Synthesis and glycosidase inhibitory activity of isourea-type bicyclic sp2-iminosugars related to galactonojirimycin and allonojirimycin

Article abstract of DOI:10.1016/j.tet.2011.10.091

A series of sp²-iminosugars featuring a fused piperidine-isourea bicyclic core and hydroxylation profiles of stereochemical complementarity with the 'classical' iminosugars galactonojirimycin and allonojirimycin have been prepared and their inh

Full text of DOI:10.1016/j.tet.2011.10.091

Tetrahedron 68 (2012) 681e689
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and glycosidase inhibitory activity of isourea-type
bicyclic sp2-iminosugars related to galactonojirimycin and allonojirimycin
a
a
b
a,
*
ꢀ
ꢀ
ꢀ
Matilde Aguilar-Moncayo , Paula Díaz-Perez , Jose M. García Fernandez , Carmen Ortiz Mellet
,
,
M. Isabel García-Moreno a
*
a
ꢀ
ꢀ
ꢀ
Departamento de Química Organica, Facultad de Química, Universidad de Sevilla, c/ Profesor García Gonzalez n 1, E-41012 Sevilla, Spain
Instituto de Investigaciones Químicas, CSICdUniversidad de Sevilla, c/ Americo Vespucio n 49, Isla de la Cartuja, E-41092 Sevilla, Spain
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
A series of sp2-iminosugars featuring a fused piperidineeisourea bicyclic core and hydroxylation profiles
of stereochemical complementarity with the ‘classical’ iminosugars galactonojirimycin and allonojir-
imycin have been prepared and their inhibitory activity evaluated against a panel of commercial gly-
cosidases. The synthetic methodology involves 2-aminooxazoline pseudo-C-nucleosides, accessible from
vic-hydroxycarbodiimide precursors, as key intermediates and is compatible with molecular diversity-
oriented strategies. Alkyl, aryl and glycosyl substituents have been incorporated in order to assess the
potential of non-glyconic interactions to modulate the enzyme selectivity. All the galactonojirimycin
Article history:
Received 13 September 2011
Received in revised form 24 October 2011
Accepted 27 October 2011
Available online 2 November 2011
Keywords:
Iminosugars
Glycosidase inhibitors
Isourea
Galactonojirimycin
Allonojirimycin
derivatives behaved as potent competitive inhibitors of
higher for aliphatic substituents (in the nM range), but the highest selectivity within
isoenzymes was achieved for a N0-glucopyranosyl pseudodisaccharide analogue. Going from
-allo configuration further increased enzyme selectivity, but strongly penalized the inhibition potency.
b
-glucosidases. The inhibition potency was
-glucosidase
-galacto to
b
D
D
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Among sp2-iminosugars, bicyclic isourea derivatives have
proven particularly interesting.13 This structural feature is present
sp2-Iminosugars are carbohydrate mimics1 characterized by the
presence of a pseudoamide-type (e.g., (thio)urea, guanidine, (thio)
carbamate, or iso(thio)urea) nitrogen atom at the position equiva-
lent to that of the endocyclic oxygen in monosaccharides.2 In these
compounds the anomeric effect is exacerbated, which confers
chemical and conformational stability to reducing derivatives as
well as to the corresponding O, S- and N-pseudoglycosides.3 This is
remarkably different to the situation encountered in classical imi-
nosugars, such as nojirimycin (NJ, 1) or galactonojirimycin (GNJ, 2)
due to the lability of acetal functions involving nitrogen (Fig. 1).4
sp2-Iminosugars related to the piperidine,5 indolizidine,6 pyrroli-
dine,7 pirrolizidine7 and nor-tropane8 (calystegine) poly-
hydroxylated alkaloids have already been reported. Notably, several
representatives exhibited potent glycosidase inhibitory activity and
higher enzyme selectivities as compared with the parent imino-
sugars. Given the multitude of roles of glycosidases in biological
and pathological processes, selective inhibitors of these enzymes
bear strong potential as chemotherapeutic agents, e.g., in the
treatment of type II diabetes mellitus,9 viral infections,10 cancer11 or
lysosomal storage diseases.12
in the natural a,a
0-trehalose mimic trehazoline (3), a nanomolar
inhibitor of trehalase.14 Trehazoline binding to the active site of
trehalase benefits from simultaneous glyconic and non-glyconic
interactions, which has been further exploited in the design of
potent analogues.15 By closing an isourea-type ring between N-5
and O-6 in nojirimycin, the incorporation of a battery of sub-
stituents become feasible, which likewise provide an opportunity
for non-glyconic contributions to enzyme binding.13,16 As a proof of
concept, the bicyclic (N-5,O-6)-N0-(n-octylimino)-NJ derivative 4
was shown to be a very potent inhibitor of
b-glucosidases, in-
cluding human b-glucocerebrosidase (GCerase), that exhibited total
anomeric selectivity.17 X-ray data demonstrated that the octyl chain
of 4 and related sp2-iminosugar NJ derivatives sits in a hydrophobic
pocket at the vicinity of the active site in
b-glucosidase while it
leads to steric clashes in the case of -glucosidase, playing thus
a
a crucial role in both the free energy of binding and the enzyme
selectivity.18 The ability of 4 to increase the lysosomal concentra-
tion of a range of GCerase mutants though a rescuing mechanism19
and its facility to cross cell membranes makes it a good candidate
as pharmacological chaperone20 for the treatment of Gaucher
disease.17,21
The synthesis developed to build the fused piperidineeisourea
skeleton (I) relies in the spontaneous rearrangement of 2-
aminooxazoline pseudo-C-nucleosides (IIb) through the transient
* Corresponding authors. E-mail address: isagar@us.es (M.I. García-Moreno).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.091

682
M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
exocyclic substituent or in the configurational pattern and the
study of the influence of such structural modifications in the in-
hibitory activity was very appealing. Here we report the synthesis
of the new isourea-type bicyclic sp2-iminosugars 6e9, related to
GNJ, and the
D
-allo configured diastereomers 10 and 11 and
their evaluation against a panel of representatives commercial
glycosidases.
2. Results and discussion
2.1. Synthesis
Structures 6e9 were selected to check the influence of the nature
(aromatic, aliphatic, polar) of the exocyclic substituent in the gly-
cosidase inhibitory activity. The synthesis of the requested amino-
oxazoline precursors started from the pivotal vic-azidoalcohol
Fig. 1. Structures of NJ (1), GNJ (2), trehazoline (3), and the bicyclic isourea-type sp2-
iminosugars 4 (a NJ analogue) and 5 (a GNJ analogue).
open-chain aldehydo form (IIa), and was purposely conceived to
allow introducing molecular diversity at a relatively low synthetic
cost (Fig. 2). In principle, both the configurational pattern and the
nature of the N0-substituent can be systematically modified. Pre-
liminary results indicated that the GNJ bicyclic isourea analogue 5
12,18a derived from 1,2-O-isopropylidene-
a-D-galactofuranose,
and involved its transformation into 2-hydroxycarbodiimide in-
termediates, which were expected to undergo spontaneous intra-
molecular ring closing by nucleophilic addition of the primary
hydroxyl to the heterocummulene group. Two different strategies
were considered for the key azide/carbodiimide transformation,23
namely (i) reduction to the corresponding aminoalcohol (13) and
conversion into a thiourea derivative that can be further desulfu-
rated, or (ii) tandem Staudigereaza-wittig-type reaction24 with
triphenylphosphine and an isothiocyanate. The later reaction has
been shown to proceed through a transient phosphazide, rather
than an iminiphosphorane, when both reagents are added simul-
taneously to the reaction mixture containing the azide substrate,
affording directly the target carbodiimide.25 Preliminary experi-
ments evidenced that the efficiency of each route was dependent on
the nature of the isothiocyanate reagent, the first one being pref-
erably for the less reactive aliphatic isothiocyanates and the second
one in the case of aromatic derivatives. The respective reaction se-
quences leading tocompounds 6e8 are depicted in Schemes 1 and 2.
adopted a similar orientation as 4 in the active site of
b-glucosidase,
being a 50e100-fold more potent inhibitor.18a
HO
HO
N3
H2/Pd-C
98%
O
O
H2N
O
O
H
H
O
O
AcO
AcO
13
Fig. 2. Retrosynthetic analysis and structures of the new isourea-type bicyclic sp2-
12
BuNCS, py
74%
iminosugars prepared and evaluated in this work.
OR
TMSO
S
While the above commented kinetic and crystallographic data
demonstrate the utmost importance of non-glyconic interactions
involving the N-substituent in the b-glucosidase inhibitory potency
O
N
O
N
H
N C N
O
H
O
H
H
and selectivity of bicyclic sp2-iminosugars, there is not much in-
formation available about the optimal requirements that this sub-
stituent must fulfil for optimal binding. Thermodynamic studies by
isothermal titration calorimetry (ITC) point to a favourable entropic
contribution in the case of the octyl chain in 4 and 5. On the other
hand, X-ray structures point to some unfulfilled potential for aro-
matic and polar interactions with amino acid residues at the vi-
cinity of the active site. It is also unclear whether or not the
observed configurational promiscuity at the position equivalent to
C-4 in monosaccharides can also occur at other positions in the six-
membered piperidine ring.
HgO
88%
O
O
AcO
AcO
16
15, R = TMS
14, R = H
TMSCl, py
80%
1) TBAF, THF
83%
2) NaOMe, MeOH
NH
O
N
O
HO
N
H
90% TFA aq
O
N
O
HO
90%
H
HO
HO
O
OH
17
6
Scheme 1. Synthesis of the isourea-type bicyclic sp2-iminosugar 6.
A second important conclusion from our pervious work in this
field is that evaluation of the sp2-iminosugar inhibitor towards
Coupling of 13 and n-butyl isothiocyanate afforded the thiourea
adduct 14 with total chemoselectivity in 74% yield. Protection of the
hydroxyl group as the corresponding trimethylsilyl ether (/15)
and subsequent desulfuration with mercury(II) oxide led to car-
bodiimide 16. Sequential fluorolysis of the silyl ether function and
deacetylation gave the 2-butylaminooxazoline derivative 17
that, after trifluoroacetic acid-catalyzed hydrolysis of the acetal
protecting group and neutralization of the reaction mixture,
commercial
b-glucosidases from bovine liver and almonds, be-
longing to the same clan A as human GCrase in the CAZy classifi-
cation,22 provides a reliable feed-back for pre-selection of suitable
candidates for most costly tests on the non-commercial human
lysosomal enzymes as well as in vitro evaluation as pharmacolog-
ical chaperones. In the light of these considerations, the prepara-
tion of a wider series of derivatives differing in the nature of the

M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
683
It is known that
b
-glucosidases are often not very sensitive to
RO
N3
TMSO
the configuration at position C-4 in glycopyranosides or the
equivalent position in iminosugar glycomimetics. For instance,
RNCS, TPP
O
O
RN
C
N
O
O
H
H
the enzyme from bovine liver can hydrolyse
b-glucopyranosides
O
O
-galactopyranosides at essentially the same rate.28 The ex-
AcO
AcO
and
b
19, R = Ph (74%)
21, R = 1-Nph (75%)
12, R = H
18, R = TMS
TMSCl, py
90%
istence of additional non-glyconic interactions can alter the rel-
ative binding affinity of epimers, as seen by the 50-fold lower Ki
value observed for 5 as compared to 4 for this enzyme.18a We
were also interested to investigate whether or not non-glyconic
interactions could drive the binding of sp2-iminosugars that do
not match the normally accepted hydroxylation patterns of the
enzyme. The presence of a hydroxyl group at C-2 in the right
configuration is known to be essential for strong binding,29 but
the possibility for the enzyme accepting epimers at C-3 cannot be
RHN
TBAF, THF
NR
N
O
HO
HO
N
H
O
O
O
1) NaOMe, MeOH
H
AcO
HO
2) 90% TFA aq
OH
O
7, R = Ph (80%)
8, R = 1-Nph (85%)
20, R = Ph (84%)
22, R = 1-Nph (83%)
Scheme 2. Synthesis of the isourea-type bicyclic sp2-iminosugars 7 and 8.
discarded. The -allo configured derivatives 10 and 11, with
D
phenyl and b-D-glucopyranosyl exocyclic substituents, were
prepared for this purpose. To the best of our knowledge, no ac-
tivity data have been reported for allonojirimycin,30 the C-3
epimer of compound 1, although the corresponding 1-deoxy and
1-homo derivative have been shown to keep some residual
activity towards several glucosidases and galactosidases.31 Com-
parison of the inhibition data for 10 and 11 with those for the
underwent spontaneous furanose/piperidine rearrangement to
yield the target bicyclic sp2-iminosugar 6 (Scheme 1).
For the synthesis of the 5-N,6-O-(N0-phenyl and N0-1-
naphtyliminomethylidene)GNJ derivatives 7 and 8, compound 12
was first transformed into the 6-O-trimethylsilyl derivative 18.
Reaction of 18 with triphenylphosphine in the presence of phenyl
or 1-naphtyl isothiocyanate afforded the carbodiimide adducts 19
and 21 in 74 and 75% yield, respectively. Further removal of the silyl
ether group (/20 and 22) and final standard deprotection of the
alcohol functions proceeded as previously to give the fully un-
protected glycomimetics 7 and 8 (Scheme 2).
D
-galacto analogues 7 and 9, bearing identical N-substituents,
should allow estimating the effect of the configurational change
in the inhibitory properties. The results (see Section 2.2 herein-
after) did not recommend the synthesis of a wider series of de-
rivatives in this case.
The synthesis of 10 and 11 was accomplished in four steps from
the previously reported 3-O-acetyl-5-azido-5-deoxy-1,2-O-iso-
The synthesis of the pseudodisaccharide derivative 9 was mo-
tivated by the previous observation that glycosyl substituents can
modulate the inhibitory activity of sp2-iminosugar glycomimetics
towards different isoenzymes, imparting selectivity.26 Similarly to
aromatic isothiocyanates, glycopyranosyl isothiocyanates are acti-
vated towards nucleophilic addition due to the global electron
withdrawing effect of the glycosyl substituent.27 Direct trans-
formation of azide 18 into the b-(1/5) carbodiimide-linked glu-
coegalacto pseudodisaccharide derivative 23 was thus performed
in 60% yield by Staudingereaza-Wittig-type reaction with triphe-
propylidene-
hydroxyl group as the trimethylsilyl ether 26. Condensation with
phenyl or 2,3,4,6-tetra-O-acetyl- -glucopyranosyl isothiocyanate
a-D
-allofuranose 25,6c after protection of the primary
b-D
provided the corresponding carbodiimides 27 and 29, respectively.
Further transformation into the oxazoline derivatives 28 and 30
and the desired bicyclic isoureas proceeded smoothly following
a parallel reaction sequence to that above discussed for GNJ de-
rivatives (Scheme 4).
nylphosphine and 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl iso-
thiocyanate.27 Oxazoline ring closing (/24) and final
rearrangement to the bicyclic sp2-iminosugar 9 parallelled the
conditions and yields of the previous syntheses (Scheme 3).
OR
OAc
O
N3
O
O
AcO
AcO
NCS
OAc
TPP, 66%
H
PhNCS, TPP
45%
O
AcO
b
OAc
O
25, R = H
TMSCl, py
90%
TMSO
N3
AcO
AcO
NCS
OTMS
26, R = TMS
OR
O
OAc
OAc
O
O
O
PhN
C N
O
H
O
TPP, 60%
AcO
AcO
H
N
C
N
O
O
AcO
H
O
OAc
AcO
OTMS
O
O
18
27
AcO
OAc
O
29
AcO
TBAF, THF
95%
TBAF, THF
85%
N C N
O
OAc
O
TBAF, THF
97%
OAc
AcO
PhHN
O
H
OAc
H
N
AcO
AcO
O
N
AcO
23
H
O
N
OAc
H
O
H
N
O
O
O
AcO
AcO
O
H
O
H
N
OAc
H
AcO
O
O
O
AcO
O
28
30
H
AcO
1) NaOMe, MeOH
2) 90% TFA aq
(62%)
24
1) NaOMe, MeOH
2) 90% TFA aq
(95%)
O
OH
1) NaOMe, MeOH
2) 90% TFA aq
(96%)
OH
HO
OH
OH
NPh
O
N
HO
OH
O
O
O
N
HO
HO
O
OH
N
N
HO
N
HO
HO
OH
HO
HO
OH
HO
HO
OH
11
10
9
Scheme 3. Synthesis of the isourea-type bicyclic sp2-iminosugar 9.
Scheme 4. Synthesis of the isourea-type bicyclic sp2-iminosugars 10 and 11.

684
M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
The 1H and 13C NMR spectra of the new sp2-iminosugars con-
3. Conclusions
firmed their bicyclic character. Thus, the high field resonance of the
pseudoanomeric carbon (C-1) (78.3e76.2 ppm) was in agreement
with the presence of the hemiaminal functionality. The vicinal 3JH,H
The synthesis of 2-aminooxazoline pseudo-C-nucleosides via
transient vic-hydroxycarbodiimides and their rearrangement into
fused piperidineeisourea bicyclic systems has been implemented
to access a small collection of six sp2-iminosugar glycomimetics.
The efficiency of the reaction sequence and the possibility of using
different monosaccharides as starting materials make the approach
very well-suited for structureeactivity relationship studies where
both the configurational pattern and the nature of pseudoaglyconic
substituents can be systematically profiled. Both features contrib-
ute decisively to glycosidase inhibition potency and selectivity.
Although in this particular case the new derivatives synthesized did
values about the six-membered ring agreed with the
(6e9) and
-allo (10, 11) configurational patterns in the 4C1 chair
conformation. In all cases, the -anomer with the pseudoanomeric
D-galacto
D
a
hydroxyl group in the axial orientation fitting the anomeric effect,
was by far the major species in D2O solution (>95%). Minor pro-
portions of the b
-anomer were detected in some cases in the 1H
NMR spectra (see Supplementary data). Purity was further con-
firmed by combustion analysis.
not improve the inhibition results against
b-glucosidases pre-
2.2. Glycosidase inhibitory activity
viously attained with the lead compound 5, discouraging further
biological tests, the body of results here collected illustrates how
the configurational, anomeric and isoenzyme selectivities are
highly sensitive to structural modifications. The possibility to op-
timize the inhibition properties by acting on the nature of non-
glyconic substituents in combination with the systematic evalua-
tion of the products against a panel of commercial glycosidases
with structural resemblance to human enzymes is particularly in-
teresting in terms of time-saving and pre-selection of candidates
for specific targets. Application of this strategy to the control of
therapeutically relevant enzymes is currently pursued in our
laboratories.
The inhibitory activity of compounds 6e11 was evaluated
against a series of glycosidases including
glucosidase (almonds), -glucosidase/ -galactosidase (bovine liver,
cytosolic), -mannosidase (Jack bean), trehalase (pig kidney),
amyloglucosidase (Aspergillus niger), -galactosidase (green coffee
a-glucosidase (yeast), b-
b
b
a
a
beans and A. niger) and isomaltase (yeast). The corresponding in-
hibition constants (Ki) are collected in Table 1. Data for the pre-
viously synthesized derivatives 4 and 5 have been also included for
comparative purposes.
Table 1
Ki values (m
M) for 4e11 against a panel of glycosidasesa
4. Experimental section
4.1. General methods
Enzyme
4
5
6
7
8
9
10
n.i. n.i. n.i. n.i. n.i.
1.5 33 346 n.i.
249 383 308 n.i. n.i
11
a
b
a
-Glucosidase (baker’s yeast) 168 n.i.b
-Glucosidase (almond)
-Galactosidase (green
coffee bean)
n.i.
1.9 0.019 0.073
n.i. n.i. 288
5
Reagents and solvents were purchased from commercial
sources and used without further purification. Optical rotations
were measured with a JASCO P-2000 polarimeter, using a sodium
b
-Gluco/
b
-galactosidase
2.7 0.052 0.2
13
5.2 956 n.i. 933
(bovine liver)
Trehalase (pig kigney)
Amyloglucosidase (A. niger)
182 n.i.
n.i. n.i.
n.i.
n.i.
n.i. n.i. n.i. n.i. 320
n.i. n.i. n.i. n.i. 609
lamp (
l
¼589 nm) at 22 ꢀC in 1 cm or 1 dm tubes. IR spectra were
a
recorded on a JASCO FTIR-410 instrument. UV spectra were
Inhibition was competitive in all cases. No inhibition was observed for any
compound at 2 mM concentration on Jack bean
a
-mannosidase, A. niger
a
-galacto-
recorded on Philips PU-8710 instrument, unit for
3
values:
mMꢁ1 cmꢁ1. NMR experiments were performed at 300 (75.5),
and 500 (125.7) MHz using Bruker DMX300, and DRX500. 1-D
TOCSY as well as 2-D COSY and HMQC experiments were car-
ried out to assist in signal assignment. In the FABMS spectra, the
primary beam consisted of Xe atoms with maximum energy of
8 keV. The samples were dissolved in m-nitrobenzyl alcohol or
thioglycerol as the matrices and the positive ions were separated
and accelerated over a potential of 7 keV. NaI was added as
cationizing agent. Thin-layer chromatography was performed on
E. Merck precoated TLC plates, silica gel 30 F245, with visualiza-
tion by UV light and by charring with 10% H2SO4 or 0.2% w/v
cerium (IV) sulfatee5% ammonium molybdate in 2 M H2SO4 or
0.1% ninhydrin in EtOH. Column chromatography was performed
sidase and baker’s yeast isomaltase.
b
n.i., no inhibition observed at 2 mM concentration of the inhibitor.
The bicyclic GNJ derivatives 6e9 strongly inhibited
did no inhibit -glucosidase at concentrations up to 2 mM, in
agreement that previously observed for compound 5, and showed
very weak inhibitoryactivity towards -galactosidase (Ki in the range
249e383
M). The influence of the nature of the N0-substituent in
enzyme selectivity was evident when comparing the -glucosidases
b-glucosidase,
a
a
m
b
from almonds and bovine liver (cytosolic). Aliphatic substituents (5
and 6) afforded the most potent inhibitors of these enzymes, fol-
lowed by the aromatic derivatives (7 and8). In all cases the selectivity
ratio between these two isoenzymes was in the range 2.6e3.5. In
sharp contrast, the pseudodisaccharide derivative 9 was a very se-
lective inhibitor of the almonds enzyme (selectivity ration ca. 30),
on Chromagel (SDS silica 60 AC.C 70e200
yses were performed at the Servicio de Microanalisis del Instituto
m
m). Elemental anal-
ꢀ
which is remarkable considering that both
the same glycosyl hydrolase GH1 family in the CaZy classification.22
Changing the hydroxylation configurational profile from -gal-
acto to -allo had a dramatic effect in the enzyme binding abilities.
Thus, the N0-phenyl derivative 10 was a totally selective but very
weak inhibitor of almonds -glucosidase (Ki 346 M, that is, ca. 70-
b
-glucosidases belong to
de Investigaciones Químicas de Sevilla, Spain. 3-O-Acetyl-5-
azido-5-deoxy-1,2-O-isopropylidene-
was prepared from
-galactose using the reported route.6c 3-O-
Acetyl-5-amino-5-deoxy-1,2-O-isopropylidene- -galactofur-
a
-
D
-galactofuranose
(12)
D
D
D
a-D
anose (13) was synthesized by catalytic hydrogenation of azide
b
m
12 following the procedure already reported.18a 3-O-Acetyl-5-
fold weaker as compared with the galactonojirimycin analogue 7).
The pseudosaccharide derivative 11 completely lost the activity
against this enzyme. Although it has been previously suggested
azido-5-deoxy-1,2-O-isopropylidene-
synthesized from 3-O-acetyl-1,2-O-isopropylidene-6-O-trityl-
-allofuranose using the previously described route.6c
a-D-allofuranose 25 was
a
-
D
that there might be, in the active site of b-glucosidase, an extra
hydrogen-bond acceptor positioned in the vicinity of the 3-position
somewhere beneath the sugar ring,32 this interaction does not
seem to be efficient enough in the case of the bicyclic sp2-imino-
sugars considered in this work.
4.2. General procedure for the inhibition assays
Inhibition constant (Ki) values were determined by spectro-
photometrically measuring the residual hydrolytic activities of the

M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
685
glycosidases against the respective o- (for
from bovine liver) or p-nitrophenyl - or
other glycosidases) or
0-trehalose (for trehalase) in the presence
of compounds 4e11. Each assay was performed in phosphate buffer
or phosphateecitrate buffer (for - or -mannosidase and amylo-
b
-gluco/
-glycopyranoside (for
b
-galactosidase
C14H25N3O6Si: C, 46.78; H, 7.01; N, 11.69. Found: C, 46.68; H, 6.65;
N, 11.46.
a
b
-D
a,a
4.4. Preparation of 3-O-acetyl-5-(3-butylcarbodiimido)-5-
deoxy-1,2-O-isopropylidene-6-O-trimethylsilyl-a-D-
a
b
glucosidase) at the optimal pH for the enzymes. The reactions were
initiated by addition of enzyme to a solution of the substrate in the
absence or presence of various concentrations of inhibitor. The
mixture was incubated for 10e30 min at 37 ꢀC or 55 ꢀC (for amy-
loglucosidase) and the reaction was quenched by addition of 1 M
Na2CO3. For trehalase the reaction was stopped by placing the
mixture over boiling water for 3 min, cooled in ice/water and
centrifuged at 12,000 rpm for 5 min for remove the denatured
galactofuranose (16)
4.4.1. 3-O-Acetyl-5-(3-butylthioureido)-5-deoxy-1,2-O-isopropy-
lidene-
-galactofuranose (14). A solution of amine 1318a (130 mg,
a-D
0.5 mmol) in pyridine (5 mL), Et3N (0.5 mL, 3.6 mmol) and n-butyl
isothiocyanate (0.6 mmol) were added and the mixture was stirred
at room temperature for 18 h. Then, the solvent was removed under
reduced pressure and the resulting residue coevaporated several
times with toluene and purified by column chromatography
(40:1/20:1 CH2Cl2/MeOH) to afford the thioureido derivative 14
protein. The concentration of D-glucose in the supernatant was
determined by the glucose oxidaseeperoxidase method (Glucose
Trinder 100, from Sigma). Reaction times were appropriate to ob-
tain 10e20% conversion of the substrate in order to achieve linear
rates. The absorbance of the resulting mixture was determined at
405 nm or 505 nm (for trehalase). Approximate values of Ki were
determined using a fixed concentration of substrate (around the KM
value for the different glycosidases) and various concentrations of
inhibitor. Full Ki determinations and enzyme inhibition mode were
determined from the slope of LineweavereBurk plots and double
reciprocal analysis. Representative examples of the Line-
weavereBurk plots, with typical profile for competitive inhibition
mode, are shown in the Supplementary data.
(139 mg, 74%). Rf 0.58 (20:1 CH2Cl2/MeOH); [
a
]
D ꢁ35 (c 1.0, CH2Cl2);
UV (CH2Cl2) 251 nm ( 13.4); IR (KBr) nmax 3362, 2959, 1746,
3
mM
1553, 1381, 1236, 1094 cmꢁ1 1H NMR (500 MHz, CDCl3, 313 K)
;
d
6.61 (br s, 1H, NH), 6.42 (br s, 1H, NH0), 5.88 (d, 1H, J1,2¼3.8 Hz, H-
1), 5.11 (d, 1H, J3,4¼3.0 Hz, H-3), 4.63 (d, 1H, H-2), 4.34 (m, 1H, H-5),
4.17 (dd, 1H, J4,5¼6.9 Hz, H-4), 3.91 (dd, 1H, J6a,6b¼11.8 Hz,
J5,6a¼3.9 Hz, H-6a), 3.83 (br d, 1H, H-6b), 3.43 (m, 2H, CH2N), 3.02
(br s, 1H, OH), 2.07 (s, 3H, MeCO), 1.55 (m, 2H, CH2CH2N), 1.57, 1.31
(2s, 6H, CMe2), 1.32 (m, 2H, CH2), 0.90 (t, 3H, 3JH,H¼7.4 Hz, CH3); 13
C
NMR (125.7 MHz, CDCl3, 313 K)
d 182.7 (CS), 170.5 (CO), 113.5
(CMe2), 105.4 (C-1), 84.6 (C-2, C-4), 77.4 (C-3), 63.1 (C-6), 56.7 (C-5),
44.6 (CH2N), 30.9 (CH2), 26.9, 25.8 (CMe2), 20.7 (MeCO), 20.0 (CH2),
13.7 (CH3); FABMS: m/z 399 (50, MþNaþ), 377 (100, MþHþ). Anal.
Calcd for C16H28N2O6S: C, 51.05; H, 7.50; N, 7.44. Found: C, 50.91; H,
7.53; N, 7.35.
4.3. Synthesis of 3-O-acetyl-5-azido-5-deoxy-1,2-O-
isopropylidene-6-O-trimethylsilyl-a-D-furanoses (18, 26)
To a solution of 3-O-acetyl-azido-5-deoxy-1,2-O-isopropylidene-
-galactofuranose 126c or 3-O-acetyl-5-azido-5-deoxy-1,2-O-iso-
4.4.2. 3-O-Acetyl-5-(3-butylthioureido)-5-deoxy-1,2-O-isopropy-
lidene-6-O-trimethylsilyl-a-D-galactofuranose (15). To a solution of
a-D
propylidene-6-O-trimethylsilyl-
a-D
-allofuranose 256c (0.57 mmol)
the thioureido derivative 14 (218 mg, 0.58 mmol) in pyridine
(5.5 mL), trimethylsilyl chloride (0.94 mL) and hexamethyldisila-
zane (1.85 mL) were added and the reaction mixture was stirred for
2 h at room temperature. The solvents were removed, and the
residue was extracted with petroleum ether, concentrated and
purified by column chromatography using 1:2 EtOAc/petroleum
ether as eluent. Yield: 208 mg (80%). Rf 0.63 (1:1 EtOAc/petroleum
in pyridine (3.7 mL), trimethylsilyl chloride (0.93 mL) and hexame-
thyldisilazane (1.84 mL) were added and the reaction mixture was
stirred at room temperature for 2 h. The solvents were removed, and
the residue was extracted with petroleum ether, concentrated and
purified by column chromatography using 1:6 EtOAc/petroleum
ether as eluent.
ether); [
(KBr) nmax 3418, 2957, 1750, 1643, 1541, 1383, 1256, 1094 cmꢁ1
NMR (300 MHz, CDCl3)
6.75 (br s, 1H, NH), 6.21 (br s, 1H, NH0),
a
]
D ꢁ27 (c 1.0, CH2Cl2); UV (CH2Cl2) 251 nm (
3
mM 18.8); IR
4.3.1. 3-O-Acetyl-5-azido-5-deoxy-1,2-O-isopropylidene-6-O-trime-
;
1H
thylsilyl-
EtOAc/petroleum ether); [
2965, 1750, 1375, 1227, 1103 cmꢁ1; 1H NMR (500 MHz, CDCl3)
a
-
D
-galactofuranose (18). Yield: 184 mg (90%). Rf 0.47 (1:6
d
a
]
D
ꢁ48 (c 1.0, CH2Cl2); IR (KBr) nmax
5.82 (d, 1H, J1,2¼3.8 Hz, H-1), 5.10 (d, 1H, J3,4¼2.3 Hz, H-3), 4.55 (d,
1H, H-2), 4.12 (m, 2H, H-4, H-5), 3.72 (m, 1H, H-6a), 3.66 (dd, 1H,
J6a,6b¼10.1 Hz, J5,6b¼5.6 Hz, H-6b), 3.38 (m, 2H, CH2N), 2.03 (s, 3H,
MeCO), 1.52 (m, 2H, CH2CH2N), 1.52, 1.25 (2 s, 6H, CMe2), 1.32
d
5.90
(d, 1H, J1,2¼3.9 Hz, H-1), 5.19 (d, 1H, J3,4¼3.0 Hz, H-3), 4.60 (d, 1H, H-
2), 4.00 (dd, 1H, J4,5¼7.4 Hz, H-4), 3.80 (dd, 1H, J6a,6b¼10.0 Hz,
J5,6a¼4.0 Hz, H-6a), 3.75 (ddd, 1H, J5,6b¼6.2 Hz, H-5), 3.70 (dd, 1H,
H-6b), 2.08 (s, 3H, MeCO), 1.59, 1.33 (2s, 6H, CMe2), 0.12 (s, 9H,
(m, 2H, CH2), 0.87 (t, 3H, 3JH,H¼7.3 Hz, CH3), 0.05 (s, 9H, SiMe3); 13
C
NMR (75.5 MHz, CDCl3) d 182.6 (CS), 169.9 (CO), 113.4 (CMe2), 105.2
SiMe3); 13C NMR (125.7 MHz, CDCl3)
d
170.0 (CO), 113.7 (CMe2),
(C-1), 84.8 (C-2, C-4), 76.9 (C-3), 62.5 (C-6), 56.4 (C-5), 45.1 (CH2N),
30.9 (CH2), 26.9, 25.8 (CMe2), 25.8 (CH2CH2N), 20.7 (MeCO), 20.0
(CH2), 13.7 (CH3), ꢁ0.74 (SiMe3); FABMS: m/z 471 (20, MþNaþ), 449
(100, MþHþ). Anal. Calcd for C19H36N2O6SSi: C, 50.86; H, 8.09; N,
6.24. Found: C, 50.79; H, 7.78; N, 6.20.
105.5 (C-1), 85.0 (C-2), 84.3 (C-4), 77.5 (C-3), 63.4 (C-5), 63.0 (C-6),
27.1, 26.3 (CMe2), 21.0 (MeCO), ꢁ0.49 (SiMe3); FABMS: m/z 382 (100,
MþNaþ). Anal. Calcd for C14H25N3O6Si: C, 46.78; H, 7.01; N, 11.69.
Found: C, 46.81; H, 6.77; N, 11.66.
4.3.2. 3-O-Acetyl-5-azido-5-deoxy-1,2-O-isopropylidene-6-O-trime-
4.4.3. 3-O-Acetyl-5-(3-butylcarbodiimido)-5-deoxy-1,2-O-iso-
thylsilyl-
EtOAc/petroleum); [
2109, 1760, 1379, 1251 cmꢁ1
a
-
D
-allofuranose (26). Yield: 184 mg (90%). Rf 0.33 (1:6
propylidene-6-O-trimethylsilyl-a-D-galactofuranose (16). To a solu-
a
]
ꢁ88 (c 1.0, CH2Cl2); IR (KBr) nmax 2936,
tion of the thiourea 15 (0.47 mmol) in CH2Cl2/H2O (1:1, 12.0 mL),
HgO (1.36 mmol) was added. The reaction mixture was stirred
vigorously at room temperature for 45 min, diluted with CH2Cl2
(10 mL), and the organic phase was separated. The aqueous phase
was extracted with CH2Cl2 (2ꢂ10 mL), and the combined organic
extracts were dried (MgSO4), and concentrated. The resulting res-
idue was purified by column chromatography using 1:3 EtOAc/
petroleum ether as eluent. Yield: 172 mg (88%). Rf 0.60 (1:2 EtOAc/
D
;
1H NMR (300 MHz, CDCl3)
d 5.80 (d,
1H, J1,2¼3.7 Hz, H-1), 4.83 (dd, 1H, J2,3¼4.7 Hz, J3,4¼3.0 Hz, H-3),
4.81 (m, 1H, H-2), 4.20 (dd, 1H, J4,5¼4.3 Hz, H-4), 3.80 (m, 1H, H-5),
3.64 (m, 2H, H-6a, H-6b), 2.11 (s, 3H, MeCO), 1.54, 1.29 (2s, 6H,
CMe2), 0.13 (s, 9H, SiMe3); 13C NMR (75.5 MHz, CDCl3)
d 169.8
(CO), 113.1 (CMe2), 103.9 (C-1), 77.4 (C-2), 76.0 (C-4), 72.9 (C-3),
63.7 (C-6), 62.3 (C-5), 27.1, 26.5 (CMe2), 20.5 (MeCO), ꢁ0.83
(SiMe3); FABMS: m/z 382 (100, MþNaþ). Anal. Calcd for
petroleum ether); [
a
]
D ꢁ51 (c 1.0, CH2Cl2); IR (KBr) nmax 2954, 2139,

686
M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
1751, 1377, 1235, 1090 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d
5.88 (d,
Et2O/petroleum ether. Yield: 215 mg (60%). Rf 0.37 (2:1 Et2O/pe-
troleum ether); [
ꢁ35 (c 1.0, CH2Cl2); IR (KBr) nmax 2953, 2143,
1751, 1643, 1375, 1229, 1107 cmꢁ1; 1H NMR (500 MHz, CDCl3)
1H, J1,2¼4.0 Hz, H-1), 5.28 (d, 1H, J3,4¼3.3 Hz, H-3), 4.58 (d, 1H, H-2),
3.96 (dd, 1H, J4,5¼6.7 Hz, H-4), 3.75 (dd, 1H, J6a,6b¼9.9 Hz,
J5,6a¼4.8 Hz, H-6a), 3.70 (m, 1H, H-5), 3.65 (dd, 1H, J5,6b¼5.7 Hz, H-
6b), 3.26 (m, 2H, CH2N), 2.09 (s, 3H, MeCO), 1.58 (m, 2H, CH2CH2N),
1.41 (m, 2H, CH2), 1.61, 1.35 (2s, 6H, CMe2), 0.93 (t, 3H, JH,H¼7.3 Hz,
a]
D
d
5.85
(d, 1H, J1,2¼3.9 Hz, H-1), 5.18 (d, 1H, J3,4¼3.5 Hz, H-3), 5.17 (t, 1H,
0
0
0
0
0
0
0
0
J2 ,3 ¼J3 ,4 ¼9.5 Hz, H-3 ), 5.09 (t,1H0, J4 ,5 ¼9.5 Hz, H-4 ), 4.95 (dd,1H,
0
0
0
J1 ,2 ¼9.5 Hz, H-2 ), 4.76 (d, 1H, H-10), 4.56 (d, 1H, H-2), 4.22 (dd, 1H,
CH3), 0.15 (s, 9H, SiMe3); 13C NMR (75.5 MHz, CDCl3)
d
169.7 (CO),
J6a ,6b ¼12.3 Hz, J5 ,6a ¼4.7 Hz, H-6a ), 4.11 (dd, 1H, J5 ,6b ¼2.2 Hz, H-
6b0), 3.97 (dd, 1H, J4,5¼7.0 Hz, H-4), 3.83 (ddd, 1H, J5,6b¼6.1 Hz,
J5,6a¼5.4 Hz, H-5), 3.73 (m, 1H, H-50), 3.71 (dd, 1H, J6a,6b¼10.5 Hz, H-
6a), 3.64 (dd, 1H, H-6b), 2.07, 2.06, 2.04, 2.00, 1.98 (5s, 15H, 5MeCO),
1.55, 1.32 (2s, 6H, CMe2), 0.12 (s, 9H, SiMe3); 13C NMR (125.7 MHz,
0
0
0
0
0
0
140.6 (NCN), 113.5 (CMe2), 105.1 (C-1), 85.3 (C-2), 83.9 (C-4), 77.3
(C-3), 63.7 (C-6), 59.5 (C-6), 46.3 (CH2N), 33.1 (CH2CH2N), 27.1, 26.4
(CMe2), 20.8 (MeCO), 20.0 (CH2), 13.6 (CH3), ꢁ0.61 (SiMe3); FABMS:
m/z 365 (70, [MþNaꢁTMS]þ), 343 (100, [MþHꢁTMS]þ). Anal. Calcd
for C19H34N2O6Si: C, 55.05; H, 8.27; N, 6.76. Found: C, 54.89; H, 8.50;
N, 6.72.
CDCl3)
d 170.7, 170.3, 169.7, 169.3, 169.2 (5 CO), 138.7 (NCN), 113.5
(CMe2), 105.1 (C-1), 85.0 (C-2), 84.9 (C-10), 83.4 (C-4), 77.2 (C-3),
73.5 (C-50), 73.1 (C-30), 72.5 (C-20), 68.2 (C-40), 63.1 (C-6), 61.9 (C-60),
59.6 (C-5), 27.0, 26.3 (CMe2), 20.7, 20.6, 20.5, 20.4, 20.3 (MeCO),
ꢁ0.66 (SiMe3); FABMS: m/z 711 (90, MþNaþ), 689 (50, MþHþ).
Anal. Calcd for C29H44N2O15Si: C, 50.57; H, 6.44; N, 4.07. Found: C,
50.38; H, 6.28; N, 3.91.
4.5. General procedure for the preparation of 3-O-acetyl-5-
alkyl (aryl, glycopyranosyl)carbodiimido-5-deoxy-1,2-O-
isopropylidene-6-O-trimethylsilyl-
a-D-furanoses via aza-
Wittig reaction (19, 21, 23, 27 and 29)
A solution of azide 18 or 26 (186 mg, 0.52 mmol) in toluene
4.5.4. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(3-phenylcarbod-
(3.5 mL) was stirred under Ar for 30 min. Then, the corresponding
phenyl, naphtyl, or 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl iso-
iimido)-6-O-trimethylsilyl-
tography, eluent toluene/1:7 EtOAc/toluene. Yield: 102 mg (45%).
Rf 0.51 (1:7 EtOAc/toluene); [
þ50 (c 1.0, CH2Cl2); IR (KBr) nmax
2962, 2134, 1752, 1386, 1251, 1099 cmꢁ1; 1H NMR (300 MHz, CDCl3)
a-D-allofuranose (27). Column chroma-
thiocyanate (0.62 mmol) and a solution of PPh3 (0.63 mmol) in
toluene (2 mL) were added at room temperature. The reaction
mixture was stirred at 80 ꢀC for 2 h and concentrated. The resulting
residue was purified by column chromatography using the eluent
indicated in each case to afford the carbodiimide derivatives 19, 21,
23, 27 and 29 as amorphous solids.
a]
D
d
7.31e7.07 (m, 5H, Ph), 5.80 (d, 1H, J1,2¼3.6 Hz, H-1), 4.92 (dd, 1H,
J3,4¼8.4 Hz, J2,3¼4.9 Hz, H-3), 4.84 (dd, 1H, H-2), 4.02 (ddd, 1H,
J4,5¼5.4 Hz, J4,6b¼1.8 Hz, H-4), 3.83 (dd, 1H, J6a,6b¼8.5 Hz,
J5,6a¼3.9 Hz, H-6a), 3.82 (ddd, 1H, J5,6b¼8.5 Hz, H-5), 3.67 (td, 1H, H-
6b), 2.14 (s, 3H, MeCO), 1.55, 1.34 (2s, 6H, CMe2), 0.06 (s, 9H, SiMe3);
4.5.1. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(3-phenylcarbodi-
13C NMR (125.7 MHz, CDCl3)
d 169.9 (CO), 139.7 (NCN), 137.8e123.8
imido)-6-O-trimethylsilyl-
matography, eluent toluene/1:7 EtOAc/toluene. Yield: 167 mg
(74%). Rf 0.50 (1:7 EtOAc/toluene); [
D ꢁ57 (c 1.0, CH2Cl2); IR (KBr)
nmax 2968, 2139, 1756, 1389, 1238, 1094 cmꢁ1 1H NMR (300 MHz,
CDCl3)
(dd, 1H, J3,4¼3.1 Hz, J2,3¼0.5 Hz, H-3), 4.58 (dd, 1H, H-2), 4.02 (dd,
1H, J4,5¼7.2 Hz, H-4), 3.96 (ddd, 1H, J5,6b¼6.3 Hz, J5,6a¼4.7 Hz, H-5),
3.80 (dd, 1H, J6a,6b¼10.5 Hz, H-6a), 3.74 (dd, 1H, H-6b), 2.07 (s, 3H,
MeCO), 1.57, 1.23 (2s, 6H, CMe2), 0.07 (s, 9H, SiMe3); 13C NMR
a
-
D
-galactofuranose (19). Column chro-
(Ph),113.0 (CMe2), 104.0 (C-1), 77.6 (C-2), 76.6 (C-4), 73.2 (C-3), 62.3
(C-6), 61.1 (C-5), 26.6, 26.5 (CMe2), 20.5 (MeCO), ꢁ0.84 (SiMe3);
FABMS: m/z 435 (90, MþHþ). Anal. Calcd for C21H30N2O6Si: C,
58.04; H, 6.96; N, 6.45. Found: C, 57.92; H, 6.77; N, 6.36.
a]
;
d
7.15e7.05 (m, 5H, Ph), 5.88 (d, 1H, J1,2¼3.9 Hz, H-1), 5.25
4.5.5. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-[3-(2,3,4,6-tetra-
O-acetyl-b-D-glucopyranosyl)carbodiimido]-6-O-trimethylsilyl-a-D-
allofuranose (29). Column chromatography, eluent toluene/1:5
EtOAc/toluene. Yield: 236 mg (66%). Rf (1:2 EtOAc/toluene) 0.42;
(125.7 MHz, CDCl3)
d
169.8 (CO), 140.2 (NCN), 137.9e124.1 (Ph),
[
a
]
D þ51 (c 1.0, CH2Cl2); IR (KBr) nmax 2943, 2149, 1760, 1371, 1236,
113.6 (CMe2), 105.3 (C-1), 85.1 (C-2), 83.7 (C-4), 77.4 (C-3), 63.4 (C-
6), 60.4 (C-5), 27.1, 26.4 (CMe2), 20.8 (MeCO), ꢁ0.67 (SiMe3);
FABMS: m/z 457 (80, MþNaþ), 435 (100, MþHþ). Anal. Calcd for
C21H30N2O6Si: C, 58.04; H, 6.96; N, 6.45. Found: C, 57.99; H, 6.91; N,
6.40.
1045 cmꢁ1. 1H NMR (500 MHz, CDCl30)
d
5.80 (d, 1H, J1,2¼3.6 Hz, H-
0
0
0
0
0
0
1), 5.18 (t, 1H, J2 ,3 ¼J3 ,4 ¼9.5 Hz, H-3 ), 5.10 (t, 1H, J4 ,5 ¼9.5 Hz, H-
40), 4.94 (dd, 1H, J1 ,2 ¼8.8 Hz, H-2 ), 4.84 (dd, 1H, J3,4¼8.2 Hz,
0
0
0
J2,3¼5.1 Hz, H-3), 4.81 (d, 1H, H-2), 4.71 (d, 1H, H-10), 4.24 (dd, 1H,
0
0
0
0
0
J6a ,6b ¼12.4 Hz, J5 ,6a ¼4.9 Hz, H-6a ), 4.17 (dd, 1H, J4,5¼40.5 Hz, H-4),
0
0
0
4.13 (dd, 1H, J5 ,6b ¼1.8 Hz, H-6b ), 3.80 (m, 2H, H-5, H-5 ), 3.73 (dd,
1H, J6a,6b¼10.2 Hz, J5,6a¼4.8 Hz, H-6a), 3.54 (dd, 1H, J5,6b¼7.2 Hz, H-
6b), 2.12, 2.08, 2.07, 2.02, 1.99 (5 s, 15H, 5MeCO), 1.53, 1.33 (2 s, 6H,
4.5.2. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(3-napht-1-ylcar-
bodiimido)-6-O-trimethylsilyl-
chromatography, eluent toluene/1:7 EtOAc/toluene. Yield:
189 mg (75%). Rf 0.46 (1:3 EtOAc/toluene); [
D ꢁ76 (c 1.0, CH2Cl2);
IR (KBr) nmax 2943, 2133, 1744, 1585, 1379, 1228, 1116 cmꢁ1; 1H NMR
(300 MHz, CDCl3, 313 K) 8.29e6.36 (m, 7H, Ph), 5.91 (d, 1H,
a-
D-galactofuranose
(21). Column
CMe2), 0.14 (s, 9H, SiMe3); 13C NMR (125.7 MHz, CDCl3)
d 170.5,
a
]
170.1, 169.8, 169.7, 169.2 (5 CO), 137.8 (NCN), 112.9 (CMe2), 104.0 (C-
1), 84.7 (C-10), 77.5 (C-2), 76.6 (C-4), 73.6 (C-50), 72.8 (C-30), 72.7 (C-
3), 72.5 (C-20), 67.9 (C-40), 62.0 (C-6), 61.7 (C-60), 60.1 (C-5), 26.6,
26.5 (CMe2), 20.8, 20.7, 20.6, 20.5, 20.4 (MeCO), ꢁ0.67 (SiMe3);
FABMS: m/z 711 (90, MþNaþ). Anal. Calcd for C29H44N2O15Si: C,
50.57; H, 6.44; N, 4.07. Found: C, 50.45; H, 6.37; N, 3.95.
d
J1,2¼4.0 Hz, H-1), 5.30 (d, 1H, J3,4¼3.0 Hz, H-3), 4.40 (dd, 1H, H-2),
4.07 (dd, 1H, J4,5¼7.0 Hz, H-4), 4.04 (ddd, 1H, J5,6b¼6.0 Hz,
J5,6a¼4.5 Hz, H-5), 3.86 (dd, 1H, J6a,6b¼10.5 Hz, H-6a), 3.80 (dd, 1H,
H-6b), 2.08 (s, 3H, MeCO), 1.66, 1.32 (2s, 6H, CMe2), ꢁ0.06 (s, 9H,
SiMe3); 13C NMR (125.7 MHz, CDCl3, 313 K)
d
169.8 (CO), 137.5
4.6. General procedure for the preparation of 2-alkyl(aryl,
glycopyranosyl)amino-4-(tetrafuranos-40-yl)-2-oxazolines (17,
20, 22, 24, 28 and 30)
(NCN), 136.7e120.8 (Ph), 113.7 (CMe2), 105.3 (C-1), 85.2 (C-2), 83.7
(C-4), 76.8 (C-3), 63.5 (C-6), 60.6 (C-5), 27.2, 26.4 (CMe2), 20.8
(MeCO), e0.64 (SiMe3); FABMS: m/z 507 (100, MþNaþ), 485 (75,
MþHþ). Anal. Calcd for C25H32N2O6Si: C, 61.96; H, 6.66; N, 5.78.
Found: C, 62.05; H, 6.80; N, 5.71.
To a solution of the corresponding carbodiimido derivative 16,
19, 21, 23, 27 and 29 (0.34 mmol) in THF (6.3 mL) at 0 ꢀC under Ar,
TBAF (1 M in THF, 0.35 mL) was added. In the case of 24 and 30, the
reaction mixture was adjusted at pH 7 using glacial AcOH. The
solution was stirred at 0 ꢀC for 25 min, then diluted with Et2O
(6 mL), washed with water or iced saturated aqueous NaHCO3
4.5.3. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-[3-(2,3,4,6-tetra-
O-acetyl-
b-D-glucopyranosyl)carbodiimido]-6-O-trimethylsilyl-a-D-
galactofuranose (23). Column chromatography, eluent 1:1/3:1

M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
687
(2ꢂ3 mL), dried (MgSO4), filtered and concentrated. The resulting
residue was purified by column chromatography using the eluent
indicated in each case to obtain the corresponding isoureas. In the
(C-4), 76.9 (C-3), 73.2 (C-30, C-50), 71.2 (C-6), 69.7 (C-20), 68.5 (C-
40), 64.2 (C-5), 62.0 (C-60), 27.2, 26.5 (CMe2), 20.9e20.8 (MeCO);
FABMS: m/z 639 (100, MþNaþ), 617 (90, MþHþ). Anal. Calcd for
C26H36N2O15: C, 50.65; H, 5.88; N, 4.54. Found: C, 50.65; H, 5.92;
N, 4.42.
ꢀ
case of 16, the resulting residue was directly Zemplen O-deacety-
lation affording the compound 17 further purification by column
chromatography.
4.6.5. (4R)-4-[(40R)-30-O-Acetyl-10,20-O-isopropylidene-
a-D-eryth-
4.6.1. (4S)-2-Butylamino-4-[(40S)-10,20-O-isopropylidene-
furanos-40-yl]-2-oxazoline (17). Column chromatography, eluent
45:5:3 EtOAc/EtOH/H2O. Yield: 85 mg (83%). Rf 0.26 (45:5:3 EtOAc/
b
-
L
-threo-
rofuranos-40-yl]-2-phenylamino-2-oxazoline (28). Column chroma-
tography, eluent 100:1/50:1 CH2Cl2/MeOH. Yield: 117 mg (95%). Rf
0.55 (20:1 CH2Cl2/MeOH); [
a
]
þ83 (c 1.0, CH2Cl2); IR (KBr) nmax
D
EtOH/H2O); [
a]
ꢁ42 (c 0.99, CH2Cl2); IR (KBr) nmax 3433, 2959,
2968, 1744, 1553, 1379, 1251, 1093 cmꢁ1; 1H NMR (500 MHz, CDCl3)
D
1740, 1559, 1396, 1261, 1092 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
5.82
d
7.36e6.97 (m, 5H, Ph), 5.81 (d, 1H, J1,2¼2.4 Hz, H-1), 4.82 (br s, 2H,
(d, 1H, J1,2¼3.8 Hz, H-1), 4.64 (t, 1H, J5,6a¼J6a,6b¼8.8 Hz, H-6a), 4.50
(dd, 1H, J2,3¼1.2 Hz, H-2), 4.46 (dd, 1H, J5,6b¼5.7 Hz, H-6b), 4.36
(ddd, 1H, J4,5¼6.8 Hz, H-5), 4.04 (dd, 1H, J3,4¼4.3 Hz, H-3), 3.77 (dd,
H-2, H-3), 4.37 (t, 1H, J5,6a¼J6a,6b¼8.4 Hz, H-6a), 4.28 (m, 2H, H-5, H-
6b), 4.15 (dd, 1H, J3,4¼7.8 Hz, J4,5¼5.4 Hz, H-4), 2.11 (s, 3H, MeCO),
1.56, 1.31 (2s, 6H, CMe2); 13C NMR (125.7 MHz, CDCl3)
d 170.5 (CO),
3
1H, H-4), 3.21 (t, 2H, JH,H¼7.0 Hz, CH2N), 1.52 (m, 2H, CH2CH2N),
158.0 (CN), 129.1e118.8 (Ph), 113.4 (CMe2), 104.3 (C-1), 79.5 (C-4),
78.0 (C-2), 74.1 (C-3), 69.3 (C-6), 66.0 (C-5), 26.9, 26.8 (CMe2), 21.0
(MeCO); FABMS: m/z 385 (80, MþNaþ), 363 (100, MþHþ). Anal.
Calcd for C18H22N2O6: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.52; H,
5.95; N, 7.52.
1.33 (m, 2H, CH3), 1.48, 1.26 (2s, 6H, CMe2), 0.92 (t, 3H, 3JH,H¼7.0 Hz,
CH3); 13C NMR (125.7 MHz, CD3OD)
d 163.9 (CN), 114.5 (CMe2),
106.4 (C-1), 89.2 (C-2), 88.4 (C-4), 76.3 (C-3), 72.4 (C-6), 61.1 (C-5),
43.5 (CH2N), 32.4 (CH2CH2N), 27.8, 27.7 (CMe2), 20.8 (CH2), 13.9
(CH3); FABMS: m/z 301 (100, MþHþ). Anal. Calcd for C14H24N2O5: C,
55.98; H, 8.05; N, 9.33. Found: C, 56.27; H, 8.01; N, 9.44.
4.6.6. (4R)-4-[(40R)-30-O-Acetyl-10,20-O-isopropylidene-
rofuranos-40-yl]-2-(2,3,4,6-tetra-O-acetyl-
-glucopyranosyl)
amino-2-oxazoline (30). Column chromatography, eluent
100:1/20:1 CH2Cl2/MeOH. Yield: 178 mg (85%). Rf 0.33 (20:1
CH2Cl2/MeOH); [
D þ42 (c 0.98, CH2Cl2); IR (KBr) nmax 3420, 2990,
1750,1540,1374,1239,1040 cmꢁ1; 1H NMR (500 MHz, CDCl3)
5.79
a-D-eryth-
b-D
4.6.2. (4S)-4-[(40S)-30-O-Acetyl-10,20-O-isopropylidene-
b-L-threofur-
anos-40-yl]-2-phenylamino-2-oxazoline (20). Column chromatog-
raphy, eluent 100:1/50:1 CH2Cl2/MeOH. Yield: 103 mg (84%). Rf
a
]
0.42 (20:1 CH2Cl2/MeOH); [
3433, 2993, 1746, 1555, 1383, 1233, 1101 cmꢁ1; 1H NMR (500 MHz,
CDCl3)
a
]
ꢁ15 (c 1.0, CH2Cl2); IR (KBr) nmax
d
D
0
0
0
0
0
(d, 1H, J1,2¼3.7 Hz, H-1), 5.24 (t, 1H, J2 ,3 ¼J3 ,4 ¼9.5 Hz, H-3 ), 5.06 (t,
0
0
0
0
0
0
d
7.24e6.95 (m, 5H, Ph), 5.85 (d, 1H, J1,2¼4.0 Hz, H-1), 5.75
1H, J4 ,5 ¼9.5 Hz, H-4 ), 4.95 (d, 1H, J1 ,2 ¼9.5 Hz, H-1 ), 4.91 (t, 1H, H-
20), 4.77 (t, 1H, J3,4¼J4,5¼4.3 Hz, H-4), 4.67 (dd, 1H, J2.3¼8.8 Hz H-3),
4.35 (dd, 1H, J6a,6b¼13.0 Hz, J5,6a¼6.7 Hz, H-6a), 4.29 (dd, 1H,
(br s,1H, NH), 5.05 (br s,1H, H-3), 4.57 (d,1H, H-2), 4.38 (m, 3H, H-5,
H-6a, H-6b), 3.99 (dd, 1H, J4,5¼6.0 Hz, J3,4¼3.5 Hz, H-4), 2.02 (s, 3H,
MeCO), 1.50, 1.24 (2s, 6H, CMe2); 13C NMR (125.7 MHz, CDCl3)
0
0
0
0
0
J6a ,6b ¼12.4 Hz, J5 ,6a ¼4.3 Hz, H-6a ), 4.23 (m, 1H, H-5), 4.20 (dd,1H,
0
0
d
169.8 (CO), 157.2 (CN), 128.9e119.5 (Ph), 113.8 (CMe2), 105.1 (C-1),
J5,6b¼6.7 Hz, H-6b), 4.14 (dd,1H, H-2), 4.08 (dd,1H, J5 ,6b ¼2.2 Hz, H-
85.3 (C-2), 85.0 (C-4), 76.6 (C-3), 68.6 (C-6), 61.9 (C-5), 27.2, 26.3
(CMe2), 20.8 (MeCO); FABMS: m/z 385 (80, MþNaþ), 363 (100,
MþHþ). Anal. Calcd for C18H22N2O6: C, 59.66; H, 6.12; N, 7.73.
Found: C, 59.47; H, 6.01; N, 7.64.
6b0), 3.76 (ddd, 1H, H-50), 2.10e1.99 (5s, 15H, 5MeCO), 1.50, 1.29 (2s,
6H, CMe2); 13C NMR (125.7 MHz, CDCl3)
d 170.9e169.7 (5 CO), 160.1
(CN), 113.3 (CMe2), 104.2 (C-1), 82.8 (C-10), 79.0 (C-2), 77.8 (C-4),
73.3 (C-50), 73.2 (C-3), 73.1 (C-30), 70.8 (C-20), 69.6 (C-6), 68.5 (C-40),
64.8 (C-5), 61,9 (C-60), 26.8, 26.7 (CMe2), 20.9e20.8 (MeCO); FABMS:
m/z 639 (40, MþNaþ), 617 (100, MþHþ). Anal. Calcd for
C26H36N2O15: C, 50.65; H, 5.88; N, 4.54. Found: C, 50.32; H, 5.65; N,
4.36.
4.6.3. (4S)-4-[(40S)-30-O-Acetyl-10,20-O-isopropylidene-
b-L-threofur-
anos-40-yl]-2-napht-1-ylamino-2-oxazoline (22). Column chroma-
tography, eluent 2:1 EtOAc/petroleum ether. Yield: 116 mg (83%). Rf
0.57 (2:1 EtOAc/petroleum ether); [
nmax 3420, 3055, 2928, 1752, 1577, 1379, 1236, 1100 cmꢁ1
(500 MHz, CDCl3) 7.76e7.02 (m, 7H, Ph), 5.90 (br s, 1H, H-1), 5.85
a
]
D ꢁ29 (c 1.0, CH2Cl2); IR (KBr)
;
1H NMR
4.7. General procedure for the preparation of 5-N,6-O-(N0-
alkyl(aryl, glycopyranosyl)iminomethylidene)nojirimycin
derivatives (6e11)
d
(br s, 1H, NH), 4.97 (br s, 1H, H-3), 4.60 (br s, 1H, H-2), 4.37 (m, 3H,
H-5, H-6a, H-6b), 4.03 (m, 1H), 2.02 (s, 3H, MeCO), 1.48, 1.34 (2s, 6H,
CMe2); 13C NMR (125.7 MHz, CDCl3)
d
169.7 (CO), 155.5 (CN),
To a solution of the corresponding 2-amino-2-oxazoline pre-
cursors 20, 22, 24, 28 and 30 (0.28 mmol) in dry MeOH (2 mL),
methanolic NaMeO (1 M, 0.1 equiv per mol of acetate) was added.
The reaction mixture was stirred at room temperature for 30 min,
then neutralized with solid CO2 and concentrated. The resulting
residue and the corresponding desacetylated derivative 17 was
treated with TFA/H2O (9:1, 2.0 mL) for 15 min, concentrated under
reduced pressure, coevaporated several times with water, neu-
tralized with Amberlite IRA-68 (OHꢁ) ion-exchange resin and
subjected to column chromatography with the eluent indicated in
each case to obtain the isoureas 6e11.
134.4e122.8 (Ph), 113.2 (CMe2), 105.6 (C-1), 86.6 (C-2, C-4), 76.9 (C-
3), 68.7 (C-6), 58.2 (C-5), 26.8, 25.8 (CMe2), 20.8 (MeCO); FABMS: m/
z 435 (30, MþNaþ), 413 (100, MþHþ). Anal. Calcd for C22H24N2O6: C,
64.07; H, 5.86; N, 6.79. Found: C, 64.09; H, 5.94; N, 6.73.
4.6.4. (4S)-4-[(40S)-30-O-Acetyl-10,20-O-isopropylidene-
anos-40-yl]-2-(2,3,4,6-tetra-O-acetyl-
-glucopyranosyl)amino-2-
b-L-threofur-
b-D
oxazoline (24). Column chromatography, eluent 100:1/20:1
CH2Cl2/MeOH. Yield: 203 mg (97%). Rf 0.40 (20:1 CH2Cl2/MeOH);
[
a
]
ꢁ25 (c 1.0, CH2Cl2); IR (KBr) nmax 3418, 2938, 1750, 1543,
D
1373, 1231, 1038 cmꢁ1
;
1H NMR (500 MHz, CDCl3)
d
5.88 (d, 1H,
0
J1,2¼3.7 Hz, H-1), 5.24 (t, 1H, J2 ,3 ¼J3 ,4 ¼9.5 Hz, H-3 ), 5.07 (t, 1H,
4.7.1. 5-N,6-O-(N0-Butyliminomethylidene)galactonojirimycin
(6). Column chromatography, eluent 10:1 MeCN/H2O. Yield: 65 mg
0
0
0
0
0
0
0
0
0
0
J4 ,5 ¼9.5 Hz, H-4 ), 4.97 (d, 1H, J1 ,2 ¼9.5 Hz, H-1 ), 4.96 (br s, 1H,
H-3), 4.89 (t, 1H, H-20), 4.56 (d, 1H, H-2), 4.33 (m, 3H,0 H-5, H-6a,
(90%). Rf 0.41 (10:1:1 MeCN/H2O/NH4OH); [
NMR (500 MHz, D2O)
a
]
D ꢁ4.1 (c 1.0, H2O); 1H
0
0
0
0
H-6b), 4.26 (dd, 1H, J6a ,6b ¼12.3 Hz, J5 ,6a ¼3.8 Hz, H-6a ), 4.08 (dd,
d
5.52 (d, 1H, J1,2¼4.0 Hz, H-1), 4.82 (t, 1H,
0
0
0
1H, J5 ,6b ¼2.6 Hz, H-6b ), 3.93 (dd, 1H, J4,5¼6.2 Hz, J3,4¼3.1 Hz, H-
J5,6a¼J6a,6b¼9.0 Hz, H-6a), 4.69 (dd, 1H, J5,6b¼7.0 Hz, H-6b), 4.49
(ddd, 1H, J4,5¼2.5 Hz, H-5), 4.02 (t, 1H, J3,4¼2.5 Hz, H-4), 3.87 (dd,
4), 3.78 (ddd, 1H, H-50), 2.07e1.99 (5s, 15H, 5MeCO), 1.55, 1.32 (2s,
6H, CMe2); 13C NMR (125.7 MHz, CDCl3)
d
170.9e169.7 (5 CO),
1H, J2,3¼10.5, H-3), 3.77 (dd, 1H, H-2), 3.30 (t, 2H, JH,H¼7.0 Hz,
3
159.6 (CN), 113.8 (CMe2), 105.4 (C-1), 86.7 (C-2), 85.2 (C-10), 83.1
CH2N), 1.48 (m, 2H, CH2CH2N), 1.25 (m, 2H, CH2), 0.81 (t, 3H,

688
M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
3JH,H¼7.0 Hz, CH3); 13C NMR (125.7 MHz, D2O)
d
158.5 (CN), 74.9 (C-
1H, J4,5¼8.4 Hz, J3,4¼2.8 Hz, H-4), 3.58 (dd, 1H, J5 ,6b ¼1.7 Hz, H-6b ),
0
0
0
0
0
0
0
0
0
1), 70.6 (C-6), 68.8 (C-3), 68.0 (C-4), 67.3 (C-2), 56.2 (C-5), 42.3
(CH2N), 30.3 (CH2CH2N),19.1 (CH2),12.8 (CH3); FABMS: m/z 283 (20,
MþNaþ), 261 (100, MþHþ). Anal. Calcd for C11H20N2O5: C, 50.78; H,
7.75; N, 10.77. Found: C, 50.66; H, 8.04; N, 10.71.
3.39 (t, 1H, J2 ,3 ¼J3 ,4 ¼9.0 Hz, H-3 ),0 3.36 (m, 1H, H-5 ), 3.33 (t, 1H,
J4 ,5 ¼9.0 Hz, H-4 ), 3.11 (t, 1H, H-2 ); 13C NMR (125.7 MHz, D2O)
0
0
0
d
157.2 (CN), 86.5 (C-10), 77.4 (C-50), 76.3 (C-30), 74.9 (C-20), 74.5 (C-
1), 72.5 (C-3), 70.2 (C-6), 69.7 (C-40), 69.6 þ(C-4), 67.4 (C-2), þ60.8 (C-
60), 50.2 (C-5); FABMS: m/z 389 (50, MþNa ), 367 (30, MþH ). Anal.
Calcd for C13H22N2O10: C, 42.62; H, 6.05; N, 7.65. Found: C, 42.34; H,
6.01; N, 7.56.
4.7.2. 5-N,6-O-(N0-Phenyliminomethylidene)galactonojirimycin
(7). Column chromatography, eluent 20:1/4:1 CH2Cl2/MeOH.
Yield: 63 mg (80%). Rf 0.45 (4:1 CH2Cl2/MeOH); [
a
]
ꢁ5.2 (c 0.58,
D
H2O); 1H NMR (500 MHz, D2O)
d
7.23e6.91 (m, 5H, Ph), 5.44 (d, 1H,
Acknowledgements
J1,2¼4.0 Hz, H-1), 4.36 (t, 1H, J5,6a¼J6a,6b¼8.3 Hz, H-6a), 4.20 (t, 1H,
J5,6b¼7.8 Hz, H-6b), 4.11 (m,1H, H-5), 3.93 (m,1H, H-4), 3.82 (dd,1H,
J2,3¼10.2 Hz, J3,4¼2.6 Hz, H-3), 3.77 (dd, 1H, H-2); 13C NMR
This study was supported by the Spanish Ministerio de Ciencia e
ꢀ
Innovacion (contract numbers SAF2010-15670 and CTQ2010-
ꢀ
ꢀ
(125.7 MHz, D2O) d 159.5 (CN),146.1e123.4 (Ph), 78.3 (C-1), 69.5 (C-
15848), the Fundacion Ramon Areces, the Junta de Andalucía
(Project P08-FQM-03711) and the European Regional Development
Funds (FEDER and FSE).
3), 68.2 (C-4), 67.7 (C-2), 66.4 (C-6), 53.6 (C-5); FABMS: m/z 303 (60,
MþNaþ), 281 (30, MþHþ). Anal. Calcd for C13H16N2O5: C, 55.71; H,
5.75; N, 9.99. Found: C, 55.74; H, 5.64; N, 9.87.
Supplementary data
4.7.3. 5-N,6-O-(N0-Napht-1-yliminomethylidene)galactonojirimycin
(8). Column chromatography, eluent 45:5:3 EtOAc/EtOH/H2O.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.10.091.
Yield: 63 mg (85%). Rf 0.36 (45:5:3 EtOAc/EtOH/H2O); [
a]
D ꢁ16.6 (c
1.0, H2O); 1H NMR (500 MHz, CD3OD, 313 K)
d
8.07e7.10 (m, 7H,
Ph), 5.74 (br s, 1H, H-1), 4.39 (d, 2H, J5,6¼7.3 Hz, H-6), 4.25 (m, 1H,
H-5), 3.95 (dd, 1H, J2,3¼9.5 Hz, J1,2¼3.5 Hz, H-2), 3.92 (m, 1H, H-4),
3.88 (dd, 1H, J3,4¼2.8 Hz, H-3); 13C NMR (125.7 MHz, D2O, 313 K)
References and notes
1. (a) Winchester, B. G. Tetrahedron: Asymmetry 2009, 20, 645e651; (b) Iminosu-
gars: From Synthesis to Therapeutic Applications; Compain, P., Martin, O. R., Eds.;
Wiley-VCH: Weinheim, Germany, 2007; (c) Iminosugars as Glycosidase In-
d
158.5 (CN), 134.2e121.5 (Ph), 76.0 (C-1), 69.1 (C-3), 68.5 (C-4),68.4
(C-6), 67.8 (C-2), 57.1 (C-5); FABMS: m/z 353 (50þNaþ), 331 (100,
MþHþ). Anal. Calcd for C17H18N2O5: C, 61.81; H, 5.49; N, 8.48.
Found: C, 61.65; H, 5.44; N, 8.46.
€
hibitors: Nojirimycin and Beyond; Stutz, A. E., Ed.; Wiley-VCH: Weinheim, Ger-
many, 1999; (d) Heightman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed. 1999, 38,
750e770; (e) Bols, M. Acc. Chem. Res. 1998, 31, 1e8; (f) Legler, G. Adv. Carbohydr.
Chem. Biochem. 1990, 48, 319e384; (g) Lillelund, V. H.; Jensen, H. H.; Liang, X. F.;
Bols, M. Chem. Rev. 2002, 102, 515e553.
4.7.4. 5-N,6-O-(N0-
actonojirimycin (9). Column chromatography, eluent 4:1 MeCN/
b-D-Glucopyranosyliminomethylidene)gal-
ꢀ
ꢀ
2. Jimenez Blanco, J. L.; Díaz Perez, V. M.; Ortiz Mellet, C.; Fuentes, J.; García
ꢀ
~
Fernandez, J. M.; Díaz Arribas, J. C.; Canada, F. J. Chem. Commun. 1997,
1969e1970.
H2O. Yield: 98 mg (96%). Rf 0.37 (6:3:1 MeCN/H2O/NH4OH); [a]
D
ꢀ
ꢀ
3. Sanchez-Fernandez, E. M.; Rísquez-Cuadro, R.; Chasseraud, M.; Ahidouch, A.;
Ortiz Mellet, C.; Ouadid-Ahidouch, H.; García Fernandez, J. M. Chem. Commun.
2010, 5328e5330.
ꢁ6.0 (c 1.0, H2O); 1H NMR (500 MHz, D2O)
d
5.46 (d, 1H, J1,2¼4.0 Hz,
0
0
0
H-1), 4.72 (d, 1H, J1 ,2 ¼9.0 Hz, H-1 ), 4.54 (t, 1H, J6a,6b¼J5,6a¼7.7 Hz,
4. (a) Johnson, C. R.; Golebiowski, A.; Sundram, H.; Miller, M. W.; Dwaihy, R. L.
Tetrahedron Lett. 1994, 36, 653e654; (b) Paulsen, H. Adv. Carbohydr. Chem. Bi-
ochem. 1968, 23, 115e232.
5. (a) García-Moreno, M. I.; Ortiz Mellet, C.; García Fernandez, J. M. Tetrahedron:
Asymmetry 1999, 10, 4271e4275; (b) Aguilar, M.; Díaz-Perez, P.; García-Moreno,
H-6a), 4.39 (t,1H, J5,6b¼7.7 Hz, H-6b), 4.22 (m, 1H, H-5), 3.94 (m, 1H,
H-4), 3.79 (dd, 1H, J2,3¼10.2 Hz, J3,4¼2.7 Hz, H-3), 3.75 (m, 1H, H-2),
0
ꢀ
0
0
0
0
3.73 (dd, 1H, J6a ,6b ¼12.4 Hz, J5 ,6a ¼2.5 Hz, H-6a ), 3.60 (dd, 1H,
ꢀ
0
0
0
0
0
0
0
0
J5 ,6b ¼4.9 Hz, H-6b ), 3.38 (t, 1H, J2 ,3 ¼0 J3 ,4 ¼9.0 Hz, H-3 ), 3.37 (m,
ꢀ
M. I.; Ortiz Mellet, C.; García Fernandez, J. M. J. Org. Chem. 2008, 73, 1995e1998.
1H, H-50), 3.31 (t, 1H, J4 ,5 ¼9.0 Hz, H-4 ), 3.19 (t, 1H, H-2 ); 13C NMR
0
0
0
ꢀ
6. (a) Díaz Perez, V. M.; García-Moreno, M. I.; Ortiz Mellet, C.; Fuentes, J.; Díaz
d
157.8 (CN), 85.5 (C-10), 77.5 (C-50), 76.1 (C-30),
~
ꢀ
Arribas, J. C.; Canada, F. J.; García Fernandez, J. M. J. Org. Chem. 2000, 65,
(125.7 MHz, D2O)
ꢀ
136e143; (b) Díaz Perez, V. M.; García-Moreno, M. I.; Ortiz Mellet, C.; García
74.7 (C-1), 73.8 (C-20), 69.4 (C-40), 69.2 (C-3), 68.2 (C-4), 68.1 (C-þ6),
67.5 (C-2), 60.6 (C-60), 54.5 (C-5); FABMS: m/z 389 (100, MþNa ),
367 (35, MþHþ). Anal. Calcd for C13H22N2O10: C, 42.62; H, 6.05; N,
7.65. Found: C, 42.29; H, 5.77; N, 7.39.
ꢀ
ꢀ
Fernandez, J. M. Synlett 2003, 341e344; (c) Díaz-Perez, P.; García-Moreno, M. I.;
ꢀ
Ortiz Mellet, C.; García Fernandez, J. M. Eur. J. Org. Chem. 2005, 2903e2913; (d)
Benltifa, M.; García-Moreno, M. I.; Ortiz Mellet, C.; García Fernandez, J. M.;
Wadouachi, A. Bioorg. Med. Chem. Lett. 2008, 18, 2805e2808; (e) Aguilar-
Moncayo, M.; García-Moreno, M. I.; Ortiz Mellet, C.; García Fernandez, J. M. J.
ꢀ
ꢀ
Org. Chem. 2009, 74, 3595e3598.
7. García-Moreno, M. I.; Rodríguez-Lucena, D.; Ortiz Mellet, C.; García Fernandez,
4.7.5. 5-N,6-O-(N0-Phenyliminomethylidene)allonojirimycin
(10). Column chromatography, eluent 10:1/4:1 CH2Cl2/MeOH.
ꢀ
J. M. J. Org. Chem. 2004, 69, 3578e3581.
ꢀ
8. (a) García Fernandez, J. M.; Ortiz Mellet, C.; Benito, J. M.; Fuentes, J. Synlett 1998,
Yield: 74 mg (95%). [
a]
ꢁ46.1 (c 0.78, H2O). Rf 0.50 (4:1 CH2Cl2/
D
316e318; (b) García-Moreno, M. I.; Benito, J. M.; Ortiz Mellet, C.; García
MeOH); 1H NMR (500 MHz, D2O)
d
7.30e6.99 (m, 5H, Ph), 5.38 (br s,
Fernandez, J. M. J. Org. Chem. 2001, 66, 7604e7614; (c) García-Moreno, M. I.;
ꢀ
ꢀ
Ortiz Mellet, C.; García Fernandez, J. M. Eur. J. Org. Chem. 2004, 1803e1809; (d)
1H, J1,2¼4.0 Hz, H-1), 4.59 (t, 1H, J5,6a¼J6a,6b¼8.1 Hz, H-6a), 4.20 (t,
1H, J5,6b¼8.8 Hz, H-6b), 4.11 (t, 1H, J2,3¼J3,4¼2.9 Hz, H-3), 4.09 (dt,
1H, J4,5¼9.9 Hz, H-5), 3.77 (br s, 1H, H-2), 3.66 (dd, 1H, H-4); 13C
ꢀ
García-Moreno, M. I.; Ortiz Mellet, C.; García Fernandez, J. M. Tetrahedron 2007,
73, 7879e7884; (e) Aguilar, M.; Gloster, T. M.; García-Moreno, M. I.; Ortiz
Mellet, C.; Davies, G. J.; Llebaria, A.; Casas, J.; Egido-Gabas, M.; García Fernan-
dez, J. M. ChemBioChem 2008, 9, 2612e2618.
9. (a) Robinson, K. M.; Begovic, M. E.; Rhinehart, B. L.; Heineke, E. W.; Ducep, J.-B.;
Kastner, P. R.; Marshall, F. N.; Danzin, C. Diabetes 1991, 40, 825e830; (b) Krentz,
A. J.; Bailey, C. J. Drugs 2005, 65, 385e411.
ꢀ
ꢀ
NMR (125.7 MHz, D2O)
d 155.7 (CN), 146.2e124.5 (Ph), 76.2 (C-1),
73.6 (C-3), 71.6 (C-4), 71.3 (C-6), 68.5 (C-2), 51.3 (C-5); FABMS: m/z
303 (100, MþNaþ). Anal. Calcd for C13H16N2O5: C, 55.71; H, 5.75; N,
9.99. Found: C, 55.77; H, 5.49; N, 9.81.
10. (a) Durantel, D.; Alotte, C.; Zoulim, F. Curr. Opin. Invest. Dr. (Bio Med Cent.) 2007,
€
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2007; p 269.
12. (a) Fan, J. Q. Biol. Chem. 2008, 389, 1e11; (b) Butters, T. D. Expert Opin. Phar-
macother. 2007, 8, 427e435; (c) Yu, Z.; Sawkar, A. R.; Whalen, L. J.; Wong, C.-H.;
Kelly, J. W. J. Med. Chem. 2007, 50, 94e100; (d) Matsuda, J.; Suzuki, O.; Oshima,
A.; Yamamoto, Y.; Noguchi, A.; Takimoto, K.; Itoh, M.; Matsuzaki, Y.; Yasuda, Y.;
Ogawa, S.; Sakata, Y.; Nanba, E.; Higaki, K.; Ogawa, Y.; Tominaga, L.; Ohno, K.;
Iwasaki, H.; Watanabe, H.; Brady, R. O.; Suzuki, Y. Proc. Natl. Acad. Sci. U.S.A.
4.7.6. 5-N,6-O-(N0-
imycin (11). Column chromatography, eluent 10:1/2:1 MeCN/
b-D-Glucopyranosyliminomethylidene)allonojir-
H2O. Yield: 63 mg (62%). Rf 0.28 (6:3:1 MeCN/H2O/NH4OH); [a]
D
ꢁ11.9 (c 1.0, H2O); 1H NMR (500 MHz, D2O)
d 5.28 (d, 1H,
0
0
0
J1,2¼4.3 Hz, H-1), 4.72 (d, 1H, J1 ,2 ¼9.0 Hz, H-1 ), 4.59 (t, 1H,
J6a,6b¼J5,6a¼8.0 Hz, H-6a), 4.19 (t, 1H, J5,6b¼8.0 Hz, H-6b), 4.04 (t, 1H,
J2,3¼J3,4¼2.8 Hz, H-3), 3.40 (m, 1H, H-5), 3.75 (dd, 1H,
0
0
0
0
0
J6a ,6b ¼12.3 Hz, J5 ,6a ¼1.8 Hz, H-6a ), 3.69 (dd, 1H, H-2), 3.60 (dd,

M. Aguilar-Moncayo et al. / Tetrahedron 68 (2012) 681e689
689
2003, 100, 15912e15917; (e) Butters, T. D.; Mellor, H. R.; Narita, K.; Dwek, R. A.;
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Fan, J.-Q. Trends Pharmacol. Sci. 2003, 24, 355e360; (c) Yu, Z.; Sawker, A. R.;
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Fernandez, J. M.; Ortiz Mellet, C. Expert Opin Ther. Pat. 2011, 21, 885e903.
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K.; García Fernandez, J. M.; Benito, J. M. J. Org. Chem. 2009, 74, 2997e3008; (b)
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€
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