Synthesis and glycosidase inhibitory activity of some N-substituted 5a-carba-β-fuco- and β-galactopyranosylamines, and selected derivatives

Article abstract of DOI:10.1016/j.bmc.2004.09.023

In the course of chemical modification of α-fucosidase inhibitors of 5a-carba-fucopyranosylamine type, an N-dodecyl derivative of the enantiomer 6-deoxy-5a-carba-β-d-galactopyranosylamine demonstrated very strong inhibition of β-galactosidase and β-glucos

Full text of DOI:10.1016/j.bmc.2004.09.023

Bioorganic & Medicinal Chemistry 12 (2004) 6569–6579
Synthesis and glycosidase inhibitory activity of some
N-substituted 5a-carba-b-fuco- and b-galactopyranosylamines,
and selected derivativesq
Seiichiro Ogawa,a,* Shigeo Fujieda,a Yuko Sakata,a Masahiro Ishizaki,a
Seiichi Hisamatsu,a Kensuke Okazaki,a Yoriko Ooki,b Midori Mori,b
Masayoshi Itohb and Takashi Korenagab
aDepartment of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Hiyoshi, Kohoku-ku,
Yokohama 223-8522, Japan
bDepartment of Chemistry, Tokyo Metropolitan University, Minami-Ohsawa, Hachioji, Tokyo 192-0397, Japan
Received 26 July 2004; revised 8 September 2004; accepted 8 September 2004
Abstract—In the course of chemical modification of a-fucosidase inhibitors of 5a-carba-fucopyranosylamine type, an N-dodecyl
derivative of the enantiomer 6-deoxy-5a-carba-b-D-galactopyranosylamine demonstrated very strong inhibition of b-galactosidase
and b-glucosidase. This finding led us to synthesize corresponding 6-hydroxy compounds, in order to elucidate structure–activity
relationships for inhibitors of this type. Among four N-alkyl-5a-carba-b-D-galactopyranosylamines prepared, the N-octyl derivative
could be demonstrated to possess moderate activity toward a- and b-galactosidases, and b-glucosidase.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and L-enantiomeric modifications, and a comparison
of their inhibitory activity regarding four glycosidases.
In addition, the corresponding 6-hydroxy derivatives
of 3 were prepared in order to elucidate roles of C-5 sub-
stituents in displaying inhibition potential (Fig. 1).
Recently, the N-octyl derivative1 2 of 5a-carba-a-
fucopyranosylamine2 (1), a-5a-CFucamine, was demon-
strated to be a very strong a-L-fucosidase inhibitor
(bovine kidney) with p-nitrophenyl-a-L-fucopyranoside,
essentially comparable with deoxyfuconojirimycin
(DFJ) in this regard. Although oxocarbenium ion tran-
sition-state analogs, the (5,5a)-unsaturated derivatives
of 1, were shown to possess weak activity, contrary to
expectation, it is of interest to note that the b-anomer3
3 also possessed high activity against a-L-fucosidase.
These results are in contrast with the a-glucosidase
inhibitory activity relationship4 observed between the
ground-state mimic validamine, and a transition-state
mimic, its unsaturated derivative, valienamine.
Chemical modification of the b-anomer 3 was carried
out by incorporation of hydrophobic alkyl and phenyl-
alkyl portions into the amino group by direct treatment
of the carba-sugar epoxide 13, a newly prepared versa-
tile precursor, with alkyl and phenylalkylamines. For
convenience, rough screening of the activity of several
racemic compounds 5a–f readily available was per-
formed, to allow estimation of the approximate enantio-
meric contribution to the activity, based on comparison
of data observed for D- and/or L-enantiomers (Fig. 2).
The antipode of 5a-carba-b-L-fucopyranosylamine
should be 6-deoxy-5a-carba-b-D-galactopyranosylam-
ine. Therefore, inhibitory activity of the racemic modifi-
cation of 5a-carba-hexopyranosylamine should be a sum
of those of its components. This consideration is based
on the assumption that each enantiomer does not inter-
fere with the competitive inhibitory action of the others.
For example, with racemic DL-5d, activity (IC50 3.8lM)
against L-fucosidase should be largely due to the
The present paper documents details for synthesis of
some N-substituted derivatives5 of 3 with both racemic
Keywords: Glycosidase inhibitor; Galactopyranosylamine.
q In this paper, the nomenclature of carba-sugar derivatives follows the
IUPAC-IUB Recommendations 1996 (Carbohydr. Res. 1997, 297, 1).
*
Corresponding author. Tel.: +81 45 566 1788; fax: +81 45 566
1789; e-mail: sogawa379@ybb.ne.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.023

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S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
obtained for the newly synthesized optically active L
-5d
(IC50 0.91lM, a-fucosidase) (Table 2). Therefore, these
results stimulated us to synthesize the corresponding
parent 6-hydroxy compounds 6a–d in order to elucidate
structure–inhibitory activity relationships for inhibitors
of this type, especially regarding the hydrophobic region
due to C-5 substituents. As indicated by N-substitution1
of the a-anomer 1, chemical modification5 of the b-ano-
mer 3 and its parent might be expected to increase its
potential.
NHX
1
NH2
OH
5
3
Me
5a
2
Me
OH
4
OH
HO
OH
HO
α-L-5aCFuc(5,5a)enamine
α-L-5aCFucamine
1: X = H
2: X = (CH2)7Me
OH
OH
HO
HO
HO
HO
HO
HO
NH2
NH2
2. Results and discussion
α-D-5aCGlc(5,5a)enamine
α-D-5aCGlcamine
Validamine
Valienamine
Treatment of 3,4-di-O-acetyl-2,6-dibromo-2,6-dideoxy-
5a-carba-b-L-glucopyranosyl bromide6 (L-7) with slight
excess of zinc dust in DMF for 2h at 80ꢁC gave 1,2-
diacetoxy-6-bromomethyl-3-cyclohexene (D-8, 59%),
together with the 6-debromo compound D-9 (32%).
When the reaction time was prolonged at a similar tem-
perature, compound D-9 was mainly obtained but in
poor yield due to its simultaneous decomposition. Com-
pound D-8 isolated by fractionation was conventionally
debrominated with tributyltinhydride in toluene in the
H
Me
N
OH
OH
HO
DFJ
HO
Me
Me
NH2
OH
OH
HO
NH2
OH
HO
OH
D-3
´
presence of AIBN to give D-9 (86%). Zemplen O-
L-3
deacetylation of D-9 with methanolic sodium methoxide,
selective benzoylation of the allylic hydroxyl group, and
subsequent mesylation afforded the 1-mesylate D-10
(64%). Direct nucleophilic substitution of D-10 with an
benzoate anion in DMF at 110ꢁC and successive depro-
tection with methanolic sodium methoxide gave
(1R,2S,6R)-1,2-dihydroxy-6-methyl-3-cyclohexene L-11
(93%). Conventional protection of L-11 with cyclohexyl-
idene group (!L-12, 85%) and epoxidation with m-
chloroperbenzoic acid in a phosphate buffer solution
(pH ꢀ 6) gave the single b-epoxide L-13 selectively in
90% yield. Rear attack of the peracid seems to be ade-
quately restricted by the presence of a bulky cyclohexyl-
idene group.7 The corresponding isopropylidene
derivatives also gave similar results, but isolation of
the products was somewhat difficult, compared to those
for L-12 and L-13.
HO
HO
OH
OH
NH2
NH2
OH
D-4
OH
HO
L-4
Figure 1. 5a-Carba-b-fucopyranosylamine (3) and b-galactopyranos-
ylamine (4).
HO
X
X
NH(CH2)nY
HO
NH(CH2)nY
OH
OH
OH
HO
L
D
Reaction of the racemate8 DL-13 with alkylamines or
phenylalkylamines in 2-propanol in sealed tubes at
120ꢁC proceeded slowly but almost regio-selectively to
afford the respective N-substituted 5a-carba-b-galacto-
pyranosylamines DL-14a–f, in 70–90% yields, which,
without isolation, were treated with 80% aqueous
acetic acid at 80ꢁC, and the resulting amine acetates
were purified over a column of Dowex-50W · 2 (H+)
resin with 1% methanolic ammonia to give the free
bases DL-5a–f almost quantitatively. The optically
active L-5b–d,f were similarly prepared from L-13
(Scheme 1).
n
5
7
9
11
2
4
3
Compd
X
H
H
H
H
H
H
OH
OH
OH
OH
Y
5a
b
c
Me
Me
Me
Me
Ph
d
e
f
Ph
6a
b
Me
Me
Me
Me
7
9
11
c
d
Figure 2. N-Substituted 5a-carba-b-fucopyranosylamines and b-
galactopyranosylamines, assayed for inhibitory activity against seven
glycosidases.
Since N-substituted 6-deoxy-5a-carba-b-galactopyranos-
ylamines have been shown to possess strong inhibitory
activity5 (see Section 4.28) against b-galactosidase and
b-glucosidase, the findings stimulated our evaluation
of inhibitory potential of the corresponding 5a-carba-
b-galactopyranosylamines 4, the structures of which
more resemble those of the substrates.
L-enantiomer, and high inhibition toward b-galactosi-
dase (IC50 0.02lM) and b-glucosidase (IC50 0.50lM)
would be probably attributable to the D-enantiomer
(Table 1). This conclusion is partially supported by data

S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
6571
Br
O
OBn
OBn
OH
O
O
a
AcO
O
NH(CH2)nMe
AcO
Br
O
8
a
Br
AcO
D-16a–d
D-15
Br
AcO
b
n
b
Me
AcO
7
a
b
c
d
3
7
9
AcO
D-6a–d
9
c
11
d
Me
MsO
Me
OBn
a
b
L-15
L-6b
BzO
NH(CH2)7Me
OH
OH
HO
O
O
e
f
11
10
L-16b
O
Scheme 2. Synthesis of N-substituted 5a-carba-b-galactopyranosyl-
amines 6a–d. Reagents and conditions: (a) alkylamines (molar equiv),
2-propanol, 120ꢁC, 2–6days; (b) 80% aq AcOH; H2, EtOH, 10% Pd/C,
Me
O
Me
O
O
O
purified by
a
column of Dowex-50W · 2 (H+) resin, with 1%
methanolic ammonia, or that of silica gel.
g
13
12
h
plex due to the restricted rotation around the secondary
amido group, so that confirmation of the structure was
rather difficult. The N-acetyl derivative was hydrolyzed
with 1M hydrochloric acid and the resulting hydrochlo-
ride was purified similarly to afford the amine D-6b
(Scheme 2).
NH(CH2)nX
Me
5a–f
OH
O
O
n
X
a
b
c
d
e
5
7
9
11
2
Me
Me
Me
Me
Ph
Ph
14a–f
The enantiomeric epoxide L-15 was prepared from 3,4,6-
tri-O-acetyl-1,5-anhydro-2-deoxy-5a-carba-D-xylo-hex-
1-enitol according to the standard procedure,9 in a 15%
over-all yield. Reaction of L-15 with a molar equivalent
of octylamine gave the N-octyl derivative, which was
deprotected usually to give L-6b.
f
4
Scheme 1. Synthesis of N-substituted 5a-carba-b-DL- and L-fucopy-
ranosylamines 5a–f. For convenience, the formulae only depict those
of the L-enantiomers. Reagents and conditions: (a) Zn powder, AcOH,
80ꢁC; (b) Bu3SnH, AIBN, toluene; (c) methanolic NaOMe; BzCl
(1.2molar equiv), pyridine; MsCl, DMAP, pyridine; (d) NaOBz, 90%
aqueous DMF, 110ꢁC; methanolic NaOMe; (e) (MeO)2C6H10, TsOH,
DMF; (f) mCPBA, CH2Cl2, phosphate buffer; (g) alkyl or phenyl-
alkylamines (molar equiv), 2-propanol, 120ꢁC, 4–10days; (h) 80% aq
AcOH, Dowex-50W · 2 (H+) resin, 1% aq NH3.
Taking advantage of the readily available free base L-3,
the N-linked carba-disaccharides11 with carba-b-fucopy-
ranosylamine residues were prepared in order to ascer-
tain enzyme-inhibitory functions of the derivatives, the
alkyl chains of which were replaced with sugar residues.
The epoxide L-13 was treated with sodium azide in
DMF to give a sole azide 17, having a 5a-carba-b-fuco-
pyranose structure. This compound was catalytically re-
duced with Pd/C to give, after elution through an acid
column with methanolic ammonia, the free base L-3
(72%). Coupling of L-3 and 1,6:3,4-dianhydro-2-azido-
2-deoxy-b-D-galactopyranose12 18 (1.6molar equiv), in
2-propanol for two weeks 120ꢁC gave a single conden-
sate 19 (93%), the structure of which was confirmed with
1,2-Anhydro-6-O-benzyl-3,4-O-isopropylidene-5a-carba-
a-D-galactopyranose9 (D-15) was chosen as the synthetic
precursor of N-alkyl-5a-carba-b-galactopyranosylam-
ines. Diequatorial cleavage at C-1 with alkylamines
was expected to occur preferentially in consideration
of the previous synthesis of N-acetyl-5a0-carba-lactos-
aminide.10 Thus, a mixture of a molar equivalent of
D-15 and alkylamines was heated in a sealed tube at
120ꢁC for 2–3weeks, producing the corresponding sec-
ondary amines D-16a–d in 50–60% yields. The protected
amine 16a was O-deisopropylidenated with aqueous ace-
tic acid, followed by hydrogenolysis with Pd/C, to give,
after purification by an acid resin column, the amine D-
6a (93%) as a white solid. Similarly, compounds D-16b–d
were converted into the corresponding amines D-6b–d.
Attempted isolation of the di-N,O-acetyl derivative of
D-16b was successful, but its NMR spectrum was com-
1
reference to the H NMR spectrum of its tetra-O-acetyl
derivative 20 conventionally prepared (Scheme 3).
Synthesis of symmetric N-linked bis(5a-carba-b-fucopy-
ranosyl) disaccharide was attempted by conventional
coupling of L-3 and L-13, affording the protected dicar-
badisaccharide 21 (40%), which was O-decyclohexylide-
nated to give a symmetric compound 22 (ꢀ100%). The
structure was fully supported by 1H NMR spectral data
(Scheme 4).

6572
S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
Me
N3
+
a
L-3
L-13
OH
O
L-13
O
a
b
HO
L-17
O
Me
N
OH
H
Me
OH
O
O
HO
O
Me
21
NH2
O
OH
+
OH
HO
b
N3
L-3
18
HO
c
3'
5'
5
3
1
1'
2'
5a
2
OH
Me4
5a'
N
H
OH
4'
6
5
O
Me
OH
OH
OR
O
HO
1
4
3
2
22
5'
1'
5a'
2'
N3
Me
3'
N
H
Scheme 4. Synthesis of N-linked 5a,5a0-dicarbadisaccharide: bis(5a-
carba-b-L-fucopyranosyl)amine 22. Reagents and conditions: (a) L-13
(1.5molar equiv), 2-propanol, 120ꢁC, three weeks; (b) 80% aq AcOH,
80ꢁC.
OR
4'
OR
RO
19: R = H
20: R = Ac
Scheme 3. Synthesis of N-linked carba-disaccharide 19 containing 5a-
carba-b-L-fucopyranose residue. Reagents and conditions: (a) excess
NaN3, DMF, 110ꢁC; (b) H2, EtOH, 10% Pd/C; (c) 18 (1.6molar
equiv), 2-propanol, 120ꢁC, two weeks.
(4) were assayed for activity in a standard manner,13
against seven glycosidases: a-galactosidase (green coffee
beans), b-galactosidase (bovine liver), a-glucosidase (Ba-
kerꢀs yeast), b-glucosidase (almond), b-glucosaminidase
(bovine kidney), a-mannosidase (Jack beans), and a-
fucosidase (bovine kidney), as listed in Tables 1 and 2.
None of the compounds were found to be inhibitors of
a-glucosidase, b-glucosaminidase, or a-mannosidase.
3. Enzyme inhibitory activity
Four N-alkyl and two N-phenylalkyl derivatives 5a–f of
5a-carba-b-fucopyranosylamine (3) and four N-alkyl
derivatives 6a–d of 5a-carba-b-galactopyranosylamine
Table 1. Inhibitory activity [IC50 (Ki) lM] of N-substituted 5a-carba-b-DL-fucopyranosylamines against three glycosidases
Compounda
Inhibitory activity, IC50 (Ki), (lM)
b-Galactosidase (bovine liver)
n
X
Y
a-Fucosidase (bovine kidney)
b-Glucosidase (almond)
DL-5a
DL-5b
DL-5c
DL-5d
DL-5e
DL-5f
5
7
9
H
H
H
H
H
H
Me
Me
Me
Me
Ph
8.2
5.5
2.7
3.8
7.5
5.1
3.7
0.73
0.7 (0.11)
0.2 (0.009)
0.02 (0.0045)
5.7
1.5 (3.2)
2.0 (0.4)
0.50 (0.53)
0.38 (0.057)
1.4 (1.2)
11
2
4
Ph
0.9 (0.046)
a All compounds did not show any notable inhibitory activity against a-galactosidase, a-glucosidase, b-glucosaminidase, and a-mannosidase.
Table 2. Inhibitory activity (IC50, lM) of 5a-carba-b-fuco- and b-galactopyranosylamines and its N-substituted derivatives against four glycosidases
Compounda
Y
Inhibitory activity, IC50 (lM)
X
n
a-Fucosidase
(bovine kidney)
a-Galactosidase
(green coffee beans)
b-Oalactosidase
(bovine liver)
b-Glucosidase
(almond)
L-3
H
H
0
0
7
9
4.3
NI
1.8
0.7
0.91
1.2
NI
NI
105
NI
NI
NI
2.8
NI
NI
NI
NI
39
NI
NI
3.7
1.0
0.46
2.4
NI
8
NI
NI
15
D-4
OH
H
H
L-5b
L-5c
L-5d
L-5f
D-6a
D-6b
L-6b
D-6c
D-6d
Me
Me
Me
Ph
H
17
H
11
4
6.1
10
H
OH
OH
OH
OH
OH
Me
Me
Me
Me
Me
3
13
14
7
5.2
NI
25
7
87
16
210
25
9
11
35
5.8
8.7
NI: no inhibition <10ꢁ3 M.
a All compounds did not show any notable inhibitory activity against a-glucosidase, b-glucosaminidase, and a-mannosidase.

S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
6573
Compared to the corresponding derivatives2 of the a-
anomers, DL-5a–f possessed about one tenth of the
inhibitory activity against a-fucosidase and the potential
was actually thought to be mainly due to the b-L-fucose-
mimicking enantiomers, as verified by assaying L-5b–d
and L-5f newly prepared.
39lM) against a-fucosidase, suggesting selectivity along
the lines observed for L-3. Since, the former compound
was initially designed for the purpose of providing a syn-
thetic precursor for N-linked N-acetyl-5a0-carba-lactos-
aminide derivatives, it is promising that this kind of
disaccharide mimic may give selective a-fucosidase inhi-
bition through further chemical modification.
It is interesting to note that compounds DL-5a–f possess
very strong activity toward both b-galactosidase and b-
glucosidase, showing marked cross-reactivities with
both enzymes as observed for aminocyclopentitol gly-
cosidase inhibitors.14,15 Their high potential could be
demonstrated to be largely attributable to the respective
D-enantiomers, that is, N-alkyl-6-deoxy-5a-carba-b-D-
galactopyranosylamines. In fact, the L-enantiomers, N-
alkyl-5a-carba-b-L-fucopyranosylamines had decreased
activity toward these two enzymes as shown for L-5b–d
and L-5f, displaying rather potent cross-reactivities with
both a-fucosidase and b-galactosidase. Among the six
derivatives, compound DL-5d, actually D-5d, was the
strongest inhibitor of b-galactosidase: Ki < 4.5nM. Fur-
thermore, compound DL-5e was demonstrated to be a
very potent inhibitor, possessing characteristic pH-
dependent activity against b-glucosidase: Ki 0.39lM at
pH5.5; Ki 0.057lM at pH6.8. These results would indi-
cate that optically active D-5e is an effective b-glucosi-
dase inhibitor fully compatible with results for
isofagomine16 (Ki 0.11lM, at pH6.8) and calystegine17
(Ki 0.75lM, pH independent). Thus, N-substituted 6-
deoxy-5a-carba-b-D-galactopyranosylamines may be
promising lead compounds for new carba-sugar-type
b-galactosidase and b-glucosidase inhibitors. The inhibi-
tory potential of 5a-carba-glycosylamines and deriva-
tives appears to be comparable to those of structurally
related 4-amino-5-(hydroxymethyl)cyclopentane-1,2,3-
triols.15 It may probably depend on close resemblance
between the structures of 5a-carba-hexopyranosylamine
and those of ground states of the corresponding sub-
strates. However, in the case of 5a-carba-hexopyrano-
sylamines, the configuration of the 4-hydroxyl function
seems to be very important for the generation of acti-
vity, although it is difficult to explain rationally the fact
that, the corresponding b-gluco isomers, the 4-epimers
of D-5b, had rather decreased3 the activity toward b-
glucosidase, in spite of close resemblance of the sub-
strate structures.
4. Experimental
4.1. General methods
Optical rotations were measured with a JASCO DIP-370
polarimeter, and [a]D values are given in
10ꢁ1 degcm2 gꢁ1 1H NMR spectra were recorded for
.
solutions in deuteriochloroform and deuteriomethanol
with internal tetramethylsilane (TMS) as a reference
with a JEOL JNM LAMBDA-300 (300MHz) instru-
ment. Mass spectra were determined with HITACHI
M-8000 ion trap mass spectrometer. TLC was per-
formed on silica gel 60 F-254 (E. Merck, Darmstadt).
The silica gel used for a column chromatography was
Wakogel C-300 (Wako Junyaku Kogyo Co., Osaka,
200–300mesh) or silica gel 60 KO (Katayama Kagaku
Kogyo Co., Osaka, 70–230mesh). Organic solutions
were dried over anhydrous Na2SO4 and concentrated
at >45ꢁC under diminished pressure.
Unless otherwise noted, when both racemic and opti-
cally active compounds were prepared, the processing
for the later were only described.
4.2. 3,4-Di-O-acetyl-1,5-anhydro-6-bromo-2,6-dideoxy-
5a-carba-D-xylo-hex-1-enitol (D-8) and 3,4-di-O-acetyl-
1,5-anhydro-2,6-dideoxy-5a-carba-D-xylo-hex-1-enitol
[(1S,2S,3R)-1,2-di-O-acetyl-3-methylcyclohex-5-ene-1,2-
diol] (D-9)
(a) 3,4-Di-O-acetyl-2,6-dibromo-2,6-dideoxy-5a-carba-
L-b-glucopyranosyl bromide6 (L-7, 46.2g, 0.10mol)
was dissolved in acetic acid (370mL), to which zinc pow-
der (33.5g, 0.51atom) was added. The suspension was
stirred vigorously for 2h at 80ꢁC. Formation of two
components was observed by TLC (1:6 EtOAc/hexane).
Excess zinc dust and precipitates were removed by filtra-
tion, and the filtrate was co-evaporated with toluene.
The products were fractionated over a silica gel column
(500g, 1:10 EtOAc/hexane) to give D-9 (7.0g, 32%) and
D-8 (17.8g, 59%) as a colorless syrup. Data for D-8:
TLC: Rf 0.28 (1:6 EtOAc/hexane). The compound was
characterized by comparison with an authentic
racemate.6
It is noteworthy that the free bases L-3 and D-418 were
found to be not very strong but selective inhibitors
against a-fucosidase and a-galactosidase, corresponding
to the respective stereochemistry of the substrates (Table
2). As demonstrated for N-octyl derivative D-6b, N-alk-
yl-5a-carba-b-D-galactopyranosylamines are moderate
a- and b-galactosidases, and b-glucosidase inhibitors,
so that the N-substituents and the hydrophobic area
conferred by the 5-methyl branching on the carbocyclic
ring are likely to control and enhance binding to the
active sites of enzymes. The L-enantiomers, as indicated
by L-6b, almost lacked inhibitory activity against all
enzymes.
22
D
Data for D-9: TLC: Rf 0.37 (1:6 EtOAc/hexane); ½aꢂ
+12 (c 1.7, CHCl3); 1H NMR (300MHz, CDCl3): d
5.80 (m, 1H, H-1), 5.44–5.51 (m, 2H, H-2, H-3), 4.94
(dd, 1H, J3,4 = 7.8Hz, J4,5 = 11.0Hz, H-4), 2.27 [ddd,
1H, J1,5a(eq) = J5,5a(eq) = 4.3Hz, J5agem = 13.8Hz, H-
5a(eq)], 2.04 and 2.08 (2s, each 3H, 2 · Ac), 1.89–2.04
[m, 2H, H-5a(ax)], 0.97 (d, 3H, J5,6 = 6.3Hz, H-6);
ITMS-ESI (positive mode): m/z 235 [M+Na]+, 251
[M+K]+.
Newly prepared carba-disaccharide-type compounds 19
and 22 only showed inhibitory activity (IC50 3.9 and

6574
S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
(b) To a solution of D-8 (0.10g, 0.34mmol) in toluene
(8.0mL) were added in turn AIBN (8mg, 0.048mmol)
and tributyltinhydride (0.27mL, 1.0mmol), and the
mixture was stirred for 10min at 120ꢁC. The reaction
mixture was evaporated to dryness, and the residue
was chromatographed on a silica gel column (7.0g,
hexane ! 1:10 EtOAc/hexane) to give D-9 (62mg,
86%).
residue was chromatographed on a silica gel column
(30g, 1:2 EtOAc/hexane) to give L-11 (0.34g, 93%) as
22
D
a
syrup: ½aꢂ
+29 (c 0.99, CHCl3); 1H NMR
(300MHz, CDCl3): d 5.73 (br d, 1H, J1,2 = 10.3Hz, H-
1), 5.47 (br d, 1H, J1,2 = 10.3Hz, H-2), 4.19 (br s, 1H,
H-3), 3.77 (br s, 1H, H-4), 3.36 (br s, 2H, 2 · OH),
1.75–1.92 (m, 3H, H-5, 2 · H-5a), 1.03 (d, 3H,
J5,6 = 6.3Hz, H-6); ITMS-ESI (positive mode): m/z
151 [M+Na]+.
4.3. 3-O-Benzoyl-1,5-anhydro-2,6-dideoxy-4-O-mesyl-5a-
carba-D-xylo-hex-1-enitol [(1S,2S,3R)-1-O-benzoyl-2-O-
mesyl-3-methylcyclohex-5-ene-1,2-diol] (D-10)
4.5. 1,5-Anhydro-3,4-O-cyclohexylidene-2,6-dideoxy-5a-
carba-L-lyxo-hex-1-enitol (L-12)
A solution of D-9 (0.94g, 4.4mmol) in methanol
(5.0mL) was treated with 1M methanolic sodium meth-
oxide (1.0mL) for 1h at room temperature, and, after
neutralization with Amberlite IR-120B (H+) resin, con-
centrated to dryness. The residual diol was dissolved
in dry pyridine (6.0mL) and a solution was stirred at
ꢁ15ꢁC, to which a solution of benzoyl chloride
(0.62mL, 5.3mmol) in pyridine (3mL) was added drop-
wise for 0.5h. The mixture was stirred for 15h at 0ꢁC.
TLC (1:10 MeOH/CHCl3) showed disappearance of
the diol and formation of a new component. To the
reaction mixture were added mesyl chloride (1.0mL,
12.9mmol) and a catalytic amount of DMAP under
stirring at 0ꢁC, and then the mixture was stirred for
17h at room temperature. Formation of one major
and one minor components was detected by TLC.
After the reaction was quenched by addition of
methanol at 0ꢁC, the mixture was diluted with EtOAc
(150mL). The solution was washed thoroughly
with 1M hydrochloric acid, saturated aqueous NaH-
CO3, and water, and dried. The organic layer was
evaporated and the residue was chromatographed on a
A mixture of the triol L-11 (2.00g, 15.6mmol), 1,1-
dimethoxycyclohexane (10mL), TsOHÆH2O (0.53g,
3mmol), and DMF (40mL) was stirred for 2h at room
temperature. The mixture was then neutralized with tri-
ethylamine and evaporated to dryness. The product was
purified with a silica gel column (20g, 1:100 EtOAc/hex-
ane) to give L-12 (2.83g, 85%) as a syrup, TLC: Rf 0.47
24
D
(1:10 EtOAc/hexane); ½aꢂ +37 (c 0.9, CHCl3); 1H NMR
(300MHz, CDCl3): d 5.71 [br ddd, 1H, J1,5a(ax) = 2.0Hz,
J1,5a(eq) = 2.4Hz, J1,2 = 6.1Hz, H-1], 5.51 (br d, 1H,
J1,2 = 6.1Hz, H-2), 4.44 (m, 1H, H-3), 4.08 (br d, 1H,
J4,5 = 5.2Hz, H-4), 1.77–1.85 (m, 2H, 2 · H-5a), 1.75
(br dd, 1H, J4,5 = 5.2Hz, J5,6 = 6.3Hz, H-5), 1.42 (m,
10H, C6H10), 1.05 (d, 3H, J5,6 = 6.3Hz, Me); ITMS-
APCI (positive mode): m/z 209 [M+H]+.
4.6. 1,2-Anhydro-3,4-O-cyclohexylidene-5a-carba-a-L-
fucopyranose (L-13)
To a stirred suspension of compound L-12 (2.83g,
13.3mmol) and a mixture of 1M aqueous NaH2PO4
(1.0mL) and 1M aqueous Na2HPO4 (1.0mL) in dichlo-
romethane (42mL), was added mCPBA (2.98g,
17.2mmol), and it was agitated vigorously for 3h at
0ꢁC. The mixture was then diluted with chloroform
(600mL), and the solution was thoroughly washed suc-
cessively with saturated aqueous sodium thiosulfate,
sodium hydrogen carbonate, and water, dried, and
evaporated. The residue was chromatographed on a sil-
ica gel column (30g, 1:80 ! 1:60 EtOAc/hexane) to give
silica gel (100g, 1:7 EtOAc/hexane) to afford D-10
22
D
(0.87g, 64%) as a colorless syrup: ½aꢂ +144 (c 0.67,
CHCl3); 1H NMR (300MHz, CDCl3): d 7.44–8.11
(m, 5H, Ph), 5.87 [dddd, 1H, J1,3 = 2.0Hz, J1,5a(ax)
=
J1,5a(eq) = 4.9Hz, J1,2 = 10.0Hz, H-1], 5.77 (ddd, 1H,
J1,3 = 2.0Hz, J2,3 = 3.5Hz, J3,4 = 7.8Hz, H-3), 5.62
[dddd, 1H, J2,5a(ax) = J2,5a(eq) = 1.8Hz, J2,3 = 3.5Hz,
J1,2 = 10.0Hz, H-2], 4.83 (dd, 1H, J3,4 = 7.8Hz, J4,5
=
11.2Hz, H-4), 2.91 (s, 3H, Ms), 2.40 [ddd, 1H,
J1,5a(eq) = J5,5a(eq) = 4.9Hz, J5agem = 17.8Hz, H-5a(eq)],
2.19 [dddd, 1H, J5,5a(eq) = 4.9Hz, J5,6 = 6.3Hz,
J5,5a(ax) = 10.0Hz, J4,5 = 11.2Hz, H-5], 2.03 [m, 1H,
H-5a(ax)], 1.20 (d, 3H, J5,6 = 6.3Hz, H-6); ITMS-ESI
(positive mode): m/z 333 [M+Na]+, 349 [M+K]+.
the epoxide L-13 (1.57g, 90%) as a crystalline solid,
24
TLC: Rf 0.67 (1:10 EtOAc/hexane); ½aꢂ +19 (c 1.0,
D
CHCl3); 1H NMR (300MHz, CDCl3): d 4.31 (br d,
1H, J3,4 = 5.6Hz, H-3), 3.98 (br d, 1H, J3,4 = 5.6Hz,
H-4), 3.23 (br s, 1H, H-1), 2.97 (br d, 1H,
J2,3 = 3.2Hz, H-2), 1.84 (m, 3H, H-5, 2 · H-5a), 1.25–
1.78 (m, 10H, C6H10), 1.04 (d, 3H, J5,6 = 4.4Hz, Me);
ITMS-APCI (positive mode): m/z 225 [M+H]+.
4.4. 1,5-Anhydro-2,6-dideoxy-5a-carba-L-lyxo-hex-1-eni-
tol [(1S,2R,3R)-3-methylcyclohex-5-ene-1,2-diol] (L-11)
4.7. N-Hexyl-5a-carba-b-DL-fucopyranosylamine (DL-5a)
A mixture of D-10 (0.87g, 2.8mmol) and sodium benzo-
ate (2.0g, 14mmol) in 90% aqueous DMF (13mL) was
stirred for 15h at 110ꢁC. After cooling, the mixture was
diluted with ethyl acetate (150mL) and the solution was
washed with water. The organic layer was evaporated
and the residue was treated with 1M methanolic sodium
methoxide (2mL) in methanol (4mL) for 3h at room
temperature. After neutralization with Amberlite IR-
120B (H+) resin, the mixture was evaporated and the
A mixture of the epoxide DL-13 (50mg, 0.22mmol), hex-
ylamine (28.7lL, 0.22mmol), and 2-propanol (0.5mL)
was heated in a sealed tube for four days at 120ꢁC,
and then evaporated. The residue was chromatographed
on a silica gel column (5.0g, 100:1 ! 20:1 CHCl3/
MeOH) to give the protected amine DL-14a (47mg,
66%). It was treated with 80% aqueous acetic acid
(1.0mL) for 2h at 80ꢁC, and the mixture was evapo-
rated. The product was purified on a column of Dow-

S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
6575
ex-50W · 2 (H+) resin (1.0g, 1% aqueous NH3) to give
the amine DL-5a (16mg, 92%) as a white powder,
TLC: Rf 0.57 (5:1 CHCl3/MeOH); 1H NMR
(300MHz, CD3OD): d 3.80 (br s, 1H, H-1), 3.59 (dd,
1H, J1,2 = J2,3 = 9.5Hz, H-2), 3.43 (br d, 1H,
J2,3 = 9.5Hz, H-3), 2.75 (dt, 1H, J = 7.1Hz,
Jgem = 11.0Hz, NHCH2), 2.39–2.51 (m, 2H, NHCH2,
H-1), 1.59–1.70 [m, 2H, H-5, H-5a(eq)], 1.45–1.51 (m,
2H, NHCH2CH2), 1.29–1.36 [m, 7H, (CH2)3CH3, H-
5a(ax)], 1.04 (d, 3H, J5,6 = 6.5Hz, H-6), 0.88 (t, 3H,
J = 6.6Hz, CH2CH3); ITMS-ESI (positive mode): m/z
246 [M+H]+.
then evaporated. The residue was chromatographed on
a silica gel column (5.0g, 80:1 ! 20:1 CHCl3/MeOH)
to give the protected amine L-14d (68mg, 93%). It was
treated with 80% aqueous acetic acid (1.0mL) for 4h
at 80ꢁC, and evaporated. The product was purified on
a column of Dowex-50W · 2 (H+) resin (1.0g, 1% aque-
ous NH3) to give the amine L-5d (35mg, 69%) as a white
24
powder, TLC: Rf 0.69 (5:1 CHCl3/MeOH); ½aꢂ +0.2 (c
D
1.4, CHCl3); 1H NMR (300MHz, CD3OD): d 3.70 (br s,
1H, H-4), 3.48 (dd, 1H, J2,3 = 9.6Hz, J1,2 = 9.7Hz, H-2),
3.29 (dd, 1H, J3,4 = 3.0Hz, J2,3 = 9.6Hz, H-3), 2.69 (dt,
1H, J = 7.3Hz, Jgem = 11.2Hz, NHCH2), 2.36–2.51 (m,
2H, H-1, NHCH2), 1.49–1.66 [m, 4H, H-5, H-5a(eq),
NHCH2CH2], 1.28–1.40 [m, 19H, (CH2)9CH3, H-
5a(ax)], 1.01 (d, 3H, J5,6 = 6.6Hz, H-6), 0.89 (t, 3H,
J = 6.6Hz, CH2CH3); ITMS-ESI (positive mode): m/z
330 [M+H]+.
4.8. N-Octyl-5a-carba-b-L-fucopyranosylamine (L-5b)
A mixture of the epoxide L-13 (50mg, 0.22mmol), octyl-
amine (36.1lL, 0.22mmol), and 2-propanol (0.5mL)
was heated in a sealed tube for four days at 120ꢁC,
and then evaporated. The residue was chromatographed
on a silica gel column (5.0g, 100:1 ! 20:1 CHCl3/
MeOH) to give the protected amine L-14b (47mg,
66%). It was treated with 80% aqueous acetic acid
(1.0mL) for 3h at 80ꢁC, and evaporated. The product
was purified on a column of Dowex-50W · 2 (H+) resin
(1.0g, 1% aqueous NH3) to give the amine L-5b (33mg,
ꢀ100%) as a white powder, TLC: Rf 0.58 (5:1 CHCl3/
4.11. N-(2-Phenylethyl)-5a-carba-b-DL-fucopyranosyl-
amine (DL-5e)
A mixture of the epoxide DL-13 (50mg, 0.22mmol), 2-
phenylethylamine (27.5lL, 0.22mmol), and 2-propanol
(0.5mL) was heated in a sealed tube for five days at
120ꢁC, and then evaporated. The residue was chromato-
graphed on a silica gel column (5.0g, 100:1 ! 20:1
CHCl3/MeOH) to give the protected amine DL-14e
(55mg, 72%). It was treated with 80% aqueous acetic
acid (1.0mL) for 3h at 80ꢁC, and evaporated. The prod-
uct was purified on a column of Dowex-50W · 2 (H+)
resin (1.0g, 1% aqueous NH3) to give the amine DL-5e
(10mg, 50%) as a white powder, TLC: Rf 0.60 (5:1
24
1
MeOH); ½aꢂ +21 (c 1.4, MeOH); H NMR (300MHz,
D
CD3OD): d 3.80 (br s, 1H, H-4), 3.59 (dd, 1H,
J1,2 = J2,3 = 9.5Hz, H-2), 3.21 (br d, 1H, J2,3 = 9.5Hz,
H-3), 2.75 (dt, 1H, J = 7.1Hz, Jgem = 11.0Hz, NHCH2),
2.39–2.51 (m, 2H, H-1, NHCH2), 1.59–1.70 [m, 2H, H-5,
H-5a(eq)], 1.29–1.36 [m, 7H, (CH2)3CH3, H-5a(ax)],
1.04 (d, 3H, J5,6 = 6.5Hz, H-6), 0.88 (t, 3H, J = 6.6Hz,
CH2CH3); ITMS-ESI (positive mode): m/z 274 [M+H]+.
1
CHCl3/MeOH); H NMR (300MHz, CD3OD): d 7.16–
7.31 (m, 5H, Ph), 3.69 (br s, 1H, H-4), 3.50 (dd, 1H,
J2,3 = 9.6Hz, J1,2 = 9.7Hz, H-2), 3.30 (br d, 1H,
J2,3 = 9.6Hz, H-3), 2.82–3.06 [m, 4H, NH(CH2)2], 2.53
4.9. N-Decyl-5a-carba-b-L-fucopyranosylamine (L-5c)
[ddd, 1H, J1,5a(eq) = 2.2Hz, J1,2 = 9.7Hz, J1,5a(ax)
=
A mixture of the epoxide L-13 (50mg, 0.22mmol), decyl-
amine (42.9lL, 0.22mmol), and 2-propanol (0.5mL)
was heated in a sealed tube for eight days at 120ꢁC,
and then evaporated. The residue was chromatographed
on a silica gel column (5.0g, 100:1 ! 20:1 CHCl3/
MeOH) to give the protected amine L-14c (53mg,
88%). It was treated with 80% aqueous acetic acid
(1.0mL) for 3h at 80ꢁC, and evaporated. The product
was purified on a column of Dowex-50W · 2 (H+) resin
(1.0g, 1% aqueous NH3) to give the amine L-5c (24mg,
11.9Hz, H-1], 1.59–1.68 [m, 2H, H-5, H-5a(eq)], 1.39
[m, 1H, H-5a(ax)], 1.02 (d, 3H, J5,6 = 6.6Hz, H-6);
ITMS-ESI (positive mode): m/z 266 [M+H]+.
4.12. N-(4-Phenylbutyl)-5a-carba-b-L-fucopyranosyl-
amine (L-5f)
A mixture of the epoxide L-13 (50mg, 0.22mmol), 4-
phenylbutylamine (34.5lL, 0.22mmol), and 2-propanol
(0.5mL) was heated in a sealed tube for six days at
120ꢁC, and then evaporated. The residue was chromato-
graphed on a silica gel column (5.0g, 100:1 ! 20:1
CHCl3/MeOH) to give the protected amine L-14f
(55mg, 66%). It was treated with 80% aqueous acetic
acid (1.0mL) for 3h at 80ꢁC, and evaporated. The prod-
uct was purified on a column of Dowex-50W · 2 (H+)
resin (1.0g, 1% aqueous NH3) to give the amine L-5f
(10mg, 54%) as a white powder, TLC: Rf 0.60 (5:1
ꢀ100%) as a white powder, TLC: Rf 0.60 (5:1 CHCl3/
24
D
MeOH), ½aꢂ
+1.4 (c 0.8, MeOH); 1H NMR
(300MHz, CD3OD): d 3.61 (br s, 1H, H-4), 3.45 (dd,
1H, J2,3 = 9.3Hz, J1,2 = 9.5Hz, H-2), 3.21 (br d, 1H,
J2,3 = 9.3Hz, H-3), 2.73 (dt, 1H, J = 7.3Hz,
Jgem = 11.5Hz, NHCH2), 2.46–2.60 (m, 2H, H-1,
NHCH2), 1.45–1.57 [m, 4H, NHCH2CH2, H-5, H-
5a(eq)], 1.21–1.34 [m, 15H, (CH2)7CH3, H-5a(ax)],
1.05 (d, 3H, J5,6 = 6.3Hz, Me); ITMS-ESI (positive
mode): m/z 302 [M+H]+.
24
D
1
CHCl3/MeOH); ½aꢂ +0.4 (c 1.5, CD3OD); H NMR
(300MHz, CDCl3): d 7.04–7.17 (m, 5H, Ph), 3.60 (br
s, 1H, H-4), 3.38 (dd, 1H, J1,2 = 9.3Hz, J2,3 = 9.5Hz,
H-2), 3.18 (br d, 1H, J2,3 = 9.5Hz, H-3), 2.29–2.69 [m,
5H, NH(CH2)2, H-1], 1.45–1.62 [m, 6H, (CH2)2Ph,
H-5, H-5a(eq)], 1.24 [br dd, 1H, J1,5a(ax) = 8.7Hz,
J5agem = 12.6Hz, H-5a(ax)], 0.92 (d, 3H, J5,6 = 6.5Hz,
H-6); ITMS-ESI (positive mode): m/z 294 [M+H]+.
4.10. N-Dodecyl-5a-carba-b-L-fucopyranosylamine (L-5d)
A mixture of the epoxide L-13 (50mg, 0.22mmol), dodec-
ylamine (40.0lL, 0.22mmol), and 2-propanol (0.5mL)
was heated in a sealed tube for 10days at 120ꢁC, and

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S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
4.13. N-Butyl-6-O-benzyl-3,4-O-isopropylidene-5a-carba-
b-D-galactopyranosylamine (D-16a)
J1,5a(ax) = 9.6Hz, H-1], 1.73 [ddd, 1H, J1,5a(eq) = 3.0Hz,
J5,5a(eq) = 3.5Hz, J5agem = 12.2Hz, H-5a(eq)], 1.49–1.66
(m, 3H, H-5, H-20), 1.24–1.46 [m, 11H, H-5a(ax),
0
0
A mixture of 1,2-anhydro-6-O-benzyl-3,4-O-isopropy-
lidene-5a-carba-a-D-galactopyranose9 D-15 (53mg,
0.18mmol), butylamine (18lL, 0.18mmol), and 2-pro-
panol was heated in a sealed tube for two days at
120ꢁC, and then evaporated to dryness. The products
were chromatographed on a silica gel column (7g,
CHCl3 ! 20:1 CHCl3/MeOH) to give the amine D-16a
(25mg, 38%) as a syrup, together with D-15 (13mg,
(CH2)5CH3], 0.89 (t, 3H, J7 ;8 ¼ 6:6Hz, CH2CH3);
ITMS-ESI (positive mode): m/z 290 [M+H]+.
4.15. 1,2-Anhydro-6-O-benzyl-3,4-O-isopropylidene-5a-
carba-a-L-galactopyranose (L-15)
This compound was prepared from 3,4,6-tri-O-acetyl-
1,5-anhydro-2-deoxy-5a-carba-D-xylo-hex-1-enitol: ½aꢂ
23
D
+22 (c 0.7, CHCl3); ITMS-ESI (positive mode); m/z
24
1
25%) recovered, ½aꢂ ꢁ3.8 (c 0.26, MeOH); H NMR
D
(300MHz, CDCl3): d 7.27–7.35 (m, 5H, Ph), 4.54 (s,
2H, CH2Ph), 4.29 (dd, 1H, J4,5 = 4.2Hz, J3,4 = 4.5Hz,
H-4), 3.89 (dd, 1H, J3,4 = 4.5Hz, J2,3 = 7.6Hz, H-3),
3.47 and 3.64 (dd, each 1H, J5,6 = 7.3Hz, J6gem = 9.0Hz,
H-6a, H-6b), 3.41 (dd, 1H, J2,3 = 7.6Hz, J1,2 = 10.7Hz,
H-2), 2.68 (br s, 1H, OH), 2.46 and 2.77 (2dt, each
1H, J = 7.2Hz, Jgem = 11.1Hz, NHCH2), 2.32 [ddd,
1H, J1,5a(eq) = 3.1Hz, J1,2 = 10.7Hz, J1,5a(ax) = 11.6Hz,
293 [M+Na]+, 309 [M+K]+, according to the standard
23
D
four-step reactions,9 ½aꢂ +28 (c 0.8, CHCl3); ITMS-
APCI (positive mode): m/z 291 [M+H]+.
4.16. N-Octyl-6-O-benzyl-3,4-O-isopropylidene-5a-
carba-b-L-galactopyranosylamine (L-16b)
A mixture of the epoxide L-15 (52mg, 0.19mmol), octyl-
amine (30.2lL, 0.19mmol), and 2-propanol (0.5mL)
was heated in a sealed tube for six days at 120ꢁC, and
then evaporated to dryness. The residue was chromato-
graphed on a silica gel column (8.0g, 50:1 ! 10:1
CHCl3/MeOH) to give the amine L-16b (50mg, 63%)
H-1], 2.05 –2.17 (m, 1H, H-5), 1.97 [ddd, 1H, J1,5a(eq)
=
3.1Hz, J5,5a(eq) = 3.4Hz, J5agem = 12.5Hz, H-5a(eq)],
1.35 and 1.49 (2s, each 3H, CMe2), 1.25–1.52 (m, 4H,
H-20, H-30), 1.14 [ddd, 1H, J1,5a(ax) = 11.6Hz,
J5,5a(ax) = 12.2Hz, J5agem = 12.5Hz, H-5a(ax)], 0.91 (t,
0
0
3H, J3 ;4 ¼ 7:2Hz, CH2CH3); ITMS-ESI (positive
as a pale yellow syrup, TLC: Rf 0.58 (1:3 EtOH/toluene);
24
D
mode): m/z 364 [M+H]+.
½aꢂ +10.6 (c 0.79, CHCl3); 1H NMR (300MHz,
CDCl3): d 7.26–7.35 (m, 5H, Ph), 4.54 (br s, 1H, H-4),
4.28 (dd, 1H, J4,5 = 3.9Hz, J3,4 = 4.4Hz, H-4), 3.89–
3.93 (m, 1H, H-3), 3.62–3.67 (m, 1H, H-6a), 3.45–3.51
(m, 2H, H-2, H-6b), 2.51 and 2.82 (2dt, each 1H,
J = 4.1Hz, Jgem = 11.0Hz, NHCH2), 2.39 [br dd,
1H, J1,5a(eq) = 3.2Hz, J1,2 = 8.5Hz, H-1], 2.13 [br dd,
1H, J4,5 = J5,5a(eq) = 3.9Hz, H-5], 1.99 [br dd, 1H,
J1,5a(eq) = 3.2Hz, J5,5a(eq) = 3.9Hz, H-5a(eq)], 1.35 and
1.49 [2s, each 3H, CMe2], 1.18–1.32 [m, 11 H,
(CH2)5CH3, H-5a(ax)], 0.88 (t, 3H, J = 6.5Hz,
CH2CH3); ITMS-ESI (positive mode): m/z 420 [M+H]+.
4.14. N-Octyl-6-O-benzyl-3,4-O-isopropylidene-5a-
carba-b-D-galactopyranosylamine (D-16b) and N-octyl-
5a-carba-b-D-galactopyranosylamine (D-6b)
A mixture of the epoxide D-15 (98mg, 0.34mmol), octyl-
amine (50lL, 0.30mmol), and 2-propanol was heated in
a sealed tube for two days at 120ꢁC, and then evapo-
rated to dryness. The products were chromatographed
on a silica gel column (10g, 60:1 CHCl3/MeOH) to give
the protected amine D-16b (73mg, 51%) as a syrup and
D-15 (41mg, 42%) recovered. Compound D-16b (73mg,
0.17mmol) was treated with acetic anhydride
(0.36mL) and pyridine (0.73mL) overnight at room
temperature.
4.17. N-Decyl-6-O-benzyl-3,4-O-isopropylidene-5a-
carba-b-D-galactopyranosylamine (D-16c)
A mixture of the epoxide D-15 (54mg, 0.18mmol),
decylamine (40lL, 0.20mmol), and 2-propanol was
heated in a sealed tube for two days at 120ꢁC, and then
evaporated to dryness. The products were chromato-
graphed on a silica gel column (8g, CHCl3 ! 25:1
The reaction mixture was evaporated, the residue was
diluted with ethyl acetate (20mL), and the solution
was washed with 1M hydrochloric acid, saturated aque-
ous NaHCO3, and water, dried, and evaporated. The
residue was chromatographed on silica gel (9g, 2:3
EtOAc/hexane) to give the N,O-acetyl derivative
(70mg, 82%) of D-16b.
CHCl3/MeOH) to give the amine D-16c (49mg, 60%)
20
as a syrup, ½aꢂ ꢁ4.2 (c 0.85, CHCl3); 1H NMR
D
(300MHz, CDCl3): d 7.30–7.35 (m, 5H, Ph), 4.54 (s,
2H, CH2Ph), 4.29 (dd, 1H, J4,5 = 4.2Hz, J3,4 = 4.5Hz,
H-4), 3.90 (dd, 1H, J3,4 = 4.5Hz, J2,3 = 7.7Hz, H-3),
3.47 and 3.64 (dd, each 1H, J5,6 = 7.5Hz, J6gem = 8.5Hz,
H-6a, H-6b), 3.44 (dd, 1H, J2,3 = 7.7Hz, J1,2 = 10.4Hz,
H-2), 3.29 (br s, 1H, OH), 2.48 and 2.78 (2dt, each
1H, J = 7.3Hz, Jgem = 11.2Hz, NHCH2), 2.35 [ddd,
1H, J1,5a(eq) = 2.9Hz, J1,2 = 10.4Hz, J1,5a(ax) = 11.4Hz,
H-1], 2.05–2.18 (m, 1H, H-5), 1.98 [ddd, 1H, J1,5a(eq)
=2.9Hz, J5,5a(eq) = 3.4Hz, J5agem = 12.5Hz, H-5a(eq)],
1.35 and 1.49 (2s, each 3H, CMe2), 1.10–1.52 [m,
Hydrolysis of the N,O-acetyl derivative (70mg,
0.14mmol) with 1M hydrochloric acid for 6h at
100ꢁC gave, after chromatography on Dowex-50W · 2
(H+) resin with 1% methanolic ammonia as eluent, the
24
D
amine D-6b (38mg, 71%) as a white powder, ½aꢂ ꢁ36
1
(c 0.44, MeOH); H NMR (300MHz, CD3OD): d 3.97
(br s, 1H, H-4), 3.63 (dd, 1H, J1,2 = 7.3Hz, J2,3
10.7Hz, H-2), 3.50 (dd, 1H, J3,4 = 6.3Hz, J2,3
=
=
10.7Hz, H-3), 3.28 and 3.51 (2dd, each 1H,
0
0
J5,6 = 2.7Hz, J6gem = 9.5Hz, H-6a, H-6b), 2.47 and
0
17H, H-5a(ax), H-20, H-90], 0.88 (t, 3H, J9 ;10
¼
0
2.71 (2dt, each 1H, J1 ;2 ¼ 7:5Hz, Jgem = 11.1Hz,
6:5Hz, CH2CH3); ITMS-ESI (positive mode): m/z 448
NHCH2), 2.41 [br dd, 1H, J1,2 = 7.3Hz,
[M+H]+.

S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
6577
4.18. N-Dodecyl-6-O-benzyl-3,4-O-isopropylidene-5a-
carba-b-D-galactopyranosylamine (D-16d)
gel column (3g, 50:1 ! 10:1 CHCl3/MeOH) to give the
24
amine L-6b (14.5mg, 62%) as a pale yellow syrup,
TLC: Rf 0.28 (1:2 EtOH/toluene); ½aꢂ +37 (c 0.89,
D
A mixture of the epoxide D-15 (48mg, 0.17mmol), dode-
cylamine (31mg, 0.17mmol), and 2-propanol was
heated in a sealed tube for two days at 120ꢁC, and then
evaporated to dryness. The products were chromato-
graphed on a silica gel column (8g, CHCl3 ! 25:1
CHCl3); 1H NMR (300MHz, CDCl3): d 3.97 (br s,
1H, H-4), 3.64 (dd, 1H, J5,6a = 7.4Hz, Jgem = 10.4Hz,
H-6a), 3.47–3.55 (m, 2H, H-3, H-6b), 3.27–3.34 (m,
1H, H-2), 2.72 (dt, 1H, J = 3.9Hz, Jgem = 11.3Hz,
NHCH2), 2.39–2.53 (m, 2H, H-1, NHCH2), 1.71–1.78
(m, 1H, H-5), 1.47–1.63 [m, 3H, NHCH2CH2, H-
5a(eq)], 1.32 [m, 11H, (CH2)5CH3, H-5a(ax)], 0.89 (t,
3H, J = 6.4Hz, CH2CH3); ITMS-APCI (positive mode):
m/z 290 [M+H]+.
CHCl3/MeOH) to give the amine D-16d (58mg, 74%)
24
as a syrup, ½aꢂ ꢁ4.1 (c 0.33, MeOH); 1H NMR
D
(300MHz, CDCl3): d 7.29–7.35 (m, 5H, Ph), 4.54 (s,
2H, CH2Ph), 4.29 (dd, 1H, J4,5 = 4.2Hz, J3,4 = 4.5Hz,
H-4), 3.90 (dd, 1H, J3,4 = 4.5Hz, J2,3 = 7.6Hz, H-3),
3.47 and 3.64 (dd, each 1H, J5,6 = 7.2Hz, J6gem = 9.0Hz,
H-6a, H-6b), 3.38 (dd, 1H, J2,3 = 7.6Hz, J1,2 = 10.7Hz,
H-2), 2.22 (br s, 1H, OH), 2.45 and 2.76 (2dt, each
1H, J = 7.2Hz, Jgem = 11.1Hz, NHCH2), 2.30 [ddd,
1H, J1,5a(eq) = 3.1Hz, J1,2 = 10.7Hz, J1,5a(ax) 12.2Hz,
H-1], 2.05–2.17 (m, 1H, H-5), 1.98 [ddd, 1H,
J1,5a(eq) = 3.1Hz, J5,5a(eq) = 3.4Hz, J5agem = 12.5Hz, H-
5a(eq)], 1.44–1.46 (m, 2H, H-20), 1.35 and 1.49 (2s, each
3H, CMe2), 1.25 [br m, 18H, (CH2)9CH3], 1.12 [ddd,
4.21. N-Decyl-5a-carba-b-D-galactopyranosylamine
(D-6c)
A mixture of D-16c (38mg, 86lmol) and 80% aqueous
acetic acid (2mL) was stirred for 4h at 80ꢁC, and then
evaporated to dryness. The residue was hydrogenated
in ethanol (2mL) in the presence of 10% Pd/C under
atmospheric pressure of hydrogen for 3h at room tem-
perature. A catalyst was removed by filtration, and the
filtrate was evaporated. The residual product was chro-
matographed on a column of Dowex-50W · 2 (H+) resin
1H, J1,5a(ax) = 12.2Hz, J5,5a(ax) = 12.2Hz, J5agem
0
=
¼
0
12.5Hz,
6:7Hz, CH2CH3); ITMS-ESI (positive mode): m/z 476
H-5a(ax)],
0.88
(t,
3H,
J11 ;12
(2.7g, 1% methanolic NH3) to give the amine D-6c
24
[M+H]+.
(20mg, 73%) as a white powder, ½aꢂ ꢀ0 (c 0.28,
D
MeOH); 1H NMR (300MHz, CD3OD): d 3.97 (br,
1H, H-4), 3.52 (dd, 1H, J1,2 = 9.0Hz, J2,3 = 9.2Hz, H-
2), 3.50 and 3.64 (dd, each 1H, J5,6 = 7.2Hz,
J6gem = 10.6Hz, H-6), 3.29 (dd, 1H, J3,4 = 2.8Hz,
J2,3 = 9.2Hz, H-3), 2.48 and 2.72 (2dt, each 1H, J =
7.1Hz, Jgem = 13.3Hz, NHCH2), 2.43 [ddd, 1H,
J1,5a(eq) = 3.1Hz, J1,2 = 9.0Hz, J1,5a(ax) = 11.2Hz, H-1],
1.74 [ddd, 1H, J1,5a(eq) = 3.1Hz, J5,5a(eq) = 3.7Hz,
J5agem = 12.2Hz, H-5a(eq)], 1.57–1.67 (m, 1H, H-5),
4.19. N-Butyl-5a-carba-b-D-galactopyranosylamine
(D-6a)
A mixture of D-16a (14mg, 40lmol) and 80% aqueous
acetic acid (2mL) was stirred for 16h at 80ꢁC, and then
evaporated to dryness. The residue was hydrogenated in
ethanol (2mL) in the presence of 10% Pd/C under
atmospheric pressure of hydrogen for 3h at room tem-
perature. A catalyst was removed by filtration, and the
filtrate was evaporated. The residual product was chro-
matographed on a column of Dowex-50W · 2 (H+) resin
1.52 (br t, 1H, J2 ;3 ¼ 6:2Hz, H-20), 1.24–1.37 [m,
0
0
15H,
0
(CH2)7CH3,
H-5a(ax)],
0.89
(t,
3H,
0
J9 ;10 ¼ 6:6Hz, CH2CH3); ITMS-ESI (positive mode):
(0.9g, 1% methanolic NH3) to give the amine D-6a
m/z 318 [M+H]+.
20
(8.6mg, 93%) as a white powder, ½aꢂ +20 (c 0.42,
D
MeOH); 1H NMR (300MHz, CD3OD): d 3.97 (br,
1H, H-4), 3.63 (dd, 1H, J1,2 = 7.3Hz, J2,3 = 10.7Hz,
H-2), 3.50 (dd, 1H, J3,4 = 6.3Hz, J2,3 = 10.7Hz, H-3),
3.28 and 3.51 (dd, 1H, J5,6 = 2.7Hz, J6gem = 9.5Hz, H-
6a, H-6b), 2.47 and 2.71 (2dt, each 1H, J = 7.5Hz,
4.22. N-Dodecyl-5a-carba-b-D-galactopyranosylamine
(D-6d)
A mixture of D-16d (49mg, 0.12mmol) and 80% aque-
ous acetic acid (2mL) was stirred for 16h at 80ꢁC,
and then evaporated to dryness. The residue was hydro-
genated in ethanol (2mL) in the presence of 10% Pd/C
under atmospheric pressure of hydrogen for 3h at room
temperature. A catalyst was removed by filtration, and
the filtrate was evaporated. The residual product was
chromatographed on a column of Dowex-50W · 2
Jgem = 11.1Hz, NHCH2), 2.41 [br dd, 1H, J1,2
=
7.3Hz, J1,5a(ax) = 9.6Hz, H-1], 1.73 [ddd, 1H,
J1,5a(eq) = 3.0Hz, J5,5a(eq) = 3.5Hz, J5agem = 12.2Hz,
H-5a(eq)], 1.49–1.66 (m, 3H, H-5, H-20), 1.24–1.46 [m,
0
0
11H, H-5a(ax), (CH2)5CH3], 0.89 (t, 3H, J7 ;8 ¼ 6:6Hz,
CH2CH3); ITMS-ESI (positive mode): m/z 234
[M+H]+.
(H+) resin (3.6g, 1% methanolic NH3) to give the
24
amine D-6d (25mg, 69%) as a white powder, ½aꢂ ꢁ45
D
4.20. N-Octyl-5a-carba-b-L-galactopyranosylamine
(L-6b)
(c 0.17, MeOH); 1H NMR (300MHz, CD3OD): d
3.97 (br, 1H, H-4), 3.47–3.55 (m, 2H, H-2, H-6), 3.64
(dd, 1H, J5,6 = 7.2Hz, J6gem = 10.6Hz, H-6), 3.29 (dd,
1H, J3,4 = 2.8Hz, J2,3 = 9.5Hz, H-3), 2.48 and 2.72
(2dt, each 1H, J = 7.4Hz, Jgem = 11.4Hz, NHCH2),
A mixture of L-16b (52mg, 0.19mmol) and 80% aqueous
acetic acid (1.0mL) was stirred for 2h at 80ꢁC, and then
evaporated to dryness. The residue was dissolved in eth-
anol (1.0mL) and the solution was hydrogenated in the
presence of a catalytic amount of 10% Pd–C in the
atmospheric pressure of hydrogen for 2h at room tem-
perature. The product was chromatographed on a silica
2.42 [ddd, 1H, J1,5a(eq) = 3.1Hz, J1,2 = 9.5Hz, J1,5a(ax)
=
9.8Hz, H-1], 1.74 [ddd, 1H, J1,5a(eq) = 3.1Hz,
J5,5a(eq) = 3.5Hz, J5agem = 12.2Hz, H-5a(eq)], 1.57–1.67
(m, 1H, H-5), 1.52 (br t, 2H, J2 ;3 ¼ 6:2Hz, H-20),
0
0
1.28–1.36 [m, 19H, (CH2)9CH3, H-5a(ax)], 0.89 (t, 3H,

6578
S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
0
0
J11 ;12 ¼ 6:7Hz, CH2CH3); ITMS-ESI (positive mode):
1H NMR (300MHz, CD3OD): d 5.38 (br s, 1H, H-1),
4.44 (br d, 1H, H-5), 4.09 (br d, 1H, H-6a), 3.82 (br s,
1H, H-3), 3.61–3.67 (m, 2H, H-6b, H-40), 3.52
(br s, 1H, H-2), 3.17–3.35 (m, 2H, H-20, H-30), 2.66
(br s, 1H, H-4), 2.46–2.54 (m, 1H, H-10), 1.51–1.74 (m,
2H, H-50, H-5a0eq), 1.05 (dd, 1H, Jgem = 13.4Hz,
m/z 346 [M+H]+.
4.23. 3,4-O-Cyclohexylidene-5a-carba-b-L-fucopyranosyl
azide (L-17)
A mixture of the epoxide L-13 (152mg, 0.66mmol) and
sodium azide (650mg, 25molar equiv) in DMF
(3.0mL) was stirred for two days at 110ꢁC. The reaction
mixture was then diluted with ethyl acetate (60mL), and
the solution was washed with saline and water, dried,
and evaporated to dryness. The residue was chromato-
graphed on silica gel (18g, 1:6 ethyl acetate/hexane) to
J5;5a ax ¼ 12:0Hz, H-5a0ax), 0.82 (d, 3H, J5,6 = 6.6Hz,
0
H-6); ITMS-APCI (positive mode): m/z 331 [M+H]+.
Compound 19 (11.4mg) was converted into the acetyl
derivative by treatment with acetic anhydride (0.5mL)
and pyridine (1.0mL) for 10h at room temperature.
The product was chromatographed on a silica gel
(1.0g, 1:7 acetone/hexane) to give the tetra-O-acetyl
give L-17 (174mg, 98%) as a solid, TLC: Rf 0.58 (1:3
24
ethyl acetate/hexane); ½aꢂ ꢁ3.1 (c 0.39, MeOH); 1H
1
derivative 20 (13.5mg, 78%) as a colorless syrup: H
NMR (300MHz, CD3OD): d 5.44 (br s, 1H, H-1),
D
NMR (300MHz, CDCl3): d 4.07 (t, 1H, J4,5 = 3.9Hz,
J3,4 = 4.7Hz, H-4), 3.89 (dd, 1H, J3,4 = 4.7Hz,
5.31 (br s, 1H, H-40), 5.19 (dd, 1H, J1 ;2 ¼ J2 ;3
¼
0
0
0
0
J2,3 = 7.4Hz, H-3), 3.51 (dd, 1H, J2,3 = 7.4Hz, J1,2
=
10:0Hz, H-20), 4.83 (br s, 1H, H-2), 4.56 (br s, 1H, H-
5), 4.09 (br d, 1H, Jgem = ꢀ7.0Hz, H-6a), 3.82 (br dd,
1H, H-6b), 3.51 (br s, 1H, H-3), 3.02–3.10 (m, 1H, H-
10), 2.71 (br s, 1H, H-4), 2.14, 2.12, 2.09, and 1.97 (4s,
each 3H, 4 · Ac), 1.75–1.82 [m, 2H, H-5, H-5a(eq)],
1.48–1.55 [m, 1H, H-5a(ax)], 0.93 (d, 3H, J5,6 = 6.6Hz,
H-6); ITMS-APCI (positive mode): m/z 499 [M+H]+.
10.3Hz, H-2), 3.25 [ddd, 1H, J1,5a(eq) = 4.0Hz,
J1,5a(ax) = 8.0Hz, J1,2 = 10.3Hz, H-1], 2.78 (s, 1H, OH),
2.14 (s, 3H, Ac), 1.93 [dddd, 1H, J5,5a(eq) = 3.4Hz,
J4,5 = 3.9Hz, J5,6 = 6.8Hz, J5,5a(ax) = 9.0Hz, H-5],
1.37–1.96 [m, 12H, C6H10, 2 · H-5a], 1.15 (d, 3H,
J5,6 = 6.8Hz, Me); ITMS-APCI (positive mode): m/z
268 [M+H]+.
4.26. N-(5a-Carba-b-L-fucopyranos-1-yl)-3,4-O-cyclo-
hexylidene-5a-carba-b-L-fucopyranosylamine (21)
Data for the 2-O-acetyl derivative: TLC: Rf 0.40 (1:5
ethyl acetate/hexane); H NMR (300MHz, CDCl3): d
5.01 (dd, 1H, J2,3 = 7.8Hz, J1,2 = 10.7Hz, H-2), 4.08
1
A mixture of L-3 (13.2mg, 0.08mmol) and L-13
(28.2mg, 0.12mmol) in 2-propanol (1mL) was heated
in a sealed tube for three weeks at 120ꢁC, and then evap-
orated to dryness. The residual product was eluted from
a column of silica gel (1.5g, 1:10 MeOH/CHCl3) to give
21 (12.9mg, 40%) as a colorless syrup: 1H NMR
(300MHz, CD3OD): d 3.99–4.02 (m, 1H, H-40), 3.73–
(dd, 1H, J3,4 = J4,5 = 4.0Hz, H-4), 3.96 (dd, 1H, J3,4
4.0Hz, J2,3 = 7.8Hz, H-3), 3.28 [ddd, 1H, J1,5a(eq)
=
=
3.9Hz, J1,5a(ax) = 8.0Hz, J1,2 = 10.7Hz, H-1], 2.14 (s,
3H, Ac), 1.79 [ddd, 1H, J1,5a(eq) = J5,5a(eq) = 4.0Hz,
J1,2 = 10.7Hz, H-5a(eq)], 1.63 [m, 2H, H-5, H-5a(ax)],
1.19–2.01 (m, 10H, C6H10), 1.17 (d, 3H, J5,6 = 6.3Hz,
Me).
3.78 (br d, 1H, J3 ;4 ¼ 4:4Hz, H-30), 3.60 (br s, 1H,
H-4), 3.20–3.37 (m, 3H, H-2, H-3, H-20), 2.37–2.48 (m,
2H, H-1, H-10), 1.79–1.85 (m, 1H, H-50), 1.26–1.68
[m, 13H, C6H10, H-5, H-5a(eq), H-5a0eq], 1.16–1.22
(m, 1H, H-5a0ax), 1.12 [br d, 1H, J5,5a(ax) = 4.1Hz,
0
0
4.24. 5a-Carba-b-L-fucopyranosylamine (L-3)
A solution of L-17 (250mg, 0.94mmol) in ethanol (5mL)
was hydrogenolyzed in the presence of a catalytic
amount of 10% Pd/C for 4h at room temperature. The
product was O-decyclohexylidenated similarly to give,
H-5a(ax)], 1.01 (d, 3H, J5 ;6 ¼ 6:8Hz, H-60), 0.92 (d,
0
0
3H, J5,6 = 6.6Hz, H-6).
after purification by acid resin column to give the
4.27. N-(5a-Carba-b-L-fucopyranosyl)-5a-carba-b-L-
fucopyranosylamine (22)
24
D
amine L-3 (109mg, 72%) as a white powder, ½aꢂ ꢁ2.2
(c 0.29, MeOH); 1H NMR (300MHz, D2O): d 3.77
(br s, 1H, H-4), 3.40 (m, 2H, H-2, H-3), 2.78 (m, 1H,
Compound 21 (42mg, 0.11mmol) was treated with 80%
aqueous acetic acid (0.5mL) for 5h at 80ꢁC, and then
co-evaporated with ethanol and toluene. The residue
was chromatographed on a column of Dowex-50W · 2
(H+) (1.0g, 1% aqueous NH3) to give 22 (12.2mg,
H-1), 1.71 (m, 1H, H-5), 1.62 [ddd, 1 H, J1,5a(eq)
=
J5,5a(eq) = 4.0Hz, J5agem = 12.7Hz, H-5a(eq)], 1.30 [ddd,
1H, J1,5a(ax) = J5,5a(ax) = J5agem = 12.7Hz, H-5a(ax)],
0.92 (d, 3H, J5,6 = 6.8Hz, H-6); ITMS-APCI (positive
mode): m/z 162 [M+H]+.
24
1
ꢀ100%) as a white powder: ½aꢂ +16 (c 0.38, MeOH); H
D
NMR (300MHz, CD3OD): d 3.68 (br s, 2H, H-4,
H-40), 3.46 (dd, 2H, J1;2 ¼ J1 ;2 ¼ 9:3Hz, J2;3
¼
0
0
4.25. 1,6-Anhydro-2-azido-4-(5a-carba-b-L-fucopyranos-
1-yl)amino-2,4-dideoxy-b-D-glucopyranose (19)
J2 ;3 ¼ 9:7Hz, H-2, H-20), 3.32 (dd, 2H, J3;4
¼
0
0
J3 ;4 ¼ 2:5Hz, J2;3 ¼ J2 ;3 ¼ 9:7Hz, H-3, H-30), 2.80
0
0
0
0
0
0
0
A mixture of L-3 (11.8mg, 0.07mmol) and 1,6:3,4-dian-
hydro-2-azido-2-deoxy-b-D-galactopyranose12 (18, 18.6
mg, 0.11mmol) in 2-propanol (1mL) was heated for
two weeks at 120ꢁC, and then evaporated to dryness.
The residue was chromatographed on a silica gel
(ddd, 2H, J1;5aðeqÞ ¼ J1 ;5a0eq ¼ 3:9Hz, J1;2 ¼ J1 ;2
¼
9:3Hz, J1;5aðaxÞ ¼ J1 ;5a0ax ¼ 12:2Hz, H-1, H-10), 1.57–
0
1.69 [m, 4H, H-5, H-50, H-5a(eq), H-5a0eq], 1.22 [br
0
dd, 2H, J1;5aðaxÞ ¼ J1 ;5a0ax ¼ 12:2Hz, J5aðaxÞ;5aðeqÞ
¼
J5a ax;5a eq ¼ 12:4Hz, H-5a(ax), H-5a0ax], 0.84 (d, 6H,
0
0
J5;6 ¼ J5 ;6 ¼ 6:6Hz, H-6, H-60); ITMS-ESI (positive
0
0
(2.0g, 1:10 MeOH/CHCl3) to give the amine 19
24
D
(22.5mg, 93%) as a syrup: ½aꢂ ꢁ37 (c 0.18, MeOH);
mode): m/z 306 [M+H]+.

S. Ogawa et al. / Bioorg. Med. Chem. 12 (2004) 6569–6579
6579
4.28. Biological assay
providing us with a sample of calystegine, Prof.
Yoshiyuki Suzuki (International University of Health
and Welfare, Otawara, Japan) for helpful discussions,
and Ms. Miki Kanto for her assistance in preparing this
manuscript.
(a) Determination of IC50: the IC50 values were deter-
mined for all inhibitors and correspond to the inhibition
concentration required for 50% inhibition of the enzyme
in our experimental conditions. a-Fucosidase (bovine
kidney) was assayed using p-nitrophenyl a-L-fucopyr-
anoside (4mM) as a substrate at pH5.5 (30mM
CH3COONa) at 37ꢁC. a-Galactosidase (green coffee
beans) was assayed using p-nitrophenyl a-D-galactopyr-
anoside as a substrate at pH6.8 (20mM, phosphate buf-
fer) at 27ꢁC. b-Galactosidase (bovine liver) assayed
similarly using p-nitrophenyl b-galactopyranoside at
37ꢁC. a-Glucosidase (Bakerꢀs yeast) was assayed simi-
larly using p-nitrophenyl a-D-glucopyranoside (4mM)
as a substrate at pH5.5 (30mM CH3COONa) at 27ꢁC.
b-Glucosidase (almond) was assayed similarly using p-
nitrophenyl b-D-glucopyranoside (4mM) as a substrate
at pH5.5 (30mM CH3COONa) at 27ꢁC. b-Glucos-
aminidase (bovine kidney) was assayed using p-nitro-
phenyl N-acetyl-b-D-glucosaminide (4mM) as a substrate
at pH5.5 (30mM CH3COONa) at 37ꢁC. a-Mannosi-
dase was assayed using p-nitrophenyl a-D-mannopyr-
anoside (4mM) as a substrate at pH5.5 (30mM
CH3COONa) at 27ꢁC.
References and notes
1. Ogawa, S.; Mori, M.; Takeuchi, G.; Doi, F.; Watanabe,
M.; Sakata, Y. Bioorg. Med. Chem. Lett. 2002, 811.
2. Ogawa, S.; Sekura, R.; Maruyama, A.; Yuasa, H.;
Hashimoto, H. Eur. J. Org. Chem. 2002, 2089.
3. Ogawa, S.; Watanabe, M.; Maruyama, A.; Hisamatsu, S.
Bioorg. Med. Chem. Lett. 2002, 12, 749.
4. Takeuchi, M.; Kamata, K.; Yoshida, M.; Kameda, Y.;
Matsui, K. J. Biochem. 1990, 108, 42; Kameda, Y.; Takai,
N.; Asano, N.; Matsui, K. Chem. Pharm. Bull. 1990, 38,
1970.
5. Ogawa, S.; Fujieda, S.; Sakata, Y.; Ishizaki, M.; Hisa-
matsu, S.; Okazaki, K. Bioorg. Med. Chem. Lett. 2003, 13,
3461.
6. Ogawa, S.; Uematsu, Y.; Yoshida, S.; Sasaki, N.; Suami,
T. J. Carbohydr. Chem. 1987, 6, 471.
7. Other than compound L-12, the tri-O-benzyl and 1,2-O-
isopropylidene derivatives were subjected to conventional
conditions for epoxidation. Exclusive selectivity was
obtained for L-12.
Test compound was added to each reaction mixture de-
scribed above and it was incubated for 1h at 27 or 37ꢁC.
After 1h, the reaction was quenched by addition of three
volumes of aqueous 0.2M sodium carbonate, and the
absorbance of the liberated p-nitrophenol was measured
at 410nm. The percentage inhibition was calculated by
the formula BA · 100, where A is the p-nitrophenol lib-
erated by the enzyme without an inhibitor and B is that
with an inhibitor.
8. Unless otherwise noted, when both racemic and optically
active compounds were subjected to reaction, the process-
ing for the latter were reported.
9. Tsunoda, H.; Ogawa, S. Liebigs Ann. Chem. 1994,
103.
10. Ogawa, S.; Hirai, K.; Odagiri, T.; Matsunaga, N.;
Yamazaki, T.; Nakajima, A. Eur. J. Org. Chem. 1998,
1099.
11. Ogawa, S.; Sekura, R.; Maruyama, A.; Odagiri, T.; Yuasa,
H.; Hashimoto, H. Carbohydr. Lett. 2000, 4, 13.
12. (a) Paulsen, H.; Koebernick, H. Chem. Ber. 1976, 109, 104;
(b) Ogawa, S.; Aso, D. Carbohydr. Res. 1993, 250,
177.
13. All biological assays were carried out in a standard
manner by Dr. Akihiro Tomoda (Hokko Chemical
Industry, Co. Ltd).
14. Greul, J. N.; Kleban, M.; Schneider, B.; Picasso, S.; Ja¨ger,
V. ChemBioChem 2001, 2, 368, and references cited
therein.
15. Dickson, G. L.; Leroy, E.; Reymond, J.-L. Org. Biomol.
Chem. 2004, 2, 1217, and references cited therein.
16. (a) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup,
T.; Lundt, I.; Bols, M. Angew. Chem., Int. Ed. Engl. 1994,
33, 1778; (b) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.;
Suhara, Y. J. Am. Chem. Soc. 1998, 120, 3007.
17. Asano, N.; Kato, A.; Oseki, K.; Kizu, H.; Matsui, K. Eur.
J. Biochem. 1995, 229, 369.
(b) Determination of Ki: the Ki values were determined
for especially potent inhibitors using Dixon graphical
method. The effects of pH on the inhibition were investi-
gated for DL-5e and reference inhibitor calystegin C,17 at
pH5.5 and 6.8, in order to compare their Ki values with
those reported for isofagomine.16 The following sub-
strate concentrations were applied: 4, 2, 1, and 0.5mM,
and the inhibitor concentrations: 0.25, 0.1, 0.05, and
0.025lgmLꢁ1 or 0.025, 0.01, 0.005, and 0.0025lgmLꢁ1
.
Acknowledgements
The authors would like to thank Drs. A. Takahashi
and A. Tomoda (Hokko Chemical Industry, Co. Ltd,
Atsugi, Japan) for the biological assays, Prof. Naoki
Asano (Hokuriku University, Kanazawa, Japan) for
18. Ogawa, S.; Funayama, S.; Okazaki, K.; Ishizuka, H.;
Sakata, Y.; Doi, F. Bioorg. Med. Chem. Lett., in press.