Synthesis, conformational analysis, and evaluation as glycosidase inhibitors of two ether-bridged iminosugars

Article abstract of DOI:10.1080/07328303.2011.630547

Two bicyclic iminosugars have been synthesized from polyhydroxylated azepanes by ring isomerization followed by debenzylative cyclization. Their conformations have been studied by NMR and molecular modeling and their glycosidase inhibition profiles were d

Full text of DOI:10.1080/07328303.2011.630547

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Synthesis, Conformational Analysis, and
Evaluation as Glycosidase Inhibitors of
Two Ether-Bridged Iminosugars
Bo Luo a , Filipa Marcelo b , Jérôme Désiré d , Yongmin Zhang a
, Matthieu Sollogoub a , Atsushi Kato c , Isao Adachi c , F. Javier
Cañada b , Jesús Jiménez-Barbero b & Yves Blériot a d
a UPMC Univ Paris 06 , Institut Parisien de Chimie Moléculaire (UMR
CNRS 7201) , FR 2769, C. 181, 4 place Jussieu, 75005 , Paris , France
b Centro de Investigaciones Biológicas, CSIC , 28040 , Madrid , Spain
c Department of Hospital Pharmacy , University of Toyama , 2630
Sugitani, Toyama , 930-0194 , Japan
d Université de Poitiers , UMR 6514, Laboratoire “Synthèse et
Réactivité des Substances Naturelles,” 4, avenue Michel Brunet,
86022 , Poitiers , France
Published online: 14 Dec 2011.
To cite this article: Bo Luo , Filipa Marcelo , Jérôme Désiré , Yongmin Zhang , Matthieu Sollogoub ,
Atsushi Kato , Isao Adachi , F. Javier Cañada , Jesús Jiménez-Barbero & Yves Blériot (2011) Synthesis,
Conformational Analysis, and Evaluation as Glycosidase Inhibitors of Two Ether-Bridged Iminosugars,
Journal of Carbohydrate Chemistry, 30:7-9, 641-654, DOI: 10.1080/07328303.2011.630547
To link to this article: http://dx.doi.org/10.1080/07328303.2011.630547
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Journal of Carbohydrate Chemistry, 30:641–654, 2011
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2011.630547
Synthesis, Conformational
Analysis, and Evaluation as
Glycosidase Inhibitors of Two
Ether-Bridged Iminosugars
Bo Luo,1 Filipa Marcelo,2 Je´roˆme De´sire´,4 Yongmin Zhang,1
Matthieu Sollogoub,1 Atsushi Kato,3 Isao Adachi,3
F. Ja v i e r C a n˜ada,2 Jesu´s Jime´nez-Barbero,2 and Yves Ble´riot1,4
1UPMC Univ Paris 06, Institut Parisien de Chimie Mole´culaire (UMR CNRS 7201), FR
2769, C. 181, 4 place Jussieu, 75005 Paris, France
2Centro de Investigaciones Biolo´gicas, CSIC, 28040 Madrid, Spain
3Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan
4Universite´ de Poitiers, UMR 6514, Laboratoire “Synthe`se et Re´activite´ des
Substances Naturelles,” 4, avenue Michel Brunet 86022 Poitiers, France
Two bicyclic iminosugars have been synthesized from polyhydroxylated azepanes by
ring isomerization followed by debenzylative cyclization. Their conformations have
been studied by NMR and molecular modeling and their glycosidase inhibition pro-
files were determined. They both adopt a chair conformation for the piperidine ring and
display weak inhibition on α-glucosidases.
Keywords Conformational analysis; Iminosugar; Glycosidase inhibitor; Glycomime-
tics
INTRODUCTION
Iminosugars, sugar mimics in which the endocyclic oxygen has been re-
placed by nitrogen, represent the most promising class of sugar analogs as
Received September 4, 2011; accepted October 5, 2011.
Address correspondence to Jesu´s Jime´nez-Barbero, Centro de Investigaciones
Biolo´gicas, CSIC, 28040 Madrid, Spain. E-mail: jjbarbero@cib.csic.es; or Yves Ble´riot,
UPMC Univ Paris 06, Institut Universitaire de France, Institut Parisien de Chimie
Mole´culaire (UMR CNRS 7201), FR 2769, C. 181, 4 place Jussieu, 75005 Paris,
France, and Universite´ de Poitiers, UMR 6514, Laboratoire “Synthe`se et Re´activite´
des Substances Naturelles,” 4, avenue Michel Brunet 86022 Poitiers, France. E-mail:
yves.bleriot@univ-poitiers.fr
641

B. Luo et al.
642
therapeutic agents.[1] Since the discovery of its most famous representative,
1-deoxynojirimycin (DNJ), a vast array of derivatives have been synthesized
and assayed on numerous pathologies.[2] Promising results have been obtained
in the field of cancer[3] and viral infections[4] including HIV,[5] but more signifi-
cantly, an N-hydroxyethyl analog, miglitol, was approved by the U.S. Food and
Drug Administration (FDA) in 1996 for the treatment of late-onset diabetes
(marketed as Glyset).[6] Additionally, an N-butyl derivative, NB-DNJ or miglu-
stat,[7] marketed as Zavesca, 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]
Most of the synthetic efforts have been directed toward five- and six-
membered as well as bicyclic iminosugars because of their natural origin
and/or therapeutic potency. Unnatural ether-bridged iminosugars have been
poorly investigated, although their restricted conformation could lead to inter-
esting biological activities. A dihydroxy-3,6-anhydro iminoheptitol 1 has been
independently reported by Kilonda[9] and Poitout.[10] Fuentes has described the
synthesis of a series of ether-bridged nortropane-like iminocyclitols 2 bearing
various substituents at C-1.[11] Interestingly, N-substituted derivatives 3 and
4 display analgesic and anti-inflammatory activities in mice and rats,[12] while
[2,2,2]-bicyclic iminosugar 5[13] shows weak inhibition on several glycosidases.
More recently, Wu disclosed the synthesis and biological evaluation of a se-
ries of N-substituted ether bridged bicyclic iminosugars 6a–d (Fig. 1).[14] We
report herein a skeletal rearrangement-based route to similar scaffolds and
their evaluation as glycosidase inhibitors.
RESULTS AND DISCUSSION
Our group has reported a new access to six-membered homoiminosugars
via the ring isomerization of polyhydroxylated azepanes.[15] In this sequence
chloromethyl piperidines were generated by ring contraction of polyhydroxy-
lated azepanes[16] and the chlorine atom was subsequently displaced with an
acetate in moderate yield to furnish target homoiminosugars after deprotec-
tion. In order to improve the substitution step, we investigated the formation
Figure 1: Structure of ether bridged bicyclic iminosugars 1–6.

Ether-Bridged Iminosugars
643
of the more reactive iodomethyl piperidine. Known azepane 7 was first treated
with mesyl chloride to yield the corresponding piperidine 8 (75% yield). Reac-
tion of 8 with NaI in DMF did not afford the corresponding iodinated piperi-
dine but the symmetrical bicyclic derivative 9 as the major product (64% yield).
Its formation can be tentatively explained by a halogen exchange to yield com-
pound A followed by an intramolecular iodine displacement by the benzyl ether
at C-6 to furnish intermediate B followed by O-debenzylation. Nitrogen partic-
ipation leading to a fused aziridinium-piperidine scaffold C can also take place
in this transformation. Final hydrogenolysis quantitatively furnished the bi-
cyclic piperidine 10 (Sch. 1). Simplification of the NMR spectra for compounds
9 and 10 supports their symmetrical structure. Similar cyclizations mediated
by iodine have been previously reported in sugar chemistry.[17] Compound 10
displays an interesting structure as it can be seen as a locked iminoxylitol,
an iminosugar with therapeutic potential in Gaucher disease.[18] It can also
be viewed as a calystegine[19] analog in which the two-carbon bridge locking
the iminosugar conformation has been replaced by a dimethyleneoxy tether
yielding a fused morpholine-iminosugar hybrid.
Scheme 1: Synthesis of bicycle 10.
A similar strategy was applied to the β-D-manno-like azepane 11. Ring
isomerization with mesyl chloride yielded the piperidine 12 (59% yield). Upon
treatment with NaI in DMF the bicyclic derivative 13 was obtained in 84%
yield. Unlike the formation of compound 10 involving the primary benzyl ether
at C-6, compound 13 results from a debenzylative cyclization involving the ben-
zyl ether at C-4. The regioselectivity of the cyclization can be rationalized by

B. Luo et al.
644
the short distance between the benzyloxy oxygen at C-4 and the carbon at C-1
(Sch. 2). Final hydrogenolysis furnished the bicycle 14, the structure of which
was studied and confirmed by NMR and molecular modeling (see conforma-
tional analysis section).
Scheme 2: Synthesis of bicycle 14.
Conformational Analysis
In order to confirm the structures of compounds 10 and 14 and deduce
their conformations in solution, NMR experiments and molecular mechanics
calculations were performed. The coupling constant analysis of iminosugar 10
indicates that it assumes a major conformation in solution that involves two
fused piperidine rings, both adopting a chair conformation. In particular, the
observed J5,6 (5.3 Hz) and J6,7b (2.0 Hz) values are only in accordance with the
calculated J values (4.6 Hz and 2.6 Hz, respectively) for such a chair/chair con-
formation and not with the other boat/chair, chair/boat, and boat/boat conform-
ers (Table 1). Moreover, the combination of a chair/chair conformation is also
the energetically preferred solution. Molecular dynamics studies were then
conducted using the same force field, using the optimized chair/chair confor-
mation (Fig. 2, left) as starting geometry. The MD simulations were carried
out over 10 ns at 298 K, with a 0.05 fs time step and a 5 ps equilibration step;
250 structures were sampled for further analysis (see Fig. 2, right). The MD
simulation showed that the chair/chair conformation is fairly stable and only
displays minor fluctuations around its basic structure.
The coupling constant analysis of iminosugar 14 indicates that the piperi-
dine ring adopts a chair conformation in solution (Fig. 3). In particular, the
observed J4,5 (4.6 Hz) and J5,6 (1.6 Hz) values agree well with the calculated

Ether-Bridged Iminosugars
645
Table 1: MM3∗-based relative energy values (kJ/mol) and calculated and
experimental vicinal coupling constant values (Hz) for the possible six-membered
ring conformations of 10
Conformation
(piperidine/morpholine)
Chair/
chair
Boat/
chair
Chair/
boat
Boat/
boat
Experimental
Relative energy (MM3∗)
J4,5 = J4,3 (Hz)
−41.2
7.1
−17.6
3.2
−21.1
7.4
0
4.3 and 1.9
7.2 and 8.0
2.3 and 8.1
7.2 and 8.7
9.1
5.3
<1.0
2.0
J5,6 = J3,2 (Hz)
4.6
8.2
4.5
J6,7a = J2,8a (Hz)
J6,7b = J2,8b (Hz)
0.9
2.6
0.8
2.7
5.2
8.6
Figure 2: Left: View of the global minimum geometry for iminosugar 10, as calculated by
MM3∗ molecular mechanics calculations. Right: Superimposition of 250 structures of 10,
sampled during the 10 ns MD simulation at 298 K and using the GB/SA solvent model for
water. Hydrogen atoms have been removed for sake of clarity (color figure available online).
Figure 3: Left: The boat conformation of 14, as calculated by MM3∗ molecular mechanics
calculations (MM). Right: The chair conformation of 14. The atomic numbering is also given
(color figure available online).

B. Luo et al.
646
Table 2: MM3∗-based relative energy values (kJ/mol) and calculated and
experimental vicinal coupling constant values (Hz) for the possible six-membered
ring conformations of 14
Conformation (piperidine)
Chair
Boat
Experimental
Relative energy (MM3∗)
J2,3 (Hz)
−13.2
1.8
0.0
1.7
3.8
1.1
0.6
0.9
7.9
4.5
11.0
1.0
3.1
J2,8a (Hz)
3.7
J2,8b (Hz)
1.1
<1.0
4.6
J4,5 (Hz)
4.9
J4,3 (Hz)
0.8
<1.0
1.6
J5,6 (Hz)
0.9
J6,7a (Hz)
6.6
6.1
J6,7b (Hz)
11.0
10.7
values (4.9 Hz and 0.9 Hz, respectively) for a chair conformation but not with
the ones corresponding to a boat conformer (Table 2).
Further evidence of the exclusive existence of this conformation in com-
pound 14 was confirmed by analysis of the NOE contacts. NOE values of
15% (H-7a/H-7b), 4% (H-6/H-7a), 4% (H-7b/H-8b), 1.5% (H-3/H-6), and 4.5%
(H-4/H-5) were obtained and the corresponding distances between key protons
calculated (Fig. 4, top right). The strong NOE observed between H-7b and H-8b
supports a chair conformation for the piperidine ring. Molecular dynamics
studies were then conducted with the same force field and using the optimized
chair conformation (Fig. 3, right) as starting geometry. The MD simulations
Figure 4: Left: 2D-NOESY (mixing time, 700 ms) of 14. Right top: The chair conformation of 14,
showing the key interproton distances (in green) observed in the NOESY spectrum. Right
bottom: Superimposition of 250 structures of 14, sampled during the 10 ns MD simulation
(MM3∗, 298 K, and using the GB/SA solvent model for water). Hydrogen atoms have been
removed for sake of clarity (color figure available online).

Ether-Bridged Iminosugars
647
Table 3: Concentration of iminosugars giving 50% inhibition of glycosidases (IC50
,
mM)
Enzyme
10
11
α-Glucosidase
Rice
177
344
50
Yeast
NIa (0%)b
β-Glucosidase
Almond
NI (15.8%)
NI (15.4%)
NI (30.2%)
NI (30.9%)
Bovine liver
α-Galactosidase
Coffee beans
β-Galactosidase
Bovine liver
306
NI (15.4%)
962
NI (12.2%)
NI (37.2%)
NI (0.4%)
NI (22.3%)
NI (23.1%)
NI (22.8%)
NI (1.5%)
NI (0%)
α-Mannosidase
Jack beans
NI (16.1%)
NI (27.9%)
NI (0%)
β-Mannosidase
Snail
α-L-Rhamnosidase
P. decumbens
α-L-Fucosidase
Bovine epididymis
β-Glucuronidase
Bovine liver
142
NI (7.1%)
NI (11.4%)
NI (3.1%)
NI (5.1%)
Trehalase
Porcine kidney
Amyloglucosidase
Aspergillus niger
β-Xylosidase
Aspergillus niger
NI (1.1%)
aNI: No inhibition (less than 50% inhibition at 1000 mM).
b(): inhibition % at 1000 mM.
cND: Not determined.
were carried out over 10 ns at 298 K, with a 0.05 fs time step and a 5 ps
equilibration step; 250 structures were sampled for further analysis (see Fig.
4, bottom right). Again, the global minimum geometry was conformationally
stable during the MD run, just displaying minor fluctuations around its basic
structure.
Glycosidase Inhibition
The two ether-bridged iminosugars 10 and 14 were assayed for their
inhibitory activity toward 14 commercially available glycosidases. They did
not inhibit the following enzymes at 1 mM concentration and optimal pH:
almond and bovine liver β-glucosidases, Jack bean α-mannosidase, Helix
pomatia snail β-mannosidase, P. decumbens α-L-rhamnosidase, bovine liver β-
glucuronidase porcine kidney trehalase, Aspergillus niger amyloglucosidase,

B. Luo et al.
648
and β-xylosidase. Compound 10 proved to be a weak inhibitor of rice α-
glucosidase (IC50 177 µM) and coffee bean α-galactosidase (IC50 306 µM),
while 14 was found to be a moderate inhibitor of yeast α-glucosidase (IC50 50
µM) and a weak inhibitor of rice α-glucosidase (IC50 344 µM), bovine liver
β-galactosidase (IC50 962 µM), and bovine epididymis α-L-fucosidase (IC50
142 µM) (see Table 3).
CONCLUSION
In conclusion, two bicyclic ether-bridged iminosugars 10 and 14 have been pre-
pared by ring isomerization of polyhydroxylated azepanes followed by deben-
zylative cyclization. They both adopt a chair-like spatial arrangement for the
polyhydroxylated piperidine ring, compound 10 displaying a 4C1 conformation
1
analogous to glucose, while 14 showed a C4 conformation analogous to man-
nose. Their glycosidase inhibition profile was determined, demonstrating that
they are weak inhibitors of α-glucosidases.
EXPERIMENTAL SECTION
General Methods
Melting points (m.p.) were determined with a Bu¨chi B-535 apparatus and
are uncorrected. Optical rotations were measured at 20 2◦C with a Perkin
Elmer Model 241 digital polarimeter, using a 10-cm, 1-mL cell. Chemical ion-
ization mass spectra (CI-MS ammonia) and fast atom bombardment mass
1
spectra (FAB-MS) were recorded on a JMS-700 spectrometer. H NMR and
13C NMR were performed on a Bruker DRX 400 spectrometer (400 MHz for
1H, 100.6 MHz for 13C). All chemical shifts (δ) are given in ppm relative to
the residual deuterated solvent signals. Coupling constants (J) are reported in
Hertz. Reactions were monitored by thin-layer chromatography (TLC) using
Merck silica gel 60 F254 precoated plates and detection by charring with sulfu-
ric acid. Flash column chromatography was performed on silica gel Merck 60
(230–400 mesh).
(2S,3R,4R,6S,5S)-1-Benzyl-3,4,5-tris(benzyloxy)-2-
((benzyloxy)methyl)azepan-6-ol 7
Rf 0.26, Cy/EtOAc, 4:1; [α]D −7.8 (c = 1.1 in CHCl3); 1H NMR (CDCl3, 400
MHz): 7.38–7.27 (m, 25H, 5 × Ph), 4.77 (d, 1H, J = 11.0 Hz, CHPh), 4.73 (d, 1H,
J = 11.7 Hz, CHPh), 4.71 (d, 1H, J = 11.0 Hz, CHPh), 4.70 (d, 1H, J = 11.4 Hz,
CHPh), 4.60 (d, 1H, J = 11.7 Hz, CHPh), 4.52 (d, 1H, J = 12.0 Hz, CHPh), 4.50
(d, 1H, J = 11.4 Hz, CHPh), 4.47 (d, 1H, J = 12.0 Hz, CHPh), 4.05–3.95 (m,
5H, NCH2Ph, H-6, H-4, H-8a), 3.89 (dd, 1H, J = 6.8, 3.2 Hz, H-3), 3.80 (dd, 1H,

Ether-Bridged Iminosugars
649
J = 5.3, 2.1 Hz, H-5), 3.69 (dd, 1H, J = 9.8, 5.3 Hz, H-8b), 3.35 (m, 1H, H-
2), 3.20 (dd, 1H, J = 14.1, 6.9 Hz, H-7a), 2.76 (dd, 1H, J = 14.1, 3.6 Hz, H-
7b). 13C NMR (CDCl3, 100 MHz): 138.48, 138.43, 138.28, 138.21 (5 × Cipso),
128.46–126.85 (25 aromatic C), 83.66 (C-2), 82.16 (C-4), 81.44 (C-3), 73.96,
73.43, 72.95, 72.87 (4 × CH2Ph), 70.27 (C-1), 67.77 (C-6), 60.82 (C-5), 59.09
(NCH2Ph), 51.83 (C-7); ESI-HRMS: Calcd for C42H46NO5 (M + H+): 644.3376.
Found 644.3383.
(2S,3R,4S,5S,6S)-1-Benzyl-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-6-
(chloromethyl) piperidine 8
To a stirred solution of alcohol 7 (61 mg, 0.09 mmol) in dry DCM (2 mL)
was added Et3N (38 µL, 0.27 mmol) at 0◦C under an atmosphere of nitro-
gen, followed by addition of MsCl (11 µL, 0.138 mmol). The reaction mixture
was stirred at 0◦C for 1 h. TLC (Cy/EtOAc, 4:1) showed a complete reaction.
The reaction mixture was concentrated under reduced pressure. Purification
by preparative thin-layer plates (Cy/EtOAc, 4:1) gave chloromethyl piperidine
8 as a clear oil (45 mg, 75%).
1
Rf 0.73, Cy/EtOAc, 4:1; [α]D +18 (c = 1.4 in CHCl3); H NMR (CDCl3,
400 MHz): 7.40–7.30 (m, 25H, 5 × Ph), 4.98 (s, 2H, CH2Ph), 4.73 (d, 1H, J
= 11.2 Hz, CHPh), 4.62 (d, 1H, J = 11.2 Hz, CHPh), 4.61 (s, 2H, CH2Ph), 4.57
(d, 1H, J = 12.0 Hz, CHPh), 4.53 (d, 1H, J = 12.0 Hz, CHPh), 4.24 (s, 2H,
CH2Ph), 3.94 (dd, 1H, J = 11.2, 5.7 Hz, H-8a), 3.86 (dd, 1H, J = 10.0, 5.8
Hz, H-7a), 3.85 (app. t, 1H, J = 5.4 Hz, H-8b), 3.78–3.71 (m, 3H, H-5, H-3,
H-4), 3.65 (dd, 1H, J = 10.0, 7.0 Hz, H-7b), 3.60 (m, 1H, J = 5.5, 2.9 Hz, H-2),
3.48 (m, 1H, H-6); 13C NMR (CDCl3, 100 MHz): 139.95, 138.81, 138.32, 138.16
(5 × Cipso), 128.34–126.98 (25 aromatic C), 79.99 (C-4), 79.70 (C-5), 78.55 (C-
3), 75.08, 73.43, 72.98, 72.71 (4 × CH2Ph), 70.07 (C-7), 61.67 (C-2), 58.06 (C-
6), 57.97 (NCH2Ph), 43.61 (C-1); ESI-HRMS (M+H+): Calcd for C42H45O4NCl:
662.3037. Found: 662.3020.
(1S,5R,6S,7R,8R)-9-Benzyl-6,7,8-tris(benzyloxy)-3-oxa-9-aza-
bicyclo[3.3.1]nonane 9
A mixture of chloromethyl piperidine 8 (31 mg, 0.054 mmol) and NaI (33
mg, 0.216 mmol) in DMF (1 mL) was stirred at 130◦C for 3 h. The reaction
mixture was concentrated under reduced pressure. The resulting residue was
purified by preparative thin-layer plates (Cy/EtOAc, 4:1) to give bicyclic deriva-
tive 9 (15 mg, 64%) as an oil.
Rf 0.5, petroleum ether/EtOAc, 9:1; 1H NMR (CDCl3, 400 MHz): 7.40–7.26
(m, 20H, 4 × Ph), 4.96 (s, 2H, 2 × CHPh), 4.69 (d, 2H, J = 11.8 Hz, 2 × CHPh),
4.60 (dd, 2H, J = 11.8 Hz, 2 × CHPh), 4.55 (t, 1H, J = 8.6 Hz, H-7), 4.00
(d, 2H, J = 11.3 Hz, H-2a, H-4a), 3.93 (m, 4H, H-6, H-8, NCH2Ph), 3.74 (d,
2H, J = 11.2 Hz, H-2b, H-4b), 2.72 (d, 2H, J = 4.7 Hz, H-1, H-5); 13C NMR

B. Luo et al.
650
(CDCl3, 100 MHz): 139.36, 138.62, 138.50 (4 × Cipso), 128.44–127.27 (20 aro-
matic C), 84.67 (C-7), 80.26 (C-6, C-8), 75.56, 72.65 (3 × CH2Ph), 61.90 (C-
2, C-4), 55.68 (NCH2Ph), 54.43 (C-1, C-5); ESI-HRMS: Calcd for C35H38NO4
(M + H+): 536.2801. Found 536.2804.
(1S,5R,6S,7R,8R)-3-Oxa-9-aza-bicyclo[3.3.1]nonan-6,7,8-triol 10
To a solution of 9 (12 mg, 0.027 mmol) in MeOH (2 mL) containing 1 M aq.
HCl (20 µL) was added 10% Pd/C (5 mg). The suspension was stirred under
H2 atmosphere for 4 h at rt, filtered through a pad of Celite, and eluted with
MeOH. The solvent was removed under reduced pressure to afford compound
10 (5.6 mg, quant.) as its hydrochloride salt.
[α]D 0 (c = 0.3 in MeOH); 1H NMR (D2O, 400 MHz): 4.27–4.20 (m, 3H, J =
13.6, 9.2 Hz, H-4a, H-2a, H-7), 3.95 (dd, 2H, J = 9.2, 5.2 Hz, H-6, H-8), 3.86
(d, 2H, J = 13.6 Hz, H-4b, H-2b), 3.58 (d, 2H, J = 5.2 Hz, H-1, H-5); 13C NMR
(D2O, 100 MHz): 74.05 (C-7), 69.56 (C-6, C-8), 61.87 (C-2, C-4), 51.53 (C-1, C-5);
ESI-HRMS: Calcd for C7H14O4N (M + H+): 176.0917. Found 176.0914.
(2R,3R,4S,5R,6R)-1-Benzyl-3,4,5-tris(benzyloxy)-2-
((benzyloxy)methyl)azepane-6-ol 11
1
Rf 0.46, Cy/EtOAc, 4:1; [α]D +26 (c = 1.1 in CHCl3); H NMR (CDCl3, 400
MHz): 7.43–7.28 (m, 25H, 5 × Ph), 4.81 (d, 1H, J = 11.6 Hz, CHPh), 4.74
(d, 1H, J = 11.4 Hz, CHPh), 4.71 (d, 2H, J = 11.6 Hz, CH2Ph), 4.63 (d, 1H,
J = 11.4 Hz, CHPh), 4.55 (d, 1H, J = 12.0 Hz, CHPh), 4.50 (d, 1H, J =
12.0 Hz, CHPh), 4.49 (d, 1H, J = 11.4 Hz, CHPh), 4.19 (dd, 1H, J = 7.0, 4.2 Hz),
4.09 (d, 1H, J = 13.9 Hz, NCHPh), 4.07 (m, 1H), 3.91 (d, 1H, J = 14.0 Hz,
NCHPh), 3.85 (dd, 1H, J = 10.0, 5.9 Hz), 3.82 (dd, 1H, J = 6.5, 4.2 Hz), 3.80
(dd, 1H, J = 7.0, 4.4 Hz), 3.75 (dd, 1H, J = 10.0, 4.1 Hz), 3.43 (br. dd, 2H,
J = 10.8, 6.1 Hz), 3.33 (br. S), 3.03 (dd, 1H, J = 13.5, 2.8 Hz), 2.92 (dd,
1H,break; J = 13.5, 7.4 Hz); 13C NMR (CDCl3, 100 MHz): 139.92, 138.51,
138.40, 138.26, 137.85 (5 × Cipso), 128.65–126.90 (25 aromatic C), 85.55 (C-
2), 83.17 (C-3), 78.63 (C-4), 73.54, 73.20, 72.72, 72.51 (4 × CH2Ph), 68.55 (C-1),
67.32 (C-6), 62.42 (C-5), 59.19 (NCH2Ph), 52.70 (C-7); ESI-HRMS: Calcd for
C35H38NO4 (M + H+): 644.3376. Found 644.3369.
(2R,3R,4S,5R,6R)-1-Benzyl-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-6-
(chloromethyl) piperidine 12
To a stirred solution of alcohol 11 (38 mg, 0.59 mmol) in dry DCM (2 mL)
was added Et3N (25 µL, 0.177 mmol) at 0◦C under an atmosphere of nitrogen,
followed by addition of MsCl (7 µL, 0.088 mmol). The reaction mixture was
stirred at 0◦C for 1 h and was concentrated under reduced pressure. Purifica-
tion by preparative thin layer chromatography (Cy/EtOAc, 4:1) furnished the
chloropiperidine 12 (23 mg, 59%) as an oil.

Ether-Bridged Iminosugars
651
1
Rf 0.68, Cy/EtOAc, 4:1; [α]D −34 (c = 1.0 in CHCl3); H NMR (CDCl3, 400
MHz): 7.29–7.08 (m, 25H, 5 × Ph), 4.72 (d, 1H, J = 11.4 Hz, CHPh), 4.58 (ddd,
3H, J = 11.6, 11.3, 11.0 Hz, 3 × CHPh), 4.47 (dd, 2H, J = 11.6 Hz, 2 × CHPh),
4.21–4.13 (m, 4H, 2 × CHPh, NCHPh, H-5), 3.96–3.90 (m, 2H, NHPh, H-3),
3.77 (dd, 1H, J = 11.1, 6.7 Hz, H-7a), 3.68 (dd, 1H, J = 6.6, 2.6 Hz, H-4), 3.64
(dd, 1H, J = 9.3, 7.2 Hz, H-8a), 3.57–3.52 (m, 2H, H-7b, H-8b), 3.19–3.15 (m,
1H, H-6), 3.06–3.02 (m, 1H, H-2); 13C NMR (CDCl3, 100 MHz): 140.86, 138.73,
138.59, 138.49, 138.40 (5 × Cipso), 128.33–126.71 (25 aromatic C), 80.87 (C-4),
75.97 (C-5), 74.67 (C-3), 73.18, 72.87, 72.73, 72.47 (4 × CH2Ph), 70.09 (C-7),
63.67 (C-2), 61.25 (C-6), 57.87 (NCH2Ph), 45.13 (C-1); ESI-HRMS (M + H+):
Calcd for C42H45O4NCl: 662.3037. Found: 662.3025.
(1S,3R,4R,5R,8R)-2-Benzyl-4,8-bis(benzyloxy)-3-((benzyloxy)methyl)-6-oxa-2-
aza-bicyclo [3.2.1]octane 13
A mixture of chloropiperidine 12 (23 mg, 0.035 mmol) and NaI (21 mg,
0.139 mmol) in DMF (1 mL) was stirred at 130◦C for 3 h. The reaction mixture
was then concentrated under reduced pressure and the resulting residue puri-
fied by preparative TLC (Cy/EtOAc, 4:1) to give bicyclic compound 13 (16 mg,
84%) as an oil.
1
Rf 0.47, Cy/EtOAc, 4:1; [α]D −2 (c = 1.0 in CHCl3); H NMR (CDCl3, 400
MHz): 7.35–7.25 (m, 20H, 4 × Ph), 4.60 (dd, 2H, J = 12.0 Hz, 2 × CHPh), 4.54
(d, 1H, J = 12.0 Hz, CHPh), 4.49–4.39 (m, 5H, 3 × CHPh, H-5, H-4), 4.02 (dd,
1H, J = 8.6, 3.2 Hz, H-9a), 3.97 (s, 2H, NCH2Ph), 3.91 (d, 1H, J = 4.8 Hz,
H-8), 3.66 (d, 1H, J = 8.6 Hz, H-9b), 3.60–3.49 (m, 2H, H-7a, H-7b), 3.29 (d,
1H, J = 3.2 Hz, H-3), 3.04 (dd, 1H, J = 9.9, 4.8 Hz, H-1); 13C NMR (CDCl3, 100
MHz): 139.86, 138.74, 138.74, 138.43 (4 × Cipso), 128.88–127.38 (20 aromatic
C), 79.15 (C-5), 79.08 (C-8), 76.06 (C-4), 73.98 (C-7), 72.98, 71.40, 70.81 (3 ×
CH2Ph), 71.70 (C-9), 61.31 (C-3), 61.14 (NCH2Ph), 60.55 (C-1); ESI-HRMS:
Calcd for C35H38O4N (M + H+): 536.2795. Found 536.2796.
(1R,3R,4R,5R,8R)-3-(Hydroxymethyl)-6-oxa-2-azabicyclo[3.2.1]octane-
4,8-diol 14
To a solution of compound 13 (16 mg, 0.030 mmol) in MeOH (3 mL) con-
taining 1M aq. HCl (20 µL) was added 10% Pd/C (16 mg). The suspension was
stirred under H2 atmosphere for 4 h at rt, filtered through a Celite plug, and
eluted with MeOH. The solvent was removed under reduced pressure to afford
bicyclic derivative 14 (4.4 mg, 70%) as its hydrochloride salt.
[α]D +6 (c = 0.34 in MeOH); 1H NMR (D2O, 400 MHz): 4.55 (d, 1H, J = 1.4
Hz, H-8), 4.20 (d, 1H, J = 4.5 Hz, H-5), 4.04 (dd, 1H, J = 11.4, 3.4 Hz, H-7a),
3.96–3.93 (m, 2H, J = 11.4, 3.4 Hz, H-7b, H-1), 3.87 (dd, 1H, J = 4.5 Hz, H-4),
3.73 (dd, 1H, J = 12.1, 6.1 Hz, H-9a), 3.56 (dd, 1H, J = 12.1, 10.6 Hz, H-9b),
3.30–3.25 (dddd, 1H, J = 10.6, 6.1, 1.4 Hz, H-3); 13C NMR (D2O, 100 MHz):

B. Luo et al.
652
80.70 (C-5), 68.22 (C-8), 67.67 (C-4), 67.13 (C-7), 59.23 (C-9), 58.41 (C-3), 57.42
(C-1); ESI-HRMS: Calcd for C7H14O4N (M + H+): 176.0917. Found 176.0916.
Conformational analysis
NMR experiments were performed on a Bruker AVANCE 500 spectrometer
at 298 K and using D2O as solvent. NMR assignments were performed using
standard COSY and NOESY experiments. The obtained NMR parameters (J
and NOE data) were compared to those expected for the possible geometries
of the molecules. Thus, all possible arrangements for the fused six-membered
rings were investigated using Macromodel 9.6[20] as implemented in the
Maestro suite of programs (version 8.5.110).[21] The conformational search cal-
culations were performed using the MM3∗ force field, with the GB/SA water
solvent model[22] at 298 K. The general PRCG (Polak-Ribiere Conjugate Gra-
dient) method for energy minimization was applied for a maximum iteration
number of 5000 or a convergence threshold of 0.05. For all the obtained ge-
ometries, the expected vicinal coupling constants were estimated from the cal-
culated torsion angles by using the empirical Karplus equation proposed by
Haasnoot et al.[23] and compared to those obtained experimentally.
Glycosidase inhibition assay
For rice α-glucosidase and rat intestinal maltase activities, the reaction
mixture contained 25 mM maltose and the appropriate amount of enzyme,
and the incubations were performed for 10 to 30 min at 37◦C. The reac-
tion was stopped by heating at 100◦C for 3 min. After centrifugation (600 g;
10 min), 0.05 mL of the resulting reaction mixture was added to 3 mL of
the Glucose CII-test Wako (Wako Pure Chemical Ind., Osaka, Japan). The ab-
sorbance at 505 nm was measured to determine the amount of the released
D-glucose. Other glycosidase activities were determined using an appropriate
p-nitrophenyl glycoside as substrate at the optimum pH of each enzyme. The
reaction mixture contained 2 mM of the substrate and the appropriate amount
of enzyme. The reaction was stopped by adding 2 mL of a 400-mM Na2CO3 so-
lution. The released p-nitrophenol was measured spectrometrically at 400 nm.
ACKNOWLEDGMENTS
Bo Luo is the recipient of a CSC Fellowship.
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