An expeditious synthesis of an analogue of (-)-steviamine by way of the 1,3-dipolar cycloaddition of a nitrile oxide with a 1-C-allyl iminosugar

Article abstract of DOI:10.1016/j.tetlet.2011.09.065

In our continuing effort to develop inhibitors of the mycobacterial galactan biosynthesis, we planned to synthesize original iminosugar-based analogues of UDP-galactofuranose by way of the 1,3-dipolar cycloaddition reaction between a 1-C-allyl iminosugar and a nitrile oxide, followed by the reductive cleavage of the resulting isooxazoline. In initial studies, it was found that this last step led in one pot to a new poly-hydroxylated indolizidine derivative closely related to the recently isolated (-)-steviamine, in good yield, by way of a sequence involving at least five individual reactions. The activity of this new compound as a glycosidase inhibitor was evaluated against a panel of glycosidases and compared to (-)-steviamine.

Full text of DOI:10.1016/j.tetlet.2011.09.065

Tetrahedron Letters 52 (2011) 6399–6402
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An expeditious synthesis of an analogue of (À)-steviamine by way of the
1,3-dipolar cycloaddition of a nitrile oxide with a 1-C-allyl iminosugar
Aleksandra Chronowska a, Estelle Gallienne a, Cyril Nicolas a, Atsushi Kato b, Isao Adachi b,
Olivier R. Martin a,
⇑
a Institut de Chimie Organique et Analytique, UMR 6005, Université d’Orléans & CNRS, rue de Chartres, BP 6759, 45067 Orléans cedex 2, France
b Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In our continuing effort to develop inhibitors of the mycobacterial galactan biosynthesis, we planned to
synthesize original iminosugar-based analogues of UDP-galactofuranose by way of the 1,3-dipolar cyclo-
addition reaction between a 1-C-allyl iminosugar and a nitrile oxide, followed by the reductive cleavage
of the resulting isooxazoline. In initial studies, it was found that this last step led in one pot to a new poly-
hydroxylated indolizidine derivative closely related to the recently isolated (À)-steviamine, in good yield,
by way of a sequence involving at least five individual reactions. The activity of this new compound as a
glycosidase inhibitor was evaluated against a panel of glycosidases and compared to (À)-steviamine.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 22 August 2011
Accepted 14 September 2011
Available online 24 September 2011
Keywords:
1,3-Dipolar cycloaddition
Iminosugar
Glycosidase inhibitor
Indolizidines
In the context of our studies on iminosugars of therapeutic
interest,1 we engaged in a research program dedicated to finding
new inhibitors of mycobacterial galactan biosynthesis. Indeed
This type of structure could be derived from the isoxazoline
moiety obtained by way of the 1,3-dipolar cycloaddition of a 1-C-
allyl iminosugar such as 4 and a nitrile oxide C, generated in situ
and carrying the aromatic group.
while
D-galactose is widely distributed in higher eucaryotes in
the pyranose form (Galp), the furanose form (Galf) is found only
in prokaryotes, protozoans, and fungi2 as well as in a few lower
eukaryotes.3 As it is specific to a number of pathogenic bacteria,
the biosynthesis of galactofuranose-containing glycans is becom-
ing an important target for the development of new antibiotics.4
The enzymes involved in the biosynthesis of galactans are UDP-
Gal mutase (UGM),5 which is responsible for the isomerization of
UDP-Galp into UDP-Galf, and two or more UDP-Galf transferases
(GlfT),6 which transfer Galf units to build the galactan core
(Scheme 1). As part of our investigations on the synthesis of poten-
tial inhibitors of these enzymes,7,8 we prepared the iminosugar-
based analogue of UDP-Galf 1, as well as a number of disaccharide
mimics such as 2 (Fig. 1) which all exhibited very weak activity on
UGM.7d We also prepared a b-linked UDP-Galf mimic 3 (Fig. 1),7b
which was found to have significant activity as an inhibitor of
UDP-Galf transferase GlfT2.9
However, precursor B was found to be highly prone to dehydra-
tion and intramolecular reductive amination reactions during the
final hydrogenolysis step, leading unexpectedly to a new indolizi-
dine iminosugar 5, which was found to be an analogue of the nat-
ural product (À)-steviamine (Fig. 2), recently isolated from Stevia
rebaudiana leaves.10 The steviamine structure was fully elucidated
by X-ray crystallography of its hydrobromide salt11 and glycosi-
dase inhibition studies12 showed it to be the first natural product
to have a (weak) inhibition effect on an a-galactosaminidase.
Thus with the initial objective of making simple UDP-Galf
analogues by means of a nitrile oxide 1,3-dipolar cycloaddition,
we first investigated the synthesis of the protected b-1-C-allyl-
1,4-dideoxy-1,4-imino-
readily obtained in seven steps from
glycosylation of -xylose into its methyl furanoside, benzylation
of the hydroxyl groups, and hydrolysis of the glycosidic bond led
to 2,3,5-tri-O-benzyl-
-xylofuranose 613 in excellent yield. Under
L
-arabinitol 4. This iminoalditol could be
D
-xylose (Scheme 3): kinetic
D
In continuation of this program, we planned to synthesize more
simple analogues of UDP-Galf having the general structure A
D
conditions we reported earlier,14 addition of benzyl carbamate di-
rectly to the free hemiacetal 6 in the presence of TMSOTf provided
the protected xylofuranosylamine 7 (52% after purification, 5 g-
scale). We then used the chain extension methodology we
developed for the synthesis of UDP-Galf mimics:7d addition of
AllylSiMe3 to the N-protected glycosylamine 7 in the presence of
shown in Scheme 2. The 1,4-dideoxy-1,4-imino-
of 1 and 3 would be replaced by a more easily accessible
D
-galactitol unit
-arabino
L
iminopentitol, as in 4, and the nucleoside moiety would be mim-
icked by a simple substituted aromatic ring.
TMSOTf led to amino
high syn-diastereoselectivity, as no trace of the other (
D
-iditol derivative 8 in excellent yield with
-gulo)
⇑
Corresponding author. Tel.: +33 238 494 581; fax: +33 238 417 281.
E-mail address: olivier.martin@univ-orleans.fr (O.R. Martin).
D
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.09.065

6400
A. Chronowska et al. / Tetrahedron Letters 52 (2011) 6399–6402
OH
OH
OH
O
UDP-Gal
mutase
O
UDP-Galf
transferase
O
OUDP
OH
O
O
OUDP
O
O
OH
HO
OH
HO
OH
HO
OH
OH
HO
OH
Galactan fragment
UDP-Galp
UDP-Galf
Scheme 1. UDP-galactofuranose and galactan biosynthesis.
O
O
HO
OH
OH
NH
NH
OH
OH
OH
HN
H
N
H
N
O
O
P
OH
H
N
N
O
N
O
O
O
P
O
O
HO
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
HO
OH
OH
1
2
3
Figure 1. Previously synthesized inhibitors of UGM and GlfT2.
OH
OBn
H
N
NZ
R
R
OH
O
O
N
HO
OH
BnO
OBn
A
B
Z
OBn
N
+
N
R
+
-
O
BnO
OBn
C
4
Scheme 2. Retrosynthetic analysis of compounds of structure A.
diastereoisomer could be observed by NMR. The stereochemistry
of the new chiral center was determined after the cyclization step.
Ring closure was achieved by a two-step sequence: mesylation of
the free alcohol function of 8, then treatment of the resulting mes-
MeO
N
N
HO
HO
ylate with tBuOK gave the desired iminosugar 4 in the L-arabino
series.
HO
OH
HO
OH
Determination of the configuration of the pseudo-anomeric car-
bon by NMR was not possible directly on compound 4, because of
the presence of Z-group rotamers. Deprotection of the nitrogen
atom in 4 was achieved selectively by hydrogenolysis under basic
conditions leading to iminosugar 9 (Scheme 4), which could be
fully characterized by NMR. NOESY experiments on this compound
confirmed unambiguously the 1,2-cis configuration with the pres-
ence of a correlation between H1 and H4.
Having the allylic iminosugar 4 in hands, we then prepared the
nitrile oxide moiety (structure C) in order to perform the 1,3-dipolar
cycloaddition reaction. Because of their facile dimerization, nitrile
oxides are usually generated in situ by the dehydration of primary
Steviamine
5
Figure 2. Chemical structures of (À)-steviamine and its synthesized analogue.
OBn
BnO
OBn
BnO
O
O
a, b, c
d
OH
OBn
NHZ
OBn
D-xylose
6
7
Z
OBn
BnO
OH OBn
N
e
f, g
BnO
OBn
BnO
OBn
OBn NHZ
H
N
OBn
NZ
8
4
1
4
Scheme 3. Reagents and conditions: (a) HCl (0.11 M), MeOH, 30 °C, 3.5 h; (b) NaH
(6 equiv), BnBr (4.5 equiv), DMF, 0 °C to rt; (c) 1 M HCl:AcOH (1:4), 80 °C, 55% (three
steps); (d) H2NCO2Bn (2 equiv), TMSOTf (1 equiv), CH2Cl2, 4 Å MS, rt, 52%; (e)
AllTMS (7 equiv), TMSOTf (1 equiv), CH3CN, À20 °C, 85%; (f) MsCl (2.1 equiv), NEt3
(2.2 equiv), CH2Cl2, 4 Å MS, rt; (g) tBuOK (2 equiv), THF, rt, 59% (two steps).
OBn
BnO
OBn
4
9
Scheme 4. Reagents and conditions: H2, 10% Pd/C, NEt3 (0.25 equiv), iPrOH, rt, 97%.

A. Chronowska et al. / Tetrahedron Letters 52 (2011) 6399–6402
6401
nitro compounds15 or by base-induced dehydrohalogenation of
Z
OBn
hydroximoyl chlorides.16 The first method was not easily applicable
to the preparation of the planned nitrile oxides, as corresponding
starting nitro compounds are not commercially available. Conse-
quently we used the second methodology from hydroximoyl chlo-
rides, which are usually prepared by chlorination or oxidation of
aldoximes. We followed Kulkarni’s procedure,17 which would pro-
vide a great diversity of nitrile oxides of structure C possessing a
methylene knuckle, from the conjugated nitrostyrene precursors.
These compounds could be conveniently prepared by the addition
of nitromethane on different commercially available substituted
benzaldehydes.18 As a first example, p-anisaldehyde 10 was effi-
ciently converted into the conjugated nitrostyrene compound
11,17b which was then submitted to the action of titanium chloride
in the presence of triethylsilane to give the desired hydroximoyl
chloride 12;17b this precursor was used as a crude material in the
cycloaddition reaction (Scheme 5).
Cl
N
+
N
HO
OMe
BnO
OBn
12 (2 equiv)
4 (1 equiv)
OBn
NZ
O
N
OMe
BnO
OBn
13
Scheme 6. Reagents and conditions: NEt3 (6 equiv), CH2Cl2, rt, 67% (3:2 mixture of
two diastereoisomers).
10
OBn
8
MeO
9
NZ
1,3-Dipolar cycloaddition was then performed at room temper-
ature by mixing the dipolarophile 4 with an excess of the hydroxy-
moyl chloride 12 in the presence of triethylamine, for in situ
generation of the corresponding nitrile oxide (Scheme 6). The reac-
tion proved to be highly regioselective in favor of the 5-substituted
isoxazoline, which is in accordance with the predictions based on
frontier orbital theory.19 However the stereoselectivity was not
as high, as two diastereoisomers were obtained in a 3:2 ratio; these
isomers were quite difficult to separate by flash chromatography
on silica gel. Nevertheless a sufficient amount of one diastereoiso-
mer was obtained to perform the final hydrogenolysis step.
With the goal of cleaving the isoxazoline and deprotecting the
iminosugar moiety, compound 13 was submitted as a single dia-
stereoisomer to catalytic hydrogenolysis under aqueous acidic
conditions, in order to favor the hydrolysis of the intermediate
imine into a ketone. Quite surprisingly, this reaction gave directly
the indolizidine derivative 520 (Scheme 7), in good yield and as a
single diastereoisomer.21 Although the formation of 5 can be read-
ily explained, this result is remarkable in that it must involve a ser-
ies of at least 9 individual steps including a 5-step sequence of
cleavage of the N–O bond, hydrolysis of the resulting imine, dehy-
7
N
4
3
6
5
HO
O
N
1
OMe
BnO
OBn
2
HO
OH
13
5
Scheme 7. Reagents and conditions: H2, 10% Pd/C, H2O (2 equiv), 1 N HCl (1 equiv),
MeOH/CH2Cl2 1:1, rt, 57%.
Table 1
Inhibition profile of 5 toward different glycosidases
Enzymes
-Glucosidase
Inhibition (%) at 1000 lM
a
Yeast
Rice
4.7
8.7
Rat intestinal maltase
Almond
24.9
10.7
37.8
19.2
34.7
6.7
0
0.6
6.5
47.8
4.4
b-Glucosidase
Bovine liver
Coffee beans
Bovine liver
Jack beans
a
-Galactosidase
b-Galactosidase
-Mannosidase
b-Mannosidase
-Fucosidase
b-Glucuronidase
a
Snail
a-
L
Bovine kidney
Bovine liver
E. coli
Porcine kidney
Aspergillus niger
Penicillium decumbens
dration to an a,b-unsaturated ketone, hydrogenation to a saturated
ketone, and stereoselective intramolecular reductive amination.
The stereochemistry of the chiral center resulting from the
reductive amination was unambiguously determined by a NOESY
experiment, which indicated by the correlations between H1, H4,
and H9 that the three protons are all on the same side of the mol-
ecule. Compound 5 is an analogue of the recently isolated natural
product (À)-steviamine,10 bearing a p-methoxybenzyl group in-
a,a
-Trehalase
Amyloglucosidase
-Rhamnosidase
7.2
23.0
a-
L
In conclusion, we have developed an efficient synthesis of pro-
tected b-1-C-allyl-1,4-dideoxy-1,4-imino- -arabinitol, an imino-
L
stead of the methyl group and having a ‘b-
L-arabino’ configuration
sugar derivative which could be used in a diversity of coupling
reactions to prepare UDP-Galf mimics. It was successfully engaged
as a dipolarophile in a 1,3-dipolar cycloaddition reaction with a ni-
trile oxide. Further elaboration of the resulting isoxazoline by
hydrogenolysis led, by way of a remarkable sequence of steps, to
a new indolizidine, which is an analogue of the natural product
(À)-steviamine. Inhibition studies showed only weak inhibitory
properties for this new compound toward a panel of glycosidases.
Further work is in progress in our laboratory to achieve the synthe-
sis of iminosugar-based analogues of UDP-galactofuranose related
to A as potential inhibitors of the mycobacterial galactan biosyn-
thesis, using nitrile oxide 1,3-dipolar cycloaddition reaction.
in the pyrrolidine instead of the ‘ -lyxo’ configuration. Because of
a
-D
its relation with the well-known glycosidase inhibitors, this new
indolizidine was tested against a panel of glycosidases. With the
exception of b-glucuronidase from Escherichia coli, for which a
weak inhibition effect was observed, compound 5 did not show
significant inhibitory properties toward other glycosidases
(Table 1).
H
O2N
Cl
a
b
O
N
OMe
HO
OMe
OMe
Acknowledgment
10
11
12
A.C. is grateful to the Conseil Régional du Centre for the award
of a stipend.
Scheme 5. Reagents and conditions: (a) AcONH4 (1.1 equiv), CH3NO2, reflux, 82%;
(b) Et3SiH (2.1 equiv), TiCl4 (2.2 equiv), CH2Cl2, rt, crude.

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A. Chronowska et al. / Tetrahedron Letters 52 (2011) 6399–6402
12. Hu, X.-G.; Bartholomew, B.; Nash, R. J.; Wilson, F. X.; Fleet, G. W. J.; Nakagawa,
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20. Characterization
data
for
5
[(1S,2S,3S,5S,8aR)-3-hydroxymethyl-5-p-
methoxybenzyl-indolizidine-1,2-diol]:
[a
]
D = À28 (c 1, CHCl3). 1H NMR
(400 MHz, MeOD, numbering as in Scheme 7) d 7.08 (d, J = 8.3 Hz, 2H, Harom),
6.83 (d, J = 8.3 Hz, 2H, Harom), 4.02 (s, 1H, H3), 3.75–3.73 (m, 4H, H5b, OCH3),
3.68 (d, J = 2.8 Hz, 1H, H2), 3.57 (dd, J = 7.2, 10.8 Hz, 1H, H5a), 3.11 (dd, J = 3.4,
12.1 Hz, 1H, H10b), 2.88 (dd, J = 2.1, 7.2 Hz, 1H, H4), 2.64 (dt, J = 2.8, 2.8, 10.0 Hz,
1H, H1), 2.50 (tt, J = 3.4, 10.8 Hz, 1H, H9), 2.37–2.28 (m, 1H, H10a), 1.75–1.72 (m,
1H, H7b), 1.61–1.52 (m, 2H, H6), 1.39–1.36 (m, 1H, H8b), 1.25–1.13 (m, 1H, H7a),
1.11–1.01 (m, 1H, H8a). 13C NMR (100 MHz, MeOD, numbering as in Scheme 7)
d 159.52 (Carom), 132.43 (Carom), 131.34 (CHarom), 114.69 (CHarom), 80.54 (C3),
79.18 (C2), 73.11 (C4), 69.09 (C1), 68.15 (C9), 65.94 (C5), 55.64 (OCH3), 42.33
(C10), 32.08 (C8), 26.35 (C6), 25.67 (C7). HRMS (ESI) calcd for C17H26NO4 [M+H]+
308.18563, found 308.18564.
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Lett. 2010, 3, 136–138.
21. When the reaction was performed on the minor stereoisomer of 13, the
reaction gave a mixture (ꢀ1:1) of 5 and of the corresponding derivative having
conserved the OH group (7-OH derivative of 5).
11. Thompson, A. L.; Michalik, A.; Nash, R. J.; Wilson, F. X.; van Well, R.; Johnson, P.;
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2009, E65, o2904–o2905.