Amphiphilic 1-deoxynojirimycin derivatives through click strategies for chemical chaperoning in N370S gaucher cells

Article abstract of DOI:10.1021/jo201125x

In Gaucher disease (GD), mutant β-glucocerebrosidases (β-GCase) that are misfolded are recognized by the quality control machinery of the endoplasmic reticulum (ER) and degraded proteolytically. Hydrophobic iminosugars can be used as pharmacological chaperones to provide an improvement in the folding of the enzyme and promote trafficking from the ER. We have developed here an efficient click procedure to tether hydrophobic substituents to N-azidopropyl-1-deoxynojirimycin. A set of 14 original iminosugars was designed and evaluated for inhibition of commercially available glucosidases. Most of the compounds were micromolar inhibitors of those enzymes. In vitro inhibition assays with the N370S β-GCase revealed that the sublibrary containing the derivatives with aromatic aglycons displayed the highest inhibitory potency. Chaperone activity of the whole set of synthetic compounds was also explored in mutant Gaucher cells. The most active compound gave a nearly 2-fold increase in enzyme activity at 20 μM, a significantly higher value than the 1.33-fold recorded for the reference compound N-nonyl-1-deoxynojirimycin (N-nonyl-DNJ). As previously reported with bicyclic sp²-iminosugars (Luan, Z.; Higaki, K.; Aguilar-Moncayo, M.; Ninomiya, H.; Ohno, K.; Garcia-Moreno, M. I.; Ortiz Mellet, C.; Garcia Fernandez, J. M.; Suzuki, Y. ChemBioChem 2009, 10, 2780), in vitro inhibition of β-GCase measured for the compounds did not correlate with the cellular chaperone activity. The potency of new iminosugar chaperones is therefore not predictable from structureactivity relationships studies based on the in vitro β-GCase inhibition. (Figure presented)

Full text of DOI:10.1021/jo201125x

ARTICLE
pubs.acs.org/joc
Amphiphilic 1-Deoxynojirimycin Derivatives through Click Strategies
for Chemical Chaperoning in N370S Gaucher Cells
Jennifer D. Diot,† Isabel Garcia Moreno,‡ Gabriele Twigg,|| Carmen Ortiz Mellet,*,‡ Karsten Haupt,§
Terry D. Butters,*,|| Josꢀe Kovensky,*,† and Sꢀebastien G. Gouin*,†,^
†Laboratoire des Glucides UMR CNRS 6219, Institut de Chimie de Picardie, Facultꢀe des Sciences, Universitꢀe de Picardie Jules Verne,
33 rue Saint-Leu, 80039 Amiens Cedex 1, France
‡Departamento de Química Orgꢀanica, Facultad de Química, Universidad de Sevilla C/Profesor García Gonzꢀalez no. 1, 41012 Sevilla,
Spain
§Laboratoire Gꢀenie Enzymatique et Cellulaire, UMR CNRS-6022, Universitꢀe de Technologie de Compiꢁegne BP 20205, 60205
Compiꢁegne Cedex, France
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU,
United Kingdom
S Supporting Information
b
ABSTRACT: In Gaucher disease (GD), mutant β-glucocereb-
rosidases (β-GCase) that are misfolded are recognized by the
quality control machinery of the endoplasmic reticulum (ER)
and degraded proteolytically. Hydrophobic iminosugars can be
used as pharmacological chaperones to provide an improve-
ment in the folding of the enzyme and promote trafficking from
the ER. We have developed here an efficient click procedure to
tether hydrophobic substituents to N-azidopropyl-1-deoxyno-
jirimycin. A set of 14 original iminosugars was designed and
evaluated for inhibition of commercially available glucosidases. Most of the compounds were micromolar inhibitors of those
enzymes. In vitro inhibition assays with the N370S β-GCase revealed that the sublibrary containing the derivatives with aromatic
aglycons displayed the highest inhibitory potency. Chaperone activity of the whole set of synthetic compounds was also explored in
mutant Gaucher cells. The most active compound gave a nearly 2-fold increase in enzyme activity at 20 μM, a significantly higher
value than the 1.33-fold recorded for the reference compound N-nonyl-1-deoxynojirimycin (N-nonyl-DNJ). As previously reported
with bicyclic sp2-iminosugars (Luan, Z.; Higaki, K.; Aguilar-Moncayo, M.; Ninomiya, H.; Ohno, K.; García-Moreno, M. I.; Ortiz
Mellet, C.; García Fernꢀandez, J. M.; Suzuki, Y. ChemBioChem 2009, 10, 2780), in vitro inhibition of β-GCase measured for the
compounds did not correlate with the cellular chaperone activity. The potency of new iminosugar chaperones is therefore not
predictable from structureꢀactivity relationships studies based on the in vitro β-GCase inhibition.
’ INTRODUCTION
iminosugar C-glycosides,9 seven-membered ring iminoalditols,10
constrained bicyclic glycomimetics,11 and multivalent deoxyno-
jirimycin analogues12,13 have been recently reported. In
particular, efforts in the chemical synthesis of the piperidinic
imino-sugars have been rewarded by the launch of Miglitol
(Glyset) and N-butyl-1-deoxynojirimycin (Zavesca), respectively,
approved for the treatment of type II diabetes and type 1 Gaucher
disease (GD).14,15
GD is the most common of the glycosphingolipidoses, a group
of inherited diseases caused by defects in the lysosomal degrada-
tion of glycosphingolipids. In GD, progressive accumulation of
glucosylceramide in macrophages occurs because of a deficient
activity of β-GCase, an enzyme which hydrolyzes glucosylcer-
amide into ceramide and glucose. The most frequent mutation,
Carbohydrates containing nitrogen in the hemiacetal ring,
the so-called “imino-sugars”, have been extensively studied since
the first synthetic examples were reported in the 1960s1ꢀ3 and
the concomitant isolation of sugar-mimic alkaloids in micro-
organisms.4 The formal replacement of the endocyclic oxygen by
a protonated nitrogen atom leads to transition-state analogues of
glycosidases, forming strong ionic interactions with the two
carboxylic acid units catalyzing the cleavage of glycosidic bonds.
Iminosugars are of particular interest in view of their therapeutic
potential in the treatment of carbohydrate-mediated diseases
such as viral infections,5,6 tumor metastasis,7 diabetes, and
lysosomal storage diseases. Several synthetic methodologies have
been developed to improve the potency of the naturally occur-
ring classes of iminosugars such as indolizidines, pyrrolidines,
or piperidines.8 Research opportunities remain, and abundant
chemical approaches to generate novel structures such as
Received: June 1, 2011
Published: August 10, 2011
r
2011 American Chemical Society
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Chart 1. (A) N-Butyl-DNJ (Zavesca); (B) Potent GCS
Inhibitor Designed by Overkleeft and Co-workers; (C) Click
Chemical Strategy Adopted for the Functionalization of
N-Azidopropyl-DNJ with a Set of Hydrophobic Substituents
(in Gray)
Scheme 1. Synthesis of Protected N-Alkyl-DNJ Derivative 4
Scheme 2. Synthesis of N-Azidopropyl-DNJ 6
N370S β-GCase, is particularly prevalent in the Ashkenazi Jewish
population. For type 1 GD, which does not affect the nervous
central system, two treatments are currently available. Enzyme
replacement therapy (ERT) is an effective treatment, although it
is costly and ineffective for the neuronopathic form of GD, as the
β-GCase fails to cross the bloodꢀbrain barrier. Alternatively,
substrate reduction therapy (SRT) is aimed at inhibiting glyco-
lipid biosynthesis to balance the deficient activity of β-GCase.
N-Butyl-1-deoxynojirimycin (N-Bu-DNJ) was identified as a
potent inhibitor of the glucosyltransferase-catalyzed biosynthesis
of glucosylceramide and was shown to prevent lysosomal glyco-
lipid storage,16 allowing novel oral treatment for non-neurono-
pathic Gaucher’s disease.17 Potent glucosylceramide synthase
(GCS) inhibitors have emerged after the registered success of
N-Bu-DNJ marketed as Zavesca (Chart 1).17 Aerts and co-workers
have shown that DNJ derivatives bearing the hydrophobic ada-
mantyl are submicromolar inhibitors of GCS using an in vitro
assay.18 Since structural information on the GCS enzyme is
lacking, the search for new inhibitors proceeds through struc-
tureꢀactivity relationship studies. Successful strategies consist
of varying the nature18or the position19 of the bulky substituent
in the N-alkyl series or the synthesis of stereoisomer analogues
of DNJ.20,21
Chaperone-mediated therapy (CMT) has recently emerged as
an appealing strategy for the treatment of lysosomal storage
disorders. Mutant β-GCases that are misfolded are recognized by
the quality control machinery of the endoplasmic reticulum (ER)
and proteolytically degraded. Iminosugars can be used as phar-
macological chaperones that bind to the active site of the enzyme
to assist its folding and proper trafficking from the ER. In
particular, iminosugar chaperones bearing hydrophobic groups
have been shown to stabilize N370S β-GCase, the most common
mutation causing Gaucher disease.22 Considering the high
potential of iminosugars for SRT and chaperone-mediated
therapy (CMT) in a wide range of lysosomal disorders,23 more
potent and selective inhibitors are still required, and we are
witnessing a rebirth of this field of research.
implemented this reaction to connect alkynyl-armed substituents
to an azido-functionalized N-propyl-DNJ core (Chart 1). The
formed triazole should not impair the binding with β-GCase as
recently observed with N-substituted aminocyclitols.26 1,2,3-
Triazoles are interesting pharmacophores in view of their topo-
logical and electronic similarities with amide bonds and their
inertness toward in vivo oxidation and reduction processes.30 We
first assessed the solution-phase binding affinities of the synthetic
iminosugars for a set of commercial glucosidases (glu). Then, we
specifically compared β-GCase inhibition profiles for the set of
iminosugars with their potency to act as chaperones and increase
the enzyme activity in Gaucher cells.
’ RESULTS AND DISCUSSION
Synthesis. We first synthesized the imino sugar core 4 using a
procedure that we implemented for the synthesis of multivalent
iminosugars.12 Diol 1, obtained from methyl α-D-glucopyrano-
side in five steps,31ꢀ33 was converted in 2 using Swern conditions
(Scheme 1).34,35 A microwave-assisted double reductive amina-
tion of crude 2 in the presence of 3-azidopropylamine 336,37 led
exclusively to the expected diastereoisomer 4.
Removal of the benzyl protective groups after CuAAC should
be problematic for compounds bearing substituents sensitive to
hydrogenolysis. With this concern, we also decided to synthesize
unprotected azido derivative 6 as a specific substrate (Scheme 2).
Attempts to selectively remove benzyl groups by the use of BCl3
were unsuccessful and led to a mixture of products. To achieve
this goal, Pearlman’s catalyst was used in acidic media to avoid
poisoning of the palladium by the amino-groups.38 The azido
group was regenerated with a copper-catalyzed diazo transfer
reaction with triflyl azide, a procedure previously reported by
Wong and co-workers on glycosamines.39
CuAAC were first performed with 4 and a set of commercially
available hydrophobic alkynes 7ꢀ14 (Table 1). The highest
yields were obtained in a mixture of dioxaneꢀwater (4:1)
with copper sulfate and sodium ascorbate as catalyst system.
Microwave irradiation was previously described to significantly
We present here an original strategy to develop a set of
iminosugars bearing hydrophobic substituent. Considering the
usefulness of the copper catalyzed azideꢀalkyne cycloaddition24
(CuAAC) for the synthesis of sugar mimetics,12,13,25ꢀ29 we
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Table 1. Chemical Structure of the Cycloadducts Obtained from Commercial Alkynes
a Pd(OH)2, H2, MeOH, HCl, rt. b Pd(OH)2, H2, MeOH, 65 ꢀC. c Yields reported for the cyclization step after purification on silica gel or HPLC.
decrease the reaction times in CuAAC.40 This method allowed a fast
and exclusive formation of the expected 1,4-disubstituted 1,2,3-
triazoles 15ꢀ22, which were identified by the large Δ(δC4 ꢀ δC5)
values (∼20 ppm) observed by 13C NMR spectroscopy for
the cycloadducts.41 We then performed a catalytic hydrogeno-
lysis of the benzyl groups in aqueous media. Although these
conditions allowed us to isolate 23 and 24 with satisfactory yields
of 61 and 65% after HPLC purification, respectively, moderate
yields were obtained with the other cycloadducts, and we
observed a dehydration of the free hydroxyl groups in 20 and
21. Compounds 15ꢀ22 were successfully deprotected at 65 ꢀC
with Pd(OH)2 in methanol42 leading to 23ꢀ30 with yields
ranging from 56 to 75% after purification.
In order to increase the chemical diversity of our library
of iminosugars, we have functionalized a set of commercial
hydrophobic alcohols with propargyl bromide. The resulting
compounds 31ꢀ34 and protected mannoside 3543 were sub-
sequently subjected to CuAAc with 4 or when the substituents
were sensitive to the subsequent hydrogenolysis with 6
(Table 2). As an illustrative example, attempts to selectively
deprotect the benzyl groups of 36 led to the simultaneous
removal of the two chlorine atoms in 75% yield. After deprotection,
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Table 2. Chemical Structure of the Cycloadducts Obtained from Alkynyl-Armed Synthetic Intermediates 31ꢀ35
a Pd(OH)2, H2, MeOH, HCl, rt. b Pd(OH)2, H2, MeOH, 65 ꢀC. c MeONa, MeOH. d Yields reported for the cyclization step after purification on silica gel
or by HPLC for 38.
compounds 37, 40, 43, and 45 were purified by preparative HPLC
on a C18 column.
β-Glucosidase from almond and amyloglucosidase were effi-
ciently inhibited by the whole set of aromatic derivatives
(23ꢀ27). Subtle chemical modifications on the position of
substituents in the aromatic substituents for 23/24 and 25/26
or in the size of cyclic alcohols substituents (from cyclopentanol
to cyclohexanol) for 29/28 do not significantly affect the binding
affinity, except for the latter couple with β-glucosidase from
bovine liver leading to worse results. Compound 38, chemically
very closed to 37, is a much more potent inhibitor of this enzyme
(Ki 9 μM vs 317 μM, respectively). Interestingly, both com-
pounds 37 and 38 are potent inhibitors of almond β-glucosidase,
with 37 also being selective for this enzyme. With a binding
constant of 590 nM for almond β-glucosidase, 43 displayed the
lowest inhibitory value.
β-GCase Inhibition. All compounds were initially screened
for β-GCase inhibition at 100 μM. N-Nonyl-1-deoxynojirimycin
(N-nonyl-DNJ), a potent chemical chaperone of N370S β-GCase,
was included as a reference (Figure 1).38 The whole set of com-
pounds bearing aromatic aglycons 23ꢀ26, 37, 38, 43 displayed
strong inhibition of β-GCase, in the same order as N-nonyl-DNJ.
Glycosidases Inhibition. To test the selectivity profile, the
new 1-DNJ derivatives (except 30 for solubility reasons) were
first tested as inhibitors against a series of commercial glycosi-
dases including α-glucosidase (yeast), β-glucosidase (almonds),
β-glucosidase (bovine liver, cytosolic), and amyloglucosidase
(Aspergillus niger). The corresponding inhibition constants (Ki)
are collected in Table 3. Inhibition data for DNJ have been also
included for comparative purposes. Weak or no inhibition of the
yeast α-glucosidase was observed in all cases (excepting for 41
showing Ki = 20 μM). The values of Ki range from 9 to 669 μM
for the β-glucosidase from bovine liver, 0.59 to 177 μM
for almond β-glucosidase, 20 μM to >2 mM for baker's yeast
α-glucosidase, and 1 to >2 mM for amyloglucosidase. A strong
influence of the nature of the exocyclic substituent on the
potency of binding β-glucosidases was observed. Amphiphilic
derivatives bearing hydrophobic N0-substituents were much
more potent than derivatives bearing polar substituents (i.e.,
compound 45 having an α-D-mannopyranosyl residue).44
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Table 3. Glycosidase Inhibitory Activities (Ki, μM) for 1-DNJ
and the Synthetic Derivative Inhibitors 5, 6, 23ꢀ29, 37, 38,
40ꢀ43, and 45 against Commercial Glycosidasesa
β-glucosidase β-glucosidase α-glucosidase amyloglucosidase
compd (bovine liver) (almond) (baker's yeast) (Aspergillus niger)
DNJ
5
42
623
256
167
158
151
66
47
177
58
25
872
n.i.
506
409
n.i.
255
28
2.1
81
9.2
20
6
23
24
25
26
27
28
29
37
38
40
41
43
45
4.5
3.5
2.8
3.6
1.9
11
13
Figure 2. β-GCase inhibition at 5 μM. Compounds were assayed at 100
and 5 μM using purified enzyme and 0.5 mM 4-MU-β-glucoside as
described in the text. Triplicate determinations were made, and the
mean and standard deviations are shown as a percentage of control
enzyme activity. NN: N-nonyl-DNJ.
9.9
27
1
43
41
20
16
25
18
669
177
317
9
88
n.i.
n.i.
684
57
55
3
Table 4. β-GCase Activity in Mutant Gaucher Cells
1
390
23
5.1
n.i.
20
fold increase in β-GCase activity
3.2
compd (μM)
0.1
1
10
20
β-GCase inhibition
14
0.59
n.i.
n.i.
6.9
38
242
124
NNa
5
1.2
1.36
1.18
1.05
1.15
1.39
1.11
1.18
1.06
1.04
1.32
0.87
1.18
0.94
0.87
0.72
0.82
1.4
1.33
1.39
1
IC50 = <5 μM
IC50 = >50 μM
IC50 = >100 μM
IC50 = <5 μM
IC50 = <5 μM
IC50 = <5 μM
IC50 = <5 μM
IC50 = <5 μM
IC50 = <50 μM
IC50 = <50 μM
IC50 = ,5 μM
IC50 = <5 μM
IC50 = ,5 μM
IC50 = ,5 μM
IC50 = <5 μM
IC50 = ∼50 μM
IC50 = >50 μM
a Inhibition was competitive in all cases. n.i.: no inhibition was observed
at 2 mM concentration of the inhibitor.
1.09
0.97
0.99
1.26
0.99
1.15
1.09
1
1.51
1.13
1.27
1.25
1.17
1.2
6
15
23
24
25
26
28
29
30
37
38
41
43
44
45
1.21
1.45
0.95
1.28
1.23
0.97
0.91
0.94
1.94
1.37
0.89
0.28
1.02
1.17
0.95
1.12
1
1.26
0.81
1.2
1.59
1.21
0.98
0.36
0.8
0.85
0.74
0.71
0.78
Figure 1. β-GCase inhibition at 100 μM. Compounds were assayed at
100 and 5 μM using purified enzyme and 0.5 mM 4-MU-β-glucoside as
described in the text. Triplicate determinations were made, and the
mean and standard deviations are shown as a percentage of control
enzyme activity. NN: N-nonyl-DNJ.
nd
nd
nd
nd
a NN: N-nonyl-DNJ.
concentration of 5 μM (Figure 2). The selected derivatives
retained their faculty to inhibit the enzyme. Compounds 23,
30, 38, and 41 and N-nonyl-DNJ were able to decrease the
β-GCase activity to less than 13% of control activity.
Variation in the nature of the aromatic substituents exerted
relatively few effects. A significant drop in the inhibitory potency
was observed for compounds lacking “clicked” appends (5, 6).
Thus, the introduction of substituents is in general beneficial, and
a large variety of chemical structures can be used to improve
affinity. The more hydrophilic derivatives 28 and 29 with free
hydroxyl groups are less potent inhibitors than the set of aromatic
ones. Additionally, unprotected hydrophilic mannoside 45 is
significantly less potent than the protected synthetic intermedi-
ate 44. It seems, therefore, that the hydrophilic behavior, rather
than the nature and topology of the substituents, has more
influence on the binding affinity with β-GCase. These results are
in accordance with recent observations made with hydrophilic
aminocyclitols45 and with bicyclic isoureas and guanidines.46
The 10 most potent inhibitors 24ꢀ26, 30, 37, 38, 40, and 41
were then evaluated for their inhibitory effect at the lower
Ability To Chaperone β-GCase. Chaperone activity of the set
of compounds in mutant Gaucher lymphoblasts with the most
common N370S mutation23 is reported in Table 4. These data
show the fold increase in β-GCase activity compared to un-
treated cells. The IC50 of the compounds for the tested enzyme
are also reported. At the highest concentration reported in
activation assays with N370S Gaucher lymphoblasts, no cyto-
toxicity was observed as measured using a MTS assay.47 The
structurally related compounds 23 and 24 are both good
inhibitors of β-GCase. However, only 23 was active at elevating
mutant enzyme in cells. With almost a 2-fold increased activity at
20 μM, compound 37 was the most active compound but was
not a better inhibitor than 23 or 24. Compound 30 was one of
the three most potent inhibitors of β-GCase in vitro but did not
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enhance enzyme activity in Gaucher cells. Compound 5, a weak
inhibitor, of β-GCase in vitro was a surprisingly good chaperone
for mutant enzyme at 10 μM. Altogether, these results suggests
that the best inhibitors were not necessary the best enzyme
chaperones. The addition of an adamantlyl group to 37 (leading
to 43) did not significantly modulate inhibition of βGCase in
vitro. However, a dramatic change in the inhibitory profile was
observed in the cellular activity of mutant βGCase, 37 being the
best chaperone of the series and 43 the most potent inhibitor.
Therefore, in vitro inhibition of βGCase did not always correlate
with the increase in mutant enzyme activity in Gaucher cells.
mmol) was carried out with Pd(OH)2 (220 mg) in MeOHꢀ1 M HCl
(4 mL, 1:1) at 1 atm for 16 h. The catalyst was filtered over a pad of
Celite, and the solvent was removed to give 5 quantitatively as a colorless
solid: [α]20D = +3 (c = 1, H2O); 1H NMR (300 MHz, D2O) δ = 4.14
(1 H, dd, J6a,6b = 13.1 Hz, J5,6a = 1.9 Hz, H-6a), 4.02 (1 H, dd, J5,6b = 2.4 Hz,
H-6b), 3.86 (1 H, ddd, J = 11.2, J 9.5 Hz, J1a,2 = 5.0 Hz, H-2), 3.70 (1 H, t,
J
4,5 = J5,6 = 9.5 Hz, H-5), 3,63 (1 H, dd, J1a,1b = 12.2 Hz, H-1a), 3.55 (2 H,
m, H-3, H-10a), 3.43ꢀ3.29 (2H, m, H-4, H-10b), 3.17 (3 H, m, H-1b, 2 ꢁ
H-30), 2.29ꢀ2.15 (2 H, m, 2 ꢁ H-20); 13C NMR (75 MHz, D2O) δ =
75.7 (C-3), 67.0 (C-5), 65.9 (C-2), 65.8 (C-4), 53.8 (C-6), 53.1 (C-1),
49.8 (C-10), 36.6 (C-30), 21.2 (C-20); HRMS (ES+) found 221.1499,
C9H21N2O4 requires 221.1501.
N-(3-Azidopropyl)-1.5-dideoxy-1,5-imino-D-glucitol (6). Pre-
paration of TfN3 Dolution. To a solution of sodium azide (289 mg,
4.45 mmol) in H2O (0.5 mL) and DCM (1.5 mL), cooled to 0 ꢀC, was
added Tf2O (148 μL, 0.98 mmol) dropwise. The mixture was stirred at
0 ꢀC for 2 h. The organic layer was separated and the aqueous layer
extracted with DCM (2 ꢁ 1.5 mL). The organic layers were combined
and washed with saturated NaHCO3 (4 mL). The organic solution
containing TfN3 was separated and used without further purification.
Diazo-Transfer Reaction. To a solution of 5 (100 mg, 0.454 mmol) in
H2O (1.5 mL) were added K2CO3 (94 mg, 0.681 mmol), copper sulfate
(1 mg, 6.26 μmol), MeOH (15 mL), and the whole TfN3 solution.
(Caution! TfN3 is explosive when not in solution.) The mixture was
stirred at rt for 24 h and quenched with glycine (350 mg) for 5 h. The
solvents were evaporated under reduced pressure, and the residue was
purified by preparative HPLC (conditions 1) to give 6 (73 mg, 66%) as a
white hygroscopic solid: [α]D = +7 (c = 0.1, MeOH); tR = 19.7 min; 1H
NMR (500 MHz, D2O) δ = 3.94ꢀ3.81 (2 H, ddd, J6a,6b = J6a,5 = 12.8 Hz,
J6b,5 1.8 Hz, 2 ꢁ H-6), 3.55 (1 H, dt, J1b,2 = J2,3 = 9.5 Hz, J1a,2 = 4.7 Hz,
H-2), 3.41ꢀ3.35 (3 H, m, H-4, 2 ꢁ H-30), 3.26 (1 H, t, J2,3 = J3,4 = 9.5
Hz, H-3), 3.02 (1H, dd, J1a,1b = 11.3 Hz, H-1a), 2.89ꢀ2.68 (2 H, m, 2 ꢁ
H-10), 2.36ꢀ2.26 (2 H, m, H-1b, H-5), 2.83ꢀ2.78 (2 H, m, 2 ꢁ H-20);
13C NMR (75 MHz, D2O) δ = 78.3 (C-3), 70.0 (C-4), 68.8 (C-2), 64.9
(C-5), 57.5 (C-6), 55.3 (C-1), 49.4 (C-30), 49.2 (C-10), 22.5 (C-20);
HRMS (ES+) found 247.1417, C9H19N4O4 requires 247.1406.
General Procedure for the Preparation of Compounds
15ꢀ22, 36, 39, 42, and 44 by CuAAC. To a solution of the azido
derivative 4 (80 mg, 0.132 mmol) and the alkyne 7 (19 mg, 0.145 mmol)
in dioxaneꢀH2O (5 mL, 4:1) were added CuSO4 (32 mg, 0.198 mmol)
and sodium ascorbate (78 mg, 0.396 mmol), and the mixture was stirred
under microwave irradiation at 80 ꢀC for 45 min. The mixture was
diluted with EtOAc (10 mL) and washed with NH4Cl satd solution (2 ꢁ
8 mL). The organic layer was dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromatographed
on a silica gel column with 4:1 EtOAcꢀcyclohexane as eluent to afford
15 (63 mg, 65%).
N-(3-(4-(2-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)-
2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (15):
[α]D = +8 (c = 0.5, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ = 8.39
(1H, dd, J= 7.7, 1.6 Hz, Har), 8.00 (1 H, s, Har), 7.36ꢀ7.08 (22 H, m, 21 ꢁ
Har, CdCHN), 6.98 (1 H, d, J = 8.2 Hz, Har), 4.98ꢀ4.80 (3 H, 3 d,
J =11.0, 10.8, 11.0 Hz, CH2Ph, CHHPh), 4.67ꢀ4.60 (2 H, 2 d, J = 11.6,
11.4 Hz, CH2Ph), 4.44ꢀ4.28 (5 H, m, CH2Ph, CHHPh, 2 ꢁ H-30), 3.93
(3 H, s, CH3), 3.67ꢀ3.48 (5 H, m, H-2, H-3, H-4, 2 ꢁ H-6), 3.03 (1 H,
dd, J1a,1b = 11.0 Hz, J1a,2 = 4.7 Hz, H-1a), 2.88ꢀ2.78 (1 H, m, H-10a),
2.64ꢀ2.56 (1 H, m, H-10b), 2.34 (1 H, d, J5,4 = J5,6 8.9 Hz, H-5), 2.18
(1 H, t, J1a,1b = J1b,2 = 11.0 Hz, H-1b), 2.06 (2 H, m, J = 7.0 Hz, 2 ꢁ H-20);
13C NMR (75 MHz, CDCl3) δ = 155.7 (CH3OCar), 143.1 (NCdCH),
139.0, 138.5, 137.8 (Car), 129.0, 128.5, 128.4, 128.0, 127.8, 127.7, 127.6,
123.4 (CHar), 121.2 (NCHdC), 119.5 (NCHdCCar), 110.9 (CHar),
87.2 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.4, 72.9 (CH2Ph), 66.1
(C-6), 64.3 (C-5), 55.5 (CH3), 54.6 (C-1), 49.3 (C-10), 48.4 (C-30), 25.8
’ CONCLUSION
In summary, we have developed an efficient synthetic meth-
odology relying on copper-mediated 1,3-dipolar cycloaddition
for the design of iminosugars bearing hydrophobic aglycons. The
procedure allowed the rapid obtention of a large set of com-
pounds from a common intermediate. The “click iminosugars”
were micromolar inhibitors of commercial β-glucosidase and
amyloglucosidase and displayed a significant selectivity for these
enzymes toward α-glucosidase. Compounds 30, 38, and 41 were
particularly potent in vitro inhibitors of β-GCase with inhibitory
activity in the same range as the reference compound N-nonyl-
DNJ. The nearly 2-fold increase of β-GCase activity in mutant
Gaucher cells at 20 μM for 37 was significantly higher than the
reference compound N-nonyl-DNJ. This chaperoning effect is
promising considering that a modest increase in activity may be
clinically useful.48 Interestingly, a discrepancy between the in
vitro inhibition of β-GCase and the chaperone activity was
observed. In vitro β-GCase inhibition results should therefore
be interpreted with caution and not directly translated into ability
for chaperoning the enzyme.
’ EXPERIMENTAL METHODS
General Chemical Reagents and Methods. All purchased
materials were used without further purification. Dichloromethane
was distilled from calcium hydride and tetrahydrofuran over sodium
and benzophenone. Flash chromatography was performed on silica
(0.040ꢀ0.060 mm pore size) using distilled solvents. Microwave
irradiation experiments were performed using a CEM-Discover system
in an open vessel. The temperature profiles were recorded using an
infrared probe. NMR chemical shifts are reported in parts per million
relative to tetramethylsilane or a residual solvent peak (CHCl3: 1H: δ =
7.26 ppm, 13C: δ = 77.2 ppm). Assignments of 1H and 13C were assisted
by 2D 1H COSY and 2D 1Hꢀ C CORR experiments. Optical rotations
13
were measured at 20 ꢀC in a 1 cm cell in the stated solvent; [α]D values
are given in 10ꢀ1 deg cm2 gꢀ1 (concentration c given as g/100 mL).
3
HPLC samples were purified on a preparative C18 (2.2 ꢁ 25 cm) or an
NH2 5 μM column using a PL-ELS 1000 photodiode array detector.
Conditions 1 (C18 column): The mobile phase was H2O (solvent A)
and MeOH (solvent B). The gradient consisted of 100% A for 5 min
then 100% A to 100% B in 35 min (22 mL/min flow rate). Conditions 2
(C18 column): The gradient consisted of 100% A for 1 min then 100% A
to 100% B in 39 min (22 mL/min flow rate). Conditions 3 (NH2
column): The gradient consisted of 100% A to 100% B in 40 min
(22 mL/min flow rate). Conditions 4 (NH2 column): the mobile phase
was H2O (solvent A) and CH3CN. The gradient consisted of 10% A to
50% A in 50 min (22 mL/min flow rate). All of the HPLC samples were
controlled by NMR and showed >95% purity.
N-(3-Aminopropyl)-1,5-dideoxy-1,5-imino-D-glucitol (5).
Conventional catalytic hydrogenation of compound 4 (200 mg, 0.330
7762
dx.doi.org/10.1021/jo201125x |J. Org. Chem. 2011, 76, 7757–7768

The Journal of Organic Chemistry
ARTICLE
(C-20); HRMS (ES+) found 761.3671, C46H50N4O5Na requires
761.3679.
N-(3-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)-
(22 H, m, 21 ꢁ Har, CdCHN), 4.97ꢀ4.79 (3 H, 3 d, J = 11.0, 10.8, 11.1
Hz, CH2Ph, CHHPh), 4.66ꢀ4.58 (2 H, 2 d, J = 11.4, 11.6 Hz, CH2Ph),
4.46ꢀ4.3 (5 H, m, CH2Ph, CHHPh, 2 ꢁ H-30), 3.94 (3 H, s, CH3),
3.69ꢀ3.57 (4 H, m, H-2, H-4, 2 ꢁ H-6), 3.51 (1 H, t, J2,3 = J3,4 = 8.4 Hz,
H-3), 3.02 (1 H, dd, J1a,1b = 11.0 Hz, J1a,2 = 4.7 Hz, H-1a), 2.88ꢀ2.80
(1 H, m, H-10a), 2.60ꢀ2.53 (1 H, m, H-10b), 2.35 (1 H, d, J4,5 = J5,6 = 8.7
Hz, H-5), 2.17 (1 H, t, J1a,1b = J1b,2 = 11.0 Hz, H-1b), 2.08 (2 H, m, J = 7.1
Hz, 2 ꢁ H-20); 13C NMR (75.5 MHz, CDCl3): δ = 158.0 (CH3OCar),
147.9 (NCdCH), 139.0, 138.5, 137.8, 134.4 (Car), 129.8 (CHar), 129.1
(Car), 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5
(CHar), 126.0 (Car), 124.5, 124.4, 120.0 (CHar), 119.4 (NCHdC),
105.9 (CHar), 87.2 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.4, 72.9
(CH2Ph), 66.4 (C-6), 64.5 (C-5), 55.4 (CH3), 54.5 (C-1), 49.3 (C-10),
48.6 (C-30), 26.1 (C-20); HRMS (ESI+) found 811.3826, C50H52N4O5Na
requires 811.3835.
2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (16):
1
[α]20 = +11 (c = 0.6, CH2Cl2); H NMR (300 MHz, CDCl3) δ =
D
7.72 (2 H, d, J = 8.8 Hz, 2 ꢁ Har), 7.53 (1 H, s, CdCHN), 7.30ꢀ7.12 (20
H, m, 20 ꢁ Har), 6.90 (2 H, d, J = 8.8 Hz, 2 ꢁ Har), 4.96ꢀ4.78 (3 H, 3 d,
J = 11.0, 10.8, 11.0 Hz, CH2Ph, CHHPh), 4.65ꢀ4.59 (2 H, 2 d, J = 11.2,
11.1 Hz, CH2Ph), 4.43ꢀ4.19 (5 H, m, CH2Ph, CHHPh, 2 ꢁ H-30), 3.82
(1 H, s, CH3), 3.66ꢀ3.44 (5 H, m, H-2, H-3, H-4, 2ꢁ H-6), 3.00 (1 H, dd,
J1a,1b = 11.1 Hz, J1a,2 = 4.4 Hz, H-1a), 2.86ꢀ2.75 (1 H, m, H-10a),
2.54ꢀ2.50 (1 H, m, H-10b), 2.33 (1 H, d, J4,5 = J5,6 = 8.3 Hz, H-5), 2.15
(1 H, t, J1a,1b = J1b,2 = 11.1 Hz, H-1b), 2.01 (2 H, quint, J = 7.0 Hz, 2 ꢁ
H-20); 13C NMR (75 MHz, CDCl3) δ = 159.6 (Car), 147.5 (NCdCH),
139.0, 138.5, 137.8 (Car), 128.5, 128.4, 128.3, 127.9, 127.8, 127.7, 127.6,
127.0 (CHar), 123.5 (NCHdCCar), 119.2 (NCHdC), 114.3 (2 ꢁ
CHar), 87.1 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.3, 72.9 (CH2Ph),
66.3 (C-6), 64.5 (C-5), 55.4 (OCH3), 54.5 (C-1), 49.3 (C-10), 48.5
(C-30), 26.0 (C-20); HRMS (ES+) found 739.3849, C46H51N4O5 re-
quires 739.3859.
N-(3-(4-(2-Fluorophenyl)-1H-1,2,3-triazol-1-yl)propyl)-2,
3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (17).
[α]D = +13 (c = 0.5, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ = 7.61
(1 H, s, CdCHN), 7.57ꢀ7.50 (2 H, m, 2 ꢁ Har), 7.39ꢀ7.14 (21 H, m,
21 ꢁ Har), 7.02 (1 H, dt, J = 7.6, 2.6 Hz, Har), 4.97ꢀ4.79 (3 H, 3 d, J =
11.1, 10.9, 11.1 Hz, CH2Ph, CHHPh), 4.70ꢀ4.60 (2 H, 2 d, J = 11.6 Hz,
CH2Ph), 4.47ꢀ4.27 (5 H, m, CH2Ph, CHHPh, 2 ꢁ H-30), 3.67 (1 H,
dd, J6a,6b = 10.5 Hz, J6a,5 = 3.1 Hz, H-6a), 3.59ꢀ3.48 (4 H, m, H-2, H-3,
H-4, H-6b), 3.01 (1 H, dd, J1a,1b = 11.0 Hz, J1a,2 = 4.3 Hz, H-1a),
2.89ꢀ2.79 (1 H, m, H-10a), 2.57ꢀ2.50 (1 H, m, H-10b), 2.34 (1 H, d,
J4,5 = J5,6 = 8.4 Hz, H-5), 2.15 (1 H, t, J1a,1b = J1b,2 = 11.0 Hz, H-1b), 2.04
(2 H, m, J = 6.8 Hz, 2 ꢁ H-20); 13C NMR (75.5 MHz, CDCl3) δ = 163.3
(d, JCꢀF = 243.7 Hz, FCar), 146.6 (NCdCH), 139.0, 138.5, 137.8
(Car), 130.6 (d, JCꢀF = 8.2 Hz, CHar), 128.6, 128.5, 128.4, 128.1, 128.0,
127.9, 127.8, 127.6 (CHar), 121.4 (d, JCꢀF = 2.6 Hz, CHar), 120.5
(NCHdC), 115.0 (d, JCꢀF = 21.0 Hz, CHar), 112.7 (d, JCꢀF = 23.3 Hz,
CHar), 87.1 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.4, 73.0
(CH2Ph), 66.5 (C-6), 64.6 (C-5), 54.5 (C-1), 49.3 (C-10), 48.7
(C30), 26.2 (C-20); HRMS (ESI+) found 749.3449, C45H47N4O4FNa
requires 749.3479.
N-(3-(4-(Cyclohexanol-1-yl)-1H-1,2,3-triazol-1-yl)propyl)-
2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (20):
1
[α]D = +12 (c = 0.6, CH2Cl2); H NMR (300 MHz, CDCl3) δ =
7.33ꢀ7.14 (21 H, m, 20 ꢁ Har, CdCHN), 4.98ꢀ4.79 (3 H, 3 d, J = 11.0,
10.9, 11.1 Hz, CH2Ph, CHHPh), 4.67ꢀ4.61 (2 H, 2 d, J = 11.3 Hz, 11.1
Hz, CH2Ph), 4.46ꢀ4.41 (3 H, m, CH2Ph, CHHPh), 4.22ꢀ4.18 (2 H, m,
2 ꢁ H-30), 3.68ꢀ3.53 (4 H, m, H-2, H-4, 2 ꢁ H-6), 3.50 (1 H, t, J2,3
=
J3,4 = 8.0 Hz, H-3), 3.01 (1 H, dd, J1a,1b = 10.8 Hz, J1a,2 = 4.2 Hz, H-1a),
2.88ꢀ2.72 (1 H, m, H-10a), 2.61ꢀ2.58 (1 H, m, H-10b), 2.34 (1 H, d,
J4,5 = J5,6 = 8.3 Hz, H-5), 2.14 (1 H, t, J1a,1b = J1b,2 = 10.8 Hz, H-1b),
2.04ꢀ1.84 (4 H, m, 2 ꢁ H-20, CH2), 1.76ꢀ1.51 (5 H, m, 2 ꢁ CH2,
CHH), 1.37ꢀ1.27 (1 H, m, CHH); 13C NMR (75 MHz, CDCl3) δ =
155.5 (NCdCH), 138.9, 138.5, 137.8 (Car), 128.6, 128.5, 128.4, 128.1,
128.0, 127.8, 127.7, 127.6 (CHar), 120.1 (NCHdC), 87.1 (C-3), 78.5,
78.3 (C-2, C-4), 75.4, 75.3, 73.4, 72.9 (CH2Ph), 69.6 (C), 66.4 (C-6),
64.4 (C-5), 54.3 (C-1), 49.4 (C-10), 48.6 (C-30), 38.3, 38.2 (CH2), 25.9
(C-20), 25.5 (CH2), 22.1 (2 ꢁ CH2); HRMS (ES+) found 731.4143,
C45H55N4O5 requires 731.4172.
N-(3-(4-(Cyclopentanol-1-yl)-1H-1,2,3-triazol-1-yl)propyl)-
2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (21):
[α]D = +5 (c = 0.5, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ =
7.35ꢀ7.14 (21 H, m, 20 ꢁ Har, CdCHN), 4.97ꢀ4.79 (3 H, 3 d, J = 11.0
Hz, 10.8 Hz, 11.0 Hz, CH2Ph, CHHPh), 4.70ꢀ4.60 (2 H, 2 d, J = 11.4
Hz, 11.6 Hz, CH2Ph), 4.50ꢀ4.36 (3 H, m, CH2Ph, CHHPh), 4.21 (2 H,
dt, J = 6.9 Hz, 6.8 Hz, 2 ꢁ H-30), 3.68ꢀ3.46 (6 H, m, H-2, H-3, H-4, 2 ꢁ
H-6), 3.00 (1 H, dd, J1a,1b = 11.1 Hz, J1a,2 = 4.3 Hz, H-1a), 2.89ꢀ2.75
(1H, m, H-10a), 2.56ꢀ2.43 (1 H, m, H-10b), 2.32 (1 H, d, J4,5 = J5,6 = 8.7
Hz, H-5), 2.15ꢀ1.90 (9 H, m, H-1b, 2 ꢁ H-20, 3 ꢁ CH2), 1.82 (2 H, m,
CH2); 13C NMR (75.5 MHz, CDCl3) δ = 154.1 (NCdCH), 138.9,
138.5, 137.8 (Car), 128.6, 128.5, 128.5, 128.4, 128.1, 128.0, 127.8, 127.7,
127.6 (CHar), 120.3 (NCHdC), 87.2 (C-3), 78.6, 78.4 (C-2, C-4), 75.4,
75.3 (CH2Ph), 73.2 (C), 73.4, 72.9 (CH2Ph), 66.4 (C-6), 64.5 (C-5),
54.3 (C-1), 49.4 (C-10), 48.6 (C-30), 41.5, 41.3 (CH2), 26.1 (C-20), 23.7
(2 ꢁ CH2); HRMS (ESI+) found 739.3859, C44H52N4O5Na requires
739.3835.
N-(3-(4-(3-Fluoro-2-methylphenyl)-1H-1,2,3-triazol-1-yl)-
propyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-gl-
1
ucitol (18): [α]D = +7 (c = 0.3, CH2Cl2); H NMR (300 MHz,
CDCl3) δ = 7.66 (1 H, d, J = 8.3 Hz, Har), 7.58ꢀ7.52 (2 H, m,
CdCHN, Har), 7.32ꢀ7.13 (20 H, m, 20 ꢁ Har), 7.04 (1 H, t, J = 8.7
Hz, Har), 4.98ꢀ4.83 (3 H, 3 d, J = 11.0, 10.8, 11.0 Hz, CH2Ph,
CHHPh), 4.70ꢀ4.60 (2 H, 2 d, J = 11.6, 11.6 Hz, CH2Ph), 4.46ꢀ4.21
(5 H, m, CH2Ph, CHHPh, 2 ꢁ H-30), 3.76ꢀ3.45 (5 H, m, H-2, H-3,
H-4, 2 ꢁ H-6), 3.01 (1 H, dd, J1a,1b = 10.9 Hz, J1a,2 = 4.3 Hz, H-1a),
2.89ꢀ2.80 (1 H, m, H-10a), 2.63ꢀ2.51 (1 H, m, H-10b), 2.36ꢀ2.32
(4 H, m, H-5, CH3), 2.16 (1 H, t, J1b,1a = J1b,2 = 10.9 Hz, H-1b), 2.04 (2
H, m, J = 6.4 Hz, 2 ꢁ H-20); 13C NMR (75.5 MHz, CDCl3) δ = 161.3
(d, JCꢀF = 244.5 Hz, Car), 147.0 (NCdCH), 139.0, 138.5, 137.8 (Car),
128.9, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.6 (CHar),
124.7 (d, JCꢀF = 7.5 Hz, CHar), 119.8 (NCHdC), 115.5 (d, JCꢀF 22.5
Hz, CHar), 87.1 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.4, 73.0
(CH2Ph), 66.5 (C-6), 64.5 (C-5), 54.5 (C-1), 49.3 (C-10), 48.6 (C-30),
26.1 (C-20), 14.6 (CH3); HRMS (ESI+) found 741.3810, C46H50-
N4O4F requires 741.3816.
N-(3-(4-(3-Methoxyestradiol)-1-yl-1H-1,2,3-triazol-1-yl)-
propyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-gl-
1
ucitol (22): [α]D = +14 (c = 0.5, CH2Cl2); H NMR (300 MHz,
CDCl3) δ = 7.33ꢀ7.10 (22 H, m, 21 ꢁ Har, CdCHN), 6.69ꢀ6.62
(2 H, m, 2 ꢁ Har), 4.98ꢀ4.80 (3 H, 3 d, J = 11.1 Hz, 10.9 Hz, 11.1 Hz,
CH2Ph, CHHPh), 4.72ꢀ4.62 (2 H, 2 d, J = 11.2 Hz, 11.1 Hz, CH2Ph),
4.48ꢀ4.42 (3 H, m, CH2Ph, CHHPh), 4.24 (2 H, dt, J = 7.0 Hz, 6.8 Hz,
2 ꢁ H-30), 3.77 (3 H, s, OCH3), 3.70ꢀ3.57 (4 H, m, H-2, H-4, 2 ꢁ
H-6), 3.51 (1 H, t, J2,3 = J3,4 = 6.7 Hz, H-3), 3.03 (1 H, dd, J1a,1b = 11.3
Hz, J1a,2 = 4.7 Hz, H-1a), 2.91ꢀ2.80 (3 H, m, CH2, H-10a), 2.61ꢀ2.52
(1 H, m, H-10b), 2.47ꢀ2.35 (2 H, m, H-5, CHH), 2.23ꢀ2.14 (3 H, m,
H-1b, CHH, CHH), 2.05ꢀ1.93 (6 H, m, 2 ꢁ H-20, 2 ꢁ CHH, CHH,
CH), 1.65ꢀ1.31 (5 H, m, 2 ꢁ CHH, CHH, 2 ꢁ CH), 1.28 (3 H, s,
CH3), 1.01 (1 H, dt, J = 12.8 Hz, 2.8 Hz, CHH); 13C NMR (75 MHz,
N-(3-(4-(6-Methoxynaphtalen-2-yl)-1H-1,2,3-triazol-1-yl)-
propyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol
(19): [α]D = +6 (c = 0.5, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ =
8.24 (1 H, s, Har), 7.87 (1 H, dd, J = .6 Hz, 1.7 Hz, Har), 7.81 (1 H, d, J =
4.8 Hz, Har), 7.78 (1 H, d, J = 5.0 Hz, Har), 7.71 (1 H, s, Har), 7.31ꢀ7.16
7763
dx.doi.org/10.1021/jo201125x |J. Org. Chem. 2011, 76, 7757–7768

The Journal of Organic Chemistry
ARTICLE
CDCl3) δ = 157.5 (CH3OCar), 153.7 (NCdCH), 138.5, 138.3, 138.0,
137.0, 132.6 (Car), 128.6, 128.5, 128.4, 128.0, 127.8, 127.7, 127.6,
126.4 (CHar), 121.4 (NCHdC), 113.8, 111.5 (CHar), 87.2 (C-3),
82.5 (C), 78.5, 78.3 (C-2, C-4), 75.4, 75.2, 73.3, 72.9 (CH2Ph), 66.3
(C-6), 64.3 (C-5), 55.2 (OCH3), 54.5 (C-1), 49.3 (C-10), 48.5 (C-30,
CH2), 47.4, 43.5, 39.6 (CH), 38.1, 33.1, 30.0, 27.5, 26.4 (CH2), 25.9
(C-20), 23.5 (CH2), 14.4 (CH3); HRMS (ES+) found 917.5207,
C58H69N4O6 requires 917.5217.
General Procedure for the Preparation of 23, 24, and 43.
Conventional catalytic hydrogenation of compound 15 (35 mg, 0.0474
mmol) was carried out with Pd(OH)2 (25 mg) in EtOHꢀ1 M HCl
(2 mL, 1:1) at 1 atm for 19 h. The catalyst was filtered over a pad of
Celite, the solvent was removed, and the residue was purified by
preparative HPLC (conditions 2) to afford 23 (11 mg, 61%) as a
white solid.
(C-10), 48.6 (C-30), 23.7 (C-20); HRMS (ES+) found 367.1781,
C17H24N4O4F requires 367.1782.
N-(3-(4-(3-Fluoro-2-methylphenyl)-1H-1,2,3-triazol-1-yl)-
propyl)-1,5-dideoxy-1,5-imino-D-glucitol (26): [α]D = +3 (c =
0.2, MeOH); tR = 26.9 (conditions 2); 1H NMR (300 MHz, D2O) δ =
7.84 (1 H, s, CdCHN), 7.24 (2 H, d, J = 7.6 Hz, 2 ꢁ Har), 6.81 (1 H, t,
J = 9.2 Hz, Har), 4.26 (2 H, m, 2 ꢁ H-30), 3.81ꢀ3.68 (2 H, m, 2 ꢁ H-6),
3.52 (1 H, dt, J1b,2 = J2,3 = 9.8 Hz, J1a,2 = 4.9 Hz, H-2), 3.36 (1 H, t, J3,4
J4,5 = 9.8 Hz, H-4), 3.21 (1 H, t, J2,3 = J3,4, H-3), 2.94 (1 H, dd, J1a,1b
=
=
10.8 Hz, H-1a), 2.74ꢀ2.66 (1 H, m, H-10a), 2.63ꢀ2.58 (1 H, m, H-10b),
2.24ꢀ2.14 (2 H, m, H-1b, H-5), 2.00ꢀ1.92 (5 H, m, 2 ꢁ H-20, CH3);
13C NMR (75 MHz, D2O): δ = 160.8 (d, JCꢀF = 243.7 Hz, Car), 146.4
(NCdCH), 128.3, 125.3 (d, JCꢀF = 17.2 Hz, CHar), 124.3 (Car), 121.1
(NCHdC), 115.2 (d, JCꢀF = 23.2 Hz, CHar), 122.0 (CHar), 78.2 (C-
3), 69.2 (C-4), 68.7 (C-2), 64.9 (C-5), 57.2 (C-6), 55.3 (C-1), 48.7 (C-
10), 48.4 (C-30), 24.0 (C-20), 13.5 (CH3); HRMS (ESI+) found
403.1767, C18H25FN4O4Na requires 403.1758.
N-(3-(4-(2-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)-
1,5-dideoxy-1,5-imino-D-glucitol (23): [α]D = ꢀ5 (c = 0.1,
1
N-(3-(4-(6-Methoxynaphtalen-2-yl)-1H-1,2,3-triazol-1-yl)-
propyl)-1,5-dideoxy-1,5-imino-D-glucitol (27): [α] D = +29 (c =
0.1, MeOH); tR = 29.5 (conditions 1); 1H NMR (300 MHz, CD3OD) δ
= 8.43 (1 H, s, CdCHN), 8.23 (1 H, s, Har), 7.90ꢀ7.79 (4 H, m, Har),
7.25 (1 H, d, J = 2.1 Hz, Har), 7.16 (1 H, dd, J = 8.9 Hz, 2.5 Hz, Har),
4.52 (2 H, t, J = 6.8 Hz, 2 ꢁ H-30), 3.93 (3 H, s, CH3), 3.84 (2 H, ddd, J =
12.7, 11.9, 3.1 Hz, 2 ꢁ H-6), 3.45 (1 H, dt, J1b,2 = J2,3 = 9.5 Hz, J1a,2 = 5.2
MeOH); tR = 24.7 min; H NMR (300 MHz, D2O) δ = 8.25 (1 H,
s, CdCHN), 7.92 (1 H, d, J = 7.8 Hz, Har), 7.42 (1 H, t, J = 8.1 Hz,
Har), 7.13 (2 H, d, J = 7.6 Hz, 2 ꢁ Har), 4.43 (2 H, t, J = 6.9 Hz, 2 ꢁ
H-30), 3.90 (3 H, s, CH3), 3.75 (2 H, s, 2 ꢁ H-6), 3.51 (1 H, dt, J1b,2
=
J2,3 = 9.5 Hz, J1a,2 = 5.0 Hz, H-2), 3.35 (1 H, t, J3,4 = J4,5 = 9.5 Hz, H-4),
3.24 (1 H, t, J2,3 = J3,4, H-3), 2.96 (1 H, dd, J1a,1b = 11.5 Hz, H-1a),
2.85ꢀ2.56 (2 H, m, 2 ꢁ H-10), 2.33ꢀ2.23 (2 H, m, H-1b, H-5), 2.12 (2
H, m, J = 6.9 Hz, 2 ꢁ H-20); 13C NMR (75.5 MHz, D2O) δ = 155.7
(CH3OCar), 143.0 (NCdCH), 130.0, 127.2 (CHar), 124.7
(NCHdC), 121.1 (CHar), 118.0 (NCHdCCar), 111.9 (CHar), 78.1
(C-3), 69.9 (C-4), 68.7 (C-2), 64.7 (C-5), 57.3 (C-6), 55.4 (OCH3),
55.2 (C-1), 48.7 (C-10), 48.4 (C-30), 23.7 (C-20); HRMS (ESI+) found
379.1993, C18H27N4O5 requires 379.1981.
Hz, H-2), 3.34 (1 H, t, J3,4 = J4,5 = 9.5 Hz, H-4), 3.23 (1 H, t, J2,3 = J3,4
,
H-3), 3.05ꢀ2.92 (2 H, m, H-1a, H-10a), 2.65ꢀ2.55 (1 H, m, H-10b),
2.21ꢀ2.10 (4 H, m, H-1b, H-5, 2 ꢁ H-20); 13C NMR (75 MHz, D2O) δ
= 159.8 (CH3OCar), 149.3(NCdCH), 136.2 (Car), 130.8 (CHar),
130.6 (Car), 128.7 (CHar), 125.4 (2 ꢁ CHar), 122.5 (NCHdC), 120.5
(CHar), 80.7 (C-3), 72.2 (C-4), 70.9 (C-2), 68.1 (C-5), 59.7 (CH3),
57.8 (C-6), 56.0 (C-1), 50.5 (C-10), 50.0 (C-30), 27.2 (C-20); HRMS
(ES+) found 429.2129, C22H29N4O5 requires 429.2138.
N-(3-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)-
1,5-dideoxy-1,5-imino-D-glucitol (24): [α]D = ꢀ6 (c = 0.2,
MeOH); tR = 23.4 min (conditions 2); 1H NMR (300 MHz, D2O) δ
= 8.03 (1 H, s, CdCHN), 7.57 (2 H, d, J = 8.6 Hz, 2 ꢁ Har), 6.96 (2 H,
d, J = 8.6 Hz, 2 ꢁ Har), 4.36 (2 H, t, J = 7.1 Hz, 2 ꢁ H-30), 3.81 (3 H, s,
N-(3-(4-(Cyclohexanol-1-yl)-1H-1,2,3-triazol-1-yl)propyl)-
1,5-dideoxy-1,5-imino-D-glucitol (28): [α]D = +4 (c = 0.2,
1
MeOH); tR = 18.8 (conditions 2); H NMR (300 MHz, D2O) δ =
7.98 (1 H, s, CdCHN), 4.45 (2 H, m, 2 ꢁ H-30), 3.81ꢀ3.72 (2 H, m,
2 ꢁ H-6), 3.53 (1 H, dt, J1b,2 = J2,3 = 9.7 Hz, J1a,2 = 4.6 Hz, H-2), 3.37
(1 H, t, J3,4 = J4,5 = 9.4 Hz, H-4), 3.26 (1 H, t, J2,3 = J3,4 = 9.2 Hz, H-3),
2.98 (1 H, dd, J1a,1b 11.5 Hz, H-1a), 2.86ꢀ2.56 (2 H, m, 2 ꢁ H-10),
2.38ꢀ2.28 (2 H, m, H-1b, H-5), 2.15ꢀ2.06 (4 H, m, 2 ꢁ H-20, CH2),
1.83ꢀ1.79 (2 H, m, CH2), 1.71ꢀ1.67 (2 H, m, CH2), 1.50ꢀ1.33 (4 H,
m, 2 ꢁ CH2); 13C NMR (75 MHz, D2O) δ = 159.4 (NCdCH), 122.7
(NCHdC), 114.7, 112.5 (CHar), 78.0 (C-3), 69.7 (C-4), 69.05 (C),
68.5 (C-2), 64.7 (C-5), 57.1 (C-6), 55.4 (C-1), 48.6 (C-10), 48.2 (C-30),
37.0 (2 ꢁ CH2), 0.24.7 (CH2), 23.7 (C-20), 22.0 (2 ꢁ CH2); HRMS
(ES+) found 393.2120, C17H30N4O5Na requires 393.2114.
CH3), 3.74 (2 H, s, 2 ꢁ H-6), 3.51 (1 H, dt, J1b,2 = J2,3 = 9.6 Hz, J1a,2
4.9 Hz, H-2), 3.34 (1 H, t, J3,4 = J4,5 = 9.6 Hz, H-4), 3.23 (1 H, t, J2,3
=
=
J3,4, H-3), 2.93 (1 H, dd, J1a,1b = 11.0 Hz, H-1a), 2.79ꢀ2.56 (2 H, m,
2 ꢁ H-10), 2.28ꢀ2.21 (2 H, m, H-1b, H-5), 2.04 (2 H, m, J = 7.1 Hz, 2 ꢁ
H-20); 13C NMR (75.5 MHz, D2O) δ = 159.0 (CH3OCar), 147.1
(NCdCH), 126.9 (2 ꢁ CHar), 122.4 (Car), 121.2 (NCHdC), 114.4
(2 ꢁ CHar), 78.2 (C-3), 69.9 (C-4), 68.8 (C-2), 64.7 (C-5), 57.4 (C-6),
55.3 (CH3), 55.2 (C-1), 48.6 (C-10), 48.5 (C-30), 23.8 (C-20); HRMS
(ESI+) found 379.1987, C18H27N4O5 requires 379.1981.
General Procedure for the Preparation of 25ꢀ30, 37, and
40. Compound 17 (36 mg, 0.0496 mmol) was dissolved in methanol
(10 mL) at 50 ꢀC. Palladium hydroxide (55 mg) was added, and the
mixture was stirred at 65 ꢀC for 24 h. The mixture was filtered over a pad
of Celite, and the filtrate evaporated under reduced pressure. The
residue was purified by preparative HPLC (conditions 2) to afford 25
(11 mg, 61%) as a white solid.
N-(3-(4-(Cyclopentanol-1-yl)-1H-1,2,3-triazol-1-yl)propyl)-
1,5-dideoxy-1,5-imino-D-glucitol (29): [α]D = +9 (c = 0.1, MeOH);
1
tR = 16.2 (conditions 2); H NMR (300 MHz, D2O) δ = 7.96 (1 H, s,
CdCHN), 4.46 (2 H, dt, J = 6.7, 6.8 Hz, 2 ꢁ H-30), 3.78ꢀ3.68 (2 H, m, 2 ꢁ
H-6), 3.55ꢀ3.49 (1 H, dt, J1b,2 =J2,3 =9.5Hz, J1a,2 = 5.1 Hz, H-2), 3.34 (1 H, t,
J3,4 = J4,5 = 9.5 Hz, H-4), 3.23 (1 H, t, J2,3 = J3,4 = 9.3 Hz, H-3), 2.94 (1 H, dd,
J1a,1b = 11.0 Hz, H-1a), 2.79ꢀ2.63 (2 H, m, 2 ꢁ H-10), 2.28ꢀ2.09 (2 H, m,
H-1b, H-5), 2.14ꢀ2.04 (6 H, m, 2 ꢁ H-20,2ꢁ CH2), 1.90ꢀ1.80 (4 H, m, 2 ꢁ
CH2); 13C NMR (75 MHz, D2O) δ = 157.8 (NCCH), 122.2 (NCHC), 78.6
(C), 78.2 (C-3), 69.9 (C-4), 68.8 (C-2), 64.6 (C-5), 58.7 (C-6), 55.2 (C-1),
48.6 (C-10), 48.5 (C-30), 39.9 (2 ꢁ CH2), 23.7 (C-20), 22.9 (2 ꢁ CH2);
HRMS (ES+) found 379.1952, C16H28N4O5Na requires 379.1957.
N-(3-(4-(2-Fluorophenyl)-1H-1,2,3-triazol-1-yl)propyl)-1,5-
dideoxy-1,5-imino-D-glucitol (25): [α]D = ꢀ4 (c = 0.2, MeOH);
tR = 24.0 (conditions 2); 1H NMR (300 MHz, D2O) δ = 8.17 (1 H, s,
CdCHN), 7.44ꢀ7.34 (3 H, m, 3 ꢁ Har), 7.08 (1 H, dt, J = 8.2 Hz, 3.2
Hz, Har), 4.41 (2 H, t, J = 7.2 Hz, 2 ꢁ H-30), 3.76 (2 H, s, 2 ꢁ H-6), 3.53
(1 H, dt, J1b,2 = J2,3 = 9.7 Hz, J1a,2 = 4.8 Hz, H-2), 3.35 (1 H, t, J3,4 = J4,5
=
9.7 Hz, H-4), 3.24 (1 H, t, J2,3 = J3,4, H-3), 2.96 (1 H, dd, J1a,1b = 11.3 Hz,
H-1a), 2.81ꢀ2.61 (2 H, m, 2 ꢁ H-10), 2.33ꢀ2.23 (2 H, m, H-1b, H-5),
2.10 (2 H, quint, J = 7.2 Hz, 2 ꢁ H-20); 13C NMR (75 MHz, D2O) δ =
162.8 (d, JCꢀF 241.3 Hz, FCar), 146.3 (NCdCH), 131.5 (Car), 130.8 (d,
JCꢀF = 8.6Hz, CHar), 122.3 (NCHdC), 121.2 (d, JCꢀF = 2.7 Hz, CHar),
115.2 (d, JCꢀF = 21.1 Hz, CHar), 112.1 (d, JCꢀF = 23.1 Hz, CHar), 78.2
(C-3), 70.4 (C-4), 68.7 (C-2), 64.7 (C-5), 57.4 (C-6), 55.2 (C-1), 48.7
N-(3-(4-(3-Methoxyestradiol-1-yl)-1H-1,2,3-triazol-1-yl)-
propyl)-1,5-dideoxy-1,5-imino-D-glucitol (30): [α]D = +36 (c =
0.5, MeOH); tR = 35.6 (conditions 2); 1H NMR (500 MHz, CD3OD)
δ = 7.85 (1 H, s, CdCHN), 7.83 (1 H, d, J = 8.6 Hz, Har), 6.64 (1 H, dd,
J = 8.5, 2.6 Hz, Har), 6.60 (1 H, d, J = 2.5 Hz, Har), 4.48 (2 H, t, J = 6.7
Hz, 2 ꢁ H-30), 3.84 (2 H, s, 2 ꢁ H-6), 3.74 (3 H, s, OCH3), 3.49 (1 H, dt,
7764
dx.doi.org/10.1021/jo201125x |J. Org. Chem. 2011, 76, 7757–7768

The Journal of Organic Chemistry
ARTICLE
J1b,2 = J2,3 = 9.7 Hz, J1a,2 = 4.2 Hz, H-2), 3.38 (1 H, t, J3,4 = J4,5 = 9.7 Hz,
H-4), 3.18 (1 H, t, J2,3 = J3,4, H-3), 3.04 (1 H, dd, J1a,1b = 11.2 Hz, H-1a),
2.94 (1 H, m, H-10a), 2.86ꢀ2.77 (2 H, m, CH2), 2.60ꢀ2.45 (2 H, m,
H-10b, CHH), 2.23ꢀ2.07 (6 H, m, H-1b, H-5, 2 ꢁ H-20, CHH, CHH),
2.05ꢀ1.88 (3 H, m, CHH, CHH, CH), 1.71ꢀ1.65 (2 H, m, CHH, CH),
1.62ꢀ1.53 (1 H, m, CHH), 1.47ꢀ1.32 (3 H, CHH, CHH, CH), 0.92 (3
H, s, CH3), 0.74 (1 H, dt, J 12.8, 3.8 Hz, CHH); 13C NMR (125 MHz,
CD3OD) δ = 159.1 (CH3OCar), 155.4 (NCdCH), 138.9, 133.8 (Car),
127.1 (CHar), 124.1 (NCHdC), 114.7, 112.5 (CHar), 83.5 (C), 80.4
(C-3), 72.0 (C-4), 70.6 (C-2), 67.9 (C-5), 59.6 (C-6), 57.6 (C-1), 55.6
(OCH3), 49.7 (C-10), 49.5 (CH), 49.3 (C-30), 48.5, 48.4 (CH), 44.5,
41.1, 38.5, 34.4, 28.7, 27.2, 27.1 (CH2), 24.6 (C-20), 14.8 (CH3); HRMS
(ES+) found 557.3351, C30H45N4O6 requires 557.3339.
N-(3-(4-(3,4-Dichlorophenoxymethyl)-1H-1,2,3-triazol-1-yl)-
propyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol
(36): [α]D = +11 (c = 0.4, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ =
8.10 (1 H, s, CdCHN), 7.33ꢀ7.10 (23 H, m, 23 ꢁ Har), 5.23 (2 H, s,
OCH2), 4.99ꢀ4.80 (3 H, 3 d, J = 11.0, 10.9, 11.0 Hz, CH2Ph, CHHPh),
4.69ꢀ4.62 (2 H, 2 d, J = 11.6, 11.4 Hz, CH2Ph), 4.47ꢀ4.37 (3 H, m,
CH2Ph, CHHPh), 4.31ꢀ4.19 (2 H, m, 2 ꢁ H-30), 3.68ꢀ3.49 (5 H, m,
H-2, H-3, H-4, 2 ꢁ H-6), 3.01 (1 H, dd, J1a,1b = 10.9 Hz, J1a,2 = 4.5 Hz,
H-1a), 2.85ꢀ2.77 (1 H, m, H-10a), 2.55ꢀ2.47 (1 H, m, H-10b), 2.32
(1 H, d, J4,5 = J5,6 = 8.4 Hz, H-5), 2.14 (1 H, t, J1a,1b = J1b,2, H-1b), 2.01 (2
H, q, J = 6.8 Hz, 2 ꢁ H-20);13C NMR (75 MHz, CDCl3) δ = 8.10 (1 H, s,
CdCHN), 7.33ꢀ7.10 (23 H, m, 23 ꢁ Har), 5.23 (2 H, s, OCH2),
4.99ꢀ4.80 (3 H, 3 d, J 11.0, 10.9, 11.0 Hz, CH2Ph, CHHPh), 4.69ꢀ4.62
(2 H, 2 d, J = 11.6, 11.4 Hz, CH2Ph), 4.47ꢀ4.37 (3 H, m, CH2Ph,
CHHPh), 4.31ꢀ4.19 (2 H, m, 2 ꢁ H-30), 3.68ꢀ3.49 (5 H, m, H-2, H-3,
H-4, 2 ꢁ H-6), 3.01 (1 H, dd, J1a,1b = 10.9 Hz, J1a,2 = 4.5 Hz, H-1a),
2.85ꢀ2.77 (1 H, m, H-10a), 2.55ꢀ2.47 (1 H, m, H-10b), 2.32 (1 H, d,
J4,5 = J5,6 = 8.4 Hz, H-5), 2.14 (1 H, t, J1a,1b = J1b,2, H-1b), 2.01 (2 H, q, J =
6.8 Hz, 2 ꢁ H-20); HRMS (ES+), found 807.3091, C46H49N4O5Cl2
requires 807.3080.
J
5,6 = 9.5 Hz, H-5), 2.02ꢀ1.91 (2 H, m, 2 ꢁ H-20); 13C NMR (125 MHz,
D2O) δ = 154.5 (CH2OCar), 142.6 (NCdCH), 133.1 (Car), 127.8
(CHar), 124.8 (NCHdC), 122.7 (CHar), 121.0 (Car), 112.2 (CHar),
78.2 (C-3), 69.7 (C-4), 68.7 (C-2), 62.5 (C-5), 64.9 (OCH2), 57.2 (C-
6), 55.4 (C-1), 48.7 (C-10), 48.5 (C-30), 24.2 (C-20); HRMS (ES+)
found 469.1029, C18H24N4O5Cl2Na requires 469.1021.
4-{[(1S,2S,4R)-Bicyclo[2.2.1]heptan-2-yloxy]methyl}-1-
[3-(2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-gluci-
tol-N-yl)propyl]-1,2,3-triazole (39): [α]D = +2 (c = 0.4,
CH2Cl2); 1H NMR (300 MHz, CDCl3) δ = 7.40 (1 H, s, CdCHN),
7.31ꢀ7.11 (20 H, m, 20 ꢁ Har), 4.94ꢀ4.79 (3 H, 3 d, J = 11.0, 10.8,
11.1 Hz, CH2Ph, CHHPh), 4.68ꢀ4.39 (9 H, m, 2 ꢁ CH2Ph, CHHPh,
2 ꢁ H-30, OCH2), 3.72ꢀ3.44 (6 H, m, H-2, H-3, H-4, 2 ꢁ H-6, CH),
3.01 (1 H, dd, J1a,1b = 11.1 Hz, J1a,2 = 4.7 Hz, H-1a), 2.86ꢀ2.75 (1 H,
m, H-10a), 2.61ꢀ2.46 (1 H, m, H-10b), 2.38ꢀ1.97 (6 H, m, H-1b, H-5,
2 ꢁ H-20, 2 ꢁ CH), 1.56ꢀ1.42 (5 H, m, CH2, 3 ꢁ CHH), 1.12ꢀ0.99
(3 H, m, 3 ꢁ CHH); 13C NMR (75 MHz, CDCl3) δ = 146.0
(NCdCH), 139.0, 138.5, 137.8 (Car), 128.5, 128.4, 128.0, 127.9,
127.8, 127.7, 127.6 (CHar), 122.6 (NCHdC), 87.2 (C-3), 82.7
(CH), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.4, 72.9 (CH2Ph), 66.3
(C-6), 64.5 (C-5), 62.1 (OCH2), 54.5 (C-1), 49.2 (C-10), 48.4 (C-30),
40.4 (CH), 39.6 (CH2), 35.3 (CH), 35.0 (CH2), 28.6 (CH2), 26.1 (C-
20), 24.7 (CH2); HRMS (ES+) found 757.4339, C47H57N4O5
requires 757.4329.
4-{[(1S,2S,4R)-Bicyclo[2.2.1]heptan-2-yloxy]methyl}-1-
[3-(1,5-dideoxy-1,5-imino-D-glucitol-N-yl)propyl]-1,2,3-tria-
zole (40): [α]D = ꢀ5 (c = 0.3, MeOH); tR = 25.9 (conditions 2); 1H
NMR (300 MHz, D2O) δ = 8.03 (1 H, s, CdCHN), 4.62 (2 H, s,
OCH2), 4.49ꢀ4.45 (2 H, m, 2 ꢁ H-30), 3.75 (2 H, s, 2 ꢁ H-6), 3.62 (1
H, d, J = 6.4 Hz, CH), 3.52 (1 H, m, dt, J1b,2 = J2,3 = 9.6 Hz, J1a,2 = 4.9 Hz,
H-2), 3.38 (1 H, t, J3,4 = J4,5 = 9.6 Hz, H-4), 3.27 (1 H, t, J2,3 = J3,4 = 9.6
Hz, H-3), 2.93 (1 H, dd, J1a,1b = 11.1 Hz, H-1a), 2.85ꢀ2.50 (2 H, m, 2 ꢁ
H-10), 2.34ꢀ2.15 (3 H, m, H-1b, H-5, CH), 2.12 (2 H, quint, J 6.9 Hz,
2 ꢁ H-20), 1.68ꢀ1.62 (1 H, m, CHH), 1.46ꢀ1.37 (4 H, m, CHH, 3 ꢁ
CHH), 1.14 (1 H, d, J 9.8 Hz, CHH), 1.04ꢀ0.99 (2 H, m, 2 ꢁ CHH);
13C NMR (125 MHz, D2O) δ = 144.5 (NCdCH), 125.0 (NCHdC),
83.0 (CH), 78.2 (C-3), 69.2 (C-4), 68.8 (C-2), 64.7 (C-5), 60.3
(OCH2), 57.4 (C-6), 55.3 (C-1), 48.6 (C-10), 48.5 (C-30), 40.1
(CH), 38.9 (CH2), 35.1 (CH), 34.3 (CH2), 27.6 (CH2), 24.1 (C-20),
23.7 (CH2); HRMS (ES+) found 397.2431, C19H33N4O5 requires
397.2451.
1-[3-(1,5-Dideoxy-1,5-imino-D-glucitol-N-yl)propyl)]-4-
{[2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy]methyl}-
1,2,3-triazole (41). Compound 6 (10 mg, 0.0406 mmol), compound 33
(9 mg, 0.0447 mmol), CuSO4 (9 mg, 0.0610 mmol), and sodium ascorbate
(24 mg, 0.122 mmol) were dissolved in dioxane (0.8 mL) and water
(0.11 mL) The mixture was stirred under microwave irradiation at 80 ꢀC for
45 min. The mixture was evaporated under reduced pressure, and the residue
purified by preparative HPLC (conditions 2) to afford 41 (11 mg, 60%):
[α]D =ꢀ9(c= 0.1, MeOH); tR = 33.5; 1HNMR(500MHz, D2O) δ=8.11
(1H, s, CCHN), 5.40(1H, s, CH2CHC), 4.71(2H, s, OCH2C), 4.59 (2 H,
m, 2 ꢁ H-30), 3.98ꢀ3.92 (2 H, m, 2 ꢁ H-6), 3.75ꢀ3.69 (3 H, m, H-2,
CH2), 3.57 (1 H, t, J3,4 = J4,5 = 9.3 Hz, H-4), 3.45 (1 H, t, J2,3 = J3,4 = 9.3 Hz,
H-3), 3.22 (1 H, dd, J1a,1b = 11.0 Hz, J1a,2 = 4.9 Hz, H-1a), 2.90ꢀ3.02 (1 H,
m, H-10a), 2.90ꢀ2.83 (1 H, m, H-10b), 2.52ꢀ2.47 (2 H, m, H-1b, H-5),
2.41ꢀ2.27 (7H, m, 2 ꢁ H-20, 2 ꢁ CH2, CHH), 2.18 (2 H, s, CH), 1.39
(3H, s, CH3), 1.25(1H, d, J=8.2Hz, CHH), 0.94 (3 H, s, CH3);13CNMR
(125 MHz, CD3OD): δ = 145.4 (C), 144.8 (NCdCH), 124.9 (NCHdC),
118.3 (CH2CHC), 78.5(C-3), 70.0 (C-4), 68.9 (C-2, CH2), 65.6 (C-5),
63.7 (OCH2), 57.4 (C-6), 55.7 (C-1), 49.3 (C-10), 48.7 (C-30), 45.8, 41.0
(CH), 38.1 (C), 31.8, 31.6 (CH2), 26.5 (CH3), 24.7 (C-20), 21.4 (CH3);
HRMS (ES+) found 451.2906, C23H39N4O5 requires 451.2920.
N-(3-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)propyl)- 1,5-
dideoxy-1,5-imino-D-glucitol (37): [α]D = 36 (c= 0.5, MeOH); tR
1
= 22.5 (conditions 2); H NMR (300 MHz, D2O) δ = 7.95 (1H, s,
CdCHN), 7.31 (2H, d, J = 8.0 Hz, 2 ꢁ Har), 7.03ꢀ6.91 (3H, m, 3 ꢁ
Har), 5.16 (2H, s, OCH2), 4.41ꢀ4.34 (2H, m, 2 ꢁ H-30), 3.78ꢀ3.68
(2H, m, 2 ꢁ H-6), 3.52 (1H, m, dt, J1b,2 = J2,3 = 9.7 Hz, J1a,2 = 4.6 Hz,
H-2), 3.35 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4), 3.27 (1H, t, J2,3 = J3,4, H-3),
2.90 (1H, dd, J1a,1b = 11.1 Hz, H-1a), 2.75ꢀ2.50 (2H, m, 2 ꢁ H-10),
2.24ꢀ2.17 (2H, m, H-1b, H-5), 2.03 (2H, quint, J = 7.2 Hz, 2 ꢁ H-20);
13C NMR (75 MHz, D2O): δ = 157.2 (OCar), 143.4 (NCdCH), 129.9
(2 ꢁ CHar), 125.1 (NCHdC), 122.0 (CHar), 115.3 (2 ꢁ CHar), 78.1
(C-3), 69.2 (C-4), 68.7 (C-2), 64.7 (C-5), 61.0 (OCH2), 57.2 (C-6),
55.4 (C-1), 48.5 (C-10), 48.2 (C-30), 23.8 (C-20); HRMS (ES+) found
379.1985, C18H27N4O5 requires 379.1981.
N-(3-(4-(3,4-Dichlorophenoxymethyl)-1H-1,2,3-triazol-1-yl)-
propyl)-1,5-dideoxy-1,5-imino-D-glucitol (38). To a solution of
the azido derivative 6 (15 mg, 0.061 mmol) and the alkyne 31 (14 mg,
0.067 mmol) in dioxaneꢀH2O (2 mL, 4:1) were added CuSO4 (15 mg,
0.0915 mmol) and sodium ascorbate (36 mg, 0.183 mmol), and the
mixture was stirred under microwave irradiation at 80 ꢀC for 45 min. The
mixture was diluted with EtOAc (10 mL) and washed with NH4Cl satd
solution (2 ꢁ 8 mL). The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was residue
purified by preparative HPLC (conditions 2) to afford 38 (16 mg,
59%): [α]D = ꢀ5 (c = 0.3, MeOH); tR = 28.7 (conditions 2); 1H NMR
(500 MHz, D2O) δ = 7.89 (1 H, s, CdCHN), 6.97 (1 H, s, Har),
6.88ꢀ6.84 (2 H, m, 2 ꢁ Har), 5.04 (2 H, s, OCH2), 4.28 (2 H, s, 2 ꢁ
H-30), 3.78ꢀ3.69 (2 H, m, 2 ꢁ H-6), 3.52ꢀ3.58 (1 H, m, H-2), 3.40
(1 H, t, J3,4 = J4,5 = 9.3 Hz, H-4), 3.25 (1 H, t, J2,3 = J3,4 = 9.3 Hz, H-3),
2.91ꢀ2.98 (1 H, m, H-1a), 2.77ꢀ2.70 (1 H, m, H-10a), 2.58ꢀ2.50 (1 H,
4-[4-Adamantylphenoxymethyl]-1-[3-(2,3,4,6-tetra-O-
benzyl-1,5-dideoxy-1,5-imino-D-glucitol-N-yl)propyl]-1,2,3-
m, H-10b), 2.17 (1 H, t, J1a,1b = J1b,2 = 10.3 Hz, H-1b), 2.14 (1 H, d, J4,5
=
7765
dx.doi.org/10.1021/jo201125x |J. Org. Chem. 2011, 76, 7757–7768

The Journal of Organic Chemistry
ARTICLE
1
triazole (42): [α]D = +6 (c = 0.3, CH2Cl2); H NMR (300 MHz,
CDCl3) δ = 7.48 (1 H, s, CdCHN), 7.33ꢀ7.16 (22 H, m, 22 ꢁ Har),
6.94 (2 H, d, J = 8.9 Hz, Har), 5.28 (2 H, s, OCH2), 4.98ꢀ4.80 (3 H, 3 d,
J = 11.0, 10.9, 11.0 Hz, CH2Ph, CHHPh), 4.65ꢀ4.59 (2 H, 2 d, J = 11.6,
11.6 Hz, CH2Ph), 4.46ꢀ4.35 (3 H, m, CH2Ph, CHHPh), 4.28ꢀ4.21
(2 H, m, 2 ꢁ H-30), 3.64ꢀ3.50 (5 H, m, H-2, H-3, H-4, 2 ꢁ H-6), 3.00
(1 H, dd, J1a,1b = 11.2 Hz, J1a,2 = 4.6 Hz, H-1a), 2.86ꢀ2.77 (1 H, m,
H-10a), 2.60ꢀ2.46 (1 H, m, H-10b), 2.32 (1 H, d, J4,5 = J5,6 = 8.7 Hz,
H-5), 2.17ꢀ2.04 (4 H, m, H-1b, 3 ꢁ CH), 2.00 (2 H, m, J = 7.2 Hz, 2 ꢁ
H-20), 1.89 (6 H, d, J = 2.6 Hz, 3 ꢁ CH2) 1.82ꢀ1.72 (6 H, m, 3 ꢁ CH2);
13C NMR (75 MHz, CDCl3) δ = 156.2 (OCar), 144.4 (NCdCH),
139.0, 138.5, 137.8 (Car), 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7,
127.6 (CHar), 126.0 (2 ꢁ CHar), 123.0 (NCHdC), 114.3 (2 ꢁ CHar),
87.2 (C-3), 78.6, 78.4 (C-2, C-4), 75.4, 75.3, 73.3, 72.9 (CH2Ph), 66.4
(C-6), 64.6 (C-5), 62.2 (OCH2), 54.5 (C-1), 49.2 (C-10), 48.5 (C-30),
43.4 (3 ꢁ CH2), 36.8 (3 ꢁ CH2), 35.7 (C), 29.1 (3 ꢁ CH), 26.1 (C-20);
HRMS (ES+) found 873.4977, C56H65N4O5 requires 873.4955.
N-(3-(4-(4-Adamantylphenoxymethyl)-1H-1,2,3-triazol-
1-yl)propyl)-1,5-dideoxy-1,5-imino-D-glucitol (43). Conven-
tional catalytic hydrogenation of compound 42 (40 mg, 0.0459 mmol)
was carried out with Pd(OH)2 (30 mg) in MeOHꢀ1 M HCl (3 mL, 1:1)
at 1 atm for 18 h. The catalyst was filtered over a pad of Celite and the
solvent was removed. The residue was purified by preparative HPLC
(conditions 2) to afford 43 (11 mg, 45%) as a white solid: [α]D = +55 (c
= 0.1, MeOH); tR = 38.0; 1H NMR (300 MHz, CD3OD) δ = 8.08 (1 H,
s, CdCHN), 7.28 (2 H, d, J = 8.9 Hz, 2 ꢁ Har), 6.94 (2 H, d, J = 8.9 Hz, 2
ꢁ Har), 5.15 (2 H, s, OCH2), 4.47 (2 H, t, J = 6.9 Hz, 2 ꢁ H-30), 3.82 (2
H, ddd, J = 12.7, 12.1, 3.4 Hz, 2 ꢁ H-6), 3.46 (1 H, dt, J1b,2 = J2,3 = 9.5 Hz,
J1a,2 = 4.8 Hz, H-2), 3.33 (1 H, t, J3,4 = J4,5 = 9.5 Hz, H-4), 3.23 (1 H, t, J2,3
= J3,4, H-3), 2.99 (1 H, dd, J1a,1b = 11.1 Hz, H-1a), 2.92ꢀ2.85 (1 H, m,
H-10a), 2.57ꢀ2.48 (1 H, m, H-10b), 2.17ꢀ2.08 (7 H, m, H-1b, H-5, 2 ꢁ
H-20, 3 ꢁ CH), 1.91 (6 H, d, J = 2.7 Hz, 3 ꢁ CH2), 1.85ꢀ1.75 (6 H, m, 3
ꢁ CH2); 13C NMR (75 MHz, CD3OD) δ = 158.3 (OCar), 146.3, 146.2
(NCdCH, Car), 127.7 (2 ꢁ CHar), 126.2 (NCHdC), 116.3 (2 ꢁ
CHar), 81.3 (C-3), 72.8 (C-4), 71.6 (C-2), 68.7 (C-5), 63.3 (OCH2),
60.4 (C-6), 58.5 (C-1), 50.4 (C-10), 49.4 (C-30), 45.4 (3 ꢁ CH2), 38.7
(3 ꢁ CH2), 37.5 (C), 31.4 (3 ꢁ CH), 27.9 (C-20); HRMS (ES+) found
513.3058, C28H41N4O5 requires 513.3077.
(c = 0.2, MeOH); tR = 11.2; 1H NMR (500 MHz, D2O) δ = 7.92 (1 H,
s, CdCHN), 4.89 (1 H, d, J1,2 = 1.4 Hz, H-100), 4.47 (2 H, quad, J = 6.8
Hz, 2 ꢁ H-30), 4.03ꢀ3.98 (1 H, m, OCHHCH2), 3.92 (1 H, s, H?),
3.86ꢀ3.81 (4 H, m, 2 ꢁ H-6, H-6a00, OCHHCH2), 3.76ꢀ3.71 (3H, m, 2
ꢁ H?, H-6b00), 3.65 (1 H, t, J = 9.3 Hz, H?), 3.57 (1 H, dt, J1b,2 = J2,3 = 9.4
Hz, J1a,2 = 4.9 Hz, H-2), 3.40 (1 H, t, J3,4 = J4,5 = 9.4 Hz, H-4), 3.35ꢀ3.32
(1 H, m, H?), 3.29 (1 H, t, J2,3 = J3,4, H-3), 3.06 (2 H, t, J = 6.0 Hz,
OCH2CH2), 3.00 (1 H, dd, J1a,1b = 11.0 Hz, J1a,2 = 4.5 Hz, H-1a),
2.85ꢀ2.77 (1 H, m, H-10a), 2.75ꢀ2.66 (1 H, m, H-10b), 2.37ꢀ2.30 (2 H,
m, H-1b, H-5), 2.15 (2 H, m, J = 6.8 Hz, 2 ꢁ H-20); 13C NMR (75.5 MHz,
D2O) δ = 145.8 (NCdCH), 125.0 (NCHdC), 100.0(C-100), 78.6 (C-3),
73.2 (C?), 71.0, 70.4 (C?), 70.3 (C-4), 69.1 (C-2), 67.0 (C?), 66.7
(OCH2CH2), 65.1 (C-5), 61.2 (C-600), 57.8 (C-6), 55.6 (C-1), 49.1 (C-
10), 48.8 (C-30), 25.6 (OCH2CH2), 24.3 (C-20); HRMS (ESI+) found
501.2169, C19H34N4O10Na requires 501.2173.
General Procedures for Inhibition Assay. The glycosidases
β-glucosidase (from bovine liver, cytosolic), α-galactosidase (from Asper-
gillus niger), α-galactosidase (from green coffee beans), β-glucosidase
(from almonds), amyloglucosidase (from Aspergillus niger), α-glucosidase
(from yeast), isomaltase (from yeast), naringinase (Penicillium decumbes),
β-mannosidase (from Helix pomatia), and α-mannosidase (from jack
bean) used in the inhibition studies, as well as the corresponding o- and
p-nitrophenyl glycoside substrates, were purchased from a commercial
supplier. Inhibitory potencies were determined by spectrophotometrically
measuring the residual hydrolytic activities of the glycosidases against
the respective o- (for β-glucosidase/β-galactosidase from bovine liver) or
p-nitrophenyl α- or β-D-glycopyranoside, in the presence of the corre-
sponding iminosugar derivative. Each assay was performed in phosphate
or phosphate-citrate (for α- or β-mannosidase or amyloglucosidase)
buffer at the optimal pH for each enzyme. The Km values for the different
glycosidases used in the tests and the corresponding working pHs
are listed herein: β-glucosidase (bovine liver), Km = 2.0 mM (pH 7.3);
α-glucosidase (yeast), Km = 0.35 mM (pH 6.8); β-glucosidase (almonds),
Km = 3.5mM(pH 7.3); α-galactosidase(coffeebeans), Km = 2.0mM(pH
6.8); amyloglucosidase (Aspergillus niger), Km = 3.0 mM (pH 5.5);
naringinase (Penicillium decumbes), Km = 2.7 mM (pH 6.8); β-mannosi-
dase (Helix pomatia), Km = 0.6 mM (pH 5.5); α-mannosidase (jack
bean), Km = 2.0 mM (pH 5.5). The reactions were initiated by addition of
enzyme to a solution of the substrate in the absence or presence of various
concentrations of inhibitor. After the mixture was incubated for 10ꢀ30
min at 37 ꢀC the reaction was quenched by addition of 1 M Na2CO3. The
absorbanceof theresulting mixture wasdeterminedat405 or505nm. The
Ki value and enzyme inhibition mode were determined from the slope of
LineweaverꢀBurk plots and double reciprocal analysis using a Microsoft
Office Excel 2003 program. Inhibition mode was competitive in all cases.
Cell Culture. HL60 cells and Gaucher lymphoblasts (N370S) were
cultured in RPMI1640 medium supplemented with 10% or 15% (v/v)
fetal bovine serum, respectively, 2 mM L-glutamine, 100 U/ml penicillin,
and 100 mg/mL streptomycin at 37 ꢀC and 5% CO2.
N-(3-(4-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxye-
thyl)-1H-1,2,3-triazol-1-yl)propyl)-2,3,4,6-tetra-O-benzyl-1,5-
dideoxy-1,5-imino-D-glucitol (44): [α]D = +30 (c = 0.2, MeOH); tR
1
= 22.2 (conditions 2); H NMR (300 MHz, D2O) δ = 7.90 (1 H, s,
CdCHN), 5.25ꢀ5.20 (2 H, m, H-200, H-300), 4.96 (1 H, d, J1,2 = 1.3 Hz,
H-100), 4.47 (2 H, t, J = 6.9 Hz, 2 ꢁ H-30), 4.32 (1 H, dd, J6a ,6b = 12.7 Hz,
0
00
000
0
0
0
0
J5 ,6a = 4.0 Hz, H-6a ), 4.09 (1 H, dd, J5 ,6b = 1.9 Hz, H-6b ), 3.97 (1 H,
m, OCHHCH2), 3.85ꢀ3.76 (3 H, m, 2 ꢁ H-6, OCHHCH2), 3.63ꢀ3.53
(3 H, m, H-2, H-400, H-500), 3.39 (1 H, t, J3,4 = J4,5 = 9.3 Hz, H-4), 3.27 (1
H, t, J2,3 = J3,4 = 9.3 Hz, H-3), 3.06ꢀ2.99 (3 H, m, H-1a, OCH2CH2),
2.92ꢀ2.62 (2 H, m, 2 ꢁ H-10), 2.40ꢀ2.25 (2 H, m, H-1b, H-5),
2.20ꢀ2.11 (11 H, 3 s, m, 3 ꢁ CH3, 2 ꢁ H-20), 2.04 (3 H, s, CH3);
13C NMR (75.5 MHz, D2O) δ = 173.5, 172.9, 172.8, 172.7 (CdO),
151.5 (NCdCH), 124.1 (NCHdC), 100.1 (C-100), 87.1 (C-3), 69.7, 69.6
(C-4,), 69.4 (), 68.6 (C-2), 68.0 (CHsucre), 66.7 (OCH2CH2), 65.6
(CHsucre), 64.9 (C-5), 61.9 (C-600), 57.1 (C-6), 55.1 (C-1), 48.8 (C-10),
48.3 (C-30), 25.1 (OCH2CH2), 24.0 (C-20), 20.2 (CH3); HRMS (ESI+)
found 669.2591, C27H42N4O14Na requires 669.2595.
β-Glucocerebrosidase Inhibition Assay. Human placental
β-glucocerebrosidase was isolated and partially purified from modified
procedure.49 Enzyme activity was measured in 50 μL of 5 mM 4-methy-
lumbelliferyl-β-glucoside in 0.1 M citrate phosphate buffer, pH 5.2
containing 0.25% sodium taurocholate, 0.1% TX100 at 37 ꢀC for 15ꢀ60
min. The reaction was stopped by the addition of 200 μL 0.5 M sodium
carbonate and the fluorescence measured at ex 350 nm, em 460 nm.
Inhibitory activity was generated for placental β-glucocerebrosidase
(Km for 4-MU-β-glucoside, 1.9 ( 0.3 mM) using 0.5 mM substrate
concentrations to obtain IC50 values.
β-Glucocerebrosidase Activation Assay. Gaucher lympho-
blasts (N370S) were cultured in the presence of various concentra-
tions of inhibitor (0ꢀ50 μM) for 3 days before β-glucocerebrosidase
activity was measured. Cells were washed twice in phosphate-
buffered saline, homogenized in water using a small dounce
N-(3-(4-(α-D-Mannopyranosyloxyethyl)-1H-1,2,3-triazol-1-
yl)propyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-
glucitol (45). To a solution of 44 (20 mg, 0.0301 mmol) in dry
MeOH (1 mL) was added methanolic NaMeO (1 M, 0.1 equiv per mol
of acetate). The reaction mixture was stirred at rt for 3 h, then
neutralized with Amberlite IRA-120 (H+) ion-exchange resin, concen-
trated, and the resulting residue was purified by preparative HPLC
(conditions 1) to afford 45 (10 mg, 65%) as a white solid: [α] D = +30
7766
dx.doi.org/10.1021/jo201125x |J. Org. Chem. 2011, 76, 7757–7768

The Journal of Organic Chemistry
ARTICLE
homogenizer, centrifuged at 800g for 5 min and the supernatant taken
for protein and ss-glucocerebrosidase activity. Protein concentration
was determined using the BCA assay (Pierce, UK) according to
manufacturer’s instructions. All enzyme activation measurements
were made using aliquots of homogenate and 5 mM 4-methylum-
belliferyl-β-glucoside in 0.1 M citrate phosphate buffer, pH 5.2
containing 0.25% sodium taurocholate, 0.1% TX100 as described
above. Bromoconduritol (500 μM to 2.5 mM) was added to some
enzyme activity determinations to confirm the specific hydrolysis of
substrate by β-glucocerebrosidase. Enzyme activation is defined as
the fold increase in enzyme activity (U/mg protein) in treated cells
compared to untreated cells.
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of compound and incubated at 37 ꢀC for 16 h. MTS reagent (20 mL) was
added to each well, and the plates were mixed and incubated at 37 ꢀC for
1 h. The absorbance was read at 490 nm using a UV max Kinetic
microplate reader to determine the amount of formazan product, which
is directly proportional to the number of living cells in culture.
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’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
compounds 15ꢀ30 and 36ꢀ45 and examples of Lineweaverꢀ
Burk plots for Ki determinations of 38 and 43. This material is
available free of charge via the Internet at http://pubs.acs.org.
(18) Overkleeft, H. S.; Renkema, G. H.; Neele, J.; Vianello, P.; Hung,
I. O.; Strijland, A.; van der Burg, A. M.; Koomen, G.-J.; Pandit, U. K.;
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Marel, G. A.; Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. J. Org.
Chem. 2007, 72, 1088–1097.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: mellet@us.es; terry.butters@bioch.ox.ac.uk; jose.kovensky@
u-picardie.fr; sebastien.gouin@univ-nantes.fr
(20) Pandey, G.; Dumbre, S. G.; Khan, M. I.; Shabab, M. J. Org.
Chem. 2006, 71, 8481–8488.
Present Addresses
^Universitꢀe de Nantes, CEISAM, Chimie Et Interdisciplinaritꢀe,
Synthꢁese, Analyze, Modꢀelisation, UMR CNRS 6230, UFR des
Sciences et des Techniques, 2, rue de la Houssiniꢁere, BP 92208,
44322 NANTES Cedex 3, France.
(21) Wennekes, T.; Meijer, A. J.; Groen, A. K.; Boot, R. G.; Groener,
J. E.; van Eijk, M.; Ottenhoff, R.; Bijl, N.; Ghauharali, K.; Song, H.;
O’Shea, T. J.; Liu, H.; Yew, N.; Copeland, D.; van den Berg, R. J.; van der
Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. J. Med. Chem. 2010,
53, 689–698.
(22) Sawkar, A. R.; Cheng, W.-C.; Beutler, E.; Wong, C.-H.; Balch,
W. E.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (24),
15428–15433.
(23) Benito, E.; García Fernꢀandez, J. M.; Ortiz Mellet, C. Expert
Opin. Ther. Patents 2011, 21, 885–903.
(24) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596. (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
(25) Ferreira, S. B.; Sodero, A. C. R.; Cardoso, M. F. C.; Lima, E. S.;
Kaiser, C. R.; Silva, F. P.; Ferreira, V. F. J. Med. Chem. 2010, 53, 2364–2375.
(26) Díaz, L. A.; Bujons, J.; Casas, J.; Llebaria, A.; Delgado, A. J. Med.
Chem. 2010, 53, 5248–5255.
’ ACKNOWLEDGMENT
This work was carried out with financial support from the
Centre National de la Recherche Scientifique, the Ministꢁere
Dꢀelꢀeguꢀe ꢁa l’Enseignement Supꢀerieur et ꢁa la Recherche, and the
Conseil Rꢀegional de Picardie. This study was also supported by
the Spanish Ministerio de Ciencia e Innovaciꢀon (SAF2010-
15670), the Fundaciꢀon Ramꢀon Areces, and the Junta de Anda-
lucía (P08-FQM-03711).
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