N-Linked carba-disaccharides as potential inhibitors of glucosidases I and II

Article abstract of DOI:10.1039/a902826i

1 On leave from the Departmcnto de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribcirao Preto, Brasil. An N-linked pseudodisaccharide 2 containing an inositol moiety in place of a glycopyranosy

Full text of DOI:10.1039/a902826i

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N-Linked carba-disaccharides as potential inhibitors of
glucosidases I and II
Ivone Carvalho† and Alan H. Haines*
School of Chemical Sciences, University of East Anglia, Norwich, UK NR4 7TJ
Received (in Cambridge) 9th April 1999, Accepted 18th May 1999
An N-linked pseudodisaccharide 2 containing an inositol moiety in place of a glycopyranosyl residue has been
synthesised to mimic the disaccharide unit, 3-O-(α--glucopyranosyl)--glucopyranose, cleaved by glucosidase II
during glycoprotein processing. Its inhibitory action towards yeast α-glucosidase was inferior to that of 1-deoxynojir-
mycin, a known inhibitor of glucosidase II. An attempt to prepare the analogous 2-N-linked compound 1 as an
inhibitor of glucosidase I led to the novel N-phenyl derivative 11 of 2-amino-2-deoxy--glucopyranose.
2. These mimic, respectively, the terminal pair and penultimate
Introduction
pair of carbohydrate residues in Glc3Man9GlcNAc2 and may be
The enzymes glucosidase I and glucosidase II are involved in
designated N-linked carba-disaccharides. Consideration of the
key steps in the processing of N-linked oligosaccharides
mechanism of glycosidase action4 suggests that such com-
(Scheme 1) by cleaving three terminal glucose residues from the
pounds might preferentially occupy the catalytic site of the
enzyme with the nitrogen atom at the site of initial protonation
OH
O
of a normal substrate. Importantly, such compounds resemble
the terminal carba-disaccharide unit in the effective α-amylase
inhibitor acarbose 3 in having a nitrogen bridge between the
two rings, one of which is carbocyclic.5,6 We now report full
details of our work which was the subject of a preliminary
communication.7
OH
O
OH
O
OH
O
OH
OH
OH
O
OH
O
HO
HO
O
OH
HO
OH
Glucosidase I
(Man) (GlcNAc) (Asn-P)
O
Glucosidase II
8
2
Results and discussion
The synthesis of 1 and 2 hinges on imine formation between the
key chiral penta-O-benzylinosose 4, accessible from the corre-
sponding chiral protected myo-inositol8 and either protected
methyl 2-amino-2-deoxy- or methyl 3-amino-3-deoxy-α--
glucopyranoside, 5 or 6, respectively, followed by stereoselective
reduction (NaBH3CN) of the imine and then deprotection
of the reduction product by catalytic hydrogenolysis of the
O-benzyl groups.
Preparation of the chiral inosose 4 involves a 9-step synthesis
from myo-inositol, in which the racemic 1,2,4,5,6-penta-O-
benzyl-myo-inositol is resolved9 through ester formation with
(1S,4R)-camphanic chloride and chromatographic separation
OH
O
and
OH
3 x
OH
OH
(Man)9 (GlcNAc)2 (Asn-P)
OH
Scheme 1
tetradeca-oligosaccharide moiety Glc3Man9GlcNAc2 of an
important intermediate N-linked oligosaccharide.1 Inhibitors
of these enzymes such as 1-deoxynojirimycin (DNJ) (for gluco-
sidase I and glucosidase II) and castanospermine (for gluco-
sidase I) cause malformation of these oligosaccharides2 and
have shown interesting anti-HIV activity, which is thought to
arise from prevention of successful completion of gp120 in the
viral coat, and hence viral reproduction.3 We considered that
specificity and inhibitory activity towards these two enzymes
might well be increased substantially by designing inhibitors
which mimic the two carbohydrate residues immediately
involved in the chain cleavage and further, that the exploration
of inhibitors containing exocyclic nitrogen at C-1, rather than
endocyclic nitrogen as found in 1-deoxynojirimycin and
castanospermine, would be fruitful. However, since simple link-
age of the two carbohydrate residues via nitrogen would afford
a glycosylamine derivative which is likely to be hydrolytically
unstable under physiological conditions, we sought to replace
the glycosyl component in such a glycosylamine by a carbo-
cyclic ring which might mimic to a great extent the topography
of -glucose and thus we arrived at the target molecules 1 and
OH OH
OH
OH
O
OH OH
OH
OH
O
OH
NH
OH
OMe
NH
OH
OH
OH
OH
OH
OMe
OH
1
2
CH2OH
CH2OH
O
CH3
OH
CH2OH
O
O
OH
OH
OH
OH
OH
NH
O
HO
O
OH
OH
OH
3
R1
O
OR OR
OR
R2
R1
O
OMe
R3
OR
OR
† On leave from the Departmento de Ciências Farmacêuticas, Facul-
dade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de
São Paulo, Ribeirão Preto, Brasil.
4 R = CH2Ph
5 R1 = R2 = OCH2Ph; R3 = NH2
6 R1 = R3 = OCH2Ph; R2 = NH2
.
J. Chem. Soc., Perkin Trans. 1, 1999, 1795–1800
1795

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OR
of the diastereoisomeric esters. Swern oxidation of the
(Ϫ)-1,2,4,5,6-penta-O-benzyl-myo-inositol gave (ϩ)-2,3,5/
4,6-pentabenzyloxycyclohexanone 4 as an analytically pure
solid in 83% yield whose spectroscopic properties agree well
with those reported10 for the racemic compound. Character-
istically, it showed carbonyl absorption at 1730 cmϪ1 and an
absorption at δC 205.78 in its 13C NMR spectrum.
RO
2'
4'
1'
H
6'
RO
RO
OR
3'
5'
RO
O
RO
N
H
OMe
16
Synthesis of the protected 2-amino gluco-compound 5
required temporary N-protection with the 2,2,2-trichloro-
ethoxycarbonyl (troc) group, introduced (Scheme 2) by reaction
OR
OR
RO
O
RO
2'
4'
N
RO
6'
3'
1'
H
RO
RO
OR2
O
OR2
OH
O
OR2
O
OR2
5'
OMe
RO
H
i
iv
v
5
OH
17
R = Bn
OH
NH2
R2O
HO
OR1
NHCO2CH2CCl3
R2O
R1O
OMe
NH
Fig. 1 Configurational isomers which can arise form reduction of the
imine 15 showing stereo-dependence of J1Ј,2Ј and J1Ј,6Ј
OR1
.
7
8
9
R1 = H, R2 = H
R1 = Me, R2 = H
10 R1 = Me, R2 = Bn
ii
-glucopyranoside 13, together with the methyl 2-azido-2-
deoxy-α--altropyranoside were first prepared by a reported
procedure14 involving ring opening of methyl 2,3-anhydro-α--
allopyranoside with azide ion and chromatographic separation
of the isomeric azides. The ratio of the gluco- (2,3-diequatorial)
to altro- (2,3-diaxial) isomers was 2.7:1, a much more favour-
able ratio with respect to the required gluco-isomer 13 than the
1:15 obtained15 by a similar reaction on the corresponding
4,6-O-benzylidene derivative, which is conformationally con-
strained and therefore forced to follow a diaxial ring opening of
the epoxide. Conventional benzylation of azide 13 gave the tri-
O-benzyl azide 14 which, on reduction of the azido group by
treatment with triphenylphosphine–THF–H2O or with LiAlH4
in diethyl ether gave the required protected 3-amino compound
6 in 70 and 75% yield, respectively.
Although a direct reductive alkylation of the amines 5 and 6
with the chiral inosose 4 would seem to offer the most direct
route to compounds of the type 1 and 2, problems were
encountered on attempting to obtain reaction of amine 5 in
this manner (see later) and yields were disappointing when the
reaction was attempted with 6. We investigated, therefore, an
indirect two-step approach involving imine formation followed
by cyanoborohydride reduction. With amine 6, the first step
was best conducted using the extremely mild conditions first
reported by Taguchi and Westheimer16 in which a solution of
the carbonyl component and the amine in benzene is stored
over molecular sieves at ambient temperature for 70 h; in this
manner the imine 15 was obtained from 4 and 6 in ~60% yield
and was then immediately reduced in DMF solution with
sodium cyanoborohydride which could afford, in theory, one or
both of the expected products 16 and 17, which are depicted in
their expected chair conformations in Fig. 1.
iii
OR1
11 R1
=
R2 = Bn
12 R1 = R2 = H
vi
Scheme 2 Reagents and conditions (and yields): i, CCl3CH2OCOCl, aq.
NaHCO3, 10 ЊC for 2 h then 20 ЊC for 19 h (72%); ii, 2.5% w/v HCl
in MeOH, reflux 8.5 h, (65%); iii, CCl3C(NH)OCH2Ph, CH2Cl2–
cyclohexane (1:2), CF3SO3H, 20 ЊC, 2.5 h (78%); iv, Zn/Cu, THF–1 
aq. KH2PO4 (5:1), 20 ЊC, 8.5 h (62%); v, 4, C6H6 (addition and evapor-
ation, × 5), 60 ЊC (79%); vi, EtOH–cyclohexene (2:1), Pd-black, reflux
under Ar, 38 h, yield not determined (air-unstable product).
of -glucosamine 7 with 2,2,2-trichloroethoxycarbonyl chlor-
ide in an aqueous solution of sodium hydrogen carbonate.11
Fischer-type glycosidation of the so-formed carbamate 8 gave
initially a mixture of α- and β-glycosides with the α-isomer 9
1
predominating as indicated by the integrated signals in the H
NMR spectrum for OMe, with the α-isomer resonating at
higher field (δH 3.39) than the β-isomer (δH 3.50). As reaction
time increased, the proportion of α-isomer increased until it
was the major component of the crude product from which it
could then be isolated in 65% yield by crystallisation. The
glycoside 9 could not be benzylated under the usual conditions
(NaH, PhCH2Br) because of instability of the N-protecting
group under basic conditions. However, O-benzylation was
successfully achieved in an acid-catalysed process using
benzyl trichloroacetimidate,12 and removal of the troc group of
the 3,4,6-tri-O-benzyl ether 10 with zinc powder in THF–
potassium dihydrogen phosphate buffer13 gave the free amine 5.
For the synthesis of 6 (Scheme 3), methyl 3-azido-3-deoxy-α-
OR
O
OR OR
OR
OR
O
Evidence that the reduction had proceeded with good stereo-
selectivity to give required product 16 was provided by the H
ii
iii
1
N3
6
N
and 13C NMR spectra but the complexity of the 1H NMR spec-
trum precluded a definitive decision regarding the absence of a
small amount of a second isomer, which later measurements on
the deprotected material suggested must have been present in a
minor amount (< 10%). Extensive investigation of the 1H NMR
spectrum at 600 MHz and a consideration of the conformations
of 16 and 17 allowed allocation of structure 16 to the pre-
dominant reduction product on the basis of the coupling pat-
tern observed for H-1 of the cyclitol. The key observation is the
coupling pattern observed for H-1 in the cyclitol ring, which for
16 would be expected to be an apparent triplet (Japparent ~3 Hz)
resulting from two similar coupling constants (J1Ј,2Ј J1Ј,6Ј ~3 Hz),
and for 17 would be expected to be a double doublet (J1Ј,2Ј
~3 Hz, J1Ј,6Ј ~9 Hz). The observed signal, assignment confirmed
by deuterium labelling, was an apparent triplet at δH 3.82 with
Japparent ~4.4 Hz.
OMe
OR
RO
OR
OMe
OR
RO
OR
13 R = H
14 R = Bn
15 R = Bn
i
iv
OR OR
OR
OR
OR
O
v
2
NH
OR
OMe
OR
OR
16 R = Bn
Scheme 3 Reagents and conditions (and yields): i, NaH, BnBr, DMF,
20 ЊC, 1 h (91%); ii, Ph3P, THF–H2O (10:1), 80 ЊC, 7 h (70%) or
LiAlH4, Et2O, 35 ЊC, 1 h (75%); iii, 4, C6H6, 4 Å molecular sieves under
N2, 20 ЊC, 70 h; iv, NaBH3CN, DMF, 20 ЊC, 70 h and 100 ЊC, 1 h (61%);
v, EtOH–cyclohexene (2:1), Pd-black, reflux 24 h (67%).
Deprotection of 16 using catalytic transfer hydrogenation17
(Pd–cyclohexene–EtOH) gave the required pseudodisaccharide
1796
J. Chem. Soc., Perkin Trans. 1, 1999, 1795–1800

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1
2. The H NMR spectrum was consistent with the expected
which is in accord with the poor inhibitory properties of 2
structure, but alongside the doublet signal at δ 4.66 for the
anomeric proton was a neighbouring doublet resonance at
δ 4.46 of very low intensity with the same spacing, which we
attribute to a small amount (~5%) of the stereoisomeric reduc-
tion product. The proportion of the latter could be reduced
significantly, but with loss in overall yield, by careful re-
chromatography on silica gel.
compared with DNJ against yeast α-glucosidase.
Experimental
1H NMR spectra were recorded at 270 MHz on a JEOL EX270
FT spectrometer, or at 600 MHz on a Varian Unity Inova spec-
trometer for samples in CDCl3 unless stated otherwise, with
Me4Si as internal standard. 13C NMR spectra were similarly
recorded at 67.9 MHz on a JEOL EX270 FT spectrometer.
Coupling constants (J-values) are given in Hz. Where appropri-
ate, signal assignments were deduced by DEPT, COSY and
HETCOR NMR experiments. Optical rotations were measured
at ambient temperature with a Perkin-Elmer model 141 polar-
imeter for solutions in CHCl3 unless stated otherwise and [α]D-
values are given in 10Ϫ1 deg cm2 gϪ1. IR spectra were recorded
on a Perkin-Elmer model 298 spectrophotometer. High-
resolution mass spectra were recorded by the EPSRC Mass
Spectrometry Service Centre at the University College of Swan-
sea. Low-resolution mass spectra and elemental analyses were
performed by Mr A. W. R. Saunders at the University of East
Anglia. TLC was performed on silica gel (Machery-Nagel) SIL
G-25UV254 and compounds on developed plates were detected
either by viewing with a UV lamp (254 nm), or by spraying with
a 10% sulfuric acid, 1.5% molybdic acid, 1% ceric (Ce4ϩ) sulfate
spray followed by heating to 150 ЊC. Column chromatography
was performed on Matrex Silica 60 (70–200 mm mesh, Fisons)
or Kieselgel 60 (70–230 mm mesh, Merck). Light petroleum
refers to the fraction with boiling range 40–60 ЊC. Where mixed
solvents were used, the ratios given are v/v. Tetrahydrofuran
and diethyl ether were pre-dried by storage over sodium wire
and obtained anhydrous by distillation from sodium metal and
benzophenone once the blue colouration due to the ketyl
radical had been achieved; dichloromethane was obtained
anhydrous by distilling from calcium hydride; methanol was
dried by distilling from the alkoxide (formed by reaction with
activated magnesium) and storage over powdered 3 Å molecular
sieves; toluene and benzene were dried by distillation from
calcium hydride and stored over 4 Å molecular sieves; triethyl-
amine was dried by distilling from calcium hydride immediately
prior to use; pyridine was obtained anhydrous by distillation
from calcium hydride and storage over potassium hydroxide
pellets. Organic solutions were dried over anhydrous MgSO4.
Reactions were maintained at Ϫ78 ЊC by means of a solid CO2–
acetone bath, and at 0 ЊC by means of an ice-bath.
An attempt to repeat the reaction sequence for the prepar-
ation of 1 by reaction of 4 and 5 gave unexpected results. No
reaction occurred on storage of a mixture of 4 and 5 in benzene
over molecular sieves for several days but repeated evaporation
of benzene from the mixture at 60 ЊC gave a product whose 1H
NMR spectrum contained a sharp two-proton singlet at δ 6.25
and a total of only six benzyl groups, pointing to aromatisation
of the former cyclitol ring. NMR spectral data and elemental
analysis indicated the compound to be methyl 3,4,6-tri-O-
benzyl-2-(2,4,6-tribenzyloxyphenylamino)-2-deoxy-α--gluco-
pyranoside 11 which can arise from an initially formed imine
by elimination of two molecules of benzyl alcohol followed
by aromatisation of the imino quinone so formed. The single
aromatic resonance in the 1H NMR spectrum requires
that the isomeric 3,4,5-tribenzyloxy structure also be con-
sidered for the product but the assignment of the 2,4,6-
tribenzyloxy structure is based on combined consideration of
(i) comparison of the estimated 13C chemical shifts for the two
isomers with those measured (especially for Ar-CNHR: calcu-
lated for 2,4,6-isomer δC 120.2, calculated for 3,4,5-isomer δC
1
140.0, observed δC 121.38) (ii) the H NMR chemical shifts of
ArH in 2,4,6-trimethoxyaniline (δH 6.05) and 3,4,5-trimethoxy-
aniline (δH 5.91) compared with that observed (δH 6.25) (iii)
the likely mechanism of formation from the supposed imine
intermediate.
Careful catalytic transfer hydrogenolysis of 11 afforded a
readily oxidised solid product, whose spectral properties sup-
ported the structure 12.
The inhibitory properties of compound 2 against yeast
α-glucosidase were compared with that of DNJ by measuring
the rate of enzymic release of 4-nitrophenol from 4-nitrophenyl
α--glucopyranoside in the absence and in the presence of
inhibitor followed by standard Lineweaver–Burk analysis.18
Compound 2 was a poor competitive inhibtor of the enzyme
(Ki ~1.5 mM) in contrast to DNJ for which we obtained Ki 30
µM. Under identical conditions, to bring about a 20% reduction
in the rate of enzymic hydrolysis of the aryl glycoside, an
approximately 100-fold greater molar concentration of 2 was
required compared with that for DNJ. The considerable differ-
ence in inhibitory properties of the two compounds towards the
enzyme is perhaps not surprising in view of their likely different
mechanisms of action as a result of the different positions of
the nitrogen atoms in the two molecules. Protonated DNJ
is likely to mimic the cyclic oxocarbenium intermediate of
glycoside hydrolysis whereas protonated 2 mimics an earlier
intermediate in the hydrolytic pathway, the O-1-protonated
glycoside. Interestingly, recent reports suggest that nitrogen-
containing inhibitors in which the N atom occupies the site of
the carbon atom at the glycosidic centre (e.g., C-1 in aldo-
pyranoses) often show high inhibitory activity.19 This carbon
atom carries a formal positive charge in one of the resonance
contributors of the intermediate oxocarbenium ion obtained.
Clearly, comparative experiments with suitable inhibitors of a
glycosidase, in which nitrogen replaces O-1, C-1 or ring-O of
a glycoside, could provide useful information on the detailed
mechanism of action of this class of enzymes.
(Ϫ)-1,2,4,5,6-Penta-O-benzyl-myo-inositol9 was prepared
from myo-inositol, 2-(2Ј,2Ј,2Ј-trichloroethoxycarbonylamino)-
2-deoxy--glucose11 8 from -glucosamine 7, and methyl
3-azido-3-deoxy-α--glucopyranoside14 13 from methyl 2,3-
anhydro-α--glucopyranoside by the literature procedures.
Yeast α-glucosidase (type IV from brewer’s yeast) was obtained
from Sigma Chemical Co.
(؉)-2,3,5/4,6-Pentabenzyloxycyclohexanone 4
A solution of oxalyl dichloride (0.17 ml, 1.95 mmol) in dichloro-
methane (4.3 ml) was cooled to Ϫ78 ЊC and dimethyl sulfoxide
(0.29 ml, 4.12 mmol) in dichloromethane (0.9 ml) was added
dropwise from a syringe over ca. 5 min. The mixture was then
stirred for 10 min at Ϫ78 ЊC and then (Ϫ)-1,2,4,5,6-penta-O-
benzyl-myo-inositol (900 mg, 1.43 mmol) dissolved in dichloro-
methane (1.72 ml) was added dropwise during ca. 5 min.
Stirring was continued at Ϫ78 ЊC for 30 min after which time
triethylamine (1.19 ml, 8.58 mmol) was added. The cooling
bath was removed and H2O (5 ml) was added at room temper-
ature. Stirring was continued for 10 min and the organic layer
was then separated. The aqueous phase was extracted with
dichloromethane (3 × 5 ml) and the combined organic layers
were washed successively with brine (5 ml), 1% aq. HCl (5 ml),
H2O (4 ml), 5% aq. Na2CO3 (5 ml) and water (4 ml). The solu-
In independent experiments performed by Dr W. McDowell,
compound 2 was assayed for inhibitory properties against
glucosidases I and II using rat liver microsomes possessing
activity in these enzymes and a labelled substrate. Unfortu-
nately, no inhibition of the activity of either of these enzymes
was observed for 2 up to a concentration of 100 µg mlϪ1, a result
J. Chem. Soc., Perkin Trans. 1, 1999, 1795–1800
1797

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tion was dried, concentrated, and the residue was subjected to
silica gel column chromatography [eluent: toluene–ethyl acetate
(15:1)] to give title chiral inosose 4 (740 mg, 83%), mp 90–92 ЊC
(Found: C, 78.2; H, 6.25. C41H40O6 requires C, 78.3; H, 6.4%);
[α]D ϩ9.2 (c 1); νmax(Nujol)/cmϪ1 1730 (CO), 1090 (COC), 745,
695 (Ar); δH (270 MHz) 3.44 (1H, dd, J2,3 2.7, J3,4 9.3, 3-H), 3.51
(1H, t, J4,5 = J5,6 = 9.3, 5-H), 3.97 (1H, d, 2-H), 4.28 (1H, t,
4-H), 4.41 (2H, s, OCH2Ph), 4.44 and 4.60 (each 1H, d, JAB
12.2, OCH2Ph), 4.54 and 4.56 (each 1H, d, JAB 11.9, OCH2Ph),
4.69 (1H, d, 6-H), 4.77 and 4.83 (each 1H, d, JAB 10.5,
OCH2Ph), 4.88 and 4.91 (each 1H, d, JAB 10.5, OCH2Ph),
7.26–7.32 (25H, m, 5 × Ph); δC (67.9 MHz) 72.61 (OCH2Ph),
72.81 (OCH2Ph), 73.36 (OCH2Ph), 75.86 (OCH2Ph), 75.99
(OCH2Ph), 79.19 (3-C), 80.84 (4-C), 81.16 (2-C), 81.93 (5-C),
83.10 (6-C), 127.58–138.43 (30 C, Ar-C), 205.78 (CO).
4.64 (each 1H, d, JAB 11.9, OCH2Ph), 4.52 and 4.62 (each 1H,
d, JAB 10.9, OCH2Ph), 4.64 (2H, s, OCH2Ph), 4.63 (1H, 1-H),
7.18–7.35 (15H, m, 3 × Ph); δC (67.9 MHz) 53.76 (3-C), 55.14
(OCH3), 68.60 (6-C), 69.86 (5-C), 72.81 (OCH2Ph), 73.53
(OCH2Ph), 74.53 (OCH2Ph), 78.95 (4-C), 79.87 (2-C), 97.03
(1-C), 127.65–138.12 (18 C, Ar-C).
Methyl 2,4,6-tri-O-benzyl-3-[(1S,2S,3S,4R,5R,6S)-2,3,4,5,6-
pentabenzyloxycyclohexylamino]-3-deoxy-á-D-glucopyranoside
16
A solution of ketone 4 (270 mg, 0.43 mmol) and amine 6 (250
mg, 0.54 mmol, 20% excess) in dry benzene (1.0 ml), main-
tained under nitrogen atmosphere, was stirred in the presence
of 4 Å molecular sieves (300 mg, powdered) for 3 days. The
mixture was filtered and the solid washed with dry benzene. The
filtrate was concentrated in vacuo and the resulting crude imine
15 was diluted in DMF (5.0 ml) and Na(CN)BH3 (185 mg) was
introduced in one portion. The reaction mixture was stirred
for 3 days at room temperature and 1 h at 100 ЊC. DMF was
removed under reduced pressure and the residue was partitioned
(water–dichloromethane). The organic phase was separated
and the aqueous phase extracted with dichloromethane. The
combined organic phase was dried, and concentrated in vacuo.
Purification of the residue by column chromatography [eluent:
hexane–ethyl acetate (3:1)] gave title compound 16 (282 mg,
61%) as a viscous, colourless oil (Found: C, 76.65; H, 6.8; N,
1.35. C69H73NO10 requires C, 77.0; H, 6.8; N, 1.3%); [α]D ϩ34 (c
0.5); νmax(film)/cmϪ1 3310 (NH), 1065 (COC), 740 and 695 (Ar);
δH (600 MHz) 3.20–3.35 (2H, m, 2- and 3-H), 3.33 (3H, s,
OCH3), 3.43–3.47 (1H, m, 4-H), 3.62 (1H, dd, J5,6a 1.9, J6a,6b
10.8, 6a-H), 3.68 (1H, dd, J5,6b 3.5, 6b-H), 3.77 (1H, br dt, J4,5
10.0, 5-H), 3.81 (1H, t, J3Ј,4Ј = J4Ј,5Ј = 9.8, 4Ј-H*), 3.82 (1H, t,
J1Ј,2Ј = J1Ј,6Ј = 4.4, 1Ј-H), 3.91 (1H, t, J5Ј,6Ј 9.8, 5Ј-H*), 3.94 (1H,
dd, 6Ј-H), 4.03 (1H, br t, J2Ј,3Ј 3.0, 2Ј-H), 4.20 (1H, dd, 3Ј-H),
4.29 and 4.57 (each 1H, d, JAB 12.0, OCH2Ph), 4.39 and 4.65
(each 1H, d, JAB 11.2, OCH2Ph), 4.46 and 4.61 (each 1H, d, JAB
12.0, OCH2Ph), 4.54 and 4.59 (each 2H, d, JAB 11.5, OCH2Ph),
4.54 and 4.64 (each 1H, d, JAB 12.0, OCH2Ph), 4.67 (1H, d, J1,2
3.6, 1-H), 4.76 and 4.79 (each 1H, d, JAB 10.8, OCH2Ph), 4.80
and 4.89 (each 1H, d, JAB 10.8, OCH2Ph), 7.03–7.38 (40H, m,
8 × Ph); δC (67.9 MHz) 55.02 (OCH3), 56.72 (1Ј-C), 58.29 (3-C),
68.64 (6-C), 69.81 (5-C), 72.82 (OCH2Ph), 72.93 (OCH2Ph),
72.99 (2 × OCH2Ph), 73.20 (OCH2Ph), 73.56 (OCH2Ph), 75.59
(OCH2Ph), 75.65 (OCH2Ph), 76.94 (2Ј-C), 79.35 (6Ј-C), 79.89
(4-C), 80.43 (2- and 3Ј-C), 82.31 (4Ј- or 5Ј-C), 82.53 (5Ј- or
4Ј-C), 97.41 (1-C), 127.00–139.40 (48 C, Ar-C).
Methyl 3-azido-2,4,6-tri-O-benzyl-3-deoxy-á-D-glucopyrano-
side 14
Methyl 3-azido-3-deoxy-α--glucopyranoside 13 (100 mg, 0.46
mmol) was dissolved in DMF (2 ml), under nitrogen atmos-
phere, and sodium hydride (110 mg of a 60% dispersion in
mineral oil, previously washed with hexane, 2.73 mmol) was
added cautiously, portionwise. After being stirred for 40 min,
the mixture was cooled (0 ЊC, ice-bath) and benzyl bromide
(0.48 ml, 4.1 mmol) was added dropwise. After 10 min, the ice-
bath was removed and the stirring was continued for 10 min at
room temperature. Methanol (0.6 ml) was then added and the
mixture was evaporated to dryness in vacuo. The residue was
purified by silica gel column chromatography [eluent: first light
petroleum and then hexane–ethyl acetate (7:3)] to give, as a
viscous oil, title compound 14 (203 mg, 91%) (Found: C, 68.6;
H, 6.3; N, 8.5. C28H31N3O5 requires C, 68.7; H, 6.4; N 8.6%);
[α]D ϩ58.2 (c 1); νmax(film)/cmϪ1 2100 (N3), 1100 and 1040
(COC), 730 and 700 (Ar): δH (270 MHz) 3.33 (3H, s, OCH3),
3.37 (1H, dd, J1,2 3.3, J2,3 9.9, 2-H), 3.43 (1H, t, J3,4 = J4,5 = 9.9,
4-H), 3.58 (1H, dd, J5,6a 3.6, J6a,6b 11.7, 6a-H), 3.66–3.74 (2H, m,
5-H and 6b-H), 3.89 (1H, t, 3-H), 4.42 and 4.78 (each 1H, d, JAB
10.7, OCH2Ph), 4.45 and 4.60 (each 1H, d, JA,B 12.0, OCH2Ph),
4.63 and 4.76 (each 1H, d, JAB 12.2, OCH2Ph), 4.58 (1H, d,
1-H), 7.18–7.39 (15H, m, 3 × Ph); δC (67.9 MHz) 55.20 (OCH3),
65.46 (3-C), 68.04 (6-C), 69.64 (5-C), 73.17 (OCH2Ph), 73.58
(OCH2Ph), 74.80 (OCH2Ph), 76.13 (4-C), 77.82 (2-C), 97.32
(1-C), 127.85–138.12 (18 C, Ar-C).
Methyl 3-amino-2,4,6-tri-O-benzyl-3-deoxy-á-D-glucopyrano-
side 6
* These assignments may be reversed, with corresponding
re-allocation of J-values.
Method 1. Triphenylphosphine (352 mg, 1.34 mmol) and
water (0.4 ml) were added to a solution of azide 14 (224 mg,
0.46 mmol) in THF (4 ml) and the mixture was heated at 80 ЊC
for 7 h and then concentrated. The residue was purified by silica
gel column chromatography [eluent: diethyl ether–triethylamine
(10:1)] to give amine 6 (148 mg, 70%) as a colourless oil.
Methyl 3-deoxy-3-[(1S,2S,3S,4R,5R,6S)-2,3,4,5,6-penta-
hydroxycyclohexylamino]-á-D-glucopyranoside 2
Pd-black catalyst (35 mg) was added to a solution of 16 (250
mg, 0.23 mmol) in ethanol–cyclohexene [6.9 ml (2:1)]. The mix-
ture was heated under reflux for 24 h, cooled, filtered, and the
filtrate was concentrated in vacuo. The residue was purified first
by silica gel column chromatography [eluent: MeOH–ethyl
acetate (1:1)] to give compound 2 in crude form. A second
purification by ion exchange resin IRA 400 (eluent: MeOH)
afforded 2 (55 mg, 67%) as a foam; [α]D ϩ92.4 (c 0.4,
MeOH); νmax(Nujol)/cmϪ1 3387 (OH, NH), 1040 (CO); δH (600
MHz; CD3OD) 2.78 (1H, t, J2,3 = J3,4 = 9.8, 3-H), 3.25 (1H, t,
J4,5 9.8, 4-H), 3.26 (1H, br t, J1Ј,2Ј = J1Ј,6Ј = 4.5, 1Ј-H), 3.32
(1H, dd, J1,2 3.7, 2-H), 3.41 (3H, s, OCH3), 3.47 (1H, t,
J3Ј,4Ј = J4Ј,5Ј = 9.1, 4Ј-H*), 3.51 (1H, m, 5-H), 3.53 (1H, t, J5Ј,6Ј
9.1, 5Ј-H*), 3.63 (1H, dd, J5,6a 5.9, J6a,6b 11.8, 6a-H), 3.67 (1H,
dd, J2Ј,3Ј 3.2, 3Ј-H), 3.77 (2H, 2 × dd, J5,6b 2.3, 6b-H and 6Ј-H),
4.04 (1H, t, 2Ј-H), 4.66 (1H, d, 1-H), 4.84 (s br, OH); δC (67.9
MHz; CD3OD) 55.45 (OCH3), 62.44 (1Ј-C), 62.82 (6-C), 64.27
(3-C), 72.20 (4-C), 72.55 (2- and 6Ј-C), 72.72 (3Ј-C), 73.24
Method 2. A solution of the azide 14 (160 mg, 0.33 mmol) in
dry diethyl ether (0.7 ml) was added dropwise to a suspension
of LiAlH4 (30 mg, 0.79 mmol) in dry diethyl ether (0.7 ml). The
reaction mixture was heated under reflux for 1 h and then
cooled (0 ЊC) and the remaining LiAlH4 was destroyed and
inorganic salts were precipitated by the successive addition of
H2O (0.03 ml), 15% aq. NaOH (0.03 ml) and then H2O (0.1 ml).
The mixture was stirred for 30 min, filtered and the solid
washed with diethyl ether. The combined organic filtrate was
dried, concentrated and the residue was purified as described in
method 1 to give oily amine 6 (113 mg, 75%) (Found: C, 72.4;
H, 7.05; N, 3.1. C28H33NO5 requires C, 72.55; H, 7.2; N, 3.0%);
[α]D ϩ51.5 (c 0.5); νmax(film)/cmϪ1 3380 (NH2), 1100 and 1030
(COC), 740 and 700 (Ar); δH (270 MHz) 1.68 (2H, br s, NH2),
3.34 (3H, s, OCH3), 3.33–3.38 (2H, m, 2- and 3-H), 3.42–3.47
(1H, m, 4-H), 3.64–3.77 (3H, m, 5-H, 6a-H, 6b-H), 4.50 and
1798
J. Chem. Soc., Perkin Trans. 1, 1999, 1795–1800

View Article Online
(2Ј-C), 74.32 (5-C), 74.57 (4Ј- or 5Ј-C), 75.27 (5Ј- or 4Ј-C),
101.05 (1-C); m/z (FAB) 356 [M ϩ H]ϩ (Found: [M ϩ H]ϩ
356.1560. C13H26NO10 requires m/z, 356.1557.
* These assignments may be reversed, with corresponding
re-allocation of J-values.
chromatography [eluent: diethyl ether–triethylamine (5:1)] to
give title compound 5 (198 mg, 62%) as a colourless oil; [α]D
ϩ101.4 (c 0.3); νmax(film)/cmϪ1 3400 (NH), 1050 (COC), 740
and 700 (Ar); δH (270 MHz) 1.46 (2H, s br, NH2), 2.81 (1 H,
dd, J1,2 3.4, J2,3 9.2, 2-H), 3.35 (3H, s, OCH3), 3.55 (1H, t,
J3,4 = J4,5 = 9.2, 4-H), 3.63 (1H, t, 3-H), 3.65–4.00 (3H, m, 5-H,
6-H2), 4.52 and 4.64 (each 1H, d, JAB 12.3, OCH2Ph), 4.52 and
4.78 (each 1H, d, JAB 10.9, OCH2Ph), 4.69 and 4.95 (each 1H,
d, JAB 11.5, OCH2Ph), 4.74 (1H, d, 1-H), 7.15–7.36 (15H, m,
3 × Ph); δC (67.9 MHz) 55.05 (OCH3), 55.90 (2-C), 68.64 (6-C),
70.83 (5-C), 73.51 (OCH2Ph), 74.66 (OCH2Ph), 75.54
(OCH2Ph), 78.79 (3-C), 84.06 (4-C), 100.64 (1-C), 127.56–
138.57 (18 C, Ar-C); m/z (EI) 463 [M]ϩ (Found: [M]ϩ, 463.2359.
C28H33NO5 requires M, 463.2359).
Methyl 2-(2Ј,2Ј,2Ј-trichloroethoxycarbonylamino)-2-deoxy-á-D-
glucopyranoside 9
Acetyl chloride (1 ml, 14.08 mmol) was added dropwise to
a suspension of 2-(2Ј,2Ј,2Ј-trichloroethoxycarbonylamino)-2-
deoxy--glucose 8 (1.0 g, 2.82 mmol) in dry methanol (20 ml).
The mixture was heated under reflux for 8.5 h, after which time
TLC [ethyl acetate–methanol (9:1)] showed that most of the
starting material was consumed. The reaction mixture was
cooled, and then neutralised by portionwise addition of lead
carbonate (1.41 g). Stirring was continued for 1 h and then the
mixture was filtered through a kieselguhr pad and the filtrate
was concentrated to dryness. Recrystallisation of the crude
product, containing mostly α-isomer, from toluene gave title
compound 9 (678 mg, 65%) as a crystalline solid, mp 135–138 ЊC
(Found: C, 31.3; H, 4.45; N, 3.5; Cl, 27.6. C10H16NO7Cl3ؒH2O
requires C, 31.1; H, 4.7; N, 3.6; Cl, 27.5%); [α]D ϩ75.4 (c 0.3,
MeOH); νmax(Nujol)/cmϪ1 3350 (OH, NH), 1730 (CO), 1055
(COC), 820 (CCl3); δH (270 MHz; CDCl3 ϩ CD3OD) 3.39 (3H,
s, OCH3), 3.46 (1H, t, J3,4 = J4,5 = 9.2, 4-H), 3.53–3.59 (1H, m,
5-H), 3.60–3.70 (2H, m, 2-, 3-H), 3.73 (1H, dd, J5,6a 5.0, J6a,6b
11.9, 6a-H), 3.85 (1H, dd, J5,6b 2.7, 6b-H), 4.68, 4.73, 4.78, 4.83
(2H, AB q, JAB 12.0, OCH2CCl3), 4.75 (1H, d, J1,2 2.7, 1-H);
δC (67.9 MHz; CDCl3 ϩ CD3OD) 55.39 (OCH3), 56.45 (2-C),
62.02 (6-C), 71.35 (4-C), 72.39 (3-C), 72.61 (5-C), 75.12
(OCH2CCl3), 95.84 (OCH2CCl3), 99.18 (1-C), 155.61 (CO).
Methyl 3,4,6-tri-O-benzyl-2-(2Ј,4Ј,6Ј-tribenzyloxyphenyl-
amino)-2-deoxy-á-D-glucopyranoside 11
Compound 5 (195 mg, 0.42 mmol) and ketone 4 (214 mg, 0.34
mmol) were dissolved in dry benzene (3 ml) and the solution
was concentrated to dryness at 60 ЊC and maintained under
reduced pressure at this temperature for 3 h. The residue was re-
dissolved in benzene (3 ml) and the process repeated four times.
The residue was purified by column chromatography [eluent:
hexane–ethyl acetate (3:1)] to give title compound 11 (230 mg,
79%) as a viscous oil (Found: C, 76.6; H, 6.4; N, 1.6. C55H55NO8
requires C, 77.0; H, 6.45; N, 1.6%); [α]D ϩ28.3 (c 0.7); νmax(film)/
cmϪ1 1050 (COC), 740, 700 (Ar); δH (270 MHz) 3.29 (3H, s,
OCH3), 3.58 (1H, t, J3,4 = J4,5 = 9.4, 4-H), 3.66 (1H, dd, J5,6a 1.7,
J6a,6b 10.3, 6a-H), 3.73 (1H, dd, J5,6b 3.7, 6b-H), 3.76–3.91 (2H,
m, 3-, 5-H), 4.14 (1H, dd, J1,2 3.3, J2,3 9.9, 2-H), 4.46 and 4.74
(each 1H, d, JAB 10.8, OCH2Ph), 4.50 and 4.60 (each 1H, d, JAB
12.2, OCH2Ph), 4.69 (2H, s, OCH2Ph), 4.85 (2H, s, OCH2Ph),
4.97 and 5.00 (each 1H, d, JAB 12.1, OCH2Ph), 4.98 (2H, s,
OCH2Ph), 4.99 (1H, d, 1-H), 6.25 (2H, s, 3Ј- and 5Ј-H), 6.93–
7.40 (30H, m, 6 × Ph); δC (67.9 MHz) 55.05 (OCH3), 58.63
(2-C), 68.91 (6-C), 70.47 (5-C), 70.58 (OCH2Ph), 70.65 (2 ×
OCH2Ph), 73.35 (OCH2Ph), 74.66 (OCH2Ph), 74.75 (OCH2-
Ph), 78.59 (3-C), 83.71 (4-C), 94.87 (3Ј- and 5Ј-C), 100.32 (1-C),
121.38 (1Ј-C, Ar-CNHR), 126.84–139.02, (36 C, Ar-C), 149.79
(2Ј- and 6Ј-C), 152.63 (4Ј-C).
Methyl 3,4,6-tri-O-benzyl-2-(2Ј,2Ј,2Ј-trichloroethoxycarbonyl-
amino)-2-deoxy-á-D-glucopyranoside 10
Triflic acid (0.01 ml) was added to a stirred suspension of com-
pound 9 (100 mg, 0.27 mmol) and benzyl trichloroacetimidate
(0.3 ml, 1.62 mmol) in cyclohexane–dichloromethane (2:1, 2.7
ml) and the mixture was stirred for 2.5 h. The reaction mixture
was then filtered and the filtrate washed successively with satur-
ated aq. NaHCO3 and water. The dried solution was evaporated
to dryness and the residue was purified by column chrom-
atography [eluent: first hexane–ethyl acetate (9:1) and then
(7:3)]. The product 10 (135 mg, 78%) was obtained as a viscous
oil which crystallised spontaneously to give a solid, mp 59–
62 ЊC; [α]D ϩ57.4 (c 0.8); νmax(film)/cmϪ1 3350 (NH), 1735 (CO),
1055 (COC), 825 (CCl3), 740, 705 (Ar); δH (270 MHz) 3.34 (3H,
s, OCH3), 3.60–3.80 (5H, m, 3-, 4-, 5-H, 6-H2), 3.86–4.10 (1H,
m, 2-H), 4.51 and 4.62 (each 1H, d, JAB 11.4, OCH2Ph), 4.51
and 4.78 (each 1H, d, JAB 10.5, OCH2CCl3), 4.63 and 4.77 (each
1H, d, JAB 12.2, OCH2Ph), 4.70 and 4.81 (each 1H, d, JAB 11.2,
OCH2Ph), 4.73 (1H, d, J1,2 3.6, 1-H), 7.16–7.33 (15H, m,
3 × Ph); δC (67.9 MHz) 55.03 (OCH3 and 2-C), 68.35 (6-C),
70.69 (5-C), 73.40 (OCH2Ph), 74.59 (OCH2Ph), 74.89 (OCH2-
CCl3), 75.12 (OCH2Ph), 78.20 (3- or 4-C), 80.44 (4- or 3-C),
95.36 (OCH2CCl3), 98.61 (1-C), 127.67–138.00 (18 C, Ar-C),
154.17 (CO); m/z (EI) 546 [M Ϫ C7H7]ϩ (Found: [M Ϫ C7H7]ϩ
546.0846. C24H27Cl3NO7 requires m/z, 546.0853).
Methyl 2-deoxy-2-(2Ј,4Ј,6Ј-trihydroxyphenylamino)-á-D-gluco-
pyranoside 12
Compound 11 (134 mg, 0.16 mmol), under argon atmosphere,
was dissolved in ethanol–cyclohexene (2:1 v/v; 4.3 ml), a
catalytic amount of Pd-black was added, and the mixture
heated under reflux for 38 h. The catalyst was separated by
decantion and the solution was transferred to a flask while being
maintained constantly under an argon atmosphere. The solvent
was removed by distillation in vacuo with intermittent introduc-
tion of argon. The pale yellow solid 12 (identified solely by its
NMR spectra) was obtained in quantitative yield and was used
directly to prepare the NMR sample, also under inert atmos-
phere; δH (270 MHz; CD3OD) 3.40 (3H, s, OCH3), 3.42 (1H, t,
J3,4 = J4,5 = 9.5, 4-H), 3.57–3.66 (1H, m, 5-H), 3.64 (1H, dd, J1,2
3.3, J2,3 10.8, 2-H), 3.71 (1H, dd, J5,6a 5.3, J6a,6b 11.9, 6a-H), 3.82
(1H, dd, J5,6b 2.3, 6b-H), 4.04 (1H, dd, 3-H), 4.63 (1H, d, 1-H),
4.90 (br s, OH), 6.00 (2H, s, 3Ј- and 5Ј-H); δC (67.9 MHz;
CD3OD) 55.93 (OCH3), 62.17 (6-C), 64.58 (2-C), 71.12 (3-C),
71.94 (4-C), 74.08 (5-C), 95.63 (3Ј- and 5Ј-C), 96.92 (1-C),
102.47 (1Ј-C, Ar-CNHR), 153.88 (2Ј- and 6Ј-C), 161.02 (4Ј-C).
Methyl 2-amino-3,4,6-tri-O-benzyl-2-deoxy-á-D-glucopyrano-
side 5
Activated zinc dust20 (1.09 g) was added to a stirred solution of
compound 10 (440 mg, 0.69 mmol) in THF (9.4 ml) and 1.0 M
potassium dihydrogen phosphate solution (1.9 ml). After
stirring for 9 h the reaction mixture was filtered through a
kieselguhr pad, concentrated and the residue was re-diluted
with dichloromethane and treated with pyridine (0.8 ml). The
mixture was stirred for 2 h and then washed with water, dried
and evaporated to dryness. The crude material was purified by
Enzyme assays
Inhibition experiments on yeast α-glucosidase (type IV from
brewer’s yeast) were performed as described previously21 at
30 ЊC and at pH 6.5 in PIPES–NaOAc buffer through
measurement of the rate of liberation of 4-nitrophenol from
4-nitrophenyl α--glucopyranoside by monitoring the absorp-
J. Chem. Soc., Perkin Trans. 1, 1999, 1795–1800
1799

View Article Online
6 E. Truscheit, W. Frommer, B. Junge, L. Muller, D. D. Schmidt and
W. Wingender, Angew. Chem., Int. Ed. Engl., 1981, 20, 744.
7 A. H. Haines and I. Carvalho, Chem. Commun., 1998, 817.
8 R. Aneja, S. Aneja, V. P. Pathak and P. T. Ivanova, Tetrahedron
Lett., 1994, 35, 6061.
9 G. R. Baker, D. C. Billington and D. Gani, Tetrahedron, 1991, 47,
3895.
tion of phenoxide anion at 400 nm. Plotting a graph of the
slopes of Lineweaver–Burk reciprocal plots of 1/v against 1/[S],
obtained in the presence of increasing amounts of inhibitor,
against corresponding inhibitor concentrations [I], to which
they are linearly related, yields an inhibition constant (Ki) from
the intercept of this graph on the [I]-axis.18
10 D. J. R. Massy and P. Wyss, Helv. Chim. Acta, 1990, 73, 1037.
11 M. Imoto, H. Yoshimura, T. Shimamoto, N. Sakaguchi, S.
Kusumoto and T. Shiba, Bull. Chem. Soc. Jpn., 1987, 60, 2205.
12 T. Iversen and D. R. Bundle, J. Chem. Soc., Chem Commun., 1981,
1240; H.-P. Wessel, T. Iversen and D. R. Bundle, J. Chem. Soc.,
Perkin Trans. 1, 1985, 2247.
13 G. Just and K. Grozinger, Synthesis, 1976, 457; J. F. Carson,
Synthesis, 1981, 269.
14 T. Morikawa, K. Tsujihara, M. Takeda and Y. Arai, Chem. Pharm.
Bull., 1982, 30, 4365.
15 R. D. Guthrie and D. Murphy, J. Chem. Soc., 1963, 5288.
16 K. Taguchi and F. H. Westheimer, J. Org. Chem., 1971, 36, 1570.
17 G. M. Anantharamaiah and K. M. Sivanandaiah, J. Chem. Soc.,
Perkin Trans. 1, 1977, 490.
Acknowledgements
We thank Mr C. MacDonald for expert help in obtaining
1H NMR spectra at 600 MHz, Mr A. W. R. Saunders for
microanalysis and low-resolution mass spectra, the Mass
Spectrometry Service Centre at Swansea for high-resolution
mass spectra and FAPESP (Brasil) for a research fellowship (to
I. C.). We also thank Dr W. McDowell of Glaxo Wellcome
Research and Development Ltd, Stevenage, UK for conducting
the inhibitory studies on glucosidase I and II.
18 I. H. Segel, Enzyme Kinetics, Wiley, New York, 1975
19 M. Bols, Acc. Chem. Res., 1998, 31, 1; Y. Ichikawa, Y. Igarashi,
M. Ichikawa and Y. Suhara, J. Am. Chem. Soc., 1998, 120, 3007;
M. Godskesen and I. Lundt, Tetrahedron Lett., 1998, 39, 5841.
20 S. Winstein and J. Sonnenberg, J. Am. Chem. Soc., 1961, 83, 3235.
21 P. A. Fowler, A. H. Haines, R. J. K. Taylor, E. J. T. Chrystal and
M. B. Gravestock, J. Chem. Soc., Perkin Trans. 1, 1994, 2229.
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