Selection of the biological activity of DNJ neoglycoconjugates through click length variation of the side chain

Article abstract of DOI:10.1039/c1ob05119a

A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively either strongly inhibit ER-α-glucosidases and ceramide glucosyltransferase or restore the activity of CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.

Full text of DOI:10.1039/c1ob05119a

Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 9 | Number 15 | 7 August 2011 | Pages 5305–5580
ISSN 1477-0520
PAPER
Boris Vauzeilles et al.
Selection of the biological activity of DNJ neoglycoconjugates
through click length variation of the side chain

Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 5373
www.rsc.org/obc
PAPER
Selection of the biological activity of DNJ neoglycoconjugates through click
length variation of the side chain†
Nicolas Ardes-Guisot,a Dominic S. Alonzi,b Gabriele Reinkensmeier,b Terry D. Butters,b Caroline Norez,c
Fre´de´ric Becq,c Yousuke Shimada,d Shinpei Nakagawa,d Atsushi Kato,d Yves Ble´riot,*‡e Matthieu Sollogoube
and Boris Vauzeilles*a
Received 21st January 2011, Accepted 21st March 2011
DOI: 10.1039/c1ob05119a
A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection
with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the
glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective
ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively
either strongly inhibit ER-a-glucosidases and ceramide glucosyltransferase or restore the activity of
CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.
Gaucher disease,9 the most prevalent lysosomal storage dis-
order, is caused by mutations in the gene encoding for glu-
cosylceramidase (or b-glucocerebrosidase, EC 3.2.1.45).10 This
enzyme is responsible for the cleavage of the glucosylceramide
sphingolipid into glucose and ceramide. Its deficiency leads to the
accumulation of glucosylceramide in the lysosome causing severe
Introduction
Iminosugars, sugar mimics in which the endocyclic oxygen has
been replaced by nitrogen, represent the most promising class
of sugar analogues as therapeutic agents.1 Since the discovery
of their most famous representative, deoxynojirimycin (DNJ) 1
(Fig. 1), a vast array of derivatives, in which the nature and
symptoms. Additionally, glucosylceramide is the precursor of
stereochemistry of the substituents on the piperidine ring has
complex glycosphingolipids involved in key human physiological
processes such as cell–cell and cell–pathogen recognition.11 To
date, two therapies that reduce glucosylceramide storage have
been approved for the treatment of Gaucher disease. Enzyme
replacement therapy (ERT) is based on the administration of
been modified extensively, has been synthesized and assayed on
numerous pathologies.2 Promising results have been obtained in
the field of cancer3 and viral infections4 including HIV,5 but more
significantly, a N-hydroxyethyl analogue, miglitol, was approved
by the FDA in 1996 for the treatment of late onset diabetes
R
Cerezymeꢀ,12 a recombinant form of glucosylceramidase, to
R
(marketed as Glysetꢀ).6 Additionally, a N-butyl derivative, NB-
supplement the defective enzyme (Fig. 1). Its main drawbacks
are its cost (over 150 000 $/year), as well as its limited use to
non-neuronopathic patients as the enzyme is unable to cross the
blood–brain barrier. The second approach, substrate reduction
therapy (SRT),13 is based on the use of a small molecule able to
inhibit the biosynthesis of glucosylceramide through inactivation
of a ceramide glucosyltransferase, glucosylceramide synthase
(GCS), which condenses UDP-glucose and ceramide. Its major
drawbacks are its limited use to patients with mild-to-moderate
type 1 Gaucher’s disease and its side effects related to its
broad-spectrum inhibition, especially to digestive glucosidases.
More recently, a third approach, the active-site-specific chaperone
(ASSC) therapy,14 has received increased attention. It is based on
the use of small molecules that partially stabilise the defective
and misfolded glucosylceramidase, thus avoiding its degradation
in the endoplasmic reticulum by the quality control mechanism
of the cell and allowing its normal trafficking to the lysosome.
Administration of sub-inhibitory concentrations of potent and
competitive glucosylceramidase inhibitors including iminosugars
and aminocyclitols has demonstrated a chaperone-like profile for
these molecules.15
R
DNJ or miglustat 2,7 marketed as Zavescaꢀ, (Fig. 1) has been
launched for the treatment of Gaucher disease, a rare lysosomal
disorder. The same compound is in phase II trials for the treatment
of cystic fibrosis.8
aCNRS and Univ. Paris Sud, Glycochimie Mole´culaire et Macromole´culaire,
ICMMO, UMR 8182, Orsay, F-91405, France. E-mail: boris.vauzeilles@
u-psud.fr; Fax: +33 1 69 15 47 15; Tel: +33 1 69 15 68 36
bGlycobiology Institute, Oxford University, South Parks Road, Oxford, UK,
OX1 3QU; Fax: +44 1 865 275 21671
cInstitut de Physiologie et Biologie Cellulaires, Universite´ de Poitiers, CNRS,
1 rue Georges Bonnet, BP633, 86022 Poitiers cedex, France
dDepartment of Hospital Pharmacy, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan
eUPMC, Univ Paris 06, Institut Parisien de Chimie Mole´culaire, (UMR
CNRS 7201), 4 place Jussieu, C. 181, F-75005 Paris, France; Fax: + 33 1
44 27 55 04
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05119a
‡ Universite´ de Poitiers, Laboratoire de Synthe`se et Re´activite´ des Sub-
stances Naturelles, UMR CNRS 6514, 4 avenue Michel Brunet, F-86022
Poitiers, France.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5373
©

Fig. 1 Glucosyl ceramide biosynthesis and catabolism, and structure of DNJ and potent glucosylceramide synthase inhibitors.
Since SRT is a relatively general and affordable approach,
glucosylceramide synthase is becoming a very attractive thera-
peutic target for an increasing number of pathological processes,
which are being correlated with excessive glycosphingolipid levels.
Therefore more potent and selective GCS inhibitors are needed
and NB-DNJ is a good starting point for the design of improved
structures. Counter-intuitively, it has been shown at the molecular
level that NB-DNJ is a ceramide mimic rather than a glucose
surrogate, molecular modelling showing good superimposition
between the ceramide and NB-DNJ.16 As a consequence, C,
N or O-alkylation of DNJ with alkyl chains of various length
has been explored to yield lipophilic 1-deoxynojirimycin-based
inhibitors as ceramide surrogates which, when C or O-alkylated,
were less efficient than reference NB-DNJ. This indicated that
N-substitution seems to be the best DNJ modification to ob-
tain potent glucosylceramide synthase inhibitors.17,18 Amongst
the great number of N-substituted DNJ analogues synthesized,
Overkleeft demonstrated that introduction of an adamantane
moiety capping of the N-alkyl chain significantly increased the
potency of the inhibitor, and AMP-DNJ 3 (N-[5-(adamantan-1-
yl-methoxy)-pentyl]-1-deoxynojirimycin, Fig. 1) proved to be 100-
fold more potent than NB-DNJ.19 From these results it is clear
that the DNJ and adamantane moieties are amongst key motifs
for strong inactivation.
Cystic fibrosis (CF) is an autosomal recessive disorder, which
is caused by mutations in the gene coding for the cystic fibrosis
transmembrane conductance regulator (CFTR). CFTR is an ABC
transporter-class protein and ion channel that transports chloride
anions across the apical membrane of epithelial cells. Mutations of
the CFTR gene affect folding and/or functioning of the chloride
channels in these cell membranes, causing CF. The most common
CF mutation, F508del, observed in more than 90% of patients,
causes misfolding of the protein, intracellular retention by the en-
doplasmic reticulum quality control and premature degradation.20
It has been shown that NB-DNJ 2 is a partial corrector of
F508del-CFTR acting as a pharmacological chaperone,21 and
that incubation with NB-DNJ 2 partially rescues the defective
F508del-CFTR function.8,21 Since, other iminosugars have shown
similar potency.22 The hypothesis to explain the mechanism of
action of NB-DNJ 2 is related to its a-glucosidase inhibition,
which disturbs the ER quality control, more specifically the
interaction between the ER lectin calnexin and F508del-CFTR.
This perturbation might allow the restoration of F508del-CFTR
to the plasma membrane. However, whether the glucosidase
inhibition of NB-DNJ is associated with the chaperone activity
has yet to be established. A host of chaperones, enzymes and
regulatory proteins control the folding, complex assembly and
ultimately exit of secretory proteins, which can be viewed as the
clients of this ER machinery and as potential corrector targets.23
As part of our ongoing program devoted to the development
of new lipophilic DNJ analogues as glucosylceramide synthase
inhibitors and F508del-CFTR correctors, we embarked on a click
strategy starting from 3 as a lead compound to design more potent
structures. Possible modifications of the structure of 3 can be as
follows: (a) tuning of the length and nature of the spacer, (b)
alteration of the hydrophobicity of the adamantyl-linker moiety,
(c) modification of stereochemistry and substitution pattern of
the iminosugar moiety, (d) variation of the attachment site of
the adamantyl-spacer on the iminosugar core. As modifications
(c) and (d) have already been explored and optimised,18,24,25 we
focused on the modification of the nature and the length of
the tether. One efficient way to access a library of AMP-DNJ
derivatives is to exploit the popular click azide–alkyne coupling
methodology that has been used to connect a vast array of natural
and unnatural patterns. Surprisingly, such an approach has only
recently been exploited in the field of iminosugars.26 This should
allow the construction of AMP-DNJ analogues in which the
key DNJ scaffold and the adamantane moiety are connected
through a triazole core bearing alkyl chains of various length
such as in analogues A and B (Fig. 2). The work reported herein
describes the synthesis and biological evaluation of a small library
of AMP-DNJ analogues in which the length of the tether and the
position and orientation of the triazole moiety have been studied.
Click27 coupling conditions have also been optimised to furnish
the iminosugar glycoconjugates in high yield.
Synthesis
We first investigated the direct N-alkylation of unprotected
DNJ
1
synthesized using well established methodology.28
5374 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

Fig. 2 General structure of the library of AMP-DNJ analogues of this study.
Disappointingly, N-alkylation through reductive amination with
performed in a screw-cap sealed tube allowed optimum conversion
as undesired reoxidation of copper(I) to copper(II) was otherwise
probably taking place during the reaction. These conditions were
applied to the efficient 1,3-cycloaddition of all complementary
adamantane and DNJ partners (Scheme 1).
All the triazole glycoconjugates were obtained in good (86%)
to excellent (100%) yields independently of the chain length of
the coupling partner. The DNJ-adamantane conjugates 12a–
d and 13a–c were then deprotected by hydrogenolysis (Pd/C,
EtOH, 1 M HCl) to afford the target glycoconjugates 14–20 in
almost quantitative yield (Fig. 3). In summary, the whole coupling
process (N-alkylation, 1,3-cycloaddition, deprotection) starting
from protected DNJ 4 and modified adamantanes 10 and 11 is
very efficient (yields ranging between 51% and 82% for the three
steps) and validates the sequence, which could be applied to other
key iminosugars that have demonstrated therapeutic potential.
functionalised aldehydes or via nucleophilic displacement of alkyl
tosylates or bromides29 afforded the desired N-alkyl DNJ deriva-
tives only in modest yields, especially for those with longer chains.30
In contrast, N-alkylation of tetra-O-benzyl-deoxynojirimycin 4,
synthesized according to Overkleeft,18 with alkyl moieties bearing
an alkyne (propargyl bromide 6a and longer chains 6b–c) or an
azido group (derivatives 5a–d) was successful but required a fine
tuning of the reaction parameters including temperature, solvent
(acetonitrile, DMF) and base (NaH, Na2CO3, K2CO3) (see the
ESI†). Reaction with alkyl tosylates in refluxing acetonitrile with
potassium carbonate as base proved to be the best conditions
to perform the N-alkylation and yielded the click precursors
7a–d and 8a–c. The copper-catalyzed azide–alkyne cycloaddition
(CuAAC)31 between these N-alkylated DNJ derivatives 7a–d and
8a–c and the adamantane partners 1032 and 11,33 obtained, respec-
tively, through alkylation of alcohol 9 with propargyl bromide or
tosylation of alcohol 9, followed by displacement with azide, was
then examined. Optimised click reaction conditions that were pre-
viously established in our laboratory regarding temperature (room
temperature), substrate concentration (0.25 M), the amounts of
copper sulfate (5%) and sodium ascorbate (15 mol%)34 were used
in a dichloromethane/water (1 : 1) solvent system. This mixture
proved better than the extensively used aqueous alcohol systems
(see the ESI†).35 Noteworthily, we observed that the reaction
Biological evaluation
The small library of DNJ-adamantane conjugates was submitted
to a series of biological evaluations, which include glycoenzyme
inhibition profiling, activity on the cellular enzymes relevant to the
treatment of Gaucher disease, as well as correction of defective
F508del-CFTR function. These experiments are summarised
below.
Scheme 1 Synthesis of DNJ-adamantane conjugates.
The Royal Society of Chemistry 2011 Org. Biomol. Chem., 2011, 9, 5373–5388 | 5375
This journal is
©

Fig. 3 Structure of the target adamantane-DNJ conjugates 14–20 synthesized (yields are indicated in brackets; left: debenzylation yield; right: overall
yield, starting from tetra-O-benzyl-deoxynojirimycin 4).
Glycoenzymes inhibition profiling
derivatives showed inactivation of coffee bean a-galactosidase,
but their behaviour towards b-galactosidases showed once again
distinct trends depending on the specific enzyme: for bovine liver
b-galactosidase, increased length of the tether led to increased
inhibition, whereas a more complex picture could be observed
for rat intestinal lactase. Finally, no inhibition of jack beans a-
mannosidase or snail b-mannosidase was observed.
Compounds 14–20 were assayed as inhibitors of a collection of
glycosidases, including glucosidases, galactosidases and mannosi-
dases, in comparison with DNJ and miglitol (Table 1). They all
display good inhibition towards a-glucosidases (from rice, rat
intestinal maltase, rat intestinal sucrase), with a trend correlating
higher inhibition with increased chain length. It is, however,
interesting to note that the shorter compounds 14 and, to a lesser
extent, 15 proved to be relatively poor inhibitors compared to
DNJ and miglitol. The inhibition profile is notably different with
respect to b-glucosidases. Whereas only compounds 17–20 inhibit
almond b-glucosidase, with potencies similar to DNJ, compound
15 is the only derivative in our library showing higher activity
than miglitol towards rat intestinal cellobiase. None of these
Cellular glycoenzymes inhibition
The compounds were used at the highest non-cytotoxic con-
centration, as determined by MTS36 cell proliferation assay, for
ER-a-glucosidase inhibition, using a free oligosaccharide (FOS)
analysis, and glycosphingolipid biosynthesis (GSL) inhibition,
following a 3 day incubation with compound in HL60 cells.
Table 1 Concentration of iminosugars 14–20 resulting in 50% inhibition of various glucosidases, in comparison with DNJ and miglitol
IC50/mM
Enzyme
14
15
16
17
18
19
20
DNJ
miglitol
a-glucosidase
rice
rat intestinal
maltase
0.32
4.5
0.24
6.2
0.084
0.59
0.15
1.8
0.047
0.29
0.030
0.16
0.025
0.37
0.05
0.36
0.099
0.59
rat intestinal
sucrase
13
7.3
1.2
2.6
0.91
0.87
3.4
16
1.0
b-glucosidase
almond
NIa
NI
NI
70
NI
1000
128
549
62
152
73
541
118
NI
85
327
378
164
rat intestinal
cellobiase
a-galactosidase
coffee beans
b-galactosidase
bovine liver
rat intestinal
lactase
NI
NI
NI
NI
NI
NI
NI
890
NI
163
NI
7.4
14
110
158
34
97
32
56
7.1
222
2.1
NI
NI
49
183
41
a-mannosidase
jack beans
b-mannosidase
snail
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
a NI: No inhibition (less than 50% inhibition at 1000 mM).
5376 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

ER-a-glucosidase inhibition
inhibition by all glycoconjugates of this series except 14. At the
lower concentration of 5 mM, compounds 19 and 20 proved to be
the most potent inhibitors of a-glucosidase II (Table 2).
Glucosidase I inhibition by NB-DNJ is a major concern regarding
long term treatments as this cellular glycosidase is involved in
the maturation of the glycan moiety of glycoproteins. Therefore
compounds that can selectively restore F508del-CFTR activity
without inhibiting a-1,2 glucosidase are highly needed.37
Ceramide glucosyltransferase inhibition
To evaluate the cellular inhibition of ceramide glucosyltransferase,
a key enzyme in the biosynthesis of glycosphingolipids,16 the com-
pounds were administered at non-toxic concentrations to HL60
cells for 3 days (see the ESI for the determination of cytotoxic
concentrations over 3 day incubation times†). Following lipid
extraction, enzymatic release of the oligosaccharide head group
and fluorescence labelling, normal phase HPLC (NP-HPLC) was
used to analyse the effects of inhibition on biosynthesis. HL60 cells
have a simple repertoire of glycolipids and the dominant species
is a mono-sialylated ganglioside, GM3.40 Inhibition of ceramide
glucosyltransferase by imino sugars results in the decrease in GM3
which was measured following HPLC separation (see the ESI,
Fig. S2†). The amount of GM3 reduction as result of inhibition
is shown in Fig. 4. At 5 mM, 19 and 20 were more inhibitory
than AMP-DNJ, a potent inhibitor developed by Overkleeft and
co-workers,19 and similar to 100 mM concentrations of NB-DNJ.
These compounds are therefore approximately 20 times more
potent in cells than NB-DNJ.
Compounds 14–18 were non-toxic at 100 mM over a 24 h
incubation time. Compounds 19 (CC50 = 150 mM) and 20 (CC50
=
50 mM) were evaluated at their highest non-toxic concentrations,
50 and 5 mM, respectively.
FOS analysis in HL60 cells was used to evaluate levels of a-
glucosidases I and II inhibition as determined by the amount
of Glc3Man5GlcNAc1 and Glc1Man4GlcNAc1 oligosaccharide,
respectively, produced in the cytosol as a result of endoplasmic
reticulum-associated protein degradation (ERAD). These FOS
species are the major tri- and mono-glucosylated FOS species
produced in response to a-glucosidase I and II inhibition in the
endoplasmic reticulum (ER), as a result of retrotranslocation via
an ERAD pathway and the actions of PNGase and cytosolic a-
mannosidase.38 The FOS species Glc3Man5GlcNAc1 eluted from
the TSKgel amide-80 column at approximately 36.5 min with a
glucose unit (GU) value of 8.26, consistent with our previously
published data (see the ESI, Fig. S1A†).38,39 The structure of all
FOS species was confirmed by a-glucosidase digestion and mass
spectrometry as reported38 (results not shown).
In control untreated HL60 cells no Glc3Man5GlcNAc1 was
produced, and using 100 mM NB-DNJ treatment for 24 h
significant tri-glucosylated FOS species was observed (Table 2).
At 5 mM concentration AMP-DNJ was totally ineffective at
inhibiting a-glucosidase I, whereas at 100 mM 18 provided the
most inhibition (487 21 pmol mg-1, Table 2). Whilst 19 and 20
showed effective a-glucosidase I inhibition, lower concentrations
(50 and 5 mM, respectively) had to be used due to cytotoxicity at
100 mM. To determine the relative inhibition of ER-glucosidases
at the same concentration of compound, FOS analysis was
repeated at a compound concentration of 5 mM and the change
in the FOS profile evaluated as the result of a-glucosidase II
inhibition and the appearance of Glc1Man4GlcNAc1 (eluting
at approximately 27.2 min, GU value 5.31, see the ESI, Fig.
S1B†). At lower concentrations of inhibitor, a-glucosidase II
activity is preferentially affected leading to the appearance of
Glc1Man4GlcNAc1 oligosaccharide.38,39 NB-DNJ showed very
little inhibition of a-glucosidase II at 5 mM when compared to
Fig. 4 Reduction in GM3 synthesis in HL60 cells following inhibitor
treatment. Compounds were incubated at the highest non-cytotoxic
concentration for 3 days with cells and the GSL-oligosaccharides separated
by NP-HPLC following enzyme release and labelling, as described in the
text.
Table 2 FOS concentrations in HL60 cells following treatment with inhibitor. Cells were treated with compounds at the concentrations shown for 24 h,
FOS extracted, fluorescently labelled and separated by NP-HPLC. Peak areas corresponding to the oligosaccharide species shown (see the ESI, Fig. S1†),
were measured and normalised to cell protein concentration. Experiments were performed in triplicate and standard deviations are shown
Inhibitor concentration
[Glc3Man5GlcNAc1]/pmol mg-1
Inhibitor concentration
[Glc1Man4GlcNAc1]/pmol mg-1
0.2% DMSO
5 mM AMP-DNJ
100 mM NB-DNJ
100 mM 14
0.0
0.0
172
24
38
105
111
487 21
190 13
16
0.2% DMSO
5 mM AMP-DNJ
5 mM NB-DNJ
5 mM 14
0.0
17.5
3.0 0.5
0.0
5.8
133
172 28
358
578 13
476 46
4
6
2
4
100 mM 15
5 mM 15
1
100 mM 16
5
8
5 mM 16
8
100 mM 17
5 mM 17
100 mM 18
5 mM 18
4
50 mM 19
5 mM 19
5 mM 20
2
5 mM 20
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5377
©

Table 3 Concentration of iminosugars 14–20 resulting in 50% inhibition
of b-glucocerebrosidase activity, in comparison with NB-DNJ and AMP-
DNJ
a strong increase of the recorded fluorescence signal after Fsk +
Gst stimulation (Fig. 5A) was observed. The signal was then fully
inhibited by the CFTR inhibitor CFTRinh-172 (see the ESI, Fig.
S4†). Fig. 5B summarizes the result obtained by iodide efflux assay,
after stimulation by Fsk + Gst on untreated or treated CF-KM4
cells. These results also showed no effect of triazoles 15–20, while
14 was active. No apparent CF-KM4 cell toxicity was observed
for triazole 14 up to 1 mM as measured by the MTT test.21
Compound
IC50/mM
AMP-DNJ
NB-DNJ
5.06 0.84
259.00 22
2.11 0.48
1.12 0.15
0.60 0.11
0.30 0.04
0.46 0.02
0.47 0.10
0.81 0.28
14
15
16
17
18
19
20
Discussion
Regarding glycosidase inhibition, the small library of
deoxynojirimycin-adamantane neoglycoconjugates exhibit potent
a-glucosidase inhibition, most of them being more potent than
DNJ or miglitol, with the exception of the shortest conjugates 14
and 15. Although the general trend observed is consistent with
the inhibition profile of DNJ and miglitol, the results clearly show
that our synthetic approach allows efficient modulation of the
inhibition profile of our inhibitors. This is easily exemplified by
the data obtained for the two structurally extreme members of our
library, the short derivative 14 and the extended compound 20.
This library also contains more efficient ER a-glucosidase
inhibitors than NB-DNJ. Since the key oligosaccharides resulting
from ER a-glucosidase inhibition are observed with all our
iminosugars we can assume an intracellular and ER localisation
of these compounds, but the extent may vary with structure.
The length of the aliphatic linker connecting the imino sugar
nitrogen and the triazole has a major influence on cellular potency
(20, 19 > 18 > 16, 17 > 15 > 14). These data confirm our
previous findings regarding the importance of alkyl chain length
between the iminosugar and a hydrophobic group,39 and that
potency reflects enzyme affinity rather than increased cellular
uptake.30c Insertion of an ether function between the triazole and
adamantyl groups does not have a significant effect on inhibitory
activity (16 = 17). A similar analysis can be invoked to explain
the increased potency towards ceramide glucosyltransferase of
compounds bearing longer chains.42 As a consequence, 19 and 20
are superior to 18, 16 and 14.
Measurement of the GM3 (see the ESI, Fig. S2†) peak area was
used to determine inhibition of GSL biosynthesis. Experiments
were conducted in triplicate and the error bars show standard
deviations.
b-Glucocerebrosidase inhibition
All compounds showed improved inhibitory potency for hu-
man placental b-glucocerebrosidase compared to NB-DNJ, as
determined by a fluorogenic assay using 4-methylumbelliferyl-
b-glucoside (Table 3). In general, extending the N-alkyl linker
chain length provided increased efficacy, where similar inhibition
constants were observed for 16, 17, 18, 19 and 20. These
data contrast with a-glucosidase inhibition where inhibition was
maximal in 19 treated cells but correlates with cellular ceramide
glucosyltransferase inhibition, showing improvement in inhibition
with N-alkyl chain length extension.
Cystic fibrosis assay
The potential corrector effect of triazoles 14–20 on CFTR function
in CF-KM4 cells41 was assessed using single-cell fluorescence
imaging. The cells were treated for 2 h with 100 mM of triazoles
14–20 and then CFTR proteins were stimulated by a cocktail of
forskolin (Fsk) + genistein (Gst). As expected, no variation of
the fluorescence was observed after the cocktail stimulation in
untreated CF-KM4 bearing the defective F508del-CFTR protein.
However, following a treatment of CF-KM4 cells with triazole 14,
Ultimately, given the higher ER-glucosidase and ceramide
glucosyltransferase inhibitions obtained here over previously
published iminosugar conjugates, our approach provides a useful
Fig. 5 (A) Functional evaluation of F508del-CFTR by DiSBAC2(3) assay in CFKM4 cells treated with 100 mM of compounds during 2 h. Histograms
report the mean of the relative fluorescence collected from separate experiments (N = 3) with a total of 36 cells. (B) F508del-CFTR activity was assayed
with the iodide efflux technique in the presence of forskolin (10 mM) + genistein (30 mM). Histograms summarizing the rescue of CFTR activity observed
on CF-KM4 cells. n = 8 for each bar. Statistical significance is *P < 0.05; **P < 0.01; ***P < 0.001; ns, non significant.
5378 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

platform for the design and synthesis of ever more potent
therapeutics for the treatment of lysosomal storage disorders.43,44
Furthermore, the library of DNJ derivatives shows high b-
glucocerebrosidase inactivation, paving the way to their potential
use as molecular chaperones in order to restore the cellular activity
of this enzyme. Interestingly, derivatives of DNJ bearing a terminal
adamantyl group have previously been described as very active
compounds in this field.45
strategy to access new iminosugar mimics with high potential as
therapeutic agents, the triazole core being well tolerated by all
the enzymes assayed in this work. This strategy allows simple and
rapid library generation,50 and has been shown to enable efficient
modulation of the biological activity of the conjugates.
Experimental
Concerning CF, miglustat 27 has been recently shown to be
a corrector of the abnormal trafficking of the mutated protein,
F508del-CFTR.46 Pre-clinical data were accumulated on various
CF models on the basis of the a-glucosidase inhibitor effect of
miglustat,8,46–48 which is now evaluated in phase II trials for the
treatment of cystic fibrosis.8 The identification of 14, which is
not an a-glucosidase inhibitor, as a corrector of F508del-CFTR
activity is thus very promising and suggests a different mecha-
nism of action compared to 2. Interestingly, other iminosugar
derivatives have recently been evaluated on CF models22,49 and
isoLAB,22 a trihydroxylated pyrrolidine, has also been found to be
a CFTR corrector while not inhibiting ER a-glucosidase. Thus,
the effects of iminosugars on CFTR involved in cystic fibrosis
are probably more complicated than previously anticipated and
certainly deserve further studies based on at least two hypotheses,
one depending on a-glucosidase inhibition in the case of miglustat,
and the second one being independent of glucosidase in the case
of isoLAB and 14.
General
NMR spectra (1H and 13C) were recorded on Bruker DRX-250,
AM-250, DRX-300, AV-360 or DRX-400 instruments. Chemical
shifts were reported in the d scale relative to CHCl3 as an internal
reference (7.26 ppm for 1H and 77.0 ppm for 13C). The following
abbreviations have been used to describe the signal multiplicity: br
(broad), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet),
h (hextet), m (multiplet), dd (doublet of doublets), dt (doublet of
triplets).
Electrospray ionisation mass spectra (MS) were measured
on a Finnigan MAT 95
S instrument. Optical rotations
were measured at 20 ◦C with a Perkin Elmer 341 polarime-
ter. Thin layer chromatography (TLC) analyses were per-
formed on commercial glass plates bearing a 0.25 mm layer
of Merck silica gel 60 F254 (Merck Art. No.5715). These
were developed using standard visualising techniques: anisalde-
hyde/heating, 2,4-dinitrophenylhydrazine/heating, potassium
permanganate/heating and UV fluorescence (254 nm). Rf values
are reported to the nearest 0.01. Mixed solvent system composi-
tions are quoted as volumetric ratios. Flash chromatography was
carried out with silica gel, Merck Type 60 (70–325 mesh ASTM)
or Merck Type 60 (230–400 mesh ASTM), adapting the method
of Still.51 Pet. ether refers to the petroleum fraction boiling in the
range 40–60 ◦C. In general, reactions were carried out in dry sol-
vents under an argon atmosphere, unless otherwise mentioned. All
other reagents were purified in accordance with the recommended
procedures,52 or used as obtained from commercial sources. NMR,
and MS data were obtained on all intermediates described herein
using chromatographically homogeneous samples.
Conclusion
A series of deoxynojirimycin-adamantane neoglycoconjugates has
been efficiently prepared by click chemistry from protected DNJ 4
and adamantane derivatives 10 and 11, allowing their connection
through triazole-based linkers of various lengths. These DNJ
derivatives showed interesting inhibition profiles towards a col-
lection of glycosidases, their selectivities being modulated by the
variation of the linker chain length. Their evaluation as correctors
of defective F508del-CFTR involved in cystic fibrosis has also
shown that DNJ-triazole-adamantane conjugate 14, displaying
the shortest spacer in the series, was able to rescue F508del-
CFTR function. Interestingly, 14 is not an ER a-glucosidase I
or a-glucosidase II inhibitor, and is a relatively poor inhibitor of
intestinal glycosidases, which makes 14 a compound to explore
further as it seems to act by a different mechanism compared
to NB-DNJ. Regarding Gaucher disease and glucosylceramide
synthase inactivation, the DNJ-triazole-adamantane conjugates
19 and 20 bearing the longest arms were approximately 20-
fold more potent than model NB-DNJ 2 and even more potent
than reference compound AMP-DNJ 3. Evaluation of the ER
a-glucosidase inhibition showed that DNJ-triazole-adamantanes
18–20 were potent a-glucosidase I inhibitors but cytotoxicity
was detected at 100 mM for the longest compounds 19 and 20.
These two derivatives also proved to be the most potent a-
glucosidase II inhibitors of this series, all AMP-DNJ triazoles
demonstrating inhibition towards this enzyme, with the important
exception of compound 14. Finally, all neoglycoconjugates proved
more potent b-glucocerebrosidase inhibitors than NB-DNJ and
AMP-DNJ, an observation relevant to their potential use as
glucocerebrosidase correctors for active-site-specific chaperone
therapy. In conclusion, click coupling is therefore a powerful
General procedure A – alkylation with bromide 6a or tosy-
lates 6b–c, 5a–d. To a solution of 2,3,4,6-tetra-O-benzyl-1-
deoxynojirimycin 4 (1.0 eq.), propargyl bromide 6a, compound
6b–c or 5a–d (1.5 eq.) in acetonitrile (0.2 M) was added potassium
carbonate (2.1 eq.). The reaction mixture was heated to 85 ◦C
(oil bath) for 12 h 30 under argon then cooled. Most of the
acetonitrile was evaporated under reduced pressure. Water and
chloroform were added to the residue and the whole mixture was
stirred for 10 min, and then partitioned. The aqueous layer was
extracted with chloroform (3¥). The combined organic extracts
were dried over sodium sulfate, filtrated and concentrated under
reduced pressure. Further purification by flash chromatography
gave the title compound.
General procedure B – 1,3-dipolar cycloaddition with 10 or 11.
To a solution of compound 7a–d or 8a–c (1.0 eq.) and 10 or
11 (1.5 eq.) in dichloromethane/water (0.25 M, 1 : 1) in a screw
cap tube was added an aqueous solution of copper(II) sulfate
(0.2 M, 0.06 eq.), followed by an aqueous solution of sodium
ascorbate (0.2 M, 0.15 eq.). The reaction mixture was purged
under argon, the cap screwed on and the solution then stirred
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5379
©

at room temperature for 18 h. An aqueous saturated solution of
sodium bicarbonate and chloroform were added. The mixture was
partitioned and the aqueous layer was extracted with chloroform
(3¥), dried over sodium sulfate and filtrated.
Rf 0.20 (Pet. Ether/EtOAc 7 : 3). 1H NMR (360 MHz, CDCl3) d
1.40–1.48 (m, 6H, 3¥CH2 Ad), 1.52–1.73 (m, 6H, 3¥CH2 Ad), 1.93–
2.01 (m, 3H, 3¥CHAd), 2.16 (dd, 1H, J1ax,1eq10.9, J1eq,210.9 Hz,
H-1eq), 2.26 (ddd, 1H, J4,59.5, J5,6a2.8, J5,6b1.8 Hz, H-5), 3.14 (dd,
1H, J1ax,1eq10.9, J1ax,24.9 Hz, H-1ax), 3.36 (dd, 1H, J2,39.1, J3,49.1
Hz, H-3), 3.54 (dd, 1H, J4,59.5, J3,49.1 Hz, H-4), 3.65 (ddd, 1H,
J1eq,210.9, J2,39.1, J1ax,24.9 Hz, H-2), 3.74 (dd, 1H, J6a,6b10.7, J5,6a2.8
Hz, H-6a), 3.91 (s, 2H, CH2Ad), 3.95 (d, 1H, J1¢a,1¢b 15.2 Hz, H-
1¢a), 3.97 (dd, 1H, J6a,6b10.7, J5,6b1.8 Hz, H-6b), 4.15 (d, 1H, J1¢a,1¢b
15.2 Hz, H-1¢b), 4.37 (d, 1H, J 10.9 Hz, CHH Bn), 4.46 (d, 1H, J
11.9 Hz, CHH Bn), 4.54 (d, 1H, J 11.9 Hz, CHH Bn), 4.62 (s, 2H,
OCH2Ph), 4.75 (d, 1H, J 11.1 Hz, CHH Bn), 4.84 (d, 1H, J 10.9 Hz,
CHH Bn), 4.90 (d, 1H, J 11.1 Hz, CHH Bn), 7.06–7.36 (m, 21H,
CH aromatic+CH triazole). 13C (90 MHz, CDCl3) d 28.1 (CHAd),
34.2 (CAd), 36.6 (CH2 Ad), 40.3 (CH2 Ad), 47.4 (C-1¢), 54.5 (C-1),
62.1 (CH2Ad), 62.9 (C-5), 66.5 (C-6), 72.6 (CH2 benzyl), 73.5
(CH2 benzyl), 75.2 (CH2 benzyl), 75.3 (CH2 benzyl), 78.5 (C-2),
78.6 (C-4), 87.1 (C-3), 124.3 (CH triazole), 127.5 (CH aromatic),
127.6 (CH aromatic), 127.8 (CH aromatic), 127.8 (CH aromatic),
127.9 (CH aromatic), 128.3 (CH aromatic), 128.3 (CH aromatic),
128.4 (CH aromatic), 128.4 (CH aromatic), 137.9 (C aromatic),
138.5 (C aromatic), 138.5 (C aromatic), 139.0 (C aromatic), 141.6
(C triazole). LRMS (ESI+) 753.4 [M+H]+, 60%, 775.4 [M+Na]+,
100%. HRMS Found 775.4199 [M+Na]+. C48H56N4O4Na requires
775.4194.
General procedure C – Pd/C catalyzed hydrogenolysis of com-
pounds 12a–b and 13a–c. A solution of compound 12a–d or 13a–
c (1.0 eq.) in ethanol (0.03 M) was acidified to pH ~2 with 1 M aq.
HCl. Argon was passed through the solution for 5 min, after which
palladium 10% wt on carbon (0.6–0.9 eq.) was added. Hydrogen
was passed through the reaction mixture for 5 min and the reaction
was stirred for 20 h under atmospheric hydrogen pressure. Pd/C
was removed by filtration over a microfilter syringe, followed by
thorough rinsing with CH3OH. The filtrate was concentrated with
co-evaporation of toluene. Further purification with either a Strata
C18-E column cartridge (H2O/ACN) or by flash chromatography
(CHCl3/CH3OH) gave the desired compound.
2,3,4,6-Tetra-O-benzyl-N-(3-prop-1-ynyl)-1-deoxynojirimycin
8a. General procedure A was applied to 2,3,4,6-tetra-O-benzyl-
1-deoxynojirimycin 4 (860 mg, 1.64 mmol) and 6a. The residue
was treated with water (2 mL) and extracted with EtOAc (3 ¥
3 mL), dried over MgSO4, filtered and concentrated. Further
purification by flash chromatography (silica/sample ratio 50 : 1;
Pet. Ether/EtOAc 9 : 1 to 4 : 6) gave 2,3,4,6-tetra-O-benzyl-N-(2-
propynyl)-1-deoxynojirimycin 8a (845 mg, 1.50 mmol, 92% yield)
as a thick oil.
N -[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]methyl-1-
deoxynojirimycin 14. Hydrogenolysis of N-[1-(adamant-1-yl-
methyl)-1H-1,2,3-triazol-4-yl]methyl-2,3,4,6-tetra-O-benzyl-1-
deoxynojirimycin 13a (258 mg, 343 mmol) following general
procedure C gave after purification by flash silica gel column
chromatography (CHCl3/CH3OH, 8 : 2) gave N-[1-(adamant-
1-yl-methyl)-1H-1,2,3-triazol-4-yl]methyl-1-deoxynojirimycin 14
(132 mg, 336 mmol, 98%) as a white solid.
Rf 0.24 (Pet. Ether/EtOAc 85 : 15). 1H NMR (360 MHz, CDCl3)
d 2.19 (t, 1H, J 2.2 Hz, H-3¢), 2.43 (ddd, 1H, J4,59.2, J5,6a2.2,
J5,6b1.8 Hz, H-5), 2.52 (dd, 1H, J1ax,1eq11.0, J1eq,210.6 Hz, H-1eq),
2.95 (dd, 1H, J1ax,1eq11.0, J1ax,24.8 Hz, H-1ax), 3.37 (dd, 1H, J1¢a,1¢b
18.0, J1a¢,3¢ 2.2 Hz, H-1¢a), 3.47 (dd, 1H, J2,39.2, J3,49.2 Hz, H-3),
3.57 (dd, 1H, J6a,610.5,J5,6a2.2 Hz, H-6a), 3.62 (dd, 1H, J3,49.2,
J4,59.2 Hz, H-4), 3.70 (ddd, 1H, J1eq,210.6, J2,39.2 Hz, J1eq,24.8 Hz,
H-2), 3.72 (dd, 1H, J1¢a,1¢b 18.0, J1¢b,3¢2.2 Hz, H-1¢b), 3.73 (dd, 1H,
J6a,6b10.5, J5,6b1.8 Hz, H-6b), 4.34 (d, 1H, J 11.0 Hz, CHH Bn),
4.42 (d, 1H, J 12.3 Hz, CHH Bn), 4.53 (d, 1H, J 12.3 Hz, CHH
Bn), 4.64 (d, 1H, J 12.3 Hz, CHH Bn), 4.68 (d, 1H, J 12.3 Hz,
CHH Bn), 4.81 (d, 1H, J 11.0 Hz, CHH Bn), 4.86 (d, 1H, J
11.0 Hz, CHH Bn), 4.96 (d, 1H, J 11.0 Hz, CHH Bn), 7.05–
7.39 (m, 20H, CH aromatic). 13C (90 MHz, CDCl3) d 42.3 (C-1¢),
55.0 (C-1), 62.1 (C-5), 64.8 (C-6), 72.7 (CH2 benzyl), 73.6 (CH2
benzyl), 74.1 (C-3¢), 75.2 (CH2 benzyl),75.4 (CH2 benzyl), 78.2
(C-2), 78.2 (C-4), 84.2 (C-2¢), 87.1 (C-3), 127.5 (CH aromatic),
127.5 (CH aromatic), 127.6 (CH aromatic), 127.8 (CH aromatic),
127.9 (CH aromatic), 128.3 (CH aromatic), 128.4 (CH aromatic),
128.4 (CH aromatic), 137.6 (C aromatic), 138.4 (C aromatic), 138.5
(C aromatic), 138.8 (C aromatic). LRMS (ESI+) 562.3 [M+H]+,
100%, 584.3 [M+Na]+, 40%. HRMS: Found 562.2951[M+H]+.
C37H40NO4 requires 562.2957.
Rf 0.22 (CHCl3/CH3OH 80 : 20). 1H NMR (300 MHz, CD3OD)
d 1.46–1.59 (m, 6H, 3¥CH2 Ad), 1.59–1.81 (m, 6H, 3¥CH2 Ad), 1.90–
2.07 (m, 3H, 3¥CHAd), 2.47–2.80 (m, 2H, H-1eq), 3.13–3.28 (m,
3H, H1ax, H-3, H-5), 3.35 (s, 2H, H-1¢), 3.49–3.77 (m, 2H, H-2,
H-4), 4.05–4.30 (m, 2H, H-6a, H-6b), 4.12 (s, 2H, CH2Ad), 4.38
(bs, OH), 8.04 (s, 1H, CH triazole). 13C (90 MHz, CD3OD) d 29.8
(CHAd), 35.4 (CAd), 37.8 (CH2 Ad), 41.4 (CH2 Ad), 48.4, 49.4, 56.0 (C-
1), 57.3, 63.2 (CH2Ad), 67.0, 70.4, 79.0 (C-3), 128.9 (CH triazole),
139.5 (C triazole). HRMS Found 393.25051 [M+H]+. C20H33N4O4
requires 393.2496. (d 0.9 ppm). LRMS (ESI+) 393.3 [M+H]+, 93%,
415.3 [M+Na]+, 100%. Found 415.2314 [M+Na]+. C20H32N4O4Na
requires 415.2321.
2,3,4,6-Tetra-O-benzyl-N-(4-but-1-ynyl)-1-deoxynojirimycin 8b.
Applying general procedure
A to 2,3,4,6-tetra-O-benzyl-1-
deoxynojirimycin 4 (846 mg, 1.62 mmol) and 6b gave after
flash chromatography on silica gel (Pet. Ether/EtOAc 9 : 1 to
4 : 6) 2,3,4,6-tetra-O-benzyl-N-(3-butynyl)-1-deoxynojirimycin 8b
(659 mg, 1.15 mmol, 71%) as a white solid.
N-[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]methyl-2,3,
4,6-tetra-O-benzyl-1-deoxynojirimycin
13a. 2,3,4,6-Tetra-
Rf 0.24 (Pet. Ether/EtOAc 85 : 15). 1H NMR (360 MHz, CDCl3)
d 1.94 (t, J 2.6 Hz, 1H, H alkyne), 2.19 (m, 2H, H-2¢), 2.33 (dd,
1H, J1ax,1eq11.0, J1eq,210.5 Hz, H-1eq), 2.39 (ddd, 1H, J4,59.6, J5,6a2.6,
J5,6b2.2 Hz, H-5), 2.88–3.02 (m, 2H, H-1¢), 3.06 (dd, 1H, J1ax,1eq11.0,
J1ax,24.8 Hz, H-1ax), 3.43 (dd, 1H, J2,39.2, J3,49.2 Hz, H-3), 3.52 (dd,
1H, J3,49.2, J4,59.2 Hz, H-4), 3.60 (dd, 1H, J6a,610.5,J5,6a2.2 Hz, H-
6a), 3.62 (ddd, 1H, J1eq,210.5, J2,39.2 Hz, J1eq,24.8 Hz, H-2), 3.67 (dd,
O-benzyl-N-(2-propynyl)-1-deoxynojirimycin 8a (316 mg,
0.562 mmol) was used in general procedure B. Further purification
of the residue by flash silica gel column chromatography (Pet.
Ether/EtOAc 8 : 2 to 4 : 6) gave N-[1-(adamant-1-yl-methyl)-
1H-1,2,3-triazol-4-yl]methyl-2,3,4,6-tetra-O-benzyl-1-deoxynoji-
rimycin 13a (383 mg, 0.509 mmol, 91% yield).
5380 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

1H, J6a,6b10.5, J5,6b2.6 Hz, H-6b), 4.36 (d, 1H, J 11.0 Hz, CHH Bn),
4.44 (d, 1H, J 12.2 Hz, CHH Bn), 4.51 (d, 1H, J 12.2 Hz, CHH
Bn), 4.63 (d, 1H, J 11.8 Hz, CHH Bn), 4.67 (d, 1H, J 11.8 Hz, CHH
Bn), 4.79 (d, 1H, J 11.0 Hz, CHH Bn), 4.85 (d, 1H, J 11.0 Hz,
CHH Bn), 4.94 (d, 1H, J 11.0 Hz, CHH Bn), 7.05–7.36 (m, 20H,
CH aromatic). 13C (90 MHz, CDCl3) d 14.0 (C-2¢), 51.1 (C-1¢),
54.4 (C-1), 63.0 (C-5), 65.8 (C-6), 69.4 (C-4¢), 72.7 (CH2 benzyl),
73.4 (CH2 benzyl), 75.1 (CH2 benzyl),75.3 (CH2 benzyl), 78.3 (C-
2), 78.4 (C-4), 82.6 (C-3¢), 87.1 (C-3), 127.4 (CH aromatic), 127.5
(CH aromatic), 127.6 (CH aromatic), 127.8 (CH aromatic), 128.3
(CH aromatic), 128.4 (CH aromatic), 137.7 (C aromatic), 138.4 (C
aromatic), 138.9 (C aromatic). LRMS (ESI+) 576.3 [M+H]+, 100%.
HRMS Found 576.3118 [M+H]+. C38H42NO4 requires 576.3114 (d
0.7 ppm). [a]2D0 8.5 (c 1.2, CH2Cl2)
3.81 (m, 2H, H-2, H-1¢b), 3.98 (dd, 1H, J6a,6b12.3, J5,6a2.7 Hz, H-
6a), 4.07 (s, 2H, CH2Ad), 4.13 (d, 1H, J6a,6b12.3 Hz, H-6b), 7.85
(s, 1H, CH triazole). 13C (90 MHz, CD3OD) d 20.9 (C-2¢), 29.7
(CHAd), 35.2 (CAd), 37.7 (CH2 Ad), 41.3 (CH2 Ad), 54.9 (C-1, C-6),
63.1 (CH2Ad), 67.7 (C-5), 67.8 (C-1¢, C-2), 68.9 (C-4), 77.9 (C-
3), 125.8 (CH triazole), 143.1 (C triazole). LRMS (ESI+) 407.3
[M+H]+, 100%, 429.3 [M+Na]+, 50%. HRMS Found 407.2651
[M+H]+. C21H35N4O4 requires 407.2658 (d -0.7 ppm)
2,3,4,6-Tetra-O-benzyl-N -(6-hex-1-ynyl)-1-deoxynojirimycin
8c. General procedure A, using 2,3,4,6-tetra-O-benzyl-1-
deoxynojirimycin 4 (728 mg, 1.39 mmol) and 6c, gave after flash
chromatography (silica/sample ratio 50 : 1; Pet. Ether/EtOAc 9 : 1
to 1 : 1) 2,3,4,6-tetra-O-benzyl-N-(5-hexynyl)-1-deoxynojirimycin
8c (621 mg, 1.03 mmol, 74%) as a white solid.
N-[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]ethyl-2,3,4,
Rf 0.18 (Pet. Ether/EtOAc 85 : 15). 1H NMR (360 MHz, CDCl3)
d 1.35–1.61 (m, 4H, H-2¢, H-3¢), 1.93 (t, 1H, J 2.6 Hz, H-6¢), 2.14
(dt, 2H, H-4¢), 2.22 (dd, 1H, J1ax,1eq10.9, J1eq,210.4 Hz, H-1eq), 2.29–
2.35 (m, 1H, H-5), 2.50–2.62 (m, 1H, H-1¢a), 2.62–2.76 (m, 1H,
H-1¢b), 3.09 (dd, 1H, J1ax,1eq10.9, J1ax,25.0 Hz, H-1ax), 3.46 (dd, 1H,
J2,39.1, J3,48.6 Hz, H-3), 3.52–3.61 (m, 1H, H-6a), 3.58 (dd, 1H,
J3,49.1, J4,59.1 Hz, H-4), 3.61–3.70 (m, 1H, H-6b), 3.65 (ddd, 1H,
J1eq,210.5, J2,39.1 Hz, J1eq,25.0 Hz, H-2), 4.43 (d, 1H, J 10.9 Hz,
CHH Bn), 4.45 (d, 1H, J 12.3 Hz, CHH Bn), 4.49 (d, 1H, J 12.3
Hz, CHH Bn), 4.65 (d, 1H, J 11.8 Hz, CHH Bn), 4.70 (d, 1H,
J 11.8 Hz, CHH Bn), 4.81 (d, 1H, J 10.9 Hz, CHH Bn), 4.88
(d, 1H, J 10.9 Hz, CHH Bn), 4.95 (d, 1H, J 10.9 Hz, CHH Bn),
7.11–7.40 (m, 20H, CH aromatic). 13C (90 MHz, CDCl3) d 18.2
(C-4¢), 22.9, 26.3 (C-2¢, C-3¢), 51.7 (C-1¢), 54.4 (C-1), 63.9 (C-5),
65.6 (C-6), 68.6 (C-6¢), 72.8 (CH2 benzyl), 73.4 (CH2 benzyl), 75.2
(CH2 benzyl),75.3 (CH2 benzyl), 78.6 (C-2), 78.7 (C-4), 84.2 (C-
5¢), 87.4 (C-3), 127.4 (CH aromatic), 127.5 (CH aromatic), 127.6
(CH aromatic), 127.8 (CH aromatic), 128.3 (CH aromatic), 128.4
(CH aromatic), 137.8 (C aromatic), 138.6 (C aromatic), 139.0 (C
aromatic). LRMS (ESI+) 604.4 [M+H]+, 100%. HRMS Found
604.3425 [M+H]+. C40H46NO4 requires 604.3427.[a]2D0 17.2 (c 1.2,
CH2Cl2).
6-tetra-O-benzyl-1-deoxynojirimycin
O-benzyl-N-(3-butynyl)-1-deoxynojirimycin 8b
13b. 2,3,4,6-Tetra-
(302 mg,
0.524 mmol) was subjected to general procedure B. Further
purification by flash silica gel column chromatography (Pet.
Ether/EtOAc 9 : 1 to 6 : 4) gave N-[1-(adamant-1-yl-methyl)-1H-
1,2,3-triazol-4-yl]ethyl-2,3,4,6-tetra-O-benzyl-1-deoxynojiri-
mycin 13b (402 mg, 0.524 mmol, 100%).
Rf 0.10 (Pet. Ether/EtOAc 85 : 15). 1H NMR (400 MHz, CDCl3)
d 1.38–1.48 (m, 6H, 3¥CH2 Ad), 1.51–1.73 (m, 6H, 3¥CH2 Ad), 1.93–
2.01 (m, 3H, 3¥CHAd), 2.29 (dd, 1H, J1ax,1eq10.9, J1eq,210.6 Hz, H-
1eq), 2.39–2.48 (m, 1H, H-5), 2.74–2.97 (m, 3H, H-2¢, H-1¢a), 3.02–
3.13 (m, 1H, H-1¢b), 3.16 (dd, 1H, J1ax,1eq10.9, J1ax,24.5 Hz, H-1ax),
3.48 (dd, 1H, J3,49.1, J2,38.8 Hz, H-3), 3.56 (dd, 1H, J3,49.1, J4,58.6
Hz, H-4), 3.58 (ddd, 1H, J1eq,210.6, J2,38.8, J1ax,24.5 Hz, H-2), 3.66–
3.75 (bs, 2H, H-6), 3.80 (d, 1H, J 13.8 Hz, NCHHAd), 3.91 (d, 1H,
J 13.8 Hz, NCHHAd), 4.43 (d, 1H, J 10.9 Hz, CHH Bn), 4.45 (s,
2H, OCH2Ph), 4.63 (s, 2H, OCH2Ph), 4.81 (d, 1H, J 11.1 Hz, CHH
Bn), 4.88 (d, 1H, J 10.9 Hz, CHH Bn), 4.96 (d, 1H, J 11.1 Hz, CHH
Bn), 7.05 (s, 1H, CH triazole), 7.12–7.37 (m, 20H, CH aromatic).
13C (100 MHz, CDCl3) d 20.9 (C-2¢), 28.0 (CHAd), 34.0 (CAd),
36.5 (CH2 Ad), 40.2 (CH2 Ad), 51.4 (C-1¢), 54.0 (C-1), 61.9 (CH2Ad),
63.7 (C-5), 66.0 (C-6), 72.6 (CH2 benzyl), 73.4 (CH2 benzyl), 75.2
(CH2 benzyl), 75.3 (CH2 benzyl), 78.4 (C-2), 78.6 (C-4), 87.2 (C-3),
122.8 (CH triazole), 127.4 (CH aromatic), 127.5 (CH aromatic),
127.6 (CH aromatic), 127.8 (CH aromatic), 128.0 (CH aromatic),
128.3 (CH aromatic), 137.8 (C aromatic), 138.3 (C aromatic),
138.4 (C aromatic), 138.9 (C aromatic), 145.0 (C triazole). LRMS
(ESI+) 767.5 [M+H]+, 100%. HRMS Found 767.4259 [M+H]+.
C49H59N4O4 requires 767.4531. [a]2D0 4.0 (c 1.210, CH2Cl2)
N-[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]butyl-2,3,4,
6-tetra-O-benzyl-1-deoxynojirimycin
13c. 2,3,4,6-Tetra-O-
benzyl-N-(5-hexynyl)-1-deoxynojirimycin 8c (303 mg, 502 mmol)
was treated according general procedure B to afford after
purification by flash silica gel column chromatography (Pet.
Ether/EtOAc
6 : 4)
N-[1-(adamant-1-yl-methyl)-1H-1,2,3-
triazol-4-yl]butyl-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin 13c
(397 mg, 499 mmol, 99% yield) as a white solid.
N-[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]ethyl-1-deo-
xynojirimycin 15. Hydrogenolysis of N-[1-(adamant-1-yl-
methyl)-1H-1,2,3-triazol-4-yl]ethyl-2,3,4,6-tetra-O-benzyl-1-deo -
xynojirimycin 13b (314 mg, 409 mmol), following procedure C,
gave after purification by flash silica gel column chromatography
(CHCl3/CH3OH 8 : 2) gave N-[1-(adamant-1-yl-methyl)-1H-
1,2,3-triazol-4-yl]ethyl-1-deoxynojirimycin 15 (159 mg, 391 mmol,
96%) as a white solid.
Rf 0.28 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.49–1.59 (m, 6H, 3¥CH2 Ad), 1.59–1.83 (m, 6H, 3¥CH2 Ad), 1.93–
2.03 (m, 3H, 3¥CH Ad), 3.11 (dd, 1H, J1ax,1eq12.3, J1eq,211.4 Hz,
H-1eq), 3.16–3.30 (m, 3H, H-5, H-2¢a, H-2¢b), 3.42 (dd, 1H, J3,49.1,
J2,39.1 Hz, H-3), 3.52–3.68 (m, 1H, H-1¢a), 3.59 (dd, 1H, J1ax,1eq12.3,
J1ax,25.0 Hz, H-1ax), 3.65 (dd, 1H, J4,59.5, J3,49.0 Hz, H-4), 3.69–
Rf 0.16 (Pet. Ether/EtOAC 7 : 3). 1H NMR (360 MHz, CDCl3)
d 1.34–1.73 (m, 16H, 6¥CH2 Ad, H-2¢, H-3¢), 1.94–2.01 (m, 3H,
3¥CHAd), 2.20 (dd, 1H, J1ax,1eq10.9, J1eq,210.4 Hz, H-1eq), 2.24–2.32
(m, 1H, H-5), 2.55–2.78 (m, 2H, H-1¢), 2.66 (t, 2H, J 7.3 Hz, H-4¢),
3.06 (dd, 1H, J1ax,1eq10.9, J1ax,24.5 Hz, H-1ax), 3.43 (dd, 1H, J2,39.1,
J3,49.1 Hz, H-3), 3.49–3.69 (m, 4H, H-6a, H-4, H-6b, H-2), 3.94
(s, 2H, CH2Ad), 4.40 (d, 1H, J 10.9 Hz, CHH Bn), 4.45 (s, 2H,
OCH2Ph), 4.62 (d, 1H, J 11.8 Hz, CHH Bn), 4.67 (d, 1H, J 11.8
Hz, CHH Bn), 4.79 (d, 1H, J 10.9 Hz, CHH Bn), 4.85 (d, 1H,
J 10.9 Hz, CHH Bn), 4.93 (d, 1H, J 10.9 Hz, CHH Bn), 7.08 (s,
1H, CH triazole), 7.09–7.36 (m, 20H, CH aromatic). 13C (90 MHz,
CDCl3) d 23.1 (C-2¢), 25.4 (C-4¢), 27.1 (C-3¢), 28.0 (CHAd), 34.1
(CAd), 36.5 (CH2 Ad), 40.2 (CH2 Ad), 52.0 (C-1¢), 54.3 (C-1), 63.0
(CH2Ad), 63.7 (C-5), 65.4 (C-6), 72.7 (CH2 benzyl), 73.3 (CH2
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5381
©

benzyl), 75.1 (CH2 benzyl),75.2 (CH2 benzyl), 78.4 (C-2), 78.4 (C-
4), 87.2 (C-3), 122.0 (CH triazole), 127.4 (CH aromatic), 127.4
(CH aromatic), 127.6 (CH aromatic), 128.0 (CH aromatic), 128.6
(CH aromatic), 137.7 (C aromatic), 138.4 (C aromatic), 138.9 (C
aromatic), 147.0 (C triazole). LRMS (ESI+) 795.5 [M+H]+, 100%.
HRMS Found 795.4854 [M+H]+. C51H62N4O4 requires 795.4844.
[a]2D0 9.7 (c 1.150, CH2Cl2)
N -(4-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-
12a.
1-yl]}butyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin
General procedure B was applied to N-(4-azidobutyl)-2,3,4,6-
tetra-O-benzyl 1-deoxynojirimycin 7a (206 mg, 0.332 mmol) and
3-(adamant-1-yl-methoxy)-prop-1-yne 10 (99 mg, 0,48 mmol).
Further purification by flash chromatography (Pet. Ether/EtOAc
8 : 2 to 4 : 6) gave N-(4-{4-[adamant-1-yl-methoxymethyl]-
1H-1,2,3-triazol-1-yl]}butyl)-2,3,4,6-tetra-O-benzyl-1-deoxyno-
jirimycin 12a (236 mg, 0.287 mmol, 86%).
N-[1-(Adamant-1-yl-methyl)-1H-1,2,3-triazol-4-yl]butyl-1-deo-
xynojirimycin 16. Hydrogenolysis of N-[1-(adamant-1-yl-
methyl)-1H-1,2,3-triazol-4-yl]butyl-2,3,4,6-tetra-O-benzyl-1-
deoxynojirimycin 13c (278 mg, 349 mmol) using general
procedure C gave after purification by flash chromatography
(CHCl3/CH3OH 8 : 2 to 7 : 3) N-[1-(adamant-1-yl-methyl)-1H-
1,2,3-triazol-4-yl]butyl-1-deoxynojirimycin 16 (144 mg, 330
mmol, 95%) which was concentrated and taken up in water then
lyophilized to afford a pale yellow solid.
Rf 0.16 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.49–1.60 (m, 6H, 3¥CH2 Ad), 1.60–1.80 (m, 6H, 3¥CH2 Ad), 1.80–
1.95 (m, 4H, H-2¢, H-3¢), 1.96–2.07 (m, 3H, 3¥CHAd), 2.84–2.94
(m, 2H, H-4¢), 3.00 (dd, 1H, J1ax,1eq11.8, J1eq,211.4 Hz, H-1eq), 3.07
(~d, 1H, H-5), 3.22–3.41 (m, 1H, H-1¢a), 3.38 (dd, 1H, J2,310.0,
J3,49.0 Hz, H-3), 3.42–3.53 (m, 1H, H-1¢b), 3.47 (dd, 1H, J1ax,1eq11.8,
J1ax,25.0 Hz, H-1ax), 3.60 (dd, 1H, J4,510.0, J3,49.0 Hz, H-4), 3.66–
3.79 (m, 1H, H-2), 3.93 (~d, 1H, J6a,6b12.3 Hz, H-6a), 4.11 (~d, 1H,
J6a,6b12.3 Hz, H-6b), 4.17 (s, 2H, CH2Ad), 8.07 (s, 1H, CH triazole).
13C (90 MHz, CD3OD) d 23.6 (C-2¢), 24.7 (C-4¢), 26.9 (C-3¢), 29.6
(CHAd), 35.3 (CAd), 37.6 (CH2 Ad), 41.1 (CH2 Ad), 54.1 (C-1¢), 55.1
(C-1), 64.1 (CH2Ad), 67.6 (C-5), 67.8 (C-2), 68.8 (C-4), 78.1 (C-
3), 127.1 (CH triazole), 146.2 (C triazole). LRMS (ESI+) 435.3
[M+H]+, 100%, 457.3 [M+Na]+, 30%. HRMS Found 435.2979
[M+H]+. C23H39N4O4 requires 435.2971.
Rf 0.22 (Pet. Ether/EtOAc 6 : 4). 1H NMR (360 MHz, CDCl3)
d 1.28–1.48 (m, 2H, H-2¢), 1.49–1.55 (m, 6H, 3¥CH2 Ad), 1.56–1.83
(m, 8H, 3¥CH2 Ad + H-3¢), 1.89–1.99 (m, 3H, 3¥CHAd), 2.12 (dd,
1H, J1ax,1eq10.7, J1eq,210.7 Hz, H-1eq), 2.28 (ddd, 1H, J4,59.1, J5,6a3.1,
J5,6b2.7 Hz, H-5), 2.46–2.53 (m, 1H, NCHH), 2.68–2.76 (m, 1H,
NCHH), 3.00 (dd, 1H, J1ax,1eq10.7, J1ax,24.5 Hz, H-1ax), 3.07 (s, 2H,
OCH2Ad), 3.44 (dd, 1H, J2,39.1, J3,49.1 Hz, H-3), 3.52 (dd, 1H,
J3,4 = J4,59.1 Hz, H-4), 3.54 (dd, 1H, J6a,6b10.6, J5,6a2.3 Hz, H-6a),
3.61 (ddd, 1H, J1eq,210.7, J2,39.1, J1ax,24.5 Hz, H-2), 3.63 (dd, 1H,
J6a,6b10.6, J5,6b2.7 Hz, H-6b), 4.22 (t, 2H, J 7.3 Hz, CH2N-triazole),
4.43 (d, 1H, J 10.9 Hz, CHH Bn), 4.43 (d, 1H, J 11.8 Hz, CHH
Bn), 4.44 (d, 1H, J 11.8 Hz, CHH Bn), 4.58 (s, 2H, OCH2-triazole),
4.61 (d, 1H, J 11.8 Hz, CHH Bn), 4.67 (d, 1H, J 11.8 Hz, CHH
Bn), 4.79 (d, 1H, J 11.2 Hz, CHH Bn), 4.87 (d, 1H, J 10.9 Hz,
CHH Bn), 4.93 (d, 1H, J 11.2 Hz, CHH Bn), 7.13–7.32 (m, 20H,
CH aromatic), 7.35 (s, 1H, CH triazole). 13C (90 MHz, CDCl3)
d 21.4 (C-2¢), 28.1 (C-3¢), 28.2 (CHAd), 34.0 (CAd), 37.2 (CH2 Ad),
39.7 (CH2 Ad), 49.9 (C-4¢), 51.3 (C-1¢), 54.3 (C-1), 64.1 (C-5), 65.3
(OCH2-triazole), 65.9 (C-6), 72.8 (CH2 benzyl), 73.4 (CH2 benzyl),
75.2 (CH2 benzyl), 75.3 (CH2 benzyl), 78.4 (C-2), 78.6 (C-4), 81.6
(OCH2Ad), 87.3 (C-3), 121.8 (CH triazole), 127.4 (CH aromatic),
127.5 (CH aromatic), 127.6 (CH aromatic), 127.8 (CH aromatic),
128.3 (CH aromatic), 128.4 (CH aromatic), 137.7 (C aromatic),
138.5 (C aromatic), 138.9 (C aromatic), 145.9 (C triazole). LRMS
(ESI+) 825.5 [M+H]+, 100%, 847.6 [M+Na]+, 20%. HRMS Found
825.4984 [M+H]+. C52H65N4O5 requires 825.4955. [a]2D0 2.9 (c 2.1,
CH2Cl2).
N -(4-Azidobutyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin
7a. 2,3,4,6-Tetra-O-benzyl-1-deoxynojirimycin
4
(188 mg,
0.359 mmol) was alkylated following general procedure A
with 5a to give after purification by flash chromatography
(Pet. Ether/EtOAc 9 : 1 to 3 : 7) 2,3,4,6-tetra-O-benzyl-N-(4-
azidobutyl)-1-deoxynojirimycin 7a (179 mg, 0.289 mmol, 80%
yield) as a thick colourless oil.
N -(4-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-1-
yl]}butyl)- 1-deoxynojirimycin 17. Hydrogenolysis of N-(4-{4-
[Adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-yl]}butyl)-2,
3,4,6-tetra-O-benzyl-1-deoxynojirimycin 12a (178 mg, 216 mmol)
gave after purification by flash silica gel column chromatography
(CHCl3/CH3OH 8 : 2) N-(4-{4-[adamant-1-yl-methoxymethyl]-
1H-1,2,3-triazol-1-yl]}butyl)-1-deoxynojirimycin 17 (97 mg,
21 mmol, 97% yield) as a pale yellow solid.
Rf 0.28 (Pet. Ether/EtOAc 4 : 1). 1H NMR (360 MHz, CDCl3)
d 1.34–1.57 (m, 4H, H-2¢, H-3¢), 2.19 (dd, 1H, J1ax,1eq11.2, J1eq,210.4
Hz, H-1eq), 2.28–2.36 (m, 1H, H-5), 2.50–2.61 (m, 1H, H-1¢a),
2.64–2.76 (m, 1H, H-1¢b), 3.08 (dd, 1H, J1ax,1eq11.2, J1ax,24.7 Hz,
H-1ax), 3.18 (t, 2H, J 6.4 Hz, H-4¢), 3.47 (dd, 1H, J3,49.1, J2,38.6 Hz,
H-3), 3.53–3.69 (m, 2H, H-6a, H-2), 3.57 (dd, 1H, J4,59.5, J3,49.1
Hz, H-4), 3.66 (dd, 1H, J6a,6b10.4, J5,6b2.7 Hz, H-6b), 4.43 (d, 1H,
J 12.2 Hz, CHH Bn), 4.45 (d, 1H, J 11.0 Hz, CHH Bn), 4.49 (d,
1H, J 12.2 Hz, CHH Bn), 4.65 (d, 1H, J 11.8 Hz, CHH Bn), 4.70
(d, 1H, J 11.8 Hz, CHH Bn), 4.82 (d, 1H, J 11.4 Hz, CHH Bn),
4.91 (d, 1H, J 11.0 Hz, CHH Bn), 4.96 (d, 1H, J 11.4 Hz, CHH
Bn), 7.12–7.36 (m, 20H, CH aromatic). 13C (90 MHz, CDCl3) d
21.3, 26.7 (C-2¢, C-3¢), 51.1 (C-4¢), 551.6 (C-1¢), 54.3 (C-1), 64.0
(C-5), 65.7 (C-6), 72.7 (CH2 benzyl), 73.6 (CH2 benzyl), 75.1 (CH2
benzyl),75.2 (CH2 benzyl), 78.4 (C-2), 78.6 (C-4), 87.3 (C-3), 127.4
(CH aromatic), 127.5 (CH aromatic), 127.6 (CH aromatic), 127.8
(CH aromatic), 127.8 (CH aromatic), 128.2 (CH aromatic), 128.3
(CH aromatic), 128.3 (CH aromatic), 137.7 (C aromatic), 138.5
(C aromatic), 139.0. LRMS (ESI+) 621.3 [M+H]+, 100%. HRMS
Found 621.3438 [M+H]+. C38H45N4O4 requires 621.3441.
Rf 0.18 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.52–1.60 (m, 6H, 3¥CH2 Ad), 1.60–1.81 (m, 8H, 3¥CH2 Ad + H-
2¢), 1.89–2.06 (m, 5H, 3¥CHAd + H-3¢), 2.49–2.68 (m, 2H, H-1eq,
H-5), 2.86–3.00 (m, 1H, H-1¢a), 3.07 (s, 2H, OCH2Ad), 3.09–3.30
(m, 3H, H-1¢b, H-1ax), 3.24 (dd, 1H, J2,39.1, J3,48.6 Hz, H-3), 3.46
(dd, 1H, J4,59.5, J3,49.1 Hz, H-4), 3.57 (ddd, 1H, J1eq,210.9, J2,39.1,
J1ax,24.9 Hz, H-2), 3.89 (dd, 1H, J6a,6b11.8, J5,6a2.7 Hz, H-6a), 3.96
(dd, 1H, J6a,6b11.8 J5,6b2.3 Hz, H-6b), 4.48 (t, 2H, J 6.8 Hz, H-
4¢), 4.58 (s, 2H, OCH2-triazole), 7.96 (s, 1H, CH triazole). 13C
(90 MHz, CD3OD) d 22.0 (C-2¢), 28.7 (C-3¢), 29.7 (CHAd), 35.1
(CAd), 38.3 (CH2 Ad), 40.7 (CH2 Ad), 50.7 (C-4¢), 53.2 (C-1), 56.0
(C-1¢), 57.1 (C-6), 65.4 (OCH2-triazole), 67.6(C-5), 69.0 (C-2),
70.2 (C-4), 79.1 (C-3), 82.7 (OCH2Ad), 124.9 (CH triazole), 146.5
(C triazole). LRMS (ESI+) 465.4 [M+H]+ 85%, 487.4 [M+Na]+,
5382 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

100%. HRMS Found 487.2892 [M+Na]+. C24H40N4O5Na requires
487.2896.
127.7 (CH benzyl), 128.0 (CH benzyl), 128.4 (CH benzyl), 128.5
(CH benzyl), 137.9 (C benzyl), 138.7 (C benzyl), 139.1 (C benzyl),
146.1 (C triazole). LRMS (ESI+) 853.6 [M+H]+, 100%. HRMS
Found 853.5258 [M+H]+. C54H69N4O5 requires 853.5268. [a]2D0 4.2
(c 1.0, CH2Cl2).
N -(6-Azidohexyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin
7b. 2,3,4,6-Tetra-O-benzyl-1-deoxynojirimycin
4
(835 mg,
1.60 mmol) was subjected to general procedure A with 5b to afford
after flash silica gel column chromatography (Pet. Ether/EtOAc
4 : 1) 2,3,4,6-tetra-O-benzyl-N-(6-azidohexyl)-1-deoxynojirimycin
7b (717 mg, 1.10 mmol, 69%).
N -(6-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-1-
yl]}hexyl-1-deoxynojirimycin
18. N-(6-{4-[Adamant-1-yl-
Rf 0.14 (Pet. Ether/EtOAc 85 : 15). 1H NMR (360 MHz, CDCl3)
d 1.09–1.48 (m, 6H, H-2¢, H-3¢, H4¢), 1.55 (~p, 2H, J 7.0 Hz, H-
5¢), 2.21 (dd, 1H, J1ax,1eq10.9, J1eq,210.9 Hz, H-1eq), 2.26–2.36 (m,
1H, H-5), 2.48–2.61 (m, 1H, H-1¢a), 2.61–2.75 (m, 1H, H-1¢b),
3.08 (dd, 1H, J1ax,1eq10.9, J1ax,24.8 Hz, H-1ax), 3.22 (t, 2H, J 7.0
Hz, H-6¢), 3.46 (dd, 1H, J2,39.1, J3,48.6 Hz, H-3), 3.52–3.70 (m,
4H, H-4, H-6a, H-2, H-6b), 4.44 (d, 1H, J 10.9 Hz, CHH Bn),
4.44 (d, 1H, J 12.3 Hz, CHH Bn), 4.49 (d, 1H, J 12.3 Hz, CHH
Bn), 4.64 (d, 1H, J 11.6 Hz, CHH Bn), 4.69 (d, 1H, J 11.6 Hz,
CHH Bn), 4.81 (d, 1H, J 10.9 Hz, CHH Bn), 4.89 (d, 1H, J 10.9
Hz, CHH Bn), 4.95 (d, 1H, J 10.9 Hz, CHH Bn), 7.10–7.39 (m,
20H, CH aromatic). 13C (90 MHz, CDCl3) d 23.7 (C-4¢), 26.6 (C-
3¢), 27.0 (C-2¢), 28.8 (C-5¢), 51.3 (C-6¢), 52.2 (C-1¢), 54.4 (C-1),
63.9 (C-5), 65.6 (C-6), 72.7 (CH2 benzyl), 73.4 (CH2 benzyl), 75.1
(CH2 benzyl), 75.2 (CH2 benzyl), 78.5 (C-2), 78.6 (C-4), 87.3 (C-3),
127.4 (CH aromatic), 127.5 (CH aromatic), 127.6 (CH aromatic),
127.8 (CH aromatic), 128.3 (CH aromatic), 128.3 (CH aromatic),
137.8 (C aromatic), 138.6 (C aromatic), 139.0 (C aromatic). LRMS
(ESI+) 649.5 [M+H]+, 100%. HRMS Found 649.3745 [M+H]+.
C40H49N4O4 requires 649.3754.[a]2D0 9.0 (c 1.1, CH2Cl2)
methoxymethyl]-1H-1,2,3-triazol-1-yl]}hexyl)-2,3,4,6-tetra-O-
benzyl-1-deoxynojirimycin 12b (319 mg, 373 mmol) was
hydrogenated following general procedure C. Further purification
by flash chromatography (CHCl3/CH3OH 8 : 2) gave N-(6-
{4-[adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-yl]}hexyl-
1-deoxynojirimycin 18 (191 mg, 362 mmol, 97%) as a white
solid.
Rf 0.22 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.29–1.37 (m, 4H, H-3¢, H-4¢), 1.54–1.55 (m, 8H, 3¥CH2 Ad + H-
2¢), 1.65–1.77 (m, 6H, 3¥CH2 Ad), 1.91–1.94 (m, 5H, 3¥CHAd, H-5¢),
2.23 (m, 1H, H-5), 2.26 (dd, 1H, J1eq,1ax11.4 Hz, J1eq,210.4 Hz, H-
1eq), 2.56–2.70 (m, 1H, H-1¢a), 2.80–2.93 (m, 1H, H-1¢b), 3.03 (dd,
1H, J1eq,1ax11.4, J1ax,24.8 Hz, H-1ax), 3.06 (s, 2H, OCH2Ad), 3.15
(dd, 1H, J2,39.4, J3,49.3 Hz, H-3), 3.37 (dd, 1H, J3,49.4, J4,58.9
Hz, H-4), 3.49 (ddd, 1H, J1eq,210.4, J2,39.4, J1ax,24.8 Hz, H-2),
3.85 (s, 1H, H-6a), 3.86 (s, 1H, H-6b), 4.41 (t, 2H, J 7.0 Hz, H-
6¢), 4.54 (s, 2H, OCH2-triazole), 7.94 (s, 1H, CH triazole). 13C
NMR (90 MHz, CD3OD) 25.0, 27.3, 27.8, 29.7, 31.2, 35, 38.3,
40.7, 51.2, 53.6, 57.3, 59.0, 65.3, 67.5, 70.3, 71.6, 80.2, 82.5, 124.8
(CH triazole), 146.3 (C triazole). LRMS (ESI+) 493.4 [M+H]+,
100%, 515.4[M+Na]+, 70%. HRMS Found 493.3386 [M+H]+.
C26H45N4O5 requires 494.3390.
N -(6-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-
1-yl]}hexyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin 12b. Cy-
cloaddition of N-(6-azidohexyl)-1-deoxynojirimycin 7b (300.8 mg,
0.464 mmol) was achieved using general procedure B. Fur-
ther purification by silica gel flash column chromatography
(Pet. Ether/EtOAc 8 : 2 to 1 : 1) gave N-(6-{4-[adamant-1-yl-
methoxymethyl]-1H-1,2,3-triazol-1-yl]}hexyl)-2,3,4,6-tetra-O-
benzyl-1-deoxynojirimycin 12b (367 mg, 0.430 mmol, 93%).
Rf 0.38 (Pet. Ether/EtOAc 8 : 2). 1H NMR (360 MHz, CDCl3)
d 1.12–1.46 (m, 6H, H-2¢, H-3¢, H-4¢), 1.52 (m, 6H, 3¥CH2 Ad),
1.61–1.76 (m, 6H, 3¥CH2 Ad), 1.86 (p, 2H, J 7.3 Hz, H-5¢), 1.93–
1.99 (m, 3H, 3¥CHAd), 2.20 (dd, 1H, J1eq,1ax10.8 Hz, J1eq,210.8 Hz,
H-1eq), 2.27–2.34 (m, 1H, H-5), 2.49–2.57 (m, 1H, H-1¢a), 2.63–
2.71 (m, 1H, H-1¢b), 3.07 (dd, 1H, J1eq,1ax10.8, J1ax,24.5 Hz, H-1ax),
3.09 (s, 2H, OCH2Ad), 3.46 (dd, 1H, J2,39.2, J3,49.1 Hz, H-3),
3.55 (dd, 1H, J6a,6b10.4, J5,6a1.8 Hz, H-6a), 3.57 (dd, 1H, J3,4 9.1,
J4,59.1 Hz, H-4), 3.65 (ddd, 1H, J1eq,210.8, J2,39.1, J1ax,24.5 Hz,
H-2), 3.66 (dd, 1H, J6a,6b10.4, J5,6a2.7 Hz, H-6b), 4.30 (t, 2H, J
7.3 Hz, H-6¢), 4.44 (d, 1H, J 10.9 Hz, CHH Bn), 4.41–4.46 (m,
1H, CHH Bn), 4.48 (d, 1H, J 12.3 Hz, CHH Bn), 4.62 (s, 2H,
OCH2-triazole), 4.65 (d, J 11.8 Hz, 1H, CHH Bn), 4.70 (d, 1H,
J 11.8 Hz, CHH Bn), 4.82 (d, J 11.2 Hz, 1H, CHH Bn), 4.89
(d, 1H, J 10.9 Hz, CHH Bn), 4.96 (d, 1H, J 11.2 Hz, CHH Bn),
7.08–7.40 (m, 20H, CH aromatic), 7.47 (s, 1H, CH triazole). 13C
NMR (90 MHz, CDCl3) 23.9, 26.5, 27.0 (C-2¢, C-3¢, C-4¢), 28.4
(CHAd), 30.4 (C-5¢), 34.2 (CAd), 37.3(CH2 Ad), 39.8(CH2 Ad), 50.3 (C-
6¢), 52.3 (C-1¢), 54.6 (C-1), 64.0 (C-5), 65.4 (OCH2-triazole), 65.8
(C-6), 72.9 (CH2 benzyl), 73.5 (CH2 benzyl), 75.3 (CH2 benzyl),
75.4 (CH2 benzyl), 78.7 (C-2), 78.8 (C-4), 81.7 (OCH2Ad), 87.5
(C-3), 122.0 (CH triazole), 127.5 (CH benzyl), 127.6 (CH benzyl),
N -(8-Azidooctyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin
7c. 2,3,4,6-Tetra-O-benzyl-1-deoxynojirimycin
4
(179 mg,
0.342 mmol), and 5c (199 mg, 0.850 mmol) were reacted according
to general procedure A to give after purification by flash chro-
matography (Pet. Ether/EtOAc 4 : 1) 2,3,4,6-tetra-O-benzyl-N-(8-
azidooctyl)-1-deoxynojirimycin 7c (161 mg, 0.238 mmol, 70%)
1
Rf (Pet. Ether/EtOAc 6 : 4). H NMR (360 MHz, CDCl3) d
1.05–1.45 (m, 10H, H-2¢, H-3¢, H-4¢, H-5¢,H-6¢), 1.58 (~p, 2H, J 7.0
Hz, H-7¢), 2.22 (dd, 1H, J1ax,1eq10.9, J1eq,210.4 Hz, H-1eq), 2.26–2.35
(m, 1H, H-5), 2.48–2.61 (m, 1H, H-1¢a), 2.61–2.74 (m, 1H, H-1¢b),
3.09 (dd, 1H, J1ax,1eq10.9, J1ax,24.5 Hz, H-1ax), 3.25 (t, 2H, J 7.0 Hz,
H-8¢), 3.45 (dd, 1H, J2,39.1, J3,49.1 Hz, H-3), 3.51–3.72 (m, 4H, H-4,
H-6a, H-2, H-6b), 4.42 (d, 1H, J 10.9 Hz, CHH Bn), 4.44 (d, 1H, J
12.3 Hz, CHH Bn), 4.49 (d, 1H, J 12.3 Hz, CHH Bn), 4.64 (d, 1H,
J 11.7 Hz, CHH Bn), 4.69 (d, 1H, J 11.7 Hz, CHH Bn), 4.81 (d,
1H, J 11.1 Hz, CHH Bn), 4.87 (d, 1H, J 11.1 Hz, CHH Bn), 4.95
(d, 1H, J 10.9 Hz, CHH Bn), 7.10–7.38 (m, 20H, CH aromatic).
13C (90 MHz, CDCl3) d 23.7, 26.6, 27.3 (C-2¢, C-3¢, C-4¢), 28.8 (C-
7¢), 29.1, 29.3 (C-5¢, C6¢), 51.4 (C-8¢), 52.4 (C-1¢), 54.4 (C-1), 63.9
(C-5), 65.6 (C-6), 72.7 (CH2 benzyl), 73.4 (CH2 benzyl), 75.1 (CH2
benzyl), 75.2 (CH2 benzyl), 78.6 (C-2), 78.6 (C-4), 87.3 (C-3), 127.4
(CH aromatic), 127.5 (CH aromatic), 127.6 (CH aromatic), 127.8
(CH aromatic), 128.3 (CH aromatic), 128.3 (CH aromatic), 137.8
(C aromatic), 138.6 (C aromatic), 139.0 (C aromatic). LRMS (EI)
677.4 [M+H]+, 100%. LRMS (ESI+) 677.4 [M+H]+, 100%. HRMS
Found 677.4055 [M+H]+. C42H53N4O4 requires 677.4067. [a]2D0 6.8
(c 1.1, CH2Cl2)
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5383
©

(C triazole). LRMS (ESI+) 521.4 [M+H]+, 100%, 543.4 [M+Na]+,
50%. HRMS Found 521.3687 4 [M+H]+. C28H49N4O5 requires
521.3703.
N -(8-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-1-
yl]}octyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin 12c. Gen-
eral procedure
B was used with N-(8-azidooctyl)-2,3,4,6-
tetra-O-benzyl-1-deoxynojirimycin 7c (309 mg, 0.457 mmol)
and 11 to afford after purification by flash chromatogra-
phy (Pet. Ether/EtOAc 8 : 2 to 1 : 1) N-(8-{4-[adamant-1-yl-
methoxymethyl]-1H -1,2,3-triazol-1-yl]}octyl)-2,3,4,6-tetra-O-
benzyl-1-deoxynojirimycin 12c (392 mg, 0.445 mmol, 97% yield).
Rf 0.20 (Pet. Ether/EtOAc 6 : 4). 1H NMR (360 MHz, CDCl3)
d 1.03–1.45 (m, 10H, H-2¢, H-3¢, H-4¢, H-5¢, H-6¢), 1.48–1.56 (m,
6H, 3 ¥ CH2 Ad), 1.58–1.75 (m, 6H, 3 ¥ CH2 Ad), 1.83–1.91 (m,
2H, H-7¢), 1.91–1.98 (M, 3H, CHAd), 2.21 (dd, 1H, J1ax,1eq10.9,
J1eq,210.9 Hz, H-1eq), 2.26–2.33 (m, 1H, H-5), 2.46–2.59 (m, 1H,
H-1¢a), 2.60–2.72 (m, 1H, H-1¢b), 3.07 (dd, 1H, J1ax,1eq10.9, J1ax,25.0
Hz, H-1ax), 3.07 (s, 2H, OCH2Ad), 3.44 (dd, 1H, J2,39.1, J3,49.1
Hz, H-3), 3.50–3.59 (m, 2H, H-4, H-6a), 3.59–3;70 (m, 2H, H-2,
H-6b), 4.32 (t, 2H, J 7.3 Hz, H-8¢), 4.41 (d, 1H, J 10.9 Hz, CHH
Bn), 4.43 (d, 1H, J 12.3 Hz, CHH Bn), 4.47 (d, 1H, J 12.3 Hz,
CHH Bn), 4.60 (s, 2H, triazole-CH2O), 4.63 (d, 1H, J 11.6 Hz,
CHH Bn), 4.68 (d, 1H, J 11.6 Hz, CHH Bn), 4.80 (d, 1H, J 10.9
Hz, CHH Bn), 4.86 (d, 1H, J 10.9 Hz, CHH Bn), 4.94 (d, 1H, J
10.9 Hz, CHH Bn), 7.09–7.37 (m, 20H, CH aromatic), 7.47 (s, 1H,
CH triazole). 13C (90 MHz, CDCl3) d 23.4, 27.3, 28.9, 29.2 (C-2¢,
C-3¢,C-4¢, C-5¢), 26.4 (C-6¢), 28.2 (CHAd), 30.3 (C-7¢), 33.9 (CAd),
37.1 (CH2 Ad), 39.6 (CH2 Ad), 50.2 (C-8¢), 52.3 (C-1¢), 54.4 (C-1),
63.6 (C-5), 65.1 (triazole-CH2O), 65.2 (C-6), 72.7 (CH2 benzyl),
73.4 (CH2 benzyl), 75.1 (CH2 benzyl), 75.2 (CH2 benzyl), 78.5 (C-
2), 78.5 (C-4), 81.5 (OCH2Ad), 87.3 (C-3), 121.8 (CH triazole),
127.4 (CH aromatic), 127.5 (CH aromatic), 127.6 (CH aromatic),
127.8 (CH aromatic), 127.9 (CH aromatic), 128.0 (CH aromatic),
128.1 (CH aromatic), 128.2 (CH aromatic), 128.3 (CH aromatic),
137.7 (C aromatic), 138.5 (C aromatic), 138.9 (C aromatic), 145.9
(C triazole). LRMS (ESI+) 881.6 [M+H]+, 100%. HRMS Found
881.5587 [M+H]+. C56H73N4O5 requires 881.5581. [a]2D0 6.4 (c 1.2,
CH2Cl2)
N -(10-Azidodecyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin
7d. 2,3,4,6-Tetra-O-benzyl-1-deoxynojirimycin
4
(189 mg,
0.360 mmol) and 5d were treated following general proce-
dure A to give after purification by flash chromatography
(Pet. Ether/EtOAc 9 : 1 to 1 : 1) 2,3,4,6-tetra-O-benzyl-N-(10-
azidodecyl)-1-deoxynojirimycin 7d (140 mg, 0.199 mmol, 55%).
Rf 0.38 (Pet. Ether/EtOAc 4 : 1). 1H NMR (360 MHz, CDCl3)
d 1.06–1.45 (m, 12H, H-2¢, H-3¢, H-4¢, H-5¢,H-6¢,H-7¢, H-8¢), 1.59
(~p, 2H, J 7.0 Hz, H-9¢), 2.21 (dd, 1H, J1ax,1eq10.9, J1eq,210.4 Hz,
H-1eq), 2.21 (dd, 1H, J4,59.5, J5,6a1.8, J5,6b2.7 Hz, H-5), 2.48–2.60
(m, 1H, H-1¢a), 2.60–2.73 (m, 1H, H-1¢b), 3.08 (dd, 1H, J1ax,1eq10.9,
J1ax,24.5 Hz, H-1ax), 3.25 (t, 2H, J 7.0 Hz, H-10¢), 3.45 (dd, 1H,
J2,39.1, J3,49.1 Hz, H-3), 3.54 (dd, 1H, J6a,6b10.4, J5,6a1.8 Hz, H-
6a), 3.58 (dd, 1H, J3,49.1, J4,59.5 Hz, H-4), 3.65 (dd, 1H, J6a,6b10.4,
J5,6b2.7 Hz, H-6b), 3.65 (ddd, 1H, J1eq,210.9, J2,39.1, J1ax,24.5 Hz,
H-2), 4.42 (d, J 10.9 Hz, 1H, CHH Bn), 4.44 (d, 1H, J 12.3 Hz,
CHH Bn), 4.48 (d, 1H, J 12.3 Hz, CHH Bn), 4.64 (d, 1H, J 11.6
Hz, CHH Bn), 4.68 (d, 1H, J 11.6 Hz, CHH Bn), 4.80 (d, 1H,
J 11.0 Hz, CHH Bn), 4.87 (d, 1H, J 10.9 Hz, CHH Bn), 4.94
(d, 1H, J 11.0 Hz, CHH Bn), 7.10–7.38 (m, 20H, CH aromatic).
13C (90 MHz, CDCl3) d 23.6, 26.7, 27.5 (C-2¢, C-3¢,C-4¢), 28.8
(C-9¢), 29.1, 29.4, 29.5 (C-5¢, C6¢, C-7¢, C-8¢), 51.5 (C-10¢), 52.4
(C-1¢), 54.5 (C-1), 63.8 (C-5), 65.4 (C-6), 72.7 (CH2 benzyl), 73.4
(CH2 benzyl), 75.1 (CH2 benzyl), 75.3 (CH2 benzyl), 78.6 (C-2),
78.6 (C-4), 87.4 (C-3), 127.4 (CH aromatic), 127.5 (CH aromatic),
127.6 (CH aromatic), 127.8 (CH aromatic), 128.3 (CH aromatic),
137.8 (C aromatic), 138.6 (C aromatic), 139.1 (C aromatic). LRMS
(ESI+) 705.4 [M+H]+, 100%. HRMS Found 705.4374 [M+H]+.
C44H57N4O4 requires 705.4380. [a]2D0 7.3 (c 1.1, CH2Cl2)
N-(10-{4-[Adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-
yl]}decyl)-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin 12d. A so-
lution of 13d (154 mg, 0.218 mmol) and 10 (67 mg, 0,327 mmol)
was subjected to general procedure B. Further purification by flash
chromatography (Pet. Ether/EtOAc 8 : 2 to 1 : 1) gave 12d (190 mg,
0.209 mmol, 96%).
N -(8-{4-[Adamant-1-yl-methoxymethyl]-1H -1,2,3-triazol-1-
yl]}octyl-1-deoxynojirimycin 19. Hydrogenolysis of N-(8-{4-
[adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-yl]}octyl)-2,3,
4,6-tetra-O-benzyl-1-deoxynojirimycin 12c (306 mg, 347 mmol)
following procedure C gave after purification by flash silica
gel column chromatography (CHCl3/CH3OH 8 : 2) N-(8-{4-
[adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-yl]}octyl-1-
deoxynojirimycin 19 (175 mg, 335 mmol, 97%) as a white solid.
Rf 0.24 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.25–1.45 (m, 8H, H-3¢, H-4¢, H-5¢, H-6¢), 1.50–1.59 (m, 6H,
3¥CH2 Ad), 1.62–1.82 (m, 8H, 3¥CH2 Ad + H-2¢), 1.86–1.98 (m, 5H,
3¥CH Ad, H-7¢), 2.98 (dd, 1H, J1ax,1eq12.3, J1eq,211.4 Hz, H-1eq),
3.00 (dd, 1H, J4,510.0, J5,6a2.3 Hz, H-5), 3.06 (s, 2H, OCH2Ad),
3.12–3.23 (m, 1H, H-1¢a), 3.31–3.41 (m, 1H, H-1¢b), 3.36 (dd, 1H,
J2,310.0, J3,49.5 Hz, H-3), 3.44 (dd, 1H, J1eq,1ax12.3, J1ax,24.5 Hz, H-
1ax), 3.59 (dd, 1H, J4,510.0, J3,49.5 Hz, H-4), 3.63–3.73 (m, 1H, H-
2), 3.89 (dd, 1H, J6a,6b12.2, J5,6a2.3 Hz, H-6a), 4.12 (d, 1H, J6a,6b12.3
Hz, H-6b), 4.41 (t, 2H, J 6.8 Hz, H-8¢), 4.54 (s, 2H, OCH2-triazole),
7.93 (s, 1H, CH triazole). 13C NMR (90 MHz, CD3OD) 24.1 (C-
2¢), 27.2, 27.4, 29.6, 29.9 (C-3¢, C-4¢, C-5¢, C-6¢), 29.7 (CH Ad), 31.1
(C-7¢), 35.1 (C Ad), 38.3 (CH2 Ad), 40.7 (CH2 Ad), 51.2 (C-8¢), 54.4 (C-
1¢), 54.9 (C-1, C-6), 65.3 (OCH2-triazole), 67.4 (C-5), 67.8 (C-2),
68.8 (C-4), 78.2 (C-3), 82.5 (OCH2Ad), 124.8 (CH triazole), 146.4
Rf 0.32 (Pet. Ether/EtOAc 6 : 4). 1H NMR (360 MHz, CDCl3) d
1.04–1.44 (m, 14H, H-2¢, H-3¢, H-4¢, H-5¢,H-6¢,H-7¢, H-8¢), 1.49–
1.55 (m, 6H, 3 ¥ CH2 Ad), 1.59–1.75 (m, 6H, 3 ¥ CH2 Ad), 1.84–1.98
(m, 5H, H-7¢, 3 ¥ CHAd), 2.21 (dd, 1H, J1ax,1eq10.9, J1eq,210.4 Hz,
H-1eq), 2.29 (dd, 1H, J4,510.4, J5,6a2.3, J5,6b2.3 Hz, H-5), 2.50–2.60
(m, 1H, H-1¢a), 2.61–2.71 (m, 1H, H-1¢b), 3.07 (s, 2H, OCH2Ad),
3.08 (dd, 1H, J1ax,1eq10.9, J1ax,24.8 Hz, H-1ax), 3.45 (dd, 1H, J2,39.1,
J3,49.1 Hz, H-3), 3.54 (dd, 1H, J6a,6b10.4, J5,6a2.3 Hz, H-6a), 3.58
(dd, 1H, J4,59.5, J3,49.1 Hz, H-4), 3.65 (ddd, 1H, J1eq,210.4, J2,39.1,
J1ax,24.8 Hz, H-2), 3.65 (dd, 1H, J6a,6b10.4, J5,6b2.3 Hz, H-6b), 4.32
(t, 2H, J 7.3 Hz, H-10¢), 4.41 (d, 1H, J 10.9 Hz, CHH Bn), 4.44 (d,
1H, J 12.3 Hz, CHH Bn), 4.48 (d, 1H, J 12.3 Hz, CHH Bn), 4.60
(s, 2H, triazole-CH2O), 4.64 (d, 1H, J 11.8 Hz, CHH Bn), 4.68 (d,
1H, J 11.8 Hz, CHH Bn), 4.80 (d, 1H, J 10.9 Hz, CHH Bn), 4.84
(d, 1H, J 10.9 Hz, CHH Bn), 4.94 (d, 1H, J 10.9 Hz, CHH Bn),
7.09–7.35 (m, 20H, CH aromatic), 7.47 (s, 1H, CH triazole). 13C
(90 MHz, CDCl3) d 23.7, 27.4, 29.0, 29.3, 29.4 (C-2¢, C-3¢,C-4¢, C-
5¢, C-6¢, C-7¢), 26.5 (C-8¢), 28.2 (CHAd), 30.3 (C-9¢), 34.0 (CAd), 37.2
(CH2 Ad), 39.6 (CH2 Ad), 50.3 (C-10¢), 52.4 (C-1¢), 54.4 (C-1), 63.8
(C-5), 65.2 (triazole-CH2O), 65.5 (C-6), 72.7 (CH2 benzyl), 73.4
5384 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

(CH2 benzyl), 75.1 (CH2 benzyl), 75.2 (CH2 benzyl), 78.5 (C-2),
78.6 (C-4), 81.5 (OCH2Ad), 87.3 (C-3), 121.8 (CH triazole), 127.3
(CH aromatic), 127.4 (CH aromatic), 127.5 (CH aromatic), 127.8
(CH aromatic), 128.2 (CH aromatic), 128.3 (CH aromatic), 128.5
(CH aromatic), 137.8 (C aromatic), 138.6 (C aromatic), 139.0 (C
aromatic), 145.9 (C triazole). LRMS (ESI+) 909.6 [M+H]+, 100%.
HRMS Found 909.5875 [M+H]+. C58H77N4O5 requires 909.5894.
[a]2D0 7.7 (c 1.1, CH2Cl2)
The released p-nitrophenol was measured spectrometrically at
400 nm.
Cell culture
HL60 were cultured in RPMI media containing 10% foetal calf
serum, 2 mM L-glutamine and 1% of a stock solution of penicillin-
streptomycin (Invitrogen). The human airway epithelial CF-KM4
cells were used to evaluate the efficacy of compounds on F508del-
CFTR function. Cells were cultured at 37 ◦C in 5% CO2 under
standard culture conditions as previously described.54
N-(10-{4-[Adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-
yl]}decyl)-1-deoxynojirimycin
20. N-(10-{4-[Adamant-1-yl-
methoxymethyl]-1H-1,2,3-triazol-1-yl]}decyl)-2,3,4,6-tetra-O-
benzyl-1-deoxynojirimycin 12d (141 mg, 155 mmol) was
subjected to general procedure C. Further purification by
flash chromatography (CHCl3/CH3OH 8 : 2) gave N-(10-{4-
[adamant-1-yl-methoxymethyl]-1H-1,2,3-triazol-1-yl]}decyl)-1-
deoxynojirimycin 20 (88 mg, 150 mmol, 97% yield) as a pale
yellow solid.
Cell proliferation assay for ER a-glucosidase assay
The effects of compounds on HL60 cell proliferation were deter-
R
mined using a Promega CellTiter 96ꢀ AQueous Cell Proliferation
Assay as described previously.42 Cells were seeded at a density of
5 ¥ 105 per mL in 96 well plates with or without a range of
concentrations of compound and incubated at 37 ◦C for 16 h.
MTS reagent (20 mL) was added to each well, the plates mixed
and incubated at 37 ◦C for 1 h. The absorbance was read at
490 nm using a UV max Kinetic microplate reader (Molecular
Devices) to determine the amount of formazan product, which
is directly proportional to the number of living cells in culture.
The mean CC50 (concentration of compound required to reduce
proliferation by 50%) and standard error was then calculated for
each compound relative to untreated cells based on the results of
three experiments.
Rf 0.26 (CHCl3/CH3OH 80 : 20). 1H NMR (360 MHz, CD3OD)
d 1.22–1.41 (m, 12H, H-3¢, H-4¢, H-5¢, H-6¢), 1.44–1.61 (m, 8H,
3¥CH2 Ad + H-2¢), 1.62–1.80 (m, 6H, 3¥CH2 Ad), 1.85–1.98 (m,
5H, 3¥CH Ad, H-9¢), 2.23–2.39 (m, 2H, H-1eq, H-5), 2.61–2.74
(m, 1H, H-1¢a), 2.84–2.95 (m, 1H, H-1¢b), 3.05 (s, 2H, OCH2Ad),
3.07 (dd, 1H, J1eq,1ax11.3, J1ax,24.8 Hz, H-1ax), 3.17 (dd, 1H, J2,39.1,
J3,49.1 Hz, H-3), 3.39 (dd, 1H, J4,59.5, J3,49.1 Hz, H-4), 3.51 (ddd,
1H, J1eq,210.7, J2,39.1, J1ax,24.8 Hz, H-2), 3.85 (dd, 1H, J6a,6b12.1,
J5,6a2.8 Hz, H-6a), 3.89 (dd, 1H, J6a,6b12.1, J5,6b2.3 Hz, H-6b), 4.40
(t, 2H, J 6.9 Hz, H-10¢), 4.54 (s, 2H, OCH2-triazole), 7.93 (s,
1H, CH triazole). 13C NMR (90 MHz, CD3OD) 25.1 (C-2¢), 27.4
(C-8¢), 28.4, 30.0, 30.5 (C-3¢, C-4¢, C-5¢, C-6¢, C-7¢), 29.7 (CH
Ad), 31.2 (C-9¢), 35.0 (C Ad), 38.3 (CH2 Ad), 40.7 (CH2 Ad), 51.3 (C-
10¢), 53.9 (C-1¢), 57.2 (C-1), 58.7 (C-6), 65.3 (OCH2-triazole), 67.4
(C-5), 70.2 (C-2), 71.5 (C-4), 80.1 (C-3), 82.4 (OCH2Ad), 124.9
(CH triazole), 146.4 (C triazole). LRMS (ESI+) 549.4 [M+H]+,
100%, 571.5 [M+Na]+, 50%. HRMS Found 549.4003 [M+H]+.
C30H53N4O5 requires 549.4016.
Free oligosaccharides (FOS) Isolation from Cells
Cells were cultured to high density (1 ¥ 107 cells mL-1) before the
medium was replaced with fresh medium containing inhibitor at
varying concentrations and the cells were seeded at a lower density
so as to achieve a high density at the end of the incubation period.
Following cell culture the medium was removed and the cells were
washed 3 times with PBS by centrifugation. Washed cells were
stored at -20 ◦C for a short time before thawing and Dounce
homogenisation in water. The conditions for extraction of FOS
were optimised to maximise recovery of FOS. The homogenate
was desalted and deproteinated by passage through a mixed bed
ion exchange column [0.2 mL AG50W-X12 (H+, 100–200 mesh)
over 0.4 mL AG3–X4 (OH-, 100–200 mesh)], pre-equilibrated with
water (5 ¥ 1 mL). The homogenate was added and collected with
4 ¥ 1 mL water washes. The extracted purified FOS was dried
under vacuum and dissolved in 30 mL water.
Other compounds
NB-DNJ was provided by Pharmacia. AMP-DNJ was a generous
gift of Prof. Robert Moriarty, University of Illinois at Chicago,
USA). Compounds were greater than 95% pure when analysed by
500 MHz NMR.
Glycosydases inhibition profiling53
The animal experimental protocols with live animals were ap-
proved by the Animal Experiments Committee of the University
of Toyama (S-2010 UH-2). Male Wistar rats with body weight of
130 g were obtained from Japan SLC, Inc. (Hamamatsu, Japan).
Brush border membranes were prepared from the rat small intes-
tine according to the method of Kessler et al., and were assayed
at pH 5.8 for rat intestinal maltase, isomaltase, sucrase, cellobiase,
and lactase using the appropriate disaccharides as substrates.
The released D-glucose was determined colorimetically using
Glucose CII-test Wako (Wako Pure Chemical Ind., Osaka, Japan).
Other glycosidase activities were determined using appropriate p-
nitrophenyl glycoside as a substrate at the optimum pH of each
enzyme. The reaction was stopped by adding 400 mM Na2CO3.
Inhibition of glycolipid biosynthesis
To determine the inhibition of ceramide glucosyltransferase
activity in a cell-based assay, HL60 cells were cultured in the
presence of various concentrations (0–100 mM) of compound for
3 days until confluence. Cells were harvested and washed with
PBS before re-suspension in water and Dounce homogenisation.
An aliquot of this homogenate was taken for protein assay. The
remainder was made 4 : 8 : 3 (v/v/v) chloroform : methanol : water
to extract glycolipids as described.55 Extracted glycolipids were
hydrolysed overnight at 37 ◦C using a preparation of ceramide
glycanase (purified in house from Hirudo medicinalis) in 20 mL
of 50 mM sodium acetate buffer, pH 5.0, containing 1 mg mL-1
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5385
©

sodium taurodeoxycholate. Glycolipid-derived oligosaccharides
were made to 30 mL with water and labelled with anthranilic
acid (2-AA) as described below. Labelled oligosaccharides were
analysed by NP-HPLC as described below.55,56
20% C (0.8 mL min-1). Samples (<50 mL) were injected in Milli-Q
water/acetonitrile (1 : 1, v/v).
Peak areas of FOS corresponding to Glc1Man4GlcNAc1 and
Glc3Man5GlcNAc1 species were analysed and used as a measure
for a-glucosidase II and a-glucosidase I inhibition, respectively,
in the presence of imino sugar.38,39 For GSL analysis, peak areas
corresponding to monosialyl-ganglioside GM3 were measured in
response to inhibitor treatment to generate inhibition constants.57
Carbohydrate fluorescent labelling
FOS and glycolipid derived oligosaccharides were labelled with
anthranilic acid as described previously (Neville et al., 2004).55
Briefly, anthranilic acid (30 mg mL-1) was dissolved in a solution
of sodium acetate trihydrate (4%, w/v) and boric acid (2% w/v) in
methanol. This solution was added to sodium cyanoborohydride
(final concentration 45 mg mL-1) and mixed to give the final
labelling mixture. 2-AA labeling mixture (80 mL) was added to
FOS samples (30 mL water) or glycolipid-derived oligosaccharides
followedby incubationat80 ◦C for 1h. The reaction was allowedto
cool to room temperature, 1 mL acetonitrile/water (97 : 3, v/v) was
added, and the mixture was vortexed. Labelled oligosaccharides
were purified by chromatography through Speed Amide 2 columns
(Applied Separations, Allentown, USA). The columns were pre-
equilibrated with 2 ¥ 1 mL acetonitrile, 2 ¥ 1 mL water followed
by 2 ¥ 1 mL acetonitrile. The samples were loaded using gravity
flow and allowed to drip through the column. The column was
washed with 2 ¥ 1 mL acetonitrile/water (95 : 5, v/v) and labelled
oligosaccharides eluted with 2 ¥ 0.75 mL water.
b-Glucocerebrosidase inhibition assay
Human placental b-glucocerebrosidase was isolated and purified
by a modified procedure of Furbish et al.58 Enzyme activity was
measured in 50 ml of 5 mM 4-methylumbelliferyl-b-glucoside
(4-MU-b-glucoside) in 0.1 M citrate phosphate buffer, pH 5.2
containing 0.25% sodium taurocholate, 0.1% TX100 at 37 ◦C for
15–60 min. The reaction was stopped by the addition of 200 ml
0.5 M sodium carbonate and the fluorescence measured at ex 350
nm, em 460 nm. Inhibition constants (IC50) were generated for
placental b-glucocerebrosidase (Km for 4-MU-b-glucoside, 1.9
0.3 mM) using 0.5 mM substrate concentration. Determinations
were made in triplicate. Data were fitted using Hill Slope plots
(Prizm software) and symmetrical standard errors determined for
each IC50 value.
F508del-CFTR restoration assay
CFTR Cl- channel activity was assayed by single-cell fluo-
rescence imaging using the potential-sensitive probe, bis-(1,3-
diethylthiobarbituric acid)trimethine oxonol.8 The results of fluo-
rescence imaging are presented as transformed data to obtain the
percentage signal variation (Fx) relative to the time of addition of
the stimulus, according to the equation: Fx = ([Ft - F0]/F0) ¥
100 where Ft and F0 are the fluorescent values at the time t
and at the time of addition of the stimulus, respectively. For
histogram representation, the values correspond to the level of
stable variation of fluorescence induced by each drug.
Purification of fluorescently labelled FOS
Labelled FOS in 50 mM Tris/HCl buffer, pH 7.2 were purified
through Concanavalin A (Con A)-Sepharose 4B column (100 mL
packed resin). The column was pre-equilibrated with 2 ¥ 1 mL
water followed by 1 mL of 1 mM MgCl2, 1 mM CaCl2 and 1 mM
MnCl2 in water and finally 2 ¥ 1 mL 50 mM TrisHCl buffer, pH
7.2. The sample was added and washed with 2 ¥ 1 mL 50 mM
TrisHCl buffer, pH 7.2. The bound FOS were then eluted with 2 ¥
1 mL hot (70 ◦C) 0.5 M methyl-a-D-mannopyranoside in 50 mM
TrisHCl buffer, pH 7.2.
Acknowledgements
Carbohydrate analysis by normal-phase high performance liquid
chromatography (NP-HPLC)
This work was supported by the ANR program “Jeunes
Chercheuses – Jeunes Chercheurs”, project number ANR-05-
JCJC-0199.
DSA, GR and TDB thank the Glycobiology Institute for
support.
ConA-Sepharose purified 2-AA-labelled free oligosaccharides and
glycolipid-derived oligosaccharides were separated by NP-HPLC
using a 4.6 ¥ 250 mM TSK gel Amide-80 column (Sigma, UK)
according to previously published methods.38,55 The chromatogra-
phy system consisted of a Waters Alliance 2695 separations module
and an in-line Waters 474 fluorescence detector set at Exl 360 ◦nm
and Eml 425 nm. All chromatography was performed at 30 C.
Solvent A was acetonitrile. Solvent B was Milli-Q water. Solvent
C was composed of 100 mM ammonium hydroxide, titrated to
pH 3.85 with acetic acid, in Milli-Q water and was prepared using
a standard 5.0 N ammonium hydroxide solution (Sigma, UK).
Gradient conditions were as follows: time = 0 min (t = 0), 71.6%
A, 8.4% B, 20% C (0.8 mL min-1); t = 6, 71.6% A, 8.4% B, 20%
C (0.8 mL min-1); t = 6, 71.6% A, 8.4% B, 20% C (0.8 mL min-1);
t = 40, 52% A, 28% B, 20% C (0.8 mL min-1); t = 41, 23% A, 57%
B, 20% C (1.0 mL min-1); t = 43, 23% A, 57% B, 20% C (1.0 mL
min-1); t = 44, 71.6% A, 8.4% B, 20% C (1.2 mL min-1); t = 59,
71.6% A, 8.4% B, 20% C (1.2 mL min-1); t = 60, 71.6% A, 8.4% B,
References
1 (a) A. E. Stu¨tz, Iminosugars as Glycosidase Inhibitors: Nojirimycin and
Beyond; Wiley-VCH: Weinheim, 1999; (b) O. R. Martin, P. Compain,
Iminosugars: From synthesis to therapeutic applications; Wiley-VCH:
Weinheim 2007.
2 N. Asano, in Glycoscience: Chemistry and Chemical Biology, B. Fraser-
Reid, K. Tatsuta, J. Thiem, ed., Springer Verlag, Heidelberg, 2008., pp
1887–1911.
3 (a) N. Zitzmann, A. S. Mehta, S. Carroue´e, T. D. Butters, F. M. Platt,
J. McCauley, B. S. Blumberg, R. A. Dwek and T. M. Block, Proc. Natl.
Acad. Sci. U. S. A., 1999, 96, 11878–11882; (b) Y. Nishimura, Curr. Top.
Med. Chem., 2003, 3, 575–591; (c) H. Fiaux, F. Popowycz, S. Favre, C.
Schu¨tz, P. Vogel, S. Gerber-Lemaire and L. Juillerat-Jeanneret, J. Med.
Chem., 2005, 48, 4237–4246.
4 (a) W. G. Laver, N. Bischofberger and R. G. Webster, Sci. Am., 1999,
280, 78–87; (b) R. A. Dwek, J. Virol., 2002, 76, 3596–3604; (c) T. M.
5386 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©

Block and R. Jordan, Antiviral Chem. Chemother., 2002, 12, 317–325;
(d) P. Greimel, J. Spreitz, A. E. Stu¨tz and T. M. Wrodnigg, Curr. Top.
Med. Chem., 2003, 3, 513–523; (e) L. A. Sorbera, J. Castanez and L.
Garc´ıa-Capdevila, Drugs Future, 2005, 30, 545–552; (f) R. J. Nash, M.
W. Carroll, A. A. Watson, G. W. J. Fleet and G. Horn, PCT Int. Appl.,
2007; Chem. Abstr., 2007, 146, p. 177231.
5 (a) J. E. Groopman, Rev. Infect. Dis., 1990, 12, 908–911; (b) I. Robina,
A. J. Moreno-Vargas, A. T. Carmona and P. Vogel, Curr. Drug Metab.,
2004, 5, 329–361.
26 P. Compain, C. Decroocq, J. Iehl, M. Holler, D. Hazelard, T. M.
Barragmn, C. Ortiz Mellet and J.-F. Nierengarten, Angew. Chem., Int.
Ed., 2010, 49, 5753–5756; J. Diot, M. I. Garcia-Moreno, S. G. Gouin,
C. Ortiz Mellet, K. Haupt and J. Kovensky, Org. Biomol. Chem., 2009,
7, 357–363; L. Diaz, J. Bujons, J. Casas, A. Llebaria and A. Delgado,
J. Med. Chem., 2010, 53, 5248–5255.
27 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
28 S. M. Marcuccio, WO 00/56713, 24 March 1999; Y. Xu, S.-R. Choi,
M.-P. Kung and H. F. Kung, Nucl. Med. Biol., 1999, 26, 833–839.
29 Bromides were obtained by nucleophilic displacement of tosylates 6a–b
and 9a–d using LiBr in acetone.
30 Modest yields have often been reported for the direct reductive
alkylation of DNJ. For, an example, see: (a) I. Lundt, A. J. Steiner,
A. E. Stu¨tz, C. A. Tarling, S. Ully, S. G. Withers and T. M. Wrodnigg,
Bioorg. Med. Chem., 2006, 14, 1737–1742. For a discussion on the
optimization of the initially modest yielding alkylation of DNJ in the
synthesis of AMP-DNJ, see: (b) T. Wennekes, B. Lang, M. Leeman, G.
A. von der Marel, E. Smits, M. Weber, J. van Wiltenburg, M. Wolberg,
J. M. F. G. Aerts and H. S. Overkleeft, Org. Process Res. Dev., 2008,
12, 414–423. For a recent report of the efficient reductive alkylation of
DNJ with various aldehydes, see: (c) A. J. Rawlings, H. Lomas, A. W.
Pilling, M. J.-R. Lee, D. S. Alonzi, J. S. S. Rountree, S. F. Jenkinson, G.
W. J. Fleet, R. A. Dwek, J. H. Jones and T. D. Butters, ChemBioChem,
2009, 10, 1101–1105.
31 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064; V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B.
Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
32 A. Robinson, J. Messbah, T. Smith and M. Foroozech, J. Undergrad.
Chem. Res., 2002, 1, 157–159.
33 T. Sasaki, S. Eguchi, T. Katada and O. Hiroaki, J. Org. Chem., 1977,
40, 3741.
6 See for example: G. S. Jacob, Curr. Opin. Struct. Biol., 1995, 5, 605–
611.
7 P. L. McCormack and K. L. Goa, Drugs, 2003, 63, 2427–2434.
8 C. Norez, F. Antigny, S. Noel, C. Vandebrouck and F. Becq, Am. J.
Respir. Cell Mol. Biol., 2009, 41, 217–225.
9 T. D. Butters, Curr. Opin. Chem. Biol., 2007, 11, 412–418.
10 H. Dvir, M. Harel, A. A. McCarthy, L. Toker, I. Silman, A. H.
Futerman and J. L. Sussman, EMBO Rep., 2003, 4, 704–709.
11 T. Kolter and K. Sandhoff, Angew. Chem., Int. Ed., 1999, 38, 1532–
1568.
12 N. W. Barton, R. O. Brady, J. M. Dambrosia, A. M. Di Bisceglie, S.
H. Doppelt, S. C. Hill, H. J. Mankin, G. J. Murray, R. I. Parker, C.
E. Argoff, R. P. Grewal and K.-T. Yu, N. Engl. J. Med., 1991, 324,
1464–1470.
13 T. Cox, R. Lachmann, C. Hollak, J. Aerts, S. van Weely, M. Hrebicek,
F. Platt, T. Butters, R. Dwek, C. Moyses, I. Gow, D. Elstein and A.
Zimran, Lancet, 2000, 355, 1481–1485.
14 J.-Q. Fan, Trends Pharmacol. Sci., 2003, 4, 260.
15 P. Compain, O. R. Martin, C. Boucheron, G. Godin, L. Yu, K. Ikeda
and N. Asano, ChemBioChem, 2006, 7, 1356–1359; M. Egido-Gabas,
D. Canals, J. Casas, A. Llebaria and A. Delgado, ChemMedChem,
2007, 2, 992–994; M. B. Tropak, G. J. Kornhaber, B. A. Rigat, G. H.
Maegawa, J. D. Buttner, J. E. Blanchard, C. Murphy, S. J. Tuske, S. J.
Coales, Y. Hamuro, E. D. Brown and D. J. Mahuran, ChemBioChem,
2008, 9, 2650–2662; Z. Luan, K. Higaki, M. Aguilar-Moncayo, H.
Ninomiya, K. Ohno, M. I. Garc´ıa-Moreno, C. Ortiz Mellet, J. M.
Garc´ıa Ferna´ndez and Y. Suzuki, ChemBioChem, 2009, 10, 2780–2792;
G. Shitter, A. J. Steiner, G. Potoschnig, E. Scheucher, M. Thonhofer,
C. A. Tarling, S. G. Withers, K. Fantur, E. Paschke, D. J. Mahuran, B.
A. Rigat, M. B. Tropak, C. Illaszewicz, R. Saf, A. E. Stu¨tz and T. M.
Wrodnigg, ChemBioChem, 2010, 11, 2026–2033; Z. Luan, K. Higaki,
M. Aguilar-Moncayo, L. Li, H. Ninomiya, E. Nanba, K. Ohno, M.
I. Garc´ıa-Moreno, C. Ortiz Mellet, J. M. Garc´ıa Ferna´ndez and Y.
Suzuki, ChemBioChem, 2010, 11, 2453–2464.
34 A. Baron, Y. Ble´riot, M. Sollogoub and B. Vauzeilles, Org. Biomol.
Chem., 2008, 6, 1898–1901.
35 This solvent system has been described to promote CuAAC between
organo-soluble azides and alkynes with more efficiency than wa-
ter/alcohol mixtures: B.-Y. Lee, S. R. Park, H. B. Jeon and K. S. Kim,
Tetrahedron Lett., 2006, 47, 5105–5109.
36 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt.
37 For a selective F508del-CFTR corrector: see ref. 22.
38 D. S. Alonzi, D. C. A. Neville, R. H. Lachmann, R. A. Dwek and T. D.
Butters, Biochem. J., 2008, 409, 571–580.
16 T. D. Butters, L. A. G. M. van den Broek, G. W. J. Fleet, T. M. Krulle,
M. R. Wormald, R. A. Dwek and F. M. Platt, Tetrahedron: Asymmetry,
2000, 11, 113–124.
17 C. Boucheron, V. Desvergnes, P. Compain, O. R. Martin, A. Lavi, M.
Mackeen, M. R. Wormald, R. A. Dwek and T. D. Butters, Tetrahedron:
Asymmetry, 2005, 16, 1747–1756.
18 T. Wennekes, R. J. B. H. N. van den Berg, W. Donker, G. A. von der
Marel, A. Strijland, J. M. F. G. Aerts and H. S. Overkleeft, J. Org.
Chem., 2007, 72, 1088–1097.
39 D. S. Alonzi, R. A. Dwek and T. D. Butters, Tetrahedron: Asymmetry,
2009, 20, 897–901.
40 H. R. Mellor, D. C. A. Neville, D. J. Harvey, F. M. Platt, R. A. Dwek
and T. D. Butters, Biochem. J., 2004, 381, 861–866.
41 For details of the human tracheal gland serous epithelial cell line CF-
KM4 derived from a CF patient homozygous for the F508del mutation,
see W. Kammouni, B. Moreau, F. Becq, R. Saleh, A. Pavirani, C.
Figarella and M. D. Merten, Am. J. Respir. Cell. Mol. Biol., 1999, 20,
684–691.
19 H. S. Overkleeft, G. H. Renkema, J. Neele, P. Vianello, I. O. Hung, A.
Strijland, A. M. von der Burg, G.-J. Koomen, U. K. Pandit and J. M.
F. G. Aerts, J. Biol. Chem., 1998, 273, 26522–26527.
20 (a) S. H. Cheng, R. J. Gregory, J. Marshall, S. Paul, D. W. Souza, G.
A. White, C. R. O’Riordan and A. E. Smith, Cell, 1990, 63, 827–834;
(b) M. J. Welsh and A. E. Smith, Cell, 1993, 2, 1251–1254.
21 S. Noel, C. Faveau, C. Norez, C. Rogier, Y. Mettey and F. Becq, J.
Pharmacol. Exp. Ther., 2006, 319, 349–359.
42 H. R. Mellor, J. Nolan, L. Pickering, M. R. Wormald, F. M. Platt,
R. A. Dwek, G. W. J. Fleet and T. D. Butters, Biochem. J., 2002, 366,
225–233.
43 R. A. Dwek, T. D. Butters, F. M. Platt and N. Zitzmann, Nat. Rev.
Drug Discovery, 2002, 1, 65–75.
44 T. Wennekes, R. J. B. H. N. van den Berg, R. G. Boot, G. A. von der
Marel, H. S. Overkleeft and J. M. F. G. Aerts, Angew. Chem., Int. Ed.,
2009, 48, 8848–8869.
22 D. Best, S. F. Jenkinson, A. W. Saville, D. S. Alonzi, M. R. Wormald,
T. D. Butters, C. Norez, F. Becq, Y. Ble´riot, I. Adachi, A. Kato and G.
W. J. Fleet, Tetrahedron Lett., 2010, 51, 4170–4174.
23 R. Sitia and I. Braakman, Nature, 2003, 426, 891–894.
24 A. Ghisaidoobe, P. Bikker, A. C. J. de Bruijn, F. D. Godschalk, E.
Rogaar, M. C. Guijt, P. Hagens, J. M. Halma, S. M. van’t Hart, S.
B. Luitjens, V. H. S. van Rixel, M. Wijzenbroek, T. Zweegers, W. E.
Donker-Koopman, A. Strijland, R. Boot, G. von der Marel, H. S.
Overkleeft, J. M. F. G. Aerts and R. J. B. H. N. van den Berg, ACS
Med. Chem. Lett., 2011, 2, 119–123.
45 See: A. R. Sawkar, S. L. Adamski-Werner, W.-C. Cheng, C.-H. Wong,
E. Beutler, K.-P. Zimmer and J. W. Kelly, Chem. Biol., 2005, 12, 1235–
1244.
46 C. Norez, S. Noel, M. Wilke, M. M. Bijvelds, H. Jorna, P. Melin, H.
De Jonge and F. Becq, FEBS Lett., 2006, 580, 2081–2086.
47 S. Noel, M. Wilke, A. G. M. Bot, H. R. De Jonge and F. Becq, J.
Pharmacol. Exp. Ther., 2008, 325, 1016–1023.
48 B. Lubamba, J. Lebacq, P. Lebecque, R. Vanbever, A. Leonard, P.
Wallemacq and T. Leal, Am. J. Respir. Crit. Care Med., 2009, 179,
1022–1028.
25 For the recent study of aza-C-glycosides with various spacers, see: T.
Wennekes, R. J. B. H. N. van den Berg, T. J. Boltje, W. E. Donker-
Koopman, B. Kuijper, G. A. von der Marel, S. Strijland, C. P. Verhagen,
J. M. F. G. Aerts and H. S. Overkleeft, Eur. J. Org. Chem., 2010, 1258–
1283.
49 F. P. da Cruz, S. Newberry, S. F. Jenkinson, M. R. Wormald, T. D.
Butters, D. S. Alonzi, S. Nakagawa, F. Becq, C. Norez, R. J. Nash, A.
Kato and G. W. J. Fleet, Tetrahedron Lett., 2011, 52, 219–223.
50 For a discussion concerning the quest for simplicity in organic synthesis
(Simplicity-Oriented Synthesis), see: P. Compain, V. Desvergnes, C.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5373–5388 | 5387
©

Ollivier, F. Robert, F. Suzenet, M. Barboiu, P. Belmont, Y. Ble´riot,
F. Bolze, S. Bouquillon, E. Bourguet, B. Braida, T. Constantieux, L.
De´saubry, D. Dupont, S. Gastaldi, F. Je´rome, S. Legoupy, X. Marat,
M. Migaud, N. Moitessier, S. Papot, F. Peri, M. Petit, S. Py, E. Schulz,
I. Tranoy-Opalinski, B. Vauzeilles, P. Vayron, L. Vergnes, S. Vidal and
S. Wilmouth, New J. Chem., 2006, 30, 823–831.
54 W. Kammouni, B. Moreau, F. Becq, A. Saleh, A. Pavirani, C. Figarella
and M. D. Merten, Am. J. Respir. Cell Mol. Biol., 1999, 20, 684–691.
55 D. C. A. Neville, V. Coquard, D. A. Priestman, D. J. M. te Vruchte, D.
J. Sillence, R. A. Dwek, F. M. Platt and T. D. Butters, Anal. Biochem.,
2004, 331, 275–282.
56 D. C. A. Neville, R. A. Dwek and T. D. Butters, J. Proteome Res., 2009,
8, 681–687.
57 H. Li, T. Liu, Y. Zhang, S. Favre, C. Bello, P. Vogel, T. D. Butters, N.
G. Oikonomakos, J. Marrot and Y. Bleriot, ChemBioChem, 2008, 9,
253–260.
51 W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43, 2923–2925.
52 W. L. F. Armarego and C. L. L. Chai, Purification of Labora-
tory, Chemicals, Fifth Edition, Butterworth Heinemann Publishers,
2003.
53 M. Kessler, O. Acuto, C. Storelli, H. Murer, M. Mu¨ller and G. Semenza,
Biochim. Biophys. Acta, Biomembr., 1978, 506, 136–154.
58 F. S. Furbish, H. E. Blair, J. Shiloach, O. G. Pentchev and R. O. Brady,
Proc. Natl. Acad. Sci. U. S. A., 1977, 74, 3560–3563.
5388 | Org. Biomol. Chem., 2011, 9, 5373–5388
This journal is
The Royal Society of Chemistry 2011
©