Synthesis and enzymatic evaluation of five-membered iminocyclitols and a pseudodisaccharide

Article abstract of DOI:10.1016/S0968-0896(00)00170-X

Described here are the synthesis of five-membered iminocyclitols with galacto-configuration and a pseudodisaccharide, and their inhibitory activities against β-galactosyltransferase, β-galactosidase and α-mannosidase. Copyright (C) 2000.

Full text of DOI:10.1016/S0968-0896(00)00170-X

Bioorganic & Medicinal Chemistry 8 (2000) 2249±2261
Synthesis and Enzymatic Evaluation of Five-Membered
Iminocyclitols and a Pseudodisaccharide
Chikako Saotome,y Yoshimi Kanie,y Osamu Kanie*,y and Chi-Huey Wong*,{
Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa,
Wako-shi, Saitama 351-0198 Japan
Received 24 January 2000; accepted 15 May 2000
AbstractÐDescribed here are the synthesis of ®ve-membered iminocyclitols with galacto-con®guration and a pseudodisaccharide,
and their inhibitory activities against b-galactosyltransferase, b-galactosidase and a-mannosidase. # 2000 Published by Elsevier
Science Ltd.
Introduction
In order to study the inhibition of glycoenzymes, gen-
eration of such compounds and evaluation of their
activities are necessary. Regarding the synthesis of imi-
nocyclitols, we have developed a general and straight-
forward synthetic process using pentofuranoses as the
starting material.3h,5 In addition, capillary zone electro-
phoresis (CZE) has been used and proven extremely
useful for the kinetic evaluation of glycoenzymes.3h,6
Glycosyltransferases and glycosidases are important
classes of enzymes responsible for the synthesis and
degradation of oligosaccharides.1 Development of spe-
ci®c inhibitors of such enzymes is of current interest as
these inhibitors may be used as modulators to control
cellular functions. Enzymatic hydrolysis of a glycosidic
bond generally takes place via general acid and base
catalysis that requires two critical residues, a proton
donor and a nucleophile (Fig. 1(A)).2 A distorted half-
chair-like transition state leading to a carboxonium ion
is considered to be involved in the reaction. A similar
mechanism is expected for the glycosyltransferase cata-
lyzed reaction where a base (designated as B in Figure
1(B)) is thought to be involved in the abstraction of the
acceptor hydroxyl proton. Five-membered iminocycli-
tols carrying hydroxyl groups with speci®c orientation
to mimic the shape and charge of the transition state of
the reacting sugar moiety have been shown to be potent
inhibitors of such enzymes.1 4 Since a cation-like tran-
sition state is expected to be involved in both the glyco-
syltransferase and glycosidase catalyzed reactions,
iminocyclitols can be used as core components for the
development of transition-state analogue inhibitors of
both families of enzymes.
We report here the chemical synthesis of several ®ve-
membered iminocyclitols with galacto con®guration (Fig.
2) and a pseudodisaccharide consisting of an iminocyclitol
and N-acetylglucosamine, and evaluations of their inhi-
bitory activities against b-galactosidase (EC 3.2.1.23), a-
mannosidase (EC 3.2.1.24) and b-galactosyltransferase
(EC 2.4.1.22) using capillary zone electrophoresis.
Results and Discussions
In order to study the mechanism of enzymes associated
with processing of oligosaccharides and to develop inhi-
bitors of such enzymes, we have selected a series of ®ve-
membered iminocyclitols 1±5 (Fig. 2). Since it appears
that compound 4 exists as an equilibrium mixture of
conformations including those mimic galactose (4a) and
mannose (4b), 4 may inhibit the enzymes associated
with the processing of these carbohydrates, where six-
membered iminocyclitols such as deoxygalactonojir-
imycin and deoxymannojirimycin have been shown to
be inhibitors of these enzymes. In addition, an extended
carbon chain at C-2 was incorporated for attachment of
another group to mimic the aglycon moiety of glyco-
sides. Also, the ring nitrogen is alkylated in order to
*Corresponding authors. Fax: +81-42-724-6317; e-mail: kokanee@li-
bra.ls.magaku.co.jp (O. Kanie); tel.: +1-858-784-2487; fax: +1-858-
784-2409; e-mail: wong@scripps.edu (C.H. Wong).
yCurrent address: Glycoscience Laboratory, Mitsubishi Kasei Institute
of Life Sciences, Minamiooya 11, Machidashi, Tokyo 194-8511 Japan.
{Permanent address: Department of Chemistry, The Scripps Research
Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
0968-0896/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0968-0896(00)00170-X

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C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
Figure 1. A. Proposed b-Gal-ase reaction. A carboxylic acid and a carboxylate residue are involved in the hydrolytic reaction. B. Schematic drawing
of b-GalT-ase reaction.
investigate the e€ect of N-substitution on the inhibition
activity.
(90%). Compound 20 obtained by reduction of the alde-
hyde using DIBAL was hydrogenated to give 4 (83%). On
the other hand, the hydroxyl group of 20 was benzylated
(21, 90%) and the Boc group was deprotected to give the
amino compound 22 (92%) for the N-alkylation reaction.
Compound 5 was obtained after methylation of 22
using formaldehyde under a reductive condition to give
23, followed by hydrogenolysis.
Synthesis of compounds 2 and 3 (Scheme 1)
Wittig reaction of 2,3,5-tri-O-benzyl-lyxo-furanose7 with
methyl (triphenylphosphoranylidene)acetate a€orded a
1:1 mixture of E- and Z-6, which yielded 6 (E) (66%)
after irradiation with a 200 W lamp. The methoxycar-
bonyl group was converted to the TBDMS protected
alcohol (8) via di-iso-butylaluminum hydride (DIBAL)
reduction followed by silylation. In order to introduce
the azide function in R-con®guration at the C-6 position,
a double inversion was carried out. The 6-OH group was
®rst chloromesylated5,8 (9, 97%) and the product was
treated with CsOAc to give 10 (75%). After removal of
the acetyl group, the OH group of 11 was again chlor-
omesylated for the second inversion (12) and the TBDMS
group was deprotected to give the allylic alcohol 13 for
Sharpless epoxidation in the presence of d-( )-diethyl
tartrate to a€ord 14 (86%). Compound 14 was treated
with NaN3 to give azide 15 (93%), which was subjected
to a reduction condition to give 16 (76%). The benzyl
groups were hydrogenolyzed to give the target com-
pound 2 (93%). Compound 3 was obtained via methy-
lation of the amino function in 16 and hydrogenolysis.
Synthesis of pseudodisaccharide 1 (Scheme 3)
Adding a leaving group, or its mimic, to the leaving group
position of these mimics is expected to increase its speci®-
city and/or anity against glycoenzymes.4e,f,10 Therefore,
we have conducted the synthesis of pseudodisaccharide 1,
using the synthesized ®ve-membered iminocyclitol, as
potential inhibitor of b-galactosyltransferase (GalT-ase),
b-galactosidase (Gal-ase) and a-mannosidase (Man-
ase). Compound 1 was designed to mimic the expected
transition-state complex in the enzymatic transforma-
tion (Figures 1 and 3), since compound 1 can be con-
sidered as a mimic of b-d-Gal-(1!4)-b-d-GlcNAc-OR,
or a-d-Man-(1!6)-b-d-GlcNAc-OR, both representing
common mammalian disaccharides. Among these
enzymes, it is known that GalT-ase has the most strict
substrate speci®city especially on the GlcNAc moiety.
For this reason, we have decided, based on the mapping
studies on the substrate speci®city of GalT-ase,11 that
the acceptor C-6 of the GlcNAc residue to be used to
connect with an iminocyclitol through a two-carbon
spacer, which would permit the formation of a hydro-
gen bond between the ammonium hydrogen and the O-4
of GlcNAc.
Synthesis of compounds 4 and 5 (Scheme 2)
In order to obtain compounds 49 and 5, the secondary
amino function of 16 was ®rst protected with the Boc
group to give 18 (72%). The C-10±C-20 bond of 18 was
then cleaved using Pb(OAc)4 to give the aldehyde 19

C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
2251
Figure 2. Structures of compounds 1±5 and their similarity to the
transition state analogues of galactopyranoside and mannopyranoside
hydrolysis.
Scheme 1. Reagents and conditions: (a) (1) Ph3P=CHCO2Me/benzene;
(2) PhSSPh, hg, cyclohexane; (b) DIBAL/CH2Cl2; (c) TBDMSCl:Et3N:
DMAP/DMF; (d) ClCH2SO2Cl-Pyr; (e) CsOAc-18-Crown-6/toluene; (f)
NaOMe; (g) M-HCl/THF; (h) t-BuOOH-Ti(O-i-Pr)4-d-( )-diethyltar-
trate-MS 4A/CH2Cl2; (i) NaN3/DMF; (j) Ph3P/THF; (k) HCHO-
NaBH3CN/MeOH; (l) H2-Pd(OH)2-C/M HCl:MeOH (1:1).
At ®rst, the N-phthaloyl protected octyl b-d-glucosami-
nide12 was treated with 1,1,2,2-tetramethoxycyclohex-
ane13 in the presence of acid catalysis in order to selec-
tively protect the 1,2-trans equatorial diol system to a€ord
24 (72%). The aldehyde obtained by Swern oxidation of
the remaining hydroxyl group was then coupled with
methyl (triphenylphosphoranylidene)acetate to give 25
(93%). Reduction of the methoxycarbonyl group using
DIBAL a€orded 26 (74%), which was then treated with
hydrazine hydrate in order to reduce the double bond
and to deprotect the phthaloyl group at once. Selective
N-acetylation of the obtained amine gave compound 27
(93%, two-steps). Swern oxidation of the product
a€orded the aldehyde 28 (90%) which was used for cou-
pling with the protected iminocyclitol 22 in the presence
of NaBH3CN to a€ord the conjugate 29 (77%). Com-
pound 29 was ®nally deprotected via hydrogenolysis of
the benzyl groups and acid hydrolysis of the cyclohex-
ane-1,2-diacetal group to give compound 1 (63%).
be the most potent inhibitor of Gal-ase with
Ki=3.3 mM. Iminocyclitols without an aglycon also
showed a potent inhibitory activity in general. Indicat-
ing that the ®ve-membered iminocyclitols may mimic
both the galacto and manno con®gurations (see Fig. 2).
Interestingly, the best inhibitor against Gal-ase was
compound 1 and the worst was 4, but the result was
opposite for the inhibition of Man-ase. Together with
this observation, the result shown in Table 1 indicates
that Gal-ase tolerates and accepts N-alkylation and
additional side-chain at C-2. On the other hand, Man-
ase tolerates neither of these modi®cations. Compound
4 was found to be a strong inhibitor of Man-ase with
apparent Ki=27 mM, compared to 43 mM for deoxy-
mannojirimycin.14
Inhibition of ꢀ-galactosyltransferase (GalT-ase)
Inhibition of glycosidases
Iminocyclitols have been considered to mimic the tran-
sition state of the donor sugar moiety of glycosyl-
transferase reactions and have been shown to inhibit
such enzymes.4 Compounds 1±5 were examined as inhi-
bitors of GalT-ase.
All compounds synthesized were found to be competitive
inhibitors of both b-galactosidase (Gal-ase) and a-man-
nosidase (Man-ase) as shown in Table 1. Of these syn-
thetic compounds, pseudodisaccharide 1 was found to

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C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
Scheme 2. Reagents and conditions: (a) (Boc)2O-Et3N/CH2Cl2; (b)
Pb(OAc)4/toluene; (c) DIBAL/CH2Cl2; (d) H2-Pd(OH)2-C/HCl-MeOH;
(e) BnBr-Ag2O-KI/DMF; (f) TFA/CH2Cl2; (g) HCHO-NaBH3CN/
MeOH.
Figure 3. Pseudodisaccharide 1 may mimic the transition states of
both b-galactosyltransferase and b-galactosidase.
Table 1. Inhibition assay results of 1±5 against b-galactosidase and
a-mannosidase
Ki (M)
Compound
b-Galactosidasea
Aspergillus oryzae
a-Mannosidaseb
Jack bean
6
3
1
2
3
4
5
3.3Â10
1.7Â10
4
4
8.5Â10
2.1Â10
4
3
3.1Â10
(IC50>4Â10
)
3
5
1.0Â10
2.7Â10
5
4
7.6Â10
1.9Â10
Ð
6
Deoxy-Gal-NJ
Deoxy-Man-NJ
4.3Â10
5
Ðc
4.3Â10
Scheme 3. Reagents and conditions: (a) 1,1,2,2,-tetramethoxycyclo-
hexane-CSA/CH(OMe)3, MeOH; (b) (1) DMSO-(COCl)2/CH2Cl2
then Et3N; (2) Ph3P=CHCO2Me/benzene; (c) DIBAL/CH2Cl2; (d) (1)
H2NNH2 H2O/EtOH; (2) Ac2O/MeOH; (e) DMSO-(COCl)2/CH2Cl2
then Et3N; (f) 22, NaBH3CN/MeOH; (g) (1) H2-Pd(OH)2-C/THF-M
HCl (2:1); (2) TFA-H2O.
aKm=0.67>mM, Vmax=0.6 mM/s/mg.
bKm=1.02 mM, Vmax=0.6 mM/s/mg.
.
Table 2. IC50 values of 1±5 against b-1,4-galactosyltransferase at
Among them, compounds 3±5 showed some inhibitory
activity, and 4 was as potent as UDP (IC50=ꢀ0.1 mM,
Table 2). The activities of these compounds were a€ec-
ted by pH of the reaction medium where stronger
activities were observed at lower pH in general, despite
that the inhibition of UDP was una€ected (Table 2). It
was suggested that the protonated form of the iminocy-
clitol has high anity to the enzyme with optimal pH
around 8.15 The pKa values of some inhibitors (4,
pKa=8.0; 5, pKa=7.0) also support the assumption.
The substrate inhibition of GalT-ase, usually occurring
at concentrations over 0.2 mM for 4-methylumberiferyl
di€erent pH.a
IC50 (M)
Compound
pH 6.5
pH 7.5
pH 8.5
1
2
3
4
5
Ða
Ða
Ða
Ða
Ða
Ða
2
>10
1.0Â10
Very weak
4
Ða
4
3
4
2.2Â10
7.5Â10
2
1.5Â10
>10
Very weak
4
5
4
UDP
1.1Â10
7.0Â10
1.3Â10
aPractically no inhibition.

C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
2253
Figure 4. A. E€ect of inhibitor 4 on the substrate inhibition of b-1,4-GalT-ase by MU-GlcNAc. B. Lineweaver±Burk plot of b-1,4-GalT-ase reaction
using UDP-Gal in the presence of compound 4 . Ki of 4 toward UDP-Gal was estimated using the equation Kampp=Km/(1+i/Ki).
Experimental
b-N-acetylglucosaminide (MU-GlcNAc) as acceptor
(Fig. 4(A)), was also a€ected by 4. At lower concentra-
tions, however, an uncompetitive type of inhibition was
General methods for synthesis
observed. In addition, compound 4 was shown to be an
uncompetitive inhibitor versus UDP-Gal (Km=94 mM)
with apparent Ki=61 mM (Fig. 4B). Synergistic e€ect
with nucleoside, which is observed for inhibition of a-l-
fucosyltransferase with azasugars,4a,f,g was not observed
in this case.
Dried solvents were used for all reactions. Solutions
were evaporated under reduced pressure at a bath tem-
perature not exceeding 50 ꢁC. Column chromatography
was performed on silica gel (Merck Kieselgel 60) or
Iatro Beads (60 m) (Dia-Iatron Laboratories Inc.) when
speci®ed. Gel permeation chromatography was per-
formed using Bio Gel P-2. Melting points (mp) were
measured with Yanaco MP-S3 micro melting point
apparatus. Optical rotations were measured in a 1.0 dm
tube with a Horiba SEPA-200 polarimeter at 25Æ1 ꢁC.
JEOL EX-270 spectrometer was used to obtain NMR
Compound 1 designed to mimic the transition-state
complex was shown not to inhibit GalT-ase at all. This
result is somewhat puzzling because the previous data
suggest that modi®cation on acceptor GlcNAc is tolerated
by the enzyme at position O-6.10b,11 A possible reason
would be the requirement of the hydrogen bond donor
at the O-6 position of the acceptor by the enzyme. In this
case, an ether-linked derivative would have potential.
Another possibility would be due to the misorientation
of 1 in the binding site or the sugar moieties contribute
little to the overall binding energy.
spectra at 25 ꢁC. H NMR (270 MHz) were recorded in
1
CDCl3 or D2O using Me4Si (d 0.00) or DOH (d 4.80) as
the internal standard. 13C NMR (67.5 MHz) spectra
were recorded in CDCl3 or D2O using Me4Si (d 0.00),
CDCl3 (d 77.00), or CD3CN (d 118.2) as the internal
standard. Some key compounds were measured with
JEOL 500 MHz spectrometer as indicated. Only partial
assignments were reported. The FAB mass and HR
FAB mass spectra were obtained on JEOL JMS HX-
110 with glycerol and 3-nitrobenzylalcohol as the
matrix. MALDITOF mass spectra were recorded on
Kratos KOMPACT MALDI III with 2,5-dihydroxy-
benzoic acid as matrix. ESI mass spectra were measured
with a triple stage quadrupole mass spectrometer (Fin-
nigan MAT TSO 700) equipped with the ESI ion
source.
Conclusion
A series of galacto-iminocyclitols and a pseudodi-
saccharide have been synthesized and evaluated as inhi-
bitors of glycoenzymes using capillary electrophoresis.
The results indicated that alkylation of the ring nitrogen
of the iminocyclitols has no signi®cant e€ect on b-galac-
tosidase. Further, the pseudodisaccharide 1 exhibited a
strong inhibition activity against b-galactosidase. The
®ve-membered iminocyclitol 4 was shown to inhibit a-
mannosidase (Ki=27 mM) and b-galactosyltransferase
(Ki=61 mM against UDP-Gal).
Materials. 2,3,5-tri-O-Benzyllyxofuranose was prepared
according to the described procedure.7 Octyl 2-deoxy-2-
phthalimido-b-d-glucopyranoside
was
synthesized
according to the literature method.12

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C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
Methyl (4R,5S,6R)-6-hydroxy-4,5,7-tribenzyloxy-2(E)-hep-
tenoate (6E). A solution of 2,3,5-tri-O-benzyllyxofur-
anose (2.29 g, 5.44 mmol) and methyl (triphenylphos-
phoranylidene)acetate (2.73 g, 8.16 mmol) in benzene
(45 mL) was heated under re¯ux overnight. After cool-
ing, the solvent was removed in vacuo and the crude
mixture was puri®ed by column chromatography (5:1
n-hexane:EtOAc). A mixture of 6 (E) and 6 (Z) was
obtained (2.40 g, 93%, E:Z=1:1). A solution of 6
(2.40 g, 5.04 mmol) and diphenyl disul®de (1.32 g,
6.05 mmol) in cyclohexane (50 mL) was irradiated with
a 200 W lamp at rt for 5 days After removal of the sol-
vent, the residue was puri®ed by column chromato-
graphy (5:1 n-hexane:EtOAc) to give 6 (E) (1.58 g, 66%
in two steps): [a]d 16.6ꢁ (c 1.2, CHCl3); 1H NMR for 6
(E) (CDCl3) d 7.38±7.20 (m, 15H, aromatic), 7.00 (dd,
1H, J=6.3, 15.8 Hz, H-3), 6.13 (dd, 1H, J=1.3, 15.8 Hz,
H-2), 4.63±4.39 (m, 6H, benzyl methylene), 4.28 (dt, 1H,
J=1.3, 6.3 Hz, H-4), 4.06±3.98 (m, 1H, H-6), 3.76 (s, 3H,
OCH3), 3.64 (dd, 1H, J=2.6, 6.3 Hz, H-5), 3.52 (dd, 1H,
J=5.9, 9.8 Hz, H-7), 3.46 (dd, 1H, J=6.3, 9.8 Hz, H-7),
2.65 (d, 1H, J=6.6 Hz, OH); 13C NMR (CDCl3) d 166.22
(C-1), 145.64 (C-3), 137.74, 137.34, 136.62, 128.48, 128.34,
128.27, 128.23, 128.09, 127.94, 127.73, 127.62, 127.54 and
127.48 (aromatic), 123.13 (C-2), 79.91 (C-5), 78.20 (C-
4), 74.13, 73.24 and 71.68 (benzyl methylene), 70.73 (C-
7), 69.42 (C-6), 51.54 (OCH3); MALDITOF MS m/z:
499 [M+Na]+. 1H NMR for 6 (Z) (CDCl3) d 7.35±7.23
(m, 15H, aromatic), 6.32 (dd, 1H, J=8.6, 11.7 Hz, H-3),
5.99 (dd, 1H, J=1.2, 11.7 Hz, H-2), 5.51 (ddd, 1H, J=1.2,
3.6, 8.6 Hz, H-4), 4.78 (d, 1H, J=11.6 Hz, benzyl methyl-
ene), 4.63±4.38 (m, 5H, benzyl methylene), 3.99 (dt, 1H,
J=2.9, 5.4 Hz, H-6), 3.74 (dd, 1H, J=2.9, 3.6 Hz, H-5),
3.69 (s, 3H, OCH3), 3.53 (dd, 1H, J=5.6, 9.6 Hz, H-7),
3.48 (dd, 1H, J=6.1, 9.5 Hz, H-7), 3.25 (d, 1H,
J=4.3 Hz, OH); 13C NMR (CDCl3) d 166.04 (C-1),
147.01 (C-3), 138.17, 137.84, 137.75, 128.44, 128.34,
128.26, 128.21, 127.89, 127.81, 127.74, 127.65 and 127.53
(aromatic), 122.62 (C-2), 79.41 (C-5), 75.81 (C-4), 73.21,
72.96 and 71.95 (benzyl methylene), 70.82 (C-7), 70.48
(C-6), 51.43 (OCH3); MALDITOF MS m/z: 499
[M+Na]+.
128.12, 127.87, 127.78, 127.71, 127.62 and 127.51 (C-3
and aromatic), 79.97 (C-5), 79.70 (C-4), 73.82, 73.23 and
70.57 (benzyl methylene), 70.91 (C-7), 69.62 (C-6), 62.46
(C-1); MALDITOF MS m/z: 471 [M+Na]+.
(4R,5S,6R)-1-tert-Butyldimethylsilyloxy-4,5,7-tribenzyl-
oxy-2(E)-hepten-6-ol (8). Compound 7 (2.72 g, 6.06
mmol) was dissolved in DMF (55 mL), to this solution
was added TBDMSCl (1.37 g, 9.1 mmol), Et3N (2.5 mL,
18 mmol) and DMAP (0.37 g, 3.0 mmol). The reaction
mixture was stirred at rt for 1 h. The mixture was dilu-
ted with EtOAc and the organic layer was washed with
H2O and brine, and dried with MgSO4. After removal
of solvent, the residue was puri®ed by column chroma-
tography (9:1 n-hexane:EtOAc) to give 8 (3.34 g, 98%)
ꢁ
1
as colorless oil: [a]d 29.6 (c 1.3, CHCl3); H NMR
(CDCl3) d 7.31±7.24 (m, 15H, aromatic), 5.85 (dt, 1H,
J=3.6, 15.5 Hz, H-2), 5.75 (dd, 1H, J=7.1, 15.5 Hz, H-
3), 4.69±4.34 (m, 6H, benzyl methylene), 4.22 (d, 2H,
J=3.6Hz, H-1), 4.13±4.03 (m, 2H, H-4 and H-6), 3.58
(dd, 1H, J=2.8, 5.8 Hz, H-5), 3.51 (d, 2H, J=5.9Hz, H-
7), 2.88 (d, 1H, J=5.9Hz, OH), 0.91 (s, 9H, OSiC(CH3)3),
0.08 (s, 6H, OSi(CH3)2); 13C NMR (CDCl3) d 134.64 (C-
2), 138.02, 128.30, 128.09, 127.78, 127.75, 127.67 and
127.57 (aromatic), 126.79 (C-3), 80.07 (C-5), 79.62 (C-4),
73.80, 73.24 and 70.51 (benzyl methylene), 70.98 (C-7),
69.67 (C-6), 62.93 (C-1), 25.86 (C(CH3)3), 18.31
(C(CH3)3), 5.28 (Si(CH3)2). Anal. calcd for C34
H46O5Si: C, 72.56; H, 8.24. Found: C, 72.29; H, 8.31.
(4R,5S,6R)-1-tert-Butyldimethylsilyloxy-6-[(chlorome-
thylsulfonyl)-oxy]-4,5,7-tribenzyloxy-2(E)-heptene (9). A
solution of compound 8 (5.97 g, 10.60 mmol) and chlor-
omethylsulfonyl chloride (1.15 mL, 13 mmol) in pyridine
(6 mL) was stirred at rt for 0.5 h, then the mixture was
diluted with EtOAc and washed with H2O and brine,
dried over MgSO4. After removal of the solvent, the
residue was puri®ed by column chromatography (9:1 n-
hexane:EtOAc) to give 9 (6.93 g, 97%) as a colorless oil:
[a]d +8.9ꢁ (c 2.1, CHCl3); 1H NMR (CDCl3) d 7.35±7.25
(m, 15H, aromatic), 5.92 (dt, 1H, J=3.3, 15.5Hz, H-2),
5.79 (dd, 1H, J=7.9, 15.5Hz, H-3), 5.20±5.12 (m, 1H, H-
6), 4.75±4.39 (m, 8H, benzyl methylene and OSO2CH2Cl),
4.23 (brs, 2H, H-1), 4.10 (t, 1H, J=8.0 Hz, H-4), 3.78±3.69
(m, 2H, H-5 and H-7), 3.50 (dd, 1H, J=3.3, 10.9 Hz, H-7),
0.92 (s, 9H, OSiC(CH3)3), 0.08 (s, 6H, OSi(CH3)2); 13C
NMR (CDCl3) d 138.02, 137.45 and 137.00 (aromatic),
135.95 (C-2), 128.52, 128.37, 128.17, 128.05, 127.99 and
127.65 (aromatic), 126.09 (C-3), 83.11 (C-6), 79.37 (C-5),
78.83 (C-4), 74.61, 73.44 and 70.35 (benzyl methylene),
69.52 (C-7), 62.77 (C-1), 54.25 (OSO2CH2Cl), 25.91
(C(CH3)3), 18.35 (C(CH3)3), 5.25 (Si(CH3)2). Anal.
calcd for C35H47O7ClSSi: C, 62.25; H, 7.02. Found: C,
61.77; H, 7.02.
(4R,5S,6R)-4,5,7-Tribenzyloxy-2(E)-hepten-1,6-diol (7).
To a solution of compound 6 (E) (133 mg, 0.28 mmol) in
CH2Cl2 was added 0.98 M solution of DIBAL in n-hex-
ane (0.86 mL, 3 equiv) at 0 ꢁC. The reaction mixture was
stirred at the temperature for 0.5 h. MeOH (0.14 mL)
was added at 0 ꢁC and the temperature was raised to rt.
Saturated NaCl (0.3 mL) was added and the mixture
was diluted with Et2O (7 mL). MgSO4 (0.72 g) was
added and the whole mixture was stirred for 1 h, then
®ltered through a celite pad. The solvent was removed in
vacuo and the crude mixture was puri®ed by column
chromatography (1:1 n-hexane:EtOAc) to give 7 (120mg,
96%): [a]d 31.6ꢁ (c 1.1, CHCl3); H NMR (CDCl3) d
(4R,5S,6S)-6-Acetoxy-1-tert-butyldimethylsilyloxy-4,5,7-
tribenzyloxy-2(E)-heptene (10). A stirred mixture of
compound 9 (1.88 g, 2.78 mmol), CsOAc (2.67 g, 14
mmol), and 18-Crown-6 (0.74 g, 2.8 mmol) in toluene
(60 mL) was heated under re¯ux overnight. After cooling
to rt, the reaction mixture was washed with H2O and
brine, dried over MgSO4 and the solvent was removed in
vacuo. The crude material was puri®ed by column
1
7.34±7.24 (m, 15H, aromatic), 5.88 (dt, 1H, J=4.9,
15.5 Hz, H-2), 5.69 (dd, 1H, J=7.4, 15.5 Hz, H-3), 4.66±
4.35 (m, 6H, benzyl methylene), 4.15±4.00 (m, 4H, H-1,
H-4, and H-6), 3.60 (dd, 1H, J=3.0, 5.6 Hz, H-5), 3.51
(d, 2H, J=5.9 Hz, H-7), 2.98 (dd, 1H, J=2.3, 5.6 Hz,
OH), 1.72 (brs, 1H, OH); 13C NMR (CDCl3) d 137.95,
137.90 and 137.84 (aromatic), 134.18 (C-2), 128.25,

C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
2255
chromatography (19:1 n-hexane:EtOAc) to a€ord 10
(1.26 g, 75%) as a colorless oil: [a]d 38.6ꢁ (c 1.0,
CHCl3); H NMR (CDCl3) d 7.32±7.24 (m, 15H, aro-
and 70.08 (benzyl methylene), 68.75 (C-7), 62.80 (C-1),
54.07 (OSO2CH2Cl), 25.88 (C(CH3)3), 18.33 (C(CH3)3),
5.28 (Si(CH3)2). Anal. calcd for C35H47O7ClSSi: C,
62.25; H, 7.02. Found: C, 62.00; H, 7.02.
1
matic), 5.77±5.74 (m, 2H, H-2 and H-3), 5.24 (dt, 1H,
J=3.3, 5.6 Hz, H-6), 4.73±4.34 (m, 6H, benzyl methyl-
ene), 4.21 (br. s, 2H, H-1), 3.93 (dd, 1H, J=4.3, 7.3 Hz,
H-4), 3.86, (t, 1H, J=5.1 Hz, H-5), 3.73 (dd, 1H, J=5.9,
11.0 Hz, H-7), 3.66 (dd, 1H, J=3.3, 11.0 Hz, H-7), 2.00
(s, 3H, OCOCH3), 0.91 (s, 9H, OSiC(CH3)3), 0.07 (s,
6H, OSi(CH3)2); 13C NMR (CDCl3) d 170.00 (CO),
138.36 and 138.17 (aromatic), 135.16 (C-2), 128.28,
128.21, 127.94, 127.69, 127.62, 127.53 and 127.40 (aro-
matic) 126.23 (C-3), 80.07 (C-5), 79.78 (C-4), 74.05, 72.96
and 70.13 (benzyl methylene), 72.25 (C-6), 68.48 (C-7),
63.04 (C-1), 25.93 (SiC(CH3)3), 21.15 (COCH3), 18.36
(SiC(CH3)3), 5.23 (Si(CH3)2). Anal. calcd for C36H48
O6Si: C, 71.49; H, 8.00. Found: C, 71.11; H, 8.04.
(4R,5S,6S)-6-[(Chloromethylsulfonyl)oxy]-4,5,7-tribenzyl-
oxy-2(E)-hepten-1-ol (13). A solution of compound 12
(2.14 g, 3.16 mmol) in THF (15 mL) was treated with N
HCl (15 mL) at rt overnight. The mixture was diluted
with EtOAc and the organic layer was washed with aq
NaHCO3 and brine. After concentration the residue was
puri®ed by column chromatography (4:1 n-hexane:
EtOAc) to a€ord 13 (1.70 g, 96%) as colorless oil: [a]d
42.9ꢁ (c 1.9, CHCl3); H NMR (CDCl3) d 7.35±7.24
1
(m, 15H, aromatic), 5.94 (dt, 1H, J=5.1, 15.5 Hz, H-2),
5.64 (ddd, 1H, J=1.0, 7.6, 15.5 Hz, H-3), 5.18 (dt, 1H,
J=3.4, 7.2 Hz, H-6), 4.72±4.30 (m, 8H, benzyl methyl-
ene and OSO2CH2Cl), 4.16 (brs, 2H, H-1), 3.96 (t, 1H,
J=6.8 Hz, H-4), 3.82 (dd, 1H, J=3.6, 5.9 Hz, H-5), 3.78
(dd, 1H, J=7.9, 11.6 Hz, H-7), 3.70 (dd, 1H, J=2.0,
11.6 Hz, H-7), 1.68 (brs, 1H, OH); 13C NMR (CDCl3) d
137.79, 137.45, and 137.23 (aromatic), 135.43 (C-2),
128.52, 128.37, 128.26, 127.96, 127.87 and 127.71 (aro-
matic), 127.20 (C-3), 84.62 (C-6), 80.93 (C-5), 78.54 (C-
4), 74.16, 73.42 and 70.42 (benzyl methylene), 68.93 (C-
7), 62.68 (C-1), 54.16 (OSO2CH2Cl); MALDITOF MS
m/z: 583 [M+Na]+.
(4R,5S,6S)-1-tert-Butyldimethylsilyloxy-4,5,7-tribenzyl-
oxy-2(E)-hepten-6-ol (11). Compound 10 (4.46 g, 7.33
mmol) was dissolved in MeOH (90 mL) and treated with
1 M solution of NaOMe (22 mL, 3 equiv) at rt for 1 h.
The solvent was removed and the residue was diluted
with EtOAc, washed with H2O and brine, and dried
over MgSO4. The crude material was puri®ed by col-
umn chromatography (19:1 n-hexane:EtOAc) to give 11
(4.00 g, 97%): [a]d 43.1ꢁ (c 1.5, CHCl3); 1H NMR
(CDCl3) d 7.33±7.24 (m, 15H, aromatic), 5.92±5.76 (m,
2H, H-2 and H-3), 4.77±4.36 (m, 6H, benzyl methylene),
4.24±4.22 (m, 2H, H-1), 4.16 (dd, 1H, J=4.3, 6.9 Hz, H-
4), 3.88±3.80 (m, 1H, H-6), 3.69 (dd, 1H, J=4.5, 7.8 Hz,
H-7), 3.63±3.61 (m, 2H, H-5 and H-7), 2.77 (d, 1H,
J=4.6 Hz, OH), 0.91 (s, 9H, OSiC(CH3)3), 0.07 (s, 6H,
OSi(CH3)2); 13C NMR (CDCl3) d 138.52, 138.43 and
138.06 (aromatic), 135.13 (C-2), 128.41, 128.34, 128.23,
128.01, 127.85, 127.71, 127.64, 127.53 and 127.46 (aro-
matic), 126.61 (C-3), 81.22 (C-4), 81.11 (C-5), 74.07,
73.39 and 70.31 (benzyl methylene), 71.09 (C-7), 71.00
(C-6), 63.16 (C-1), 25.93 (C(CH3)3), 18.38 (C(CH3)3),
5.17 (Si(CH3)2); MALDITOF MS m/z: 585 [M+Na]+.
(2R,3S,4S,5S,6S) - 6 - [(Chloromethylsulfonyl)oxy] - 2,3 -
epoxy-4,5,7-tribenzyloxy-heptan-1-ol (14). A solution of
Ti(O-i-Pr)4 (2.9 mL, 9.8 mmol) and d-( )-diethyltar-
trate (2.33 g, 11 mmol) in CH2Cl2 (50 mL) was stirred at
25 ꢁC for 0.5 h in the presence of MS 4 A. To this
mixture was added a solution of compound 13 (2.71 g,
4.83 mmol) in CH2Cl2 (5 mL), and the mixture was stir-
red at the temperature for 0.5 h. 5 M solution of t-
BuOOH (2.9 mL, 15 mmol) was added and the mixture
was stirred at the same temperature for 90 h. Dimethyl
sul®de (1.3 mL) was added at 25 ꢁC and stirred for
1.5 h at the temperature. 10% Solution of tartaric acid
(15 mL) and Et2O (30 mL) were added at 25 ꢁC and
stirred for 0.5 h at the temperature, and for 0.5 h at rt
The solution was ®ltered through a Celite pad, and the
solvent was removed. The residue was dissolved in Et2O
(30 mL) and stirred with 10% NaOH solution (25 mL)
at 0 ꢁC for 0.5 h. The organic layer was washed with
H2O and brine, dried and concentrated. The crude
mixture was puri®ed by column chromatography (2:1
n-hexane:EtOAc) to a€ord 14 (2.25 g, 86%) as colorless
(4R,5S,6S)-1-tert-Butyldimethylsilyloxy-6-[(chloro-methyl-
sulfonyl)-oxy]-4,5,7-tribenzyloxy-2(E)-heptene (12).
A
mixture of compound 11 (2.28 g, 4.06 mmol) and chlor-
omethylsulfonyl chloride (440 mL, 4.9 mmol) in pyridine
(5 mL) was stirred at rt for 0.5 h. The mixture was dilu-
ted with EtOAc, washed with H2O and brine, and dried
over MgSO4. After removal of the solvent, the residue
was puri®ed by column chromatography (19:1 n-hex-
ane:EtOAc) to give 12 (2.74 g, quant) as colorless oil:
oil: [a]d 19.0ꢁ (c 1.2, CHCl3); H NMR (CDCl3) d
1
[a]d 42.3ꢁ (c 1.7, CHCl3); H NMR (CDCl3) d 7.35±
7.37±7.26 (m, 15H, aromatic), 5.24 (dt, 1H, J=2.5,
8.0 Hz, H-6), 4.83±4.43 (m, 8H, benzyl methylene and
OSO2CH2Cl), 3.87 (dd, 1H, J=2.5, 7.0 Hz, H-5), 3.81
(dd, 1H, J=2.6, 12.9 Hz, H-1), 3.75 (dd, 1H, J=8.0,
11.0 Hz, H-7), 3.60 (dd, 1H, J=2.5, 11.0 Hz, H-7), 3.55
(dd, 1H, J=3.5, 12.5 Hz, H-1), 3.36 (t, 1H, J=7.0 Hz,
H-4), 3.23 (dd, 1H, J=2.3, 7.0 Hz, H-3), 2.99 (dt, 1H,
J=2.3, 3.5 Hz, H-2); 13C NMR (CDCl3) d 137.27,
137.07, 136.95, 128.48, 128.39, 128.23, 128.14, 127.98
and 127.84 (aromatic), 84.55 (C-6), 79.21 (C-5), 78.06
(C-4), 73.80, 73.41 and 72.31 (benzyl methylene), 68.70
(C-7), 60.72 (C-1), 56.32 (C-3), 55.33 (C-2), 54.05
(OSO2CH2Cl).
1
7.24 (m, 15H, aromatic), 5.89 (dt, 1H, J=4.3, 15.5 Hz,
H-2), 5.68 (dd, 1H, J=7.6, 15.5 Hz, H-3), 5.22 (dt, 1H,
J=2.3, 8.3 Hz, H-6), 4.71±4.52 (m, 7H, benzyl methylene
and OSO2CH2Cl), 4.30 (d, 1H, J=11.6 Hz, benzyl
methylene), 4.23 (brm, 2H, H-1), 3.92 (t, 1H, J=7.3 Hz,
H-4), 3.81 (dd, 1H, J=2.3, 7.3 Hz, H-5), 3.79 (dd, 1H,
J=7.3, 11.6 Hz, H-7), 3.65 (dd, 1H, J=2.3, 11.6 Hz, H-
7), 0.92 (s, 9H, OSiC(CH3)3), 0.09 (s, 6H, OSi(CH3)2);
13C NMR (CDCl3) d 137.75, 137.48 and 137.29 (aro-
matic), 135.98 (C-2), 128.45, 128.36, 128.28, 128.12,
127.94, 127.89, 127.80 and 127.64 (aromatic), 125.82 (C-
3), 84.85 (C-6), 81.17 (C-5), 78.53 (C-4), 74.29, 73.32,

2256
C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
(2R,3S,4S,5S,6R)-6-Azide-2,3 epoxy-4,5,7-tribenzyloxy-
heptan-1-ol (15). Compound 14 (2.23 g, 3.86 mmol) was
dissolved with DMF (50 mL) and treated with NaN3
(0.64 g, 9.8 mmol) at 70 ꢁC for 1 h. The mixture was
diluted with EtOAc at rt and washed with H2O and
brine, dried and the solvent was removed. The residue
was puri®ed by column chromatography (2:1 n-hexane:
EtOAc) to give 15 (1.76 g, 93%) as colorless oil: [a]d
C7H15NO5: C, 43.52; H, 7.83; N, 7.25. Found: C, 43.18;
H, 7.68; N, 6.95.
(10R,2R,3R,4S,5R)-N-Methyl-[3,4-dibenzyloxy-5-benzy-
loxymethyl-2-(10,20-dihydroxy-ethyl)]-pyrrolidine
(17).
To a solution of 16 (46 mg, 0.12 mmol) dissolved in
MeOH (1 mL) at 0 ꢁC was added a 37% formaldehyde
solution (0.1 mL, 1.2 mmol) and NaBH3CN (22 mg,
0.33 mmol). The mixture was stirred at rt overnight. The
reaction mixture was added H2O, extracted with CHCl3
and dried over MgSO4. After removal of the solvent, the
residue was puri®ed by column chromatography (99:2:1
CHCl3:MeOH:Et3N) to yield 17 (33 mg, 71%) as color-
less oil: [a]d +32.9ꢁ (c 0.9, CHCl3); 1H NMR (CDCl3) d
7.31±7.24 (m, 15H, aromatic), 4.72±4.45 (m, 6H, benzyl
methylene), 3.97±3.91 (m, 2H, H-3 and H-4), 3.84±3.77
(m, 3H, H-10 and H-100), 3.57±3.50 (m, 3H, H-20 and H-
5), 2.82 (t, 1H, J=3.6 Hz, H-2), 2.75 (brs, 2H, OH), 2.51
(s, 3H, CH3); 13C NMR (CDCl3) d 138.29, 138.20,
138.13, 128.39, 127.85, 127.62 and 127.44 (aromatic),
79.05 (C-3 or C-4), 77.52 (C-3 or C-4), 73.33, 72.76 and
72.69 (benzyl methylene), 70.53 (C-2), 68.88 (C-10),
66.40 (C-100), 64.17 (C-20), 63.92 (C-5), 36.95 (CH3).
18.5ꢁ (c 1.0, CHCl3); H NMR (CDCl3) d 7.34±7.23
1
(m, 15H, aromatic), 4.83±4.41 (m, 6H, benzyl methyl-
ene), 3.84±3.78 (m, 2H, H-1 and H-6), 3.72 (dd, 1H,
J=2.3, 8.2 Hz, H-5), 3.65 (dd, 1H, J=7.2, 9.2 Hz, H-7),
3.57±3.52 (m, 3H, H-1, H-4 and H-7), 3.26 (dd, 1H,
J=2.3, 6.6 Hz, H-3), 3.09 (dt, 1H, J=2.3, 3.6 Hz, H-2);
13C NMR (CDCl3) d 137.56, 137.38, 128.52, 128.41,
128.18, 128.05, 127.84, 127.71, 127.55 and 127.42 (aro-
matic), 78.73 (C-4), 78.46 (C-5), 74.66, 73.32 and 72.90
(benzyl methylene), 69.17 (C-7), 60.68 (C-1), 60.63 (C-
6), 56.17 (C-3), 55.62 (C-2); MALDITOF MS m/z: 512
[M+Na]+.
(10R,2R,3R,4S,5R)-[3,4-Dibenzyloxy-5-benzyloxymethyl-
2-(10,20-dihydroxy-ethyl)]-pyrrolidine (16). A solution of
compound 15 (510 mg, 1.04 mmol) and triphenylpho-
sphine (0.42 g, 1.6 mmol) in THF (10 mL) was stirred at
rt for 4 days After removal of the solvent, the residual
mixture was puri®ed by column chromatography (98:1:1
CHCl3:MeOH:Et3N) to a€ord 16 (367mg, 76%), as col-
orless powder: mp 95ꢁC; [a]d +36.2ꢁ (c 0.5, CHCl3); 1H
NMR (CDCl3) d 7.33±7.28 (m, 15H, aromatic), 4.69±
4.42 (m, 6H, benzyl methylene), 4.07 (t, 1H, J=4.0 Hz,
H-4), 3.96 (dd, 1H, J=4.0, 7.3 Hz, H-3), 3.71±3.59 (m,
5H, H-10, H-20 and H-100), 3.51±3.40 (m, 2H, H-2 and H-
5), 3.13 (brs, 2H, OH); 13C NMR (CDCl3) d 138.20,
137.88, 137.41, 128.41, 128.37, 128.28, 127.82 and
127.69 (aromatic), 80.43 (C-3), 77.70 (C-4), 73.32, 73.23
and 72.40 (benzyl methylene), 71.91 (C-10) 69.00 (C-100),
64.26 (C-20), 62.23 (C-2 or C-5), 59.10 (C-2 or C-5).
Anal. calcd for C28H33NO5: C, 72.55; H, 7.17; N, 3.02.
Found: C, 72.03; H, 7.21; N, 2.89.
(10R,2R,3R,4S,5R)-N-Methyl-[3,4-dihydroxy-5-hydroxy-
methyl-2-(10,20-dihydroxy-ethyl)ethyl]-pyrrolidine (3). To
compound 17 (22 mg, 0.045 mmol) dissolved in MeOH
(0.5 mL) and 1 M HCl (0.5 mL) was added a catalytic
amount of 20% Pd(OH)2 on C. The reaction mixture
was stirred under H2 atmosphere at rt for 3 days After
®ltration and evaporation of the solvent, the residue was
puri®ed on a column of Iatro Beads (50:50:1 CHCl3:
MeOH:28% NH3) to a€ord 3 (7 mg, 73%); mp 147 ꢁC;
[a]d +20.9ꢁ (c 0.6, H2O). Compound 3 was further
puri®ed for inhibition assay using Sep-Pak PLUS CM,
regenerated with M HCl (10 mL) and water (20 mL),
eluted with water (20 mL) and 10% NH3 (10 mL). The
latter eluent containing 3 was ®ltered through a Millex
1
GV ®lter and lyophilized. H NMR (D2O) d 4.21±4.17
(m, 2H, H-3 and H-4), 3.97±3.92 (m, 2H, H-10 and H-
100), 3.80 (dd, 1H, J=4.4, 12.2 Hz, H-100), 3.69 (dd, 1H,
J=4.4, 11.7 Hz, H-20), 3.63 (dd, 1H, J=7.5, 11.7 Hz, H-
20), 3.18 (brd, 1H, J=4.4 Hz, H-5), 2.87 (s, 1H, H-2),
2.49 (s, 3H, CH3); 13C NMR (CDCl3) d 70.57 (C-2),
70.13 (C-3 or C-4), 69.35 (C-3 or C-4), 68.56 (C-10),
64.87 (C-5), 62.48 (C-20), 56.15 (C-100), 34.19 (CH3);
HRFAB MS calcd for C8H17NO5; [M+H]+ 208.1185;
found: m/z 208.1185.
(10R,2R,3R,4S,5R)-[3,4-Dihydroxy-5-hydroxymethyl-2-
(10,20-dihydroxy-ethyl)]-pyrrolidine (2). A solution of 16
(15 mg, 0.03 mmol) in MeOH (0.5 mL) and M HCl
(0.5 mL) was stirred with a catalytic amount of 20%
Pd(OH)2 on C under H2 atmosphere at rt overnight.
The crude material, obtained after removal of the cata-
lyst and the solvent, was treated with Dowex 1X8 (OH )
eluted with water to give 2 (5.7 mg, 93%); mp 134 ꢁC; [a]d
+24.6ꢁ (c 1.2, H2O). Compound 2 was further puri®ed
for inhibition assay using Sep-Pak PLUS CM, regener-
ated with M HCl (10 mL) and water (20 mL), eluted
with water (20 mL) and 10% NH3 (10 mL). The latter
eluent containing 2 was ®ltered through a Millex GV
(10R,2R,3R,4S,5R)-N-Butyloxycarbonyl-[3,4-dibenzyl-
oxy-5-benzyloxymethyl-2-(10,20-dihydroxy-ethyl)]-pyrroli-
dine (18). To a solution of 16 (183 mg, 0.39 mmol) in
CH2Cl2 (4 mL) and Et3N (66 mL, 0.47 mmol), (Boc)2O
(220 mL, 0.96 mmol) was added at 0 ꢁC and the mixture
was stirred at rt overnight. The reaction mixture was
diluted with CH2Cl2 and washed with 10% citric acid,
saturated NaHCO3, and water, dried over MgSO4, and
concentrated. The resulting material was puri®ed by
column chromatography (1:1 n-hexane:EtOAc) to
a€ord 18 (160 mg, 72%) as colorless oil: [a]d +8.6ꢁ (c
1
®lter and lyophilized. H NMR (D2O) d 4.23 (dd, 1H,
J=4.3, 8.1 Hz, H-3), 4.19 (t, 1H, J=4.3 Hz, H-4), 3.84±
3.80 (m, 1H, H-10), 3.80 (dd, 1H, J=6.5, 11.1 Hz, H-100),
3.73 (dd, 1H, J=3.6, 11.9 Hz, H-20), 3.66 (dd, 1H,
J=6.5, 11.1 Hz, H-100), 3.63 (dd, 1H, J=7.3, 11.9 Hz, H-
20), 3.34 (dt, 1H, J=3.5, 6.5 Hz, H-5), 3.13 (dd, 1H,
J=5.2, 8.1 Hz, H-2); 13C NMR (D2O) d 72.15 (C-3),
71.49 (C-10), 71.27 (C-4), 62.49 (C-20), 60.48 (C-2), 59.29
(C-5 or C-100), 59.20 (C-5 or C-100). Anal. calcd for
1
2.7, CHCl3); H NMR (CDCl3) d 7.32±7.23 (m, 15H,
aromatic), 4.74±4.43 (m, 6H, benzyl methylene), 4.26
(dd, 1H, J=6.3, 8.9 Hz, H-20 or H-100), 4.18±4.08 (m,

C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
2257
3H, H-3, H-4 and H-5), 3.85±3.80 (m, 2H, H-2 and
OH), 3.55±3.46 (m, 3H, H-20 and H-100), 3.25 (brs, 1H,
H-10), 3.11 (brt, 1H, J=8.6 Hz, OH), 1.43 (s, 9H,
C(CH3)3); 13C NMR (CDCl3) d 156.10 (NCOO),
138.37, 138.28, 138.08, 128.28, 128.23, 128.18, 127.82,
127.58, 127.51, 127.46, 127.39 and 127.28 (aromatic),
81.12 (C(CH3)3), 79.08 (C-3 or C-4), 77.32 (C-3 or C-4),
73.14 and 72.13 (benzyl methylene), 71.93 (C-10 and
benzyl methylene), 70.10 (C-20 or C-100), 62.95 (C-2),
62.33 (C-20 or C-100), 58.91 (C-5), 28.26 (C(CH3)3);
MALDITOF MS m/z: 586 [M+Na]+.
give 4 (7 mg, 83%); [a]d +34.8ꢁ (c 0.7, H2O). Com-
pound 4 was further puri®ed for inhibition assay using
Sep-Pak PLUS CM, regenerated with M HCl (10 mL)
and water (20 mL), eluted with water (20 mL) and 10%
NH3 (10 mL). The latter eluent containing 4 was ®ltered
1
through a Millex GV ®lter and lyophilized. H NMR
(D2O) d 4.19 (t, 1H, J=4.2 Hz, H-4), 4.00 (dd, 1H, J=4.2,
8.4 Hz, H-3), 3.80 (dd, 1H, J=6.7, 11.0Hz, H-100), 3.76
(dd, 1H, J=3.8, 11.6 Hz, H-10), 3.65 (dd, 1H, J=5.9,
11.6 Hz, H-10), 3.65 (dd, 1H, J=6.7, 11.0 Hz, H-100),
3.33 (dt, 1H, J=3.8, 6.7 Hz, H-5), 3.15 (ddd, 1H,
J=3.8, 5.9, 8.4 Hz, H-2); 13C NMR (D2O) d 71.99 (C-
3), 70.53 (C-4), 60.53 (C10 or C-100), 60.01 (C-2), 59.43
(C-10 or C-100), 58.75 (C-5); HRFAB MS calcd for
C6H13NO4: [M+H]+ 164.0923; found: m/z 164.0920.
Other physical data are also in good agreement to those
of reported values.8
(2S,3R,4S,5R)-N-Butyloxycarbonyl-(3,4-dibenzyloxy-5-
benzyloxymethyl)-pyrrolidine-2-carbaldehyde (19). To a
solution of compound 18 (1.02 g, 1.80 mmol) in toluene
(20 mL), Pb(OAc)4 (1.34 g, 2.7 mmol) was added. The
reaction mixture was stirred for 1.5 h at rt, then diluted
with Et2O, ®ltered through a Celite pad, and con-
centrated. The resulting residue was puri®ed by column
chromatography (9:1 n-hexane:EtOAc) to a€ord 19
(0.86 g, 90%): [a]d +1.7ꢁ (c 1.1, CHCl3); 1H NMR
(CDCl3) showed 19 exist as 1:1 mixture of two conforma-
tional isomers. 1H NMR: d 9.48 (d, 1H, J=1.7 Hz,
CHO), 9.40 (d, 1H, J=2.6 Hz, CHO), 7.32±7.24 (m,
30H), 4.75±4.36 (m, 12H), 4.34±4.26 (m, 4H), 4.22±4.05
(m, 2H), 4.02±3.92 (m, 4H), 3.73±3.68 (m, 2H), 1.40,
1.43 (s, each 3H, C(CH3)3); 13C NMR (CDCl3) d 198.42
and 198.06 (CHO), 154.50 and 153.06 (NCOO),137.2±
138.6, 127.3±128.8 (aromatic), 81.21 and 81.13
(C(CH3)3), 78.64, 78.19, 77.31 and 77.20 (C-3 and C-4),
73.17, 72.96, 72.89, 72.60, 72.54 and 72.42 (benzyl
methylene), 69.60, 69.15, 68.47, 58.01, 57.79, 28.21 and
28.12 (C(CH3)3).
(2R,3R,4S,5R)-N-Butyloxycarbonyl-(3,4-dibenzyloxy-
2,5-dibenzyloxymethyl)-pyrrolidine (21). To a solution
of 20 (63 mg, 0.12 mmol) in DMF (1 mL) was succes-
sively added Ag2O (160 mg, 0.69 mmol), BnBr (86 mL,
0.69 mmol), and KI (62 mg, 0.37 mmol) at 0 ꢁC. The
reaction mixture was stirred at rt for 1 h, then diluted
with EtOAc. After ®ltration through a Celite pad, the
mixture was extracted with EtOAc, the organic layer
was washed with H2O and dried over MgSO4, and con-
centrated. The residue was puri®ed by column chroma-
tography (9:1 n-hexane:EtOAc) to yield 21 (66 mg,
90%): [a]d 0.7ꢁ (c 0.9, CHCl3); 1H NMR (CDCl3)
showed 21 exists as a 2:1 mixture of two conformational
isomers. Due to the complex pattern of the spectrum, the
assignment was not made. d 7.57±7.18 (m, 20H), 4.77±
4.38 (m), 4.13±4.29 (m), 4.07±3.98 (m), 3.85 (d, minor,
J=4.3 Hz), 3.75 (d, minor, J=8.6Hz), 3.61±3.51 (m), 3.35
(dd, minor, J=6.9, 9.6 Hz), 1.43 (s), 1.39 (s); 13C NMR
(CDCl3) d 154.05, 153.46, 137.9±139.0, 126.9±128.4, 80.50,
79.88, 79.80, 79.40, 77.22, 76.73, 73.16, 72.80, 72.71, 72.44,
72.33, 72.18, 71.93, 70.40, 69.13, 68.99, 68.18, 61.57,
61.49, 58.13, 57.63, 28.4; MALDITOF MS m/z: 646
[M+Na]+.
(2R,3R,4S,5R)-N-Butyloxycarbonyl-(3,4-dibenzyloxy-5-
benzyloxymethyl-2-hydroxymethyl)-pyrrolidine (20). To
a solution of 19 (0.86 g, 1.62 mmol) in CH2Cl2 (20 mL)
cooled to 0 ꢁC was added a 0.98 M solution of DIBAL
in n-hexane (3.3 mL, 2 equiv) and the resulting mixture
was stirred for 1.5 h. MeOH (0.5 mL) was added at 0 ꢁC
and the temperature was raised to rt. Saturated NaCl
(1 mL) was added and the mixture was diluted with
Et2O (27 mL). MgSO4 (2.80 g) was added and the whole
mixture was stirred for 1 h, then ®ltered through a Celite
pad. The solvent was removed in vacuo and the crude
mixture was puri®ed by column chromatography (2:1 n-
hexane:EtOAc) to give 20 (0.79 g, 92%) as colorless oil:
[a]d +7.8ꢁ (c 1.4, CHCl3); 1H NMR (CDCl3) d 7.32±7.23
(m, 15H, aromatic), 4.77±4.44 (m, 6H, benzyl methyl-
ene), 4.16±4.11 (m, 3H), 3.98 (brs, 1H), 3.83 (brs, 1H),
3.64 (brs, 3H), 1.43 (s, 9H, C(CH3)3); 13C NMR (CDCl3)
d 155.65 (NCOO), 138.40, 138.20, 138.02, 128.25, 128.16,
127.75, 127.62, 127.53, 127.48 and 127.31 (aromatic),
80.68 (C(CH3)3), 79.41 (C-3 or C-4), 77.20 (C-3 or C-4),
73.14, 72.45 and 72.17 (benzyle methylene), 69.74 (C-10
or C-100), 64.17 (C-2 or C-5), 64.06 (C-10 or C-100), 58.71
(C-2 or C-5), 28.3 (C(CH3)). Anal. calcd for
C32H39NO6: C, 72.02; H, 7.37; N, 2.62. Found: C,
71.54; H, 7.44; N, 2.56.
(2R,3R,4S,5R)-3,4-Dibenzyloxy-2,5-dibenzyloxymethyl-
pyrrolidine (22). Compound 21 (219 mg, 0.35 mmol) was
treated with TFA:CH2Cl2 (1:2 v/v, 6 mL) at rt for 1 h.
The resulting solution was added saturated NaHCO3
and extracted with CH2Cl2. The combined organic layers
were dried over MgSO4, ®ltered, evaporated, and pur-
i®ed by column chromatography (50:50:1 n-hexane:
EtOAc-Et3N) to a€ord 22 (170 mg, 92%): [a]d +40.2ꢁ
1
(c 0.9, CHCl3); H NMR (CDCl3) d 7.32±7.24 (m, 20H,
aromatic), 4.72 (d, 1H, J=11.9 Hz, benzyl methylene),
4.61±4.41 (m, 7H, benzyl methylene), 4.05 (t, 1H,
J=4.4 Hz, H-4) 3.83 (dd, 1H, J=4.4, 6.3 Hz, H-3), 3.70
(dd, 1H, J=6.3, 9.0 Hz, H-10 or H-100), 3.62 (dd, 1H,
J=7.3, 9.0 Hz, H-10 or H-100), 3.54±3.44 (m, 4H, H-2, H-
5 and H-10 or H-100), 2.19 (brs, 1H, NH); 13C NMR
(CDCl3) d 138.53, 138.20, 138.10, 128.16, 128.09,
127.66, 127.60, 127.49, 127.46, 127.42 and 127.33 (aro-
matic), 80.59 (C-3), 77.72 (C-4), 73.16, 72.99 and 72.02
(benzyl methylene), 70.63 (C-10 or C-100), 69.83 (C-10 or
C-100), 59.77 (C-2 or C-5), 58.76 (C-2 or C-5); MAL-
DITOF MS m/z: 524 [M+H]+.
(2R,3R,4S,5R)-3,4-Dihydroxy-2,5-dihydroxymethyl-pyr-
rolidine (4). Hydrogenolysis of 20 (29 mg, 0.05 mmol)
was carried out as described for the synthesis of 2 to

2258
C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
(2R,3R,4S,5R)-N-Methyl-(3,4-dibenzyloxy-2,5-benzyloxy-
methyl)-pyrrolidine (23). To a solution of 22 (41 mg,
0.08 mmol) in MeOH (1mL) at 0ꢁC was added a 37%
formaldehyde solution (0.1 mL, 1.2 mmol) and NaBH3CN
(10 mg, 0.15 mmol). The mixture was stirred at rt over-
night. The reaction mixture was added H2O, extracted
with CHCl3 and dried with MgSO4. After removal of
the solvent, the residue was puri®ed by column chro-
matography (1:1 n-hexane:EtOAc) to yield 23 (36 mg,
85%): [a]d +10.8 (c 1.2, CHCl3); H NMR (CDCl3) d
7.32±7.23 (m, 20H, aromatic), 4.64±4.42 (m, 8H, benzyl
methylene), 4.01 (t, 1H, J=5.6 Hz, H-4), 3.86±3.81 (m,
3H, H-3 and H-100), 3.49±3.32 (m, 3H, H-10 and H-5),
3.01±2.96 (m, 1H, H-2), 2.53 (s, 3H, CH3); 13C NMR
(CDCl3) d 138.67, 138.43, 138.24, 128.32, 128.28,
128.19, 127.73, 127.67, 127.64, 127.58, 127.49, 127.42
and 127.37 (aromatic), 79.62 (C-3), 77.91 (C-4), 73.28,
73.22, 72.31 and 71.75 (benzyl methylene), 69.67 (C-10),
68.09 (C-2), 67.65 (C-100), 64.83 (C-5), 37.11 (CH3).
Anal. calcd for C35H39NO4: C, 78.18; H, 7.31; N, 2.60.
Found: C, 77.75; H, 7.42; N, 2.58.
and 21.22 (cyclohexyl-C and CH2(CH2)6CH3), 14.00
((CH2)7CH3). Anal. calcd for C30H43NO9: C, 64.15; H,
7.72; N, 2.49. Found: C, 63.96; H, 7.76; N, 2.47.
Octyl 2,6,7-trideoxy-7-methoxycarbonyl-3,4-O-[(10S,20S)-
dimethoxycyclohexylidene]-2-phthalimido-ꢀ-D-glucooct-6
(E)-enopyranoside (25). To
a solution of DMSO
(220 mL, 3.5 mmol) in CH2Cl2 (6 mL) was added a 2 M
solution of oxalyl chloride in CH2Cl2 (0.9 mL,
1.8 mmol) at 78 ꢁC and the mixture was stirred at the
temperature for 0.5 h. To this mixture was added a
solution of compound 24 (507 mg, 0.90 mmol) in
CH2Cl2 (4 mL) and the mixture was stirred at the tem-
perature for 0.5 h. Et3N (1.0 mL, 7.2 mmol) was added
and the mixture was stirred at rt for 15 min, then the
mixture was diluted with CH2Cl2 and washed with H2O,
dried over MgSO4, and evaporated in vacuo. The
resulting crude aldehyde was used for the next step
without further puri®cation. The solution of crude
aldehyde and methyl (triphenylphosphoranylidene)ace-
tate (0.45 g, 1.3 mmol) in benzene (10 mL) was heated
under re¯ux overnight. After cooling, the solvent was
removed in vacuo and the crude mixture was puri®ed by
column chromatography (5:1 n-hexane-EtOAc). E-iso-
mer 25 (492 mg, 93% in two steps) was obtained as a
sole product: mp 79 ꢁC; [a]d +104.3ꢁ (c 0.9, CHCl3); 1H
NMR (CDCl3) d 7.91±7.72 (m, 4H, aromatic), 7.11 (dd,
1H, J=4.0, 15.8 Hz, H-6), 6.24 (dd, 1H, J=1.7, 15.8 Hz,
H-7), 5.20 (d, 1H, J=8.3 Hz, H-1), 4.88 (dd, 1H, J=9.9,
11.2 Hz, H-3), 4.34±4.27 (m, 2H, H-2 and H-5), 3.82 (dt,
1H, J=6.3, 9.6 Hz, CH2(CH2)6CH3), 3.76 (s, 3H,
COOCH3), 3.66 (t, 1H, J=9.9 Hz, H-4), 3.41 (dt, 1H,
J=6.6, 9.6 Hz, CH2(CH2)6CH3), 3.13 and 3.06 (s, each
3H, OCH3), 1.80±0.97, (m, 20H, CH2(CH2)6CH3 and
cyclohexyl-H), 0.81 (t, 3H, J=7.1 Hz, (CH2)7CH3); 13C
NMR (CDCl3) d 168.37, 167.37 and 166.63 (CO),
142.41 (C-6), 134.05, 133.96, 131.79, 131.57, 123.67 and
123.00 (aromatic), 121.78 (C-7), 98.80 (C-1 and
COCH3), 72.09 (C-4 and C-5), 69.78 (CH2(CH2)6CH3),
66.78 (C-3), 53.89 (C-2), 51.61 (COOCH3), 46.96 and
46.76 (OCH3), 31.61, 29.26, 29.06, 26.88, 25.73, 22.54
and 21.26 (cyclohexyl-H and CH2(CH2)7CH3), 14.00
((CH2)7CH3). Anal. calcd for C33H45NO10: C, 64.37; H,
7.37; N, 2.27. Found: C, 64.36; H, 7.43; N, 2.38.
ꢁ
1
(2R,3R,4S,5R)-N-Methyl-(3,4-dihydroxy-2,5-hydroxy-
methyl)-pyrrolidine (5). Compound 5 was synthesized
from 23 (30mg, 0.055 mmol) according to the procedure
described for the synthesis of compound 2 to a€ord 5
(9mg, 91%): [a]d-16.6ꢁ (c 0.4, H2O); H NMR (D2O) d
1
4.25 (t, 1H, J=5.6Hz, H-4), 4.07 (t, 1H, J=5.1Hz, H-3),
3.92 (dd, 1H, J=5.1, 11.8 Hz, H-100), 3.79 (dd, 1H,
J=4.4, 11.8 Hz, H-100), 3.76 (dd, 1H, J=5.1, 11.8 Hz, H-
10), 3.68 (dd, 1H, J=5.1, 11.8 Hz, H-10), 3.12 (q, 1H,
J=5.1 Hz, H-5), 2.95 (q, 1H, J=5.1 Hz, H-2), 2.50 (s,
3H, CH3); 13C NMR (D2O) d 71.58 (C-3), 69.78 (C-4),
69.23 (C-2), 65.21 (C-5), 59.35 (C-10), 56.53 (C-100), 34.59
(CH3); HRFAB MS calcd for C7H15NO4 [M+H]+
178.1079; found: m/z 178.1096
Octyl 2-deoxy-3,4-O-[(10S,20S)-dimethoxycyclohexylidene]-
2-phthalimido-ꢀ-D-glucopyranoside (24).
A
catalytic
amount of CSA was added to a solution of octyl 2-deoxy-
2-phthalimido-b-d-glucopyranoside (115mg, 0.27mmol),
1,1,2,2-tetramethoxy cyclohexane (92 mg, 0.45mmol) and
trimethyl orthoformate (0.5 mL) in MeOH (3 mL), and
the mixture was heated under re¯ux overnight. After
neutralization with NaHCO3, the solvent was removed
in vacuo and the crude material was puri®ed by column
chromatography (9:1 n-hexane:EtOAc) to give 24
(111mg, 72%) as white powder and the unreacted starting
material (11 mg, 9%): mp 119 ꢁC; [a]d +113.7ꢁ (c 0.9,
Octyl 2,6,7-trideoxy-3,4-O-[(10S,20S)-dimethoxycyclohexy-
lidene]-2-phthalimido-ꢀ-D-glucooct-6(E)-enopyranoside
(26). To a solution of compound 25 (483 mg, 0.78
mmol) in CH2Cl2 (10 mL) was slowly added a 0.98 M
solution of DIBAL (5.6 mL, 7 equiv) at 78 ꢁC. The
reaction mixture was stirred at the temperature for 3 h.
MeOH (0.9 mL) was added at 78 ꢁC and the tempera-
ture was raised to rt. Saturated NaCl (1.8 mL) was added
and the mixture was diluted with Et2O (45 mL). MgSO4
(4.7 g) was added and the whole mixture was stirred for
1.5 h, then ®ltered through a Celite pad. The solvent was
removed in vacuo and the crude mixture was puri®ed by
column chromatography (1:1 n-hexane: EtOAc) to give
26 (344 mg, 74%) and the starting material 25 (123 mg,
26%): mp 156 ꢁC; [a]d +106.9ꢁ (c 1.3, CHCl3); 1H
NMR (CDCl3) d 7.90±7.70 (m, 4H, aromatic), 6.11 (dt,
1H, J=5.4, 15.7 Hz, H-7), 5.86 (dd, 1H, J=5.4, 15.7 Hz,
H-6), 5.18 (d, 1H, J=8.3 Hz, H-1), 4.85 (dd, 1H, J=9.9,
1
CHCl3); H NMR (CDCl3) d 7.91±7.69 (m, 4H, aro-
matic), 5.18 (d, 1H, J=8.3 Hz, H-1), 4.86 (dd, 1H,
J=9.9, 11.6 Hz, H-3), 4.28 (dd, 1H, J=8.3, 11.6 Hz, H-
2), 3.94 (t, 2H, J=9.9 Hz, H-4 and H-6), 3.85±3.70 (m,
3H, H-5, H-6 and CH2(CH2)6CH3), 3.39 (dt, 1H, J=6.6,
9.6 Hz, CH2(CH2)6CH3), 3.23 and 3.05 (s, each 3H,
OCH3), 2.02 (brs, 1H, OH) 1.81±0.89, (m, 20H, CH2
(CH2)6CH3 and cyclohexyl-H), 0.81 (t, 3H, J=7.1 Hz,
((CH2)7CH3); 13C NMR (CDCl3) d 168.39 and 167.44
(CO), 134.03, 133.94, 131.79, 131.59, 123.65 and 123.0
(aromatic), 99.07, 98.78 and 98.53 (C-1 and COCH3),
73.95 (C-5), 69.96 (CH2(CH2)6CH3), 68.27 (C-4), 66.51
(C-3), 61.33 (C-6), 54.09 (C-2), 46.92 and 46.70
(COCH3), 31.59, 29.26, 29.06, 26.90, 25.72, 22.52, 21.30

C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
2259
11.2 Hz, H-3), 4.30 (dd, 1H, J=8.3, 11.5 Hz, H-2), 4.22-
4.08 (m, 3H, H-5 and H-8), 3.82 (dt, 1H, J=6.1, 9.9 Hz,
CH2(CH2)6CH3), 3.65 (t, 1H, J=9.9 Hz, H-4), 3.39 (dt,
1H, J=6.6, 9.9 Hz, CH2(CH2)6CH3), 3.16 and 3.05 (s,
each 3H, OCH3), 1.74±0.88 (m, 20H, CH2(CH2)6CH3
and cyclohexyl-H), 0.81 (t, 3H, J=7.1 Hz, (CH2)7CH3);
13C NMR (CDCl3) d 168.30 and 167.35 (CO), 133.93
(C-7), 133.84, 132.76, 131.64, 131.46, 123.49 and 122.86
(aromatic), 125.88 (C-6), 98.56 (C-1 and COCH3), 73.13
(C-5), 72.24 (C-4), 69.53 (CH2(CH2)7CH3), 66.61 (C-3),
62.70 (C-8), 53.98 (C-2), 46.74 and 46.51 (OCH3), 31.47,
29.11, 28.93, 26.78, 25.63, 22.39, 21.17 and 21.12
(cyclohexyl-C and CH2(CH2)6CH3), 13.87 ((CH2)7CH3).
Anal. calcd for C32H45NO9: C, 65.40; H, 7.72; N, 2.38.
Found: C, 64.93; H, 7.70; N, 2.36.
(ddd, 1H, J=5.6, 7.9, 10.2 Hz, H-2), 2.70±2.45 (m, 2H,
H-7), 2.31±2.20 (m, 1H, H-6), 1.96 (s, 3H, COCH3),
1.80±1.23 (m, 21H, H-6, CH2(CH2)6CH3 and cyclo-
hexyl-H), 0.87 (t, 3H, J=6.6 Hz, (CH2)7CH3); 13C
NMR (CDCl3) d 201.92 (CHO), 170.28 (NHCOCH3),
99.44 (C-1), 98.56 and 98.40 (COCH3), 72.90 (C-4 or C-
5), 71.99 (C-4 or C-5), 70.05 (CH2(CH2)6CH3), 67.33
(C-3), 56.84 (C-2), 46.79 and 46.65 (OCH3), 40.06 (C-7),
31.74, 29.45, 29.24, 26.92, 26.85, 25.82, 23.63, 23.51,
22.57, 21.28 and 20.97 (C-6, CH2(CH2)6CH3 and cyclo-
hexyl-C), 14.02 ((CH2)7CH3). Anal. calcd for C26H45
NO8: C, 62.50; H, 9.08; N, 2.80. Found: C, 62.02; H,
9.10; N, 2.94.
Octyl 2-acetamido-2,6,7,8-tetradeoxy-3,4-O-[(10S,20S)-di-
methoxycyclohexylidene]-8-[(200R,300R,400S,500R)-300,400-di-
benzyloxy - 200,500 - dibenzyloxymethylpyrrolidinyl] - ꢀ - D -
glucooctpyranoside (29). A solution of 22 (21 mg,
0.04 mmol) and 28 (14 mg, 0.03 mmol) in MeOH
(0.9 mL) was added NaBH3CN (4.5 mg, 0.07 mmol) at
0 ꢁC and stirred at rt overnight. To the mixture was
added H2O, extracted with CHCl3 and dried with
MgSO4. After removal of the solvent, the residue was
puri®ed by column chromatography (66:33:1 CHCl3:
MeOH:Et3N) to give azasugar 22 (8 mg, 37%) and con-
densate 29 (22 mg, 77% based on 28, 54% based on 22):
[a]d +41.0ꢁ (c 1.3, CHCl3); 1H NMR (CDCl3) d 7.36±7.23
(m 20H, aromatic), 5.59 (d, 1H, J=6.9Hz, NH), 5.05 (d,
1H, J=7.9 Hz, H-1), 4.63±4.46 (m, 10H), 3.96 (t, 1H,
J=5.9Hz), 3.86±3.76 (m, 3H), 3.64±3.58 (m, 1H), 3.50±
3.37 (m, 4H), 3.23±3.05 (m, 2H), 3.19 and 3.17 (s, each
3H, OCH3), 3.02±2.91 (m, 2H), 2.61±2.50 (m, 1H), 1.96 (s,
3H, NHCOCH3), 1.88±1.68 (m, 8H), 1.53±1.50 (m, 6H),
1.39±1.23 (m, 16H), 0.86 (t, 3H, J=6.4 Hz, (CH2)7CH3);
13C NMR (CDCl3) d 170.02 (NHCO), 138.85, 138.81,
138.29, 128.32, 128.19, 128.10, 127.62, 127.53, 127.46,
127.40 and 127.21 (aromatic), 99.41 (C-1), 98.51 and 98.3
(COCH3), 79.43, 78.11, 73.73, 73.24, 73.05, 72.29, 71.93,
71.43, 70.78, 69.82, 67.93, 67.33, 61.55, 57.04, 49.83, 46.79
and 46.58 (OCH3), 31.79, 29.53, 29.35, 29.25, 27.01, 26.92,
25.95, 24.35, 23.69 (NHCOCH3), 22.63, 21.33, 14.07
((CH2)7CH3); ESI MS m/z: 1007.8 [M+H]+.
Octyl 2-acetamido-2,6,7-trideoxy-3,4-O-[(10S,20S)-dimeth-
oxycyclohexylidene]-ꢀ-D-glucooctpyranoside (27). A solu-
tion of 26 (116mg, 0.20mmol) and hydrazine mono-
hydrate (0.3 mL) in EtOH (3 mL) was heated under re¯ux
overnight. After cooling, the solvent was removed in
vacuo. To the solution of the residue in MeOH (3 mL)
was added Ac2O (0.3 mL) and the mixture was stirred at
rt overnight. After removal of the solvent, the residue
was puri®ed by column chromatography (1:3 n-hexane:
EtOAc) to give 27 (92 mg, 93%): [a]d +83.8ꢁ (c 0.8,
1
CHCl3); H NMR (CDCl3) d 5.71 (d, 1H, J=6.9Hz,
NH), 5.13 (d, 1H, J=7.9 Hz, H-1), 4.64 (t, 1H,
J=10.2 Hz, H-3), 3.84 (dt, 1H, J=6.6, 9.6 Hz, CH2
(CH2)6CH3), 3.66 (t, 2H, J=6.1Hz, H-8), 3.57±3.41 (m,
3H, H-4, H-5, and CH2(CH2)6CH3), 3.18 and 3.17 (s,
each 3H, OCH3), 3.09 (ddd, 1H, J=6.9, 7.9, 10.2 Hz, H-
2), 1.97 (s, 3H, NHCOCH3), 2.10±2.00, 1.90±1.23 (m,
24H, H-6, H-7, CH2(CH2)6CH3 and cyclohexyl-H), 0.87
(t, 3H, J=6.4 Hz, (CH2)7CH3); 13C NMR (CDCl3) d
170.76 (NHCO), 99.82 (C-1), 98.85 and 98.71 (COCH3),
74.00 (C-4 or C-5), 72.27 (C-4 or C-5), 70.37 (CH2
(CH2)6CH3), 67.87 (C-3), 62.79 (C-8), 57.09 (C-2), 47.08
and 46.94 (OCH3), 23.79 (NHCOCH3), 32.06, 29.81,
29.58, 29.54, 28.99, 27.24, 27.19, 26.17, 22.90 and 21.60
(C-6, C-7, cyclohexyl-C and CH2(CH2)6CH3), 14.34
((CH2)7CH3).
Octyl 2-acetamido-2,6,7-trideoxy-3,4-O-[(10S,20S)-dime-
thoxycyclohexylidene]-ꢀ-D-glucooctdialdo-1,5-pyranoside
(28). To a solution of DMSO (16 mL, 0.26 mmol) in
CH2Cl2 (0.5 mL) was added 2 M solution of oxalyl
chloride in CH2Cl2 (66 mL, 0.13 mmol) at 78 ꢁC and
the mixture was stirred at the temperature for 0.5 h. To
this mixture was added a solution of compound 27
(33 mg, 0.07 mmol) in CH2Cl2 (1 mL) and the mixture
was stirred at the temperature for 0.5 h. Et3N (74 mL,
0.53 mmol) was added and the mixture was stirred at rt
for 15 min, then the mixture was diluted with CH2Cl2
and washed with H2O, dried over MgSO4. After
removal of the solvent, the residue was puri®ed by col-
umn chromatography (1:2 n-hexane:EtOAc) to give 28
Octyl 2-acetamido-2,6,7,8-tetradeoxy-8-[(20R,30R,40S,50R)-
30,40-dihydroxy-20,50-dihydroxymethylpyrrolidinyl]-ꢀ-D-glu-
cooctpyranoside (1).
A
solution of 29 (16 mg,
0.016 mmol) in THF (1 mL) and M HCl (0.5 mL) was
stirred with 20% Pd(OH)2 on C under H2 atmosphere at
rt for 3 days After ®ltration, removal of the solvent
a€orded a crude debenzylated compound. The resulting
compound was used for the next step without further
puri®cation. The residue was treated with TFA:H2O
(3:2 v/v, 1 mL) at rt overnight. After removal of the
solvent, the residue was puri®ed by a column of Iatro
Beads (3:1 CHCl3:MeOH) to yield 1 (5.2 mg, 63% in
two steps); [a]d +47.8ꢁ (c 0.7, H2O). Compound 1 was
further puri®ed for inhibition assay using Sep-Pak
PLUS C18, washed with MeOH (20 mL) and water
(20 mL), eluted with water (20 mL) and MeOH (20 mL).
The latter eluent containing 1 was ®ltered through a
Millex GV ®lter and lyophilized. 1H NMR (D2O) d 4.50
(d, 1H, J=8.4 Hz, H-1), 4.36 (t, 1H, J=3.8 Hz, H-40),
4.28 (dd, 1H, J=3.8, 9.3 Hz, H-30), 4.01 (dd, 1H, J=5.3,
(30 mg, 90%): [a]d +100.9ꢁ (c 0.9, CHCl3); H NMR
1
(CDCl3) d 9.77 (s, 1H, CHO), 5.88 (brd, 1H, J=5.6Hz,
NH), 5.11 (d, 1H, J=7.9 Hz, H-1), 4.63 (t, 1H,
J=10.2 Hz, H-3), 3.79 (dt, 1H, J=6.6, 9.6 Hz, CH2
(CH2)6CH3), 3.54±3.42 (m, 3H, H-4, H-5 and CH2
(CH2)6CH3), 3.19 and 3.16 (s, each 3H, OCH3), 3.05

2260
C. Saotome et al. / Bioorg. Med. Chem. 8 (2000) 2249±2261
12.1 Hz, H-1000), 3.99 (dd, 1H, J=3.4, 12.5 Hz, H-100),
3.92 (dd, 1H, J=5.3, 12.1 Hz, H-1000), 3.87±3.82 (m, 2H,
H-8 and CH2 (CH2)6CH3), 3.78 (ddd, 1H, J=3.5, 5.2,
8.0 Hz, H-50), 3.70±3.64 (m, 4H, H-2, H-8, H-20 and H-
100), 3.59 (dt, 1H, J=6.4, 10.3 Hz, CH2(CH2)6CH3),
3.49, (dd, 1H, J=8.9, 10.3 Hz, H-3), 3.40±3.35 (m, 1H,
H-5), 3.27 (t, 1H, J=9.2 Hz, H-4), 2.03 (s, 3H,
NHCOCH3), 1.55±1.52, 1.28±1.27 (m, 16H), 0.86 (t, 3H,
J=6.9 Hz, (CH2)7CH3); 13C NMR (D2O) d 174.70
(NHCO), 101.27 (C-1), 74.66 (C-5), 73.95 (C-3), 73.70
(C-4), 71.47, 70.98, 70.73, 70.35, 62.48, 61.99, 58.35,
57.76, 56.04, 31.34, 28.83, 28.74, 28.59, 25.35, 22.45,
22.25, 13.63 ((CH2)7CH3); HRFAB MS calcd for
C24H46N2O9: [M+H]+ 507.3281; found: m/z 507.3286.
Galactosyltransferase assay
Materials. b-Galactosyltransferase (EC 2.4.1.22 from
bovine, recombinant, Spodoptera frugiperda; Calbio-
chem-Novabiochem Co. LaJolla, CA), UDP-galactose
(UDP-Gal), and uridine 50-diphosphonate (UDP) were
from Sigma Chemical Co. (St Louis, MO, USA).
Cacodylic acid sodium salt was from Nacalai Tesque
Inc. (Kyoto, Japan). 4-Methyl umbelliferyl 2-acetam-
ido-2-deoxy-b-d-glucopyranoside (MU-GlcNAc) and
cacodylic acid were from Wako Pure Chemical Ltd.
(Osaka, Japan).
Condition of capillary zone electrophoresis. Assays were
performed on a Beckman P/ACE System 5010 (USA),
which was equipped with a 57 cmÂ75 mm i.d. fused silica
capillary. Detection was carried out by on-column
measurement at 7.0 cm from the cathode. The laser-
induced ¯uorescence (LIF) detector equipped with He-
Cd Laser (IK series) from Kimmon Electro Co., Ltd.
(Japan) was used. A band-pass ®lter for excitation at
325 nm was used. The capillary was pretreated or
regenerated with 0.1 M KOH, washed with double
deionized water and separation bu€er prior to each
injection. Samples were loaded automatically by pres-
sure injection for 5 s (30.0 nL). Electrophoresis was
performed at 15 kV using 50 mM sodium borate as
electrolyte at a constant temperature of 35 ꢁC and was
detected at 375 nm (em).
Glycosidase assay
Materials. The sources of enzymes and substrates are as
follows: b-galactosidase (EC 3.2.1.23) from Aspergillus
oryzae, a-mannosidase (EC 3.2.1.24) from jack bean and
p-nitrophenyl-b-d-galactopyranoside (PNP-Gal) were
from Sigma Chemical Co. (St. Louis, MO, USA); p-
nitrophenyl-a-d-mannopyranoside (PNP-Man) was
from Nacalai Tesque. Inc. (Kyoto, Japan). 1-Deoxy-
galactonojirimycin was purchased from Toronto
Research Chemicals Inc. (North York, ON, Canada)
and 1-deoxymannojirimycin was purchased from Sigma
Chemical Co. (St Louis, MO, USA). Double deionized
water was prepared from a Milli-Q system from Milli-
pore Corp. (Milford, MA, USA).
Kinetic analysis of galactosyltransferase. Incubations
were performed in a total volume of 30 mL. Unless
otherwise stated, reaction mixtures contained 0.1 M
Cacodylate bu€er (pH as supeci®ed in Table 2 and Fig-
ure 4(A,B), 10 mM MnCl2, various amounts of UDP±
Gal (20±100 mM), various amounts of MU±GlcNAc
(10±1700 mM), various amounts of inhibitors and
0.8 mU GalTase. After incubation for 10 min at 37 ꢁC,
the reaction was terminated by the addition of 10 mL of
0.12 M EDTA in 30 mM sodium borate.
Condition of capillary zone electrophoresis. Assays were
performed on a Waters Quanta 4000E capillary electro-
phoresis system, which was equipped with a 53cmÂ75mm
i.d. fused silica capillary. Detection was carried out by on-
column measurement of UV absorption at 405 nm at
7.5 cm from the cathode. The capillary used was pre-
treated or regenerated with 0.1 M KOH (2 min) and the
separation bu€er before each injection. Samples were
loaded by means of hydrostatic pressure at 10 cm height
for 30 s (ca. 38.4nL). Electrophoresis was performed at
20kV using 50mM sodium borate (pH 9.4) as electrolyte
at a constant temperature of 37ꢁC. Electropherograms
were recorded on a Millennium 2010 system from Milli-
pore Corp.
Acknowledgements
We are grateful to Dr. Yoshitaka Nagai, Director of the
Glycobiology Research Group and Dr. Tomoya
Ogawa, Coordinator of the group, Frontier Research
Program (FRP) of the RIKEN Inst. for their continued
support and encouragement of our research. This
research was supported in part by the Science and
Technology Agency of the Japanese Government
through the FRP.
Kinetic analysis of ꢀ-galactosidase. Incubations were
performed in a total volume of 20 mL. Unless otherwise
stated, reaction mixtures contained 50 mM phosphate
bu€er (pH 7.0), various amounts of PNP±Gal (0.5±
3.0 mM), various amounts of inhibitors and b-galacto-
sidase. After incubation for 10 min at 37 ꢁC, the reaction
was terminated by addition of 20 mL of 0.2 M sodium
carbonate.
References and Notes
Kinetic analysis of ꢁ-mannosidase. Incubations were
performed in a total volume of 20 mL. Unless otherwise
stated, reaction mixtures contained 50 mM acetate buf-
fer (pH 5.0), various amounts of PNP±Man (0.7±
2.6 mM), various amounts of inhibitors and 1.05 mU of
a-mannosidase. After incubation for 10 min at 25 ꢁC,
the reaction was terminated by addition of 20 mL of
0.2 M sodium carbonate.
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