Stereoselective synthesis of 2-acetamido-1,2-dideoxynojirimycin (DNJNAc) and ureido-DNJNAc derivatives as new hexosaminidase inhibitors

Article abstract of DOI:10.1039/c5ob00507h

2-Acetamido-1,2-dideoxyiminosugars are selective and potent inhibitors of hexosaminidases and therefore show high therapeutic potential for the treatment of various diseases, including several lysosomal storage disorders. A stereoselective synthesis of 2-

Full text of DOI:10.1039/c5ob00507h

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Journal Name
RSCPublishing
ARTICLE
Stereoselective Synthesis of 2-Acetamido-1,2-
dideoxynojirimycin (DNJNAc) and Ureido-DNJNAc
Derivatives as New Hexosaminidase Inhibitors
Cite this: DOI: 10.1039/x0xx00000x
Alex de la Fuente,a Teresa MenaꢀBarragán,b Ronald A. FarrarꢀTobar,a
Xavier Verdaguer,a,c José M. García Fernández,d Carmen Ortiz Mellet,*,b
Antoni Riera,*,a,c
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
2ꢀAcetamidoꢀ1,2ꢀdideoxyiminosugars are selective and potent inhibitors of hexosaminidases
and therefore show high therapeutic potential for the treatment of various diseases, including
www.rsc.org/
several lysosomal storage disorders.
dideoxynojirimycin (DNJNAc), the iminosugar analog of
A
stereoselective synthesis of 2ꢀacetamidoꢀ1,2ꢀ
ꢀacetylglucosamine, with high
N
overall yield is here described. This novel procedure further allowed accessing ureidoꢀ
DNJNAc conjugates through derivatization of the endocyclic amine on a key pivotal
intermediate. Remarkably, some of the ureidoꢀDNJNAc representatives behaved as potent and
selective inhibitors of βꢀhexosaminidases, including the human enzyme, being the first
examples of neutral sp2ꢀiminosugarꢀtype inhibitors reported for these enzymes. Moreover, the
amphiphilic character of the new ureidoꢀDNJNAc is expected to confer better drugꢀlike
properties
.
synthesis11,12. Most of these representatives are piperidine
derivatives, such as 2ꢀacetamidoꢀ1,2ꢀdideoxynojirimycin
Introduction
.
13–16
14,17
Since the isolation of nojirimycin in 1966, iminosugars
—sugar analogs where the oxygen ring atom has been
replaced by a nitrogen— have attracted an exponential
interest as mimics of the transition state of the enzymatic
hydrolysis of glycosidic substrates.1,2 Their ability to act
as inhibitors of a great diversity of carbohydrate
processing enzymes, including glycosidases, glycosyl
transferases, nucleosideꢀprocessing enzymes and
glycogen phosphorylases, and the broad variety of
biological and pathological processes in which
carbohydrates are involved make iminosugars invaluable
tools in glycobiology and promising candidates for the
development of glycotherapies.3–5 In fact, some
iminosugars are already marketed drugs, such as miglitol
(DNJNAc,
galacto epimers (DGJNAc,
4
)
and its manno (DMJNAc,
5
)
or
6
7
),18,19 although acetamido
20,21
iminosugars with fiveꢀ (e.g.
)
or sevenꢀmembered
ring skeletons (e.g. 8)
22 have also been described. Several
of these compounds have proven to be highly selective
inhibitors of hexosaminidases —the enzymes cleaving
off amino sugar residues from oligosaccharides and
glycoconjugates— with inhibition constant (Ki) values in
the low micromolar to nanomolar range. This property
makes them potentially useful in the treatment of several
diseases involving abnormal levels of
Oꢀlinked
glucosamine (GlcNAc) in glycoproteins, including
diabetes, Parkinson’s, osteoarthritis, and some cancers.23–
27
Furthermore, at subinhibitory concentrations
(Glyset) and Nꢀbutylꢀ1ꢀdeoxynojirimycin (Zavesca), used
competitive inhibitors of the hexosaminidases are able to
promote the correct folding of mutant diseaseꢀassociated
lysosomal enzymes, thus bearing promise for the
development of pharmacological chaperone therapies28
against some lysosomal storage disorders.20,29–31 Many
studies have addressed the mechanism of action of these
compounds, showing that the acetamido group is
essential for their activity and selectivity.32–34
Most synthetic approaches to iminosugars are based on
the chiral pool thus making these processes rather
long.35–39 This is also the case for acetamido
iminosugars,13,16,40,41 with a few exceptions limited to the
for the treatment of type II diabetes mellitus and type 1
Gaucher disease respectively.6
Iminosugars reduced at Cꢀ1 and bearing an acetamido
group at the position equivalent to Cꢀ2 in the parent
monosaccharides,
namely
2ꢀacetamidoꢀ1,2ꢀ
dideoxyiminosugars, have been the focus of considerable
attention in recent years. Several representatives of
acetamido iminosugars, for instance pochonicine (1
)7,
9,10
siastatine B (
2
),8 or nagstatine (
3
)
have been isolated
from natural sources while derivatives from those and
other compounds have been obtained by chemical
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stereoselective
synthesis
of
2ꢀacetamidoꢀ1,2ꢀ
and the manno
42
dideoxyallonojirimycin (DAJNAc,
diastereomer DMJNAc (
).17 Herein, we report a new
stereoselective total synthesis of the gluco counterpart
DNJNAc ( ) and its regioisomer 3ꢀacetamidoꢀ1,3ꢀ
9
)
5
4
dideoxyaltronojirimycin (29). Moreover, the preparation
of a series of ureidoꢀDNJAc derivatives as examples of
2ꢀacetamido
sp2ꢀiminosugars,
has
also
been
accomplished. Characterized by the incorporation of a
pseudoamideꢀtype nitrogen atom with high sp2ꢀ
hybridation character in the ring 43–45, this subtype of
glycomimetics, from which nagstatine
3 can be
considered a natural representative, has previously shown
an unprecedented potential for fine tuning the inhibitory
potency and selectivity towards glycosidases by
modulating the basicity of the Nꢀfunctionality and the
nature of the exocyclic moiety.46 In our case, the
evaluation of the new ureidoꢀDNJNAc against a panel of
Scheme 1. Retrosynthetic analysis for the preparation of
DNJNAc (
4) and ureidoꢀDNJNAc conjugates (10) from the
common bicyclic precursor 11
.
glycosidases
allowed
the
identification
of
Optically pure carbamate 11 was prepared in multigram
scale from pentaꢀ1,4ꢀdienꢀ3ꢀol, and the allylic alcohol
group was subsequently protected as the corresponding
hexosaminidase inhibitors with an amphiphilic character
and a greatly reduced basicity, features that make these
compounds better suited as drug candidates.
benzyl ether 12 48
We first considered the epoxidation of
.
the double bond in 12 followed by regioselectively ringꢀ
opening by azide anion to introduce the amino
substituent. Deceivingly, classical methodologies using
m
ꢀchloroperoxybenzoic acid (MCPBA) or H2O2 proved
inefficient while harsher oxidant methods such as
CF3CO3H51,52 or oxone49 generated inseparable 1:1
mixtures of the corresponding epoxides in moderate
yields. We hypothesized that the rigid bicyclic skeleton
of 12 was probably responsible for the low reactivity
observed. However, although hydrolysis of the cyclic
carbamate by treatment with 6M NaOH at reflux,
followed by in situ Cbzꢀprotection of the endocyclic
amine afforded the monocylic derivative 13 in
satisfactory yield, all attempts at diastereoselective
epoxidation of 13 failed, regardless of the epoxidation
methodology used. Various combinations of Nꢀcarbamate
and Oꢀester/ether protecting groups were also assayed
without success. In view of these results, we explored the
use of cyclic sulfates as an alternative to epoxides,53,54 an
approach that has been applied successfully in other
iminosugar syntheses.55,56
Figure 1. Structure of some acetamido iminosugars.
The protection of the primary alcohol of 13 as a
benzoate,
followed
by
Sharpless
asymmetric
Results and discussion
dihydroxylation of the intermediate ester (14), yielded a
90:10 mixture (HPLC) of diastereomeric diols, from
which the major isomer 15 was isolated in 62% yield
(Scheme 2). Treatment of 15 with thionyl chloride gave a
mixture of sulfites that was oxidized without further
purification with RuCl3/NaIO4 to the corresponding
cyclic sulfate 16, which was obtained as a single
diasteroisomer in 80% overall yield (two steps).
Treatment of 16 with NaN3 at 50ºC gave an inseparable
mixture of the azidoalcohols 17 and 18. Attempts to
quantify the relative proportion of the two compounds at
this stage by NMR or HPLC failed. The two
azidoalcohols were hypothesized to be the result of the
Our approach to DNJNAc,
4 and the ureidoꢀDNJNAc
derivatives 10 is shown in Scheme 1. An appropriate
protecting group scheme was needed to introduce the
urea fragment in the last steps. The protected compounds
were prepared by introducing the amino function by
nucleophilic ring opening of an epoxide or a cyclic
sulfate obtained from the key intermediate 11, which is
readily accessible by Sharpless asymmetric epoxidation
of 2,4ꢀpentadienꢀ1ꢀol.47 This intermediate has been
widely used for the synthesis of various iminosugars17,48–
50 including our recent synthesis of DAJNAc ( ).42
9
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nuclephilic attack of the azide anion at C2 (gluco
ꢀ
and facile separation of the two regioisomers encouraged
configuration) and C3 (altroꢀconfiguration) positions. us to look for a shorter route to synthetize the mixture of
Sequential treatment of this mixture with NaOMe, in 19 and 20. Direct dihydroxylation of carbamate 12 using
order to cleave the benzoate group, and NaH regenerated K2OsO4·2H2O/NMO afforded 21 in satisfactory yield
the 2ꢀoxazolidinone ring, affording a 1:1 mixture of the and diastereoselectivity (78%, 85:15) (Scheme 3).
bicyclic azidoalcohols 19 and 20 (Scheme 2). Although Sharpless asymmetric dihydroxylation conditions
no selectivity was achieved during the cyclic sulfate increased both the yield and diasteroselectivity, affording
opening reaction, both carbamates 19 and 20 were easily diol 21 in 94% yield and nearly complete
separated by column chromatography, which yielded diasteroselectivity, as observed by 1HꢀNMR.17 The
crystalline compounds that could be analyzed by Xꢀray corresponding cyclic sulfate 22 was obtained in 80%
diffraction,
stereochemistry (Figure 2).
†
thus
confirming
the
proposed yield by reaction of diol 21 with SOCl2/TEA followed by
in situ oxidation with NaIO4/RuCl3, as in the previous
case. Attempts to perform direct sulfation of 21 using
SO2Cl2/TEA57 also afforded 22 but in lower yields.
OH
OBn
OBn
1) NaOH 6M
reflux
BnBr, NaH
DMF, rt
89%
OH
O
O
N
N
N
2) CbzCl,
NaHCO3
70%
Cbz
O
O
11
12
13
OBn
OBn
OBz
(DHQD)2Phal, K2OsO4
K3[Fe(CN)6], K2CO3
HO
HO
BzCl, TEA
DMAP
OBz
Cbz
N
N
ACN:H2O 1:1
62%, d.r. 9:1
Cbz
14
86%
15
OBn
1) NaN3
1) SOCl2, TEA
2) NaIO4, RuCl3
80%
O
acetone:H2O, 50ºC
O
O
OBz
Cbz
17/18
S
N
O
2) H2SO4 20% aq
16
OBn
85%
OBn
N3
HO
N3
1) NaOMe / MeOH
2) NaH, DMF, rt
O
O
N
N
HO
O
Scheme 3. Synthesis of cyclic sulfate from 12 followed by
ringꢀopening with sodium azide.
O
19
70 % (2 steps)
rr 1:1
20
Scheme 2. Synthesis of the azido intermediates 19 and 20.
Regioselective ringꢀopening reactions of the key
precursor 22 using NaN3 as the nucleophile were
extensively studied and are summarized in Table 1. We
expected that the presence of the benzyl group at the C4
position would sterically hinder approaching of the azide
anion nucleophile to C3, directing the attack to the C2
position (iminosugar numbering). The reaction did not
take place in acetonitrile (entry 1) but proceeded in N,N
ꢀ
dimethylformamide (entries 2 and 3). Thus, treatment of
sulfate 22 with sodium azide in DMF, followed by acidic
hydrolysis (to cleave the intermediate residual sulfate),
gave a 2:1 mixture of azidoalcohols in 70% yield (entry
2). However, increasing the temperature and the
equivalents of NaN3, led to a dramatic decrease in yield
and a total loss of selectivity (entry 3). In an attempt to
improve the regioselectivity, the reaction was performed
in acetone/water (entry 4 and 5), observing that fewer
equivalents of azide allowed similar ratios. The use of
lower temperatures (40ºC), even fewer equivalents of
azide (1.2) and a longer reaction time (16 h), afforded
higher yields, but also at the expenses of a total loss of
Figure 2. Xꢀray analysis of azido alcohols 19 and 20.†
We envisaged that carbamate 19 would be an excellent
precursor in the synthesis of DNJNAc ( ) and ureidoꢀ
4
DNJAc derivatives 10. The straightforward purification
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regioselectivity (entry 6). Conversely, portionꢀwise
addition of sodium azide increased the regioselectivity,
but with a significant decrease in yield (entry 7).
According to our objective of obtaining derivatives of 4,
conditions of entry 5 were chosen for scaling up
purposes.
Table 1. Optimization of the ringꢀopening reaction of sulfate 22 with
sodium azide.
NaN3 Yield 19/20
Entry Solvent
T/ºC
50
t/h
3
a
Eq.
/%
1
2
ACN
DMF
DMF
3
ꢀ
ꢀ
50
3
4
4
70
30
68
62
81
43
1.9:1
0.9:1
1:1
3
120
50
1
Acetone/
H2O 2:1
Acetone/
H2O 2:1
Acetone/
H2O 2:1
Acetone/
H2O 2:1
4
3
3
Scheme 4. Synthesis of DNJNAc (4).
5b
6
50
6
2
1.8:1
1:1
40
16
1.2
7c
50
7
3
3:1
a Relation determined w/w after purification
b The reaction was performed at multigram scale using these
conditions.
c Portionwise addition of NaN3
The synthesis of DNJNAc
4 from azidoalcohol 19 is
depicted in Scheme 4. Protection of the secondary
alcohol by treatment with BnBr/NaH gave 23 in 90%
yield. Azide reduction with H2 and Pd/C followed by in
situ acetylation with Ac2O/pyridine afforded acetamide
24 in almost quantitative yield. This compound was then
subjected to basic hydrolysis of the 2ꢀoxazolidinone ring
to give 25 in 92% yield. Final hydrogenolysis of the
Scheme
dideoxyaltronojirimycin (29).
5.
Synthesis
of
3ꢀacetamidoꢀ1,3ꢀ
benzyl protecting groups gave DNJNAc (4) in 96% yield.
The spectroscopic data of this compound were consistent
It has been described that modifications of the acetamide
moiety in DNJNAc lead to a dramatic decrease in the
inhibitory activity against hexosaminidases,40 while
modifications at the endocyclic amine are well tolerated.
with previously reported data.18 The total synthesis of
DNJNAc (4) from 11 was thus accomplished in 10
synthetic steps achieving a 23% overall yield.
Although some 3ꢀacetamido iminosugar derivatives have
been reported we could not find precedents of evaluation
of their properties as glycosidase inhibitors.41,58,59 We
thus considered it of interest to apply the above synthetic
Indeed, the incorporation of hydrophobic
Nꢀalkyl
substituents has been previously investigated,18,60 and
found to lead to an improvement of the inhibitory
potency which is consistent with the presence of a
hydrophobic pocket in the vicinity of the active site of
the enzyme.61 All DNJNAc analogs reported to date keep
the basic character of the piperidine glyconeꢀlike
skeleton, generally considered a favorable structural
feature to promote strong binding to the enzyme.
However, it has been demonstrated that higher
glycosidase affinities and, especially, improved
sequence to azido alcohol 20, i.e. benzylation (
azide reduction and acetylation of the resulting amine
→26),
(
(
→
→
27), basic hydrolysis of the cyclic carbamate group
28) and final hydrogenolysis of the benzyl protecting
groups.
In
this
manner,
3ꢀacetamidoꢀ1,3ꢀ
dideoxyaltronojirimycin 29 was prepared in excellent
overall yield (Scheme 5).
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Scheme 6. Synthesis of ureidoꢀDNJNAc derivatives 10a-d
.
selectivities can be achieved by the interplay of neutral
glyconeꢀtype cores and substituents that provide
additional nonꢀglycone interactions.62,63 Transmuting the
endocyclic sp3ꢀamine nitrogen into a sp2ꢀhybridized
pseudoamide functionality by introduction of amide,
urea, thiourea or guanidinium moieties has proven
particularly successful in this respect.64–68 For instance,
Evaluation of the glycosidase inhibitory activity of the
DNJNAc regioisomer 29 and the ureidoꢀDNJNAc
derivatives 10aꢀd, in comparison with the parent
acetamido iminosugar 4, confirmed their total selectivity
towards hexosaminidases among a panel that included
the following: βꢀglucosidases (almonds and bovine liver),
αꢀglucosidase (yeast), αꢀmannosidase (jack bean), βꢀ
mannosidase (Helix pomatia), trehalase (pig kidney),
amyloglucosidase (Aspergillus niger), αꢀrhamnosidase
(naringinasa; Penicillium decumbens), αꢀgalactosidase
(green coffee), βꢀgalactosidase (E. coli), and isomaltase
(yeast). Compound 29 was a much weaker inhibitor than
DNJNAc, confirming that even when hexosaminidases
are relatively promiscuous regarding the configurational
pattern of iminosugarꢀtype ligands, the location of the
acetamido group next to the anomeric position is critical
to ensure strong enzyme binding. Gratifyingly, all
N
ꢀ(N’ꢀbutylaminocarbamoyl)ꢀ1ꢀdeoxynojirimycin,
the
urea analog of the marketed drug Zavesca, was found to
be a very selective inhibitor of bovine liver
βꢀ
galactosidase.67 The sp2ꢀhybridized character is also
observed in some natural products such as kifunensine, a
potent inhibitor of class I αꢀmannosidase.69,70 To check
this strategy for the particular case of hexosaminidases,
we synthesized a series of ureidoꢀDNJNAc derivatives
(
10). In addition to a much lower basic character at the
endocyclic nitrogen, conversion of an amine into a urea
offers flexibility in the choice of substituents, which can
be taken advantage of to optimize the inhibitory capacity
and the pharmacokinetic behavior.
The oxazolidinone ring of azido alcohol 19 was
hydrolyzed under the usual conditions and the endocyclic
amine was protected in situ using Boc2O/NaHCO3 to
give azidoalcohol 30. Concomitant azide reduction and
cleavage of the benzyl group were accomplished by
ureidoꢀDNJNAc derivatives 10aꢀd behaved as µM
inhibitors of the three hexosaminidases assayed in this
work, namely those from human placenta, bovine kidney,
and jack beans.
benzyl; 10a 10b and 10d) led to a slight decrease in the
inhibitory potency as compared with , with inhibition
constant ( i) values in the 56ꢀ20 µM range for the human
enzyme. The ’ꢀphenyl derivative 10c was an about one
order of magnitude stronger inhibitor of the
hexosaminidases as compred with the ’alkyl
counterparts. Notably, the inhibition potency against the
human enzyme surpassed that of by over 3ꢀfold. This
result is remarkable considering the much lower basicity
of 10c as compared with . The data suggest the
N’ꢀalkyl substituents (nꢀbutyl, nꢀoctyl or
,
4
K
hydrogenation in methanol/acetic acid. The resulting vic
ꢀ
N
aminoalcohol was acetylated without further purification
by treatment with Ac2O in pyridine to afford acetamide
N
31 in 83% yield (2 steps). Next, the
selectively cleaved using TFA, and the resulting cyclic
amine was reacted in situ with ꢀbutyl, ꢀoctyl, phenyl or
NꢀBoc group was
4
n
n
benzyl isocyanate in the presence of triethylamine (TEA)
to give the corresponding urea adducts 32a-d in 70ꢀ85%
yield. Final deacetylation using a saturated solution of
ammonia in MeOH gave the target ureidoꢀDNJNAc
derivatives 10a-d (Scheme 6).
4
involvement of the urea NH proton in hydrogen bonding
in the complex of ureidoꢀDNJNAc with the
hexosaminidases,
compensating
the
electrostatic
interactions operating in the case of the basic iminosugar,
as
previously
demonstrated
for
other
The
sp2ꢀ
OBn
OBn
complexes.71
higher
1) NaOH 6M
iminosugar:glycosidase
HO
HO
reflux
OH
Boc
hydrogen bond donor capability of arylureas as compared
with alkylureas, due to the electron withdrawing
character of the aromatic ring, is consistent with the
observed activity trend. Most interestingly, the
amphiphilic character of the compounds is expected to
confer better drugꢀlike properties. Altogether, the results
reported herein are promising for the further
development of therapeutic agents for βꢀGlcNAcaseꢀ
related diseases.
O
N
N
2) Boc2O, imid.
N3
N3
O
19
30
90 %
OAc
1) H2, Pd /C
AcOH
AcO
2) Ac2O, pyr
83 %
OAc
Boc
N
MeOH, 60ºC
AcHN
31
OH OH
OAc OAc
H
Conclusions
HO
AcHN
NH3
AcO
AcHN
1) TFA, CH2Cl2
2) RNCO, TEA
H
Here we have described a new stereoselective synthesis
of 2ꢀacetamidoꢀ1,2ꢀdideoxynojirimycin (DNJNAc), the
iminosugar analog of Nꢀacetylglucosamine, with high
N
N
N
N
MeOH
R
R
O
O
overall yield. The strategy is based on the stereoselective
ringꢀopening of cyclic sulfates derived from the key
intermediate 11, which was conveniently prepared by a
multigram procedure based on Sharpless epoxidation.
This novel procedure gave access to the advanced
10a R = n-Bu
81 %
85 %
80 %
70 %
32a R = n-Bu
61 %
75 %
58 %
76 %
R = n-Oct
R = Ph
10b
10c
10d
R = n-Oct
R = Ph
32b
32c
32d
R = Bn
R = Bn
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intermediate 19 which provided us with the necessary inhibitors of hexosaminidases described to date. These
protecting group arrangement to synthesize sp2ꢀ compounds were potent inhibitors of βꢀGlcNAcase and,
iminosugar conjugates through derivatization of the given their amphiphlic character, they are expected to
endocyclic amine by reaction with isocyanates. These show acceptable drugꢀlike properties.
new ureidoꢀDNJNAc derivatives are the first neutral
Table 2. Inhibition constants (Ki, µM)a against commercial βꢀNꢀacetylglucosaminidases 10a-d and 29 determined from thee
slope of LineweaverꢀBurk plots and double reciprocal analysis compared with previously reported values for DNJNAc (4).18
10a
10b
10c
10d
4
29
R = n-Bu
R = n-Oct
R = Ph
R = Bn
Enzyme origin
Human placenta
7.0 ± 0.3
7.4 ± 0.3
2.9 ± 0.2
427 ± 20
524 ± 40
130 ± 10
56 ± 5
138 ± 10
26 ± 3
33 ± 3
82 ± 5
19 ± 2
2.1 ± 0.1
4.1 ± 0.2
1.1 ± 0.1
20 ± 1
24 ± 2
10 ± 1
Bovine kidney
Jack Bean
aInhibition was competitive in all cases.
(2
R,3S)-3-Benzyloxy-N-benzyloxycarbonyl-2-
Experimental
General
hydroxymethyl-3,6-dihydropyridine (13)
NaOH 6M (2.86 mL, 17.10 mmol) was added to a solution of
12 (421 mg, 1.71 mmol) in MeOH : H2O 9:1 (18 mL), and the
reaction was heated at reflux for 20 h. Solvents were removed
at low pressure. The resulting white solid was dissolved in THF
(30 mL) and H2O (3 mL) and cooled at 0 ºC. NaHCO3 (432 mg,
5.14 mmol) and CbzCl (0.39 mL, 2.57 mmol) were added and
the reaction was stirred at 0 ºC for 4 h. H2O (10 mL) was then
added, and the crude product was extracted with EtOAc (3x 15
mL), dried over MgSO4 and purified on SiO2 using
All commercial reagents were used without further purification.
Nonꢀaqueous reactions were performed out under nitrogen
atmosphere. Dry tetrahydrofuran, dichloromethane, and diethyl
ether were obtained using a Solvent Purification System (SPS).
Other solvents were used with no further purification. All
reactions were monitored by TLC analysis using Merck 60
F254 silica gel on aluminum sheets. Silica gel chromatography
was performed by using 35ꢀ70 mm silica or an automated
chromatography system. NMR spectra were recorded at room
temperature on a 400 MHz instrument. 1H and 13CꢀNMR
spectra were referenced to the residual peaks of the deuterated
solvent. The following abbreviations were used to define the
multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m,
hexane/EtOAc to yield 13 (427 mg, 70%) as a colorless oil.
[α]20 = +50.3 (c=1.05, CHCl3). HꢀNMR (400 MHz, CDCl3,
1
D
δ/ppm): 7.32ꢀ7.25 (m, 10H), 5.91 (m, 2H), 5.18 (m, 2H), 4.78
(m, 2H), 4.52 (m, 2H), 4.40 (m, 2H), 3.96 (m, 1H), 3.55 (m,
3H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 157.2 (CO), 138.1
(C), 137.1 (C), 128.5 (CH), 128.4 (CH), 127.9 (CH), 127.8
(CH), 127.6 (CH), 122.8 (CH), 70.4 (CH2), 69.3 (CH), 67.4
(CH2), 61.5 (CH), 54.6 (CH2), 40.9 (CH2). IR (film, νmax / cmꢀ
1): 3442, 1700, 1412, 1344, 1236. HRMS (ES): calcd. for
C21H25NO4: 354.1705 found 354.1703.
multiplet; and br, broad signal. The chemical shifts (
expressed in ppm and the coupling constants ( ), in Hertz (Hz).
IR spectra were recorded either by preparing a KBr pastille or
by depositing a film of the product on a NaCl window.
Absorptions are given in wavenumbers (cmꢀ1). Melting points
δ) are
J
(2R,3S)-2-Benzoyloxymethyl-3-benzyloxy-N-
benzyloxycarbonyl-3,6-dihydropyridine (14)
were recorded in
a
Büchi Mꢀ540 apparatus without
recrystallization of the final solids. Optical rotations were
measured at room temperature (25°C). Concentration is
expressed in g/100 mL and solvent is expressed for each case in
brackets. The cell was 10 cm long and had 1 mL of capacity.
Measuring λ was 589 nm, which corresponds to a sodium lamp.
High Resolution Mass Spectrometry were conducted using
nanoelectrospray technique..
DMAP (10 mg, 0.08 mmol), TEA (0.29 mL, 2.04 mmol), and
benzoyl chloride (0.19 mL, 1.63 mmol) were added to a
solution of 13 (288 mg, 0.82 mmol) in CH2Cl2 (10 mL) and the
reaction was stirred at r.t. until no starting material was
observed by TLC. Solvent was removed under low pressure and
the crude was purified by chromatography on silica gel using
hexane/ethyl acetate to give 14 (316 mg, 86%) as a colorless
Preparation of 1147 1248 and 2117,48 was done following
,
oil. [α]20 = +15.6 (c=1.05, CHCl3). 1HꢀNMR (400 MHz,
D
literature procedures. Starting material 11 was 99% ee.
Syntheses and characterizations of compounds in Scheme 5,
and derivatives 32b-d and 10b-d can be found in the supporting
information.
CDCl3, 55 ºC, δ/ppm): 7.93 (d,
7.5, 1.5 Hz, 1H), 7.39 (tt, = 7.5, 1.5 Hz, 2H), 7.33 – 7.17 (m,
10H), 6.00 (br, 1H), 5.94 (m, 1H), 5.17 (d, = 12.5 Hz, 1H),
5.02 (br, 2H), 4.65 (br, 1H), 4.54 (m, 1H), 4.46 (m, = 14.0,
J = 7.5 Hz, 2H), 7.55 (tt, J =
J
J
J
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 11
Journal Name
Organic & Biomolecular Chemistry
View ArticAle ROnTliInCeLE
DOI: 10.1039/C5OB00507H
8.0 Hz, 1H), 4.26 (s, 2H), 3.95 (s, 1H), 3.71 (d,
J
= 19.0 Hz,
J = 11.0 Hz, 1H), 4.40 (br, 1H), 4.32 (t, J = 7.0 Hz, 1H), 3.41
1H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 166.1 (CO), 166.0* (br, 1H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 165.8 (CO),
(CO), 156.0 (CO), 155.8* (CO), 137.8 (C), 136.4 (C), 133.1 154.9 (CO), 135.9 (C), 135.3 (C), 133.4 (CH), 129.6 (CH),
(C), 133.1* (C), 129.6 (CH), 129.6 (CH), 129.1 (CH), 128.4 129.2 (C), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH),
(CH), 128.0 (CH), 127.9 (CH), 127.9 (CH), 127.8 (CH), 127.7 128.2 (CH), 81.7 (CH), 74.5 (CH), 73.7 (CH2), 70.8 (CH), 68.4
(CH), 127.6 (CH), 127.5 (CH), 122.6 (CH), 122.3* (CH), 70.4 (CH2), 61.7 (CH2), 53.9 (CH), 40.1 (CH2). IR (film, νmax / cmꢀ
(CH2), 70.3* (CH2), 69.3 (CH), 69.1* (CH), 67.3 (CH2), 67.2* 1): 1714, 1397, 1271, 1212, 1112, 1098, 982, 712, 699. HRMS
(CH2), 62.5 (CH2), 62.2* (CH2), 52.0 (CH), 51.0* (CH), 40.9 (ES): calcd. for C28H31N2O9S : 571.1745, found 571.1747.
(CH2), 40.5* (CH2) *Rotamers. IR (film, νmax / cmꢀ1): 3039,
4-
O-Benzyl-2,3-O-(cyclic sulfate)-5N,6O-(cyclic carbamate)-
2943, 1720, 1702, 1421, 1414, 1272, 1068. HRMS (ES): calcd.
for C28H27NO5Na: 480.1781, found 480.1781.
1-deoxymannojirimycin (22)
Diol 21 (3.18 g, 11.38 mmol) was dissolved in THF (120 mL)
and cooled to 0ºC. Triethyl amine (6.66 mL, 47.81 mmol) was
added and after 10 min SOCl2 (2.5 mL, 38.72 mmol) was added
6-
O-Benzoyl-4-O-benzyl-5-N-benzyloxycarbonyl-1-
deoxymannojirimycin (15)
(DHQD)2Phal (46 mg, 0.06 mmol), K2OsO4 (10 mg, 0.03 dropwise. The reaction was stirred at 0ºC for 1 h, then treated
mmol), K2CO3 (288 mg, 2.08 mmol) and K3[Fe(CN)6] (693 g, with water (9 mL) and extracted with CH2Cl2 (3x15 mL). The
2.08 mmol) were dissolved in ACN:H2O 1:1 (6 mL). The solvent was removed under reduced pressure to give an orange
reaction was cooled to 0 ºC and CH3SO2NH2 (69 mg, 0.69 oil which was dissolved in a 1:1:1 ACN:CCl4:H2O mixture (90
mmol) was then added. After 10 min,a solution of 14 (318 mg, mL) and cooled to 0ºC. RuCl3 (35 mg, 0.17 mmol) and NaIO4
0.69 mmol) in ACN:H2O 1:1 (6 mL) was added and the mixture (4.87 g, 22.77 mmol) were added and the reaction was stirred at
was left to warm to r.t. and stirred until no starting material was 0ºC vigorously for 4h. Treatment consisted of the addition of
observed by TLC. The reaction was treated with Na2SO3 (400 Et2O (20 mL) and H2O (20 mL). The organic phase was washed
mg) and stirred for 60 min. It was then extracted with EtOAc with NaHCO3 (1x20 mL) and brine (1x10 mL), dried over
(3x 10mL) and the organic phase was washed with brine (1x MgSO4, and purified by chromatography on silica gel using
10mL), dried with MgSO4, and purified by chromatography on hexane/ethyl acetate and increasing the polarity ratio to give 22
silica gel using hexane:EtOAc to give 15 (212 mg, 62%) as one as a white foam (3.12 g, 80%). [α]20 = +47.6 (c=0.5, CHCl3).
D
1
diastereomer as a sticky white foam. [α]20 = ꢀ20.0 (c=0.76, Mp: 157ꢀ158 ºC. HꢀNMR (400 MHz, CDCl3, δ/ppm): 7.45 –
D
1
CHCl3). HꢀNMR (400 MHz, CDCl3, δ/ppm): 7.93 (d,
J
= 7.5 7.31 (m, 5H), 5.18 (m, 1H), 4.95 (dd,
= 7.5 Hz, 2H), 7.32 (d, = 11.5 Hz, 1H), 4.65 (d, = 11.5 Hz, 1H), 4.45 (d,
= 11.5 Hz, 1H), 4.94 (br, 1H), 4.86 16.0 Hz, 1H), 4.36 (dd, = 9.5, 8.0 Hz, 1H), 3.98 (dd, = 9.5,
= 11.5, 9.5 Hz, 1H), 4.59 (br, 1H), 4.47 (d, 8.0 Hz, 1H), 3.84 (dd, = 9.5, 4.5 Hz, 1H), 3.59 – 3.50 (m,
= 12.0 Hz, 1H), 4.41 (dd, , = 12.0, 5.0 Hz, 1H), 4.17 (br, 1H), 3.36 (dd,
1H), 4.07 (s, 1H), 4.03 (br, 1H), 3.75 (t, = 2.5 Hz, 1H), 3.14 CDCl3, δ/ppm): 156.1 (CO), 136.1 (C), 128.9 (CH), 128.8
(dd,
J = 8.0, 4.5 Hz, 1H), 4.91
Hz, 2H), 7.54 (t,
– 7.11 (m, 10H), 5.07 (d,
(br, 1H), 4.76 (dd,
J
= 7.5 Hz, 1H), 7.38 (t,
J
J
J
J =
J
J
J
J
J
,
J
J
J
= 16.0, 3.0 Hz, 1H). 13CꢀNMR (100 MHz,
J
J
= 13.0, 11.0 Hz, 1H), 2.95 (br, 1H). 13CꢀNMR (100 MHz, (CH), 128.6 (CH), 86.7 (CH), 79.1 (CH), 75.2 (CH), 74.4
CDCl3, δ/ppm): 166.4 (CO), 156.2 (CO), 137.4 (C), 136.2 (C), (CH2), 65.4 (CH2), 54.3 (CH), 39.9 (CH2). IR (film, νmax / cmꢀ
133.0 (C), 129.8 (CH), 129.6 (CH), 128.4 (CH), 128.4 (CH), 1): 2902, 1755, 1439, 1389, 1214, 1070, 1014. HRMS (ES):
128.3 (CH), 128.2 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), calcd. for C14H16NO7S: 342.06420, found 342.06516. EA:
75.5 (CH), 71.3 (CH2), 69.5 (CH), 67.5 (CH2), 64.7 (CH), 61.7 Anal. calcd. for C14H15NO7S: C, 49.26%; H, 4.43%; N, 4.10%;
(CH2), 52.3 (CH), 39.4 (CH2). IR (film, νmax / cmꢀ1): 3428, S, 9.39%; found C, 48.92%; H, 4.48%; N, 4.25%; S, 9.41%
2911, 1719, 1699, 1450, 1429, 1274, 1068. HRMS (ES): calcd.
for C28H30NO7: 492.2017, found 492.2024.
2-Azido-4-
dideoxynojirimycin (19) and 3-Azido-4-
(cyclic carbamate)-1,3-dideoxyallonojirimycin (20)
NaN3 (1.06 g, 16.31 mmol) and 22 (2.78 g, 8.16 mmol) were
O
-benzyl-5
N
,6
O
-(cyclic
carbamate)-1,2-
O
-benzyl-5N,6O-
6-
O-Benzoyl-4-O-benzyl-5-N-benzyloxycarbonyl-2,3-O-
(cyclic sulfate)-1-deoxymannojirimycin (16)
TEA (0.25 mL, 1.80 mmol) was added to a solution of 15 (211 dissolved in acetone: H2O 2:1 (135 mL) and heated at 50 ºC for
mg, 0.43 mmol) in THF (12 mL) cooled at 0ºC and SOCl2 (100 6 h. After removal of the acetone at low pressure, Et2O (80 mL)
µL, 1.54 mmol) was then added dropwise. The reaction was and 20% aq H2SO4 (60 mL) were added and the mixture was
stirred at 0ºC until no starting material was observed by TLC. stirred at r.t. for 24 h. The reaction was diluted with H2O (30
H2O (5 mL) was then added and the crude was extracted with mL), extracted with EtOAc (3x 50 mL), washed with aqueous
CH2Cl2 (3x 5 mL) and dried over MgSO4. The solvent was NaHCO3 (2x 15 mL), dried over MgSO4, and purified by
removed under low pressure and the obtained oil was dissolved chromatography on silica gel using hexane/ethyl acetate and
in ACN:CCl4:H2O 1:1:1 mixture (7.5 mL) and cooled at 0ºC. increasing the polarity ratio to obtain 19 (979 mg, 39%) and 20
RuCl3 (9 mg, 0.04 mmol) and NaIO4 (184 mg, 0.86 mmol) (554 mg, 22%) as white solids.
1
were added and the reaction was allowed to stir at 0ºC until no 19: [α]20 = +74.0 (c=0.37, CHCl3). Mp: 194ꢀ197 ºC. HꢀNMR
D
starting material was observed by TLC. H2O (5mL) and Et2O (5 (400 MHz, CDCl3, δ/ppm): 7.44 – 7.29 (m, 5H), 4.89 (d,
J =
mL) were added, and the reaction was stirred 10 min. The 11.6 Hz, 1H), 4.72 (d,
J
= 11.6 Hz, 1H), 4.30 (dd,
J
J
J
= 9.0, 8.0
= 9.0, 4.5
= 9.5, 8.0,
organic phase was separated and washed with NaHCO3 (1x Hz, 1H), 4.06 (dd,
5mL) and brine (1x 5mL), and dried with MgSO4. The solvent Hz, 1H), 3.59 (dt,
J
J
= 13.5, 6.0 Hz, 1H), 3.87 (dd,
= 9.5, 3.5 Hz, 1H), 3.53 (ddd,
was removed under low pressure. Purification by 4.5 Hz, 1H), 3.44 (ddd,
J
= 11.0, 9.5, 6.0 Hz, 1H), 3.29 (t,
= 3.5 Hz, 1H), 2.71 (dd, = 13.5, 11.0
Hz, 1H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 156.4 (CO),
J =
chromatography on silica gel using hexane/ethyl acetate gave 9.4 Hz, 1H), 2.76 (d,
J
J
16 (190 mg, 75%) as one diastereomer as colorless oil. [α]20
=
D
1
ꢀ16.3 (c=0.63, CHCl3). HꢀNMR (400 MHz, CDCl3, δ/ppm): 137.3 (C), 128.8 (CH), 128.6 (CH), 128.2 (CH), 80.1 (CH),
7.87 (dd,
7.44 (t,
1.5 Hz, 1H), 5.14 (d,
= 7.0 Hz, 1H), 4.79 (d,
J
= 7.5, 1.0 Hz, 2H), 7.58 (tt,
= 7.5 Hz, 2H), 7.34 – 7.24 (m, 9H), 7.19 (tt,
= 11.5 Hz, 1H), 5.08 (br, 2H), 5.02 (t,
= 11.5 Hz, 1H), 4.65 (m, 2H), 4.55 (d, 305.12443, found 305.12464. EA: Anal. calcd. for C14H16N4O4:
J
= 7.5, 1.0 Hz, 1H), 77.6 (CH), 75.1 (CH2), 65.6 (CH2), 60.6 (CH), 56.5 (CH), 42.6
= 6.0, (CH2). IR (film, νmax / cmꢀ1): 3366, 2919, 2118, 1709, 1438,
1253, 1108, 1085. HRMS (ES): calcd. for C14H17N4O4:
J
J
J
J
J
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

Organic & Biomolecular Chemistry
Page 8 of 11
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ARTICLE
Journal Name
DOI: 10.1039/C5OB00507H
C, 55.26%; H, 5.30%; N, 18.41%; found C, 55.35%; H, 5.35%;
2-Acetamido-3,4-di-
O
-benzyl-1,2-dideoxynojirimycin (25)
N, 18.51%.
NaOH 6M (0.35 mL, 2.11 mmol) was added to a solution of 24
1
20: [α]20 = +51.2 (c=0.43, CHCl3). Mp: 148ꢀ152 ºC. HꢀNMR (87 mg, 0.21 mmol) in MeOH : H2O 9:1 (8 mL) and the
D
(400 MHz, CDCl3, δ/ppm): 7.44 – 7.32 (m, 5H), 4.74 (d,
11.5 Hz, 1H), 4.52 (d, = 11.5 Hz, 1H), 4.35 (dd, = 8.5, 6.5 added and the reaction was extracted with EtOAc (3x 5 mL),
Hz, 1H), 4.17 (t,
(d,
J
=
reaction was stirred at reflux for 4 h. H2O (5 mL) was then
J
J
J
= 3.0, 1H), 4.03 (m, 2H), 3.92 (m, 2H), 3.68 dried over MgSO4, and purified by chromatography on silica
J
= 14.5 Hz, 2H), 3.25 (dd,
J
= 14.5, 1.5 Hz, 1H). 13CꢀNMR gel using CH2Cl2/MeOH to give 25 (75 mg, 92%) as a white
(100 MHz, CDCl3, δ/ppm): 158.7 (CO), 136.8 (C), 128.7 (CH), solid. [α]20 = ꢀ19.7 (c=0.08, CH3OH). Mp: 210ꢀ212 ºC. Hꢀ
1
D
128.4 (CH), 128.3 (CH), 74.8 (CH), 71.8 (CH2), 67.5 (CH), NMR (400 MHz, CD3OD, δ/ppm): 7.34 – 7.24 (m, 10H), 4.81
65.9 (CH2), 59.9 (CH), 53.2 (CH), 42.7 (CH2). IR (film, νmax
cmꢀ1): 3294, 2919, 2099, 1727, 1447, 1088, 1067. HRMS (ES): = 18.0 Hz, 1H), 3.97 (m, 1H), 3.79 (dd,
calcd. for C14H17N4O4: 305.12443, found 305.12450. EA: Anal. 3.68 (dd, = 11.0, 5.0 Hz, 1H), 3.44 (m, 2H), 3.04 (dd,
/
(dd,
J
= 11.0, 1.5 Hz, 2H), 4.73 (d,
J
= 18.0 Hz, 1H), 4.67 (d,
J
J
= 11.0, 2.5 Hz, 1H),
J
J =
calcd. for C14H16N4O4: C, 55.26%; H, 5.30%; N, 18.41%; found 12.0, 5.0 Hz, 1H), 2.55 (m, 1H), 2.44 (t,
J = 12.0 Hz, 1H), 1.88
C, 55.48%; H, 5.40%; N, 18.42%.
2-Azido-3,4-di- -benzyl-5 ,6 -(cyclic
dideoxynojirimycin (23)
(s, 3H). 13CꢀNMR (100 MHz, CD3OD, δ/ppm): 173.1 (CO),
140.2 (C), 139.8 (C), 129.4 (CH), 129.3 (CH), 128.9 (CH),
128.7 (CH), 128.7 (CH), 128.6 (CH), 86.4 (CH), 81.5 (CH),
O
N
O
carbamate)-1,2-
A solution of 19 (330 mg, 1.08 mmol) in DMF (8 mL) was 76.0 (CH2), 62.6 (CH), 62.2 (CH2), 53.3 (CH), 49.3 (CH2), 22.9
added via cannula to a suspension of NaH (40 mg, 1.62 mmol) (CH3). IR (film, νmax / cmꢀ1): 3275, 2933, 1650, 1554, 1072,
in DMF (8.5 mL) cooled at 0ºC. After 10 min, benzyl bromide 1027. HRMS (ES): calcd. for C22H29N2O4: 385.21218, found
(0.18 mL, 1.52 mmol) was added drop wise and the reaction 385.21223. EA: Anal. calcd. for C22H28N2O4+ 3/2 H2O: C,
was allowed to stir at r.t. until no starting material was observed 64.21%; H, 7.59%; N, 6.81%; found C, 64.52%; H, 7.08%; N,
by TLC. H2O (5 mL) was then added and the reaction was 6.42%.
extracted with CH2Cl2 (3x 5 mL), dried over MgSO4, and
2-Acetamido-1,2-dideoxynojirimycin (DNJNAc, 4)
purified by chromatography on silica gel using hexane/EtOAc To a solution of 25 (20 mg, 0.05 mmol) in MeOH (4 mL) was
to give 23 (384 mg, 90%) as a white solid. [α]20 = +53.4 added Pd/C (9 mg, 0.008 mmol) and the reaction was charged
D
(c=0.49, CHCl3). Mp: 110ꢀ112 ºC. 1HꢀNMR (400 MHz, CDCl3, with H2 (55 barg) and stirred at 60 ºC for 20 h. Palladium was
δ/ppm): 7.43 – 7.22 (m, 10H), 4.91 (dt,
4.61 (d, = 11.5 Hz, 1H), 4.23 (dd, = 9.0, 8.0 Hz, 1H), 4.04 chromatography on silica gel using CH2Cl2/MeOH/NH3
(dd, = 13.5, 5.5 Hz, 1H), 3.70 (dd, = 9.0, 4.5 Hz, 1H), 3.60 – 72.5:25:2.5 to give
(12 mg, 96%) as a white solid. [α]20
3.40 (m, 3H), 3.34 (t, = 9.0 Hz, 1H), 2.67 (dd,
= 13.5, 10.5 +7.9 (c=0.15, H2O). Mp: 210ꢀ212 ºC. 1HꢀNMR (400 MHz,
Hz, 1H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 156.4 (CO), CD3OD, δ/ppm): 3.81 (dd,
= 11.0, 3.0 Hz, 1H), 3.73 (m, 1H),
137.3 (C), 137.2 (C), 128.7 (CH), 128.5 (CH), 128.5 (CH), 3.63 (dd, = 11.0, 6.0 Hz, 1H), 3.24 (m, 2H), 3.11 (dd,
128.2 (CH), 128.2 (CH), 85.1 (CH), 80.0 (CH), 76.1 (CH2), 12.5, 5.0 Hz, 1H), 2.46 (ddd, = 9.5, 6.0, 3.0 Hz, 1H), 2.38 (dd,
= 12.5, 11.0 Hz, 1H), 1.96 (s, 3H). 13CꢀNMR (100 MHz,
J = 10.5, 9.5 Hz, 3H), then filtrated over Celite and the crude was purified by
J
J
J
J
4
=
D
J
J
J
J
J =
J
75.1 (CH2), 65.4 (CH2), 60.7 (CH), 56.8 (CH), 43.0 (CH2). IR
J
(film, νmax / cmꢀ1): 2917, 2110, 1761, 1425, 1091. HRMS (ES): CD3OD, δ/ppm): 173.6 (CO), 77.7 (CH), 73.9 (CH), 62.8
calcd. for C21H23N4O4: 395.1714, found 395.1706. EA: Anal. (CH2), 62.7 (CH), 53.9 (CH), 49.1 (CH2), 22.7 (CH3). IR (film,
calcd. for C21H22N4O4: C, 63.95%; H, 5.62%; N, 14.20%; found νmax/cmꢀ1): 3287, 2917, 1638, 1559, 1437, 1373, 1096, 1040.
C, 63.85%; H, 5.50%; N, 14.06%.
2-Acetamido-3,4-di- -benzyl-5
dideoxynojirimycin (24)
Pd/C (18 mg, 0.02 mmol) was added to a solution of 23 (111
HRMS (ES): calcd. for C8H17N2O4: 205.11828, found
205.11784.
O
N,6O-(cyclic carbamate)-1,2-
2-Azido-4-
O-benzyl-5-N-benzyloxycarbonyl-1,2-
dideoxynojirimycin (30)
mg, 0.28 mmol) in EtOAc (5 mL) and the reaction was charged NaOH 6M (2 mL, 11.89 mmol) was added to a solution of
with H2 (5 barg) and stirred at r.t. for 20h. Palladium was compound 19 (301 mg, 0.99 mmol) in MeOH:H2O 9:1 (20 mL)
filtered with MeOH over Celite and solvents were removed and the reaction was heated at reflux for 15 h. Solvent was then
under low pressure. The obtained colorless oil was dissolved in removed under low pressure and the crude was redissolved in
pyridine (2 mL) and Ac2O (48 ꢁL, 0.39 mmol) was added. The EtOAc:NaHCO3 saturated aqueous 1:1 (14 mL). After 30 min
reaction was stirred at r.t. for 16h. H2O (5 mL) was then added of stirring, Boc2O (436 mg, 1.91 mmol) was added and the
and the reaction was extracted with EtOAc (3x 5 mL), dried crude was allowed to stir for 24 h. The crude was treated with
over MgSO4, and purified by chromatography on silica gel water (6 mL), extracted with EtOAc (3x5 mL), dried with
using hexane/EtOAc to give 24 (110 mg, 95%) as a white solid. MgSO4, and purified by chromatography on silica gel using
1
[α]20 = +106.5 (c=0.31, CHCl3). Mp: 213 – 215 ºC. HꢀNMR hexane/ethyl acetate to give 30 (334 mg, 90%) as a colorless
D
(400 MHz, CDCl3, δ/ppm): 7.44 – 7.28 (m, 10H), 5.33 (d,
5.0 Hz, 1H), 4.92 (d, = 11.5 Hz, 2H), 4.66 (dd, = 13.0, 11.5 CDCl3, δ/ppm): 7.38 – 7.28 (m, 5H), 4.83 (d,
Hz, 2H), 4.25 (dd, = 9.0, 8.0 Hz, 1H), 4.04 (dd, = 13.0, 5.0 4.72 (d, = 11.5 Hz, 1H), 4.00 (d, = 11.0 Hz, 1H), 3.97 ꢀ 3.87
Hz, 1H), 3.76 – 3.52 (m, 4H), 3.39 (m, 1H), 2.82 (m, 1H), 1.78 (br, 2H), 3.84 (dd, = 14.0, 4.5 Hz, 1H), 3.68 (t, = 7.0 Hz,
(s, 3H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 170.4 (CO), 1H), 3.57 (t,
= 7.0 Hz, 1H), 3.53 (br, 1H), 3.39 (td, = 7.5,
156.4 (CO), 137.9 (C), 137.4 (C), 128.8 (CH), 128.7 (CH), 4.5 Hz, 1H), 3.29 (s, 1H), 3.18 (dd, = 14.0, 7.5 Hz, 1H), 1.47
J
=
oil. [α]20 = ꢀ36.1 (c=0.67, CHCl3). 1HꢀNMR (400 MHz,
D
J
J
J = 11.5 Hz, 1H),
J
J
J
J
J
J
J
J
J
128.4 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 81.9 (CH), (s, 9H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 154.9 (CO),
81.2 (CH), 75.0 (CH2), 74.9 (CH2), 65.6 (CH2), 56.9 (CH), 50.5 138.0 (C), 128.6 (CH), 128.0 (CH), 128.0 (CH), 81.5 (s), 77.7
(CH), 42.7 (CH2), 23.3 (CH3). IR (film, νmax / cmꢀ1): 3299, (C), 75.4 (CH), 74.1 (CH2), 60.8 (CH), 60.7 (CH2), 60.6 (CH),
2946, 1749, 1652, 1521, 1088. HRMS (ES): calcd. for 45.7 (CH2), 28.3 (CH3). IR (film, νmax / cmꢀ1): 3404, 2968,
C23H27N2O5: 411.19145, found 411.19214. EA: Anal. calcd. for 2107, 1668, 1422, 1367, 1250, 1162. HRMS (ES): calcd. for
C23H26N2O5+ ½H2O: C, 65.86%; H, 6.49%; N, 6.68%; found C, C18H27N4O5: 379.19760, found 379.19777.
65.85%; H, 6.13%; N, 6.50%.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 9 of 11
Journal Name
Organic & Biomolecular Chemistry
View ArticAle ROnTliInCeLE
DOI: 10.1039/C5OB00507H
J
= 7.5 Hz, 3H). 13CꢀNMR (100 MHz, CD3OD, δ/ppm): 172.7
(CO), 161.8 (CO), 71.7 (CH), 70.3 (CH), 61.9 (CH), 61.8
2-Acetamido-3,4-6-tri-
dideoxynojirimycin (31)
O-acetyl-5-N-benzyloxycarbonyl-1,2-
Pd/C (160 mg, 0.15 mmol) and acetic acid (0.43 mL, 7.55 (CH2), 51.6 (CH), 41.6 (CH2), 41.0 (CH2), 33.3 (CH2), 22.9
mmol) were added to a solution of 30 (571 mg, 1.51 mmol) in (CH3), 21.1 (CH2), 14.2 (CH3). IR (film, νmax / cmꢀ1): 3353,
degassed MeOH (15 mL) and the reaction was charged with H2 2923, 1623, 1540, 1469, 1418, 1021. HRMS (ES): calcd. for
(15 barg) and stirred at 60ºC for 16h. Palladium was filtered C13H26N3O5: 304.18670, found 304.18727.
over Celite and solvents were removed under low pressure. The
colorless obtained oil was redissolved in pyridine (10 mL) and
Ac2O (1.58 mL, 15.02 mmol) was added. The reaction was
stirred at r.t. for 16h. H2O (5 mL) was then added and the
reaction was extracted with CH2Cl2 (3x 5 mL), dried over
MgSO4 and purified by chromatography on silica gel using
hexane/EtOAc to give 31 (538 mg, 83%) as a colorless oil.
General Procedures for Inhibition Assay
The glycosidases ꢀglucosidase (from yeast), amyloglucosidase
(from Aspergillus niger), isomaltase (from yeast),
glucosidases (from almond and bovine liver), naringinase
Penicillium decumbes), ꢀgalactosidase (from green coffee
beans), ꢀgalactosidase (from Escherichia coli), ꢀmannosidase
(from jack bean), βꢀmannosidase (from Helix pomatia),
acetylglucosaminidases (from human placenta, bovine kidney
and jack bean) used in the inhibition studies, as well as the
corresponding oꢀ or pꢀnitrophenyl glycoside substrates, were
α
β
ꢀ
(
α
β
α
βꢀNꢀ
[α]20 = ꢀ7.2 (c=2.3, CHCl3). 1HꢀNMR (400 MHz, CD3OD,
D
δ/ppm): 6.12 (d,
4.61 (t, = 7.5 Hz, 1H), 4.38 (dd,
(dd, = 11.5, 6.5 Hz, 1H), 4.14 – 4.05 (m, 2H), 3.32 (dd,
J
= 8.5 Hz, 1H), 4.94 (m, 1H), 4.92 (m, 1H),
J
J
= 11.5, 8.5 Hz, 1H), 4.23
J
J =
purchased from Sigma Chemical Co. Inhibitory potencies were
determined by spectrophotometrically measuring the residual
hydrolytic activities of the glycosidases against the respective
15.0, 3.0 Hz, 1H), 2.11 (s, 6H), 2.06 (s, 3H), 1.99 (s, 3H), 1.47
(s, 9H). 13CꢀNMR (100 MHz, CD3OD, δ/ppm): 170.4 (CO),
168.9 (CO), 168.6 (CO), 168.2 (CO), 155.5 (CO), 80.8 (C),
67.8 (CH), 67.0 (CH), 59.9 (CH2), 52.9 (CH), 46.1 (CH), 39.3
(CH2), 28.2 (CH3), 23.3 (CH3), 20.8 (CH3), 20.8 (CH3), 20.7
(CH3). IR (film, νmax / cmꢀ1): 3333, 2975, 1745, 1687, 1369,
1223, 1046. HRMS (ES): calcd. for C19H31N2O: 431.20241,
found 431.20239.
o
ꢀ
(for
glycopyranoside (for
galactosidases, ꢀmannosidases and
nitrophenylꢀ
β
ꢀgalactosidases) or
p
ꢀnitrophenyl
ꢀglucosidases,
ꢀmannosidases) or
αꢀ or βꢀDꢀ
α
ꢀglucosidases,
β
αꢀ
α
β
p
ꢀ
N
ꢀacetylꢀβꢀDꢀglucosaminide/galactosaminide (for
hexosaminidases), in the presence of corresponding
iminosugars. Each assay was performed in phosphateꢀcitrate
2-Acetamido-3,4-6-tri-O-acetyl-5-N-(N’-
butylaminocarbonyl)-1,2-dideoxynojirimycin (32a)
(for
αꢀ
or
β
ꢀmannosidase, amyloglucosidase or
βꢀNꢀ
acetylglucosaminidase at pH 5.5 or 3.5) or in phosphate buffer
(at pH 7.3 or 6.8 for the other glycosidases) at the optimal pH
for each enzyme. The Km values for the different glycosidases
used in the tests and the corresponding working pHs are listed
herein: αꢀglucosidase (yeast), Km = 0.35 mM (pH 6.8);
amyloglucosidase (Aspergillus niger), Km = 3.0 mM (pH 5.5);
isomaltase (from yeast), Km = 1.0 mM (pH 6.8); βꢀglucosidase
TFA (0.48 mL, 6.31 mmol) was added to a solution of 31 (90
mg, 0.21 mmol) in CH2Cl2 (8 mL) and the reaction was stirred
at r.t. until no starting material was observed by TLC. Solvent
was removed under reduced pressure and the resulting oil was
dissolved in CH2Cl2 (8 mL). TEA (0.23 mL, 1.64 mmol) and
butyl isocyanate (71 µl, 0.63mmol) were added and the reaction
was heated at reflux for 4h. H2O (5 mL) was then added and the
reaction was extracted with CH2Cl2 (3x 5 mL), dried over
MgSO4, and purified by chromatography on silica gel using
(almond), Km = 3.5 mM (pH 7.3); βꢀglucosidase (bovine liver),
Km = 1.0 mM (pH 7.3); naringinase (Penicillium decumbes), Km
= 2.7 mM (pH 6.8); αꢀgalactosidase (coffee beans), Km = 2.0
CH2Cl2/MeOH to give 32a (73 mg, 81%) as a colorless oil.
mM (pH 6.8); βꢀgalactosidase (from Escherichia coli),
Km =
1
[α]20 = ꢀ63.5 (c=2.31, CHCl3). HꢀNMR (400 MHz, CDCl3,
D
0.12 mM (pH 7.3); αꢀmannosidase (jack bean), Km = 2.0 mM
(pH 5.5); βꢀmannosidase (Helix pomatia), Km = 0.6 mM (pH
δ/ppm): 6.52 (d,
J
= 8.0 Hz 1H), 5.04 (t,
J
= 5.5 Hz 1H), 4.99 (t,
J
= 3.0 Hz 1H),4.88 (m, 1H), 4.43 (dd,
J = 11.0, 7.5 Hz, 1H),
5.5); βꢀ
0.34 mM (pH 5.5); βꢀ
kidney), Km = 0.48 mM (pH 5.5); βꢀ
N
ꢀacetylglucosaminidase (from human placenta), Km
ꢀacetylglucosaminidase (from bovine
ꢀacetylglucosaminidase
=
4.24 (t,
J
= 6.5Hz, 1H), 4.12 (dd,
=5.0 Hz, 1H), 3.26 (dd,
Hz, 1H) , 3.20 (m, 2H), 2.09 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H),
1.95 (s, 3H), 1.47 (p, = 7.0 Hz, 2H), (h, = 7.0 Hz, 2H), 0.90
(t,
= 7.5 Hz, 3H). 13CꢀNMR (100 MHz, CDCl3, δ/ppm): 171.0
J
= 11.0, 7.5 Hz, 1H) 4.03 (q,
N
J
= 3.5 Hz, 1H), 3.95 (d,
J
J
= 15.0, 3.0
N
(from jack bean), Km = 0.49 mM (pH 5.5). The reactions were
initiated by addition of enzyme to a solution of the substrate in
the absence or presence of various concentrations of inhibitor.
After the mixture was incubated for 10–30 min at 37 ºC (or 55
ºC for amyloglucosidase), the reaction was quenched by
addition of 1 M Na2CO3. The absorbance of the resulting
mixture was determined at 405 nm. The Ki value and enzyme
inhibition mode were determined from the slope of
LineweaverꢀBurk plots and double reciprocal analysis using
Microsoft Office Excel 2007 program. Inhibition mode was
competitive in all cases.
J
J
J
(CO), 169.6 (CO), 168.8 (CO), 168.7 (CO), 159.0 (CO), 68.0
(CH), 67.0 (CH), 61.0 (CH2), 54.0 (CH), 47.0 (CH), 40.8
(CH2), 39.1 (CH2), 32.1 (CH2), 23.2 (CH3), 20.8 (CH3), 20.8
(CH3), 20.7 (CH3), 20.0 (CH2), 13.7 (CH3). IR (film, νmax / cmꢀ
1): 3364, 2958, 2932, 1749, 1652, 1539, 1370, 1225, 1043.
HRMS (ES): calcd. for C19H32N3O8: 430.21819, found
430.21839.
2-Acetamido-5-N-(N’-butylaminocarbonyl)-1,2-
dideoxynojirimycin (10a)
Compound 32a (73 mg, 0.17 mmol) was dissolved in a NH3
saturated MeOH solution (4 mL) and the reaction was stirred at
r.t. for 18h. Solvent was removed under low pressure, and the
crude was purified by chromatography on silica gel using
CH2Cl2/MeOH to give 10a (32 mg, 61%) as a slightly yellow
solid. [α]20D = +25.0 (c=1.63, CH3OH). Mp: 70ꢀ72 ºC. 1HꢀNMR
(400 MHz, CD3OD, δ/ppm): 3.94 (m, 1H), 3.91 – 3.81 (m,
Acknowledgements
We thank the Spanish Ministerio de Economia
y
Competitividad (CTQ2011ꢀ23620, SAF2013ꢀ44021R and
CTQ2010ꢀ15848), the Junta de Andalucía (Project FQMꢀ1467),
and IRB Barcelona for financial support. Coꢀfinacing by the
European Union (FEDER and FSE) is also acknowledged. A.F.
and T. M.ꢀB. thank the Ministerio de Ciencia e Innovación and
the Junta de Andalucía for doctoral and postꢀdoctoral
fellowships, respectively. Technical assistance from the
CITIUS (Universidad de Sevilla) is recognized.
3H), 3.76 (dd,
J
= 11.0, 4.5 Hz, 1H), 3.72 (t,
J = 4.5 Hz, 1H),
3.60 (t, = 4.5 Hz, 1H), 3.35 (dd,
J
J
= 13.5, 3.0 Hz, 1H), 3.24 –
3.07 (m, 2H), 1.95 (s, 3H), 1.48 (m, 2H), 1.37 (m, 2H), 0.93 (t,
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J. Name., 2012, 00, 1-3 | 9

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