An efficient approach to N-acetyl-D-glucosaminuronic acid-based sialylmimetics as potential sialidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.08.064

The synthesis of Neu5Ac2en mimetics as inhibitors of Vibrio cholerae sialidase is reported. A novel approach to the synthesis of β-glycosides of N-acetyl-d-glucosaminuronic acid, in six steps and good overall yield from N-acetyl-d-glucosamine, has been de

Full text of DOI:10.1016/j.bmcl.2004.08.064

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5555–5558
An efficient approach to N-acetyl-D-glucosaminuronic acid-based
sialylmimetics as potential sialidase inhibitors
Maretta C. Mann, Robin J. Thomson and Mark von Itzstein*
Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia
Received 19 July 2004; revised 31 August 2004; accepted 31 August 2004
Available online 18 September 2004
Abstract—A novel approach to the synthesis of b-glycosides of N-acetyl-D-glucosaminuronic acid, in six steps and good overall yield
from N-acetyl-D-glucosamine, has been developed. The key synthetic step was the Lewis acid mediated O-glycosidation of methyl
1,3,4-tri-O-pivaloyl-N-acetyl-D-glucosaminuronate (11). Elaboration of glucosaminuronides 15 and 18 provided novel sialylmimet-
ics 21 and 22, which showed inhibition of Vibrio cholerae sialidase.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Sialidases form a large and widely studied group of en-
zymes, which catalyse the release of sialic acids from gly-
coconjugates by hydrolysis of the sialosyl glycosidic
bond. The interest in sialidases has largely been due to
their involvement in microbial pathogenesis, making
them potential targets for therapeutic intervention.1–3
The unsaturated sialic acid, 5-acetamido-2,6-anhydro-
H OH
COOH
O
HO
R
H
HO
OH
1 R = NHAc, Neu5Ac2en
COOH
O
R
AcHN
O
3,5-dideoxy-D-glycero-D-galacto-non-2-enonic
acid
(Neu5Ac2en, 1), is a naturally-occurring sialidase inhib-
itor. We have been interested in the synthesis of mimet-
ics of Neu5Ac2en,4 which are both readily accessible
and have the potential for relatively facile functional
group modification. Previously, we reported4 the synthe-
sis of a mimetic of Neu5Ac2en in which the glycerol side
chain of the sialic acid was replaced by an isopropyl
ether, to give the novel sialidase inhibitor 2. Compound
2 was found to inhibit both influenza virus and V. chol-
erae sialidases.4 This interesting result has led us to de-
velop a method that facilitates rapid access to a series
of mimetics 3 from a common, advanced intermediate.
It was envisaged that this series would include deriva-
tives with functionalities of varying size and hydrophob-
icity that replace the glycerol side chain of Neu5Ac2en
(1), for evaluation as sialidase inhibitors. Described
herein is our preliminary work towards the synthesis
of a series of Neu5Ac2en mimetics 3.
OH
2 R = CHMe2
3 R = alkyl
The desired C-6 ether Neu5Ac2en mimetics 3 can be
considered as b-O-glycosides of 4,5-unsaturated 2-acet-
amido-D-glucuronic acid, and are accessible via N-acet-
yl-b-D-glucosaminuronides 4 (Fig. 1). The most widely
used approach to the preparation of b-D-glucosami-
nuronides, such as 4, involves the selective oxidation4–6
of the C-6 hydroxyl of a preformed N-acyl-b-D-glucos-
aminide, such as 5. This strategy was previously applied
to the synthesis of Neu5Ac2en mimetic 2 from N-acetyl-
D-glucosamine.4 An alternative approach is to begin
with a uronic acid 6, to introduce the nitrogen substitu-
ent at C-2, and then to form the glycoside. This is typi-
cally a more lengthy approach, but nevertheless has
been used for the synthesis of various hexosaminuronide
derivatives7,8 in moderate yield from D-glucurono-6,3-
lactone.
A strategy to form N-acyl-b-D-glucosaminuronides,
such as 4, which to the best of our knowledge has
not been explored, is the direct O-glycosidation of an
Keywords: Sialic acids; Sialidases; Carbohydrates.
*
Corresponding author. Tel.: +61 7 5552 7016; fax: +61 7 5552 9040;
e-mail: m.vonitzstein@griffith.edu.au
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.064

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M. C. Mann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5555–5558
OH
OR
COOH
COOH
O
R
AcHN
a
O
O
HO
HO
PivO
PivO
O
O
OH
OPiv
=
O
HO
R
NHAc
NHAc
OH
NHAc
8
9 R = Tr
10 R = H
3
b
COOMe
c, d
O
PivO
PivO
COOPG
O
OPiv
PGO
PGO
NHAc
OR
11
NHAc
4
Scheme 1. Reagents and conditions: (a) (i) Ph3CCl (1.2equiv),
pyridine, ca. 100ꢁC, 20min, (ii) tBuCOCl (3.6equiv), DMAP,
0ꢁC ! rt, 6d (83%); (b) 80% aq AcOH, 65ꢁC, 1h (92%); (c) cat.
TEMPO, cat. KBr, Bu4NBr, 10–15% NaOCl, satd aq NaHCO3, sat aq
NaCl, CH2Cl2, rt, 75min; (d) SOCl2 (0.6equiv), CH(OMe)3 (1.2equiv),
MeOH, rt, 30min (76% over two steps).
OH
COOPG
O
O
PGO
PGO
PGO
OR
OPG
PGO
OPG
6
NHAc
5
COOPG
O
PGO
PGO
OPG
NHAc
7
COOMe
O
COOMe
O
a
PivO
PivO
PivO
PivO
PG = protecting group or H; R = aglycone
Figure 1. Approaches to C-6 ether Neu5Ac2en mimetics 3.
OPiv
NHAc
O
HN
12
11
Me
N-acylglucosaminuronate, such as 7. This template is
readily accessible from an N-acylglucosamine (as de-
scribed below) and is set up for b-glycosidation using
anchimeric assistance from the C-2 acylamido group.9
A glycosyl bromide derived from an N-acylglucosaminur-
onate was used for N-glycosidation by Timoshchuk and
Kulinkovich in their synthesis of glucosaminuronic acid-
based nucleoside analogues.10,11 A strategy employing
such an aminouronic acid-based intermediate offers
advantages over the others described in that it is poten-
tially more versatile, with the aglycone functionality
being introduced at a later stage in the reaction
sequence, allowing rapid access to a wide range of deriv-
atives from a common glycosyl donor intermediate 7.
COOMe
O
b
PivO
OR
PivO
NHAc
13
R = aglycone
Scheme 2. Reagents and conditions: (a) TMSOTf (1.1equiv),
˚
ClCH2CH2Cl, 50ꢁC, 3d; (b) (i) 3A MS, rt, 30min, (ii) ROH (3equiv),
24h, rt (76–94%, based on recovered a-11 or 14).
COOMe
COOMe
a
O
O
PivO
PivO
OR
OR
NHAc
15 R = ethyl
18 R = 3-pentyl
PivO
NHAc
23 R = ethyl (99%)
24 R = 3-pentyl (82%)
Our approach to the formation of C-6 ether Neu5Ac2en
mimetics 3 from readily available N-acetyl-D-glucos-
amine (GlcNAc, 8) is detailed in Schemes 1–3, with
the key step being the b-selective O-glycosidation of
the N-acetylglucosaminuronate intermediate 11. The
synthesis of N-acetylglucosaminuronate intermediate
11 is outlined in Scheme 1. A sequence of selective trityl-
ation of the primary hydroxyl group of GlcNAc (8),
pivaloylation of the remaining hydroxyl groups to give
9, and detritylation, led to the compound, 10, with a free
primary hydroxyl group. The pivaloylation reaction was
found to be quite slow, even in the presence of DMAP,
requiring six days for conversion of the 6-O-tritylated
starting material to 9. Monitoring of the reaction over
time indicated that the C-4 hydroxyl group was the last
to be pivaloylated.
COOH
b
O
OR
HO
NHAc
21 R = ethyl (86%)
22 R = 3-pentyl (75%)
Scheme 3. Reagents and conditions: (a) DBU (2equiv), CH2Cl2, rt,
18h; (b) 50% aq MeOH, aq NaOH, pH13, 18h.
where the corresponding acetylated derivative showed
some loss of acetate groups. The sequence of detrityl-
ation of 9 followed by TEMPO-mediated oxidation of
the primary hydroxyl group was found to be signifi-
cantly higher yielding (approximately 70% yield over
two steps) than the previously reported10 one-pot con-
version of acetylated or benzoylated 6-O-trityl-GlcNAc
to the corresponding uronic acid using CrO3/H2SO4
(35% yield). The key intermediate 11 was thus obtained
in 58% overall yield from GlcNAc (8) in a reaction
sequence amenable to scale-up.
Conversion of 10 to the corresponding uronic acid was
carried-out using a TEMPO-mediated oxidation under
basic conditions.12 The pivaloyl protecting groups
proved to be stable under the basic reaction conditions,
All new compounds gave satisfactory spectral and analytical data.

M. C. Mann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5555–5558
5557
Table 1. b-Glycosidation of 11 with various alcohols
in CH2Cl2 gave the unsaturated derivatives 23 and 24, in
99% and 82% yield, respectively. Finally, base-catalysed
deprotection gave 21 and 22, in 86% and 75% yield,
respectively, following HPLC purification of the crude
products.
Alcohol
Product Isolated Yield (%)
yield (%) based on
recovered
a-11 and 14
Ethanol
15
16
17
18
19
64
77
53
78
56
64
80
88
82
94
76
81
Compounds 21 and 22 were tested for inhibitory activity
against V. cholerae sialidase, in comparison with
Neu5Ac2en (1), using a fluorimetric assay.16,17 Interest-
ingly, the 3-pentyl derivative 22, containing the larger
hydrophobic side chain, showed greater inhibition
(90% at 1mM; estimated Ki = 1 · 10ꢁ4 M) compared to
the ethyl derivative 21 (71% at 1mM; estimated
Ki = 5 · 10ꢁ4 M), although weaker inhibition than
Isobutanol
Isopropanol
3-Pentanola
Cyclopentanola
1,2-O-Isopropylidene-glycerol 20
a Some co-elution with unreacted a-11 during chromatographic
purification.
Neu5Ac2en
(1)
(97%
at
1mM;
estimated
Ki = 3 · 10ꢁ5 M). These results suggest that the glycerol
side chain binding pocket of V. cholerae sialidase can
accommodate a hydrophobic side chain of similar or
slightly larger size compared to the glycerol side chain
of Neu5Ac2en (1).
Glycosidation13 of 11 was carried-out in one-pot by in
situ generation of the oxazolinium ion 12 from 11 (a/b
ratio = 1.5:1.0) in the presence of TMSOTf14 over three
days, followed by reaction of 12 with an alcohol, as
shown in Scheme 2. This procedure was used to produce
a range of b-glycosides 13, that were isolated in 53–78%
yield (Table 1). Conversion of 11–13 was generally in the
order of 80%, with unreacted 11 recovered solely as the
a-anomer, and a small amount (ꢀ6%) of oxazoline 14
also produced. Based on the recovered a-11 and 14,
yields ranged from 76% to 94% (Table 1). The use of
freshly distilled TMSOTf and 1,2-dichloroethane re-
sulted in optimal conversion of 11 (e.g., 88% conversion
in reaction of 11 with isobutanol). The overall yield for
the synthesis of O-glycosides 13 from GlcNAc (8) was
31–45% over six steps.
In conclusion, we have developed an efficient and versa-
tile synthetic approach for the formation of C-6 ether
Neu5Ac2en mimetics 3 from GlcNAc (8). Further work
on a wider range of sialylmimetics and their biological
evaluation will be reported in due course.
Acknowledgements
We gratefully acknowledge the financial support of the
Australian Research Council (M.v.I., Federation
Fellowship) and the National Health and Medical Re-
search Council of Australia, and Griffith University
for the award of a Postgraduate Research Scholarship
and the Institute for Glycomics, Griffith University,
for an Institute Postgraduate Award to M.C.M. The
Alexander von Humboldt Stiftung is gratefully acknowl-
edged for the award of a von Humboldt Forschungsp-
reis to M.v.I.
COOMe
O
PivO
PivO
O
N
14
Me
Formation of the oxazolinium ion 12 from 11, and par-
ticularly from the a-anomer of 11, was found to be
slower than the corresponding formation of oxazolin-
ium ion from an anomeric mixture of peracetylated4,14
or perpivaloylated GlcNAc. This is in line with the
reported15 destabilising effect of the C-5 alkoxycarbonyl
group on the formation of a C-1 cation. The slower con-
version of the a-pivaloate (a-11) to the intermediate oxa-
Supplementary data
1H and 13C NMR data for all compounds can be found,
in the online version, at doi:10.1016/j.bmcl.2004.08.064.
1
zolinium ion 12 (as seen by H NMR analysis of the
reaction mixture over time) is consistent with observa-
tions on other 2-acetamido hexoses,9 and is presumably
due to the 1,2-cis relationship to the neighbouring C-2
acetamido group and consequent lack of anchimeric
assistance.9 Examination of the reaction conditions for
the formation of the oxazolinium ion 12 from a-11,
showed that neither an increase in the amount of
TMSOTf, nor in the length of the reaction appreciably
increased the production of 12 (or of 14 when the reac-
tion was quenched with triethylamine).
References and notes
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The conversion of the glycosides 15 and 18 to the corre-
sponding sialylmimetics 21 and 22, was achieved in two
steps (Scheme 3). b-Elimination of 15 and 18 using DBU

5558
M. C. Mann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5555–5558
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reaction was filtered through Celiteꢂ, the residue was
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13. Typical glycosidation procedure: TMSOTf (60lL,
0.33mmol, 1.1equiv) was added to a stirred solution of
an anomeric mixture of 11 (152mg, 0.30mmol) in anhy-
drous 1,2-dichloroethane (1.5mL) under Ar. The clear
yellow solution was warmed to an oil bath temperature of