gem-Diamine 1-N-iminosugars of L-fucose-type, the extremely potent L- fucosidase inhibitors

Article abstract of DOI:10.1016/S0968-0896(99)00289-8

An efficient route from D-ribono-γ-lactone to gem-diamine 1-N- iminosugars of L-fucose-type, a new family of glycosidase inhibitor, has been developed in a formation of a gem-diamine 1-N-iminopyranose ring by the Mitsunobu reaction of an aminal as a key step. The analogues were proved to be the extremely potent inhibitors against α-L-fucosidase (IC₅₀ ~3 ng mL^-1, K(i) ~5 x 10^-9 M). The present study has shown that a cyclic methanediamine generated in media affects glycosidases as a real active-form of the gem-diamine 1-N-iminosugars of L-fucose-type. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00289-8

Bioorganic & Medicinal Chemistry 8 (2000) 343±352
gem-Diamine 1-N-Iminosugars of L-Fucose-type, the Extremely
Potent L-Fucosidase Inhibitors
Eiki Shitara, Yoshio Nishimura,* Fukiko Kojima and Tomio Takeuchi
Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Received 16 July 1999; accepted 23 October 1999
AbstractÐAn ecient route from d-ribono-g-lactone to gem-diamine 1-N-iminosugars of l-fucose-type, a new family of glyco-
sidase inhibitor, has been developed in a formation of a gem-diamine 1-N-iminopyranose ring by the Mitsunobu reaction of an
aminal as a key step. The analogues were proved to be the extremely potent inhibitors against a-l-fucosidase (IC50 ꢀ3 ng mL 1, Ki
9
ꢀ5Â10 M). The present study has shown that a cyclic methanediamine generated in media a€ects glycosidases as a real active-
form of the gem-diamine 1-N-iminosugars of l-fucose-type. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
On the other hand, an a(1!3)-linked l-fucose residue
in sialyl Lewis X tetrasaccharide (sLeX, 3) expressed on
the surface of leukocyte and some kinds of tumor cells is
essential for their adhesion to the endothelial basement
membrane through cell-surface endothelial-leukocyte
adhesion molecules (ELAMs) (Fig. 2).18±21 It was also
suggested that fucosidase in invasive human ovarian
carcinoma cell mediates degradation of the sub-
endothelial extracellular matrix.22 Furthermore, N-
methyl and 5-carboxymethyl-1-pentyl derivatives (4 and
5) of 1,5-dideoxy-1,5-imino-l-fucitol (6), a-l-fucosidase
inhibitors have been shown to inhibit the cytopathic
e€ect of human immunode®ciency virus (HIV) and yield
of infectious virus (Fig. 3).23,24 These ®ndings have led
us to an intensive search for small molecules as fucosi-
dase inhibitors and as potential drug candidates for the
treatment of reperfusion injury and other in¯ammatory
disorders, HIV infection and tumor metastasis. We have
recently communicated the synthesis of novel l-fucose-
type 1-N-iminosugars, (2S,3S,4R,5R)-2-acetamido-5-
methylpiperidine-3,4-diol (7) and (2S,3S,4R,5R)-5-
methyl-2-tri¯uoroacetamidopiperidine-3,4-diol (8) (Fig.
4).25 We now report full details of the syntheses together
with the evaluation as l-fucosidase inhibitors and the
relationships between structures and inhibitory activities
for these candidates and their analogues, (2S,3S,
4R,5R)-5-methyl-2-trichloroacetamidopiperidine-3,4-diol
(9), (2S,3S,4R,5R)-5-methyl-2-phthalimidopiperidine-
3,4-diol (10), (2S,3S,4R,5S)-5-methyl-2-tri¯uoroacet-
amidopiperidine-3,4-diol (11), (2S,3S,4R)-5-methylene-
2-tri¯uoroacetamidopiperidine-3,4-diol (12), (2R,3S,4R,
5R)-2-amino-5-methylpiperidine-3,4-diol (13) and (2R,
3S,4R,5R)-2-amino-N-acetyl-5-methylpiperidine-3,4-diol
(14) (Fig. 4).
Current interest of glycosidase inhibitors has been
directed toward new tools for unlabeling how glyco-
conjugates such as glycoproteins, glycolipids and
proteoglycans regulate biological functions, and toward
new drugs for the treatment of diseases associated with
glycoconjugates biosynthesis and degradation, including
cancer, metastasis of tumors, in¯ammatory disorders,
viral and bacterial infections and so forth.1,2 Various
types of inhibitors have been designed based on the
mechanism of the enzyme-assisted hydrolysis of glyco-
sidic bonds and the structural reminiscent of natural
inhibitors.3,4 Among the e€ective glycosidase inhibitors
are pyranoses and furanoses with the ring oxygen
replaced by an imino group. In the course of our study
on glycosidase inhibitors, we proposed a new family of
glycosidase inhibitor, gem-diamine 1-N-iminosugars (1)
in which an anomeric carbon atom is replaced by a
nitrogen.5,6 We considered that the protonated gem-
diamine 1-N-iminosugars may mimic the putative gly-
cosyl cation intermediate 2 formed during enzymatic
glycosidic hydrolysis (Fig. 1).7±11 They have shown
highly potent and speci®c inhibition against glyco-
sidases, and some of them have also shown potent
suppression of experimental and spontaneous lung
metastasis of tumor cells in mice.5,6,12±17
Keywords: gem-diamine 1-N-iminosugar; l-fucosidase inhibitor;
(2S,3S,4R,5R)-2-tri¯uoroacetamido-5-methylpiperidine-3,4-dial; cyclic
methanediamine.
*Corresponding author. Tel.: +81-3-3441-4173 ext. 838; fax: +81-3-
3441-7589; e-mail: mcrf@bikaken.or.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00289-8

344
E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
Figure 3. 1,5-Dideoxy-1,5-imino-l-fucitol (6) and analogues.
Figure 1. gem-Diamine 1-N-iminosugar (1) and glycopyranosyl cation
(2).
Results and Discussion
Synthesis
We have recently developed an ecient method of
synthesis of multifunctionalized gem-diamine 1-N-imio-
sugars.3 This methodology was employed conveniently
for this synthesis. The synthesis of the pivotal inter-
mediate, aminal 23 began with the known lactam 1626
which was converted into the diol 18 upon sodium boro-
hydride reduction and removal of the protecting group
in good yield. Selective protection of the hydroxymethyl
group in 18 followed by the Dess±Martin oxidation27
gave the ketone 20 in 95% yield. The Wittig reaction of
20 with methylenetriphenylphosphorane a€orded the
methylene 21 which was transformed into the mono-
alcohol 22 by removal of the protecting group in good
yield. Stereoselective introduction of the hydroxyl group
at C(2) was best achieved by the Swern oxidation28 to
give the key intermediate 23 as a sole product in 82%
yield. The stereochemistry of 23 was established by its
1H NMR spectrum. The 1H NMR spectrum of 23
shows protons of C(2), C(3) and C(4) at d 5.68 (d,
J<2 Hz), 4.41 (dd, Jꢀ2.0 and 7.3 Hz) and 4.74 (d,
J=7.3 Hz), respectively, indicative of an equatorial
hydrogen at C(2). The same stereochemical outcome
controlled by an anomeric e€ect29 as those of the pre-
vious gem-diamine 1-N-iminosugar syntheses5,6 was
observed. This stereochemistry was later con®rmed by
X-ray crystallographic analysis of the hydrogenated
compound 25. Replacement of the aminal hydroxy
group of 23 to the amino group was successfully carried
out by the Mitsunobu reaction30 (PPh3, diethyl azodi-
carboxylate, phthalimide) in DMF to yield the imi-
nophthalimide 24 in an excellent yield. Catalytic
hydrogenation of 24 with palladium on carbon in
methanol gave the desired product 25, its epimer 26 and
the rearrangement derivative 27 in 75, 5 and 18% yield,
respectively. Hydrogenation of 27 under the same con-
dition also gave eciently 25 in good yield. However,
Figure 4. gem-Diamine 1-N-iminosugars of l-fucose-type.
the prolonged reaction period in reduction of 24 was
rather inecient. Compound 25 was crystallized from a
mixture of toluene and n-hexane to yield a single crystal
for X-ray di€raction analysis. The X-ray crystallo-
graphic analysis clearly indicated the desired absolute
stereochemistry and a boat conformation (Fig. 5). On
the other hand, the stereochemistry of 26 was estimated
1
by comparison of the H NMR spectra of 26 and its
derivatives (32 and 33) with those of 25 and its deriva-
tives (28 and 30), respectively. The large coupling con-
stants (J5,6ax=11.5±12.4 Hz) and the small coupling
constants (J4,5=2.4±3.4 Hz and J2,3=1.5±2.0 Hz) of the
1H NMR spectra of 25, 28 and 30 indicate the same
conformation as the B3,7±11 boat conformation of 25
clari®ed by X-ray crystallographic analysis (Fig. 5),
while the coupling constants (J5,6ax=12.7±13.7 Hz,
1
J4,5=6.0±9.3 Hz and J2,3=1.5±2.0 Hz) of the H NMR
spectra of 26, 32 and 33 suggest their half-chair con-
formation. These results suggest that the methylene
group of 24 easily rearranges under catalytic hydro-
genation with palladium on carbon, and that hydro-
genation may take place predominantly from the less
sterically hindered side (a) of the double bond of 27 with
Figure 2. Sialyl Lewis X (3).

E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
345
Figure 5. ORTEP drawing of compound 25.
respectively. The coupling constants (J2,3=5.9, J3,4=
2.7, J4,5=7.8, J5,6=4.4 and J5,6=8.3 Hz) of the 1H
NMR spectrum of 11 suggest its skew-boat conformation.
Biological activity
The inhibitory activities of synthesized 1-N-iminosugars
(7±12) against glycosidases are summarized in Table 1.
As expected, the l-fucose-type tri¯uoroacetamide (8)
showed very strong, speci®c inhibition against a-l-
fucosidase from bovine kidney, and the acetamide 7 also
a€ected moderately the enzyme. Compound 8 was also
proved to be a competitive inhibitor by Lineweaver±
Burk plot and, the Ki value of 8 was elucidated as
Figure 6. Possible mechanism of the sterochemical outcome in cata-
lytic hydrogenation of 24.
a boat conformation to lead 25 rather than from the less
sterically hindered side (b) of the methylene group of 24
with a di€erent boat conformation to lead 26 (Fig. 6).
Hydrazinolysis of 25 a€orded the amine 28 in 99%
yield. Conventional acetylation, tri¯uoroacetylation and
trichloroacetylation furnished the acetamide 29, the tri-
¯uoroacetamide 30 and the trichloroacetamide 31 in
excellent yields, respectively. Simultaneous removal of
both the isopropylidene and t-butyloxycarbonyl groups
in 29, 30 and 31 with 4 M hydrogen chloride in dioxane
resulted in the desired l-fucose-type 2-acetamido-, 2-
tri¯uoroacetamido- and 2-trichloroacetamido-1-N-imino-
sugars 7, 8 and 9 in excellent yields, respectively. And
another l-fucose-type 2-phthalimido-1-N-iminosugar
10 was also similarly obtained from 25 in 94%. The
large coupling constants (10.3±12.5 Hz) between H-2
and H-3 and between H-5 and H-6ax in 1H NMR
9
5Â10 M by Dixon plot. On the other hand, 7 and
8 showed no signi®cant inhibition against all other
d-glycosidases. Strikingly, the l-fucose-type tri-
chloroacetamide (9) and phthalimide (10) also a€ected
very potently a-l-fucosidase equivalent to the tri-
¯uoroacetamide (8). These results suggested that the
common intermediate derived from these analogues 8, 9
and 10 in the media might a€ect the enzyme as the real
active-form. Then, we examined thoroughly the time-
dependent alteration of the structures of the analogues
1
8, 9 and 10 in H NMR spectra. As expected, 8, 9 and
10 were proved to be unstable in the conditions of the
medium of citrate-phosphate bu€er (pH 6.3, 37 ꢁC) for
l-fucosidase inhibition assay as well as acetate bu€er
(pH 5.0, 37ꢁC) for other glycosidase inhibition assays.
1
spectra of 7, 8 and 9 are clearly indicative of their C4
conformation. On the other hand, 6-deoxy-d-altrose-
type 1-N-iminosugar 11, the epimer of 8 and 5-methyl-
ene-d-arabino-hexopyranose-type 1-N-iminosugar 12,
the 5-methylene isomer of 8 were prepared by the simi-
lar sequences of reaction from 26 and 24 in good yields,
1
Their H NMR spectra were suggestive of the existence
1
of the common intermediate of the C4-conformational
methanediamine 13. Compound 8, 9 and 10 in methanol
1
and water also showed similar H NMR spectra after a

346
E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
Scheme 1. (a) NaBH4, EtOH, 0 ꢁC to rt, (b) n-Bu4NF, THF, rt, (c) t-BuMe2SiCl, imidazole, DMF, rt, (d) Dess±Martin periodinane, CH2Cl2, (e)
Ph3PCH3Br, (Me3Si)2NLi, THF, 0 ꢁC to rt, (f) n-Bu4NF, THF, rt, (g) (COCl)2, DMSO, Et3N, CH2Cl2, 78 ꢁC to rt, (h) phthalimide, Ph3P, DEAD,
.
DMF, rt, (i) H2/Pd-C, MeOH, rt, (j) H2NNH2 xH2O, MeOH, rt, (k) Ac2O, DMAP, Py, rt, (l) (CF3CO)2O, Py, CH2Cl2, rt, (m) CCl3COCl, Py,
CH2Cl2, 0 ꢁC, (n) 4 M HCl/dioxane, 0 ꢁC to rt, (o) MeOH, 50 ꢁC, (p) Ac2O, Py, MeOH, rt.
few days, indicative of the presence of the same metha-
nediamine 13. Next we undertook the isolation of the
common intermediate. After treatment of 8 in methanol
at 50 ꢁC for 13 h, evaporation of the solvent gave the
(C-2), supportive of the cyclic methanediamine. Fur-
thermore, conventional N-acetylation (acetic anhydride,
pyridine, CH3OH) of 13 a€orded the acetamide 14 dif-
ferent from 7 as a sole product. The structure of 14 was
established by the H NMR, IR and mass spectra, and
the coupling constants of 1H NMR spectrum (J2,3
=
J3,4=3.7, J5,6eq=4.4 and J5,6ax=13.3 Hz) also suggested
the boat conformation of 14. However, it is not clear at
this stage why the ring imino-group was selectively
acetylated to give 14. Interestingly, 13 showed very
1
1
pure methanediamine 13. The H NMR spectrum of 13
shows the similar coupling constants as those shown in
1
the spectrum of 8, indicating the C4 conformation of
the cyclic methanediamine. The 13C NMR spectrum of 13
also shows peaks at d 14.21 (5-CH3), 33.52 (C-5), 44.26
(C-6), 72.25 (C-3 or C-4), 72.99 (C-4 or C-3) and 88.86

E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
Table 1. Inhibitory activity of 7±14 against glycosidases
347
IC50 (M)
Enzyme
7
8
9
10
11
12
13
14
7
8
9
8
6
7
8
4
a-l-Fucosidasea
4.8Â10
(0.11) j
1.1Â10
(0.003) j
9.0Â10
1.3Â10
(0.004) j
NT
NT
NT
NT
NT
NT
NT
1.8Â10
(0.50) j
NT
NT
NT
NT
NT
NT
NT
7.0Â10
(0.20) j
NT
NT
NT
NT
NT
NT
NT
1.6Â10
(0.003) j
>2.7Â10
(>50) j
NT
(0.003) j
NTk
NT
4
5
4
4
4
4
4
4
4
5
4
4
4
4
4
4
4
4
5
4
4
4
4
4
4
4
4
a-d-Glucosidaseb
1.8Â10
4.7Â10
5.4Â10
b-d-Glucosidasec
1.0Â10
1.2Â10
1.4Â10
NT
NT
NT
NT
NT
NT
NT
NT
a-d-Mannosidased
>2.2Â10
>2.2Â10
>2.2Â10
>2.2Â10
>2.2Â10
>2.2Â10
>2.2Â10
>1.8Â10
>1.8Â10
>1.8Â10
>1.8Â10
>1.8Â10
>1.8Â10
>1.8Â10
NT
NT
NT
NT
NT
NT
NT
>5.5Â10
>5.5Â10
>5.5Â10
>5.5Â10
>5.5Â10
>5.5Â10
>5.5Â10
b-d-Mannosidasee
a-d-Galactosidasef
b-d-Galactosidasef
b-d-Glucuronidaseg
a-d-N-Acetylgalactosaminidaseh
b-d-N-Acetylglucosaminidase j
NT
NT
NT
NT
NT
NT
aBovine kidney.
bBaker's yeast.
cAlmonds.
dJack beans.
eSnail.
fEscherichia coli.
gBovine liver.
hChicken liver.
iBovine epididymis.
jIC50 (mg/mL).
kNot tested.
strong inhibition against a-l-fucosidase equivalent to 8,
9 and 10, while 14 showed no inhibition against the
enzyme. These results support the above speculation of
which the common intermediate 13 a€ect the enzyme in
the media, and seem also to support the hypothesis of
which the protonated gem-diamine 1-N-iminosugars
may mimic the presumed glycosyl cation (2) in the
transition state of enzymatic reaction as shown in
Figure 1. On the other hand, the acetamide 7 was stable
in both methanol and water, also suggestive of the weak
inhibition against l-fucosidase. Compounds 11 and 12
also inhibited weakly a-l-fucosidase. These results indi-
cate that the 5-methyl group, its stereochemistry and the
1C4-conformation play the important roles as the major
factors for inhibition against l-fucosidase.
1H NMR spectra were recorded with a Jeol GX-400
spectrometer. Chemical shifts are expressed in d values
(ppm) with tetramethylsilane (d 0.00) for CDCl3, with
CD2HOD (3.30) for CD3OD, and with HDO (d 4.65)
for D2O as an internal standard. The mass spectra were
taken by Jeol SX102 for FAB and by Hitachi M-1200H
for APCI (atmospheric pressure chemical ionization).
General procedures for enzyme inhibition assay
The enzymes, a-l-fucosidase (bovine kidney), b-glucur-
onidase (bovine liver), a-glucosidase (baker's yeast), b-
glucosidase (almond), a-mannosidase (jack beans), b-
mannosidase (snail), a-galactosidase (Escherichia coli),
b-galactosidase (E. coli), a-N-acetylgalactosaminidase
(chicken liver), and b-N-acetylglucosaminidase (bovine
epididymis) were purchased from Sigma Chemical Co.
a-l-Fucosidase was assayed using p-nitrophenyl a-l-
In summary, an ecient synthetic route involving the
formation of a gem-diamine 1-N-iminopyranose ring by
the Mitsunobu reaction of an aminal as a key step to
l-fucose-type gem-diamine 1-N-iminosugars, from a
readily available d-ribono-g-lactone has been developed
and has produced the extremely potent inhibitors of
a-l-fucosidase. The present study indicates that the
cyclic methanediamines may generally a€ect the
enzymes as the real active-forms of the glycosidase
inhibitors of the gem-diamine 1-N-iminosugars. That
these gem-diamine 1-N-iminosugars are highly potent
inhibitors of a-l-fucosidase further supports the
hypothesis of our design of the new type inhibitor.
3
fucopyranoside (1.5Â10 M) as a substrate at pH 6.3
(0.025 M citrate-phosphate bu€er). b-Glucuronidase
was assayed using phenolphthalein mono-b-glucuronic
4
acid (3.3Â10 M) as a substrate at pH 5.0 (0.1 M ace-
tate bu€er). a- and b-glucosidases were assayed using
3
p-nitrophenyl a-d-glucopyranoside (1.5Â10 M) and
3
b-d-glucopyranoside (2Â10 M) as substrates at pH
6.3 (0.025 M citrate-phosphate bu€er) and 5.0 (0.025 M
acetate bu€er), respectively. a- and b-Mannosidases
were assayed using p-nitrophenyl a-d-mannopyranoside
3
3
(2Â10 M) and b-d-mannopyranoside (2Â10 M) as
substrates at pH 4.5 (0.05 M acetate bu€er) and 4.0
(0.05 M acetate bu€er), respectively. b-Galactosidase
Experimental
was assayed using p-nitrophenyl b-d-galactopyranoside
3
(2Â10
M) at pH 4.0 (0.025 M citrate-phosphate
General methods
bu€er). a-N-Acetylgalactosaminidase was assayed using
p-nitrophenyl N-acetyl-a-d-galactosaminide (1Â10 3 M)
as a substrate at pH 4.0 (0.025 M citrate-phosphate
bu€er). b-N-Acetylglucosaminidase was assayed using
Melting points were determined with a Yamato appa-
ratus and were uncorrected. Optical rotations were
measured with a Perkin±Elmer Model 241 polarimeter.

348
E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
3
p-nitrophenyl N-acetyl-b-d-glucosaminide (1Â10 M)
at pH 4.0 (0.025 M citrate-phosphate bu€er). The reac-
tion mixture contained 0.5 mL of bu€er, 0.1 mL of
substrate solution and water or aqueous solution con-
taining the test compound. The mixture was incubated
at 37 ꢁC for 3 min, and 0.01 mL of enzyme was added.
After 0.5±1 h of reaction, 1.0 mL of 0.3 M glycine-
sodium hydroxide bu€er (pH 10.5) was added and the
absorbance of the liberated nitrophenol or pheno-
lyphthalein measured at 400 or 525 nm, respectively.
The percentage inhibition was calculated by the formula
(A B)/AÂ100, where A is the nitrophenol liberated by
the enzyme without an inhibitor and B is that with an
inhibitor. The IC50 value is the concentration of inhi-
bitor at 50% of enzyme activity.
1-(t-Butoxycarbonyl)amino-5-O-(t-butyldimethylsilyl)-1-
deoxy-3,4-O-isopropylidene-D-ribitol (19). To a solution
of 18 (1.0 g, 3.43 mmol) in D MF (10 mL) were added
imidazole (491 mg, 7.21 mmol) and TBDMSCl (543
mg, 3.60 mmol), and the mixture was stirred at room
temperature for 2 h. After quenching with H2O, eva-
poration of the solvent gave an oil. The oil was dis-
solved in EtOAc, and the solution was washed with
water, dried over MgSO4, and ®ltered. Evaporation of
the ®ltrate gave an oil, which was subjected to column
chromatography on silica gel. Elution with n-hex-
27
d
ane:EtOAc (9:1) gave 19 as an oil (1.38 g, 99%): ‰aŠ
ꢁ
1
+8.9 (c 0.99, CHCl3); H NMR (CD3OD) d 0.11 (6H,
s, (CH3)2 of t-butyldimethylsilyl), 0.91 (9H, s, (CH3)3 of
t-butyldimethylsilyl), 1.31 and 1.40 (3H each, s, (CH3)3
of isopropylidene), 1.43 (9H, s, COOC(CH3)3), 3.03
(1H, dd, J=13.9 and 7.1 Hz, H-5), 3.44 (1H, dd,
J=13.9 and 3.2 Hz, H-50), 3.70±3.77 (2H, m, H-1 and
H-4), 3.92±3.98 (2H, m, H-1 and H-3), 4.21 (1H, br dd,
J=11.5 and 5.6 Hz, H-2); IR (CHCl3) 1710 (CˆO),
1510 (NH) cm 1; FABMS m/z 406.3 (M+H)+, 350.2,
306.3, 292.2, 248.2, 142.1, 73.1, 57.1. Anal. C19H39
NO6Si (C, H, N).
1-(t-Butoxycarbonyl)amino-2,5-di-O-(t-butyldimethylsilyl)-
1-deoxy-3,4-O-isopropylidene-D-ribitol (17). To a solu-
tion of 1626 (15.0 g, 37.4 mmol) in EtOH (500 mL) was
added NaBH4 (7.07 g, 186.8 mmol) at 0 ꢁC, and the
mixture was stirred at room temperature for 43 h. After
quenching with H2O, the mixture was further stirred for
30 min. Evaporation of the solvent gave a solid, which
was dissolved in CHCl3. The solution was washed with
H2O, dried over MgSO4, and ®ltered. Evaporation of
the ®ltrate gave an oil, which was subjected to column
D-erythro-1-(t-Butoxycarbonyl)amino-5-O-(t-butyldimethyl-
silyl)-1-deoxy-3,4-O-isopropylidene-2-pentosulitol (20).
To a solution of 19 (5.0 g, 12.3 mmol) in CH2Cl2
(100 mL) was added Dess±Martin periodinane (7.83 g,
18.5 mmol), and the mixture was stirred at room tem-
perature for 4 h. After dilution with CHCl3, the solution
was washed with a saturated aqueous NaHCO3 and
H2O, dried over MgSO4, and ®ltered. Evaporation of
the ®ltrate gave an oil, which was subjected to column
chromatography on silica gel. Elution with toluene:ethyl
27
d
acetate (3:1) gave 17 as an oil (15 g, 99%): ‰aŠ
13.6ꢁ
(c 0.91, CHCl3); 1H NMR (CDCl3) d 0.14 (6H, s,
-Si(CH3)2), 0.34 (3H, s, CH3 of isopropylidene), 0.91
(9H, s, -SiC(CH3)3), 1.43 (12H, s, COOC(CH3)3 and
CH3 of isopropylidene), 3.32 (2H, br t, J=6.2 Hz, H-5),
3.39 (1H, br t, J=6.2 Hz, -OH), 3.59 and 3.72 (1H each,
dt, J=12 and 6.2 Hz, H-1), 4.08 (1H, t, J=6.2 Hz, H-3),
4.12 (1H, br q, J=6.2 Hz, H-4), 4.20 (1H, q, J=6.2 Hz,
H-2) and 5.84 (1H, br t, J=6.4 Hz, -NHCO-); IR
(CHCl3) 1720 (CˆO), 1520 (NH) cm 1; FABMS m/z
406 (M+H)+, 350, 306, 292, 248, 73, 57.41. Anal.
C19H39NO5Si (C, H, N).
chromatography on silica gel. Elution with n-hex-
26
d
ane:EtOAc (9:1) gave 20 as an oil (4.78 g, 96%): ‰aŠ
ꢁ
1
35.8 (c 0.53, MeOH); H NMR (CDCl3) d 0.03 and
0.04 (3H each, s, (CH3)2 of t-butyldimethylsilyl), 0.86
(9H, s, (CH3)3 of t-butyldimethylsilyl), 1.35 and 1.56
(3H each, s, CH3 of isopropylidene), 1.44 (9H, s,
COOC(CH3)3), 3.68 (1H, dd, J=11.7 and 2.4 Hz, H-1),
3.75 (1H, dd, J=11.7 and 3.4 Hz, H-10), 4.27 (2H, d,
J=4.4 Hz, H-5), 4.38±4.45 (1H, m, H-2), 4.56 (1H, d,
J=8.3 Hz, H-3) and 5.23 (1H, br s, NH); IR (CHCl3)
1700 (CˆO), 1500 (NH) cm 1; FABMS m/z 404.3
(M+H)+, 348.3, 304.3, 290.2, 246.2, 140.1, 73.1, 57.1.
Anal. C19H37NO6Si (C, H, N).
1-(t-Butoxycarbonyl)amino-1-deoxy-3,4-O-isopropylidene-
D-ribitol (18). A solution of n-Bu4NF in THF (1 M,
67.3 mL) was added to a solution of 17 (13.6 g, 33.6
mmol) in THF (200 mL), and the mixture was stirred at
room temperature for 1 h. Evaporation of the solvent
gave an oil, which was dissolved in CHCl3. The solution
was washed with water, and the aqueous phase was
extracted three times with CHCl3. The organic phases
were combined, dried over MgSO4, and ®ltered. Eva-
poration of the ®ltrate gave an oil, which was subjected
to column chromatography on silica gel. Elution with
1-(t-Butoxycarbonyl)amino-1,2-dideoxy-5-O-(t-butyldi-
methylsilyl)-3,4-O-isopropylidene-2-methylene-D-erythro-
pentitol (21). To a solution of methylenetriphenylphos-
phorane, prepared from methyltriphenylphosphonium
bromide (16.92 g, 47.4 mmol) and lithium bis(tri-
methylsilyl)amide (1.0 M, 45 mL) in THF (50 mL) from
0 ꢁC to room temperature, was added a solution of 20
(4.78 g, 11.8 mmol) in THF (10 mL) at 0 ꢁC, and the
resulting mixture was stirred for 30 min. After quench-
ing with acetic acid (2.94 mL, 47.4 mmol), the mixture
was further stirred for 30 min. Evaporation of the sol-
vent gave a solid, which was dissolved in CHCl3. The
solution was washed with H2O, dried over MgSO4, and
®ltered. Evaporation of the ®ltrate gave an oil, which
was subjected to column chromatography on silica gel.
Elution with n-hexane:EtOAc (9:1) gave 21 as an oil
CHCl3:MeOH (19:1) gave 18 as an oil (9.2 g, 94%):
27
d
‰aŠ +37.7ꢁ (c 0.94, CHCl3); 1H NMR (CD3OD) d 1.32
and 1.40 (3H each, s, CH3 of isopropylidene), 1.43 (9H,
s, COOC(CH3)3), 3.40 (1H, dd, J=13.9 and 7.3 Hz, H-
5), 3.44 (1H, dd, J=13.9 and 3.4 Hz, H-50), 3.62 (1H,
dd, J=11.2 and 6.3 Hz, H-1), 3.71 (1H, ddd, J=9.3, 7.3
and 3.4 Hz, H-4), 3.81 (1H, dd, J=11.2 and 5.9 Hz, H-
10), 3.96 (1H, dd, J=9.3 and 6.4 Hz, H-3) and 4.25 (1H,
br dd, J=12.2 and 6.4 Hz, H-2); IR (CHCl3) 1680
(CˆO), 1510 (NH) cm 1; FABMS m/z 292.4 (M+H)+,
236.3, 178.2, 154.1, 136.1, 120.1, 107.1, 57.1. Anal.
C13H25NO6 (C, H, N).

E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
349
28
d
1
(3.83 g, 81%): ‰aŠ
43.5ꢁ (c 0.87, CHCl3); H NMR
(2S,3S,4S)-N-(t-Butoxycarbonyl)-3,4-O-isopropylidene-
5-methylene-2-phthalimidopiperidine-3,4-diol (24). To
the mixture of 23 (500 mg, 1.75 mmol), triphenyl-
phosphine (1.38 g, 5.26 mmol) and phthalimide (773 mg,
5.26 mmol) in D MF (10 mL) was added dropwise di-
ethyl azodicarboxylate (0.837 mL, 5.26 mmol) under
stirring, and the resulting mixture was stirred at room
temperature overnight. Addition of water and evapora-
tion of the solvent gave an oil, which was dissolved in
EtOAc. The solution was washed with saturated aque-
ous NaCl solution, dried over MgSO4, and ®ltered.
Evaporation of the ®ltrate gave an oil, which was sub-
jected to column chromatography on silica gel. Elution
with toluene:EtOAc (19:1) gave 24 as a foam (692 mg,
(CDCl3) d 0.04 and 0.06 (3H each, s, (CH3)2 of t-butyl-
dimethylsilyl), 0.88 (9H, s, (CH3)3 of t-butyldimethyl-
silyl), 1.36 (3H, s, CH3 of isopropylidene), 1.44 (12H, s,
CH3 of isopropylidene and COOC(CH3)3), 3.44 (1H,
dd, J=9.6 and 3.9 Hz, H-1), 3.55 (1H, br t, J=9.6 Hz,
H-10), 3.69 (1H, dd, J=15.1 and 3.9 Hz, H-5), 3.94 (1H,
dd, J=15.1 and 6.8 Hz, H-50), 4.18±4.23 (1H, m, H-2),
4.66 (1H, d, J=5.9 Hz, H-3), 5.15 and 5.33 (1H each, br
s, methylene) and 5.39 (1H, br s, NH); IR (CHCl3) 1710
(CˆO), 1510 (NH) cm 1; FABMS m/z 402.3 (M+H)+,
346.3, 330.2, 288.2, 230.1, 226.3, 186.2, 154.1, 138.1,
73.1, 57.1. Anal. C20H39NO5Si (C, H, N).
28
95%): ‰aŠ +57.6ꢁ (c 0.85, CHCl3); H NMR (CDCl3)
1
1-(t-Butoxycarbonyl)amino-1,2-dideoxy-3,4-O-isopropyl-
idene-2-methylene-D-erythro-pentitol (22). A solution of
n-Bu4NF in THF (1 M, 24.7 mL) was added to a solu-
tion of 21 (8.28 g, 20.6 mmol) in THF (100 mL), and the
mixture was stirred at room temperature for 30 min.
Evaporation of the solvent gave an oil, which was dis-
solved in CHCl3. The solution was washed with H2O,
dried over MgSO4, and ®ltered. Evaporation of the ®l-
trate gave an oil, which was subjected to column chro-
d
d 1.40 (3H, s, CH3 of isopropylidene), 1.49 (12H, s, CH3
of isopropylidene and COOC(CH3)3), 3.82 and 4.48
(1H each, d, J=14.7 Hz, H-6, 60), 4.75 (1H, d, J=6.4
Hz, H-3), 5.00 (1H, d, J=6.4 Hz, H-4), 5.19 and 5.32
(1H each, br s, methylene), 6.35 (1H, br s, H-2) and
7.72±7.84 (4H, m, phthalimido); IR (CHCl3) 1775
1
(CˆO), 1720 (CˆO) cm
; FABMS m/z 415.3
(M+H)+, 359.2, 315.2, 168.2, 148.1, 110.1, 57.1. Anal.
C22H26N2O6 (C, H, N).
matography on silica gel. Elution with CHCl3:MeOH
28
d
(24:1) gave 22 as an oil (5.98 g, 99%): ‰aŠ
49.3ꢁ
(c 0.94, CHCl3); 1H NMR (CDCl3) d 1.38 and 1.49
(3H each, s, CH3 of isopropylidene), 1.45 (9H, s,
COOC(CH3)3), 2.93 (1H, br t, J=6.3 Hz, -OH), 3.49
(1H, br quintet, J=5.9 Hz, H-1), 3.56±3.65 (1H, m, H-
10), 3.69 (1H, dd, J=16.8 and 6.1 Hz, H-50), 3.81 (1H,
dd, J=16.8 and 5.9 Hz, H-5), 4.31 (1H, br dd, J=6.3
and 5.9 Hz, H-2), 4.68 (1H, d, J=6.3 Hz, H-3), 4.93
(1H, br s, NH), 5.17 and 5.33 (1H each, br s, methylene);
IR (CHCl3) 1710 (CˆO), 1520 (NH) cm 1; FABMS m/z
288.3 (M+H)+, 232.2, 174.2, 154.1, 112.1, 57.1. Anal.
C14H25NO5 (C, H, N).
(2S,3S,4R,5R)-N-(t-Butoxycarbonyl)-3,4-O-isopropyl-
idene-5-methyl-2-phthalimidopiperidine-3,4-diol (25) and
(2S,3S,4R,5S)-N-(t-butoxycarbonyl)-3,4-O-isopropyl-
idene-5-methyl-2-phthalimidopiperidine-3,4-diol (26) and
(2S,3S)-N-(t-butoxycarbonyl)-4,5-didehydro-3,4-O-iso-
propylidene-5-methyl-2-phthalimidopiperidine-3,4-diol
(27). A solution of 24 (3.69 g, 8.69 mmol) in MeOH
(200 mL) was stirred with 10% palladium on carbon
(1 g) under atmosphere of hydrogen at room tempera-
ture for 5 h. After removal of catalysts, evaporation of
the solvent gave an oil, which was subjected to column
chromatography on silica gel. Elution with toluene:
AcOEt (9:1) gave 25 (2.73 g, 75%) and 26 (0.18 g, 5%)
as a solid, and 27 (0.64 g, 18%) as a foam. Compounds
25 and 26 were crystallized from toluene-n-hexane to
give their colorless crystals.
(2R,3R,4S)-N-(t-Butoxycarbonyl)-3,4-O-isopropylidene-
5-methylenepiperidine-2,3,4-triol (23). Dimethyl sulfox-
ide (1.78 mL, 25.1 mmol) was added to the stirred
solution of oxalyl chloride (1.09 mL, 12.5 mmol) in
CH2Cl2 (20 mL) at 78 ꢁC, and the mixture was stirred
for 20 min. After addition of a solution of 22 (900 mg,
3.13 mmol) in CH2Cl2 (24 mL) at 78 ꢁC within 5 min,
the mixture was stirred for 20 min. After addition of
triethylamine (8.73 mL, 62.6 mmol), the mixture was
stirred at the same temperature for 15 min, and then the
mixture was allowed to warm to room temperature.
After quenching with water, the mixture was extracted
with CH2Cl2. The extract was washed with water, dried
over MgSO4, and ®ltered. Evaporation of the ®ltrate
gave an oil, which was subjected to column chromato-
26
d
25: Mp 157±158 ꢁC; ‰aŠ
32.9ꢁ (c 0.49, CHCl3); H
1
NMR (CDCl3) d 1.08 (3H, d, J=6.8 Hz, 5-CH3), 1.36
and 1.48 (3H each, s, CH3 of isopropylidene), 1.38 (9H,
s, COOC(CH3)3), 2.34±2.44 (1H, m, H-5), 3.19 (1H, br
t, J=11.5 Hz, H-6ax), 3.50 (1H, dd, J=11.5 and 4.6 Hz,
H-6eq), 4.33 (1H, dd, J=7.3 and 3.4 Hz, H-4), 4.63 (1H,
dd, J=7.3 and 2.0 Hz, H-3), 6.05 (1H, d, J=2.0 Hz, H-
2) and 7.71±7.83 (4H, m, phthalimido); IR (KBr) 1770
1
(CˆO), 1720 (CˆO), 1700 (CˆO), 1610 (CˆO) cm
;
FABMS m/z 417.3 (M+H)+, 361.2, 317.2, 214.2, 170.2,
148.1, 112.1, 57.1: Anal. C22H28N2O6 (C, H, N).
graphy on silica gel. Elution with toluene:acetone (19:1)
29
d
gave 23 as a foam (734 mg, 82%): ‰aŠ
9.9ꢁ (c 0.45,
26
d
CHCl3); 1H NMR (CDCl3) d 1.37 and 1.44 (3H each, s,
CH3 of isopropylidene), 1.48 (9H, s, COOC(CH3)3),
3.17 (1H, br s,-OH), 3.82 and 4.19 (1H each, d, J=14.2
Hz, H-6, 60), 4.41 (1H, dd, J=7.3 and 2.0 Hz, H-3), 4.74
(1H, d, J=7.3 Hz, H-4), 5.25 and 5.33 (1H each, br s,
methylene) and 5.68 (1H, br s, H-2); IR (KBr) 1690
(CˆO) cm 1; FABMS m/z 286 (M+H)+, 271.1, 268.2,
230.1, 212.1, 168.1, 154.1, 110, 57.1. Anal. C14H23NO5
(C, H, N).
26: Mp 137±138 ꢁC; ‰aŠ +16.9ꢁ (c 0.47, CHCl3); H
1
NMR (CDCl3) d 1.12 (3H, d, J=6.8 Hz, 5-CH3); 1.33
and 1.51 (3H each, s, CH3 of isopropylidene), 1.44 (9H,
s, COOC(CH3)3), 1.78±1.88 (1H, m, H-5), 2.98 (1H, br
t, J=13.0 Hz, H-6ax), 3.90±4.02 (1H, m, H-6eq), 4.26
(1H, dd, J=8.6 and 5.4 Hz, H-4), 4.30 (1H, dd, J=5.4
and 1.5 Hz, H-3), 6.47 (1H, s, H-2) and 7.74±7.89 (4H,
m, phthalimido); IR (KBr) 1770 (CˆO), 1720 (CˆO),
1700 (CˆO), 1610 (CˆO) cm 1; FABMS m/z 417.3

350
E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
(M+H)+, 361.2, 317.2, 214.2, 170.2, 148.1, 112.1, 57.1.
Anal. C22H28N2O6 (C, H, N).
Compound 28 (120 mg, 0.419 mmol) was dissolved in
CH2Cl2 (2 mL), and to the solution were added pyridine
(0.1 mL), (CF3CO)2O (0.1 mL) and 4-dimethylamino-
pyridine (10 mg) at 0 ꢁC. The mixture was stirred at 0 ꢁC
for 30 min. Evaporation of the solvent gave an oil,
which was dissolved in ethyl acetate. The solution was
washed with saturated aqueous NaCl solution, dried
over MgSO4, and ®ltered. Evaporation of the solvent
gave an oil, which was subjected to column chromato-
28
d
27: ‰aŠ +55.5ꢁ (c 0.95, CHCl3); H NMR (CDCl3) d
1
1.31 (9H, s, COOC(CH3)3), 1.43 and 1.58 (3H each, s,
CH3 of isopropylidene), 1.78 (3H, br s, 5-CH3), 3.96 and
4.37 (1H each, d, J=15.1 Hz, H-6, 60), 4.95 (1h, br d,
J=6.8 Hz, H-3), 5.81 (1H, d, J=6.8 Hz, H-2) and 7.73±
7.88 (4H, m, phthalimido); IR (CHCl3) 1780 (CˆO),
1720 (CˆO), 1700 (CˆO) cm 1; FABMS m/z 415.3
(M+H)+, 359.2, 313.2, 255.2, 219.1, 168.2, 154.1,
148.1, 110.1, 57.1. Anal. C22H26N2O6 (C, H, N).
graphy on silica gel. Elution with toluene:EtOAc (10:1)
25
d
gave 30 as a solid (159 mg, 99%): ‰aŠ
37.2ꢁ (c 0.46,
CHCl3); 1H NMR (CDCl3) d 1.08 (3H, d, J=6.8 Hz, 5-
CH3), 1.35 (3H, s, CH3 of isopropylidene), 1.46 (12H, s,
CH3 of isopropylidene and COOC(CH3)3), 1.82±1.95
(1H, m, H-5), 3.01 (1H, br t, J=12.4 Hz, H-6ax), 3.39
(1H, dd, J=12.4 and 3.9 Hz, H-6eq), 4.32 (1H, dd,
J=6.8 and 2.4 Hz, H-4), 4.52 (1H, dd, J=6.8 and 2.0
Hz, H-3) and 5.77 (1H, br s, H-2); IR (KBr) 1720
(CˆO), 1680 (CˆO), 1530 (NH) cm 1; FABMS m/z
383.2 (M+H)+, 327.2, 214.2, 170.2, 154.1, 112.1, 57.1.
Anal. C16H25F3N2O5 (C, H, N).
Synthesis of 25 from 27
Compound 25 was synthesized similarly from 27 as in
the preparation of 25 from 24; the yield was 75%.
(2R,3S,4R,5R)-2-Amino-N-(t-butoxycarbonyl)-3,4-O-iso-
propylidene-5-methylpiperidine-3,4-diol (28). To a solu-
tion of 25 (83 mg, 0.20 mmol) in MeOH (5 mL) was
added hydrazine hydrate (0.5 mL), and the mixture was
stirred at room temperature overnight. After dilution
with CHCl3, the resulting precipitates were ®ltered o€.
The ®ltrate was washed with water, dried over MgSO4,
and ®ltered. Evaporation of the ®ltrate gave an oil,
which was subjected to column chromatography on
silica gel. Elution with CHCl3-MeOH (25:1) gave 28 as a
(2R,3S,4R,5R)-N-(t-Butoxycarbonyl)-2-trichloroacetam-
ido-3,4-O-isopropylidene-5-methylpiperidine-3,4-diol (31).
Compound 28 (17 mg, 0.0594 mmol) was dissolved in
CH2Cl2 (3 mL), and to the solution were added pyridine
(19.2 mL, 0.238 mmol) and trichloroacetyl chloride
(13.3 mL, 0.119 mmol) at 0 ꢁC. The mixture was stirred
at 0 ꢁC for 30 min. After dilution with CH2Cl2 (15 mL),
the solution was washed with water, dried over MgSO4,
and ®ltered. Evaporation of the ®ltrate gave an oil,
which was subjected to column chromatography on
silica gel. Elution with toluene:EtOAc (10:1) gave 31 as
28
d
ꢁ
1
foam (57 mg, 99%): ‰aŠ +14.3 (c 1.27, CHCl3); H
NMR (CD3OD) d 1.00 (3H, d, J=6.8 Hz, 5-CH3), 1.31
and 1.35 (3H each, s, CH3 of isopropylidene), 1.47 (9H,
s, COOC(CH3)3), 2.28±2.38 (1H, m, H-5), 2.99 (1H, br
t, J=12.2 Hz, H-6ax), 3.22 (1H, br dd, J=12.2 and 4.9
Hz, H-6eq), 4.30 (1H, br dd, J=7.8 and 2.4 Hz, H-4),
26
d
1
a foam (25.6 mg, 99%): ‰aŠ
21.2ꢁ (c 0.5, MeOH); H
4.33 (1H, dd, J=7.8 and 1.5 Hz, H-3) and 4.90 (1H, d,
1
NMR (CDCl3) d 1.08 (3H, d, J=6.8 Hz, 5-CH3), 1.36
(3H, s, CH3 of isopropylidene), 1.47 (12H, s, CH3 of
isopropylidene and COOC(CH3)3), 1.81±1.93 (1H, m,
H-5), 3.01 (1H, br t, J=12.1 Hz, H-6ax), 3.42 (1H, dd,
J=12.1 and 3.4 Hz, H-6eq), 4.34 (1H, dd, J=6.4 and
2.0 Hz, H-4), 4.56 (1H, br d, J=6.4 Hz, H-3), 5.75 (1H,
br s, H-2) and 6.60 (1H, br s, -NHCO-); IR (KBr) 1720
(CˆO), 1660 (CˆO), 1520 (NH) cm 1; FABMS m/z
433 (M+2H)+, 431 (M)+, 377, 375.16, 359.15, 270.31,
214.26, 170.24, 154.16, 57.10. Anal. C16H25 N2O5Cl3 (C,
H, N).
J=1.5 Hz, H-2); IR (CHCl3) 1680 (CˆO) cm
;
FABMS m/z 287 (M+H)+, 270.2, 214.2, 170.1, 112.1,
57.1. Anal. C14H26N2O4 (C, H, N).
(2R,3S,4R,5R)-2-Acetamido-N-(t-butoxycarbonyl)-3,4-
O-isopropylidene-5-methylpiperidine-3,4-diol (29). To a
solution of 28 (25 mg, 0.087 mmol) in CH2Cl2 (1 mL)
were added pyridine (0.1 mL), acetic anhydride (0.1 mL)
and 4-dimethylaminopyridine (2 mg), and the mixture
was stirred at room temperature for 4 h. Evaporation of
the solvent gave an oil, which was dissolved in ethyl
acetate. The solution was washed with water, dried over
MgSO4, and ®ltered. Evaporation of the ®ltrate gave an
oil, which was subjected to column chromatography on
silica gel. Elution with toluene:acetone (3:1) gave 29 as a
(2S,3S,4R,5R)-2-Acetamido-5-methylpiperidine-3,4-diol
(7). Compound 29 (20 mg, 0.0608 mmol) was dissolved
in ether (2 mL), and to the solution was added 4 M HCl
in 1,4-dioxane (0.4 mL) at 0 ꢁC. The mixture was stirred
at room temperature for 3 h. After addition of diethyl
ether, the resulting precipitates were taken by cen-
trifugation and washed with diethyl ether three times to
27
d
1
foam (29 mg, 99%): ‰aŠ
34.3ꢁ (c 0.57, CHCl3); H
NMR (CDCl3) d 1.05 (3H, d, J=6.8 Hz, 5-CH3), 1.33
and 1.35 (3H each, s, CH3 of isopropylidene), 1.46 (9H,
s, COOC(CH3)3), 1.98 (3H, s, -COCH3), 1.88±2.01 (1H,
m, H-5), 3.01 (1H, br t, J=12.3 Hz, H-6ax), 3.32 (1H, dd,
J=12.3 and 3.9 Hz, H-6eq), 4.26 (1H, dd, J=7.3 and 2.0
give 7 as a colorless solid of its hydrochloride (8.8 mg,
32.3ꢁ (c 0.41, MeOH); 1H NMR (CD3OD)
27
d
65%): ‰aŠ
d 1.05 (3H, d, J=6.8 Hz, 5-CH3), 1.95±2.06 (1H, m, H-5),
2.05 (3H, s, COCH3), 2.92 (1H, dd, J=12.3 and 3.9 Hz,
H-6eq), 3.05 (1H, br t, J=12.3 Hz, H-6ax), 3.72 (1H, dd,
J=10.3 and 2.4 Hz, H-3), 3.84±3.87 (1H, br t, J=ꢀ2.0 Hz,
H-4) and 4.91 (1H, d, J=10.3 Hz, H-2); IR (KBr) 1660
(CˆO), 1550 (NH) cm 1; FABMS m/z 189.2 (M+H)+,
176.1, 154.1, 137.1, 120.1, 107, 89, 77. Anal. C8H16
Hz, H-4), 4.53 (1H, br d, J=7.3 Hz, H-3) and 5.73 (2H,
1
br s, H-2 and -NHCO-); IR (CHCl3) 1680 (CˆO) cm
;
FABMS m/z 329.3 (M+H)+, 273.2, 214.2, 170.2, 154.1,
112.1, 57.1. Anal. C16H28N2O5 (C, H, N).
(2R,3S,4R,5R)-N-(t-Butoxycarbonyl)-2-tri¯uoroacetam-
ido-3,4-O-isopropylidene-5-methylpiperidine-3,4-diol (30).
.
N2O3 HCl (C, H, N). Calcd. Cl, 15.78; found Cl, 16.11.

E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
351
(2S,3S,4R,5R)-2-Tri¯uoroacetamido-5-methylpiperidine-
3,4-diol (8). Compound 8 as its hydrochloride was syn-
thesized similarly from 30 as in the preparation of 7
isopropylidene), 1.47 (9H, s, COOC(CH3)3), 1.82±1.93
(1H, m, H-5), 2.79 (1H, br s, H-6), 3.78 (1H, br d,
J=13.7 Hz, H-60), 4.01 (1H, br t, J=6.0 Hz, H-4), 4.24
(1H, br dd, J=ꢀ6.0 and ꢀ2.0 Hz, H-3), 5.80 (1H, br s,
H-2); IR (CHCl3) 1740 (CˆO), 1700 (CˆO), 1540 (NH)
cm 1; FABMS m/z 383.2 (M+H)+, 327.2, 214.2, 170.2,
154.1, 112.1, 57.1. Anal. C16H25N2O5F3 (C, H, N).
28
from 29; the yield was 97%: ‰aŠ
42.3ꢁ (c 0.46,
d
1
MeOH); H NMR (CD3OD) d 1.06 (3H, d, J=6.8 Hz,
5-CH3), 1.95±2.06 (1H, m, H-5), 2.97 (1H, dd, J=12.5
and 4.4 Hz, H-6eq), 3.09 (1H, br t, J=12.5 Hz, H-6ax),
3.84 (1H, dd, J=10.3 and 2.4 Hz, H-3), 3.89 (1H, br t,
J=2.4 Hz, H-4) and 4.99 (1H, d, J=10.3 Hz, H-2); IR
(KBr) 1730 (CˆO), 1555 (NH) cm 1; FABMS m/z
243.2 (M+H)+, 154.1, 137.1, 120.1, 107.1, 89, 77. Anal.
(2S,3S,4R,5S)-2-Tri¯uoroacetamido-5-methylpiperidine-
3,4-diol (11). Compound 11 as its hydrochloride was
synthesized similarly from 33 as in the preparation of 7
26
C8H13N2O3F3 HCl (C, H, N). Calcd. Cl, 12.72; found
Cl, 13.04%.
from 29; the yield was 67.8%: ‰aŠ 22.4ꢁ (c 0.091,
.
d
1
MeOH); H NMR (CD3OD) d 1.11 (3H, d, J=6.8 Hz,
5-CH3), 2.18±2.30 (1H, m, H-5), 2.86 (1H, dd, J=13.2
and 8.3 Hz, H-6ax), 3.30 (1H, dd, J=13.2 and 4.4 Hz,
H-6eq), 3.75 (1H, dd, J=7.8 and 2.7 Hz, H-4), 3.98 (1H,
dd, J=5.6 and 2.7 Hz, H-3) and 5.23 (1H, d, J=5.6 Hz,
H-2); IR (KBr) 1730 (CˆO), 1560 (NH) cm 1; FABMS
m/z 243.2 (M+H)+, 154.1, 136.1, 130.2, 107.1, 89, 77.
(2S,3S,4R,5R)-2-Trichloroacetamido-5-methylpiperidine-
3,4-diol (9). Compound 9 as its hydrochloride was syn-
thesized similarly from 31 as in the preparation of 7
22
from 29: the yield was 77%: ‰aŠ
37.8ꢁ (c 0.23,
d
1
MeOH); H NMR (CD3OD) d 1.07 (3H, d, J=6.8 Hz,
5-CH3), 1.96±2.02 (1H, m, H-5), 2.99 (1H, dd, J=12.5
and 4.4 Hz, H-6eq), 3.07 (1H, br t, J=12.5 Hz, H-6ax),
3.88±3.91 (1H, br t, J=ꢀ2.0 Hz, H-4), 3.94 (1H, dd,
J=10.3 and 2.4 Hz, H-3); IR (KBr) 1720 (CˆO), 1530
(NH) cm 1; FABMS m/z 293 (M+2H)+, 291.08 (M)+,
170.18, 154.09, 136.09, 130.13, 112.06, 107.04, 89.03,
.
Anal. C8H13N2O3F3 HCl (C, H, N). Calcd. Cl, 12.72;
found Cl, 13.07%.
(2R,3S,4S)-2-Amino-N-(t-butoxycarbonyl)-3,4-O-isopro-
pylidene-5-methylenepiperidine-3,4-diol (34). Compound
34 was synthesized similarly from 24 as in the prepara-
26
.
77.04. Anal. C8H13N2O3Cl3 HCl (C, H, N). Calcd. Cl,
43.04; found Cl, 43.47%.
tion of 28 from 25; the yield was 72%: ‰aŠ +4.0ꢁ (c
d
0.93, CHCl3); 1H NMR (CDCl3) d 1.36 and 1.45
(3H each, s, CH3 of isopropylidene), 1.48 (9H, s,
COOC(CH3)3), 3.84 (1H, d, J=14.2 Hz, H-6), 4.25 (1H,
dt, J=14.2 and 2.0 Hz, H-60), 4.40 (1H, dd, J=7.6 and
2.0 Hz, H-4), 4.70 (1H, d, J=7.6 Hz, H-3), 5.07 (1H, br
s, H-2) and 5.30 and 5.36 (1H each, br s, methylene); IR
(CHCl3) 1680 (CˆO) cm 1; FABMS m/z 268.1 (M-
NH2)+, 212.1, 168.1, 154.1, 110, 57. Anal. C14H24N2O4
(C, H, N).
(2S,3S,4R,5R)-5-Methyl-2-phthalimidopiperidine-3,4-diol
(10). Compound 10 as its hydrochloride was synthesized
similarly from 25 as in the preparation of 7 from 29; the
23
d
yield was 94%: [aŠ
19.6ꢁ (c 0.25, MeOH); H NMR
1
(CD3OD) d 1.11 (3H, d, J=6.8 Hz, 5-CH3), 2.14±2.23
(1H, m, H-5), 3.10 (1H, dd, J=12.2 and 4.4 Hz, H-6eq),
3.23 (1H, br t, J=12.2 Hz, H-6ax), 3.99 (1H, br t,
J=ꢀ2.0 Hz, H-4), 4.54 (1H, dd, J=10.3 and 2.4 Hz, H-
3), 5.44 (1H, d, J=10.3 Hz, H-2) and 7.88±8.00 (4H, m,
phthalimido); IR (KBr) 1780 (CˆO), 1720 (CˆO), 1690
(CˆO) cm 1; FABMS m/z 277.19 (M+H)+, 265.17,
202.22, 170.17, 154.10, 136.09, 130.13, 107.04, 89.03,
(2R,3S,4S)-N-(t-Butoxycarbonyl)-2-tri¯uoroacetamido-
3,4-O-isopropylidene-5-methylenepiperidine-3,4-diol (35).
Compound 35 was synthesized similarly from 34 as in
26
d
the preparation of 30 from 28; the yield was 89.2%: ‰aŠ
1
.
77.04. Anal. C14H16N2O4 HCl (C, H, N). Calcd. Cl,
11.34; found Cl, 11.63%.
50.8ꢁ (c 0.87, CHCl3); H NMR (CDCl3) d 1.37 (3H,
s, CH3 of isopropylidene), 1.48 (12H, s, CH3 of iso-
propylidene and COOC(CH3)3), 3.87 (1H, d, J=13.7
Hz, H-6), 4.25 (1H, dt, J=13.7 and 2.0 Hz, H-60), 4.44
(1H, br s, H-4), 4.74 (1H, d, J=7.8 Hz, H-3), 5.41 and
5.45 (1H each, s, methylene), 6.09 (1H, dd, J=7.8 and
1.5 Hz, H-2) and 6.26 (1H, br s, -NHCO-); IR (CHCl3)
1730 (CˆO), 1690 (CˆO), 1520 (NH) cm 1; FABMS
m/z 381.1 (M+H)+, 325.1, 279.1, 212.1, 168.1, 154.1,
110, 57.1. Anal. C16H23N2O5F3 (C, H, N).
(2S,3S,4R,5R)-2-Amino-N-(t-butoxycarbonyl)-3,4-O-iso-
propylidene-5-methylpiperidine-3,4-diol (32). Compound
32 was synthesized similarly from 26 as in the prepara-
28
tion of 28 from 25; the yield was 67.1%: ‰aŠ +58.9ꢁ (c
d
0.54, CHCl3); 1H NMR (CD3OD) d 1.00 (3H, d, J=6.4
Hz, 5-CH3), 1.35 and 1.46 (3H each, s, CH3 of iso-
propylidene), 1.48 (9H, s, COOC(CH3)3), 1.71±1.83
(1H, m, H-5), 2.67 (1H, br t, J=12.7 Hz, H-6ax), 3.72
(1H, m, H-6eq), 3.78 (1H, dd, J=9.3 and 4.9 Hz, H-4),
3.99 (1H, dd, J=4.9 and 1.8 Hz, H-3) and 5.38 (1H, br
s, H-2); IR (CHCl3) 1680 (CˆO) cm 1; FABMS m/z
270.2 (M-NH2)+, 214.2, 170.1, 112.1, 57.1. Anal.
C14H26N2O4 (C, H, N).
(2S,3S,4S)-2-Tri¯uoroacetamido-5-methylenepiperidine-
3,4-diol (12). Compound 12 as its hydrochloride was
synthesized similarly from 35 as in the preparation of 7
26
d
from 29; the yield 50.6%: ‰aŠ
27.1ꢁ (c 0.09, MeOH);
1H NMR (CD3OD) d 3.69 (1H, d, J=13.2 Hz, H-6),
3.89 (1H, dd, J=9.3 and 2.9 Hz, H-3), 4.00 (1H, d,
J=13.2 Hz, H-60), 4.48 (1H, d, J=2.9 Hz, H-4), 5.24
(2S,3S,4R,5R)-N-(t-Butoxycarbonyl)-2-tri¯uoroacetamido-
3,4-O-isopropylidene-5-methylpiperidine-3,4-diol (33).
Compound 33 was synthesized similarly from 32 as in
(1H, d, J=9.3 Hz, H-2) and 5.35 and 5.43 (1H each, s,
1
methylene); IR (KBr) 1725 (CˆO), 1550 (NH) cm
;
27
d
the preparation of 30 from 28; the yield was 77.9%: ‰aŠ
FABMS m/z 241.2 (M+H)+, 168.2, 154.1, 136.1, 128.1,
ꢁ
1
.
110.1, 89, 77. Anal. C8H11N2O3F3 HCl (C, H, N).
Calcd. Cl, 12.82; found Cl, 13.17%.
+50.3 (c 0.54, CHCl3); H NMR (CDCl3) d 1.05 (3H,
d, J=6.8 Hz, 5-CH3), 1.36 and 1.49 (3H each, CH3 of

352
E. Shitara et al. / Bioorg. Med. Chem. 8 (2000) 343±352
(2R,3S,4R,5R)-2-Amino-5-methylpiperidine-3,4-diol (13).
Compound 8 (30 mg) was dissolved in MeOH (3 mL),
and the solution was stirred at 50 ꢁC for 13 h. Evapora-
Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1992; Vol. 10,
pp 495±583.
5. Nishimura, Y.; Kudo, T.; Kondo, S.; Takeuchi, T. J. Anti-
biot. 1994, 47, 101.
6. Nishimura, Y. In Studies in Natural Products Chemistry;
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pp 75±121.
7. Cordes, E. H.; Bull, H. G. Chem. Rev. 1974, 74, 581.
8. Perkins, S. J.; Johnson, L. N.; Philips, D. C.; Dwek, R. A.
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9. Sinnott, M. L. Chem. Rev. 1990, 90, 1171.
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Ichikawa, Y.; Porco, J. A., Jr.; Wong, C.-H. J. Am. Chem.
Soc. 1991, 113, 6187.
11. Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J.
Am. Chem. Soc. 1998, 120, 3007.
12. Nishimura, Y.; Satoh, T.; Kondo, S.; Takeuchi, T.; Aze-
taka, M.; Fukuyasu, H.; Iizuka, Y.; Shibahara, S. J. Antibiot.
1994, 47, 840.
13. Satoh, T.; Nishimura, Y.; Kondo, S.; Takeuchi, T.; Aze-
taka, M.; Fukuyasu, H.; Iizuka, Y.; Ohuchi, S.; Shibahara, S.
J. Antibiot. 1996, 49, 321.
14. Nishimura, Y.; Satoh, T.; Kudo, T.; Kondo, S.; Takeuchi,
T. Bioorg. Med. Chem. 1996, 4, 96.
15. Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeu-
chi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y. J. Am. Chem.
Soc. 1996, 118, 3051.
16. Shitara, E.; Nishimura, Y.; Kojima, F.; Takeuchi, T. J.
Antibiot. 1999, 52, 348.
17. Nishimura, Y.; Shitara, E.; Adachi, H.; Takeuchi, T.;
Nakajima, M. J. Org. Chem., in press.
18. Lowe, J. B.; Stoolman, L. M.; Nair, R. P.; Larsen, R. D.;
Berhend, T. L.; Marks, R. M. Cell 1990, 63, 475.
19. Berg, E. L.; Robinson, M. K.; Mansson, O.; Butcher, E.
C.; Magnani, J. L. J. Biol. Chem. 1991, 266, 14869.
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Natl. Cancer Inst. 1991, 83, 1321.
tion of the solvent gave 13 as an oil of its hydrochloride
24.9ꢁ (c 0.15, MeOH); 1H NMR
26
d
(19.5 mg, 99%): ‰aŠ
(CD3OD) d 1.04 (3H, d, J=6.8 Hz, 5-CH3), 1.95±2.02
(1H, m, H-5), 2.99±3.04 (2H, m, H-6, 60), 3.55 (1H, dd,
J=8.8 and 2.5 Hz, H-3), 3.84 (1H, br t, J=2.5 Hz, H-4)
and 4.42 (1H, d, J=8.8 Hz, H-2); 13C NMR (CD3OD,
100 MHz) d 14.21 (CH3), 33.52 (C-5), 44.26 (C-6), 72.25
(C-3 or C-4), 72.99 (C-4 or C-3) and 88.86 (C-2); IR
(KBr) 3360, 2940, 1460, 1010 cm 1; FABMS m/z 130.2
(M-NH2)+, 107.0, 89.0, 77.1.
(2R,3S,4R,5R)-N-Acetyl-2-amino-5-methylpiperidine-
3,4-diol (14). To a solution of 13 (26.2 mg, 0.143 mmol)
in MeOH (30 mL) were added acetic anhydride (0.2
mL) and pyridine (12.8 mL, 0.158 mmol), and the mix-
ture was stirred at room temperature for 20 h. Eva-
poration of the solvent gave an oil, which was subjected
to column chromatography on silica gel. Elution with
CHCl3±MeOH (50:1) gave 14 as an oil (14 mg, 51.9%):
24
‰aŠ +0.84ꢁ (c 0.45, MeOH); 1H NMR (CD3OD) d 0.99
d
and 1.01 (total 3H, d each, J=6.8 Hz, 5-CH3), 1.62±
1.72 and 1.73±1.83 (total 1H, m each, H-5), 2.17 and
2.18 (total 3H, s each, COCH3), 2.64 and 3.12 (total 1H,
br t each, J=13.3 Hz, H-6ax), 3.37 and 4.09 (total 1H,
dd each, J=13.3 and 4.4 Hz, H-6eq), 3.45 and 3.59
(total 1H, br t each, J=3.7 Hz, H-3), 3.73 (1H, br s, H-
4) and 5.07 and 5.72 (total 1H, d each, J=3.7 Hz, H-2);
IR (CHCl3) 1640 (CˆO) cm 1; APCI±MS m/z 189
(M+H)+, 172 (M-NH2)+, 154, 130.
21. Osborn, L.; Hession, C.; Tizard, R.; Vassallo, C.;
Luhowskyj, S.; Chi-Rosso, G.; Lobb, R. Cell 1989, 59, 1203.
22. Niedbala, M. J.; Madiyalakan, R.; Matta, K.; Crickard,
K.; Sharma, M.; Bernacki, R. J. Cancer Res. 1987, 47, 4634.
23. Winchester, B.; Barker, C.; Baines, S.; Jacob, G. S.; Nam-
goong, S. K.; Fleet, G. Biochem. J. 1990, 265, 277.
24. Karpas, A.; Fleet, G. W. J.; Dwek, R. A.; Petursson, S.;
Namgoong, S. K.; Ramsden, N. G.; Jacob, G. S.; Rademacher,
T. W. Proc. Natl. Acad. Sci. USA 1988, 85, 9229.
25. Nishimura, Y.; Shitara, E.; Takeuchi, T. Tetrahedron Lett.
1999, 40, 2351.
Acknowledgements
The authors are grateful to Dr. S. Kondo for his helpful
discussion and encouragement. Thanks are due to Ms.
H. Nakamura for X-ray crystallographic analysis. We
also express appreciation to Ms. M. Kaneda for her
technical assistance.
References and Notes
26. Kudo, T.; Nishimura, Y.; Kondo, S.; Takeuchi, T. J.
Antibiot. 1992, 45, 954.
1. Karlsson, G. B.; Butters, T. D.; Dwek, R. A.; Platt, F. M. J.
Biol. Chem. 1993, 268, 570.
2. Winchester, B.; Fleet, G. W. J. Glycobiology 1992, 2, 199.
3. Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res.
1993, 26, 182.
27. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
28. Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem.
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29. Lemieux, R. U.; Kulling, R. K.; Bernstein, H. J.; Schneider,
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30. For a review, see: Mitsunobu, O. Synthesis 1981, 1±28.
4. Nishimura, Y. In Studies in Natural Products Chemistry;