The spirocyclopropyl moiety as a methyl surrogate in the structure of l-fucosidase and l-rhamnosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.10.010

Nitrogen-in-the-ring analogues of l-fucose and l-rhamnose were prepared, which feature a spirocyclopropyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a titanium-mediated aminocyclopropanation of a glycononitrile

Full text of DOI:10.1016/j.bmc.2009.10.010

Bioorganic & Medicinal Chemistry 17 (2009) 8020–8026
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The spirocyclopropyl moiety as a methyl surrogate in the structure
of L-fucosidase and L-rhamnosidase inhibitors
Morwenna S. M. Pearson a, Nicolas Floquet b, Claudia Bello c, Pierre Vogel c, Richard Plantier-Royon a,
Jan Szymoniak a, Philippe Bertus d, Jean-Bernard Behr a,
*
a Université de Reims Champagne-Ardenne, Institut de Chimie Moléculaire de Reims, CNRS UMR 6229, UFR des Sciences Exactes et Naturelles, BP 1039, 51687 Reims Cedex 2, France
b Institut des Biomolécules Max Mousseron (I.B.M.M.), CNRS UMR 5247, Université Montpellier 1, Université Montpellier 2, Faculté de Pharmacie, 15 rue Charles Flahault,
BP 14491, 34093 Montpellier Cedex 5, France
c Laboratoire de Glycochimie et Synthèse Asymétrique, Ecole Polytechnique Fédérale de Lausanne (EPFL), BCH CH-1015 Lausanne, Switzerland
d Unité de Chimie Organique Moléculaire et Macromoléculaire, CNRS and Université du Maine, UMR 6011—UCO2M, 72085 Le Mans Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2009
Revised 28 September 2009
Accepted 6 October 2009
Available online 12 October 2009
Nitrogen-in-the-ring analogues of L-fucose and L-rhamnose were prepared, which feature a spirocyclo-
propyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a tita-
nium-mediated aminocyclopropanation of a glycononitrile as the key step. Four new spirocyclopropyl
iminosugar analogues were generated, which displayed some activity towards
rhamnosidase.
L-fucosidase and L-
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Iminosugars
Glycosidases
Inhibition
Spiro compounds
1. Introduction
The processing of L-fucose and L-rhamnose, that is, their transfer
to or their trimming from a glycoconjugate, is mainly assumed by
two classes of enzymes: glycosidases (EC 3.2.1) and glycosyltrans-
ferases (EC 2.4.1). Inhibition of these enzymes is an important task,
not only to understand the biological functions of 6-deoxyhexoses,
but also to develop new therapeutic agents.9 Iminosugars, which
feature the same hydroxyl configurations as the natural carbohy-
drate substrates, are generally found to interact strongly with the
Fucose and rhamnose are the most abundant 6-deoxyhexoses
found in nature.
drates from all organisms. In bacterial cells it is a constituent of
polysaccharides forming the cell wall.1 In animal cells,
-fucose is
L-Fucose is commonly found in complex carbohy-
L
a fundamental component of N- and O-linked glycoproteins or gly-
colipids, which play role in intercellular adhesion and recognition
processes.2 More specifically,
L
-fucose is present in ABH blood
group antigens and in some Lewis-type oligosaccharides.3 In sialyl
Lewisx, for instance,
-fucose plays a key role in the recognition by
corresponding glyco-enzymes.10 For instance,
L-fuconojirimycin
1,11 its deoxy analogue DFJ 212 or the pyrroline 313 (Fig. 1) are po-
L
tent inhibitors of fucosidase with Ki’s in the low lM to the nM
E-selectins present on endothelial cells, an event that mediates
leukocytes’ extravasation in tissues.4 In addition, fucosylated oligo-
saccharides could be involved in the pathogenesis of many dis-
range.
Some pyrrolidine derivatives bearing aromatic substituents
have also shown good inhibitory properties and good selectivity
towards
the lack of
(DRJ) 4, the piperidine analogue of
C-5 epimer of 4 (compound 5) displayed an astonishing strong ef-
fect on rhamnosidase. To rationalize these results, it was hypothe-
sized that the methyl group at C-5 in the structures of iminosugars
4 or 5 appears to modulate the inhibition by modifying the confor-
mation of the piperidine ring.15 By analogy, we attempted to con-
trol the conformation of 6-deoxyhexoses mimics by introducing a
constrained spirocyclopropyl moiety instead of the natural methyl
substituent.
eases.5,6
L
-Rhamnose is widely distributed in plants and bacteria
a-L
-fucosidases.14 An important exception to this rule is
as a component of the cell walls.7 Rhamnosides are part of various
natural bioactive compounds like cytotoxic saponins, plant gly-
coalkaloids and macrolides as well as terpenol or flavonoid glyco-
L
-rhamnosidase inhibition by deoxyrhamnojirimycin
L
-rhamnose.15 By contrast, the
sides.7d,e
L-Rhamnose is also a fundamental component of the
surface antigens of many microorganisms and has been identified
as a structural element of bacterial virulence factors.8
* Corresponding author. Tel.: +33 326 91 32 38; fax: +33 326 91 31 66.
E-mail address: jb.behr@univ-reims.fr (J.-B. Behr).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.010

M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
8021
H
N
Me Me
H3C
HO
O
OH
OH
H3C
H3C
HO
R
N
O
OH
OH
O
O
HO
CN
OH
HO
HO
OH
OH
OH
O
O
D-lyxose
1 : R = OH (Fuconojirimycin)
2 : R = H (DFJ)
L-Fucose
3
Me Me
a)
13
R1
H
R2
N
H3C
HO
H3C
HO
O
OH
OH
N
RO OR
OH
HO OH
CN
b)
N
HO
OH
OH
OH
OH
O
O
4 : R1 = H , R2 = CH3 (DRJ)
O
O
6
L-Rhamnose
Me Me
5 : R1 = CH3 , R2 = H
Me Me
15a : R = Piv (21% for 3 steps)
15b : R = Ac (35% for 3 steps)
15c : R = Bz (47% for 3 steps)
14
H
N
H
N
N
N
Scheme 2. Preparation of nitrile 13. Reagents and conditions: (a) (i) Me2CO, H2SO4,
rt, (ii) H2NOHꢁHCl, MeONa, MeOH, rt; (b) RCl, pyridine, 70 °C.
HO
OH
OH
HO
OH
HO
HO
OH
OH
10
OH
7
8
9
the selective protection of
D-lyxose as its 2,3-O-isopropylidene
derivative using standard protocols,19 followed by condensation
with hydroxylamine to give the oxime intermediate 14 in a quan-
titative manner. We next envisioned to dehydrate the crude oxime
14 by treatment with an excess of acyl chloride, to obtain the cor-
responding nitrile and to protect simultaneously the free hydroxyl
groups. Whereas the reaction of oxime 14 with pivaloyl chloride
proved difficult and afforded the target compound 15a in only
21% isolated yield, acetyl chloride was slightly more efficient and
afforded 15b in 35% yield. Best results were finally obtained with
benzoyl chloride, giving the dibenzoyl nitrile derivative 15c in
Figure 1. Structures of iminosugars 1–10.
We reported in a preliminary communication the synthesis of
spirocyclopropyl-DRJ 9, using a titanium-mediated aminocyclo-
propanation of nitriles as the key reaction (Scheme 1).16 Herein,
we report the extension of the method to the preparation of new
fucose and rhamnose analogues 7, 8, and 10 featuring such a spiro-
cyclopropyl moiety, as well as their inhibition potencies towards
glycosidases.
47% overall yield in three steps from D-lyxose.
2. Results
However, cyclopropanation of 15c with EtMgBr and MeTi(Oi-
Pr)3 did not afford the expected primary amine 16 (Scheme 3).
The N-benzoyl cyclopropylamine 17 was obtained instead as the
major compound in 49% yield. Obviously, a selective migration of
the C-4 O-benzoyl moiety to the so-formed primary amine has oc-
curred during the reaction. Such an acyl transfer has recently been
observed in the analogous reaction sequence applied to tetra-
2.1. Synthesis of spirocyclopropyl-DFJ 7
For the synthesis of compound 7, we envisioned to follow a se-
quence of reactions similar to that used for the synthesis of 9. The
starting sugar is
distribution to reach the targeted
product. However, the synthesis of the key intermediate 2,3:4,5-
D
-lyxose, which features the required hydroxyl
benzoyl-b-D
-glucosyl cyanide.16f Nevertheless, compound 17 was
L
-fuco configuration of the final
isolated in preparative amount and was used as a key intermediate
for the synthesis of spirocyclopropyl-DFJ 7. The complete removal
of the N- and O-benzoyl protecting groups was achieved under ba-
sic conditions in good yield (80%) to afford the partially protected
amine 18 (Scheme 4). On the basis of what was experienced in the
di-O-isopropylidene-D-lyxononitrile 13 via the known dithioace-
tal17 proved difficult and unsatisfactory yields were obtained when
using literature protocols (Scheme 2). To overcome these difficul-
ties, the preparation of a new fully protected lyxononitrile was at-
tempted. We turned our attention to the elaboration of acyl
L
-rhamno series,16a the direct cyclization of 18 to the correspond-
derivatives 15a–c, which could be readily obtained from D-lyxose.
ing piperidine was not attempted. N-Boc protection was conducted
first to preclude side-reactions during the treatment with an acti-
vating agent. Thus, the primary amine 18 was treated with Boc2O
to give compound 19 in 58% yield. The primary alcohol was then
selectively converted to the corresponding mesylate 20 by careful
addition of MsCl at ꢀ20 °C in the presence of NEt3. Simultaneous
deprotection of the Boc and acetal protecting groups were carried
Additionally, compounds 15 feature an orthogonal protection pat-
tern, which could simplify the last steps of the reaction sequence.
Benzoyl or pivaloyl protecting groups were previously demon-
strated to be compatible with selective and efficient cyclopropana-
tion of the nitrile functionality.18 Thus, synthesis of 15 started with
Me Me
Me Me
EtMgBr + MeTi(Oi-Pr)3
HO
O
O
O
O
O
OH
a)
CN
BzO OBz
BzO OBz
O
BzO OH
NH2
NH2
b)
5
9
CN
NHBz
steps
80%
(i-PrO)2Ti
THF
O
O
O
O
HO
OH
+
O
O
O
O
O
L-arabinose
Me Me
Me Me
then BF3•OEt2
Me Me
Me Me
Me Me
12
11
15c
16
17 (49%)
Scheme 1. Preparation of spirocyclopropyl-DRJ 9. Reagents: (a) (i) HS–(CH2)3–SH,
H+, (ii) Me2CO, H+, (iii) MeI, CaCO3, (iv) H2NOHꢁHCl, (v) MsCl, pyridine; (b) EtMgBr,
MeTi(Oi-Pr)3, then BF3ꢁOEt2.
(not observed)
Scheme 3. Cyclopropanation of nitrile 15c.

8022
M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
sponding aldehyde, which spontaneously cyclized to give hemia-
HO OH
HO OH
minal 23. Compound 23 proved stable at room temperature and
was purified by silica gel chromatography (EtOAc/petroleum ether,
9/1, v/v; 81% isolated yield). According to the 1H and 13C NMR spec-
tra (CDCl3), 23 appeared as a single anomer. The configuration at
the C-2 stereocenter was attributed according to the J2,3 coupling
constant (J2,3 ca. 0 Hz), which is characteristic of a trans relation-
ship between both hydrogen atoms for this series of compounds.21
Deprotection of 23 under acidic conditions (1 M HCl) and purifica-
tion by ion-exchange chromatography afforded pyrroline 8 in 61%
yield.
A similar sequence of reactions then allowed access to the epi-
meric pyrroline 10 which features the rhamnose-like configura-
tion. Oxidative cleavage of the diol 24, which was prepared
according to the literature protocol,16a gave the corresponding
aldehyde 25 in 90% yield. The trans configured isopropylidene pro-
tecting group in compound 25 probably hampers the spontaneous
cyclization at this stage. Nevertheless, complete deprotection of 25
(1 M HCl) followed by ion-exchange chromatography afforded the
expected pyrroline 10 in 90% isolated yield.
NH2
NHBoc
a)
b)
17
80%
58%
O
O
O
O
Me Me
Me Me
18
19
MsO OH
O
MsO OH
NHBoc
.
NH2
HCl
d)
c)
83%
O
HO OH
21
Me Me
20
H
N
NH2
O
+
HO
OH
HO
OH
OH
7 (31%)
22 (50%)
Scheme 4. Preparation of spirocyclopropyl-DFJ 7. Reagents and conditions: (a)
NaOH, EtOH–H2O, 80 °C, 24 h; (b) Boc2O, Et3N, THF, 50 °C, 24 h; (c) MsCl, Et3N,
CH2Cl2, ꢀ20 °C; (d) HCl 2 N, MeOH, 50 °C, 2 h then K2CO3.
2.3. Enzymatic assays
Iminosugars 7–10 were assayed against
ginase ( -rhamnosidase) and a panel of 12 other glycosidases
(Table 1). Spirocyclopropyl-DFJ 7 was a potent and very specific
inhibitor of -fucosidase (Ki = 18 M) whereas spirocyclopro-
-rhamnosidase (72% inhi-
a-L-fucosidase, narin-
a
-L
out with diluted hydrochloric acid. The so-obtained ammonium
salt 21 was next treated with a base to induce the required
intramolecular nucleophilic displacement to afford piperidine 7.
However, treatment of 21 with ammonia afforded the C-cyclopro-
pylamine furanoid 22 as the only cyclization product. Such a cyclo-
etherification process has been frequently observed as an
unexpected side-reaction with activated 1,4-diols, due to kineti-
cally favored formation of the five-membered cycle over the six-
membered one.20 Investigation of the reaction conditions (nature
of the base, temperature) to produce 7 were conducted. Finally,
preparation of iminosugar 7 was best achieved by treatment of
21 with K2CO3 at 0 °C (31% yield from 20). Both products 7 and
22 were easily separated by flash chromatography over silica gel
and the targeted 5-spirocyclopropyl-deoxyfuconojirimycin 7 was
further purified by ion-exchange chromatography on Dowex
50WX-8 resin (elution with 0.8 M NH4OH).
a
-L
l
pyl-DRJ 9 was slightly active against a-L
bition at 1 mM).
Surprisingly, the fuco-configured pyrroline 8 was a weak inhib-
itor of -fucosidase (76% inhibition at 1 mM) but displayed a
strong affinity for the rhamnosidase (Ki = 57 M). In contrast, the
a
-L
l
rhamno-designed pyrroline 10 did slightly inhibit both the fucosi-
dase (19% inhibition at 1 mM) and the rhamnosidase (72% inhibi-
tion at 1 mM). Concerning the other glycosidases tested,
compounds 7–10 were inactive at the tested concentration of
1 mM towards coffee beans
Aspergillus oryzae b-galactosidase, yeast and rice
Aspergillus niger amyloglucosidase, almonds b-glucosidase, Jack
beans -mannosidase, snail b-mannosidase, A. niger b-xylosidase,
a-galactosidase, Escherichia coli and
a-glucosidase,
a
Jack beans and bovine kidney b-N-acetyl-glucosaminidase.
2.2. Synthesis of pyrrolines 8 and 10
3. Discussion
The reaction sequence applied to the preparation of pyrroline 8
is depicted in Scheme 5. First, oxidative cleavage of the previously
prepared diol 19 with silica-supported NaIO4 afforded the corre-
We designed spirocyclopropyl iminosugars as glycosidase
inhibitors because these bicyclic structures show all properties
necessary, that is, (i) an intramolecular nitrogen atom that may
be protonated to mimic the oxocarbenium intermediate, (ii) hydro-
xyl repartition analogous to that of the natural substrate, and (iii)
an additional group that could induce favorable interactions in
the active site of the enzyme. Indeed, a large hydrophobic pocket
surrounds the methyl substituent of fucose in its binding site, as
observed in crystal structures extracted from the protein data
bank. The cyclopropyl substituent could easily enter this site and
favorably interfere with the respective amino acids. Furthermore,
we anticipated that the presence of the small spirocyclic moiety
would induce conformational modifications in compounds 7–10,
which could promote novel and more potent binding interactions.
To further explore the SAR within this family of compounds, their
inhibition potencies were compared with known inhibitors from
the literature (Table 1).
Boc
N
HO
2
5
N
b)
a)
3 4
19
61%
81%
O
O
HO OH
Me Me
23
8
HO OH
H
NHBoc
O
NHBoc
N
a)
b)
90%
O
O
O
O
90%
HO OH
Me Me
Me Me
Concerning the six-membered iminosugars, fuconojirimycin 1,
the accurate copy of fucose with nitrogen in the ring, is one of
24
25
10
the most potent inhibitor of
a-L-fucosidase known so far with
Scheme 5. Synthesis of pyrrolines 8 and 10. Reagents and conditions: (a) NaIO4–
Ki = 1 nM. Its 1-deoxy analogue DFJ 2 retains strong inhibition
SiO2, CH2Cl2, rt; (b) 1 M HCl, EtOH, then ion-exchange chromatography.

M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
8023
Table 1
CH3
N
Inhibitory activities of compounds 7–10 and structurally related analogues from the
H
H
H
literaturea,b
H3C
HO
N
N
N
PMP
HO
PMP
HO
HO
Compound
a
-
L
-Fucosidase (bovine
a-L-Rhamnosidase (Penicillium
decumbens)
kidney)
OH
OH
OH
HO
27
Ki 0.03 µM
Figure 2. Structures of rhamnosidase inhibitors 26–29.24
H
OH
Me
HO
N
R
1 (R = OH) (0.001
2 (R = H) (0.003
l
M)11
l
M)11
ND
ND
26
Ki 5.5 µM
28
Ki 3.2 µM
29
Ki 3.0 µM
OH
OH
N
Me
(0.010
l
M)13
ND
NI
OH
OH
HO
3
In the fucose series, SAR studies have demonstrated that the
most active pyrrolidine structures retain the natural all-cis orienta-
tion of the substituents at positions C-3, C-4, and C-5 of the five-
membered ring as found in 3.25 In agreement with these results,
H
N
83% (18
76%
lM)
HO
OH
the fuco configured 8 was active against
its rhamno counterpart was not.
a-L-fucosidase whereas
7
N
87% (57
NIc,22
lM)
4. Conclusion
OH
HO
8
R2
H
N
Synthesis of unprecedented spirocyclopropyl-piperidines and -
pyrrolines in the -fuco and -rhamno series was achieved from glyc-
4 (R1 = H, R2 = CH3)
NIc,22
R1
L
L
(1.0
l
M)15
ononitriles by using Ti-mediated aminocyclopropanation as the key
step. Thus, piperidine analogues of DRJ and DFJ with a spirocyclopro-
pyl moiety in the place of the methyl group were prepared, respec-
5 (R1 = CH3, R2 = H) ND
HO
OH
OH
N
Me
tively, from
lyxose in eight steps and 3% overall yield. Spirocyclopropyl-DFJ dis-
played a potent and specific inhibition towards -fucosidase
(Ki = 18 M), however lower than the corresponding methyl deriva-
tive (Ki = 3 nM). In the -rhamno series, spirocyclopropyl DRJ exhib-
ited some inhibition towards -rhamnosidase. Preparation of new
pyrroline analogues was also achieved. The -fuco-designed pyrro-
line showed an unexpected inhibition of -rhamnosidase
(Ki = 57 M). However, our results suggest that the replacement of
L-arabinose in 11 steps and 20% overall yield and from D-
ND
NI
(0.14
72%
l
M)21a
OH
OH
HO
6
L
H
N
l
L
HO
L
OH
L
9
L
N
l
19%
72%
the methyl substituent by a spirocyclopropyl group reduces the
OH
HO
inhibitory potency towards the corresponding enzymes.
10
a
Percentage of inhibition at 1 mM concentration, Ki (in brackets), NI = no inhi-
5. Experimental
5.1. General
bition at 1 mM concentration of inhibitor, ND = not determined.
b
All the inhibition are competitive.
c
Lysosomal a-L-fucosidase.
All reactions were performed under argon. The solvents were
dried and distilled prior to use. Silica Gel 60 F254 (0.2 mm) was used
for TLC plates, detection being carried out by spraying with an alco-
holic solution of phosphomolybdic acid or an aqueous solution of
KMnO4 (2%)/Na2CO3 (4%), followed by heating. Flash column chro-
(Ki = 3 nM) despite the loss of one hydroxyl function. The replace-
ment of the methyl substituent by the spirocycle in the structure of
DFJ reduces the potential for inhibiting
a-L-fucosidase by ca. four
orders of magnitude (Ki = 18 M). Interestingly, the same struc-
l
tural modification, when applied to DRJ 4 (0% inhibition of rham-
nosidase at 1 mM), allows to recover some activity (72%
inhibition at 1 mM for compound 9). However, spirocyclopropyl-
DRJ 9 is still a weaker inhibitor of rhamnosidase than epi-DRJ 5
matography was performed over Silica Gel M 9385 (40–63 lm) Kie-
selgel 60. NMR spectra were recorded on a 250 MHz or a 500 MHz
spectrometer (250 MHz or 500 MHz for 1H, 62.8 MHz or 125 MHz
for 13C, as indicated). Chemical shifts are expressed in parts per mil-
lion (ppm) using TMS as internal standard. IR spectra were recorded
with an IRTM plus MIDAC spectrophotometer and are expressed in
cmꢀ1. Optical rotations were determined at 20 °C with a Perkin–El-
mer Model 241 polarimeter in the specified solvents. High resolu-
tion mass spectra (HRMS) were performed on Q-TOF Micro
micromass positive ESI (CV = 30 V).
(99% inhibition at 1 mM, Ki = 1.0 lM).
In contrast to piperidine iminosugars, where epimers often
show a total loss of their inhibitory potencies, a variety of configu-
rations is tolerated with polyhydroxypyrrolidines, which can be
interpreted in terms of lower steric demand.23 This is illustrated
in Figure 2 with pyrrolidine-type rhamnosidase inhibitors 26–29,
the best of which display various configurations and substitution
patterns. In the present work, both spirocyclopropyl-pyrrolines 8
5.2. Chemistry
and 10 display inhibition potencies against
a-
L-rhamnosidase. Sur-
prisingly, the fuco configured 8 is a potent inhibitor of
a-L-rham-
5.2.1. 4,5-Di-O-benzoyl-2,3-O-isopropylidene-D-lyxononitrile
nosidase, even more active than the rhamno-designed 10.
15c
However, pyrrolines 8 or 10 do not inhibit naringinase as effec-
To a suspension of
D-lyxose (4.60 g, 30.6 mmol) in acetone
tively as the methyl analogue 6 (Ki = 0.14 lM) or as the pyrrol-
(50 mL) was added concentrated H2SO4 (130
mixture was stirred at room temperature for 12 h and the solution
l
L, 2.45 mmol). The
idines 26–29.24 Here again, the spirocyclopropyl substituent
seems to be detrimental.
was basified by adding K2CO3. After filtration of the precipitate, the

8024
M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
filtrate was concentrated under reduced pressure to give 2,3-O-iso-
propylidene- -lyxofuranose (5.80 g, quant.) as a pure 30/70 mix-
ture of anomers and b (white foam).
IR (KBr):
1277 cmꢀ1
m = 3355, 3065, 2985, 2934, 1721, 1655, 1487, 1453,
D
.
a
1H NMR (250 MHz, CDCl3): d = 7.98–7.95 (m, 2H, Ar-H), 7.65–
7.54 (m, 2H, Ar-H), 7.40–7.25 (m, 6H, Ar-H), 4.52–4.35 (m, 3H,
H3, H4a, H4b), 4.18 (d, J = 7.4 Hz, 1H, H2), 4.0 (d, J = 7.4 Hz, 1H,
H1), 1.29 (s, 3H, CH3), 1.23 (s, 3H, CH3), 1.20 (m, 1H, CH2), 1.08
(m, 1H, CH2), 0.90–0.75 (m, 2H, CH2) ppm.
Rf (a/b) = 0.50 and 0.60 (100% EtOAc).
½
a 2D0
ꢂ
= +16 (c 0.328, H2O); lit.: ½a D21
ꢂ
= +18 (c 0.10, H2O).19b
1H NMR (250 MHz, CD3OD): d = 5.19 (br s, 0.7H, H1), 4.98 (br s,
0.3H, H1), 4.74 (m, 1H, H2), 4.57 (m, 0.3H, H3), 4.50 (m, 0.7H, H3),
4.30–4.00 (m, 0.9H, H4, H5), 3.85–3.65 (m, 2.1H, H4, H5), 1.45 (s,
0.9H, CH3), 1.37 (s, 2.1H, CH3), 1.31 (s, 0.9H, CH3), 1.26 (s, 2.1H,
CH3) ppm.
13C NMR (62.8 MHz, CDCl3): d = 167.9 (C@O), 166.8 (C@O),
133.4 (1 Ar-C), 131.3 (1 Ar-C), 129.8, 129.7, 128.5, 127.0 (10 Ar-
C), 108.6 (C(CH3)2), 80.2 (C1), 76.4 (C2), 67.6 (C4), 67.4 (C3), 32.0
(C–NH), 26.5 (CH3), 24.3 (CH3), 12.9 (CH2), 10.6 (CH2) ppm.
HRMS (ESI) calcd for C24H28NO6 [M+H]+: 426.1917; found:
426.1914.
13C NMR (62.8 MHz, CD3OD): d = 113.9 (C(CH3)2), 108.0 (0.3C1),
102.6 (0.7C1), 87.6, 82.0, 81.6 (0.7C2, 0.7C3, 0.7C4), 86.8, 82.4, 79.8
(0.3C2, 0.3C3, 0.3C4), 66.7 (0.3C5), 61.6 (0.7C5), 26.8 (1.4CH3), 25.3
(0.6CH3) ppm.
To
a
solution of hydroxylamine hydrochloride (1.33 g,
5.2.3. (1R)-1-C-(1-Aminocyclopropyl)-1,2-O-isopropylidene-D-
threitol 18
19.2 mmol) in MeOH (10 mL) was added a suspension of sodium
methoxide (1.17 g, 21.6 mmol) in MeOH (5 mL). The mixture was
stirred at room temperature for 5 min then at 0 °C for 10 min.
The precipitate was filtered and a solution of 2,3-O-isopropyli-
Cyclopropylamine 17 (140 mg, 0.33 mmol) was dissolved in a
4:1 mixture of ethanol and water (10 mL) and aqueous 3 M NaOH
solution (3 mL) was added. The mixture was stirred at 80 °C for
24 h then solvents were evaporated under reduced pressure. The
residue was taken up in a 7:3 mixture EtOAc/MeOH then filtered
through a pad of silica gel. Concentration of the filtrate gave an or-
ange oil which was purified by flash chromatography (30% MeOH/
EtOAc) to yield compound 18 (57 mg, 80%; pale yellow oil).
Rf = 0.20 (30% MeOH/EtOAc).
dene-D-lyxofuranose (1.52 g, 7.99 mmol) in MeOH (5 mL) was
added to the filtrate. The mixture was refluxed for 2 h then concen-
trated under reduced pressure. The crude oxime was then dis-
solved in pyridine (20 mL) and the solution cooled to 0 °C.
Benzoyl chloride (9.27 mL, 79.9 mmol) was slowly added and the
mixture was stirred at 70 °C for 72 h. Cold water (30 mL) was
added dropwise and the aqueous phase was extracted with EtOAc
(4 ꢃ 20 mL). The combined organic layers were dried over anhy-
drous MgSO4, filtered and concentrated under reduced pressure.
Purification by flash chromatography (20% EtOAc/petroleum ether)
yielded nitrile 15c (1.49 g, 47% for the three steps) as white crystal-
line plates.
½
a 2D0
IR (KBr):
ꢂ
= ꢀ25 (c 0.488, MeOH).
m
= 3417, 1593, 1386 cmꢀ1
.
1H NMR (250 MHz, CD3OD): d = 4.28 (d, J = 8.0 Hz, 1H, H2), 3.96
(t, J = 6.7 Hz, 1H, H3), 3.66 (d, J = 8.0 Hz, 1H, H1), 3.60 (d, J = 6.7 Hz,
2H, H4), 1.52 (s, 3H, CH3), 1.33 (s, 3H, CH3), 0.84 (m, 1H, CH2), 0.76–
0.58 (m, 3H, CH2) ppm.
Rf = 0.35 (20% EtOAc/petroleum ether).
13C NMR (62.8 MHz, CD3OD): d = 109.5 (C(CH3)2), 82.3 (C1), 77.3
(C2), 70.7 (C3), 64.6 (C4), 33.7 (C–NH2), 26.4 (CH3), 24.0 (CH3), 12.4
(CH2), 8.2 (CH2) ppm.
½
a2H0gð436Þꢂ = +3 (c 0.5, CHCl3); mp = 136 °C.
IR(KBr):m .
= 3090,3002,2988, 2942, 1720, 1451, 1266, 1061 cmꢀ1
1H NMR (250 MHz, CDCl3): d = 8.20–8.00 (m, 4H, Ar-H), 7.55–
7.40 (m, 6H, Ar-H), 5.83 (dt, J = 5.0, J = 5.0, J = 6.2 Hz, 1H, H4),
4.98 (d, J = 5.6 Hz, 1H, H2), 4.70 (d, J = 5.0 Hz, 2H, H5), 4.61 (dd,
J = 5.6, J = 6.2 Hz, 1H, H3), 1.64 (s, 3H, CH3), 1.42 (s, 3H, CH3) ppm.
13C NMR (62.8 MHz, CDCl3): d = 165.8 (C@O), 165.6 (C@O),
133.6 (1 Ar-C), 133.5 (1 Ar-C), 130.1, 129.9, 129.5, 128.7, 128.6
(10 Ar-C), 116.2 (C1), 113.1 (C(CH3)2), 76.4 (C3), 70.1 (C4), 65.3
(C2), 62.9 (C5), 26.9 (CH3), 25.8 (CH3) ppm.
HRMS (ESI) calcd for C10H19NO4Na [M+Na]+: 240.1212. found:
240.1214.
5.2.4. (1R)-1-C-[1-(N-tert-Butyloxycarbonyl)aminocyclopropyl]-
1,2-O-isopropylidene-
To a solution of the aminodiol 18 (82 mg, 0.38 mmol) in THF
(2 mL) at room temperature was added Et3N (58 L, 0.42 mmol).
D-threitol 19
l
HRMS (ESI): m/z calcd for C22H21NO6Na [M+Na]+: 418.1267;
found: 418.1277.
Anal. Calcd for C22H21NO6: C, 66.83; H, 5.35; N, 3.54. Found: C,
66.89; H, 5.25; N, 3.48.
At 0 °C, a solution of Boc2O (83 mg, 0.38 mmol) in THF (1 mL)
was added dropwise and the mixture was stirred at 50 °C for
24 h. Water (3 mL) was then added and the resulting aqueous
phase was extracted with EtOAc (3 ꢃ 5 mL). The combined organic
fractions were dried over anhydrous MgSO4 and filtered. After
removing of the solvent under reduced pressure, the residue was
purified by flash chromatography (80% EtOAc/petroleum ether)
to give the diol 19 (70 mg, 58%) as a colorless oil.
5.2.2. (1R)-1-C-[1-(N-Benzoyl)-aminocyclopropyl]-4-O-benzoyl-
1,2-O-isopropylidene-D-threitol 17
To a solution of nitrile 15c (455 mg, 1.15 mmol) in dry THF
(10 mL) under N2 atmosphere was added a solution of MeTi(Oi-
Pr)3 (414 lL, 1.73 mmol). The yellow solution was stirred at room
temperature for 10 min and ethylmagnesium bromide (1.05 mL,
1.65 M in THF, 1.73 mmol) was added dropwise over a period of
15 min. After 1 h, BF3ꢁOEt2 (291
Rf = 0.28 (80% EtOAc/petroleum ether).
½
a 2D0
IR (film):
ꢂ
= ꢀ6 (c 0.450, CHCl3).
m
= 3372, 2980, 2932, 1699, 1168 cmꢀ1
.
1H NMR (250 MHz, CDCl3): d = 5.91 (br s, 1H, NH), 4.13 (m, 1H,
H2), 4.08 (m, 1H, H3), 3.87 (d, J = 7.1 Hz, 1H, H1), 3.76 (dd, J = 6.3,
J = 11.2 Hz, 1H, H4a), 3.67 (dd, J = 4.6, J = 11.2 Hz, 1H, H4b), 3.00
(br s, 1H, OH), 1.48 (s, 3H, C(CH3)2), 1.41 (s, 9H, C(CH3)3), 1.32 (s,
3H, C(CH3)2), 0.93 (m, 1H, CH2), 0.86–0.65 (m, 3H, CH2) ppm.
13C NMR (62.8 MHz, CDCl3): d = 156.1 (C@O), 108.2 (C(CH3)2),
80.3 (C1), 78.9 (C(CH3)3), 76.4 (C2), 68.7 (C3), 65.8 (C4), 31.7 (C–
NH), 28.3 (C(CH3)3), 26.3 (CH3), 24.1 (CH3), 13.2 (CH2), 10.4 (CH2)
ppm.
lL, 2.30 mmol) was added and
the solution was stirred at room temperature for further 1 h. Water
(10 mL) then 1 M aqueous solution of HCl (1 mL) were added. The
mixture was then basified with 3 M aqueous NaOH solution and
the aqueous phase was extracted with EtOAc (3 ꢃ 10 mL). Com-
bined organic fractions were washed with brine, dried over anhy-
drous MgSO4, filtered and concentrated under reduced pressure to
give a crude brown oil. Purification by flash chromatography (50%
EtOAc/petroleum ether) afforded 17 (240 mg, 49% yield) as a yel-
low oil.
HRMS (ESI) calcd for C15H27NO6Na [M+Na]+: 340.1736; found:
340.1732.
Anal. Calcd for [C15H27NO6 + 1/3H2O]: C, 55.71; H, 8.62; N, 4.33.
Found: C, 55.69; H, 8.75; N, 4.26.
Rf = 0.30 (50% EtOAc/petroleum ether).
½
a 2D0
= +2 (c 0.482, CHCl3).
ꢂ

M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
8025
5.2.5. (1R)-1-C-[1-(N-tert-Butyloxycarbonyl)aminocyclopropyl]-
1,2-O-isopropylidene-4-O-methanesulfonyl- -threitol 20
To a solution of diol 19 (60 mg, 0.19 mmol) and Et3N (48
0.34 mmol) in CH2Cl2 (2 mL) at ꢀ20 °C was slowly added MsCl
(15
L, 0.19 mmol). The solution was stirred 15 min at ꢀ20 °C.
13C NMR (62.8 MHz, CD3OD): d = 93.8 (C2), 80.9 (C3), 78.5 (C4),
75.1 (C5), 35.2 (C–NH2), 14.4 (CH2), 10.7 (CH2) ppm.
MS (ESI): m/z = 160.1 [M+H+]. HRMS (ESI): m/z calcd for
C7H14NO3 [M+H]+: 160.0974; found: 160.0979.
D
lL,
l
5.2.8. (2R,3R,4R)-N-tert-Butyloxycarbonyl-3,4-O-isopropyl-
idene-5-spirocyclopropyl-2,3,4-trihydroxypyrrolidine 23
NaIO4/SiO2 (0.5 g) was slowly added to a solution of diol 19
(70 mg, 0.22 mmol) in CH2Cl2 (10 mL) and the suspension was stir-
red 30 min at room temperature. Silica gel was filtered (elution:
100% EtOAc) and the solution was concentrated under reduced
pressure. Purification by flash chromatography (10% EtOAc/petro-
leum ether) gave pyrrolidine 23 (51 mg, 81%) as a colorless oil.
Rf = 0.45 (20% EtOAc/petroleum ether).
Water (2 mL) was added and the aqueous phase was extracted
with CH2Cl2 (2 ꢃ 2 mL). The combined organic fractions were dried
over anhydrous MgSO4, filtered and concentrated under reduced
pressure to give a crude yellow residue. Purification by flash chro-
matography (50% EtOAc/petroleum ether) afforded 20 (50 mg,
83%) as white crystals and recovered starting material 19 (12 mg).
Rf = 0.36 (50% EtOAc/petroleum ether).
½
a 2D0
ꢂ
= ꢀ4 (c 0.582, CHCl3); mp = 159 °C.
IR (KBr): 3362, 3266, 2979, 2929, 1696, 1518, 1374, 1182 cmꢀ1
.
½
a 2D0
IR (film):
ꢂ
= +93 (c 1.02, CHCl3).
1H NMR (250 MHz, CDCl3): d = 5.60 (br s, 1H, NH), 4.40–4.22 (m,
3H, H3, H4a, H4b), 4.17 (d, J = 7.5 Hz, 1H, H2), 4.02 (d, J = 7.5 Hz,
1H, H1), 3.09 (s, 3H, CH3S), 2.95 (d, J = 6.3 Hz, 1H, OH), 1.49 (s,
3H, CH3), 1.41 (s, 9H, C(CH3)3), 1.33 (s, 3H, CH3), 1.13 (m, 1H,
CH2), 0.98 (m, 1H, CH2), 0.90–0.75 (m, 2H, CH2).
m
= 3440, 2980, 2936, 1674, 1393 cmꢀ1
.
1H NMR (250 MHz, CDCl3): d = 5.53 (br s, 1H, H2), 4.55 (d,
J = 6.0 Hz, 1H, H3 or H4), 4.27 (d, J = 6.0 Hz, 1H, H3 or H4), 1.45
(s, 3H, CH3), 1.43 (s, 9H, C(CH3)3), 1.30 (s, 3H, CH3), 1.06 (s, 1H,
CH2), 0.90–0.70 (m, 3H, CH2) ppm.
13C NMR (62.8 MHz, CDCl3): d = 156.2 (C@O), 108.7 (C(CH3)2),
79.7 (C1), 79.5 (C(CH3)3), 75.7 (C2), 71.4 (C4), 67.0 (C3), 37.8
(CH3S), 31.8 (C–NH), 28.5 (C(CH3)3), 26.5 (CH3), 24.3 (CH3), 13.5
(CH2), 10.9 (CH2).
13C NMR (62.8 MHz, CDCl3): d = 154.0 (C@O), 111.8 (C(CH3)2),
87.8 (C2), 85.9, 82.5 (C3, C4), 81.1 (C(CH3)3), 46.0 (C5), 28.4
(C(CH3)3), 26.9 (C(CH3)2), 25.6 (C(CH3)2), 12.0 (CH2), 6.2 (CH2) ppm.
HRMS (ESI) calcd for C14H23NO5 Na [M+Na]+: 308.1474; found:
308.1477.
HRMS (ESI): m/z calcd for C16H29NO8NaS [M+Na]+: 418.1512;
found: 418.1514.
Anal. Calcd for [C14H23NO5 + 1/4H2O]: C, 58.01; H, 8.17; N, 4.83.
Found: C, 58.16; H, 8.14; N, 4.82.
Anal. Calcd for C16H29NO8S: C, 48.59; H, 7.39; N, 3.54. Found: C,
49.00; H, 7.52; N, 3.39.
5.2.9. (3S,4R)-3,4-Dihydroxy-5-spirocyclopropyl-D-pyrroline 8
5.2.6. 5-C-Spirocyclopropyl-(5-demethyl-1-deoxy)-L-fuconojiri-
mycin 7
A 1 M HCl solution (1 mL) was added to a solution of 23 (38 mg,
0.13 mmol) in EtOH (0.3 mL) and the mixture was stirred 2 h at
room temperature. Solvents were evaporated under reduced pres-
sure to give the corresponding iminosugar as a single anomer. Puri-
fication on Dowex 50WX-8 (H+ form) by elution with a 3% aqueous
solution of NH4OH afforded imine 8 as a pure compound (10 mg,
61%, yellow oil) after lyophilization.
A 2 M HCl solution (1 mL) was added to a solution of 20 (40 mg,
0.10 mmol) in MeOH (1 mL) and the mixture was stirred 30 min at
room temperature. After evaporation of the solvents, water (2 mL)
and K2CO3 were added (pH 12). The solution was stirred at room
temperature for 30 min and concentrated under reduced pressure.
The residue was taken up in MeOH, the precipitate was filtered and
the filtrate was concentrated under reduced pressure to give a
crude 1/1 mixture of cyclopropylamine 22 and iminosugar 7 as
mesylate salts. The two products were separated by flash chroma-
tography on silica gel (CH2Cl2/MeOH/NH4OH/H2O: 6/4/0.5/0.5) and
each compound was purified on Dowex 50WX-8 (H+ form) by elut-
ing with a 3% NH4OH solution.
½
a 2D0
IR (film):
ꢂ
= +104 (c 0.096, MeOH).
m
= 3371, 2924, 2854, 1562, 1068 cmꢀ1
.
1H NMR (250 MHz, CD3OD): d = 7.47 (s, 1H, H2), 4.77 (d, J = 6.1 Hz,
1H, H3), 3.74 (d, J = 6.1 Hz, 1H, H4), 1.20–0.80 (m, 4H, CH2) ppm.
13C NMR (62.8 MHz, CD3OD): d = 169.5 (C2), 79.4 (C3), 74.2 (C4),
60.5 (C5), 12.6 (CH2), 10.0 (CH2) ppm.
HRMS (ESI) calcd for C6H10NO2 [M+H]+: 128.0712; found:
128.0714.
Spirocyclopropyl iminosugar 7 (5 mg, 31% for the two steps, col-
orless foam).
Rf = 0.40 (CH2Cl2/MeOH/NH4OH/H2O: 6/4/0.5/0.5).
½
a 2D0
IR (KBr):
ꢂ
= +12 (c 0.10, MeOH).
5.2.10. (3S)-3-C-[(N-tert-Butyloxycarbonyl)-1-aminocyclopro-
pyl]-2,3-O-isopropylidene-D-glyceraldehyde 25
m
= 3363, 2926, 1212, 1040 cmꢀ1
.
1H NMR (250 MHz, CD3OD): d = 3.73 (ddd, J = 5.1, J = 9.1,
J = 10.6 Hz, 1H, H2), 3.45 (dd, J = 2.6, J = 9.1 Hz, 1H, H3), 3.12 (br
s, 1H, H4), 2.94 (dd, J = 5.1, J = 13.1 Hz, 1H, H1a), 2.39 (dd,
J = 10.6, J = 13.1 Hz, 1H, H1b), 0.65–0.51 (m, 3H, CH2), 0.44 (m,
1H, CH2) ppm.
NaIO4/SiO2 (0.83 g) was slowly added to a solution of diol 24
(114 mg, 0.36 mmol) in CH2Cl2 (10 mL) and the suspension was
stirred 1 h at room temperature. SiO2 was filtered (elution: 100%
EtOAc) and the solution was concentrated under reduced pressure.
Purification by flash chromatography (40% EtOAc/petroleum ether)
gave aldehyde 25 (92 mg, 90%) as white crystals.
13C NMR (62.8 MHz, CD3OD): d = 76.7 (C4), 76.2 (C3), 69.9 (C2),
50.2 (C1), 41.2 (C5), 11.7 (CH2), 11.0 (CH2) ppm.
HRMS (ESI): m/z calcd for C7H14NO3 [M+H]+: 160.0974; found:
160.0972.
Rf = 0.33 (40% EtOAc/petroleum ether).
½
a 2D0
ꢂ
= ꢀ25 (c 1.2, CHCl3); mp = 135 °C.
IR (KBr): = 3364, 2988, 2934, 2836, 1729, 1700, 1512,
1168 cmꢀ1
m
.
5.2.7. (2R,3R,4R)-2-(1-Aminocyclopropyl)-3,4-dihydroxytetra-
hydrofurane 22 (8 mg, 50% for the two steps, colorless foam)
Rf = 0.45 (CH2Cl2/MeOH/NH4OH/H2O: 6/4/0.5/0.5).
1H NMR (250 MHz, CDCl3): d = 9.76 (s, 1H, H1), 5.10 (br s, 1H,
NH), 4.61 (d, J = 6.3 Hz, 1H, H2), 3.74 (d, J = 6.3 Hz, 1H, H3), 1.45
(s, 3H, C(CH3)2), 1.39 (s, 9H, C(CH3)3), 1.30 (s, 3H, CH3), 1.05–0.90
(m, 2H, CH2), 0.90–0.75 (m, 2H, CH2) ppm.
½
a 2D0
ꢂ
= +7 (c 0.24, H2O). IR (film):
m = 3355, 2922, 1605, 1072,
1046 cmꢀ1
.
13C NMR (62.8 MHz, CDCl3): d = 201.0 (C1), 155.9 (C@O), 110.8
(C(CH3)2), 82.9, 81.2 (C2, C3), 79.8 (C(CH3)3), 33.2 (C–NH), 28.4
(C(CH3)3), 26.8 (C(CH3)2), 26.2 (C(CH3)2), 12.7 (CH2), 11.4 (CH2) ppm.
1H NMR (250 MHz, CD3OD): d = 4.06 (br s, 1H, H3), 3.97 (br s,
1H, H4), 3.89 (dd, J = 3.5, J = 9.5 Hz, 1H, H5a), 3.81 (d, J = 9.5 Hz,
1H, H5b), 3.11 (d, J = 2.9 Hz, 1H, H2), 0.73–0.55 (m, 4H, CH2) ppm.

8026
M. S. M. Pearson et al. / Bioorg. Med. Chem. 17 (2009) 8020–8026
HRMS (ESI) calcd for C14H24NO5 [M+H]+: 286.1654; found:
286.1652.
Anal. Calcd for [C14H23NO5 + 1/2H2O]: C, 57.13; H, 8.22; N, 4.76.
(b) Fernandez-Rodriguez, J.; Ayude, D.; de la Cadena, M. P.; Martinez-Zorzano,
V. S.; de Carlos, A.; Caride-Castro, A.; de Castro, G.; Rodriguez-Berrocal, F. J.
Cancer Detect. Prev. 2000, 24, 143.
7. (a) Perez, S.; Rodriguez-Carvajal, M. A.; Doco, T. Biochimie 2003, 85, 5084; (b)
Tonetti, M.; Sturla, L.; Bisso, A.; Zanardi, D.; Benatti, U.; deFlora, A. Biochimie
1998, 80, 923; (c) Yu, H.; Gong, J.; Zhang, C.; Jin, F. Chem. Pharm. Bull. 2002, 50,
175; (d) Oda, Y.; Saito, K.; Ohara-Takada, A.; Mori, M. J. Biosci. Bioeng. 2002, 94,
321; (e) Avigad, G. In Plant Carbohydrates 1, Encyclopedia of Plant Physiology;
Loewus, F. A., Tanner, W., Eds.; Springer, 1982. pp 217–347.
8. Deng, L.; Kasper, D. L.; Krick, T. P.; Wessels, M. R. J. Biol. Chem. 2000, 275,
7497.
9. (a)Iminosugars: From Synthesis to Therapeutic Applications; Compain, P., Martin,
O. R., Eds.; John Wiley and Sons: Chichester, UK, 2007; (b) Robina, I.; Moreno-
Vargas, A. J.; Carmona, A. T.; Vogel, P. Curr. Drug Metab. 2004, 5, 329; (c) Behr, J.-
B. Curr. Med. Chem. (Anti-infective Agents) 2003, 2, 173.
10. (a)Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond; Stütz, A. E.,
Ed.Wiley-VCH; Weinheim: Germany, 1999; (b) Djebaili, M.; Behr, J.-B. J. Enzym.
Inhib. Med. Chem. 2005, 20, 123; (c) Gautier-Lefebvre, I.; Behr, J.-B.; Guillerm,
G.; Muzard, M. Eur. J. Med. Chem. 2005, 1255; (d) Compain, P.; Martin, O. R.
Bioorg. Med. Chem. 2001, 9, 3077.
11. Dubernet, M.; Defoin, A.; Tarnus, C. Bioorg. Med. Chem. Lett. 2006, 16, 1172.
12. Winchester, B.; Barker, C.; Baines, S.; Jacob, G. S.; Namgoong, S. K.; Fleet, G.
Biochem. J. 1990, 265, 277.
Found: C, 57.27; H, 7.92; N, 4.77.
5.2.11. (3S,4S)-3,4-Dihydroxy-5-spirocyclopropyl-D-pyrroline
10
A 1 M HCl solution (3 mL) was added to a solution of 25
(115 mg, 0.40 mmol) in EtOH (1 mL) and the mixture was stirred
for 2 h at room temperature. Solvents were evaporated under re-
duced pressure to give the corresponding iminosugar as a 50:50
mixture of anomers. Purification on Dowex 50WX-8 (H+ form) by
elution with a 3% aqueous solution of NH4OH afforded imine 10
as a pure compound (45 mg, 90%, yellow oil).
½
a 2D0
IR (KBr):
ꢂ
= +23 (c 0.574, MeOH).
m
= 3403, 1652, 1024 cmꢀ1
.
1H NMR (250 MHz, CD3OD): d = 7.44 (s, 1H, H2), 4.55 (d,
J = 3.3 Hz, 1H, H3), 3.80 (d, J = 3.3 Hz, 1H, H4), 1.25–1.02 (m, 2H,
CH2), 0.90–0.72 (m, 2H, CH2) ppm.
13. Chevrier, C.; LeNouen, D.; Neuburger, M.; Defoin, A.; Tarnus, C. Tetrahedron Lett.
2004, 45, 5363.
13C NMR (62.8 MHz, CD3OD): d = 168.2 (C2), 86.0 (C3), 80.7 (C4),
59.3 (C5), 13.8 (CH2), 9.1 (CH2) ppm.
14. (a) Robina, I.; Moreno-Vargas, A. J.; Fernández-Bolaños, J. G.; Fuentes, J.;
Demange, R.; Vogel, P. Bioorg. Med. Chem. Lett. 2001, 11, 2555; (b) Moreno-
Vargas, A. J.; Robina, I.; Demange, R.; Vogel, P. Helv. Chim. Acta 2003, 86, 1894;
(c) Moreno-Vargas, A. J.; Carmona, A. T.; Mora, F.; Vogel, P.; Robina, I. Chem.
Commun. 2005, 4949; (d) Moreno-Clavijo, E.; Carmona, A. T.; Vera-Ayoso, Y.;
Moreno-Vargas, A. J.; Bello, C.; Vogel, P.; Robina, I. Org. Biomol. Chem. 2009, 7,
1192; (e) Behr, J.-B. Tetrahedron Lett. 2009, 50, 4498.
15. Davis, B. G.; Hull, A.; Smith, C.; Nash, R. J.; Watson, A. A.; Winkler, D. A.;
Griffiths, R. C.; Fleet, G. W. J. Tetrahedron: Asymmetry 1998, 9, 2947.
16. For the synthesis of 9, see: (a) Pearson, M. S. M.; Plantier-Royon, R.; Szymoniak,
J.; Bertus, P.; Behr, J.-B. Synthesis 2007, 3589; for previous attempts towards
spirocyclopropyl-pyrrolidines, see: (b) Laroche, C.; Plantier-Royon, R.;
Szymoniak, J.; Bertus, P.; Behr, J.-B. Synlett 2006, 223; (c) Laroche, C.; Behr, J.-
B.; Szymoniak, J.; Bertus, P.; Schütz, C.; Vogel, P.; Plantier-Royon, R. Bioorg. Med.
Chem. 2006, 14, 4047; for general aspects of the cyclopropanation of nitriles,
see: (d) Bertus, P.; Szymoniak, J. Chem. Commun. 2001, 1792; (e) Bertus, P.;
Szymoniak, J. Synlett 2007, 1346; for the cyclopropanation of benzoylated
glucose, see: (f) Bertus, P.; Szymoniak, J.; Jeanneau, E.; Docsa, T.; Gergely, P.;
Praly, J.-P.; Vidal, S. Bioorg. Med. Chem. Lett. 2008, 18, 4774.
HRMS (ESI) calcd for C6H10NO2 [M+H]+: 128.0712; found:
128.0715.
5.3. Glycosidase inhibition
The enzymatic assays were performed as follows: 0.01–0.5 unit/
mL of enzyme (1 unit = 1 mol of glycoside hydrolyzed/min), pre-
incubated for 10 min at 20 °C with the inhibitor, and increasing
concentration of aqueous solution of the appropriate p-nitrophenyl
glycoside substrates (buffered to the optimum pH of the enzyme)
were incubated for 20 min at 37 °C.26 The reaction was stopped
by the addition of 100 lL of 0.3 M sodium borate buffer pH 9.8.
The p-nitrophenolate formed was quantified at 405 nm, and IC50
values were calculated. Double-reciprocal (Lineweaver–Burk) plots
were used to determine the inhibition characteristics of active
compounds.
17. Köln, O.; Redlich, H. Synthesis 1996, 826.
18. Laroche, C.; Behr, J.-B.; Bertus, P.; Szymoniak, J.; Plantier-Royon, R. Eur. J. Org.
Chem. 2005, 5084.
19. (a) Kaskar, B.; Heise, G. L.; Michalak, R. S.; Vishnuvajjala, B. R. Synthesis 1990,
1031; (b) Barbat, J.; Gelas, J.; Horton, D. Carbohydr. Res. 1991, 219, 115.
20. (a) Behr, J.-B.; Guillerm, G. Tetrahedron Lett. 2007, 48, 2369; (b) Taylor, C. M.;
Barker, W. D.; Weir, C. A.; Park, J. H. J. Org. Chem. 2002, 67, 4466.
21. (a) Provencher, L.; Steensma, D. H.; Wong, C.-H. Bioorg. Med. Chem. 1994, 2,
1179; (b) Behr, J.-B.; Defoin, A.; Mahmood, N.; Streith, J. Helv. Chim. Acta 1995,
78, 1166; (c) Behr, J.-B.; Chevrier, C.; Defoin, A.; Tarnus, C.; Streith, J.
Tetrahedron 2003, 59, 543; (d) Merino, P.; Delso, I.; Tejero, T.; Cardona, F.;
Marradi, M.; Faggi, E.; Parmeggiani, C.; Goti, A. Eur. J. Org. Chem. 2008, 2929;
Behr, J.-B.; Pearson, M. S. M.; Bello, C.; Vogel, P.; Plantier-Royon, R. Tetrahedron:
Asymmetry 2008, 19, 1829.
22. Fairbanks, A. J.; Carpenter, N. C.; Fleet, G. W. J.; Ramsden, N. G.; Cenci di
Bello, I.; Winchester, B. G.; Al-Daher, S. S.; Nagahashi, G. Tetrahedron 1992,
48, 3365.
23. Chevrier, C.; Defoin, A.; Tarnus, C. Bioorg. Med. Chem. 2007, 15, 4125.
24. Compound 26, see: Ref. 21a; compound 27, see: Håkansson, A. E.; van Ameijde,
J.; Horne, G.; Nash, R. J.; Wormald, M. R.; Kato, A.; Besra, G. S.; Gurcha, S.; Fleet,
G. W. J. Tetrahedron Lett. 2008, 49, 179; compound 28, see: Kim, J. H.; Curtis-
Long, M. J.; Seo, W. D.; Lee, J. H.; Lee, B. W.; Yoon, Y. J.; Kang, K. Y.; Park, K. H.
Bioorg. Med. Chem. Lett. 2005, 15, 4282; compound 29, see: Chapman, T. M.;
Courtney, S.; Hay, P.; Davis, B. G. Chem. Eur. J. 2003, 9, 3397.
Acknowledgments
The authors gratefully acknowledge the Conseil Régional Cham-
pagne-Ardenne for a post-doctoral fellowship (M.S.M.P.) and J. Kel-
ler, S. Lanthony and D. Harakat for technical assistance.
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