Synthesis of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 and C-9 positions and their behaviour towards sialidase from influenza virus and pig liver membrane

Article abstract of DOI:10.1016/S0008-6215(00)00267-6

The synthesis of novel 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues structurally varied at C-4 and C-9 by transformation from versatile key intermediates and their inhibitory activity against sialidase from influenza virus A and pig liver membrane are described.

Full text of DOI:10.1016/S0008-6215(00)00267-6

www.elsevier.nl/locate/carres
Carbohydrate Research 330 (2001) 31–41
Synthesis of 2-deoxy-2,3-didehydro-N-acetylneuraminic
acid analogues modified at the C-4 and C-9 positions
and their behaviour towards sialidase from influenza
virus and pig liver membrane
Kiyoshi Ikeda*,a,1 Kimihiko Sano,a Masaki Ito,b Megumi Saito,b Kazuya Hidari,c
Takashi Suzuki,c Yasuo Suzuki,c Kiyoshi Tanaka*a,2
aDepartment of Synthetic Organic Chemistry and Medicinal Chemistry, School of Pharmaceutical Sciences,
Uni6ersity of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
bDepartment of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences,
Uni6ersity of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
cDepartment of Biochemistry, School of Pharmaceutical Sciences, Uni6ersity of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
Received 31 May 2000; accepted 10 September 2000
Abstract
The synthesis of novel 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues structurally varied at C-4 and
C-9 by transformation from versatile key intermediates and their inhibitory activity against sialidase from influenza
virus A and pig liver membrane are described. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Sialic acid; Sialidase inhibitor; Cyanomethyl group; Influenza virus sialidase; Mammalian sialidase
1. Introduction
against influenza virus.2 Several groups have
previously reported the inhibition of various
sialidase activities by a variety of 2-deoxy-
2,3 - didehydro - N - acetylneuraminic acid
(Neu5Ac2en, 1) derivatives.3 Compound 1 is
postulated to be a transition-state analogue
binding to the active site of sialidase.4 By
molecular modelling techniques, von Itzstein
and his co-workers reported the design and
biological evaluation of 4-amino-2,3-didehy-
dro-2,4-dideoxy-N-acetylneuraminic acid (4-
amino-Neu5Ac2en, 2) and its guanidino
analogue (4-guanidino-Neu5Ac2en, 3), which
exhibited selective inhibition for the viral over
mammalian sialidase.5 As a part of our ongo-
ing program aimed at the synthesis of new
sialidase inhibitors, we reported the chemoen-
Influenza virus sialidase is a surface enzyme
that is essential for infection of the virus.
Sialidase catalyzes the hydrolysis of a-glyco-
sidic bonds to the terminal sialic acid residues
of surface glycoconjugates on hemagglutinin
during virus budding and on the host cell
virus receptor to assist virus release and pre-
vent aggregation.1 Therefore, a sialidase has
been considered as a suitable and effective
target for designing chemotherapeutic agents
1 *Corresponding author. Tel.: +81-54-2645742; fax: +
81-54-2645745; e-mail: ikeda@ys2.u-shizuoka-ken.ac.jp
2 *Corresponding author.
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00267-6

32
K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
zymatic synthesis of 5,9-di-N-acyl-2,3-didehy-
dro-2,3-dideoxyneuraminic acids6a and 5,9-di-
N-acetyl-4-carbamoylmethyl-2,3-didehydro-
2,3-dideoxyneuraminic acid6b and their behav-
ior towards the sialidase from the influenza
virus. In this study, in order to ascertain the
importance of the 4- and 9-hydroxyl groups in
N-acetylneuraminic acid, replacement of the
hydroxyl groups at C-4 in N-acetylneuraminic
acid analogues by the cyanomethyl, carba-
moylmethyl, amidinomethyl, methoxyimino-
methyl, and thiocarbamoylmethyl groups was
carried out. Furthermore, the hydroxyl group
at C-9 was also modified by azido or N-acetyl
groups. Here we describe the synthesis of
these novel 2-deoxy-2,3-didehydro-N-acetyl-
neuraminic acid analogues modified at the C-4
and C-9 positions and their inhibitory activi-
ties against sialidase from both viral and
mammalian sources (Scheme 1).
the C-4 modified Neu5Ac2en (4a–c) com-
pounds was accomplished as shown in Scheme
2. The Zemple´n O-deacetylation of 78 with 0.1
M NaOMe in MeOHꢀCH2Cl2, followed by
protection by an isopropylidene group at C-
8,9 by dry acetone and IR120 (H+) resin gave
compound 8 in 81% yield. Selective introduc-
tion of the cyanomethyl group at C-4 of 8 by
BrCH2CN, Ag2O, and t-Bu4NI,9 and succes-
sive removal of the isopropylidene group with
aqueous 80% AcOH furnished the triol 9 in
47% overall yield. Acetylation of 9 with Ac2O
and pyridine provided compound 10 in 98%
yield. The methylene protons of the
1
cyanomethyl group in the H NMR spectrum
of 10 resonated at l 4.11, 4.35 (d, Jgem 16.5
Hz).
Compound 10 was transformed by treat-
ment with dimethyl(methylthio)sulfonium
triflate (DMTST) and then 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU)10 to give the key
1
compound 11 in 97% yield, showing in its H
NMR spectrum, a one-proton doublet at l
6.17 (J3,4 3.5 Hz, H-3) in 97% yield.
2. Results and discussion
The key intermediate 11 thereby in hand
was transformed into 4a–c using methodology
similar to that described by Malet and Hinds-
gaul.7 Thus, initial deacetylation of 11 and
hydrolysis of the methyl ester were carried out
with 0.1 M KOH–MeOH, and chromato-
graphic purification on silica gel gave the
cyanomethyl compound 4a {FABMS: m/z 353
[M+Na]+} in 49% yield. Next, for the syn-
thesis of the carbamoylmethyl compound 4b,
Compounds 11, 15 and 17 bearing a
cyanomethyl group on C-4 position were se-
lected as the key intermediates for the prepa-
ration of 4a–c, 5a–d, and 6a–d in our
synthesis. The cyanomethyl group is a ver-
satile functional group for the preparation of
sugar amides and amidine salts,7 which may
potentially induce new electrostatic and/or hy-
drogen-bonding interactions. The synthesis of
Scheme 1.

/
3
(
3
Scheme 2.

/
3
(
3
Scheme 3.

K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
35
Scheme 4.
groups, respectively. Acetylation of 13 with
Ac2O and pyridine provided compound 14 in
95% yield, which was similarly treated with
DMTST and then DBU to give 15 {FABMS:
m/z 454 [M+H]+} in 99% yield. In the same
way as described for the preparation of 4a–c,
compound 15 was converted into the corre-
11 was treated with 0.1 M NaOMe–MeOH,
followed by aqueous NaHCO3 to give 4b
{FABMS: m/z 371 [M+Na]+, 387 [M+K]+}
in 55% yield in two steps. For the synthesis of
the amidinomethyl compound 4c, 11 was suc-
cessively treated with 0.1 M NaOMe–MeOH,
anhydrous NH4Cl, and then 0.1 M KOH–
MeOH to afford 4c {FABMS: m/z 349 [M+
H]+, 371 [M+Na]+} in 57% yield in three
steps.
For the synthesis of 5a–d bearing an azide
group at C-9, compound 9 was converted into
the common intermediate 15 (Scheme 3).
Thus, selective tosylation at C-9 of 9 with
p-toluenesulfonyl chloride in pyridine–
CH2Cl2 (64% yield) and subsequent treatment
of 12 with NaN3 and 18-crown-611 in DMF
afforded 13 in 74% yield. The IR spectrum of
13 showed absorption bands at 2106 and 1736
cm−1, characteristic of the azido and ester
sponding
cyanomethyl
compound
5a
{FABMS: m/z 396 [M+K]+}, carbamoyl-
methyl compound 5b {FABMS: m/z 396
[M+Na]+}, and amidinomethyl compound
5c {FABMS: m/z 345 [M]+} in 80, 67, and
60% yields, respectively. Additionally, 15 was
treated with 0.1 M NaOMe–MeOH, and then
0.1 M KOH–MeOH to give 5d {FABMS: m/z
411 [M+Na]+} in 76% yield.
For the synthesis of 6a–d having an NAc
group at C-9 (Scheme 4), the transformation
to the common intermediate 17 from 15 was
examined. After unfruitful attempts to reduce

36
K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
the azide group of 15 using Pd–C, H2, selec-
tive reduction of the azide group of 15 was
effected with simultaneous acetylation by
AcSH–pyridine12 giving 16 {FABMS: m/z 504
[M+H]+, 526 [M+Na]+} in 79% yield.
Compound 16 was treated with 0.1 M KOH–
MeOH to give thiocarbamoylmethyl com-
pound 6a {FABMS: m/z 406 [M+H]+, 428
[M+Na]+} in 67% yield. The azido group of
15 was successfully reduced by use of PPh3,13
and subsequent acetylation with Ac2O and
pyridine afforded compound 17 in 85% yield.
In the same way as already described,
compound 17 was transformed into the
corresponding cyanomethyl derivative 6b
{FABMS: m/z 470 [M+H]+}, the car-
bamoylmethyl compound 6c {FABMS: m/z
412 [M+Na]+}, and amidinomethyl com-
pound 6d {FABMS: m/z 391 [M+2 H]+} in
41, 60, and 52% yields, respectively.
The behaviour of the compounds newly
synthesized in this study towards sialidase
from the influenza virus A/Memphis/1/
71(H3N2)14 and pig liver membrane15 was ex-
amined in comparison to Neu5Ac2en (1)
(Table 1). As may be seen from the table,
compounds 5d, 6c, and 6d showed comparable
inhibitory activities against both enzymes, al-
though the degree of inhibition was less than
that of 1. Interestingly, compound 6a exhib-
ited selective inhibition against the viral over
mammalian sialidase, whereas the inhibitory
effects of 5b,c were only on mammalian
sialidase.
In conclusion, the synthesis of 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid analogues
modified at the C-4 and C-9 positions was
achieved via structurally similar common key
intermediates. The compounds thus synthe-
sized showed inhibitory activities against the
influenza virus and mammalian sialidase.
3. Experimental
General procedure.—Optical rotations were
measured with a JASCO DIP-140 (Japan) dig-
ital polarimeter. IR spectra were recorded on
1
a JASCO IR-810 (Japan) spectrometer. H
NMR spectra were recorded with a JEOL EX
270 (Japan) instrument. Chemical shifts are
expressed in ppm relative to Me4Si (l=0) in
CDCl3, CD3OD and in D2O referenced to
HOD (4.85 ppm) as the internal standard.
Fast-atom-bombardment (FAB) mass spectra
were obtained with a JEOL SX-102 (Japan)
mass spectrometer in the positive-ion mode
using an NBA matrix. High-resolution mass
spectra (HR-MS) were recorded on a JEOL
JMS-700 (Japan) instrument under Fab condi-
tions. Column chromatography was per-
formed on Silica Gel Merck 60 (70–230 mesh)
and LH-20 (Pharmacia). All reactions were
monitored by TLC (Silica Gel 60-F254, E.
Merck, Germany) by charring after spraying
with 5% H2SO4 in MeOH and then heating.
Methyl (phenyl 5-acetamido-3,5-dideoxy-
Table 1
Inhibition of sialidase by analogues of Neu5Ac2en modified
at the C-4 and C-9 positions a
Sialidase
A/Memphis/1/71(H3N2)
4a ++
p.g. liver membrane
8,9-O-isopropylidene-2-thio-
D
-glycero-i- -
D
galacto-2-nonulopyranosid)onate (8).—To a
soln of 7 (6.79 g, 11.6 mmol) in dry MeOH
(150 mL) was added 0.2 M NaOMe (20 mL),
and the mixture was stirred for 2 h at 0 °C,
and neutralized by the addition of Amberlite
IRC-50 (3.5 g) resin, and the mixture was
filtered and the filtrate concd. The residue was
dissolved in dry acetone (100 mL) with Am-
berlite IRA120 (H+) (5 g) resin and the mix-
ture was stirred for 13 h at rt, filtered, and
concd. Purification of the residue by column
chromatography on silica gel with 10:1
CH2Cl2–MeOH yielded 8 (4.25 g, 81%),
+
4b ++
4c ++
5a
+
+
+
5b
5c
++
++
++
5d ++
6a ++
6b
+
+
6c ++
6d ++
++
++
1
++++
++++
a Inhibition of sialidase at 1.0 mM inhibitor; +, B10%;
++, 10–30%; +++, 30–90%; ++++, \90%.

K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
37
Ac2O (2.5 mL). The mixture was stirred at
0 °C for 30 min, and then stirred overnight at
rt. The soln was diluted with CH2Cl2 and
successively washed with water, 1 M HCl, aq
satd NaHCO3, and brine, dried (MgSO4), and
then concd. The residue was chromatographed
on silica gel using 20:1 CH2Cl2–MeOH to give
10 (410 mg, 98%); [h]D −38.5° (c 1.0, CHCl3);
[h]D−169.6° (c 0.30, CHCl3); wmax (CHCl3):
1
1736, 1663, 851, and 779 cm−1; H NMR
(CDCl3) l: 1.40, 1.42 (s, each 3 H, Me2C),
2.08 (t, 1 H, J3ax,3eq =J3ax,4 =13.5 Hz, H-3ax),
2.08 (s, 3 H, AcN), 2.80 (dd, 1 H, J3eq,4 4.5 Hz,
H-3eq), 3.44 (s, 3 H, MeO), 3.57 (m, 1 H,
H-7), 3.93 (q, 1 H, J4,5=J5,6 =J5,NH =8.6 Hz,
H-5), 3.97 (m, 1 H, H-8), 3.98 (dd, 1 H, J8,9a
5.1, J9a,9b 10.5 Hz, H-9a), 4.07 (ddd, 1 H,
H-4), 4.15 (dd, 1 H, J6,7 =2.0 Hz, H-6), 4.27
(m, 1 H, H-9b), 6.33 (d, 1 H, NH), 7.24–7.38,
and 7.51–7.54 (m, 5 H, Ph); positive ion
FABMS (NBA): m/z 456 [M+H]+, 478
[M+Na]+.
1
wmax (CHCl3): 1740 and 1684 cm−1; H NMR
(CDCl3) l: 1.98 (s, 3 H, AcN), 2.02, 2.08, 2.11
(s, 9 H, AcO), 2.76 (dd, 1 H, J3ax,3eq 13.8, J3eq,4
4.5 Hz, H-3eq), 3.62 (s, 3 H, MeO), 3.93 (ddd,
1 H, J4,5 =J5,6=J5,NH=10.0 Hz, H-5), 3.99–
4.12 (m, 2 H, H-4, H-9a), 4.11, 4.35 (d, each 1
H, Jgem 16.5 Hz, ꢀOCH2CN), 4.48 (dd, 1 H,
J8,9b 1.9, J9a,9b 12.4 Hz, H-9b), 4.70 (dd, 1 H,
J6,7 2.4 Hz, H-6), 4.97–5.00 (m, 1 H, H-8),
5.47–5.49 (m, 1 H, H-7), 5.59 (d, 1 H, NH),
and 7.34–7.52 (m, 5 H, Ph); positive ion
FABMS (NBA): m/z 581 [M+H]+, 603
[M+Na]+.
Methyl (phenyl 5-acetamido-4-O-cyano-
methyl - 3,5 - dideoxy - 2 - thio -
D
- glycero - i - -
D
galacto-2-nonulopyranosid)onate (9).—Com-
pound 8 (686 mg, 1.5 mmol) was dissolved in
CH2Cl2 (20 mL) and to the mixture were
,
added 4 A molecular sieves (1.0 g) and bromo-
cyanonitrile (540 mg, 4.5 mmol). After stirring
for 1 h, to the mixture were added Ag2O (1.04
g, 4.5 mmol) and n-Bu4NI (550 mg, 1.5 mmol)
and the mixture was stirred for 2 days in the
dark under Ar. The insoluble materials were
filtered off through Celite 545 and the filtrate
was concd to dryness. The residue was chro-
matographed on silica gel using 20:1 CH2Cl2–
MeOH to give the 4-O-cyanomethyl com-
pound, which was dissolved in 80% AcOH (10
mL) and heated at 80–90 °C for 0.5 h and
concd to dryness. Purification of the residue
by column chromatography on silica gel with
10:1 CH2Cl2–MeOH gave 9 (319 mg, 47%);
[h]D−88.7° (c 0.68, CHCl3); wmax (CHCl3):
Methyl 5-acetamido-7,8,9-tri-O-acetyl-2,6-
anhydro - 4 - O - cyanomethyl - 3,5 - dideoxy -
glycero -
DMTST (147 mg, 0.57 mmol) was added to a
D
-
D
- galacto - non - 2 - enonate (11).—
stirred mixture of compound 10 (110 mg, 0.19
,
mmol) and 4 A molecular sieves (0.5 g) in dry
CH2Cl2 (4 mL) at −20 °C under Ar. After
stirring for 2 h at the same temperature, a soln
of DBU (87 mg, 0.57 mmol) in dry CH2Cl2
(0.5 mL) was added at −20 °C, and the mix-
ture was stirred for 1 h at the same tempera-
ture, and then overnight at rt. The precipitates
were filtered off through Celite 545 and the
filtrate was washed with aq satd NaHCO3 and
brine, dried (MgSO4), and concd. Column
chromatography on silica gel with 10:1
CH2Cl2–MeOH gave 11 (87 mg, 97%) as an
amorphous powder, [h]D +10° (c 1.2, CHCl3);
3427, 1738, and 1661 cm−1
;
1H NMR
(CDCl3) l: 1.99 (t, 1 H, J3ax,3eq =J3ax,4=13.9
Hz, H-3ax), 2.06 (s, 3 H, AcN), 2.78 (dd, 1 H,
J3eq,4 3.8 Hz, H-3eq), 3.55 (s, 3 H, MeO),
3.72–3.82 (m, 3 H, H-8, H-9a,b), 4.00–4.06
(m, 2 H, H-4, H-6), 4.28, 4.42 (d, each 1 H,
Jgem 16.5 Hz, ꢀOCH2CN), 4.48 (m, 1 H, H-5),
4.70 (m, 1 H, H-7), 7.24–7.38, and 7.53–7.56
(m, 5 H, Ph); positive ion FABMS (NBA):
m/z 455 [M+H]+, 477 [M+Na]+.
1
wmax (CHCl3): 1748, 1686, and 1526 cm−1; H
NMR (CDCl3) l: 2.05 (s, 3 H, AcN), 2.13,
2.19 (s, 9 H, AcO), 3.87 (s, 3 H, MeO), 4.21
(dd, 1 H, J8,9a 7.8, J9a,9b 14.6 Hz, H-9a),
4.21–4.35 (m, 2 H, H-4, 6), 4.40–4.50 (m, 1
H, H-5), 4.44, 4.55 (d, each 1 H, Jgem 16.5 Hz,
ꢀOCH2CN), 4.61 (dd, 1 H, J8,9b 3.5 Hz, H-9b),
5.41 (m, 1 H, H-8), 5.87 (d, 1 H, JNH,5 8.4 Hz,
NH), and 6.13 (d, 1 H, J3,4 3.5 Hz, H-3);
positive ion FABMS (NBA): m/z 471 [M+
H]+.
Methyl (phenyl 5-acetamido-7,8,9-tri-O-
acetyl-4-O-cyanomethyl-3,5-dideoxy-2-thio-
D
-
glycero-i- -galacto-2-nonulopyranosid)onate
D
(10).—Compound 9 (330 mg, 0.72 mmol) was
dissolved in pyridine (5 mL) and treated with

38
K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
5-Acetamido-4-O-amidinomethyl-2,6-anhy-
dro-3,5-dideoxy- -glycero- -galacto-non-2-
5-Acetamido-2,6-anhydro-4-O-cyanomethyl-
D
D
3,5-dideoxy-
D
-glycero- -galacto-non-2-enonic
D
enonic acid (4c).—Compound 11 (47 mg, 0.10
mmol) was dissolved in dry MeOH (4 mL)
and methanolic 0.1 M NaOMe (4 mL) was
added to the mixture at 0 °C under Ar. After
being stirred for 12 h at rt, anhyd NH4Cl (32
mg, 0.60 mmol) was added to the reaction
mixture and the whole was heated for 2 h at
50 °C and evaporated. The residue was dis-
solved in H2O, desalted and the aq soln was
concd to dryness. The residue was dissolved in
a 1:1 soln of 0.1 M KOH in MeOH (2 mL) at
0 °C and the mixture was stirred for 12 h at
the rt. The mixture was adjusted to pH 2 with
Amberlite 120 (H+), the resin was filtered
through Celite 545, the filtrate was desalted,
and the aq soln was evaporated. The residue
was chromatographed on silica gel using
65:35:5 CHCl3 –MeOH–H2O to give 4c (20
mg, 57%), after lyophilization; 1H NMR
(D2O): l 2.08 (s, 3 H, AcN), 3.66 (dd, 1 H,
J8,9a 5.9, J9a,9b 11.6 Hz, H-9a), 3.67 (m, 1 H,
H-7), 3.92 (dd, 1 H, J8,9b 2.7 Hz, H-9b),
3.96–4.00 (m, 1 H, H-8), 4.11, 4.24 (d, each 1
H, Jgem 15.9 Hz, ꢀOCH2C(ꢁNH)NH2), 4.27–
4.29 (m, 1 H, H-5), 4.50 (m, 1 H, H-4), and
5.82 (d, 1 H, J3,4 2.2 Hz, H-3); positive ion
FABMS (NBA): m/z 349 [M+H]+, 371
[M+Na]+. HRFABMS Anal. Calcd for
C13H22N3NaO8: m/z 371.1303 [M+Na]+,
Found: 371.1305.
acid (4a).—Compound 11 (41 mg, 0.087
mmol) was dissolved in a 1:1 soln of 0.1 M
KOH in MeOH (4 mL). The mixture was
stirred for 15 h at rt and neutralized with
Amberlite 120 (H+). The precipitates were
filtered off through Celite 545 and the filtrate
was concd. The residue was chromatographed
on silica gel using 6:6:1 CHCl3 –MeOH–H2O
1
to give 4a (14 mg, 49%) as a powder; H
NMR (D2O): l 2.10 (s, 3 H, AcN), 3.54–3.70
(m, 2 H, H-8, H-9a), 3.91, 4.29 (d, each 1 H,
Jgem 11.9 Hz, ꢀOCH2CN), 3.93–3.99 (m, 2 H,
H-7, H-9b), 4.24 (br t, 1 H, J4,5 =J5,6=11.1
Hz, H-5), 4.49–4.53 (m, 2 H, H-4, H-6), and
5.82 (d, 1 H, J3,4 2.2 Hz, H-3); positive ion
FABMS (NBA): m/z 353 [M+Na]+. HR-
FABMS Anal. Calcd for C13H18N2NaO8: m/z
353.0961 [M+Na]+, Found: 353.0958.
5-Acetamido-2,6-anhydro-4-O-carbamoyl-
methyl-3,5-dideoxy-
D
-glycero- -galacto-non-
D
2-enonic acid (4b).—Compound 11 (66 mg,
0.14 mmol) was dissolved in dry MeOH (3
mL), and methanolic 0.1 M NaOMe (3 mL)
was added to the mixture at 0 °C under Ar.
After being stirred for 12 h at rt, satd aq
NaHCO3 (1 mL) was added to the mixture,
which was stirred for 5 days at rt. The mixture
was adjusted to pH 2 with Amberlite 120
(H+), the resin was filtered through Celite 545,
the filtrate was subjected to gel filtration, and
the aq soln was concd. The residue was dis-
solved in a 1:1 soln of 0.1 M KOH in MeOH
(3 mL) at 0 °C and the mixture was stirred for
12 h at rt. The mixture was adjusted to pH 2
with Amberlite 120 (H+), the resin was filtered
through Celite 545, and the filtrate was de-
salted with an AC Micro Acylizer G1 and the
resulting aq soln was concd. The residue was
chromatographed on silica gel using 65:35:5
CHCl3–MeOH–H2O to give 4b (27 mg, 55%),
Methyl (phenyl 5-acetamido-4-O-cyano-
methyl-3,5-dideoxy-9-p-tolyesulfonyl-2-thio-
D
-
glycero-i-
D
-galacto-2-nonulopyranosid)onate
(12).—To a soln of 9 (550 mg, 1.2 mmol) in
pyridine (5 mL) and CH2Cl2 (10 mL) was
added p-toluenesulfonyl chloride (686 mg, 3.6
mmol) at 0 °C under Ar, and the mixture was
stirred for 12 h at rt, and then concd. The
residue was chromatographed on silica gel
using 10:1 CH2Cl2–MeOH to give 12 (470 mg,
64%); [h]D−79.2° (c 0.12, CHCl3); wmax
1
after lyophilization; H NMR (D2O): l 2.10
1
(s, 3 H, AcN), 3.66–3.71 (m, 2 H, H-8, H-9a),
3.90–3.97 (m, 1 H, H-9b), 4.13, 4.26 (d, each
1 H, Jgem 15.7 Hz, ꢀOCH2CONH2), 4.29–4.33
(m, 1 H, H-5), 4.52 (m, 1 H, H-4), and 5.98
(br s, 1 H, H-3); positive ion FABMS (NBA):
m/z 371 [M+Na]+. HRFABMS Anal. Calcd
for C13H20N2NaO9: m/z 371.1067 [M+Na]+,
Found: 371.1052.
(CHCl3): 1738 and 1654 cm−1; H NMR
(CDCl3) l: 1.95 (dd, 1 H, J3ax,3eq 13.9, J3ax,4
10.9 Hz, H-3ax), 2.11 (s, 3 H, AcN), 2.44 (s, 3
H, MePh), 2.80 (dd, 1 H, J3eq,4 4.3 Hz, H-3eq),
3.50 (s, 3 H, MeO), 3.94–4.14 (m, 3 H, H-8,
H-9a,b), 3.97 (ddd, 1 H, J4,5=J5,6 =J5,NH
=
10.6 Hz, H-5), 4.07 (ddd, 1 H, H-4), 4.15, 4.39
(d, each 1 H, J 16.7 Hz, ꢀOCH2CN), 6.02 (br

K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
39
Methyl 5-acetamido-7,8-di-O-acetyl-2,6-an-
hydro - 9 - azido - 4 - O - cyanomethyl - 3,5,9 - tri-
s, 1 H, NH), and 7.33–7.80 (m, 5 H, PhS);
positive ion FABMS (NBA): m/z 609 [M+
H]+.
deoxy -
D
- glycero - - galacto - non - 2 - enonate
D
Methyl (phenyl 5-acetamido-9-azido-4-O-
(15).—Transformation of 14 (30 mg, 0.053
mmol) was carried out by the same procedure
as described for 11 to give compound 15 (24
mg, 99%) as an amorphous powder, [h]D +41°
(c 0.68, CHCl3); wmax (CHCl3): 2108, 1744,
cyanomethyl-3,5-dideoxy-2-thio-
D
-glycero- -i-
D
galacto-2-nonulopyranosid)onate (13).—To a
soln of 12 (669 mg, 1.1 mmol) in DMF (10 mL)
was added sodium azide (357 mg, 5.6 mmol)
and 18-crown-6 (146 mg, 0.56 mmol) and the
mixture was heated at 60 °C with stirring for 20
h under Ar. The mixture was allowed to cool
to rt, and concd to dryness. The residue was
chromatographed on silica gel using 10:1
CH2Cl2 –MeOH to give 13 (390 mg, 74%);
[h]D−89.5° (c 1.0, CHCl3); wmax (CHCl3): 3430,
1
1680, and 1525 cm−1; H NMR (CDCl3) l:
2.01 (s, 3 H, AcN), 2.09, 2.14 (s, each 3 H,
AcO), 3.49 (dd, 1 H, J8,9a 7.6, J9a,9b 13.5 Hz,
H-9a), 3.80 (dd, 1 H, J8,9b 4.0 Hz, H-9b), 3.82
(s, 3 H, MeO), 4.16 (q, 1 H, J4,5=J5,6 =
JNH,5=8.6 Hz, H-5), 4.43, 4.46 (d, each 1 H,
Jgem 16.2 Hz, ꢀOCH2CN), 4.35–4.47 (m, 2 H,
H-4, 6), 5.20 (m, 1 H, H-8), 5.50 (m, 1 H,
H-7), 5.79 (d, 1 H, NH), and 6.09 (d, 1 H, J3,4
3.3 Hz, H-3); positive ion FABMS (NBA):
m/z 454 [M+H]+.
1
2106, 1736, 1671, and 1553 cm−1; H NMR
(CDCl3) l: 1.97 (dd, 1 H, J3ax,3eq 13.9, J3ax,4 10.9
Hz, H-3ax), 2.13 (s, 3 H, AcN), 2.76 (dd, 1 H,
J3eq,4 4.6 Hz, H-3eq), 3.49 (dd, 1 H, J9a,9b 12.5,
J8,9a 7.8 Hz, H-9a), 3.56 (s, 3 H, MeO), 3.63
(dd, 1 H, J8,9b 3.5 Hz, H-9b), 3.84–3.95 (m, 1
H, H-5), 3.98–4.46 (m, 4 H, H-4, 6, 7, 8), 4.16,
4.42 (d, each 1 H, Jgem 16.5 Hz, ꢀOCH2CN),
and 6.08 (d, 1 H, JNH,5 8.6 Hz, NH); positive
ion FABMS (NBA): m/z 480 [M+H]+.
5-Acetamido-2,6-anhydro-9-azido-4-O-cya-
nomethyl-3,5,9-trideoxy-
D
-glycero- -galacto-
D
non-2-enonic acid (5a).—Transformation of
15 (40 mg, 0.088 mmol) was performed as
described for 4a to give compound 5a (26 mg,
1
80%); H NMR (D2O): l 2.09 (s, 3 H, AcN),
Methyl (phenyl 5-acetamido-7,8-di-O-ace-
tyl-9-azido-4-O-cyanomethyl-3,5-dideoxy-2-
3.53–3.57 (m, 1 H, H-9a), 3.65–3.70 (m, 2 H,
H-7, H-9b), 4.09–4.15 (m, 1 H, H-5), 4.12,
4.24 (d, each 1 H, Jgem 15.9 Hz, ꢀOCH2CN),
4.48 (br s, 1 H, H-4), and 5.81 (br s, 1 H,
H-3); positive ion FABMS (NBA): m/z 396
[M+K]+.
thio-
D
-glycero-i- -galacto-2-nonulopyrano-
D
sid)onate (14).—Compound 13 (70 mg, 0.146
mmol) was dissolved in pyridine (2 mL) and
treated with Ac2O (1 mL). The mixture was
stirred at 0 °C for 30 min, and then overnight
at rt. The soln was diluted with CH2Cl2 and
successively washed with water, 1 M HCl, aq
satd NaHCO3, and brine, and dried (MgSO4),
and finally concd to dryness. The residue was
chromatographed on silica gel using 10:1
CH2Cl2 –MeOH to give 14 (78 mg, 95%);
[h]D−94.3° (c 0.75, CHCl3); wmax (CHCl3):
5-Acetamido-2,6-anhydro-9-azido-4-O-car-
bamoylmethyl - 3,5,9 - trideoxy -
D
- glycero - -
D
galacto-non-2-enonic acid (5b).—Transforma-
tion of 15 (40 mg, 0.088 mmol) was performed
as described for 4b to afford compound 5b (22
1
mg, 67%); H NMR (D2O): l 2.10 (s, 3 H,
AcN), 3.55 (dd, 1 H, J8,9a 5.7, J9a,9b 13.2 Hz,
H-9a), 3.66–3.72 (m, 1 H, H-7), 3.68 (dd, 1 H,
J8,9b 2.4 Hz, H-9b), 4.08, 4.23 (d, each 1 H, Jgem
13.2 Hz, ꢀOCH2CONH2), 4.09–4.25 (m, 2 H,
H-6, 8), 4.22–4.29 (m, 1 H, H-5), 4.50 (br s, 1
H, H-4), and 5.83 (br s, 1 H, H-3); positive ion
FABMS (NBA): m/z 396 [M+Na]+. HR-
FABMS Anal. Calcd for C13H19N5NaO8: m/z
396.1132 [M+Na]+, Found: 396.1132.
1
2105, 1746, 1682, and 760 cm−1; H NMR
(CDCl3) l: 1.92 (s, 3 H, AcN), 2.00, 2.03 (s, 6
H, AcO), 2.69 (dd, 1 H, J3ax,3eq 13.9, J3eq,4 4.6
Hz, H-3eq), 3.23 (dd, 1 H, J9a,8 9.2, J9a,9b 13.5
Hz, H-9a), 3.54 (dd, 1 H, J8,9b 2.3 Hz, H-9b),
3.57 (s, 3 H, MeO), 4.11 (q, 1 H, J4,5 =J5,6=
JNH,5=10.6 Hz, H-5), 5.38 (m, 1 H, H-4), 4.15,
4.39 (d, each 1 H, Jgem 16.7 Hz, ꢀOCH2CN),
4.61 (dd, 1 H, J6,7 2.3 Hz, H-6), 4.95 (m, 1 H,
H-8), 5.46 (m, 1 H, H-7), and 7.31–7.36 (m, 5
H, PhS); positive ion FABMS (NBA): m/z 564
[M+H]+.
5 - Acetamido - 4 - O - amidinomethyl - 2,6 - an-
hydro - 9 - azido - 3,5,9 - trideoxy -
D
- glycero - -
D
galacto-non-2-enonic acid (5c).—Transforma-
tion of 15 (24 mg, 0.053 mmol) described for

40
K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
1
2.12 (s, each 3 H, AcO), 3.32 (ddd, 1 H,
8,9a=JNH,9a =5.7, J9a,9b 14.6 Hz, H-9a), 3.84
4c gave compound 5c (12 mg, 60%); H NMR
(D2O): l 2.10 (s, 3 H, AcN), 3.53–3.58 (m, 1
H, H-9a), 3.67–3.70 (m, 2 H, H-7, H-9b),
4.10–4.32 (m, 4 H, H-5, H-8, ꢀOCH2C(ꢁ
NH)NH2), 4.52 (br s, 1 H, H-4), and 5.93 (br
s, 1 H, H-3); positive ion FABMS (NBA): m/z
373 [M]+. HRFABMS Anal. Calcd for
C13H19N5NaO8: m/z 396.1369 [M+Na]+,
Found: 396.1158.
J
(s, 3 H, MeO), 3.89 (ddd, 1 H, J8,9b 3.5, JNH,9b
6.2 Hz, H-9b), 4.17 (dd, 1 H, J4,3 2.7, J4,5 5.1
Hz, H-4), 4.32, 4.58 (d, each 1 H, Jgem 16.7
Hz, ꢀOCH2CN), 4.35–4.39 (m, 2 H, H-5, 6),
5.05 (m, 1 H, H-8), 5.46 (br s, 1 H, H-7), 6.08
(d, 1 H, H-3), 6.77 (dd, 1 H, NH), and 6.83 (d,
1 H, JNH,5 8.4 Hz, NH); positive ion FABMS
(NBA): m/z 504 [M+H]+, 526 [M+Na]+.
5,9 - Diacetamido - 2,6 - anhydro - 3,5,9 - tri-
5 - Acetamido - 2,6 - anhydro - 9 - azido - 3,5,9-
trideoxy-4-O-methoxyiminomethyl- -glycero-
D
D-galacto-non-2-enonic acid (5d).—Com-
deoxy-4-O-thiocarbamoylmethyl-
D
-glycero- -
D
pound 15 (40 mg, 0.088 mmol) was dissolved
in dry MeOH (2 mL) and methanolic 0.1 M
NaOMe (0.6 mL) was added to the mixture at
0 °C under Ar. After being stirred for 12 h at
rt, the mixture was adjusted to pH 2 with
Amberlite 120 (H+), the resin was filtered
through Celite 545, the filtrate was subjected
to gel filtration, and then the aq soln was
concd. The residue was dissolved in a 1:1 soln
of 0.1 M KOH in MeOH (2 mL) at 0 °C and
the mixture was stirred for 12 h at rt. The
mixture was adjusted to pH 2 with Amberlite
120 (H+), the resin was filtered through Celite
545, the filtrate was subjected to gel filtration
and the aq soln was concd. The residue was
chromatographed on silica gel using 65:35:5
CHCl3–MeOH–H2O to give 5d (26 mg, 76%),
galacto-non-2-enonic acid (6a).—Transforma-
tion of 16 (40 mg, 85 mmol) was performed as
described for 4a to give the compound 6a (23
1
mg, 67%); H NMR (D2O) l: 2.00, 2.04 (s,
each 3 H, AcN), 3.27 (dd, 1 H, J8,9a 7.3, J9a,9b
14.0 Hz, H-9a), 3.50–3.60 (m, 2 H, H-7, 9b),
3.92–3.98 (m, 1 H, H-8), 4.43–4.47 (m, 1 H,
H-4), 4.23–4.29 (m,
4
H, H-5, H-6,
ꢀOCH2CN), and 5.74 (d, 1 H, J3,4 2.2 Hz,
H-3), positive ion FABMS (NBA): m/z 406
[M+H]+, 428 [M+Na]+. HRFABMS Anal.
Calcd for C15H23N3NaO8S: m/z 428.1104
[M+Na]+, Found: 428.1096.
Methyl 5,9-diacetamido-2,6-anhydro-4-O-
cyanomethyl - 3,5,9 - trideoxy -
D
- glycero - -
D
galacto-non-2-enonate (17).—To a soln of 15
(16 mg, 0.035 mmol) in 4:3:1 1,4-dioxane–
MeOH–H2O (1.0 mL) was added PPh3 (14
mg, 0.053 mmol) at rt and the mixture was
stirred for 18 h. After removal of the solvent,
the residue was chromatographed on silica gel
using 10:1 CH2Cl2–MeOH to give 17 (14 mg,
1
after lyophilization; H NMR (D2O): l 2.10
(s, 3 H, AcN), 3.58–3.70 (m, 3 H, H-7, H-
9a,b), 3.80 (s, 3 H, ꢀOCH3), 4.27–4.35 (m, 4
H, H-5, H-8, ꢀOCH2C(ꢁNH)OCH3), 4.52–
4.60 (m, 1 H, H-4), and 5.96 (br s, 1 H, H-3);
positive ion FABMS (NBA): m/z 411 [M+
1
85%); [h]D +21.3° (c 0.36, CHCl3); H NMR
Na]+.
HRFABMS
Anal.
Calcd
for
(CDCl3) l: 1.98, 2.00 (s, each 3 H, AcN), 2.05,
2.13 (s, each 3 H, AcO), 3.31 (ddd, 1 H, J8,9a
4.6, JNH,9a 5.6, J9a,9b 14.6 Hz, H-9a), 3.75–3.89
(m, 1 H, H-9b), 3.83 (s, 3 H, MeO), 4.26 (1 H,
ddd, J4,5 =J5,6=J5,NHAc=8.9 Hz, H-5), 4.35,
4.47 (d, each 1 H, Jgem 16.7 Hz, ꢀOCH2CN),
4.41 (dd, 1 H, J3,4 3.0 Hz, H-4), 4.46 (dd, 1 H,
J6,7 3.8 Hz, H-6), 5.08 (ddd, 1 H, J8,7 3.8 Hz,
H-8), 5.42 (dd, 1 H, H-7), 6.09 (d, 1 H, H-3),
6.25 (br s, 1 H, NH), and 6.56 (br s, 1 H,
NH); positive ion FABMS (NBA): m/z 470
[M+H]+, 492 [M+Na]+.
C14H22N5NaO8: m/z 411.1366 [M+Na]+,
Found: 411.1381.
Methyl 5,9-diacetamido-7,8-di-O-acetyl-2,6-
anhydro - 3,5,9 - trideoxy - 4 - O - thiocarbamoyl-
methyl -
D
- glycero - - galacto - non - 2 - enonate
D
(16).—To a soln of 15 (47 mg, 0.10 mmol) in
pyridine (0.5 mL) and CH2Cl2 (0.5 mL) was
added thioacetic acid (38 mg, 0.50 mmol) at rt
under Ar, and the mixture was stirred for 12
h. After removal of the solvent, the residue
was chromatographed on silica gel using 10:1
CH2Cl2 –MeOH to give 16 (40 mg, 79%);
[h]D+101.8° (c 0.80, CHCl3); wmax (CHCl3):
5,9-Diacetamido-4-O-cyanomethyl-2,6-an-
hydro-3,5-dideoxy-
D
-glycero- -galacto-non-2-
D
1731, 1665, and 1524 cm−1
;
1H NMR
enonic acid (6b).—Transformation of 17 (31
(CDCl3) l: 1.96, 1.98 (s, each 3 H, AcN), 2.04,
mg, 0.066 mmol) was performed as described

K. Ikeda et al. / Carbohydrate Research 330 (2001) 31–41
41
1
for 4a to give compound 6b (10 mg, 41%); H
NMR (D2O) l: 2.20, 2.25 (s, each 3 H, AcN),
3.48 (dd, 1 H, J8,9a 7.3, J9a,9b 14.0 Hz, H-9a),
3.75 (dd, 1 H, J8,9b 10.5 Hz, H-9b), 4.16 (br s,
1 H, H-8), 4.36–4.50 (m, 3 H, H-5, 6, 7), 4.33,
4.46 (d, each 1 H, Jgem 16.5 Hz, ꢀOCH2CN),
4.64 (m, 1 H, H-4), and 5.97 (d, 1 H, J3,4 1.9
Hz, H-3); positive ion FABMS (NBA): m/z
470 [M+H]+, 492 [M+Na]+.
Tomoko Terada, Institute for Chemical Re-
search, Kyoto University, for the measure-
ment of HR-MS.
References
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5,9-Diacetamido-2,6-anhydro-4-O-carba-
moylmethyl-3,5-dideoxy-
D
-glycero- -galacto-
D
non-2-enonic acid (6c).—Transformation of
17 (30 mg, 0.064 mmol) as described for 4b
1
gave compound 6c (15 mg, 60%); H NMR
(D2O) l: 2.21, 2.25 (s, each 3 H, AcN), 3.44–
3.53 (m, 1 H, H-9a), 3.72–3.96 (m, 1 H,
H-9b), 4.13–4.45 (m, 6 H, H-5, H-6, H-7,
H-8, ꢀOCH2CONH2), 4.64 (br s, 1 H, H-4),
and 5.98 (d, 1 H, J3,4 3.0 Hz, H-3); positive
ion FABMS (NBA): m/z 412 [M+Na]+. HR-
FABMS Anal. Calcd for C15H23N3NaO9: m/z
412.1332 [M+Na]+, Found: 412.1320.
5,9-Diacetamido-2,6-anhydro-4-O-amidino-
methyl-3,5-dideoxy-
D
-glycero- -galacto-non-
D
2-enonic acid (6d).—Transformation of 17 (28
mg, 0.060 mmol) as described for 4c afforded
1
the compound 6d (12 mg, 52%); H-NMR
(D2O) l: 2.20, 2.24 (s, each 3 H, AcN), 3.44–
3.52 (m, 1 H, H-9a), 3.72–3.81 (m, 1 H,
H-9b), 3.88–3.96 (m, 1 H, H-8), 4.13–4.44 (m,
5 H, H-5, H-6, H-7, ꢀOCH2C(ꢁNH)NH2),
4.61 (t, 1 H, J3,4=J4,5=7.8 Hz, H-4), and
5.97 (d, 1 H, H-3), positive ion FABMS
(NBA): 391 m/z [M+2 H]+. HRFABMS
Anal. Calcd for C15H25N4NaO8: m/z 412.1570
[M+Na]+, Found: 412.1555.
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Acknowledgements
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The authors thank Marukin Shoyu Co.,
Ltd. (Kyoto, Japan) for a generous gift of
Neu5Ac. And also we wish to thank Mrs
.