Synthesis and glycosidase inhibitory activity of pseudo-di-(or tri-)saccharides

Article abstract of DOI:10.1016/S0040-4020(03)00257-6

The synthesis of pseudo-di-(or tri-)saccharides with a D-mannoazepane or a L-gulopiperidine skeleton either N-linked to a D-gluco-C-furanoside or to D-mannitol, and evaluation of their inhibitory activities against α- or β-D-glucosidase, α-D-mannosidase a

Full text of DOI:10.1016/S0040-4020(03)00257-6

Tetrahedron 59 (2003) 2693–2700
Synthesis and glycosidase inhibitory activity
of pseudo-di-(or tri-)saccharides
*
` `
Isabelle McCort, Michele Saniere and Yves Le Merrer
´ ´
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite Rene Descartes, UMR 8601,
`
45 rue des Saints-Peres, 75270 Paris Cedex 06, France
Received 17 January 2003; revised 17 February 2003; accepted 18 February 2003
Abstract—The synthesis of pseudo-di-(or tri-)saccharides with a D-mannoazepane or a L-gulopiperidine skeleton either N-linked to a
D-gluco-C-furanoside or to D-mannitol, and evaluation of their inhibitory activities against a- or b-D-glucosidase, a-D-mannosidase and
a-L-fucosidase are described. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
sidases we wished to explore N-substituted iminosugars
linked to a sugar analog aglycon moiety by a non-
hydrolyzable bond.11 Modulation of the aglycon part was
carried out using the 1,6-dideoxy-1,6-imino-D-mannitol 1
and 1,5-dideoxy-1,5-imino-L-gulitol 2, parent iminosugars,
which have moderate selectivities and binding affinities
for a-L-fucosidase with Ki values of 2812 and 22 mM,
respectively.13 Simple affinity measurements would allow
us to identify any increased selectivity obtained by the
addition of an aglycon substituent. In that context, we have
focused on the azadisaccharides 3, 4 and their trisaccharides
analogs 5, 6 with a D-gluco-C-furanoside or a D-mannitol
derived unit as aglycon parts, respectively (Fig. 2). To our
knowledge, neither azepane nor tetrahydrofuran have been
reported in the elaboration to pseudosaccharides. We report
here full results concerning the synthesis of these com-
pounds and evaluation as glycosidase inhibitors.
The implication of interactions between cell surface
carbohydrates and protein receptors in many important
biological events has focused attention on carbohydrate
mimics in recent years.1,2 Glycosidases are enzymes
involved in the biosynthesis of such glycoproteins, as well
as several other important processes such as the catabolism
of glycoconjugates and digestion. Inhibitors of these
enzymes have potential in the treatment of viral infections,
cancer, diabetes and other metabolic disorders.3 Interesting
naturally occurring glycosidase inhibitors are iminosugar
analogs (Fig. 1) such as DMDP (2,5-bis(hydroxymethyl)-
3,4-dihydroxy-pyrrolidine) and DNJ (1-deoxynojirimycin).3
However, a significant deterrent to the use of iminosugars as
therapeutic tools is the fact that they often inhibit a broad
range of glycosidases leading to detrimental side effects.
Recent research has shown that the affinity of an iminosugar
can be significantly increased by inclusion of an appropriate
aglycon moiety,4 for example MDL 25,6375 is a potent
specific glycosidase inhibitor and an antidiabetic drug lead.6
2. Results and discussion
Recently, we have delineated a convenient access to
polyhydroxylated heterocycles from C2-symmetrical bis-
epoxides A (3,4-O-acetonide) and B (3,4-di-O-benzyl)
In order to expand on the newer generation of stable
inhibitors7–10 with increased specificity for selected glyco-
Figure 1.
Keywords: pseudo-saccharides; glycosidases; mannitol; azepane; piperidine; C-furanoside.
*
Corresponding author. Tel.: þ33-1-42-86-21-77; fax: þ33-1-42-86-83-87; e-mail: yves.le-merrer@biomedicale.univ-paris5.fr
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00257-6

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I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
Figure 2.
derived from D-mannitol. From these compounds, after
opening of a first epoxide moiety at the least substituted
site, three different evolutions can occur: (i) opening at the
second epoxy function of A by an other equivalent of
nucleophile resulting in a C2-symmetric acyclic compound;
(ii) O-cyclization according to a 5-exo-tet process mainly
with bis-epoxide B giving tetrahydrofuran skeleton;
(iii) nucleophile-cyclization after acid–base exchange
between the alkoxide and the introduced nucleophile
affording a 6- and 7-membered ring according to the
regioselectivity of the second epoxide opening (A or B).
Taking advantage of this flexible strategy which allows
access to a great variety of carbohydrate mimics, such as
imino-,13 thio-,14 guanidino-15 and aminothiazolino16
-sugars and amino-methyl-C-furanoside17 derivatives, dis-
playing various configurations, our efforts were now
directed towards the synthesis of the pseudo-aza-di-(or tri-)-
saccharides (Fig. 2).
On one hand, elaboration to the azadisaccharides 3 and 4
required the N-cyclization of the flexible 3,4-di-O-benzyl
D-manno bis-epoxide B by the primary amino function of
the aminomethyl-C-furanoside 8. The latter resulted from
the O-cyclization of the bis-epoxide B by the azide ion
followed by reduction. On the other hand, elaboration to
azatrisaccharides 5 and 6 involved the bis-nucleophile
Scheme 1. Reagents and conditions: (i) NaN3, SiO2, CH3CN, K, 48 h, 95%; (ii) Ac2O, DMAP, 2 h for 7b, 91% or TBDMSCl, imidazole, DMF, 15 h for 7c,
95%; (iii) H2, Pd black in EtOAc for 8a (94%), 8b (65%), 8c (95%), or in AcOH for 9 (90%).

I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
2695
Scheme 2. Reagents and conditions: (i) Bu4NF, THF, rt, 10 h, for 12 (85%), 13 (80%); (ii) H2, Pd black, AcOH, for 3 (70%), for 4 (75%).
opening of the more rigid 3,4-O-acetonide D-manno bis-
epoxide A by the secondary heterocyclic amine having an
azepane or a piperidine skeleton 1 and 2, respectively.
These heterocyclic amines 1 and 2 are the result of the
N-cyclization of the bis-epoxide B by benzylamine and
subsequent hydrogenolytic removal of both N,O-benzyl
protecting groups.
Aminocyclization of a second bis-epoxide B with the
primary amine function of 8a, 8b or 8c has been studied in
methanol at room temperature. Although aminocyclization
cleanly occurred with all the amines 8, in the case of amino
derivatives 8a or 8b, we were not able to separate the
mixture of the corresponding adducts by flash chroma-
tography, even after peracetylation of the mixture. With the
silyl derivative 8c a mixture of N-methylfurano azepane 10
and N-methylfurano piperidine 11 was obtained, which
could be easily separated by flash chromatography in 40 and
30% yields, respectively (Scheme 2). Removal of the silyl
group of 10 or 11 with tetrabutylammonium fluoride
(TBAF) furnished 12 or 13 in 85 and 80% yields,
respectively. Finally, total hydrogenolysis of benzyl pro-
tecting groups in the presence of palladium black in acetic
acid gave the desired azadisaccharides 3 and 4, which were
isolated as their acetate salts after purification by flash
chromatography in 70 and 75% yields, respectively.
Synthesis of pseudo-azadisaccharides were achieved as
outlined in Schemes 1 and 2. Reaction of the bis-epoxide B
with sodium azide and silica gel under heating enabled the
one step preparation of the azidomethyl-D-gluco-C-furano-
side 7a in 95% yield (Scheme 1). The azide function was
cleanly reduced to an amine with dihydrogen in the presence
of palladium black in ethyl acetate to afford compound 8a in
which the primary alcohol function is free (94% yield).
Although the presence of this hydroxyl group does not
prevent the aminocyclization, we chose to protect the
primary alcohol function because we assume that the
separation of the mixture of 6 and 7-membered bicycles
could be difficult.
Elaboration to the azatrisaccharides 5 and 6 required the bis-
epoxide opening with the sterically hindered heterocyclic
amines 1 and 2. For this purpose, the reactivity of
piperidine, as a model, was evaluated on the 3,4-O-
acetonide- and 3,4-di-O-benzyl-D-manno-bis-epoxides A
and B, respectively (Scheme 3).
Protection of the primary alcohol function of 7a by an
acetate group (acetic anhydride in the presence of DMAP),
or by silyl ether group (tert-butyldimethylsilyl chloride and
imidazole) led to 7b or 7c in high yields. The azide function
was then reduced in the same conditions as above to give
amine 8b or 8c with 65 and 95% yields, respectively.
Furthermore, total reduction of 7a, by using acetic acid
instead of ethyl acetate as solvent, furnished the aminosugar
9 (90% yield).
Treatment of the O-acetonide bis-epoxide A, with piperi-
dine as solvent or with 2 equiv. of piperidine in methanol
at room temperature, furnished the acyclic C2-symmetric
1,6-bis-N-piperidino-1,6-dideoxy-D-mannitol derivative 14
in 90% yield. The flexible di-O-benzyl-bis-epoxide B with
piperidine, as reagent and solvent, at room temperature for 4
days led to the acyclic C2-symmetric derivative 15 (80%),
while with 2 equiv. of piperidine in refluxing acetonitrile the
cyclic N-piperidinomethyl-tetrahydrofuran 16 was isolated
in 85% yield. Interestingly, after nucleophilic opening of
bis-epoxide B by a secondary amine, O-cyclization occurred
only by a 5-exo-tet process: no tetrahydropyran (6-endo-tet
cyclization) was isolated after flash chromatography. From
bis-epoxide B, secondary amine induced regioselective
O-cyclization whereas primary amines (benzylamine,18
tryptamine19 and aminomethyl-C-furanoside 8) always led
to a 1:1 mixture of N-cyclization by 6-exo-tet and 7-endo-tet
processes.
Taking advantage of the mild experimental conditions with
2 equiv. of secondary amine in methanol at room tempera-
ture, we have carried out the nucleophilic opening of
the 3,4-O-acetonide-D-manno-bis-epoxide A by the
Scheme 3. Reagents and conditions: (i) neat piperidine, 4 days; (ii)
piperidine (2 equiv.), MeOH, 208C. (iii) piperidine (2 equiv.), CH3CN, K.

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I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
It should be noted on one hand, that the efficient strategy
described here could also be carried out from the L-ido bis-
epoxide as well as from other polyhydroxylated amines and
iminosugars in order to obtain a larger family of
polysaccharide analogs to study the glycosidase active
sites. On the other hand, the synthesis of pseudo-
azatrisaccharides could be achieved following the described
strategy for pseudo-azadisaccharides by repeating the
process after transforming the primary hydroxy group of
the tetrahydrofuran skeleton into a primary amine. Further-
more, a straightforward preparation of pseudo-azadi-
saccharides could be obtained after nucleophilic opening
of the 3,4-di-O-benzyl-D-manno-bis-epoxide B by secon-
dary amines thanks to a preference of O-cyclization
(5-membered ring) over N-cyclization (7-membered ring).
5. Experimental
Scheme 4. Reagents and conditions: (i) 1 or 2 (2 equiv.) MeOH, 12 h, 208C
for 17 or 18; (ii) CF3CO2H/H2O 4:1, 208C for 5 or 6.
5.1. General directions
polyhydroxylated azepane 1 and piperidine 2 (Scheme 4).
Under the previous conditions, each of these heterocyclic
secondary amines 1 or 2 cleanly reacted with the bis-
epoxide A to afford the protected azatrisaccharides 17 or 18,
respectively. Finally, hydrolysis of the acetonide protecting
group in the presence of trifluoroacetic acid at room
temperature, followed by purification by flash chroma-
tography gave access to the pseudo-trisaccharides 5 and 6 in
50% and 75% overall yields, respectively.
Prior to use, tetrahydrofuran (THF) and diethyl ether (Et2O)
were distilled from sodium–benzophenone and dichloro-
methane (CH2Cl2) from CaH2. CH2Cl2 and ethyl acetate
1
(EtOAc) were filtered on K2CO3 prior to use. H NMR
(250 MHz) and 13C NMR (63 MHz) spectra were recorded
on a Bruker AM 250. Chemical shifts (d) are reported in
ppm. IR spectra were recorded on a Perkin–Elmer 783
Infrared Spectrophotometer. Specific rotations were
measured on a Perkin–Elmer 241C polarimeter with
sodium (589 nm) or mercury (365 nm) lamp. Mass spectra
´
were recorded by the Service de Spectrometrie de Masse,
Ecole Normale Superieure, Paris. All reactions were carried
3. Inhibition studies
´
out under argon atmosphere, and were monitored by thin-
layer chromatography with Merck 60F-254 precoated silica
(0.2 mm) on glass. Flash chromatography was performed
with Merck Kieselgel 60 (200–500 mm); the solvent
systems were given v/v. Spectroscopic (1H and 13C NMR,
MS) and/or analytical data were obtained using chromato-
graphically homogeneous samples.
The new aza-di-(or tri-)saccharides 3–6 (Fig. 2) were
screened against four common glycosidases (a-D-gluco-
sidase, b-D-glucosidase, a-D-mannosidase and a-L-fucosi-
dase) following the protocol as previously reported.20
The pseudo-disaccharides 3 and 4 were totally inactive on
all the glycosidases studied (Ki¼1023 M) and consequently
less potent inhibitors than their parent iminosugars 1 and
2,12,13 respectively.
5.2. 2,5-Anhydro-6-azido-3,4-di-O-benzyl-6-deoxy-D-
glucitol (7a)
Whereas pseudo-trisaccharide 6 showed no inhibition
against all the 0 glycosidases, the 1,6-dideoxy-1,6-bis-N-
(10,60-dideoxy-1 ,60-imino-D-mannitol)-D-mannitol 5 dis-
played competitive and selective inhibition towards a-L-
fucosidase with a Ki¼15 mM; no inhibition was found at
1 mM on the other enzymes assayed.
To a solution of 1,2:5,6-dianhydro-3,4-di-O-benzyl-D-
mannitol B (1.0 g, 3.06 mmol) in acetonitrile (15 mL)
were successively added sodium azide (2.98 g,
45.8 mmol) and silica gel (3 g). After 48 h at reflux, the
mixture was cooled and concentrated in vacuo. Purification
by column chromatography (CH2Cl2/acetone, 97:3, Rf 0.3)
yielded the azidomethyl-D-gluco-C-furanoside 7a (1.07 g,
95%) as an oil. [a]2D0¼þ68 (c 0.9, CHCl3). IR (neat)
Thus, compound 5 has a Ki value in the same order of
magnitude of the azepane 1 of D-manno configuration but
the selectivity was increased towards a-L-fucosidase since
a-D-glucosidase was not inhibited, as by 1.12
nOH¼3440 cm21; nN3¼2120 cm21
;
1H NMR (CDCl3) d
2
3
3.37 (dd, J6a,6b¼12.8 Hz, J6a,5¼5.8 Hz, 1H, H-6a), 3.42
2
3
(dd, J6b,6a¼12.8 Hz, J6b,5¼5.1 Hz, 1H, H-6b), 3.84 (dd,
2J1a,1b¼12 Hz, 3J1a,2¼4.7 Hz, 1H, H-1a), 3.90 (dd, 2J1b,1a
¼
3
3
12 Hz, J1b,2¼5.5 Hz, 1H, H-1b), 3.95 (dd, J4,3¼1.8 Hz,
4. Conclusion
3J4,5¼3.5 Hz, 1H, H-4), 4.0–4.2 (m, 3H, H-5, H-3, H-2),
2
4.45, 4.59 (AB, JAB¼11.8 Hz, 2H, CH2Ph), 4.52, 4.55
We have synthesized azadi-(and tri-)saccharides which have
been evaluated as glycosidase inhibitors and shown that the
azatrisaccharide 5 displayed complete selectivity towards
a-L-fucosidase.
(AB, 2JAB¼11.9 Hz, 2H, CH2Ph), 7.2–7.4 (m, 10H, Harom);
13C NMR (CDCl3) d 52.5 (C-6), 61.7 (C-1), 72.1 (OCH2Ph),
80.9, 82.1, 83.6, 83.8 (C-2, C-3, C-4, C-5), 127.7, 128.2,
128.6, 128.7, 137.2, 137.4 (Carom). Anal. calcd for

I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
2697
1
C20H23N3O4: C, 65.03; H, 6.28; N, 11.37; found: C, 64.95;
H, 6.35; N, 11.53.
Rf 0.3). [a]2D0¼þ38 (c 0.98, CH2Cl2). H NMR (CDCl3)
d 2.0–2.3 (brs, 3H, NH2, OH exchangeable), 2.82
(dd, J6a,6b¼13.4 Hz, J6a,5¼5.6 Hz, 1H, H-6a), 2.93
2
3
2
3
5.3. Hydroxyl group protection of 7a
(dd, J6b,6a¼13.4 Hz, J6b,5¼3.9 Hz, 1H, H-6b), 3.8–3.9
(m, 3H), 3.95 (dd, 3J¼4.8, 2.3 Hz, 1H), 4.05–4.15 (m,
2
5.3.1. 1-O-Acetyl-2,5-anhydro-6-azido-3,4-di-O-benzyl-
6-deoxy-D-glucitol (7b). To a solution of 7a (100 mg,
0.271 mmol) in anhydrous CH2Cl2 (2 mL) was added
successively 4-dimethylaminopyridine (233 mg, 1.91 mmol)
and acetic anhydride (177.6 mL, 1.88 mmol). After stirring
2 h at room temperature, the mixture was hydrolyzed,
extracted with CH2Cl2 and dried (MgSO4). After evapo-
ration to dryness and purification by column chroma-
tography (cyclohexane/EtOAc, 8:2, Rf 0.2) compound 7b
(102 mg, 91%) was obtained as an oil. [a]2D0¼þ67 (c 1.4,
2H) 4.48, 4.60 (AB, JAB¼11.9 Hz, 2H, CH2Ph), 4.54,
2
4.60 (AB, JAB¼12.1 Hz, 2H, CH2Ph), 7.2–7.4 (m, 10H,
Harom).
5.4.2. 1-O-Acetyl-6-amino-2,5-anhydro-3,4-di-O-benzyl-
6-deoxy-D-glucitol (8b). Obtained with 65% yield after
purification by column chromatography (CH2Cl2/MeOH,
97:3, Rf 0.3). [a]2D0¼þ6 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 1.7–2.0 (brs, 2H, NH2 exchangeable), 2.05
(s, 3H, COCH3), 2.75–3.0 (m, 2H, H-6), 3.8–4.7 (m,
10H, H-1, H-2, H-3, H-4, H-5, CH2Ph), 7.1–7.4 (m, 10H,
Harom).
1
CH2Cl2). IR (neat) nN3¼2100 cm21, nCO¼1740 cm21; H
3
NMR (CDCl3) d 2.05 (s, 3H, COCH3), 3.37 (d, J6,5
¼
5.8 Hz, 2H, H-6), 3.90 (dd, 3J4,3¼1.2 Hz, 3J4,5¼2.9 Hz, 1H,
3
3
H4), 3.98 (dd, J3,2¼3.5 Hz, J3,4¼1.2 Hz, 1H, H-3), 4.07
5.4.3. 6-Amino-2,5-anhydro-3,4-di-O-benzyl-6-deoxy-1-
O-tert-butyldimethylsilyl-D-glucitol (8c). Obtained with
95% yield after purification by column chromatography
(CH2Cl2/MeOH, 97:3, Rf 0.3). [a]2D0¼þ55 (c 1.0, CH2Cl2).
1H NMR (CDCl3) d 0.07 (s, 6H, Si(CH3)2), 0.9 (s, 9H,
Si(CH3)3) 2.85 (m, 2H, H-6), 3.75–4.05 (m, 6H, H-1, H-2,
(dt, J5,6a¼3J5,6b¼5.8 Hz, J5,4¼2.9 Hz, 1H, H-5), 4.2–4.3
3
3
2
3
(m, 2H, H-2, H-1a), 4.41 (dd, J1b,1a¼14.7 Hz, J1b,2
¼
7.3 Hz, 1H, H-1b), 4.45 (s, 2H, CH2Ph), 4.43, 4.56 (AB,
2JAB¼11.9 Hz, 2H, CH2Ph), 7.2–7.4 (m, 10H, Harom);
CIMS (NH3) m/z: 429 (Mþþ18).
2
H-3, H-4, H-5), 4.44, 4.48 (AB, JAB¼12 Hz, 2H, CH2Ph),
2
5.3.2. 2,5-Anhydro-6-azido-3,4-di-O-benzyl-6-deoxy-1-
O-tert-butyldimethylsilyl-D-glucitol (7c). To a solution of
7a (470 mg, 1.27 mmol) in dimethylformamide (6 mL) was
added successively imidazole (340 mg, 2.54 mmol) and
tert-butyldimethylsilyl chloride (385 mg, 2.54 mmol). After
stirring 15 h at room temperature, the mixture was
hydrolyzed with brine, extracted with CH2Cl2 and dried
(MgSO4). After evaporation to dryness and purification by
column chromatography (cyclohexane/EtOAc, 97:3, Rf 0.3)
compound 7c (584 mg, 95%) was obtained as an oil.
[a]2D0¼þ55 (c 1.0, CH2Cl2). 1H NMR (CDCl3) d 0.1 (s, 6H,
Si(CH3)2), 0.9 (s, 9H, Si(CH3)3), 3.32 (dd, 2J6a,6b¼12.6 Hz,
4.54, 4.56 (AB, JAB¼12.2 Hz, 2H, CH2Ph), 7.2–7.4 (m,
10H, Harom); 13C NMR (CDCl3) d 25.5 (SiCH3), 18.6
(SiC(CH3)3), 26.7 (SiC(CH3)3), 31.9 (C-6), 60.9 (C-1), 71.7,
72.0 (OCH2Ph), 81.8, 82.2, 83.6, 84.7 (C-2, C-3, C-4, C-5),
126.5, 127.6, 127.7, 128.4, 129.5, 137.7, 138.0 (Carom); MS
(CI, NH3) 458 (Mþþ1).
5.4.4. 6-Amino-2,5-anhydro-6-deoxy-D-glucitol (9).
Obtained with 90% yield after purification by column
chromatography (CH2Cl2/MeOH/H2O/AcOH, 7:3:0.6:0.3,
Rf 0.35). [a]2D0¼þ25 (c 1.0, H2O). H NMR (D2O) d 3.15
1
2
3
(dd, J6a,6b¼13 Hz, J6a,5¼3.5 Hz, 1H, H-6a), 3.24 (dd,
3J6a,5¼5.6 Hz, 1H, H-6a), 3.35 (dd, J6b,6a¼12.6 Hz,
2J6b,6a¼13 Hz, 3J6b,5¼6.5 Hz, 1H, H-6b), 3.85 (dd, 2J1a,1b
¼
2
2
3
2
3J6b,5¼6.2 Hz, 1H, H-6b), 3.81 (dd, J1a,1b¼10.2 Hz,
12 Hz, J1a,2¼4 Hz, 1H, H-1a), 3.94 (dd, J1b,1a¼12 Hz,
3J1a,2¼5.5 Hz, 1H, H-1a), 3.84 (m, 1H, H-4), 3.90 (dd,
3J1b,2¼7.5 Hz, 1H, H-1b), 4.0 (dd, J5,6a¼ 3.5 Hz, J5,6b
¼
3
3
2J1b,1a¼10.2 Hz, J1b,2¼6.7 Hz, 1H, H-1b), 3.95–4.05 (m,
6.5 Hz, 1H, H-5), 4.1–4.3 (m, 3H, H-2, H-3, H-4); 13C
NMR (D2O) d 44.5 (C-6), 63.0 (C-1), 79.0, 81.7, 84.0, 84.3
(C-2, C-3, C-4, C-5); MS (CI, NH3) 164 (Mþþ1).
3
3
3
2H, H-3, H-5), 4.09 (ddd, J2,1b¼6.6 Hz, J2,1a¼5.6 Hz,
2
3J2,3¼3.9 Hz, 1H, H-2), 4.44, 4.47 (AB, JAB¼12 Hz, 2H,
CH2Ph), 4.55 (s, 2H, CH2Ph), 7.2–7.4 (m, 10H, Harom); 13
C
NMR (CDCl3) d 25.5 (SiCH3), 18.1 (SiC(CH3)3), 25.7
(SiC(CH3) 3), 52.5 (C-6), 60.7 (C-1), 71.4, 71.9 (OCH2Ph),
81.8, 82.0, 83.9, 84.0 (C-2, C-3, C-4, C-5), 127.4, 127.6,
127.7, 128.2, 137.4 (Carom); CIMS (NH3) m/z: 501 (Mþþ18,
100%), 484 (Mþþ1, 30%).
5.5. Preparation of pseudo-azadisaccharides 3 and 4
5.5.1. N-[60-(20,50-Anhydro-30,40-di-O-benzyl-60-deoxy-10-
O-tert-butyldimethylsilyl-D-glucitol)]-3,4-di-O-benzyl-
1,6-dideoxy-1,6-imino-D-mannitol (10) and N-[60-(20,50-
anhydro-30,40-di-O-benzyl-60-deoxy-10-O-tert-butyldi-
methylsilyl-D-glucitol)]-3,4-di-O-benzyl-1,5-dideoxy-1,5-
imino-L-gulitol (11). To a solution of 8c (165 mg,
0.361 mmol) in methanol (2 mL) was added bis-epoxide B
(118 mg, 0.361 mmol). After stirring 8 days at room
temperature, evaporation to dryness and purification by
column chromatography (CH2Cl2/MeOH, 97:3) compounds
10 (85 mg, 40% yield, Rf 0.35) and 11 (113 mg, 30% yield,
Rf 0.3) were obtained.
5.4. General procedure for azido group reduction
To a suspension of Pd black (38 mg) in EtOAc (1 mL), was
added the compound (0.12 mmol) to be reduced in EtOAc
(1 mL). After stirring 1 h under hydrogen (1 atm) at room
temperature, the catalyst was filtered through a celite pad
and the solution was concentrated in vacuo. When acetic
acid was used instead of EtOAc, the benzyl ethers were
hydrogenolyzed.
Compound 10. [a]2D0¼þ13 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 0.06 (s, 6H, Si(CH3)2), 0.88 (s, 9H, Si(CH3)3),
2.77 (dd, 2J¼11.2 Hz, 3J¼6 Hz, 4H), 2.93 (dd,02J¼13.4 Hz,
3J¼3.5 Hz, 2H), 3.7–4.0 (m, 10H, H-10, H-2 , H-30, H-40,
5.4.1. 6-Amino-2,5-anhydro-3,4-di-O-benzyl-6-deoxy-D-
glucitol (8a). Obtained with 94% yield after purification
by column chromatography (CH2Cl2/MeOH, 9:1 then 8:2,

2698
I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
H-50, H-2, H-3), 4.42 (s, 2H, CH2Ph), 4.55 (m, 2H, CH2Ph),
the catalyst was filtered through a celite pad and the solution
was concentrated in vacuo. Purification was performed
by flash chromatography (CH2Cl2/MeOH/H2O/AcOH,
7:3:0.6:0.3, Rf 0.35, 70% yield) for compound 3 or
(CH2Cl2/MeOH/H2O/AcOH, 5:5:1:0.5, Rf 0.4, 75% yield)
for compound 4.
2
4.62, 4.75 (AB, JAB¼11.6 Hz, 4H, CH2Ph), 7.1–7.4 (m,
20H, Harom); 13C NMR (CDCl3) d 25.4 (SiCH3), 18.2
(SiC(CH3)3), 25.9 (SiC(CH3)3), 57.1 (C-1), 60.6, 62.0 (C-10,
C-60), 68.6 (C-2), 71.7, 72.2, 73.4 (OCH2Ph), 80.7,
81.7, 81.8, 85.6 (C-3, C-20, C-30, C-40, C-50), 127.7, 127.9,
128.4, 137.7, 138.0, 138.4 (Carom); MS (CI, NH3) 784
(Mþþ1).
5.5.6. N-[60-(20,50-Anhydro-60-deoxy-D-glucitol)]-1,6-
dideoxy-1,6-imino-D-mannitol, acetate (3). [a]2D0¼þ2 (c
Compound 11. [a]2D0¼þ8 (c 1.0, CH2Cl2). 1H NMR
1.0, D2O), [a] ¼þ4. 1H NMR (D2O) d 2.18 (s, 3H, CH3),
20
Hg
(CDCl3) d 0.04 (s, 6H, Si(CH3)2), 0.88 (s, 9H, Si(CH3)3),
2.37 (d, J¼11.3 Hz, 1H), 2.42 (dd, J¼11.7, 2.1 Hz, 1H),
2
3
0
0
0
0
0
2.83 (m, 2H, H-1) 2.91 (dd, J6 a,6 b¼14.1 Hz, J6 a,5
¼
¼
2.55–2.75 (m, 2H), 2.98 (brd, J¼10.8 Hz, 1H), 3.10 (d,
6.5 Hz, 1H, H-60a), 3.11 (dd, J6 b,6 a¼14.1 Hz, J6 b,5
J¼11.1 Hz, 1H), 3.7–4.0 (m, 6H, containing at 3.77 (dd,
2
3
0
0
0
5.6 Hz, 1H, H-60b), 3.22 (dt, J5,6a¼3J5,6b¼7.7 Hz,
3J5,4¼5.6 Hz, 1H, H-5), 3.5–3.65 (m, 2H, H-3, H-6),
3.7–4.05 (m, 9H, H-10, H-20, H-30, H-40, H-50, H-2, H-4,
H-6), 4.42 (s, 2H, CH2Ph), 4.55 (s, 2H, CH2Ph), 4.58, 4.64
2J¼11.9 Hz, J¼6.8 Hz, 1H)), 4.1–4.2 (m, 2H), 4.2–4.3
3
3
(m, 2H). 13C NMR (D2O) d 26.2 (CH3), 54.4, 59.1, 61.9,
63.9 (CH2), 79.9, 82.4, 83.2, 83.5, 83.6, 84.0, 87.3 (CH); MS
(FAB) 309 (Mþþ1).
2
(AB, JAB¼11.7 Hz, 2H, CH2Ph), 4.63, 4.68 (AB,
2JAB¼11.8 Hz, 2H, CH2Ph), 7.2–7.4 (m, 20H, Harom); 13C
NMR (CDCl3) d 25.4 (SiCH3), 18.3 (SiC(CH3)3), 25.9
(SiC(CH3)3), 50.3 (C-1), 57.8, 60.7 (C-6, C-60, C-10), 61.5
(C-5), 67.7 (C-2), 71.6, 72.1, 72.4, 73.2 (OCH2Ph), 75.5,
78.2 (C-3, C-4), 81.9, 82.8, 85.7 (C-20, C-30, C-40, C-50),
127.7, 128.4, 137.7, 138.1 (Carom); MS (CI, NH3) 784
(Mþþ1).
5.5.7. N-[60-(20,50-Anhydro-60-deoxy-D-glucitol)]-1,5-di-
deoxy-1,5-imino-L-gulitol, acetate (4). [a]2D0¼þ5 (c 1.0,
1
D2O). H NMR (D2O) d 1.98 (s, 3H, CH3), 3.28 (m, 1H),
3.4–4.4 (m, 15H), containing at 3.64 (dd, 2J¼14.3 Hz,
3J¼7.8 Hz, 1H)); MS (FAB) 309 (Mþþ1).
5.6. Nucleophilic opening of the bis-epoxide by
piperidine
5.5.2. General procedure for desilylation. Silyl ether 10 or
11 (0.13 mmol) was stirred during 10 h at room temperature
with a solution of tetrabutylammonium fluoride (1.5 equiv.)
in THF (2 mL, 1 M). After evaporation to dryness and
purification by column chromatography (CH2Cl2/MeOH,
96:4), compound 12 (85% yield, Rf 0.35) or 13 (80% yield,
Rf 0.4) was obtained.
5.6.1. 1,6-Dideoxy-1,6-di-N-piperidino-3,4-O-methylethyl-
A
idene-D-mannitol
(14).
D-manno-Bis-epoxide
(0.16 mmol) in 2 mL of piperidine was stirred during
4 days at room temperature. After evaporation to dryness
and purification by column chromatography (CH2Cl2/-
MeOH/ NH3, 8:2:0.3, Rf 0.5) compound 14 was obtained
with 90% yield. [a]2D0¼þ24 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 1.2–1.7 (m, 18H, containing at 1.31 (s, C(CH3)2),
CH2–CH2–CH2N), 2.2–2.7 (m, 12H, H-1, CH2–CH2N),
3.6–4.2 (m, 6H, H-2, H-3, OH); MS (CI, NH3) 357
(Mþþ1).
5.5.3. N-[60-(20,50-Anhydro-30,40-di-O-benzyl-60-deoxy-D-
glucitol)]-3,4-di-O-benzyl-1,6-dideoxy-1,6-imino-D-man-
1
nitol (12). [a]D20¼þ9 (c 1.0, CH2Cl2). H NMR (CDCl3) d
2.7–3.250(m, 6H, H-1, H-60), 3.7–4.2 (m, 10H, H-2, H-3,
H-10, H-2 , H-30, H-40, H-50), 4.4–4.8 (m, 8H, CH2Ph), 7.1–
7.4 (m, 20H, Harom); 13C NMR (CDCl3) d 57.8 (C-1), 61.6,
62.0 (C-60, C-10), 61.9 (C-2), 71.8, 71.9, 73.5 (OCH2Ph),
80.6, 81.8, 83.4, 85.1 (C-20, C-30, C-40, C-50, C-3), 127.6,
127.8, 128.2, 128.4, 137.3, 137.5, 138.4 (Carom); MS (CI,
NH3) 670 (Mþþ1).
5.6.2. 3,4-Di-O-benzyl-1,6-dideoxy-1,6-di-N-piperidino-
D-mannitol (15). D-manno-Bis-epoxide B (0.16 mmol) in
2 mL of piperidine was stirred during 4 days at room
temperature. After evaporation to dryness and purifi-
cation by column chromatography (CH2Cl2/MeOH/NH3,
95:5:0.15, Rf 0.35) compound 15 was obtained with 80%
5.5.4. N-[60-(20,50-Anhydro-30,40-di-O-benzyl-60-deoxy-D-
glucitol)]-3,4-di-O-benzyl-1,5-dideoxy-1,5-imino-L-guli-
yield. [a]2D0¼þ10.5 (c 1.0, CH2Cl2). H NMR (CDCl3) d
1
1.3–1.7 (m, 12H, CH2–CH2–CH2N), 2.2–2.7 (m, 12H,
H-1, CH2–CH2N), 3.69 (d, 3J3,2¼6.1 Hz, 2H, H-3), 3.8–4.2
1
tol (13). [a]2D0¼þ5 (c 1.0, CH2Cl2). H NMR (CDCl3) d
2
3
2
0
0
0
0
2.83 (m, 2H, H-1), 2.91 (dd, J6 a,6 b¼14.3 Hz, J6 a,5
¼
(m, 4H, H-2, OH), 4.66, 4.75 (AB, JAB¼11.4 Hz, 4H,
6.5 Hz, 1H, H-60a), 3.1–3.25 (m, 2H, H-600b, H-5), 3.5–3.65
(m, 02H, H-3, H-6a), 3.7–4.15 (m, 9H, H-1 , H-20, H-30, H-40,
H-5 , H-2, H-4, H-6b), 4.40–4.75 (m, 8H, CH2Ph), 7.15–
7.4 (m, 20H, Harom); 13C NMR (CDCl3) d 50.5 (C-1), 57.6,
58.2 (C-60, C-6), 61.9 (C-5), 67.7 (C-2), 71.9, 72.0, 72.5,
73.1 (OCH2Ph), 75.5, 78.0 (C-3, C-4), 80.5, 82.3, 83.7, 84.7
(C-20, C-30, C-40, C-50), 127.8, 128.5, 137.4, 137.5, 138.1
(Carom); MS (CI, NH3) 670 (Mþþ1); HRMS for C40H48O8N
calcd 670.3379; found 670.3372.
CH2Ph), 7.15–7.4 (m, 10H, Harom); MS (CI, NH3) 497
(Mþþ1).
5.6.3. 2,5-Anhydro-3,4-di-O-benzyl-6-deoxy-6-N-piperi-
dino-D-glucitol (16). A solution of D-manno-bis-epoxide B
(32.6 mg, 0.1 mmol) and piperidine (7 mL, 2 equiv.) in
acetonitrile (0.2 mL) was refluxed for 2 h. After concen-
tration in vacuo and purification by column chromatography
(CH2Cl2/MeOH/NH3, 98:2:1, Rf 0.3), compound 16 was
obtained with 85% yield. 1H NMR (CDCl3) d 1.2–1.7
(m, 6H, CH2–CH2–CH2N), 2.2–2.7 (m, 6H, CH2N, H-6),
3.7–4.4 (m, 6H, H-1, H-2, H-3, H-4, H-5) 4.5–4.7 (m, 4H,
CH2Ph); 13C NMR (CDCl3) d 23.9 (N–(CH2)2–CH2), 25.7
(N–CH2–CH2), 55.9 (N–CH2–CH2), 60.8, 61.6 (C-6,
5.5.5. General procedure for benzyl deprotection. To a
suspension of Pd black in acetic acid was added the
compound 12 or 13 to be reduced in acetic acid (w/w). After
stirring 3 days under hydrogen (1 atm) at room temperature,

I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
2699
C-1), 71.8, 71.2 (OCH2Ph), 79.8, 80.4, 84.0, 84.1 (C-2, C-3,
C-4, C-5), 127.4, 127.8, 128.1, 128.4, 137.6, 138.0 (Carom);
MS (CI, NH3) 412 (Mþþ1).
Inhibition studies were displayed as previously reported.20
Briefly, assays were run in 50 mM citrate-phosphate buffer,
pH 6.5, at 378C, using the corresponding p-nitrophenyl-a-
glycosides in a total volume of 0.2 mL. The amount of
enzyme in each assay was adjusted so that less than 10% of
the substrate would be consumed.
5.7. Preparation of pseudo-azatrisaccharides 5 and 6
5.7.1. General procedure for nucleophilic opening of A.
To a solution of bis-epoxide A (0.215 mmol) in methanol
(1 mL) was added azepane 1 or piperidine 2 (2 equiv.),
and the mixture was stirred during 12 h at room tempera-
ture. After evaporation to dryness and purification by
column chromatography (CH2Cl2/MeOH/NH3, 8:3:1) com-
pounds 17 or 18 were obtained in 70 or 95% yields,
respectively.
For Ki determinations, substrates were at a concentration
range of 0.2–5 KM and inhibitors were added to final
concentration between 1023 and 1028 M. The inhibition
studies were performed by prewarming the solutions 5 min
prior to the addition of enzyme to initiate the reaction. Each
enzymatic reaction was quenched with 3 mL of 0.2 M
Na2CO3 after an incubation time of 5 min. The concen-
tration of liberated p-nitrophenoxide was determined by
measuring the optical absorbance at 400 nm. Dissociation
constants for inhibitors were calculated in the absence or
presence of inhibitors according to the Lineweaver–Burk
method.
5.7.1.1. 1,6-Dideoxy-1,6-di-N-(10,60-dideoxy-10,60-
imino-D-mannitol)-3,4-O-methylethylidene-D-mannitol
(17). [a]2D0¼ þ22 (c 1.0, H2O). H NMR (D2O) d 1.49 (s,
1
6H, C(CH3)2), 2.6–3.15 (m, 12H, containing at 2.71 (dd,
3
2
2J¼13.4 Hz, J¼9.4 Hz, 2H) and at 2.87 (dd, J¼13.6 Hz,
3J¼6.8 H0z, 4H), H-1, H-10), 3.9–4.25 (m, 12H, H-2, H-3,
H-20, H-3 ); 13C NMR (D2O) d 29.1 (CH3), 59.4 (C-10), 63.9
(C-1), 71.9, 72.2, 75.4 (C-2, C-20, C-30), 82.9 (C-3), 113.5
(C(CH3)2); MS (CI, NH3) 513 (Mþþ1).
References
5.7.1.2. 1,6-Dideoxy-1,6-di-N-(10,50-dideoxy-10,50-
imino-L-gulitol)-3,4-O-methylethylidene-D-mannitol
1. Varki, A. Glycobiology 1993, 3, 97–130.
2. Chapleur, Y. Carbohydrate Mimics: Concepts and Methods;
Wiley/VCH: New York, 1998.
1
(18). [a]2D0¼ þ38 (c 1.0, D2O). H NMR (D2O) d 1.48 (s,
6H, C(CH3)2), 2.7–3.1 (m, 10H, containing at 2.75 (dd,
3
2
2J¼12.1 Hz, J¼8.2 Hz, 20H) an0d at 2.88 (dd, J¼13.9 Hz,
3J¼8.3 Hz, 2H), H-1, H-1 , H-5 ), 3.8–4.15 (m, 14H, H-2,
H-3, H-20, H-30, H-40, H-60); 13C NMR (D2O) d 29.1 (CH3),
54.5, 58.2, 61.5 (C-1, C-10, C-60), 63.4, 69.5, 71.5, 73.0 (C-2,
C-20, C-30, C-40, C-50), 83.2 (C-3), 113.4 (C(CH3)2); MS (CI,
NH3) 513 (Mþþ1).
3. Watson, A. A.; Fleet, G. W. J.; Asano, N.; Molyneux, R. J.;
Nash, R. J. Phytochemistry 2001, 56, 265–295, and references
therein.
4. Ashry, E. S. H.; Rashed, N.; Shobier, A. H. S. Pharmazie
2000, 55, 331–347, and references therein.
5. Anzeveno, P. B.; Creemer, L. J.; Daniel, J. K.; King, C.-H. R.;
Liu;, P. S. J. Org. Chem. 1989, 54, 2539–2542.
6. Rhinehart, B. L.; Robinson, K. M.; Liu, P. S.; Payne, A. J.;
Wheatley, M. E.; Wagner, S. R. J. Pharmacol. Exp. Ther.
1987, 241, 915–920.
5.7.2. General procedure for acetonide hydrolysis.
Compounds 17 or 18 were stirred during 12 h in an aqueous
solution of trifluoroacetic acid (1/4 v/v) at room tempera-
ture. After evaporation to dryness and purification by
column chromatography (CH2Cl2/MeOH/NH3, 8:7:2, com-
pounds 5 (Rf 0.2) or 6 (Rf 0.15) were obtained with 70 or
80% yields, respectively.
7. Dong, W.; Jespersen, T.; Bols, M.; Skrydstrup, T.; Sierks, M.;
Biochemistry 1996, 35, 2788–2795.
8. Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J. Am.
Chem. Soc. 1997, 119, 4856–4865.
5.7.2.1. 1,6-Dideoxy-1,6-di-N-(10,60-dideoxy-10,60-
imino-D-mannitol)-D-mannitol (5). [a]2D0¼þ6 (c 1.0,
9. Johns, B. A.; Johnson, C. R. Tetrahedron Lett. 1998, 39,
749–752.
1
2
3
D2O). H NMR (D2O) d 2.80 (dd, J¼13 Hz, J¼8.4 Hz,
2H), 2.95–3.15 (m, 10H), 3.85–4.05 (m, 6H, containing 0at
3.98 (brs, 4H)), 4.19 (m, 4H); 13C NMR (D2O) d 59.5 (C-1 ),
65.0 (C-1), 70.3, 74.8 (C-2, C-3), 71.7, 75.4 (C-20, C-30);.
MS (CI, NH3) 473 (Mþþ1).
10. Satome, C.; Kanie, Y.; Kanie, O.; Wong, C.-H. Bioorg. Med.
Chem. 2000, 8, 2249–2261, and references therein.
`
11. Le Merrer, Y.; Saniere, M.; McCort, I.; Dupuy, C.; Depezay,
J.-C. Tetrahedron Lett. 2001, 42, 2661–2663.
12. A Ki¼4.6 mM was reported: Wong, C.-H.; Halcomb, P. L.;
Ichikawa, Y.; Kajimoto, T. Angew. Chem. Int. Ed. Engl. 1995,
34, 521–546.
5.7.2.2. 1,6-Dideoxy-1,6-di-N-(10,50-dideoxy-10,50-
imino-L-gulitol)-D-mannitol (6). [a]2D0¼þ28 (c 1.0,
D2O). 1H NMR (D2O)
d
2.82 (dd, 2J¼12.3 Hz,
13. Le Merrer, Y.; Poitout, L.; Depezay, J.-C.; Dosbaa, I.;
Geoffroy, S.; Foglietti, M.-J. Bioorg. Med. Chem. 1997, 5,
519–533.
3J¼7.7 Hz, 2H), 2.9–3.1 (m, 8H), 3.75–4.05 (m, 10H),
4.05–4.15 (m, 4H); 13C NMR (D2O) d 56.9, 57.8, 62.8 (C-1,
C-10, C-60), 60.8 (0C-50), 64.9, 68.1, 68.4, 72.0, 72.8 (C-2, C-
3, C-20, C-30, C-4 ); MS (CI, NH3) 473 (Mþþ1).
14. Le Merrer, Y.; Fuzier, M.; Dosbaa, I.; Foglietti, M.-J.;
Depezay, J.-C. Tetrahedron 1997, 53, 16731–16746.
15. Le Merrer, Y.; Gauzy, L.; Gravier-Pelletier, C.; Depezay, J.-C.
Bioorg. Med. Chem. 2000, 8, 307–320.
5.8. Inhibition studies
16. Gauzy, L.; Le Merrer, Y.; Depezay, J.-C.; Damour-Barbalat,
D.; Mignani, S. Tetrahedron Lett. 1999, 40, 3705–3708.
17. Poitout, L.; Le Merrer, Y.; Depezay, J.-C. Tetrahedron Lett.
1995, 36, 6887–6890.
All enzymes, a-D-glucosidase from Bacillus stearothermo-
philus (EC 3.2.1.20), b-D-glucosidase from almonds (EC
3.2.1.21), a-D-mannosidase from jack beans (EC 3.2.1.24)
and a-L-fucosidase from bovine kidney (EC 3.2.1.51) and
all substrates were purchased from Sigma.
18. Poitout, L.; Le Merrer, Y.; Depezay, J.-C. Tetrahedron Lett.
1994, 35, 3293–3296.

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I. McCort et al. / Tetrahedron 59 (2003) 2693–2700
´
20. McCort, I.; Fort, S.; Dureault, A.; Depezay, J.-C. Bioorg. Med.
19. Damour, D.; Barreau, M.; Blanchard, J.-C.; Burgevin, M.-C.;
Doble, A.; Herman, F.; Pantel, G.; James-Surcouf, E.;
Vuilhorgne, M.; Mignani, S.; Poitout, L.; Le Merrer, Y.;
Depezay, J.-C. Bioorg. Med. Chem. Lett. 1996, 6, 1667–1672.
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