
For the preparation of 8 using 4-bromobutylonitrile and Ag2O,
compound 6 (200 mg, 0.58 mmol) was dissolved in anhydrous
DMF (5 mL) and stirred for 1 h with freshly activated MS 4 Å

K. Ikeda et al. / Bioorg. Med. Chem. 16 (2008) 6783–6788
6787
(0.30 g). To the mixture were added 4-bromobutylonitrile (522 mg,
3.53 mmol), Ag2O (670 mg, 2.89 mmol), and TBAI (110 mg,
0.29 mmol), and the mixture was allowed to stir for 12 h in the
dark under argon. Insoluble materials were filtered through a Cel-
ite 545 and the filtrate was concentrated to dryness. The residue
was chromatographed by silica gel with CHCl3–MeOH (50:1) to
give compound 8 (154 mg, 64%).
3.1.5. Methyl 5-Acetamido-2,6-anhydro-4-O-(3-thiocarbamoyl-
propyl)-3,5-dideoxy-7,8,9-tri-O-acetyl-D-glycero-D-galacto-non-
2-enonate (12)
To a solution of compound 10 (170 mg, 0.341 mmol) and thio-
acetic acid (129 mg, 1.70 mmol) was added BF3ꢀOEt2 (126 mg,
0.89 mmol) at 0 °C and the mixture was stirred for 12 h at room
temperature. The reaction mixture was concentrated to dryness.
The residue was chromatographed by silica gel with CHCl3–MeOH
(50:1) to give 11 (26 mg, 33%). 1H NMR (500 MHz, CDCl3) d:1.96–
2.00 (m, 2H), 1.96, 2.02, 2.02, 2.06 (s, each 3H), 2.71 (dd, 2H,
J = 6.9, 7.5 Hz), 3.54 (m, 1H), 3.69 (m, 1H), 3.79 (s, 3 H), 3.88 (dd,
1H, J = 4.0, 4.0 Hz), 4.13 (dd, 1H, J = 7.5, 12.0 Hz), 4.34 (dd, 1H,
J = 4.6, 6.9 Hz), 4.40 (ddd, 1H, J = 4.0, 4.6, 4.6 Hz), 4.51 (dd, 1H,
J = 4.0, 12.0 Hz), 5.46 (ddd, J = 3.5, 7.5, 4.0 Hz), 5.60 (dd, 1H,
J = 6.9, 3.5 Hz), 5.96 (br d, 1H, J = 4.6 Hz), 6.20 (d, 1H, J = 4.0 Hz),
7.66 (br s, 1H), 8.14 (br s, 1H). 13C NMR (126 MHz, CDCl3) d:
20.7, 20.8, 20.9, 23.2, 29.2, 41.6, 47.0, 52.6, 61.8, 67.3, 68.3, 69.7,
71.3, 76.0, 108.2, 143.3, 162.0, 170.1, 170.3, 170.3, 170.8, 210.3. Po-
sitive-ion FABMS (NBA): m/z 533 [M+H]+, 555 [M+Na]+. Positive-
3.1.2. Methyl 5-Acetamido-2,6-anhydro-4-O-(2-cyanoethyl)-3,
5-dideoxy-7,8,9-tri-O-acetyl-D-glycero-D-galacto-non-2-enonate
(9)
Compound 7 (104 mg, 0.261 mmol) was dissolved in 80% aque-
ous AcOH (3 mL). After stirring for 1 h at 80 °C, the reaction solu-
tion was concentrated to dryness, the residue was dissolved in a
solution of pyridine (3 mL) and acetic anhydride (1.5 mL) at 0 °C
and the mixture was allowed to stir for 12 h at room temperature.
The reaction mixture was evaporated to dryness. The resulting res-
idue was chromatographed by silica gel with CHCl3–MeOH (50:1)
to give 9 (116 mg, 92%).
1H NMR (500 MHz, CDCl3) d: 1.96, 2.02, 2.02, 2.09 (s, each 3H),
2.61 (dd, 2H, J = 5.7, 6.3 Hz), 3.73–3.85 (m, 2H), 3.77 (s, 3H), 4.14
(dd, 1H, J = 7.5, 12.0 Hz), 4.19–4.20 (m, 2H), 4.41 (dd, 1H, J = 6.9,
5.2 Hz), 4.52 (dd, 1H, J = 3.5, 12.0 Hz), 5.34 (ddd, 1H, J = 7.5, 3.5,
4.6 Hz), 5.51 (dd, 1H, J = 5.2, 4.6 Hz), 5.96 (br s, 1H), 6.10 (d, 1H).
13C NMR (126 MHz, CDCl3) d 19.0, 20.7, 20.7, 20.9, 23.2, 47.0,
52.6, 61.9, 63.4, 68.0, 69.9, 72.9, 76.0, 108.3, 117.9, 144.2, 161.9,
170.0, 170.1, 170.4, 170.7. Positive-ion FABMS (NBA): m/z 485
[M+H]+, 507 [M+Na]+. Positive-ion HR-FABMS (NBA) (m/z) Calcd
for C21H29N2O11 [M+H]+: 485.1771, Found 485.1786.
ion HR-FABMS (NBA) (m/z) Calcd for C22H33N2O11S
[M+H]+:
533.1805, Found 533.1793.
3.1.6. 5-Acetamido-2,6-anhydro-3,5-dideoxy-4-O-(3-thiocarba-
moylpropyl)- -glycero- -galacto-non-2-enonic acid (5)
D
D
A solution of 12 (142 mg, 0.267 mmol) in 0.1 M KOH–MeOH
(1:1) (2 mL) was allowed to stir at room temperature for 15 h,
and then adjusted to pH 2–3 by Amberlite IRA-120 (H+). The resin
was filtered off and the filtrate was evaporated to dryness. The res-
idue was chromatographed by silica gel with CHCl3/MeOH/H2O
(65:35:5, v/v), and then desalted with an AC Micro Acylizer G1 to
give 5 (73 mg, 70%) after lyophilization from a H2O suspension.
1H NMR (500 MHz, D2O) d:1.87–1.93 (m, 2H), 1.97 (s, 3H), 2.57–
2.65 (m, 2H), 3.50–3.57 (m, 3H), 3.66 (m, 1H), 3.78 (dd, 1H,
J = 2.3, 12.0 Hz), 3.83 (ddd, 1H, J = 5.8, 8.0, 2.3 Hz), 4.11 (dd, 1H,
J = 8.6, 10.9 Hz), 4.18 (d, 1H), 4.28 (dd, 1H, J = 2.3, 8.6 Hz), 5.96 (d,
1H). Positive-ion FABMS (NBA): m/z 393 [M+H]+.
3.1.3. Methyl 5-Acetamido-2,6-anhydro-4-O-(3-cyanopropyl)-3,
5-dideoxy-7,8,9-tri-O-acetyl-D-glycero-D-galacto-non-2-enonate
(10)
The reaction was carried out using compound 8 (61 mg,
0.148 mmol) in a manner similar to the preparation of 9 using so-
dium hydride as a base to give 10 (99%). 1H NMR (500 MHz, CDCl3)
d: 1.86–1.91 (m, 2H), 1.98, 2.04, 2.04, 2.11 (s, each 3H), 2.39–2.52
(m, 2H), 3.64 (ddd, 1H, J = 5.2, 5.7, 12.0 Hz), 3.76 (ddd, 1H, J = 5.7,
6.3, 12.0 Hz), 3.80 (s, 3H), 4.06 (dd, 1H, J = 3.4, 6.3 Hz), 4.16 (dd,
1H, J = 7.4, 12.0 Hz), 4.24 (ddd, 1H, J = 6.3, 6.9, 8.6 Hz), 4.38 (dd,
1H, J = 6.9, 5.2 Hz), 4.53 (dd, 1H, J = 3.4, 12.0 Hz), 5.36 (ddd, 1H,
J = 7.4, 3.4, 4.1 Hz), 5.52 (dd, 1H, J = 5.2, 4.1 Hz), 5.70 (d, 1H,
J = 8.6 Hz), 6.13 (d, 1H, J = 3.4 Hz). 13C NMR (126 MHz, CDCl3)
d:14.1, 20.7, 20.8, 20.9, 23.3, 25.5, 47.2, 52.6, 61.9, 66.6, 67.9,
70.0, 73.0, 76.1, 108.5, 119.5, 143.9, 162.0, 169.9, 170.1, 170.2,
170.6. Positive-ion FABMS (NBA): m/z 499 [M+H]+, 521 [M+Na]+.
3.1.7. Methyl 5-acetamido-2,6-anhydro-4-O-(2-thiocarbamoyl-
ethyl)-3,5-dideoxy-D-glycero-D-galacto-non-2-enonate (13)
To a solution of compound 11 (64 mg, 0.124 mmol) in MeOH
(3 mL) was added trimethylsilyl chloride (18 mg, 0.161 mmol) at
room temperature and the mixture was allowed to stir for 3 h at
the same temperature. The reaction mixture was concentrated to
dryness. The residue was chromatographed by silica gel with
CHCl3/MeOH/H2O (65:35:5, v/v) to give 13 (37 mg, 76%). 1H NMR
(500 MHz, D2O) d:1.97 (s, 3H), 2.72–2.81 (m, 2H), 3.53–3.57 (m,
2H), 3.73 (s, 3H), 3.78 (dd, 1H, J = 2.9, 12.0 Hz), 3.82 (ddd, 1H,
J = 8.6, 2.9, 5.8 Hz), 3.87 (m, 1H), 3.99 (m, 1H), 4.10 (dd, 1H,
J = 9.2, 10.9 Hz), 4.21 (d, 1H), 4.29 (dd, 1H, J = 2.3, 9.2 Hz), 6.11 (d,
1H, J = 2.3 Hz). Positive-ion FABMS (NBA): m/z 393 [M+H]+. Posi-
tive-ion HR-FABMS (NBA) (m/z) Calcd for C15H25N2O8S [M+H]+:
393.1332, Found 393.1337.
Positive-ion HR-FABMS (NBA) (m/z) Calcd for
C22H31N2O11
[M+H]+: 499.1928, Found 499.1951.
3.1.4. Methyl 5-Acetamido-2,6-anhydro-4-O-(2-thiocarbamoy-
lethyl)-3,5-dideoxy-7,8,9-tri-O-acetyl-D-glycero-D-galacto-non-
2-enonate (11)
Compound 9 (74 mg, 0.152 mmol) was dissolved in thioacetic
acid (2 mL). To the solution was added benzylamine (1 mL), and
the mixture was stirred for 48 h at room temperature. The reaction
mixture was concentrated to dryness. The residue was chromato-
graphed by silica gel with CHCl3–MeOH (50:1) to give 11 (26 mg,
33%). 1H NMR (500 MHz, CDCl3) d:1.97, 2.04, 2.06, 2.11 (s, each
3H), 2.61 (m, 2H), 3.81 (s, 3H), 3.90–3.94 (m, 2H), 4.03 (m, 1H),
4.18 (dd, 2H, J = 8.0, 12.0 Hz), 4.35 (dd, 1H, J = 5.7, 6.3 Hz), 4.39 (dd,
1H, J = 6.3, 5.2 Hz), 4.55 (dd, 1H, J = 8.0, 3.4, 12.0 Hz), 5.45 (ddd, 1H,
J = 8.0, 3.4, 4.6 Hz), 5.55 (dd, 1H, J = 5.2, 4.6 Hz), 5.64 (d, 1H), 6.20
(d, 1H), 7.53 (br s, 1H), 7.99 (br s, 1H). 13C NMR (126 MHz, CDCl3)
d: 20.6, 20.7, 20.9, 23.2, 45.4, 46.8, 52.6, 61.6, 67.8, 68.0, 69.7, 72.3,
75.8, 107.5, 143.4, 161., 170.0, 170.1, 170.4, 170.7, 207.7. Positive-
ion FABMS (NBA): m/z 519 [M+H]+, 541 [M+Na]+.
3.1.8. 5-Acetamido-2,6-anhydro-3,5-dideoxy-4-O-(3-thiocarba-
moylethyl)-D-glycero-D-galacto-non-2-enonic acid (4)
A solution of 13 (8.4 mg, 0.021 mmol) in 0.01 M potassium
phosphate buffer (pH 7.0) (2 mL) was incubated with PLE (Sigma,
7.0 mg) at 35 °C for 22 h, the insoluble materials were filtered off
and the filtrate was evaporated to dryness. The residue was chro-
matographed by silica gel with CHCl3/MeOH/H2O (65:35:5, v/v),
and then desalted with an AC Micro Acylizer G1 to give 5
(7.9 mg, quant.) after lyophilization from a H2O suspension. 1H
NMR (500 MHz, D2O) d:1.86 (s, 3H), 2.46 (m, 2H), 3.46 (m, 2H),
3.70 (m, 2H), 3.78 (m, 1H), 3.98 (m, 1H), 4.16 (m, 3H), 5.98 (d,
1H, J = 2.9 Hz). Positive-ion FABMS (NBA): m/z 401 [M+Na]+.

6788
K. Ikeda et al. / Bioorg. Med. Chem. 16 (2008) 6783–6788
3. (a) Ikeda, K.; Sato, M.; Torisawa, Y. Curr. Med. Chem. 2004, 3, 339; (b) Wilson, J.
Acknowledgements
C.; Thomson, R. J.; Dyason, J. C.; Florio, P.; Quelch, K. J.; Abo, S.; von Itzstein, M.
Tetrahedron: Asymm. 2000, 11, 53; (c) Smith, P. W.; Starkey, I. D.; Howes, P. D.;
Sollis, I. L.; Keeling, S. P.; Cherry, P. C.; von Itzstein, M.; Wu, W. Y.; Jin, B. Eur. J.
Med. Chem. 1996, 31, 143.
We thank Dr. Takeshi Sugai (Keio University) for helpful discus-
sion and encouragement. This work was financially supported in
part by a Grant-in Aid for Scientific Research No. 19590103 from
the Ministry of Education, Science, Sports, and Culture of Japan.
This work was partly supported by the Japan Health Science Foun-
dation. The authors thank Japan Food & Liquor Alliance, Inc. (Kyoto,
Japan) for the generous gift of Neu5Ac.
4. (a) Beau, J. M.; Schauer, R. Eur. J. Biochem. 1980, 106, 531; (b) Zbiral, E.;
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