An efficient synthesis of C3 C-alkylated Neu5Ac2en derivatives

Article abstract of DOI:10.1016/j.tetlet.2012.12.064

C3-modified Neu5Ac2en derivatives can be used to study the flexible 150-loop region of influenza virus sialidases and also provide a new template for the development of next-generation sialidase inhibitors. This Letter describes an efficient synthetic route towards the large scale synthesis of C3 C-alkylated Neu5Ac2en derivatives. The key intermediate, a 3-eq-allyl-Neu5Ac derivative, is formed by stereoselective addition of the allyl group in an equatorial configuration at C3, regardless of the stereochemistry of the bromohydrin precursor.

Full text of DOI:10.1016/j.tetlet.2012.12.064

Tetrahedron Letters 54 (2013) 1198–1201
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An efficient synthesis of C3 C-alkylated Neu5Ac2en derivatives
⇑
Santosh Rudrawar, Mauro Pascolutti, Beenu Bhatt, Robin J. Thomson, Mark von Itzstein
Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland 4222, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
C3-modified Neu5Ac2en derivatives can be used to study the flexible 150-loop region of influenza virus
sialidases and also provide a new template for the development of next-generation sialidase inhibitors.
This Letter describes an efficient synthetic route towards the large scale synthesis of C3 C-alkylated
Neu5Ac2en derivatives. The key intermediate, a 3-eq-allyl-Neu5Ac derivative, is formed by stereoselec-
tive addition of the allyl group in an equatorial configuration at C3, regardless of the stereochemistry
of the bromohydrin precursor.
Received 13 October 2012
Revised 29 November 2012
Accepted 13 December 2012
Available online 25 December 2012
Keywords:
Influenza
Ó 2012 Elsevier Ltd. All rights reserved.
Sialidase
Carbohydrate
Neu5Ac2en
Sialic acid
Influenza is a significant human disease, as evidenced by sea-
sonal epidemics that exact a high toll in morbidity and mortality,
and worldwide pandemics, for example the 2009 ‘swine-origin’
influenza A pandemic. Two influenza virus-specific drugs, RelenzaÒ
and TamifluÒ, target the viral enzyme sialidase, and are effective
against all wild type influenza virus strains.1 Resistance develop-
ment2 to the widely used drug, TamifluÒ, however, has fuelled a
drive to better understand fundamental aspects of sialidase
inhibitor interactions and to develop new sialidase-inhibitor anti-
influenza drugs. Recent X-ray crystal structures of a sub-group of
influenza A virus sialidases, that includes the N1 sub-types associ-
ated with currently circulating and pandemic influenza A viruses,
indicate the flexibility of a protein loop (the ‘150-loop’) adjacent
to the enzyme active site.3 When this loop is in an ‘open’ conforma-
tion, a significant cavity is formed adjacent to the active site.3
Development of probes and inhibitors which bind the extended
sialidase active site is an area of growing interest.4–7 Our work in
this area has shown that suitable functionality introduced at the
C3 position of the general sialidase inhibitor Neu5Ac2en (1), can
extend into the 150-cavity and lock the flexible 150-loop in an
open conformation.6 C3-Modified Neu5Ac2en derivatives such as
2 (a micromolar inhibitor of a number of N1 sialidases6) provide
tools to study the 150-loop region of influenza virus sialidases,
and also provide a new template for the development of more po-
tent sialidase inhibitors.
The synthesis of C3-substituted Neu5Ac2en derivatives was until
recently an unexplored field. For the synthesis of 2, a reaction path-
way was developed6 in which an allyl group was initially installed
at C3 on the saturated template, giving 3-eq-allyl N-acetylneuram-
inic acid (Neu5Ac) derivative 6 (Scheme 1). Subsequent manipula-
tion of 6 to introduce a halide at C2 allowed the generation of the
corresponding C2–C3 unsaturated derivative by base-promoted
beta-elimination of HCl.6 The key 3-eq-allyl-Neu5Ac derivative 6
is prepared via radical-mediated allylation of the trans-2,3-diequa-
torial bromohydrin 4.6,8 A low isolated yield (38%) of the required
bromohydrin 4,6 however, was a bottle-neck in this synthesis.
In addition, a moderate yield of the allylation reaction to give 6,6
led us to further investigate the synthetic strategy to 3-eq-allyl-
Neu5Ac derivative 6 that would provide efficient, and scalable ac-
cess to novel C3 C-alkylated Neu5Ac2en derivatives.
Herein, we report a synthesis of C3-substituted Neu5Ac2en
derivatives that is successful on a multi-gram scale.
The high-yielding, regioselective, bromohydroxylation of pro-
tected Neu5Ac2en 3, using N-bromosuccinimide (NBS) and water
in the presence of a co-solvent, gives a mixture of trans-2,3-diequa-
torial 4 and trans-2,3-diaxial 5 bromohydrins with the ratio varying
from 1:0.1 to 1:3.9 The stereoselectivity of the NBS-mediated
⇑
Corresponding author. Tel.: +61 7 5552 7025; fax: +61 7 5552 9040.
E-mail address: m.vonitzstein@griffith.edu.au (M. von Itzstein).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.12.064

S. Rudrawar et al. / Tetrahedron Letters 54 (2013) 1198–1201
1199
AcO AcO
AcO AcO
CO2Me
OH
OH
H
H
H
O
O
CO2Me
Bu3SnAll, AIBN
AcO
AcO
toluene, 100 °C, 8 h
57% (Ref. 6)
AcHN
Br
AcHN
AcO AcO
AcO
AcO
H
O
CO2Me
4
6
NBS
AcO
66%
MeCN-H2O,
AcHN
80 °C, 30 min.
AcO
AcO AcO
AcO AcO
4
5
ratio 1:2)
95% (
/
OH
OH
O CO2Me
H
H
O
CO2Me
3
Bu3SnAll, AIBN
X
AcO
AcO
toluene, 100 °C, 8 h
AcHN
Br
AcHN
H
AcO
AcO
5
7
Scheme 1.
bromohydroxylation is governed by solvent composition, reaction
temperature and the stereoelectronic nature of the substituent at
C4.10 The two bromohydrins formed are, however, difficult to sepa-
rate on silica gel in a number of different solvent combinations, and
require several chromatographic purifications to isolate the pure
isomers. Given that the trans-2,3-diaxial bromohydrin 5 is always
formed in the bromohydroxylation of 3, we examined the reactivity
of 5 towards C3 allylation (Scheme 1). Reaction of 5 at 100 °C in tol-
uene with allyltributyltin using AIBN as the free-radical initiator
afforded a single C3-allylated product in 66% yield. Interestingly,
the main product was assigned as the C3 equatorially-allylated
derivative 6, rather than the C3 axially-substituted product 7, based
on the large J3,4 value (10.5 Hz) of the product, and comparison with
NMR data for 6 prepared previously from the C3 equatorial bro-
mohydrin.6,8 The yield of 6 was slightly improved when compared
to reaction with trans-2,3-diequatorial bromohydrin 4 (57%6) under
thesamereactionconditions, and importantly wasreproducibleona
large scale (up to 25 g, 44 mmol, of 5).
Scheme 2.
Table 1
Reaction conditions evaluated for C3 allylation of bromohydrins 4/5 using Bu3SnAll in
the presence of AIBNa
Precursor trans-2,3-diequatorial 4 and trans-2,3-diaxial 5 bro-
mohydrins each give rise to the same C3 equatorially-substituted
product 6, indicating that the stereoselectivity of the free-radical
reaction is evidently not influenced by the configuration of the
activating group (bromide) on the C3 position. Substituents adja-
cent to the radical centre in glycopyranosyl radicals influence the
direction of attack on that centre:11 where the radical centre is
flanked by equatorial substituents there is a marked preference
for an equatorial product; where the substituents are axial, the
preference is for axial attack.11 In the case of the radical species
8 formed from bromohydrins 4/5, the bulkiest substituents b to
the radical centre (4-OAc, 2-CO2Me) are equatorial. The formation
of the equatorially allylated product 6 is therefore consistent with
the reported11 trend.
Entry
Solvent and reaction conditionsb
Outcomec (yield %)
4/5
6
9
1
2
3
4
5
6
Toluene, 100 °C, 8 h
Benzene, 80 °C, 10 h
THF, 60 °C, 10 h
1,4-Dioxane, 100 °C, 8 h
MeCN, 80 °C, 10 h
—
—
5
10
15
18
68
79
8
20
40
26
22
8
80
35
35
40
1,2-Dichloroethane, 80 °C, 10 h
a
All reactions were carried out on 0.17 mmol (0.1 g) of a mixture of 4/5 mixture
(ratio = 1:2), except for entries 1 (1.75 mmol, 1 g) and 2 (0.35 mmol, 0.2 g).
Reaction composition: 4/5 (0.17 mmol), Bu3SnAll (0.84 mmol), AIBN
(0.017 mmol), anhydrous solvent (8 mL).
b
c
Isolated yields.
To investigate if the yield of 6 could be further improved, we
studied the effects of solvent on the outcome of the allylation
reaction. The use of toluene (chosen6 to achieve reaction at high
temperature) as a solvent for the free-radical allylation of
bromohydrin 4 always proceeded with the formation of de-
brominated Neu5Ac derivative 9 as a by-product. (e.g., 22% under
the optimised reaction conditions; Table 1, entry 1).
Presumably, the formed radical at the C3 position abstracts a
hydrogen atom from the solvent (toluene). Bromohydrin mixture
4/5 was therefore reacted with allyltributyltin in alternative sol-
vents (benzene, THF, 1,4-dioxane, acetonitrile, 1,2-dichloroethane)
(Scheme 2). The results of these reactions are summarised in
Table 1. Interestingly, replacing toluene with anhydrous benzene
led to an increase in reaction yield (79%, up from 68%), with the
de-brominated derivative 9 formed in only ꢀ8% yield (Table 1, en-
try 2). Reaction in THF, 1,4-dioxane, acetonitrile or 1,2-dichloroeth-
ane (DCE), however, resulted in less than satisfactory yields of 6
(Table 1, entries 3–6). While reaction in benzene gave the most
In light of this result, the reactivity of bromohydrin mixture 4/5
towards the allylation reaction was examined. Reaction of a mix-
ture of 4/5 (ratio 1:2) at 100 °C in toluene with allyltributyltin in
the presence of AIBN afforded the single C3 equatorially-allylated
product 6 in 68% yield (Scheme 2, Table 1, entry 1). Using the
bromohydrin mixture, the overall yield of C3 C-allyl derivative
6 from glycal 3 over two steps was 63%, a substantial increase
in yield over that previously achieved (22%6) via trans-2,3-diequ-
atorial bromohydrin 4.

1200
S. Rudrawar et al. / Tetrahedron Letters 54 (2013) 1198–1201
AcO AcO
OH
H
O
CO2Me
AcO
AcHN
Br
AcO
4 / 5 mixture
Bu3SnAll, AIBN
toluene, 100 °C, 8 h
68%
63% over two steps
(22% Ref. 6)
Approach I
Approach II
AcO AcO
AcO AcO
AcO AcO
H
Grubbs' cat. 2nd gen.
4-methylstyrene, CH2Cl2
OH
H
O CO2Me
OH
CO2Me
OAc
H
O
CO2Me
O
Ac2O, DMAP, py
AcO
AcO
AcHN
AcO
AcHN
R
rt, 16 h
94%
40 °C, 48 h
85%
AcHN
H
H
H
AcO
AcO
AcO
6
10
15 R = p-CH3-C6H4
i) AcCl, MeOH, CH2Cl2, 0 °C to rt, 48 h
ii) DBU, CH2Cl2, rt, 16 h
Ac2O, DMAP, py
rt, 16 h
95%
OAc
71% over 2 steps
AcO AcO
AcO AcO
H
AcO AcO
Grubbs' cat. 2nd gen.
olefin, CH2Cl2
i) AcCl, MeOH, CH2Cl2
H
O
H
CO2Me
O
CO2Me
O
CO2Me
0 °C to rt, 48 h
AcO
AcO
AcHN
AcO
AcHN
R
R
40 °C, 24–48 h
ii) DBU, CH2Cl2
rt, 16 h
AcHN
H
12 92%
13 95%
14 96%
AcO
AcO
11
AcO
12 74% over 2 steps
12 R = p-CH3-C6H4
13 R = p-F-C6H4
16 R = p-CH3-C6H4
14 R = p-CF3-C6H4
Scheme 3.
satisfactory outcome, its use for large-scale reactions is disfa-
voured due to its known toxicity.12
product 15 (85%) in a similar high yield to the reactions carried out
on unsaturated derivative 11. Subsequent processing of 15 as for
6—2-O-acetylation, 2-b-chlorination, and subsequent DBU-cata-
lysed b-elimination of HCl—provided the 2,3-unsaturated deriva-
tive 12 (70% yield over 3 steps from 15). The overall yield of 12
from 6 via either Approach I or II was approximately 60%.
In summary, an efficient synthetic route to the synthesis of C3
C-alkylated Neu5Ac2en derivatives has been developed. The for-
mation of key 3-eq-allyl-Neu5Ac derivative 6, required for the later
introduction of the C2–C3 double bond, is facilitated by a highly
stereoselective addition of the allyl group in an equatorial configu-
ration at C3, regardless of the stereochemistry of the bromohydrin
precursor. This outcome provides a significant improvement over
our previously reported synthesis of these derivatives.6 Subse-
quent elaboration provides access to C3 C-alkylated Neu5Ac2en
derivatives. This pathway is amenable to scale-up, with reaction
conditions optimised to prepare the C3 C-allyl Neu5Ac2en deriva-
tive 6 on a 15 g scale. This work provides access to a new inhibitor
template for exploration of influenza virus sialidases and other
Neu5Ac2en-recognising proteins.
We next explored two approaches (Scheme 3) for the conver-
sion of 6 into C3 C-alkylated Neu5Ac2en derivatives. Approach I
(which was carried-out on the indicated scale) provided the ad-
vanced stage key intermediate, 3-allyl-Neu5Ac2en derivative 11.
This can be further elaborated through manipulation of the allyl
substituent to prepare a diverse range of compounds to explore
structure–activity relationships. Acetylation of the C2 hydroxyl
group of 6 (29 mmol; Ac2O, DMAP, anhydrous pyridine) gave
2-O-acetate derivative 10 in 94% yield (Scheme 3, approach I).
Unsaturation was introduced between C2 and C3 using a two step
reaction sequence: conversion of 10 (14 mmol) into the 2-b-chloro
derivative by reaction with in situ generated HCl gas, followed by
treatment with DBU in CH2Cl2 affording the desired 3-allyl-Neu5Ac-
2en derivative 116 in 71% yield over two steps. In this larger-scale
reaction, the yield of 11 from 10 was substantially increased over
that previously reported (46%6), by extending the reaction time
from 86 to 16 h.
To illustrate the synthetic potential of 11, we examined its
cross-metathesis reactions with aromatic olefins to give C3-
extended Neu5Ac2en derivatives. The cross-metathesis reaction of
11 (7.6 mmol) with 4-methylstyrene using the second-generation
Grubbs catalyst (15 mol %) in CH2Cl2 at 40 °C provided 12 in 92%
yield. For this larger scale reaction, as compared to the original
synthesis,6 addition of the ruthenium catalyst in two batches
(10 mol % at the beginning of the reaction and 5 mol % after 24 h)
and extension of the reaction time from 24 to 48 h resulted in an
improved yield of 12 (92%, compared to 69%6). Similarly, cross-
metathesis reactions of 11 (0.4 mmol scale) with 4-fluorostyrene
and 4-(trifluoromethyl)styrene proceeded smoothly to give cou-
pled products 13 (95%) and 14 (96%), respectively.
Acknowledgments
This work was supported by the Honda Foundation (Australia)
through a Grant to the Institute for Glycomics.
Supplementary data
Supplementary data (experimental details and data for new
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.tetlet.2012.12.064.
In approach II (Scheme 3), the reaction sequence was explored
in reverse, beginning with olefin cross-metathesis of 3-allyl-
Neu5Ac derivative 6 (6 mmol) with 4-methylstyrene. This afforded
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