Synthesis and evaluation as glycosidase inhibitors of 2,5-imino-D- glucitol and 1,5-imino-D-mannitol related derivatives

Article abstract of DOI:10.1016/S0968-0896(99)00267-9

Selectively functionalized 2,5-imino-D-glucitol and 1,5-imino-D-mannitol derivatives were synthesized and tested as precursors of hydrolytically resistant pseudo-disaccharides. Among them N-acetyl-6-amino-6-deoxy-2,5- imino-D-glucitol (11) and N-acetyl-6-amino-6-deoxy-1,5-imino-D-mannitol (12) were found potent and specific inhibitors against β-D-glucosidase and α-L- fucosidase, respectively. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00267-9

Bioorganic & Medicinal Chemistry 8 (2000) 135±143
Synthesis and Evaluation as Glycosidase Inhibitors of
2,5-Imino-D-glucitol and 1,5-Imino-D-mannitol Related Derivatives
Isabelle McCort,* Sebastien Fort, Annie Dureault* and Jean-Claude Depezay
Â
Universite Rene Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, associe au CNRS,
Â
Â
Á
45 rue des Saints-Peres, 75270 Paris, Cedex 06, France
Received 8 July 1999; accepted 7 September 1999
AbstractÐSelectively functionalized 2,5-imino-d-glucitol and 1,5-imino-d-mannitol derivatives were synthesized and tested as
precursors of hydrolytically resistant pseudo-disaccharides. Among them N-acetyl-6-amino-6-deoxy-2,5-imino-d-glucitol (11) and
N-acetyl-6-amino-6-deoxy-1,5-imino-d-mannitol (12) were found potent and speci®c inhibitors against b-d-glucosidase and a-l-
fucosidase, respectively. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
carbon±nitrogen and carbon±sulfur linked pseudo-
aza-disaccharides12 and the preparation of valuable
intermediates for coupling reactions. For instance we
have recently reported the preparation of bisubstrates in
which a 2,5-imino-d-glucitol is coupled at the pseudo-
anomeric carbon through an ester or amide link with
glucose (Scheme 1).13
Glycosidases are involved in several metabolic pathways
and the development of selective inhibitors is an impor-
tant strategy towards the treatment of numerous dis-
eases. The family of N-containing carbohydrate mimics
commonly referred to as azasugars constitute a major
class of transition-state analogue inhibitors of glycosi-
dases.1±4 Monosaccharide and azaglycoside mimics are
at the moment the targets of much synthetic e€ort and
stable aza-analogues of glycoconjugates have potential
as selective inhibitors of targeted glycosidase-mediated
processes.
We report here, the synthesis and evaluation as glycosi-
dase inhibitors of various derivatives related to 6-
amino-6-deoxy-2,5-imino-d-glucitol (2), 6-amino-6-
deoxy-1,5-imino-d-mannitol (3) and 2,5-imino-d-gluci-
tol (4). Both 2 and 3 are related to known potent gly-
cosidases inhibitors, respectively, 2,5-imino-d-glucitol
(4),14 inhibitor of a- and b-glucosidases and d-1-deoxy-
mannojirimycin 5, a more potent inhibitor of a-l-fuco-
sidase (Ki 5 mM) than of a-d-mannosidase15 (Scheme 2).
In order to generate stable structures a few solutions
have been recently proposed; in particular pseudo aza-
disaccharides have been prepared, by replacing the
interglycosidic oxygen atom by a non hydrolysable
bond,5±7 or by linking the aglycone directly to the
intracyclic nitrogen atom.8
Results and Discussion
The divergent methodology we have developed to pre-
pare enantiomerically pure functionally diverse pyrroli-
dines and piperidines consists of a double nucleophilic
opening of C-2 symmetric bis-aziridines of l-ido con®g-
uration.9±11 This strategy allows a one step access to
The ring-opening of d-mannitol derived bis-aziridine 1
by nucleophiles is a versatile method for the selective
synthesis of either polyhydroxylated pyrrolidines bear-
ing a free functional group at C-19±11 or piperidines
functionalized at C-211 (Scheme 3). In aprotic solvents,
the ring-opening proceeds mainly with complete
selectivity at the primary carbon (path m), followed by
subsequent 5-exo cyclization leading to the pyrrolidine
ring. If the ring opening of 1 proceeds with SN1 char-
acteristics, nucleophilic attack takes place selectively at
the secondary carbon (path n) and the formation of the
piperidines 3 of d-manno con®guration is favored.
Keywords: C2 symmetric bis-aziridine; azasugars; b-d-glucosidase
inhibitor; a-l-fucosidase inhibitor.
*Corresponding authors. Tel.: +33-1-42-86-21-77; fax: +33-1-42-
86-83-87; e-mail: dureault@biomedicale.univ-paris5.fr or mccort@
biomedicale.univ-paris5.fr
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00267-9

136
I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
opening at the primary carbon (Scheme 3, path m)
followed by Ag+ promoted rearrangement.10,11 Com-
pound 6 could be converted, alternatively, into the
known iminoglucitol 4, the carbohydrate mimics 9a±d
and the cyclic urea 10 (Scheme 5).
Tri¯uoracetic acid-mediated deprotection of 6 a€orded
13, which was N-acetylated providing 14. Derivatives 6,
13 and 14 could be converted alternatively into the aza-
sugars 9a±c by hydrogenolysis on Pd black in acetic
acid. The oxazolidinone 13 is converted into the cyclic
urea 15 in basic medium and subsequent hydrogenolysis
yielded 10. Nitrous deamination of 13 using iso-
amylnitrite, led to a 1:1 mixture of diastereoisomeric
carbamate-protected iminoglucitols 16 and 17, both
precursors of the 2,5-iminoglucitol 4. Compounds 16
and 17 could be chromatographically separated and
hydrogenolysis of 16 led to the bicyclic oxazolidone 9d.
Compounds 4 and 9d are identical to compounds pre-
pared previously following a di€erent strategy.17 The
pyrrolidines 13 and 16 are useful intermediates for
aglycon coupling at C-6, while the urea 15 enables cou-
pling at C-1.
Scheme 1.
The protected 6-deoxy 6-amino iminoalditols 6, 7 and 8
can be prepared alternatively from bis-aziridine 1
(Scheme 4). These compounds constitute suitable pre-
cursors for the construction of bisubstrates in which the
unnatural sugar moiety is coupled with the aglycon
either at C-1 or C-6 for pyrrolidines and either at C-2 or
C-6 for piperidines. The amino group at C-6 constitutes
an useful anchor group as a means of immobilization on
polymer supports either for chromatographic anity
puri®cation or solid phase synthesis.16 We also took
advantage of having easily accessible derivatives bearing
a nitrogen function at C-6 to study the importance of
the hydroxyl group at C-6 for the inhibitors 4 and 5, in
particular the e€ect of its replacement by an acetamido
group.
Preparation of the orthogonally protected pyrrolidines 7
and piperidines 8 has been achieved by the reaction of
bis-aziridine 1 with hydroxylated reagents,11 which
regioselectivity highly depends on the catalyst. The
opening of the ®rst aziridine-ring at the primary carbon
followed by 5-exo tet heterocyclization yields the pyr-
rolidines (Scheme 3, path m), while the formation of the
piperidines results from the preliminary attack at the
secondary carbon followed by 6-exo tet heterocycliza-
tion (path n).
We have tested, after total deprotection various azasu-
gars related to 2 and 3 in order to probe the importance
of the di€erent functions for the biological activity.
The synthesis of the cyclic carbamate-protected pyrroli-
dine 6 is achieved in 75% yield from 1 by Li2NiBr4
Reaction of bis-aziridine 1 with acetic acid enabled the
one step preparation of 1-O-acetyl pyrrolidine 7a (pre-
cursor of 2 and 11) in 60% yield along with 27% of 8a,
whereas the reaction of 1 with allyl alcohol, at room
temperature, under ytterbium tri¯ate catalysis, selec-
tively yielded the piperidine 8b (55%) precursor of 3 and
12 along with 7b (27%) (Scheme 6). We have shown
that decreasing the temperature improved the selectivity
towards 8 but to the detriment of the chemical yield.
The sodium reduction followed by tri¯uoracetic acid-
mediated deprotection of 7a yielded 2. We have also
carried out the selective acetylation of the diamine 18
giving 19, which presents a selectively free endocyclic
amino function, in 65% yield. Total deprotection of the
hydroxy groups of 19 with Na in NH3 gave pyrrolidine
1118 (Scheme 6).
Scheme 2.
Scheme 3.

I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
137
Scheme 4.
Scheme 5. i: H2, Pd black, AcOH.
Tri¯uoracetic acid-mediated deprotection of 8b gave the
diamine 20 whose selective acetylation led to 21 in 76%
yield, suitable for coupling at the endocyclic nitrogen.
Total deprotection of the hydroxy groups of 20 and 21
using Na in NH3 gave, respectively, piperidines 3 and 12.
On the other hand, we were able to carry out the selec-
tive reductive deprotection of the hydroxyl group at C-2
of 8b giving 22 in 75%, using tributyltin hydride under

138
I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
Scheme 6. i: TFA, CH2Cl2; ii: Na, NH3; iii: Ac2O, Et3N; iv: Bu3SnH, ZnCl2, Pd(Ph3)4 cat.
Pd(Ph3)4 catalysis.19 Furthermore the same piperidine
22, a valuable intermediate for coupling reactions, was
obtained by the opening of 1 with water.11
experiment (6.5), compounds 2 and 3, positively
charged at the primary amino function, are not suitable
mimics of the oxocarbonium transition state. The pKa
values of the two amino functions in 2 or 3 have been
calculated, using the Pallas 2.0 program ®le, to be=9.8
for the primary ammonium at C-6 and respectively 5.15
and 5.92 for the intracyclic ammonium. It seems that no
electrostatic interaction occurs between the positively
charged ammonium at C-6 and a negatively carboxylate
group in the active site.
Inhibition Studies
Bicyclic azasugars 9a±9d, 10, azafuranoses analogues 2
and 11, and azapyranoses 3 and 12 (Table 1) have been
screened against ®ve common glycosidases (a-glucosi-
dase, b-glucosidase, a-galactosidase, a-mannosidase, a-
l-fucosidase). Compounds 9a, 9c, 9d and 10, which do
not present any positive charge either at the pseudo-
anomeric carbon or at the intramolecular nitrogen,
showed unsurprisingly poor inhibition of a-glucosidase
and were totally inactive on the other glycosidases stu-
died. In addition to these electronic reasons, the oxazo-
lidinone ring linking the pseudoanomeric center to the
ring nitrogen of the pyrrolidine enhances the rigidity of
the mimic and may sterically obstruct the access of the
substrate to the active site. Therefore, bicyclic com-
pounds 9 do not allow any SAR concerning the sub-
stituent at the C-6 position of the pseudo azasugar.
Compounds 11 and 12 showed competitive inhibition
towards b-glucosidase and a-l-fucosidase, respectively.
The 6-acetylamino analogue 11 of d-iminoglucitol 4,
positively charged at the intracyclic nitrogen proved to
be a good (Ki=10 mM) and selective inhibitor against b-
glucosidase. Iminoglucitol 414 inhibited a-glucosidase
(Kiꢀ1 mM) but also b-glucosidase and a-galactosidase
with one order of magnitude lower. Pyrrolidine 11
mimic of a glucosyl cation displayed no inhibition
3
against the other enzymes assayed (Ki >10 M). The
selectivity may arise from a speci®c interaction between
the acetylamino group and the active asite of the b-glu-
cosidase. Apparently, with the other enzymes, either this
interaction is not favorable or the sterical hindrance
interferes with binding to the active sites.
Compounds 2 and 3 displayed no inhibition towards the
2
®ve enzymes tested (Ki >10 M). At the pH of the
Due to the inherent structural similarity of a-l-fucosi-
dase and d-mannosidase inhibitors, many compounds
inhibit both groups of enzymes and therefore, due to
this lack of selectivity cannot be used for practical
applications. Interestingly, piperidine 12 of d-manno
con®guration was found to display potent and selective
inhibition towards a-l-fucosidase (Ki 4 mM) and dis-
played no inhibition against the other enzymes assayed
Table 1. Inhibition study of glycosidases by azasugars 2±3 and 9±12
a
Inhibitor
IC50
Inhibitor
Ki
9a
9b
9c
9d
10
8 mM
NIb
6 mM
7 mM
3.5 mM
2
11
3
>1 0 3 Mc
10 mM b-glucosidase
>1 0 3 Mc
3
12
4 mM a-l-fucosidase
(Ki >10 M). A similar selectivity had been observed
for the parent deoxymannojirimycin (DMJ),15 a potent
a-l-fucosidase (Ki=5 mM) and a poor inhibitor of jack
bean a-mannosidase (750 mM).
aDetermined on a-glucosidase.
bNo inhibition.
cOn the ®ve glycosidases assayed.

I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
139
Conclusion
added dropwise acetic anhydride (1 equiv) and triethy-
lamine (1 equiv). After 2 h at this temperature, the mix-
ture was concentrated and the residue was puri®ed by
¯ash chromatography as speci®ed.
Diversely funtionalized pyrrolidines and piperidines
deriving from 2 and 3 have been synthesized as pre-
cursors for the elaboration of complex glycosides and
have been evaluated as glycosidase inhibitors. For the
active compounds 11 and 12, we have shown that the
replacement of hydroxyl at C-6 of the known inhibitors
4 and 5 by acetamido group does not harm the activity.
Procedure D. Hydroxyl deprotection by sodium ammonia.
To a solution of the compound (0.14 mmol) in liquid
ammonia (25 mL) was added sodium until the blue
color was persistent. The reaction mixture was stirred
under ammonia re¯ux for 2 h. The mixture was then
neutralized with NH4Cl until disparition of the blue
color. Ammonia was evaporated under a stream of
argon and the residue diluted with CH3OH or with
CH3OH and AcOH, ®ltered through Celite and con-
centrated. The crude product was further puri®ed by
column-chromatography on Kieselgel.
This synthetic work is currently being extended to new
bisubstrates using, in particular the d-mannitol 3 core.
Experimental
Chemistry
General directions. Prior to use, tetrahydrofuran (THF)
and diethyl ether (Et2O) were distilled from sodium-
benzophenone and dichloromethane (CH2Cl2) from
P2O5. CH2Cl2 and ethyl acetate (EtOAc) were ®ltered
3,4-Di-O-benzyl-6-[(tert-butyloxycarbonyl)amino]-2,5-
[(1-oxycarbonyl)imino]-2,5,6-trideoxy-D-glucitol (6). At
0 ꢁC, to a stirred solution of 1 (569 mg, 1.08 mmol) in
THF (16.4 mL) was added dropwise Li2NiBr4 (0.4 M in
THF, 2.7 mL, 1.08 mmol). The mixture was stirred for
4 h at this temperature, then overnight at 4 ꢁC. The
mixture was then hydrolyzed with a bu€er solution
KH2PO4/K2HPO4 (1 M, 15 mL) and extracted with
CH2Cl2 (3Â20 mL). The combined organic layers were
dried (MgSO4) and concentrated in vacuo. To the crude
product containing the monobromo aziridine and the
bromopyrrolidine dissolved in DMF was added AgNO3
(360 mg, 2 equiv), then the mixture was heated at 70 ꢁC
for 9 h. After ®ltration on a Celite bed, the solvent was
evaporated in vacuo. The crude product was puri®ed by
column chromatography to yield 6 as a colourless oil
(380 mg, 75%); Rf 0.25 (cyclohexane:EtOAc, 7:3). [a]d
1
on K2CO3 prior to use. H NMR (250 MHz) and 13C
NMR spectra were recorded in CDCl3 (unless otherwise
indicated) on a Brucker AM 250 (63 MHz). Chemical
shifts are reported in d (ppm) and coupling constants (J)
are given in Hertz. Speci®c rotations were measured on
a Perkin±Elmer 241C polarimeter with sodium (589 nm)
or mercury (365 nm) lamps. UV spectra were recorded
on a Uvicon 860 Spectrophotometer. Mass spectra were
recorded by the Service de Spectrometrie de Masse,
Universite Pierre et Marie Curie, Paris. All reactions
were carried out under argon atmosphere, and were
monitored by thin-layer chromatography with Merck
60F-254 precoated silica (0.2 mm) on glass. Flash chro-
matography was performed with Merck Kieselgel 60
(200±500 mm) unless otherwise noted; the solvent sys-
tems were given v/v. Spectroscopic (1H and 13C NMR,
MS) and/or analytical data were obtained using chro-
matographically homogeneous samples.
1
+14 (c 2.7, CH2Cl2). H NMR (CDCl3) d 1.39 (s, 9H,
CH3), 3.5±3.75 (m containing brd, 3H, J3,2=3 Hz, H-6,
H-5, H-3), 3.8±4.0 (m, 1H, H-60), 4.03 (brs, 1H, H-4),
4.20±4.70 (m, 7H, H-2, H-1, H-10, OCH2Ph), 5.80 (m,
1H, NH), 7.1±7.45 (m, 10Harom); 13C NMR (CDCl3) d
28.4 (CH3), 39.9 (C-6), 62.1 (C-1), 62.7, 65.3 (C-2, C-5),
71.0, 72.2 (OCH2Ph), 79.2 (CqBoc), 79.4, 85.6 (C-3,
C-4), 127.7, 127.9, 128.1, 128.6 (CHarom), 137.0, 137.2
(Cqarom), 156.3, 158.2 (CO). CIMS, NH3: m/z (%) 469
(10) (MH+), 369 (100) (MH+ Boc).
General synthetic procedures
Procedure A. Hydrogenolysis of benzyl ethers. To a sus-
pension of Pd black (50 mg) in AcOH (2.5 mL), was
added the compound (0.15 mmol) to be hydrogenolyzed
in AcOH (2 mL). After stirring overnight under 1 atm.
hydrogen and at room temperature, the catalyst was ®l-
tered through a Celite pad and the solution was evapo-
rated in vacuo.
6-[(tert-Butyloxycarbonyl)amino]-2,5-[(1-oxycarbonyl)-
imino]-2,5,6-trideoxy-D-glucitol (9b). Compound
6
(64 mg, 0.136 mmol) was deprotected according to
procedure A, to yield 9b as a white solid after ¯ash
chromatography puri®cation (33 mg, 84%); Rf 0.20
(CH2Cl2:MeOH, 95:5). [a]d +28 lit.18 +27.3 (c 0.4,
Procedure B. Boc deprotection. TFA (20 equiv per N-
tert-butyloxycarbonyl group) was added dropwise to a
solution of Boc-protected amine in the same volume of
CH2Cl2 at 0 ꢁC. After stirring for 2 h at this tempera-
ture, the volatile components were evaporated and the
residue was treated with dry Et2O. If a ®lterable solid
was obtained, the precipitate was collected, washed with
dry Et2O and dried under vacuum. Otherwise Et2O was
evaporated and the amorphous solid dried under
vacuum and used directly in the subsequent step.
ꢁ
1
CHCl3), mp 45±47 C. H NMR (CD3OD) d 1.45 (s,
9H, CH3), 3.40 (t, 1H, H-5), 3.64 (m, 2H, H-6, H-60),
3.78 (brs, 1H, H-3), 4.09 (brs, 1H, H-4), 4.45 (m, 3H, H-
1, H-10, H-2), J5,6=J5,6 =6 Hz; 13C NMR (CD3OD) d
0
28.7 (CH3), 40.5 (C-6), 64.0 (C-1), 64.7, 67.1, (C-2, C-5),
80.4 (CqBoc), 74.7, 83.8 (C-3, C-4), 158.1, 160.3 (CO).
HRMS calcd for C12H21NO6 (MH+) 289.1399, found
289.1406.
6-Amino-3,4-di-O-benzyl-2,5-[(1-oxycarbonyl)imino]-2,5,6-
trideoxy-D-glucitol, TFA (13). Compound 6 (475 mg,
1.014 mmol) was deprotected according to procedure B
Procedure C. Selective acetylation. To a solution of
TFA salt in CH2Cl2 (9 10 2 M), cooled at 10 ꢁC, were

140
I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
to yield 13 as a white solid (406 mg, 83%), mp 151±
4.30 (m, 1H, OH), 4.4±4.65 (m, 4H, OCH2Ph), 4.75 (brs,
0
153 ꢁC; [a]d +26 (c 1.0, CH2Cl2). H NMR (CDCl3) d
1H, NH), 7.1±7.45 (m, 10Harom), J6,6 =J5,6 =8.8 Hz,
1
0
3.15±3.6 (m, 2H, H-6, H-60), 3.49 (m, 1H), 3.64 (s, 1H),
3.99 (brd, 1H, J=10 Hz, 1H), 4.26±4.46 (m, 5H), 4.55
(AB, J=12 Hz, 1H), 4.57 (A0B0, J=12 Hz, 1H), 7.10±
7.40 (m, 10Harom), 8.25 (brs, 3H, NH+3 ).
J1,1 =12 Hz, J1 ,2=8.8 Hz; 13C NMR (CDCl3) d 44.6
(C-6), 59.3 (C-1), 62.7, 63.0 (C-2, C-5), 72.2, 72.3
(OCH2Ph), 84.1, 86.4 (C-3, C-4), 127.6, 128.0, 128.1,
128.6 (CHarom), 137.4 (Cqarom), 163.3 (CO).CIMS,
NH3: m/z (%) 369 (100) (MH+).
0
0
6-Amino-2,5-[(1-oxycarbonyl)imino]-2,5,6-trideoxy-D-gluc-
itol, AcOH (9a). Compound 13 (72.38 mg, 0.15 mmol)
was deprotected according to procedure A to yield 9a as
a very hygroscopic solid after ¯ash chromatography
puri®cation (34 mg, 91%); Rf 0.35 (CH2Cl2:MeOH:
6-Amino-2,5-[(6-aminocarbonyl)imino]-2,5,6-trideoxy-D-
glucitol (10). Compound 15 (44.3 mg, 0.12 mmol) was
deprotected according to procedure A. Column chro-
matography puri®cation (CH2Cl2:MeOH, 85:15 then
80:20), a€orded 10 as a white solid (19 mg, 84%); Rf
0.25 (CH2Cl2:MeOH, 8:2), mp 142±145 ꢁC; [a]d 52.5
(c 0.2, MeOH). 1H NMR (CD3OD) d 3.44 (dd, 1H,
J=7 Hz, 8.8), 3.64 (dd, 1H, J=5, 10.7 Hz), 3.66 (t, 1H,
J=9 Hz), 3.78 (ddd, 1H, J=5, 7, 9 Hz), 3.90 (t, 1H,
J=5 Hz), 4.01±4.13 (m, 3H), CIMS, NH3: m/z (%) 189
(100) (MH+).
1
H2O:AcOH, 7:3:0.6:0.3). [a]d +14 (c 0.5, MeOH). H
NMR (CD3OD) d 1.96 (s, 3H, CH3), 3.48 (m, 2H), 3.64
(m, 1H), 3.83 (m, 1H), 4.09 (s, 1H), 4.4±4.55 (m, 3H);
13C NMR (CD3OD) d 25.5 (CH3), 42.7 (C-6), 66.4 (C-
1), 66.9, 67.6 (C-2, C-5), 76.3, 86.2 (C-3, C-4), 163.2,
182.2 (CO). HRMS calcd for C7H13N2O4 (MH+)
189.0875, found 189.0873.
6-Acetylamino-3,4-di-O-benzyl-2,5-[(1-oxycarbonyl)imino]-
2,5,6-trideoxy-D-glucitol (14). To a solution of 13
(72.38 mg, 0.15 mmol) in DMF (375 mL) and acetic
anhydride (150 mL, 1.58 mmol) was added dropwise
Et3N (21 mL, 0.15 mmol). After 1 h stirring at room
temperature, the volatile components were evaporated.
After addition of water (7 mL) and CH2Cl2 (15 mL),
decantation, extraction with CH2Cl2 (3Â15 mL), the
combined organic layers were dried (MgSO4) and con-
centrated in vacuo to yield quantitatively 14 (61.5 mg);
Rf 0.12 (cyclohexane:EtOAc, 1:1), which was used in
the next step without further puri®cation. 1H NMR
(CDCl3) d 1.87 (s, 3H, CH3), 3.45±3.7 (m containing
brd, 3H, J3,2=3 Hz, H-6, H-5, H-3), 4.0±4.15 (m con-
taining brs, 2H, H-4, H-60), 4.2±4.7 (m, 7H, H-2, H-1,
H-10, OCH2Ph), 6.92 (m, 1H, NH), 7.1±7.45 (m,
10Harom).
Nitrous deamination of 13. To a solution of 13 (370 mg,
0.77 mmol) in THF (4 mL) and isoamylnitrite (533 mL,
5 equiv) was added dropwise Et3N (50 mL, 0.5 equiv).
The solution mixture was re¯uxed for 1 h 30 min then
cooled to room temperature. After addition of water
(3 mL) and CH2Cl2 (4 mL), decantation, extraction
(3Â6 mL), the combined organic layers were dried
(MgSO4) and concentrated in vacuo.The products were
further separated by column chromatography (cyclo-
hexane:EtOAc, 7:3) a€ording 16 (70 mg, 25%); Rf 0.16
and 17 (70 mg, 25%); Rf 0.12 as colourless oils.
3,4-Di-O-benzyl-2,5-[(1-oxycarbonyl)imino]-2,5-dideoxy-
D-glucitol (16). [a]d +53 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 3.7 (d, 1H, J=3.5 Hz), 3.72 (dt, 1H, J=2.7,
8.5 Hz), 3.8 (m, 1H), 3.89 (dt, 1H, J=2.7, 12.5 Hz), 3.97
(m, 1H), 4.29 (ddd, 1H, J=3.5, 5, 8.5 Hz), 4.33±4.63 (m,
7H), 7.1±7.5 (m, 10Harom); 13C NMR (CDCl3) d 61.9,
62.9 (C-1, C-6), 62.9, 67.1 (C-2, C-5), 71.3, 72.0
(OCH2Ph), 79.1, 84.6 (C-3, C-4), 127.7, 127.8, 128.3,
128.7 (CHarom), 136.8, 136.9 (Cqarom), 159.1 (CO).
CIMS, NH3: m/z (%) 370 (100) (MH+).
6-Acetylamino-2,5-[(1-oxycarbonyl)imino]-2,5,6-trideoxy-
D-glucitol (9c). Compound 14 (61.5 mg, 0.15 mmol) was
deprotected according to procedure A to yield quanti-
tatively 9c (34.5 mg) as a white solid after ¯ash chro-
matography; Rf 0.25 (CH2Cl2:MeOH, 9:1); mp 148±
151 ꢁC; [a]d +67.5 (c 0.51, CH3OH). 1H NMR
(CD3OD) d 2.0 (s, 3H, CH3), 3.43 (t, 1H, H-5), 3.78 (m,
3H, H-3, H-6, H-60), 4.12 (s, 1H, H-4), 4.39 (m, 3H, H-
3,4-Di-O-benzyl-2,5-[(6-oxycarbonyl)imino]-2,5-dideoxy-
D-glucitol (17). [a]d 9 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 3.9±4.2 (m, 5H), 4.43±4.74 (m, 7H), 7.15±7.35
(m, 10Harom); 13C NMR (CDCl3) d 58.7, 63.1 (C-2, C-
5), 64.7, 71.9, 72.4 (CH2O), 74.4, 85.5 (C-3, C-4), 127.9,
128.4, 128.7 (CHarom), 136.3 (Cqarom), 160.5 (CO).
CIMS, NH3: m/z (%) 370 (100) (MH+).
2, H-1, H-10), J5,6=J5,6 =6 Hz. 13C NMR (CD3OD) d
0
24.7 (CH3), 41.0 (C-6), 65.9 (C-1), 66.5, 68.1 (C-2, C-5),
76.6, 85.6 (C-3, C-4), 162.0 (COO), 175.0 (CON).
HRMS calcd for C9H15N2O5 (MH+) 231.0981, found
231.0982.
2,5-Dideoxy-2,5-[(1-oxycarbonyl)imino]-D-glucitol (9d).
Compound 16 (70 mg, 0.19 mmol) was deprotected
according to procedure A. Chromatographic puri®ca-
tion (CH2Cl2:MeOH, 9:1), a€orded 9d (30 mg, 83%) as
6-Amino-3,4-di-O-benzyl-2,5-[(6-aminocarbonyl)imino]-
2,5,6-trideoxy-D-glucitol (15). To a solution of 13
(60 mg, 0.128 mmol) in CH2Cl2 (0.4 mL) was added
dropwise Et3N (21.5 mL, 1.2 equiv). After 1 h stirring
at room temperature the volatile components were eva-
porated. Compound 15 was obtained quantitatively
(45.5 mg) as a colourless oil after column chromato-
graphy; Rf 0.20 (cyclohexane:EtOAc, 2:8), [a]d 24.5
1
a colourless oil; Rf 0.28; [a]d +34 (c 1.0, MeOH). H
NMR (CD3OD) d 3.49 (dt, 1H, H-5), 3.72 (brs, 1H, H-
3), 3.95 (dd, 1H, H-6), 4.09 (dd, 1H, H-60), 4.17 (brs,
1H, H-4), 4.44 (m, 3H, H-2, H-1, H-10), J2,3
=
0
0
J4,5=1 Hz, J5,6=4.8 Hz, J5,6 =4.5 Hz, J6,6 =11.5 Hz;
13C NMR (CD3OD) d 59.7, 64.3 (C-1, C-6), 64.9, 68.2
(C-2, C-5), 74.4, 83.8 (C-3, C-4), 160.5 (CO). CIMS,
CH4: m/z (%) 190 (100) (MH+).
1
(c 0.66, CH2Cl2). H NMR (CDCl3) d 3.28 (brt, 1H, H-
6), 3.54 (t, 1H, H-60), 3.75±3.96 (m, 4H, H-2, H-4, H-1,
H-5), 4.0 (dd, 1H, J=3.5Hz, 5, H-3,), 4.20 (dd, 1H, H-10),

I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
141
Opening of the N-Boc bis-aziridine 1 by acetic acid.
Compound (920 mg, 1.75 mmol) in acetic acid
2-O-Allyl-6-[(tert-butyloxycarbonyl)amino]-3,4-di-O-ben-
zyl-1,5-[tert-butyloxycarbonyl)imino]-1,5,6-trideoxy-D-
mannitol (8b). [a]d 28.5 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 1.42, 1.44 (s, 18H, CH3), 3.06 (m, 1H, H-1),
3.24 (m, 1H, H-6), 3.51 (brs, 1H, H-4), 3.63 (m, 2H),
3.82 (brs, 1H, H-3), 3.9±4.24 (m, 3H, containing
OCH2CH=CH2), 4.25±4.9 (m, 6H, H-5, NH, OCH2Ph),
5.15 (dd, 1H, J=1.5, 10.5 Hz, OCH2CH=CH2), 5.26
(dd, 1H, J=1.5, 16 Hz, OCH2CH=CH2), 5.89 (m, 1H,
OCH2CH=CH2), 7.1±7.45 (m, 10Harom); 13C NMR
(CDCl3) d 28.3, 28.4 (CH3), 36.9, 39.3 (C-1, C-6), 53.2
(C-5), 70.0, 71.1 (OCH2Ph), 73.5 (OCH2CH=CH2),
73.1, 75.1, 75.7 (C-2, C-3, C-4), 79.1, 80.0 (CqBoc), 116.5
(OCH2CH=CH2), 127.4, 127.6, 127.7, 128.3 (CHarom),
134.8 (OCH2CH=CH2), 137.9, 138.3 (Cqarom), 155.8
(CO). CIMS, NH3: m/z (%) 583 (100) (MH+).
1
(17.5 mL) was stirred for 2 h at room temperature. After
concentration in vacuo, the products were further sepa-
rated by column chromatography (cyclohexane:EtOAc,
4:1), a€ording 7a; Rf 0.33 (612 mg, 60%) and 8a; Rf 0.23
(275 mg, 27%) as colourless oils.
1-O-Acetyl-3,4-di-O-benzyl-6-[(tert-butyloxycarbonyl)-
amino]-2,5-[(tert-butyloxycarbonyl)imino]-2,5,6-trideoxy-
D-glucitol (7a). [a]d 11 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 1.42, 1.44 (s, 18H, CH3), 2.01 (s, 3H, OAc),
3.3±3.6 (m, 2H, H-6, H-60), 3.75 (m, 1H, H-5), 3.98 (t,
1H, H-4), 4.07 (t, 1H, H-3), 4.14 (dd, 1H, H-1), 4.31 (m,
2H, H-10, H-2), 4.54, 4.57 (AB, 2H, OCH2Ph), 4.61 (s,
2H, OCH2Ph), 5.33 (m, 1H, NH), 7.2±7.4 (m, 10Harom),
0
J1,1 =10.2 Hz, J1,2=4.5 Hz, J3,4=J4,5=5.5 Hz, JAB
=
12 Hz; 13C NMR (CDCl3) d 21.0 (CH3CO), 28.3, 28.4
(CH3Boc), 42.8 (C-6), 57.2, 61.5 (C-2, C-5), 62.4 (C-1),
72.5, 72.8 (OCH2Ph), 79.1, 80.9 (CqBoc), 81.5, 83.0 (C-3,
C-4), 127.8, 128.0, 128.4, 128.5 (CHarom), 137.3, 137.8
(Cqarom), 155.1, 156.1 (COBoc), 170.7 (COAc).
1-O-Acetyl-6-amino-3,4-di-O-benzyl-2,5-imino-2,5,6-tri-
deoxy-D-glucitol, TFA (18). Compound 7a (370 mg,
0.63 mmol) was deprotected according to procedure B
to yield quantitatively 18 (387 mg) as an amorphous
solid, which was used in the next step without further
puri®cation. 1H NMR (CDCl3) d 1.98 (s, 3H, CH3),
0
0
3.31 (m, 1H, H-6, J6,6 =13 Hz), 3.46 (brt, 1H, H-6 ),
2-O-Acetyl-6-[(tert-butyloxycarbonyl)amino]-3,4-di-O-
benzyl-1,5-[(tert-butyloxycarbonyl)imino]-1,5,6-trideoxy-
D-mannitol (8a). [a]d 10 (c 1.0, CH2Cl2). 1H NMR
(CDCl3) d 1.40, 1.42 (s, 18H, CH3), 2.02 (s, 3H, OAc),
3.18 (brt, 2H, J=12 Hz, H-1, H-6), 3.49 (dd, 1H, H-4,
J=1.3, 3.5 Hz), 3.6±3.8 (m, 1H, H-60), 3.88 (brs, 1H, H-
3), 4.09 (m, 1H, H-10), 4.30±4.75 (m, 6H, OCH2Ph, H-5,
NH), 5.01 (ddd, 1H, H-2, J=3, 4.7, 11 Hz); 13C NMR
(CDCl3) d 20.8 (CH3CO), 28.1, 28.3 (CH3Boc), 36.9, 39.2
(C-1, C-6), 53.0 (C-5), 67.9 (C-2), 71.0, 73.3 (OCH2Ph),
74.6 (C-3, C-4), 79.0, 80.1 (CqBoc), 127.4, 127.5, 127.7,
128.2, 128.3 (CHarom), 137.5, 137.7 (CqBoc), 155.5, 155.8
(COBoc), 169.7 (COAc).
3.78 (s, 1H), 3.9±4.05 (m, 2H), 4.09 (m, 1H), 4.3±4.6 (m,
6H, containing OCH2Ph), 7.1±7.4 (m, 10Harom).
2-O-Allyl-6-amino-3,4-di-O-benzyl-1,5-imino-1,5,6-tri-
deoxy-D-mannitol, TFA (20). Piperidine 8a (97.4 mg,
0.17mmol) was deprotected according to procedure B to
yield 20 (101mg, 100%) as an amorphous solid, which
was used in the next step without further puri®cation.
1H NMR (CDCl3) d 3.0 (m, 1H), 3.25±3.6 (m, 3H),
3.62±3.8 (m, 2H), 3.9 (brs, 2H), 4.01, 4.08 (dd, 2H,
J=5.5, 12.7 Hz, OCH2CH=CH2), 4.55±4.85 (m, 4H,
OCH2Ph), 5.15±5.35 (m, 2H, OCH2CH=CH2), 5.87 (m,
1H, OCH2CH=CH2), 7.1±7.4 (m, 10Harom); 13C NMR
(CDCl3) d 38.6 (C-6), 43.0 (C-1), 55.5 (C-5), 71.2, 72.7
(OCH2Ph), 74.8 (OCH2CH=CH2), 69.6, 74.6, 79.0 (C-2,
C-3, C-4), 118.4 (OCH2CH=CH2), 127.8, 128.0, 128.6
(CHarom), 133.8 (OCH2CH=CH2), 136.8, 137.2 (Cqarom).
Opening of the N-Boc bis-aziridine 1 by allyl alcohol. To
1 (131 mg, 0.25 mmol) in allyl alcohol (2.5 mL) was
added ytterbium tri¯ate (15 mg, 0.1 equiv) at room
temperature and the mixture was stirred for 2 h at this
temperature. After concentration the products were
further separated by column chromatography (toluene:
EtOAc, 95:5) on Kieselgel [60 and 60 H (5±40 mm), 1/1],
a€ording 7b; Rf 0.35 (39.3 mg, 27%) and 8b; Rf 0.25
(80.2 mg, 55%) as colourless oils.
1-O-Acetyl-6-N-acetylamino-3,4-di-O-benzyl-2,5-imino-
2,5,6-trideoxy-D-glucitol (19). Pyrrolidine 18 (135 mg,
0.22 mmol) was selectively acetylated according to pro-
cedure C to yield 19 (61 mg, 65%) as a white solid after
puri®cation by ¯ash chromatography (CH2Cl2:MeOH:
NH4OH, 95:5:0.1); Rf (CH2Cl2:MeOH, 95:5), mp 79 ꢁC;
1-O-Allyl-[(tert-butyloxycarbonyl)amino]-3,4-di-O-benzyl-
2,5-[(tert-butyloxycarbonyl)imino]-2,5,6-trideoxy-D-gluc-
1
[a]d +21 (c 0.61, CHCl3). H NMR (CDCl3) d 1.75 (s,
itol (7b). [a]d
10.5 (c 0.67, CH2Cl2). 1H NMR
3H, CH3CON), 2.0 (s, 3H, CH3CO2), 2.7 (brs, 1H, NH),
3.2±3.65 (m, 3H), 3.73 (s, 1H), 3.87 (brs, 1H), 4.2 (dd,
1H, J=7.5, 11 Hz), 4.29 (dd, 1H, J=5.2, 11 Hz), 4.36,
4.51 (AB, 2H, J=11.7 Hz, OCH2Ph), 4.49 (s, 2H,
OCH2Ph), 6.35 (brs, 1H, NHAc), 7.1±7.4 (m, 10Harom);
13C NMR (CDCl3) d 20.9, 23.0 (CH3), 42.3 (C-6), 59.3,
62.2, 63.9 (C-1, C-2, C-5), 71.9 (OCH2Ph), 82.1, 84.4 (C-
3, C-4), 127.6, 127.8, 128.1, 128.5 (CHarom), 137.3, 137.6
(Cqarom), 170.6, 170.9 (CO). HRMS calcd for C24H31
N2O5 (MH+) 427.2232, found 427.2228.
(CDCl3) d 1.41, 1.43 (s, 18H, CH3), 3.38 (dt, 1H,
0
J6,6 =13.5 Hz, J6,5=4.5 Hz, H-6), 3.6±3.75 (m, 4H con-
taining H-60 and H-5), 3.9±4.25 (m, 4H containing
OCH2CH=CH2), 4.50±4.75 (m, 4H, OCH2Ph), 5.12
(dd, 1H, J=1.5, 10 Hz, OCH2CH=CH2), 5.24 (dd, 1H,
J=1.5, 11 Hz, OCH2CH=CH2), 5.8±5.95 (m, 2H, NH,
OCH2CH=CH2), 7.2±7.4 (m, 10Harom); 13C NMR
(CDCl3) d 28.3, 28.5 (CH3), 42.3 (C-6), 58.0, 60.1 (C-2,
C-5), 67.3 (C-1), 72.1, 72.7, 72.9 (OCH2Ph, OCH2CH
=CH2), 78.7, 80.6 (CqBoc), 81.7, 83.1 (C-3, C-4),
117.1 (OCH2CH=CH2), 127.8, 128.4 (CHarom), 134.6
(OCH2CH=CH2), 137.8, 138.0 (Cqarom), 155.1, 156.1
(CO). CIMS, NH3: m/z (%) 583 (100) (MH+).
6-N-Acetylamino-2-O-allyl-3,4-di-O-benzyl-1,5-imino-1,5,6-
trideoxy-D-mannitol (21). The TFA salt 20 (48.8 mg,
0.8 mmol) was acetylated according to procedure C to

142
I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
yield 21 (20 mg, 73%) as a colourless oil after puri®ca-
tion by column chromatography (CH2Cl2:MeOH:
NH4OH, 95:5:1); Rf 0.5 (CH2Cl2:MeOH, 9:1). [a]d
17.5 (c 0.98, CH2Cl2). 1H NMR (CDCl3) d 1.86 (s, 3H,
CH3), 2.35±2.6 (m, 2H, H-1, H-5), 3.0±3.2 (m, 2H, H-10,
1H, H-6), 2.83 (dd, 1H, H-60), 2.95 (m, 1H, H-5), 3.28
(m, 1H, H-2), 3.65 (dd, 1H, H-1), 3.73 (dd, 1H, H-4),
3.78 (dd, 1H, H-10), 3.95 (dd, 1H, H-3), J1,1 =11 Hz,
0
0
J1,2=6 Hz, J1 ,2=5.8 Hz, J2,3=4.8 Hz, J3,4=2.5 Hz,
0
0
J4,5=3.7 Hz, J5,6 =4.5 Hz, J5,6=6.3 Hz, J6,6 =13 Hz.
13C NMR (CD3OD) d 44.7 (C-6), 62.1 (C-1), 63.0, 67.4
(C-2, C-5), 79.0, 82.0 (C-3, C-4). CIMS, NH3: m/z (%)
163 (100) (MH+).
H-6), 3.5 (m, 2H, H-3, H-4), 3.6±3.75 (m containing
0
0
ddd, 2H, J=4.6 Hz, J6,6 =13.5 Hz, H-6 , H-2), 4.11 (m,
2H, OCH2CH=CH2), 4.61, 4.91 (AB, 2H, J=10.8,
OCH2Ph), 4.63, 4.7 (A0B0, 2H, J=11.8 Hz, OCH2Ph),
5.15±5.30 (m, 2H, OCH2CH=CH2), 5.8 (m, 1H, NH),
6-N-Acetylamino-2,5-imino-2,5,6-trideoxy-D-glucitol (11).
Compound 19 (60 mg, 0.14 mmol) was deprotected
according to procedure D. Flash column chromato-
graphy using a 7:3:0.6:0.3 mixture of CH2Cl2:MeOH:
H2O:AcOH as eluent; Rf 0.50, gave 11 as AcOH salt
(29 mg, 78%). [a]d +2 (c 1.0, MeOH), [a]365 +12 (c 1.0;
MeOH). A sample was neutralized from a Dowex 1X8
and characterized; [a]d +26 (c 0.4, H2O), lit18: [a]d
5.9 (m, 1H, OCH2CH=CH2), 7.2±7.4 (m, 10Harom); 13
C
NMR (CDCl3) d 22.7 (CH3), 39.3 (C-6), 44.7 (C-1), 58.4
(C-5), 70.9, 72.1 (OCH2Ph), 70.8, 75.0, 81.6 (C-2, C-3,
C-4), 74.9 (OCH2CH=CH2), 117.9 (OCH2CH=CH2),
127.7, 127.8, 128.2, 128.4 (CHarom), 134.2 (OCH2CH
=CH2), 137.6, 137.8 (Cqarom), 171.7 (CO). CIMS, NH3:
m/z (%) 425 (100) (MH+).
1
+27.2 (c 0.23; H2O). H NMR (D2O) d 2.05 (s, 3H,
6-[(tert-Butyloxycarbonyl)amino]-3,4-di-O-benzyl-1,5-
[tert-butyloxycarbonyl)imino]-1,5,6-trideoxy-D-mannitol
CH3), 3.09 (m, 1H, H-5), 3.36 (m, 1H, H-2), 3.38 (dd,
1H, H-6), 3.49 (dd, 1H, H-60), 3.71 (dd, 1H, H-1), 3.83
(dd, 1H, H-10), 3.86 (dd, 1H, H-4), 4.16 (dd, 1H, H-3),
(22). To
a solution of allyl ether 17 (26.3 mg,
0
0
J1,1 =11 Hz, J1,2=6 Hz, J1 ,2=5.5 Hz, J2,3=5 Hz, J3,4
0.045 mmol) in 1 mL of anhydrous THF was added
anhydrous zinc chloride (16.5 mg 0.12 mmol). After
15 min stirring at 20 ꢁC, tetrakis (triphenylphosphine)
palladium (0) (10.4 mg, 0.009 mmol) was added and the
stirring carried on for 15 min. Tributyltin hydride was
then added dropwise (49 mL, 0.18 mmol) and reaction
mixture allowed to stir for 1 h. Organic solvent was
removed and the mixture was quenched by addition of
water (0.5 mL), 0.1 N HCl (1 mL), and EtOAc (2.5 mL).
The ethyl acetate phase was washed with 2 mL of satd
NaHCO3 (aq), dried (MgSO4), ®ltered, and evaporated.
Chromatographic puri®cation (EtOAc:cyclohexane, 2:3)
yielded 22 as a colourless oil (18.6 mg, 75%); Rf 0.50;
=
0
0
3 Hz, J4,5=5.5 Hz, J5,6 =6.3 Hz, J5,6=12 Hz, J6,6
=
14 Hz. 11 AcOH: 13C NMR (CD3OD) d 20.7, 22.5
(CH3), 42.1 (C-6), 61.7 (C-1), 63.0, 67.5 (C-2, C-5), 77.4,
79.7 (C-3, C-4), 172.5, 174.6 (CO). HRMS calcd for
C8H17N2O4 (MH+) 205.1188, found 205.1177.
.
6-Amino-1,5-imino-1,5,6-trideoxy-D-mannitol (3). Com-
pound 20 (38.8 mg, 0.063 mmol) was deprotected
according to procedure D. Puri®cation was performed
by ¯ash column chromatography using a 5:5:0.5:0.5
mixture of CH2Cl2:MeOH:H2O:AcOH as eluent; Rf
0.05. The residue was further eluted from a Dowex CG-
400 (OH-) ion-exchange resin to a€ord 3 (6.6 mg, 65%).
1
[a]d 23 (c 0.26, CH2Cl2). H NMR (CDCl3) d 1.41,
1
1.42 (s, 18H, CH3), 2.13 (d, 1H, OH), 2.86 (t, 1H, H-1),
3.05±3.25 (m, 1H, H-6), 3.5±3.8 (m, 3H, H-4, H-60, H-
3), 3.87 (m, 1H, H-2), 4.05 (m, 1H, H-10); 4.3±4.75 (m,
6H, OCH2Ph, H-5, NH), 7.1±7.3 (m, 10H, Harom),
[a]d 37 (c 0.67, MeOH). H NMR (CD3OD) d 2.33
(m, 1H, H-5), 2.71 (m, 2H, H-1, H-6), 2.98 (m, 2H, H-
10, H-60), 3.36 (m, 2H, H-3, H-4), 3.84 (m, 1H, H-2),
J1,1 =J6,6 =13 Hz; 13C NMR (CD3OD) d 44.1 (C-6),
50.7 (C-1), 63.1 (C-5), 70.8 (C-2), 72.3, 76.7 (C-3, C-4).
CIMS, NH3: m/z (%) 163 (100) (MH+).
0
0
0
J1,1 =J1,2=12 Hz, JOH,2=9.7 Hz, J2,3=2.8 Hz, J3,4
=
3 Hz; 13C NMR (CDCl3) d 28.3, 28.4 (CH3), 38.9, 40.3
(C-1, C-6), 52.7 (C-5), 64.4 (C-2) 71.2, 73.4 (OCH2Ph),
73.7, 77.3 (C-3, C-4), 79.3, 80.2 (CqBoc), 127.6, 127.8,
128.3, 128.4, 128.7 (CHarom), 137.2, 137.7 (Cqarom),
156.1, 155.9 (CO).
6-N-Acetylamino-1,5-imino-1,5,6-trideoxy-D-mannitol (12).
Compound 21 (61.56 mg, 0.1405 mmol) was deprotected
according to procedure D. After evaporation of ammo-
nia the residue was diluted with a solution of CH3OH:
AcOH and ®ltered. Flash chromatography using a 7:3:
0.6:0.3 mixture of CH2Cl2:MeOH:H2O:AcOH as eluent;
Rf 0.30, followed by neutralization from a Dowex
1X8 gave the title compound 12 (24 mg, 81%). [a]d 30
6-[(tert-Butyloxycarbonyl)amino]-2,5-[tert-butyloxycar-
bonyl)imino]-2,5,6-trideoxy-D-glucitol (23). Compound
7a (56.5 mg, 0.097 mmol) was deprotected according to
procedure D to yield 23 as a foam after ¯ash chroma-
tography puri®cation (35 mg, 87%); Rf 0.23 (EtOAc),
1
(c 0.8, H2O). H NMR (D2O) 2.07 (s, 3H, CH3) 2.6
1
[a]d +13.5 (c 2.0, CH2Cl2). H NMR (CDCl3) d 1.39,
(ddd, 1H, H-5), 2.75 (dd, 1H, H-1), 3.01 (dd, 1H, H-10),
3.39 (dd, 1H, H-6), 3.48 (t, 1H, H-4), 3.56 (dd, 1H,
1.43 (s, 18H, CH3), 3.44 (m, 2H), 3.59 (m, 1H), 3.7±5.0
(m, 8H containing OH), 5.45, 5.7 (brs, NH). CIMS,
CH4: m/z (%) 363 (100) (MH+).
H-60), 3.6 (dd, 1H, H-3), 4.03 (m, 1H, H-2), J1,1
=
=
0
0
0
0
J6,6 =14.4 Hz, J1,2=1.5 Hz, J1 ,2=2.5 Hz, J2,3=J5,6
3 Hz, J3,4=J4,5=9.5 Hz, J5,6=7.3 Hz; 13C NMR (D2O)
d 24.8 (CH3), 43.4 (C-6), 51.3 (C-1), 62.2 (C-5), 72.0 (C-
2), 73.0 (C-4), 77.1 (C-3), 177.6 (CO).
6-Amino-2,5-imino-2,5,6-trideoxy-D-glucitol (2). Com-
pound 23 (30 mg, 0.08 mmol) was deprotected according
to procedure B to yield the title compound 2 after
chromatography puri®cation; Rf 0.10 (CH2Cl2:MeOH:
H2O:AcOH, 5:5:1:0.5) followed by neutralization from
an Amberlite IRA-400 (11 mg, 82%), [a]d +12 (c 0.33,
Inhibition studies
All enzymes, a-glucosidase type IV from brewers yeast
(EC 3.2.1.20), b-glucosidase from almonds (EC 3.2.1.21),
1
MeOH), [a]365 +54.5. H NMR (CD3OD) d 2.75 (dd,

I. McCort et al. / Bioorg. Med. Chem. 8 (2000) 135±143
143
a-galactosidase from Aspergillus niger (EC 3.2.1.22), a-
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.
2. Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res.
1993, 26, 182.
3. Nishimura, Y. In Studies in Natural Products Chemistry,
Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1992; Vol. 10, pp
495±583.
4. de Raadt, A.; Eckhart, C. W.; Ebner, M.; Stutz, A. E.
Topics. Curr. Chem. 1997, 187, 157 and references cited therein.
5. Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J.
Am. Chem. Soc. 1997, 119, 4856.
6. Baudat, A.; Vogel, P. J. Org. Chem. 1997, 62, 6252.
7. Martin, O.; Saavedra, O. J. Org. Chem. 1996, 61, 6987.
8. Dong, W.; Jesperen, T.; Bols, M.; Skrydstrup, T.; Sierks, M.
R. Biochemistry 1996, 35, 2788.
9. Fitremann, J.; Dureault, A.; Depezay, J. C. Tetrahedron
Lett. 1994, 35, 1201.
10. Campanini, L.; Dureault, A.; Depezay, J. C. Tetrahedron
Lett. 1995, 36, 8015.
11. McCort, I.; Dureault, A.; Depezay, J. C. Tetrahedron Lett.
1996, 37, 7717.
12. Campanini, L.; Dureault, A.; Depezay, J. C. Tetrahedron
Lett. 1996, 37, 5095.
13. McCort, I.; Dureault, A.; Depezay, J. C. Tetrahedron Lett.
1998, 39, 4463.
14. Liu, K. K.-C.; Kajimoto, T.; Chen, L.; Ichikawa, Y.;
Wong, C.-H. J. Org. Chem. 1991, 56, 6280.
15. Winchester, B.; Barker, C.; Baines, S.; Jacob, G. S.; Nam-
goong, S. K.; Fleet, G. Biochem. J. 1990, 265, 277.
16. Legler, G.; Lullau, E.; Kappes, E.; Kastenholz, F. Biochi-
mica et Biophysica Acta 1991, 89.
17. Wong, C.-H.; Provencher, L.; Porco, J. A., Jr.; Jung, S.-H.;
Wang, Y.-F.; Chen, L.; Wang, R.; Steensma, D. H. J. Org.
Chem. 1995, 60, 1492.
Assays were run in 50 mM citrate±phosphate bu€er, pH
6.5, at 37 ꢁC, 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.
For IC50 determinations, substrate concentrations were
the KM values and inhibitors in 10 mL MeOH were
incorporated to give ®nal concentrations ranging from
0.1 to 10 KM. For Ki determinations, substrates, as their
salt form, were at a concentration range of 0.2±5 KM
and inhibitors were added to ®nal concentration
3
between 10 and 10 8 M. The inhibition studies were
performed by prewarming the solutions 5 min prior to
add 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 concentration of
liberated p-nitrophenoxide was determined by measur-
ing the optical absorbance at 400 nm. Dissociation con-
stants for inhibitors were calculated in the absence or
presence of inhibitors according to Linearwearer±Burck
method.
References
18. Kang, S. H.; Ryu, D. H. Tetrahedron Lett. 1997, 38, 607.
19. Yin, H.; Franck, R.; Chen, S.; Quigley, G. J.; Todaro, L. J.
Org. Chem. 1992, 57, 644.
1. Winchester, B.; Fleet, G. W. Glycobiology 1992, 2, 199.