Flexible Synthesis and Biological Activity of Uronic Acid-Type gem-Diamine 1-N-Iminosugars: A New Family of Glycosidase Inhibitors

Article abstract of DOI:10.1021/jo982448c

An efficient and flexible synthetic route to four gem-diamine 1-N-iminosugars of uronic acid-type (D-glucuronic, D-mannuronic, L-iduronic, and L-guluronic acid), a new family of glycosidase inhibitor, from l-galactono-1,4-lactone have been developed in an enantiodivergent fashion through a sequence involving as the key steps (a) the formation of gem-diamine 1-N-iminopyranose ring by the Mitsunobu reaction of an aminal and (b) the introduction of a carboxylic acid group by the Wittig reaction of a ketone, hydroboration and oxidation, and the Sharpless oxidation. D-Glucuronic and D-mannuronic acid-type 1-N-iminosugars, (3S,4R,5R,6R)- and (3S,4R,5R,6S)-4,5-dihydroxy-6-trifluoroacetamido-3-piperidinecarboxylic acid, were proven to be potent inhibitors for β-D-glucuronidase (IC₅₀ 6.5 × 10^-8M) and to affect human heparanase (endo-β-glucuronidase).

Full text of DOI:10.1021/jo982448c

2
J . Org. Chem. 2000, 65, 2-11
Articles
F lexible Syn th esis a n d Biologica l Activity of Ur on ic Acid -Typ e
gem -Dia m in e 1-N-Im in osu ga r s: A New F a m ily of Glycosid a se
In h ibitor s
Yoshio Nishimura,*,† Eiki Shitara,† Hayamitsu Adachi,† Minako Toyoshima,‡
Motowo Nakajima,‡ Yoshiro Okami,† and Tomio Takeuchi†
Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, J apan, and
Discovery Research, Takarazuka Research Institute, Novartis Pharma K. K., 10-66 Miyuki-cho,
Takarazuka 665-8666, J apan
Received December 15, 1998 (Revised Manuscript Received November 2, 1999 )
An efficient and flexible synthetic route to four gem-diamine 1-N-iminosugars of uronic acid-type
(D-glucuronic, D-mannuronic, L-iduronic, and L-guluronic acid), a new family of glycosidase inhibitor,
from l-galactono-1,4-lactone have been developed in an enantiodivergent fashion through a sequence
involving as the key steps (a) the formation of gem-diamine 1-N-iminopyranose ring by the
Mitsunobu reaction of an aminal and (b) the introduction of a carboxylic acid group by the Wittig
reaction of a ketone, hydroboration and oxidation, and the Sharpless oxidation. D-Glucuronic and
D-mannuronic acid-type 1-N-iminosugars, (3S,4R,5R,6R)- and (3S,4R,5R,6S)-4,5-dihydroxy-6-
trifluoroacetamido-3-piperidinecarboxylic acid, were proven to be potent inhibitors for â-D-
glucuronidase (IC50 6.5 × 10-8M) and to affect human heparanase (endo-â-glucuronidase).
In tr od u ction
Currently, specific inhibitors of glycosidases aid in
developing an understanding of the metabolism of oli-
gosaccharides in glycoconjugates, functional domains for
carbohydrate protein interactions involved in a variety
of biological functions such as immune response, onco-
genesis, metastasis of tumors, viral and bacterial infec-
tions, differentiation of neuronal cells, and so forth.1 The
possible applications in antimetastatic,2 antitumor,3 an-
tiviral,4 antibacterial,5 or immunoregulatory agents6
stimulate interest into the relationships between struc-
tures and specific biological functions. In the course of
our study on glycosidase inhibitors, we proposed a new
class of glycosidase inhibitors, gem-diamine 1-N-imino-
sugars (1),7 in which an anomeric carbon atom is replaced
by a nitrogen (Figure 1). We considered that the proto-
nated form of gem-diamine 1-N-iminosugars (1) may
mimic glycopyranosyl cation (3), the putative transition
state of enzymatic glycosidic hydrolysis (Figure 1). We
have also demonstrated that gem-diamine 1-N-imino-
F igu r e 1. gem-Diamine 1-N-iminosugars (1 and 2) and
glycopyranosyl cation (3), the putative transition state of
enzymatic glycosidic hydrolysis.
sugars (1), especially 2-trifluoroacetamido-1-N-iminosug-
ars (2) (Figure 1), are highly potent and specific glycosi-
dase inhibitors7a-c and that some of them show the potent
suppression of experimental and spontaneous pulmonary
metastasis of tumor cells in mice.7a,b,8
On the other hand, uronic acids (D-glucuronic acid (4)
and L-iduronic acid (5)) (Figure 2) are the major con-
stituents of the mammalian glycosaminoglycans (hyalu-
† Institute of Microbial Chemistry.
‡ Takarazuka Research Institute.
(4) (a) Gruters, R. A.; Neefjes, J . J .; Terswetle, M.; de Goede, R. E.
Y.; Tulp, A.; Huisman, H. G.; Miedema, F.; Ploegh, H. L. Nature 1987,
330, 74. (b) Walker, B. D.; Kawalski, M.; Goh, W.; Kozarsky, K.;
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J . Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8120. (c) von Itzstein, M.;
Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J . C.; J in, B.; Phan, T. V.;
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10.1021/jo982448c CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/17/1999

Uronic Acid-Type gem-Diamine 1-N-Iminosugars
J . Org. Chem., Vol. 65, No. 1, 2000
3
ronic acid, dermatan sulfate, heparan sulfate, heparin,
and chondroitin sulfate) of endotherial basement mem-
branes and extracellular matrix. â-D-Glucuronidase and
R-L-iduronidase are known to degrade the mammalian
glycosaminoglycans,9,10 and heparanase (end-â-D-glucu-
ronidase) activity in murine B16 melanoma cells cor-
relates with lung colonization ability by degradation of
heparan sulfate.11a Furthermore, glucuronidase inhibitors
inhibit pulmonary colonization of tumor cells in their
syngeneic host.11b,c Alginate-like polysaccharides, the
major constituents of glycocalyx biofilm, secreted by
mucoid strains of Pseudomonas aeruginosa isolated from
patients with cystic fibrosis12 are also composed of uronic
acids (D-mannuronic acid (6) and L-guluronic acid (7))
(Figure 2).13 They are known to interfere with the host
defense mechanisms,14 to provide a barrier against
penetration of drugs,15 and to complicate the bronchial
obstruction.16 Inhibitors of alginate biosynthetic enzymes
may prove useful in chemotherapy of P. aeruginosa
infections in cystic fibrosis patients. We now report the
F igu r e 2. Structures of uronic acids.
F igu r e 3. Structures of uronic acid-type gem-diamine 1-N-
iminosugars.
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extension of our study on glycosidase inhibitor of gem-
diamine 1-N-iminosugar to the divergent synthesis of
uronic acid-type 1-N-iminosugars, D-glucuronic acid (8),
L-iduronic acid (9), D-mannuronic acid (10), and L-
guluronic acid (11) from L-galactono-1,4-lactone (12)
(Figure 3).
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1994, 47, 101. (b) Nishimura, Y. In Studies in Natural Products
Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1995; Vol. 16,
p 75. (c) Nishimura, Y.; Satoh, T.; Kudo, T.; Kondo, S., Takeuchi, T.
BioMed. Chem. 1996, 4, 91. (d) For another type of 1-N-iminosugar
inhibitor, see: J espersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup,
T.; Lundt, I.; Bols, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1778.
Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J . Am. Chem. Soc.
1998, 120, 3007.
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M.; Fukuyasu, H.; Iizuka, Y.; Shibahara, S. J . Antibiot. 1994, 47, 840.
(b) Satoh, T.; Nishimura, Y.; Kondo, S.; Takeuchi, T.; Azetaka, M.;
Fukuyasu, H.; Iizuka, Y.; Ohuchi, S. J . Antibiot. 1996, 49, 321. (c)
Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.; Azetaka,
M.; Fukuyasu, H.; Iizuka, Y. J . Am. Chem. Soc. 1996, 118, 3051. (d)
Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.; Azetaka,
M.; Fukuyasu, H.; Iizuka, Y. J . Med. Chem. 1997, 40, 2626.
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Res. Commun. 1971, 42, 340. (b) Bach, G.; Friedman, R.; Weissman,
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Chem. 1991, 266, 9661.
Resu lts a n d Discu ssion
Syn th esis. Since our interest in this class of inhibitors
against metabolic enzymes of glycuronides encompassed
four enantiomerically pure stereoisomers, we developed
a flexible divergent strategy utilizing L-galactono-1,4-
lactone (12) as a chiral source (Scheme 1). We have
recently reported an efficient approach to the multifunc-
tionalized gem-diamine 1-N-iminosugars.7 This method-
ology, conveniently modified, should allow the introduc-
tion of the diastereomeric amino and carboxylic acid
substituents at C-2 and C-5, respectively, into the
versatile aminal 24 (Scheme 1). The synthesis of the
aminal 24 began with the known 5,6-O-isopropylidene-
L-galactono-1,4-lactone (13),17 which was converted into
the diol 15 upon protection and lithium aluminum
hydride reduction in good yield. Selective protection of
the hydroxymethyl group with the tert-butyldiphenylsilyl
(TBDPS) group in 15 followed by the Dess-Martin
oxidation18 gave the ketone 17 in 93% yield. One-carbon
extension of 17 was carried out by the Wittig reaction
with methylenetriphenylphosphorane to afford the me-
thylene 18 in 96% yield. Removal of the isopropylidene
group gave the diol 19, which was successfully trans-
formed into the monoalcohol 20 upon the Luche reduc-
tion19 of the labile aldehyde intermediate obtained by
periodate oxidation. Conversion of the hydroxyl group to
the amino function proved troublesome until it was
discovered that the corresponding sulfonate was con-
verted into the azido 21 by one-pot reaction in situ in
89% yield. Selective reduction of the azide group with
metal hydrides such as sodium and lithium borohydride,
(12) Govan, J . R. W.; Harris, G. S. Microbiol. Sci. 1986, 3, 302.
(13) Evans, L. R.; Linker, A. J . Bacteriol. 1973, 116, 915.
(14) (a) Hoiby, N. Acta Pathol. Microbiol. Scand. Sect. B Microbiol.
1974, 82, 551. (b) Baltimore, R. S.; Mitchell, M. J . Infect. Dis. 1980,
141, 238. (c) Schwarzmann, S.; Boring, J . R., III. Infect. Immun. 1971,
3, 762. (d) Oliver, A. M.; Weir, D. M. J . Clin. Lab. Immunol. 1983, 10,
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B Microbiol. 1973, 81, 298.
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1978, 4, 233. (b) Slack, M. P. E.; Nichols, W. W. Lancet 1981, 2, 502.
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Dale, D. C.; Pennington, J . E. Ann. Intern. Med. 1975, 82, 819. (b)
Burns, M. W.; May, J . R. Lancet 1968, 1, 270. (c) Diaz, F.; Mosovich,
L. L.; Neter, E. J . Infect. Dis. 1970, 121, 269.
(17) Morgenlie, S. Carbohydr. Res. 1982, 107, 137.
(18) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155.
(19) (a) Luche, J .-L. J . Am. Chem. Soc. 1978, 100, 2226. (b) Luche,
J .-L.; Rodriguez-Hahn, L.; Crabbe´, P. J . Chem. Soc., Chem. Commun.
1978, 601. (c) Gemal, A. L.; Luche, J .-L. J . Am. Chem. Soc. 1981, 103,
5454.

4
J . Org. Chem., Vol. 65, No. 1, 2000
Sch em e 1. Syn th esis of Ur on ic Acid -Typ e gem -Dia m in e 1-N-Im in osu ga r sa
Nishimura et al.
a
Key: (a) CH3OCH2Cl, n-Bu4NI, i-Pr2NEt, 70 °C, 81%; (b) LiAlH4, THF, 100%; (c) t-bu(Ph2)SiCl, i-Pr2NEt, DMAP, CH2Cl2, 99.7%; (d)
Dess-Martin periodinane, CH2Cl2, 93%; (e) Ph3PCH3Br, n-BuLi, THF, -78 °C, 96%; (f) 80% AcOH, rt, 99%; (g) NaIO4, CH3CN/H2O;
NaBH4, CeCl3, MeOH, 88%; (h) MsCl, py; NaN3, DMF, 88.7%; (i) Te, NaBH4, EtOH; (t-BuCO)2O, i-Pr2NEt, DMF, 88%; (j) n-Bu4NF, THF,
100%; (k) (COCl)2, DMSO, CH2Cl2, 93%; (l) PPh3, DEAD, phthalimide, DMF, 25: 61.4%; 26: 20%.
lithium aluminum hydride, diisobutylaluminum hydride,
and K- and L-Selectride proceeded unfavorably to yield
a complicated mixture. This problem was circumvented
by catalytic hydrogenation with sodium hydrogentellu-
ride (NaTeH)20 generated in situ from tellurium and
sodium borohydride in ethanol. The desired amide 22 was
obtained effectively by the subsequent protection with a
tert-butyloxycarbonyl (t-Boc) group in 88% yield. Removal
of a TBDPS group and the Swern oxidation21 afforded
the pivotal intermediate 24 as an epimeric mixture in
93% yield. Replacement of the aminal hydroxyl group to
the amino group was achieved by the Mitsunobu reac-
tion22 (PPh3, diethyl azodicarboxylate, phthalimide) in
N,N′-dimethylformamide (DMF) to give both the desired
epimers of iminophthalimides 25 and 26 in 61 and 20%,
respectively. Epimer 25 was crystallized from n-hexane
to yield a single crystal for X-ray diffraction analysis. The
X-ray analysis clearly indicated the desired absolute
stereochemistry and a boat conformation (Figure 4).
Another epimer, 26, was consequently assigned its
absolute stereochemistry and also its presumed a boat
conformation by its 1H NMR spectrum. Hydroboration
of 25 with borane-methyl sulfide complex followed by
oxidation with hydrogen peroxide efficiently gave the
D-gluco isomer 27 and the L-idulo isomer 28 in 17 and
77% yield, respectively (Scheme 2). Hydrazinolysis of 27
and conventional trifluoroacetylation furnished the tri-
F igu r e 4. ORTEP drawing of compound 25.
fluoroacetamide 29 in 90% yield. Conversion of the
hydroxymethyl group of 29 to the carboxylic acid was best
achieved by ruthenium tetraoxide-catalyzed oxidation in
a solvent system of CH3CN/CCl4/H2O developed by
Sharpless et al.23 in 91% yield. Simultaneous removal of
both the methoxymethyl (MOM) and t-Boc groups in 30
with 4 M hydrogen chloride in dioxane afforded quanti-
(20) (a) Suzuki, H.; Takaoka, K. Chem. Lett. 1984, 1733. (b) Izawa,
T.; Nishimura, Y.; Kondo, S. Carbohydr. Res. 1991, 211, 137.
(21) (a) Mancuso, A. J .; Huang, S.-L.; Swern, D. J . Org. Chem. 1978,
43, 2480. (b) Nishimura, Y.; Wang, W.; Kondo, S.; Aoyagi, T.; Umezawa,
H. J . Am. Chem. Soc. 1988, 110, 7249.
(22) For a review, see: Mitsunobu, O. Synthesis 1981, 1.
(23) (a) Carlson, P. H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K.
B. J . Org. Chem. 1981, 46, 3936. (b) Satoh, T.; Nishimura, Y.; Kondo,
S.; Takeuchi, T. Carbohydr. Res. 1996, 286, 173.

Uronic Acid-Type gem-Diamine 1-N-Iminosugars
J . Org. Chem., Vol. 65, No. 1, 2000
5
Sch em e 2. Syn th esis of Ur on ic Acid -Typ e gem -Dia m in e 1-N-Im in osu ga r sa
a
Key: (a) BH3‚Me2S, THF; H2O2, 2 M NaOH/H2O, 27: 16.6% 28: 77.1%; 33: 50%, 34: 38%; (b) H2NNH2.xH2O, MeOH; (CF3CO)2O,
py, CH2Cl2, 29: 90%; 31: 87%; 36: 88%; 40: 79%; (c) RuO2, NaIO4, CCl4/CH3CN/H2O, 30: 91%; 32: 90%; 38: 92%; 42: 87%; (d) 4 M
HCl/dioxane, 8: 99.7%; 9: 99%; 10: 99.7%; 11: 91%; (e) t-Bu(Me2)SiCl, imidazole, DMF, 35: 91%; 39: 100%; (f) n-Bu4NF, THF, 37: 93%;
41: 100%.
Ta ble 1. In h ibitor y Activities (IC50 (M)) of 8, 9, 10, 11,
tatively the final D-glucuronic acid-type 2-trifluoroaceta-
a n d 45 a ga in st Glycosid a ses
mido-1-N-iminosugar 8 as a hydrochloride. The 1H NMR
enzyme
8
9
10
11
45
spectrum of D-glucuronic acid-type 8 shows the large
coupling constants (J 2,3 ) J 3,4 ) 9.3 Hz, J 4,5 ) 10.5 Hz
and J 5,6ax ) 13.0 Hz) and a small coupling constant (J 5,6eq
â-glucuronidasea 6.5 × 10-8 1.3 × 10-4 6.5 × 10-8 NIi 6.5 × 10-8
R-glucosidadeb
â-glucosidasec
NI
NI
NI
NI NI
9.8 × 10-5 NI
3.6 × 10-5 NI 1.3 × 10-5
4
) 4.4 Hz), clearly indicative of the C1-conformation and
R-mannosidased NI
â-mannosidasee NI
â-galactosidasef NI
R-N-acetylgalact- NI
osaminidaseg
NI
NI
NI
NI
NI
NI
NI
NI
NI NI
the correct stereochemistry. The same sequence of reac-
tions also successfully resulted in L-iduronic acid-type
2-trifluoroacetamido-1-N-iminosugar 9 from 28. The
NI NI
NI 1.3 × 10-6
NI NI
coupling constants (J 2,3 ) J 3,4 ) 6.4 Hz, J 4,5 ) J 5,6eq
)
â-N-acetylgluc-
NI
NI
NI
NI NI
osaminidaseh
1
3.9 Hz and J 5,6ax ) 7.3 Hz) of the H NMR spectrum of
L-iduronic acid-type 9 are indicative of the boat confor-
mation. On the other hand, D-mannuronic acid-type and
L-guluronic acid-type 2-trifluoroacetamido-1-N-iminosug-
ars (10 and 11) were straightforwardly obtained from 26
by a similar sequence of structure changes varying in the
protection of the hydroxymethyl groups of 33 and 34 with
a tert-butyldimethylsilyl (TBDMS) group prior to hy-
drazinolysis of the phthalimide group for improvement
in yield. The protons of H-2 (d, J 2,3 ) 2 Hz), H-3 (dd, J 3,4
) 4 Hz), H-4 (t, J 4,5 ) 4 Hz), and H-5 (dt, J 5,6ax ) 4 Hz
a
b
d
Bovine liver. Bakers’ yeast. c Almonds. J ack beans. e Snail.
f Aspergillus niger. Chicken liver. Bovine epididymis. NI: no
g
h
i
inhibition at 3.3 × 10-3 M.
F igu r e 5. Effect of resonance contributors (43 and 44) to the
putative glycosyl cation intermediate formed during hydrolysis
by â-D-glucuronidase.
1
and J 5,6eq ) 1.6 Hz) appear in the H NMR spectrum of
D-mannuronic acid 10. This indicated that 10 has the
4
boat conformation as opposed to the C1 conformation of
D-glucuronic acid-type gem-diamine 1-N-iminosugar 8
affected â-D-glucuronidase very potently. This is rational-
ized that 8 should closely mimic a glycopyranosyl cation
43, one of the resonance contributors (the chairlike and
the flattened conformational cation 43 and 44, respec-
tively) of the putative glycopyranosyl cation intermediate
formed during hydrolysis by â-D-glucuronidase (Figure
5).7d,24 It is interesting that D-mannuronic acid-type gem-
diamine 1-N-iminosugar 10 also inhibited very strongly
â-D-glucuronidase. On the other hand, the conformation
of 10 is indicated to be a boat conformation by its 1H NMR
D-glucuronic acid 8. On the other hand, the 1H NMR
spectrum of L-guluronic acid 11 shows the protons of H-2
(d, J 2,3 ) 1.7 Hz), H-3 (dd, J 3,4 ) 3.9 Hz), H-4 (dd, J 4,5
)
2.9 Hz), and H-5 (ddd, J 5,6eq ) 5.4 Hz and J 5,6ax ) 13.0
Hz), clearly indicating the 1C4 conformation with the
exact stereochemistry. This is distinct from the boat
conformation of L-iduronic acid type 9.
Biologica l Activity. The inhibitory activities of uronic
acid-type gem-diamine 1-N-iminosugars (present 8-11
and previous 45) for â-glucuronidase (bovine liver),
R-glucosidase (bakers’ yeast), â-glucosidase (almonds),
R-mannosidase (jack beans), â-mannosidase (snail), â-ga-
lactosidase (Aspergillus niger), R-N-acetylgalactosamini-
dase (chicken liver), and â-N-acetylglucosaminidase (bo-
vine epididymis) are summarized in Table 1. As expected,
(24) (a) Cordes, E. H.; Bull, H. G. Chem. Rev. 1974, 74, 581. (b)
Perkins, S. J .; J ohnson, L. N.; Philips, D. C.; Dwek, R. A. Biochem. J .
1981, 193, 553. (c) Sinnott, M. L. Chem. Rev. 1990, 90, 1171. (d)
Kajimoto, T.; Liu, K. K.-C.; Pederson, R. L.; Zhong, Z.; Ichikawa, Y.;
Porco, J . A., J r.; Wong, C.-H. J . Am. Chem. Soc. 1991, 113, 6187.

6
J . Org. Chem., Vol. 65, No. 1, 2000
Nishimura et al.
Ta ble 2. In h ibitor y Activities (IC50) of 8, 10, a n d 45
a ga in st Hu m a n Hep a r a n a sea
compd
IC50 (µM)
8
9
10.55 ( 1.01
NI
10
11
45
28.99 ( 11.41
NI
1.02 ( 0.29
a
NI: no inhibition at 3.3 mM. Buffer: 50 mM AcNA, pH 4.2,
0.02% CHAPS. Enzyme: heparanase 0.26 µg protein/tube. Sub-
strate: FITC-Heparan sulfate 0.5 µL (5 µg HS). Incubation time:
37 °C, 2 h.
F igu r e 6. Structures of aza-sugars and δ-lactams.
â-glucuronidase from bovine liver. Furthermore, the
weak activities against heparanase compared with exo-
â-glucuronidase indicate that heparanase should recog-
nize simultaneously glucuronic acid and the adjacent
glycoses on the both sides. Compounds 8, 10, and 45 are
also shown to be moderate glucosidase inhibitors, and
45 shows good inhibition against galactosidase, indicating
that even a charged carboxyl function assists the binding
to these enzymes. On the other hand, as expected,
L-uronic acid-type gem-diamine 1-N-iminosugars 9 and
11 showed no remarkable inhibition against these D-
sugar hydrolases. These results indicate that glycohy-
drolases recognize precisely the absolute configurations
of gem-diamine 1-N-iminosugars corresponding to D- and
L-sugars for specificity and potency. Compounds 8-11
and 45 were also assayed in vitro for inhibition of
biosynthesis of glycocalyx bifilm (alginate-like polysac-
charides) composed of D-mannuronic acid (6) and L-
guluronic acid (7) produced by Pseudomonas aeruginosa
A3. All compounds showed no inhibition at a dose of 3.3
× 10-3M (see the Experimental Section). However, it is
not clear at this stage whether D-mannuronic acid-type
and L-guluronic acid-type 1-N-iminosugars (10 and 11)
may inhibit the metabolism of glycocalyx biofilm or
penetrate through the cell-membrane of Pseudomonas
aeruginosa. Further evaluation of biological activities
(antimetastatic, antibacterial, etc.) of these analogues are
now in progress.
In summary, an efficient and flexible synthetic route
to uronic acid-type gem-diamine 1-N-iminosugars from
a readily available L-galactono-1,4-lactone has been
developed and has produced the highly potent inhibitors
of â-glucuronidase. The present gem-diamine 1-N-imi-
nosugars may contribute to the study of the involvement
of carbohydrates in diseases associated with oligosaccha-
ride biosynthesis and degradation and also to the devel-
opment of drugs for these diseases. That these gem-
diamine 1-N-iminosugars are potent inhibitors of â-glucur-
onidase further supports the hypothesis of our design of
the new type inhibitor.
F igu r e 7. Structures of uronic acid- and glycose-type gem-
diamine 1-N-iminosugars.
spectrum. These facts suggest that 10 is highly likely a
mimic of the another glycopyranosyl cation, the flattened
conformational cation 44 in the course of the enzymatic
reaction (Figure 5). This result is also compatible with
the well-known results25 in which mannojirimycin (51)
and D-mannono-δ-lactam (53) show somewhat stronger
inhibitory activities against glucosidase than those of
nojirimycin (50) and D-glucono-δ-lactam (52), respectively
(Figure 6). It was also previously reported that D-
galacturonic acid-type gem-diamine 1-N-iminosugar 45
is proved to be a potent glucuronidase inhibitor (Table
1).26 In the case of gem-diamine 1-N-iminosugar glycosi-
dase inhibitors, hydrolases recognizing the D-gluco-con-
figuration such as D-glucosidase, N-acetyl-D-glucosamini-
dase, and D-glucuronidase similarly recognize the D-gal-
acto-configuration, IC50 (M, almond â-glucosidase) 4.7 ×
10-7 (46)27 and 4.4 × 10-7 (47);28 IC50 (M, bovine epid-
idymis-N-acetyl-â-D-glucosaminidase) 1.9 × 10-6 (48)7c
and 1.2 × 10-5 (49);28 IC50 (M, bovine liver â-glucu-
ronidase) 6.5 × 10-8 (8) and 6.5 × 10-8 (45) (Figure 7).26
These results indicate that the binding group corre-
sponding to the 4-OH in these enzymes is not important
for specificity of the inhibitors. Compounds 8-11 and 45
were also evaluated the inhibitory activity against re-
combinant human heparanase (endo-â-glucuronidase)
from human melanoma A375M cell transfected with
pBK-CMV expression vectors containing the heparanase
cDNA.29 Heparanase cleaves the â-1,4-linkage between
glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc)
in heparan sulfate.9c As shown in Table 2, 8, 10, and 45
showed inhibition against the enzyme. The result also
proves the relationships between structure and activity
similar to those discussed on the inhibition against exo-
Exp er im en ta l Section
5,6-O-Isop r op ylid en e-2,3-d i-O-(m eth oxym eth yl)-L-ga -
la cton o-1,4-la cton e (14). To a solution of 1317 (6 g, 27.5
mmol) in N,N-diisopropylethylamine (57.5 mL, 330 mmol)
were added chloromethyl methyl ether (12.5 mL, 110 mmol)
and tetra-N-butylammonium iodide (10.1 g, 27.5 mmol), and
the mixture was stirred at 70 °C for 3 h. Evaporation of the
solvent gave an oil, which was dissolved in CHCl3. The solution
was washed with H2O, dried over MgSO4, and filtered.
Evaporation of the filtrate gave an oil, which was subjected
to flash column chromatography on silica gel. Elution with
toluene-acetone (4:1) gave 14 as a colorless solid (7.6 g, 90%).
The solid was crystallized from n-hexane to give colorless
(25) (a) Niwa, T.; Inouye, S.; Tsuruoka, T.; Koaze, Y.; Niida, T. J .
Antibiot. 1970, 34, 966. (b) Reese, E. T.; Parrish, F. W. Carbohydr.
Res. 1971, 18, 381. (c) Niwa, T.; Tsuruoka, T.; Goi, H.; Kodama, Y.;
Itoh, J .; Inouye, S.; Yamada, Y.; Niida, T.; Nobe, M.; Ogawa, Y. J .
Antibiot. 1984, 37, 1579. (d) Legler, G.; J u¨lich, E. Carbohydr. Res. 1984,
128, 61.
(26) Nishimura, Y.; Kudo, T.; Kondo, S.; Takeuchi, T. J . Antibiot.
1992, 45, 963.
(27) Shitara, E.; Nishimura, Y.; Takeuchi, T. J . Antibiot. 1999, 52,
348.
(28) Shitara, E.; Nishimura, Y.; Kojima, F.; Takeuchi, T. Bioorg.
Med. Chem. 1999, 7, 1241.
(29) Toyoshima, M.; Nakajima, M. J . Biol. Chem. 1999, 274, 24153.

Uronic Acid-Type gem-Diamine 1-N-Iminosugars
J . Org. Chem., Vol. 65, No. 1, 2000
7
crystals of 14: [R]23 +13.7° (c 0.3, CHCl3); mp 58 °C; NMR
(7:1) gave 18 as an oil (6.5 g, 96%): [R[23D -34.5° (c 0.7, CHCl3);
1H NMR (400 MHz, CDCl3) δ 1.07 (9H, s), 1.40 and 1.44 (3H,
s each), 3.24 and 3.32 (3H, s each), 3.61 (1H, t, J ) 7.8 Hz),
3.69 (1H, dt, J ) 3.9, 5.6 Hz), 3.72-3.85 (2H, m), 4.14 (1H,
dd, J ) 6.3, 7.8 Hz), 4.30 (1H, d, J ) 3.9 Hz), 4.50-4.65 (5H,
m), 5.31 and 5.47 (1H, s each) and 7.20-7.80 (10H, m). Anal.
Calcd for C30H44O7Si: C, 66.12; H, 8.17. Found: C, 65.73; H,
7.93.
D
(CDCl3, 400 MHz) δ 1.38 and 1.41 (3H, s each), 3.41 and 3.43
(3H, s each), 3.99 (1H, dd, J ) 6.8, 7.3 Hz), 4.11 (1H, dd, J )
6.8, 7.3 Hz), 4.20 (1H, dd, J ) 2.6, 6.8 Hz), 4.38 (1H, dt, J )
2.6, 6.8 Hz), 4.47 (1H, t, J ) 7.0 Hz), 4.53 (1H, d, J ) 7.0 Hz),
6.84 and 6.83 (2H, ABq, J ) 6.8 Hz) and 4.78 and 5.06 (2H,
ABq, J ) 6.6 Hz). Anal. Calcd for C13H22O8: C, 50.97; H, 7.24.
Found: C, 50.94; H, 7.25.
1,2-O-Isop r op ylid en e-4,5-d i-O-(m eth oxym eth yl)-D-ga -
la ctitol (15). To a solution of 14 (10.35 g, 33.8 mmol) in THF
(200 mL) was added LiAlH4 (2.6 g, 68.5 mmol), and the mixture
was stirred at room temperature for 1 h. After the mixture
was quenched with a saturated aqueous Na2SO4 solution, an
organic phase was taken by decantation. Evaporation of the
solvent gave an oil, which was subjected to flash column
chromatography on silica gel. Elution with toluene-acetone
6-O-(ter t-Bu t yld ip h en ylsilyl)-3-d eoxy-4,5-d i-O-(m et h -
oxym eth yl)-3-m eth ylen e-D-xylo-h exitol (19). Compound 18
(6.5 g, 15.5 mmol) was dissolved in 80% acetic acid (325 mL),
and the solution was stirred at room temperature overnight.
Evaporation of the solvent gave an oil, which was subjected
to flash column chromatography on silica gel. Elution with
toluene-acetone (5:1) gave 19 as an oil (5.8 g, 99%): [R]23
D
-31.5° (c 0.82, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.13 (9H,
s), 2.50 (1H, br t, J ) 5.4 Hz), 3.11 (1H, d, J ) 3.9 Hz), 3.33
and 3.35 (3H, s each), 3.71 (1H, br t, J ) 5.4 Hz), 3.75-3.90
(3H, m), 4.21 (1H, br q, J ) 3.9 Hz), 4.35 (1H, d, J ) 2.9 Hz),
4.65 and 4.68 (2H, ABq, J ) 6.8 Hz), 4.69 and 4.72 (2H, ABq,
J ) 6.8 Hz), 5.44 and 5.48 (1H, s each) and 7.35-7.80 (10H,
m). Anal. Calcd for C27H40O7Si: C, 64.25; H, 7.99. Found: C,
64.02; H, 8.06.
(4:1) gave 15 as an oil (10.5 g, 100%): [R]23 -5.4° (c 1.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.37 and 1.45 (3H, s
each), 2.59 (1H, d, J ) 8.3 Hz), 3.42 (1H, m), 3.43 and 3.47
(3H, s each), 3.65∼3.80 (3H, m), 3.83 (1H, ddd, J ) 6.6, 11.7
Hz), 3.97 (1H, t, J ) 7.6 Hz), 4.02 (1H, dt, J ) 2.0, 6.6 Hz),
4.06 (1H, t, J ) 7.8 Hz), 4.31 (1H, dt, J ) 1.5, 7.6 Hz), 4.68
and 4.81 (2H, ABq, J ) 6.4 Hz) and 5.73 and 4.77 (2H, ABq,
J ) 5.8 Hz). Anal. Calcd for C13H26O8: C, 50.31; H, 8.44.
Found: C, 50.00; H, 8.52.
2-Deoxy-6-O-(ter t-b u t yld ip h en ylsilyl)-3,4-d i-O-(m et h -
oxym eth yl)-2-m eth ylen e-D-th r eo-p en titol (20). To a solu-
tion of 19 (4.2 g, 11 mmol) in CH3CN (110 mL) was added a
solution of NaIO4 (2.36 g, 11 mmol) in H2O (24 mL), and the
mixture was stirred at room temperature for 1 h. After dilution
with ethyl acetate (200 mL), the resulting inorganic material
was filtered off. The filtrate was washed with H2O, dried over
MgSO4, and filtered. Evaporation of the filtrate gave an oil
(3.73 g), which was dissolved in CH3OH (96 mL). To the
solution were added CeCl3 (3.9 g, 15.8 mmol) and NaBH4 (592
mg, 15.6 mmol), and the mixture was stirred at room temper-
ature for 1 h. After dilution with ethyl acetate (200 mL), the
resulting precipitates were filtered off. The filtrate was washed
with H2O, dried over MgSO4, and filtered. Evaporation of the
filtrate gave a viscous oil, which was subjected to flash column
chromatography on silica gel. Elution with toluene-acetone
6-O-(ter t-Bu tyld ip h en ylsilyl)-1,2-O-isop r op ylid en e-4,5-
d i-O-(m eth oxym eth yl)-D-ga la ctitol (16). To a solution of 15
(8.8 g, 28.4 mmol) in CH2Cl2 (172 mL) were added TBDPSCl
(9.6 mL, 36.9 mmol), N,N-diisopropylethylamine (20.3 mL,
116.5 mmol), and DMAP (4.3 g, 35.2 mmol), and the mixture
was stirred at room temperature for 1 h. After the mixture
was quenched with H2O, evaporation of the solvent gave an
oil. The oil was dissolved in CHCl3, and the solution was
washed with water, dried over MgSO4, and filtered. Evapora-
tion of the filtrate gave an oil, which was subjected to flash
column chromatography on silica gel. Elution with toluene-
acetone (7:1) gave 16 as an oil (12 g, 99.7%): [R[23 -10.2° (c
D
1
0.83, CHCl3); H NMR (400 MHz, CDCl3) δ 1.06 (9H, s), 1.37
and 1.45 (3H, s each), 3.09 (1H, d, J ) 7.6 Hz), 3.22 and 3.34
(3H, s each), 3.70 (1H, dt, J ) 2.20, 7.6 Hz), 3.77 (1H, dd, J )
2.0, 7.6 Hz), 3.8-3.9 (2H, m), 3.9-4.1 (2H, m), 4.10 (1H, ddd,
J ) 2.0, 5.6, 6.8 Hz), 4.32 (1H, dt, J ) 2.0, 7.6 Hz), 4.62 and
4.72 (2H, ABq, J ) 6.6 Hz), 4.71 and 4.83 (2H, ABq, J ) 6.6
Hz) and 7.3-7.7 (10H, m). Anal. Calcd for C29H44O8Si: C,
63.47; H, 8.08. Found: C, 63.24; H, 8.06.
(15:1) gave 20 as an oil (3.4 g, 88%): [R[23 -41.3° (c 1.1,
D
1
CHCl3); H NMR (400 MHz, CDCl3) δ 1.06 (9H, s), 2.41 (1H,
br t, J ) ∼6 Hz), 3.72-3.82 (2H, m), 3.87 (1H, dd, J ) 4.4, 4.9
Hz), 4.05-4.420 (2H, m), 4.4 (1H, d, J ) 4.4 Hz), 4.58 and
4.63 (2H, ABq, J ) 6.6 Hz), 4.66 and 4.71 (2H, ABq, J ) 6.6
Hz), 5.23 (1H, s), 5.30 (1H, s) and 7.3-7.8 (10H, m). Anal.
Calcd for C26H38O6Si: C, 65.79; H, 8.07. Found: C, 66.20; H,
8.23.
D-th r eo-D-glycer o-6-O-(ter t-Bu t yld ip h en ylsilyl)-1,2-O-
isop r op ylid e n e -4,5-d i-O-(m e t h oxym e t h yl)-3-h e xosu li-
tol (17). To a solution of 16 (59.2 g, 139 mmol) in CH2Cl2 (800
mL) was added Dess-Martin periodinane (68.6 g, 161.7 mmol),
and the mixture was stirred at room temperature for 2 h. After
dilution with a large amount of ether, the solution was washed
with a saturated aqueous NaHCO3 solution and H2O, dried
over MgSO4, and filtered. Evaporation of the filtrate gave an
oil, which was subjected to flash column chromatography on
silica gel. Elution with toluene-acetone (7:1) gave 17 as an
oil (55 g, 93%): [R]23D -5.1° (c 0.76, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 1.07 (9H, s), 1.40 and 1.48 (3H, s each), 3.17 and
3.34 (3H, s each), 3.75-3.85 (2H, m), 4.12-4.23 (2H, m), 4.26
(1H, t, J ) 8.0 Hz), 4.47 and 4.54 (2H, ABq, J ) 6.8 Hz), 4.65
(1H, d, J ) 2.4 Hz), 4.68 and 4.71 (2H, ABq, J ) 6.6 Hz), 4.91
(1H, dd, J ) 5.9, 8.0 Hz) and 7.3-7.7 (10H, m). Anal. Calcd
for C29H42O8Si: C, 63.71; H, 7.74. Found: C, 63.94; H, 7.79.
6-O-(t-Bu tyldiph en ylsilyl)-3-deoxy-1,2-O-isopr opyliden e-
4,5-di-O-(m eth oxym eth yl)-3-m eth ylen e-D-xylo-h exitol (18).
To a solution of methylenetriphenylphosphorane, prepared
from methyltriphenylphosphonium bromide (8.9 g, 24.9 mmol)
and n-butyllithium (1.6 M/L, 15.6 mL) in tetrahydrofuran (140
mL) from -78 °C to room temperature, was added a solution
of 17 (6.8 g, 16.1 mmol), and the resulting mixture was stirred
from -78 °C to room temperature overnight. After being
quenched with saturated aqueous NH4Cl solution, the mixture
was extracted with ether (3 × 100 mL). The extracts were
washed with H2O, dried over MgSO4, and filtered. Evaporation
of the filtrate gave an oil, which was subjected to flash column
chromatography on silica gel. Elution with toluene-acetone
1-Azid o-1,2-d id eoxy-6-O-(ter t-b u t yld ip h en ylsilyl)-3,4-
d i-O-(m eth oxym eth yl)-2-m eth ylen e-D-th r eo-p en titol (21).
To a solution of 20 (26.3 g, 75 mmol) in a mixture of CH2Cl2
(263 mL) and pyridine (53.8 mL, 665 mmol) was added CH3-
SO2Cl (7.72 mL, 99.7 mmol), and the mixture was stirred at
room temperature for 1 h. After addition of NaN3 (36 g, 554
mmol), evaporation of the solvent gave a viscous oil, which
was suspended in DMF (263 mL). The resulting mixture was
stirred at room temperature for 1 h. Evaporation of the solvent
gave a viscous oil, which was dissolved in CHCl3 (500 mL).
The solution was washed with H2O, dried over MgSO4, and
filtered. Evaporation of the filtrate gave an oil, which was
subjected to flash column chromatography on silica gel. Elution
with toluene-acetone (10:1) gave 21 as an oil (25 g, 88.7%):
[R[23D -4.8° (c 0.66, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.13
(9H, s), 3.32 and 3.38 (3H, s each), 3.7-4.0 (5H, m), 4.48 (1H,
d, J ) 3.9 Hz), 4.66 and 4.69 (2H, ABq, J ) 6.8 Hz), 4.67 and
4.73 (2H, ABq, J ) 6.8 Hz), 5.37 (1H, s), 5.41 (1H, s) and 7.4-
7.8 (10H, m). Anal. Calcd for C26H37N3O5Si: C, 62.49; H, 7.46;
N, 8.41. Found: C, 62.51; H, 7.52; N, 8.37.
6-O-(ter t-Bu tyld ip h en ylsilyl)-1-ter t-bu tyloxyca r bon yl-
a m id o-1,2-d id eoxy-3,4-d i-O-(m et h oxym et h yl)-2-m et h yl-
en e-D-th r eo-p en titol (22). To a suspension of tellurium
powder (885 mg, 6.9 mmol) in EtOH (39 mL) was added NaBH4
(315 mg, 8.3 mmol), and the mixture was refluxed with stirring
under argon for 1 h. To the resulting purple solution was added
dropwise a solution of 21 (1.73 g, 4.6 mmol) in EtOH (12 mL)
with stirring, and the mixture was stirred at room temperature

8
J . Org. Chem., Vol. 65, No. 1, 2000
Nishimura et al.
for 1 h. After dilution with H2O (20 mL), the mixture was
stirred under air until the purple color disappeared. The
resulting insoluble materials were filtered off, and the filtrate
was extracted with CH2Cl2. The extracts were washed with
H2O, dried over MgSO4, and filtered. Evaporation of the
solvent gave an oil, which was subjected to flash column
chromatography on silica gel. Elution with CHCl3-CH3OH (10:
1) gave an oil (1.47 g). To a solution of the resulting oil in DMF
(34 mL) were added i-Pr2NEt (3.2 mL, 18 mmol) and di-tert-
butyl dicarbonate (2.0 mL, 8.7 mmol), and the mixture was
stirred at room temperature overnight. Evaporation of the
solvent gave an oil, which was subjected to flash column
chromatography on silica gel. Elution with CHCl3-CH3OH (50:
1) gave 22 as an oil (1.82 g, 88%): [R]23D -34.1° (c 0.51, CHCl3);
1H NMR (400 MHz, CDCl3) δ 1.06 (9H, s), 1.44 (9H, s), 3.27
and 3.31 (3H, s each), 3.70-3.80 (5H, m), 4.35 (1H, br s), 4.57
and 4.63 (2H, ABq, J ) 6.8 Hz), 4.65 and 4.69 (2H, ABq, J )
6.6 Hz), 4.81 (1H, br s), 5.18 (1H, d, J ) <1.0 Hz), 5.20 (1H, s)
and 7.30-7.70 (10H, m). Anal. Calcd for C31H47NO7Si: C,
64.89; H, 8.26. Found: C, 64.95; H, 8.36.
was subjected to the column chromatography on silica gel.
Elution with toluene-acetone (15:1) gave a colorless solid of
25 (8.86 g, 61.4%) and 26 as a colorless solid (2.74 g, 20%). 25
was crystallized from n-hexane to give colorless crystals of 25.
25: [R]23 +98.5° (c 0.47, CHCl3); mp 99 °C; NMR (CDCl3,
D
400 MHz) δ 1.41 (9H, s), 3.02 and 3.43 (3H, s each), 4.28 (1H,
dd, J ) 1.5, 9.8 Hz), 4.33 (1H, dd, J ) 6.8, 9.8 Hz), 4.43 (1H,
dq, J ) 1.9, 16.3 Hz), 4.61 and 4.85 (2H, ABq, J ) 6.8 Hz),
4.68 (1H, br d, J ) 16.3 Hz), 4.73 and 4.75 (2H, ABq), 5.18
and 5.28 (1H, br s each), 5.99 (1H, d, J ) 6.8 Hz) and 7.70∼7.90
(4H, m). Anal. Calcd for C23H30N2O8: C, 59.73; H, 6.54; N, 6.06.
Found: C, 59.81; H, 6.47; N, 5.94.
26: [R]23 -33.7° (c 0.81, CHCl3); NMR (CD3OD, 400 MHz,
D
40 °C) δ 1.38 (9H, br s), 3.30 and 3.43 (3H, s each), 4.02 (1H,
dd, J ) 6.6, 8.5 Hz), 4.33 and 4.56 (1H, br d each, J ) 14.2
Hz), 4.65 and 4.69 (2H, ABq, J ) 6.8 Hz), 4.47 and 4.79 (2H,
ABq, J ) 6.8 Hz), 4.78 (1H, br d, J ) 6.6 Hz), 5.23 (1H, br s),
5.29 (1H, dq, J ) 1.5 Hz), 6.51 (1H, br d, J ) 8.5 Hz) and
7.80-7.95 (4H, m). Anal. Calcd for C23H30N2O8: C, 59.73; H,
6.54; N, 6.06. Found: C, 59.53; H, 6.36; N, 5.90.
1-ter t-Bu t yloxyca r b on yla m id o-1,2-d id eoxy-3,4-d i-O-
(m eth oxym eth yl)-2-m eth ylen e-D-th r eo-p en titol (23). A
solution of n-Bu4NF (1 M/L, 4.63 mL) was added to a solution
of 22 (1.77 g, 3.9 mmol) in THF (35 mL), and the mixture was
stirred at room temperature for 1 h. Evaporation of the solvent
gave an oil, which was subjected to flash column chromatog-
raphy on silica gel. Elution with CHCl3-CH3OH (20:1) gave
(+)-(2S ,3R ,4R ,5R )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym et h yl-3,4-d i-O-(m et h oxym et h yl)-2-p h t h a lim id o-
3,4-p ip er id in ed iol (27) a n d (+)-(2S,3R,4R,5S)-N-(ter t-Bu -
t y lo x y c a r b o n y l)-5-h y d r o x y m e t h y l-3,4-d i -O -(m e t h -
oxym eth yl)-2-p h th a lim id o-3,4-p ip er id in ed iol (28). To a
solution of 25 (6.37 g, 13.8 mmol) in THF (170 mL) was added
a 2 M solution of BH3‚(CH3)2S in THF (1 M/L, 18.4 mL, 18.4
mmol), and the mixture was stirred at room temperature
overnight. To the mixture were added 2 M solution of NaOH
in water (26.5 mL) and then 30% solution of H2O2 in water
(17 mL), and the mixture was stirred at room temperature
for 2 h. After dilution with CH2Cl2, the solution was washed
with a saturated solution of NaCl in water, dried over MgSO4,
and filtered. Evaporation of the filtrate gave an oil, which was
subjected to the column chromatography on silica gel. Elution
with toluene-acetone (10:1) gave 27 as a solid (1.1 g, 16.6%)
and 28 as a solid (5.1 g, 77.1%).
23 as an oil (1.32 g, 100%): [R]23 -13° (c 0.53, CHCl3); 1H
D
NMR (400 MHz, CDCl3) δ 1.44 (9H, s), 2.88 (1H, t, J ) 6.1
Hz), 3.39 and 5.48 (3H, s each), 3.55-3.85 (5H, m), 4.25 (1H,
d, J ) 5.9 Hz), 4.58 and 4.63 (2H, ABq, J ) 6.6 Hz), 4.7 and
4.8 (2H, ABq, J ) 6.8 Hz), 4.84 (1H, br s) and 5.2 (2H, s). Anal.
Calcd for C15H29NO7: C, 53.71; H, 8.72; N, 4.18. Found: C,
53.47; H, 8.75; N, 3.93.
5-ter t-Bu t yloxyca r b on yla m id o-4,5-d id eoxy-2,3-d i-O-
(m eth oxym eth yl)-4-m eth ylen e-D-th r eo-R- a n d -â-p en to-
p yr a n ose (24). Dimethyl sulfoxide (14.5 mL, 200 mmol) was
added to the stirred solution of oxalyl chloride (9.0 mL, 100
mmol) in CH2Cl2 (280 mL) at -65 °C, and the mixture was
stirred for 5 min. After addition of a solution of 23 (11.2 g,
33.4 mmol) in CH2Cl2 (24 mL) at -60 °C within 5 min, the
mixture was stirred for 15 min. After addition of triethylamine
(70 mL, 500 mmol), the mixture was stirred at the same
temperature for 15 min, and then the mixture was allowed to
warm to room temperature. After being quenched with water,
the mixture was extracted with CH2Cl2. The extract was
washed with water, dried over MgSO4, and filtered. Evapora-
tion of the filtrate gave an oil, which was subjected to flash
column chromatography on silica gel. Elution with toluene-
acetone (10:1) gave 24 as an oil (10.4 g, 93%): 1H NMR (400
MHz, CDCl3) δ 1.48 (9H, s, (R and â)), 2.70-2.80 (1/3H, br s,
(R)), 2.75-3.00 (2/3H, br s, (â)), 3.39 and 3.41 (3/3H each, s
each, (R)), 3.43 and 3.45 (6/3H each, s each, (â)), 3.52 (2/3H,
dd, J ) 3.7, J ) 9.8 Hz, (â)), 3.75 (2/3H, br d, J ) 13.7 Hz,
(â)), 3.70-3.85 (1/3H, br m, (R)), 3.97 (1/3H, br s, (R)), 4.29
(1/3H, d, J ) 3.4 Hz, (R)), 4.20-4.45 (1H, br m, (R and â)),
4.47 (2/3H, d, J ) 9.8 Hz, (â)), 4.59 and 4.70 (2/3H, ABq, J )
6.8 Hz, (R)), 4.70 and 4.73 (2/3H, ABq, J ) 6.8 Hz, (R)), 4.74
and 4.82 (4/3H, ABq, J ) 6.8 Hz, (â)), 4.78 and 4.86 (4/3H,
ABq, J ) 6.8 Hz, (â)), 5.09 and 5.19 (2/3H each, br s each,
(â)), 5.17 and 5.35 (1/3H each, br s each, (R)), 5.55∼5.75 (1/
3H, br m, (R)) and 5.80-5.90 (2/3H, br m, (â)).
(+)-(2S ,3R ,4R )-N -(t er t -Bu t yloxyca r b on yl)-3,4-d i-O-
(m et h oxym et h yl)-5-m et h ylen e-2-p h t h a lim id o-3,4-p ip e-
r id in ed iol (25) a n d (-)-(2R,3R,4R)-N-(ter t-Bu tyloxyca r -
b o n y l )-3 ,4 -d i -O -(m e t h o x y m e t h y l )-5 -m e t h y l e n e -2 -
p h th a lim id o-3,4-p ip er id in ed iol (26). To the mixture of 24
(10.4 g, 31.2 mmol), triphenylphosphine (24.6 g, 93.8 mmol),
and phthalimide (14 g, 93.8 mmol) in DMF (274 mL) was
added dropwise diethyl azodicarboxylate (14.8 mL, 94 mmol)
under stirring, and the resulting mixture was stirred at room
temperature overnight. Addition of water and evaporation of
the solvent gave an oil, which was dissolved in ether. The
solution was washed with water, dried over MgSO4, and
filtered. Evaporation of the solvent gave a viscous solid, which
27: [R]23 +109.5° (c 0.9, CHCl3); NMR (CDCl3, 400 MHz)
D
δ 1.39 (9H, s), 2.05∼2.15 (1H, m), 2.75-2.85 (1H, br s), 2.87
and 3.41 (3H, s each), 3.48 (1H, dd, J ) 5.4, 9.7 Hz), 3.53 (1H,
dt, J ) 7.3, 11.7 Hz), 3.79 (1H, dt, J ) 5.9, 11.7 Hz), 3.95 (1H,
dd, J ) 5.1, 14.7 Hz), 4.02 (1H, br d, J ) 14.7 Hz), 4.53 (1H,
t, J ) 9.7 Hz), 4.51 and 4.72 (2H, ABq, J ) 6.8 Hz), 4.69 and
4.82 (2H, ABq, J ) 6.6 Hz), 5.70 (1H, d, J ) 9.7 Hz) and 7.70-
7.90 (4H, m). Anal. Calcd for C23H32N2O9: C, 57.49; H, 6.71;
N, 5.83. Found: C, 57.90; H, 6.83; N, 5.99.
28: [R]23 +67° (c 0.53, CHCl3); NMR (CDCl3, 400 MHz) δ
D
1.37 (9H, s), 2.53 (1H, m), 3.14 and 3.31 (3H, s each), 3.63 (1H,
t, J ) 13.5 Hz), 3.70-3.80 (2H, m), 3.96 (1H, dd, J ) 6.3, 7.3
Hz), 4.04 (1H, dd, J ) 6.8, 13.5 Hz), 4.40 (1H, dd, J ) 5.7, 7.3
Hz), 4.60 and 4.72 (2H, ABq, J ) 6.4 Hz), 4.72 and 4.75 (2H,
ABq, J ) 6.8 Hz), 5.92 (1H, d, J ) 5.7 Hz) and 7.65-7.85 (4H,
m). Anal. Calcd for C23H32N2O9: C, 57.49; H, 6.71; N, 5.83.
Found: C, 57.30; H, 6.66; N, 5.67.
(-)-(2S ,3R ,4R ,5R )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym eth yl-3,4-d i-O-(m eth oxym eth yl)-2-tr iflu or oa ceta -
m id o-3,4-p ip er id in ed iol (29). To a solution of 27 (447 mg,
0.93 mmol) in CH3OH (5.6 mL) was added hydrazine hydrate
(0.98 mL, 31.5 mmol), and the mixture was stirred at room
temperature overnight. After dilution with CHCl3, the result-
ing precipitates were filtered off. The filtrate was washed with
water, dried over MgSO4, and filtered. Evaporation of the
filtrate gave an oil, which was subjected to flash column
chromatography on silica gel. Elution with CHCl3-CH3OH (10:
1) gave an oil (355 mg). The oil was dissolved in CH2Cl2 (4
mL), and to the solution were added pyridine (0.18 mL, 2.2
mmol) and (CF3CO)2O (0.16 mL, 1.1 mmol) at -20 °C. The
mixture was stirred at room temperature for 30 min. After
dilution with CH2Cl2, the solution was washed with water,
dried over MgSO4, and filtered. Evaporation of the filtrate gave
an oil. The oil was subjected to preparative thin-layer chro-
matography on silica gel developed with CHCl3-CH3OH (15:
1) to give 29 as an oil (375 mg, 90%): [R]23 -26.8° (c 0.99,
D
CHCl3); NMR (CDCl3, 400 MHz) δ 1.48 (9H, s), 2.25-2.35 (1H,
m), 2.75 (1H, t, J ) 12.7 Hz), 3.42 and 3.44 (3H, s each), 3.59

Uronic Acid-Type gem-Diamine 1-N-Iminosugars
J . Org. Chem., Vol. 65, No. 1, 2000
9
(1H, dd, J ) 8.8, 10.7 Hz), 3.65 (1H, dd, J ) 5.9, 10.7 Hz),
3.79 (1H, br s), 3.94 (1H, br d, J ) 12.7 Hz), 4.07 (1H, br s),
4.67 and 4.75 (2H, ABq, J ) 6.8 Hz), 4.72 and 4.77 (2H, ABq),
6.28 (1H, br d, J ) 6.3 Hz) and 8.00 (1H, br s). Anal. Calcd for
t y lo x y c a r b o n y l)-5-h y d r o x y m e t h y l-3,4-d i -O -(m e t h -
oxymethyl)-2-phthalimido-3,4-piperidinediol(34).Procedures
used were similar to those used for the preparation of 27 and
28 from 25; the yields of 33 and 34 were 50 and 38%,
respectively.
C
17H29N2O8F3: C, 45.74; H, 6.55; N, 6.28. Found: C, 45.59; H,
6.40; N, 6.13.
33: [R]23 +27.4° (c 0.72, CHCl3); NMR (CDCl3, 400 MHz);
D
(+)-(3S,4R,5R,6S)-N-(ter t-Bu tyloxyca r bon yl)-2,3-d id e-
h yd r o-4,5-d i(m eth oxym eth oxy)-6-tr iflu or oa ceta m id o-3-
p ip er id in eca r boxylic Acid (30). To a solution of 29 (285 mg,
0.66 mmol) in a mixture of CCl4 (3.8 mL) and CH3CN (3.8 mL)
were added a solution of NaIO4 (293 mg, 1.4 mmol) in water
(5.8 mL) and RuO2 (10 mg, 0.075 mmol). The mixture was
stirred at room temperature for 2 h. The phases were sepa-
rated. The aqueous phase was extracted three times with
EtOAc. To the combined organic extracts was added 2-propanol
(0.2 mL). The mixture was stirred at room temperature for 1
h. The mixture was washed with water, dried over MgSO4,
and filtered. Evaporation of the filtrate gave a solid. The solid
was subjected to preparative thin-layer chromatography on
silica gel developed with CHCl3-CH3OH (7:1) to give 30 as a
solid (275 mg, 91%): [R]22D +9.2° (c 1.0, CHCl3); NMR (CDCl3,
400 MHz) δ 1.48 (9H, s), 2.81 (1H, br m), 3.22 (1H, br dd, J )
2.7, 15 Hz), 3.38 and 3.44 (3H, s each), 3.84 (1H, br t, J ) 2.6
Hz), 4.35-4.55 (2H, br m), 4.58 and 4.64 (2H, ABq), 4.80 (2H,
s), 6.84 (1H, br d, J ) 6.8 Hz) and 8.02 (1H, br d, J ) 6.8 Hz).
Anal. Calcd for C17H27N2O9F3: C, 44.35; H, 5.91; N, 6.08.
Found: C, 44.51; H, 5.57; N, 5.80.
δ 1.45 (9H, s), 2.45-2.60 (1H, br m), 3.60-3.70 (1H, m), 3.70-
3.85 (1H, br m), 3.95-4.05 (1H, m), 4.05-4.25 (1H, br m), 4.14
(1H, dd, J ) 7.6, 9.8 Hz), 4.61 (2H, s), 4.67 (1H, dd, J ) 5.9,
9.8 Hz), 4.74 and 4.78 (2H, ABq, J ) 6.3 Hz), 6.72 (1H, br s)
and 7.70-7.90 (4H, m). Anal. Calcd for C23H32N2O9: C, 57.49;
H, 6.71; N, 5.83. Found: C, 57.59; H, 6.54; N, 5.65.
34: [R]23 +37.2° (c 0.74, CHCl3); NMR (CDCl3, 400 MHz);
D
δ 1.41 (9H, s), 1.75-1.95 (1H, m), 2.91 (1H, br t, J ) 6.8 Hz),
3.55-3.80 (2H, m), 3.90∼4.15 (2H, m), 4.64 (1H, dd, J ) 7.0,
9.8 Hz), 4.46 (1H, br t, J ) 9.8 Hz), 4.58 and 4.74 (2H, ABq, J
) 6.8 Hz), 4.70 and 4.87 (2H, ABq, J ) 6.3 Hz), 6.64 (1H, br
s) and 7.70-7.90 (4H, m). Anal. Calcd for C23H32N2O9: C,
57.49; H, 6.71; N, 5.83. Found: C, 57.70; H, 6.61; N, 5.55.
(+)-(2R,3R,4R,5R)-N-(ter t-Bu t yloxyca r b on yl)-5-(ter t-
bu tyld im eth ylsiloxym eth yl)-3,4-d i-O-(m eth oxym eth yl)-
2-p h th a lim id o-3,4-p ip er id in ed iol (35). To a solution of 33
(228 mg, 0.475 mmol) in DMF (4.5 mL) were added imidazole
(80.7 mg, 1.19 mmol) and TBDMSCl (107.2 mg, 0.71 mmol),
and the mixture was stirred at room temperature for 1 h. After
the mixture was quenched with water, evaporation of the
solvent gave an oil, which was dissolved in CHCl3. The solution
was washed with water, dried over MgSO4, and filtered.
Evaporation of the filtrate gave an oil, which was subjected
to preparative thin-layer chromatography on silica gel. Elution
with toluene-acetone (15:1) gave an oil of 35 (257 mg, 91%):
(+)-(3S,4R,5R,6R)-4,5-Dih yd r oxy-6-tr iflu or oa ceta m id o-
3-p ip er id in eca r b oxylic Acid (8). Compound 30 (220 mg,
0.48 mmol) was dissolved in HCl in 1,4-dioxane (4 M, 6 mL),
and the mixture was kept at room temperature overnight.
After addition of Et2O, the resulting precipitates were taken
by centrifugation and washed with Et2O three times to give a
[R]23 +40.5° (c 0.66, CHCl3); NMR (CHCl3, 400 MHz) δ 0.09
D
(6H, s), 0.93 (9H, s), 1.41 (9H, s), 1.96 (1H, m), 3.33 and 3.42
(3H, s each), 3.44 (1H, dd, J ) 12.2, 13.5 Hz), 3.50-3.75 (1H,
br m), 3.75-4.25 (2H, br m), 4.00 (1H, dd, J ) 7.1, 9.0 Hz),
4.27 (1H, dd, J ) 5.4, 13.5 Hz), 4.52 and 4.59 (2H, ABq, J )
6.0 Hz), 4.71 and 4.81 (2H, ABq, J ) 6.6 Hz), 6.40-6.65 (1H,
br m) and 7.55-7.80 (4H, m). Anal. Calcd for C29H46N2O9Si:
C, 58.56; H, 7.80; N, 4.71. Found: C, 58.25; H, 7.84; N, 4.57.
(+)-(2R,3R,4R,5R)-N-(ter t-Bu t yloxyca r b on yl)-5-(ter t-
bu tyld im eth ylsiloxym eth yl)-3,4-d i-O-(m eth oxym eth yl)-
2-tr iflu or oa ceta m id o-3,4-p ip er id in ed iol (36). Procedures
used were similar to those used for the preparation of 29 from
colorless solid of 8 as its hydrochloride (147 mg, 99.7%): [R]22
D
+23° (c 0.16, H2O); NMR (D2O, 400 MHz) δ 2.74 (1H, ddd, J
) 4.4, 10.5, 13.0 Hz), 3.24 (1H, t, J ) 13.0 Hz), 3.53 (1H, dd,
J ) 4.4, 13.0 Hz), 3.74 (1H, t, J ) 9.3 Hz), 3.85 (1H, dd, J )
9.3, 10.5 Hz) and 5.02 (1H, d, J ) 9.3 Hz). Anal. Calcd for
C8H11N2O5F3‚1HCl‚1H2O: C, 29.41; H, 4.32; N, 8.58; Cl, 10.85.
Found: C, 29.58; H, 4.09; N, 8.42; Cl, 11.09.
(+)-(2S ,3R ,4R ,5S )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym eth yl-3,4-d i-O-(m eth oxym eth yl)-2-tr iflu or oa ceta -
m id o-3,4-p ip er id in ed iol (31). Procedures used were similar
to those used for the preparation of 29 from 27; the yield was
27; the yield was 88%: [R]23 +38.6° (c 0.59, CHCl3); NMR
D
87%: [R]23 +22.8° (c 1.3, CHCl3); NMR (CDCl3, 400 MHz) δ
(CDCl3, 400 MHz) δ 0.05 and 0.06 (3H, s each), 0.89 (9H, s),
1.49 (9H, s), 1.65-1.80 (1H, m), 2.78 (1H, t, J ) 14.0 Hz), 3.50-
3.70 (3H, m), 3.75 (1H, dd, J ) 4.9, 9.3 Hz), 4.02 (1H, dd, J )
4.9, 14.0 Hz), 4.71 and 4.84 (2H, ABq, J ) 6.6 Hz), 4.74 and
4.76 (2H, ABq, J ) 6.8 Hz), 6.25 (1H, t, J ) 4.9 Hz) and 6.58
(1H, br d, J ) ∼4.9 Hz). Anal. Calcd for C23H43N2O8SiF3: C,
49.27; H, 7.73; N, 5.00. Found: C, 49.15; H, 7.52; N, 4.88.
(+)-(2R ,3R ,4R ,5R )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym eth yl-3,4-d i-O-(m eth oxym eth yl)-2-tr iflu or oa ceta -
m id o-3,4-p ip er id in ed iol (37). Procedures used were similar
to those used for the preparation of 23 from 22; the yield was
D
1.49 (9H, s), 2.05-2.15 (1H, m), 3.22 (1H, dd, J ) 3.4, 14.7
Hz), 3.41 (6H, s), 3.62 (1H, dd, J ) 6.4, 11.4 Hz), 3.73 (1H, t,
J ) 2.2 Hz), 3.81 (1H, dd, J ) 9.3, 11.4 Hz), 3.89 (1H, br s),
4.02 (1H, br d, J ) 14.7 Hz), 4.65 and 4.72 (2H, ABq, J ) 6.8
Hz), 4.73 (2H, s), 6.18 (1H, br d, J ) 8.3 Hz) and 8.02 (1H, br
d, J ) 8.3 Hz). Anal. Calcd for C17H29N2O8F3: C, 45.74; H,
6.55; N, 6.28. Found: C, 46.04; H, 6.29; N, 6.22.
(+)-(3R,4R,5R,6S)-N-(ter t-Bu tyloxyca r bon yl)-2,3-d id e-
h yd r o-4,5-d i(m eth oxym eth oxy)-6-tr iflu or oa ceta m id o-3-
p ip er id in eca r boxylic Acid (32). Procedures used were
similar to those used for the preparation of 30 from 29; the
93%: [R]23 +43.3° (c 1.5, CHCl3); NMR (CDCl3, 400 MHz) δ
D
yield was 90%: [R]23 +13.5° (c 0.55, CHCl3); NMR (CDCl3,
1.47 (9H, s), 1.76 (1H, m), 2.99 (1H, dd, J ) 12.3, 13.6 Hz),
3.17 (1H, br s), 3.39 and 3.47 (2H, s each), 3.57 (1H, br t, J )
11.6 Hz), 3.67 (1H, t, J ) 9.6 Hz), 3.82 (1H, dd, J ) 5.2, 9.6
Hz), 3.85-4.05 (2H, m), 4.67 and 4.68 (2H, ABq, J ) 4.4 Hz),
4.77 and 4.92 (2H, ABq, J ) 6.6 Hz) and 6.34 (1H, br t, J )
5.2 Hz). Anal. Calcd for C17H29N2O8F3: C, 45.74; H, 6.55; N,
6.28. Found: C, 45.43; H, 6.31; N, 5.83.
D
400 MHz) δ 1.48 (9H, s), 3.05 (1H, br d, J ) 12.0 Hz), 3.19
(1H, br t, J ) 12.0 Hz), 3.38 and 3.43 (3H, s each), 3.84 (1H,
br s), 4.36 (1H, br s), 4.15-4.40 (1H, br m), 4.65-4.80 (4H,
m), 6.26 (1H, br s) and 8.07 (1H, br d, J ) 7.8 Hz). Anal. Calcd
for C17H27N2O9F3: C, 44.35; H, 5.91; N, 6.08. Found: C, 44.30;
H, 5.93; N, 5.99.
(+)-(3R,4R,5R,6R)-4,5-Dih yd r oxy-6-tr iflu or oa ceta m ido-
3-p ip er id in eca r boxylic Acid (9). Procedures used were
similar to those used for the preparation of 8 from 30; the yield
(+)-(3S,4R,5R,6R)-N-(ter t-Bu tyloxyca r bon yl)-2,3-d id e-
h yd r o-4,5-d i-O-(m eth oxym eth oxy)-6-tr iflu or oa ceta m id o-
3-p ip er id in eca r boxylic Acid (38). Procedures used were
similar to those used for the preparation of 30 from 29; the
was 99%: [R]22 +30° (c 0.2, H2O); NMR (D2O, 400 MHz) δ
D
3.20 (1H, dt, J ) 3.9, 7.3 Hz), 3.27 (1H, dd, J ) 3.9, 13.7 Hz),
3.50 (1H, dd, J ) 7.3, 13.7 Hz), 4.07 (1H, t, J ) 6.4 Hz), 4.14
(1H, dd, J ) 3.9, 6.4 Hz) and 5.14 (1H, d, J ) 6.4 Hz). Anal.
Calcd for C8H11N2O5F3‚1HCl‚1H2O: C, 29.41; H, 4.32; N, 8.58;
Cl, 10.85. Found: C, 29.52; H, 4.23; N, 8.44; Cl, 11.08.
(+)-(2R ,3R ,4R ,5R )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym et h yl-3,4-d i-O-(m et h oxym et h yl)-2-p h t h a lim id o-
3,4-p ip er id in ed iol (33) a n d (+)-(2R,3R,4R,5S)-N-(ter t-Bu -
yield was 92%: [R]23 +10.7° (c 0.93, CHCl3); NMR (CDCl3,
D
400 MHz) δ 1.47 (9H, s), 2.67 (1H, br m), 3.18 (1H, br t, J )
12.0 Hz), 3.10 and 3.39 (3H, s each), 3.83 (1H, dd, J ) 5.4, 9.0
Hz), 3.95-4.20 (4H, m), 4.60-4.85 (4H, m), 6.25 (1H, t, J )
5.4 Hz) and 7.58 (1H, br s). Anal. Calcd for C17H27N2O9F3: C,
44.35; H, 5.91; N, 6.08. Found: C, 44.51; H, 6.03; N, 6.11.
(-)-(3S,4R,5R,6S)-4,5-Dih yd r oxy-6-tr iflu or oa ceta m id o-
3-p ip er id in eca r boxylic Acid (10). Procedures used were

10 J . Org. Chem., Vol. 65, No. 1, 2000
Nishimura et al.
Ta ble 3.
dose (µg/mL)
compd
100
20
4
0.8
0.16
0.03
0.006
0
erythromycin
Ga
Lb
G
L
G
L
G
L
G
L
(
(
+++
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
0
60
50
8
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
>100
+++
+++
+++
+++
+++
9
10
11
45
G
L
a
b
G: +++ ) full growth of Pseudomonas aeruginosa A3 detected by MTT. L: length (mm) of alginate slime.
similar to those used for the preparation of 8 from 30; the yield
was 99.7%: [R]22D -13° (c 0.14, H2O); NMR (D2O, 400 MHz) δ
2.82 (1H, dt, J ) 1.6, 4.0 Hz), 3.40 (1H, dd, J ) 4.0, 13.2 Hz),
3.57 (1H, dd, J ) 1.6, 13.2 Hz), 3.95 (1H, dd, J ) 2, 4.0 Hz),
4.43 (1H, t, J ) 4 Hz) and 5.36 (1H, d, J ) 2 Hz). Anal. Calcd
for C8H11N2O5F3‚1HCl‚1H2O: C, 29.41; H, 4.32; N, 8.58; Cl,
10.85. Found: C, 29.67; H, 4.08; N, 8.34; Cl, 11.21.
similar to those used for the preparation of 8 from 30; the yield
was 91%: [R]22 -2° (c 0.14, H2O); NMR (D2O, 400 MHz) δ
D
3.19 (1H, ddd, J ) 2.9, 5.4, 13.0 Hz), 3.37 (1H, dd, J ) 5.4,
13.0 Hz), 3.41 (1H, t, J ) 13.0 Hz), 3.99 (1H, dd, J ) 1.7, 3.9
Hz), 4.32 (1H, t, J ) 2.9, 3.9 Hz) and 5.31 (1H, d, J ) 1.7 Hz).
Anal. Calcd for C8H11N2O5F3‚1HCl‚1H2O: C, 29.41; H, 4.32;
N, 8.58; Cl, 10.85. Found: C, 29.74; H, 4.15; N, 8.32; Cl, 10.97.
Gen er a l P r oced u r es for En zym e In h ibition Assa y. The
enzymes, â-glucuronidase (bovine liver), R-glucosidase (bakers’
yeast), â-glucosidase (almond), R-mannosidase (jack beans),
â-mannosidase (snail), â-galactosidase (Aspergillus niger), R-N-
acetylgalactosaminidase (chicken liver), and â-N-acetylglu-
cosaminidase (bovine epididymis) were purchased from Sigma
Chemical Co. â-Glucuronidase was assayed using phenol-
phthalein mono-â-glucuronic acid (3.3 × 10-4M) as a substrate
at pH 5.0 (0.1 M acetate buffer). R- and â-glucosidases were
assayed using p-nitrophenyl R-D-glucopyranoside (1.5 × 10-3M)
and â-D-glucopyranoside (2 × 10-3M) as substrates at pH 6.3
(0.025 M citrate-phosphate buffer) and 5.0 (0.025 M acetate
buffer), respectively. R- and â-mannosidases were assayed
using p-nitrophenyl R-D-mannopyranoside (2 × 10-3M) and
â-D-mannopyranoside (2 × 10-3M) as substrates at pH 4.5 (0.05
M acetate buffer) and 4.0 (0.05 M acetate buffer), respectively.
â-Galactosidase was assayed using p-nitrophenyl â-D-galac-
topyranoside (2 × 10-3M) at pH 4.0 (0.025 M citrate-
phosphate buffer). R-N-Acetylgalactosaminidase was assayed
using p-nitrophenyl N-acetyl-R-D-galactosaminide (1 × 10-3M)
as a substrate at pH 4.0 (0.025 M citrate-phosphate buffer).
â-N-Acetylglucosaminidase was assayed using p-nitrophenyl
N-acetyl-â-D-glucosaminide (1 × 10-3M) at pH 4.0 (0.025 M
citrate-phosphate buffer). The reaction mixture contained 0.5
mL of buffer, 0.1 mL of substrate solution, and water or
aqueous solution containing the test compound. The mixture
was incubated at 37 °C for 3 min, and 0.01 mL of enzyme was
added. After 30 min of reaction, 1.0 mL of 0.3 M glycine-
sodium hydroxide buffer (pH 10.5) was added, and the absor-
bance of the liberated nitrophenol or phenolyphthalein mea-
sured at 400 or 525 nm, respectively. The percent inhibition
was calculated by the formula (A - B)/A × 100, 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 inhibitor at 50% of enzyme activity.
(-)-(2R,3R,4R,5S)-N-(ter t-Bu t yloxyca r b on yl)-5-(ter t-
bu tyld im eth ylsiloxym eth yl)-3,4-d i-O-(m eth oxym eth yl)-
2-p h th a lim id o-3,4-p ip er id in ed iol (39). Procedures used
were similar to those used for the preparation of 35 from 33;
the yield was 100%: [R]23 -24.3° (c 0.8, CHCl3); δ 0.07 and
D
0.08 (3H, s each), 0.91 (9H, s), 1.39 (9H, s), 2.25∼2.35 (1H, br
m), 3.32 and 3.40 (3H, s each), 3.65 (1H, t, J ) 9.6 Hz), 3.74
(1H, br d, J ) 13.2 Hz), 3.91 (1H, dd, J ) 3.0, 9.8 Hz), 3.90-
4.05 (1H, br m), 4.26 (1H, br d, J ) 13.2 Hz), 4.50 (1H, dd, J
) 5.2, 10.4 Hz), 4.60 and 4.63 (2H, ABq, J ) 6.8 Hz), 4.71 and
4.75 (2H, ABq, J ) 6.8 Hz), 6.50-6.65 (1H, br m) and 7.60-
7.85 (4H, m). Anal. Calcd for C29H46N2O9Si: C, 58.56; H, 7.80;
N, 4.71. Found: C, 58.46; H, 7.80; N, 4.64.
(+)-(2R,3R,4R,5S)-N-(ter t-Bu t yloxyca r b on yl)-5-(ter t-
bu tyld im eth ylsiloxym eth yl)-3,4-d i-O-(m eth oxym eth yl)-
2-tr iflu or oa ceta m id o-3,4-p ip er id in ed iol (40). Procedures
used were similar to those used for the preparation of 29 from
27; the yield was 79%: [R]25 +1.1° (c 1.08, CHCl3); NMR
D
(CDCl3, 400 MHz) δ 0.05 and 0.06 (3H, s each), 0.89 (9H, s),
1.48 (3H, s), 2.10-2.30 (1H, m), 2.70 (1H, br t, J ) 13.4 Hz),
3.40 and 3.16 (3H, s each), 3.35-3.55 (2H, m), 3.74 (1H, br s),
3.92 (1H, dd, J ) 4.6, 13.4 Hz), 4.06 (1H, br s), 4.55-4.80 (4H,
m), 6.27 (1H, br d, J ) 6.3 Hz) and 8.14 (1H, br s). Anal. Calcd
for C23H43N2O8SiF3: C, 49.27; H, 7.73; N, 5.00. Found: C,
49.45; H, 7.88; N, 4.90.
(+)-(2R ,3R ,4R ,5S )-N -(t er t -Bu t yloxyca r b on yl)-5-h y-
d r oxym eth yl-3,4-d i-O-(m eth oxym eth yl)-2-tr iflu or oa ceta -
m id o-3,4-p ip er id in ed iol (41). Procedures used were similar
to those used for the preparation of 23 from 22; the yield was
100%: [R]23 +35.8° (c 0.54, CHCl3); NMR (CDCl3, 400 MHz)
D
δ 1.48 (9H, s), 3.04 (1H, dt, J ) 4.0, 7.3 Hz), 3.40 (3H, s), 3.42
(3H, s), 4.04 (1H, dd, J ) 4.0, 14.3 Hz), 3.78 (1H, dd, J ) 7.3,
14.3 Hz), 4.09 (1H, dd, J ) 4.0, 6.3 Hz), 4.14 (1H, dd, J ) 2.9,
6.3 Hz), 4.55-4.80 (4H, m), 6.03 (1H, brdd, J ) 2.9, 5.9 Hz)
and 8.40 (1H, br s). Anal. Calcd for C17H29N2O8F3: C, 45.74;
H, 6.55; N, 6.28. Found: C, 45.45; H, 6.52; N, 6.20.
Hep a r a n a se Assa y. Recombinant human heparanase ac-
tivity was determined toward fluorescein isothiocyanate (FITC)
labeled heparan sulfate (FITC-HS). The assay was carried out
essentially by the method described by Toyoshima and Naka-
jima.29
In h ibition of Biofilm P r od u ction by in Vitr o Assa y
w ith Micr op la te. Test compounds (e.g., six compounds) with
serial dilution (0-100 µg/mL) are put into eight holes of each
lane of a 48 hole microplate (8 holes × 6 lanes) containing
Miller-Hinton medium. The test organisms producing biofilm
(e.g., alginate) are inoculated and incubated at 37 °C for 18 h,
and the minimum inhibitory concentration (MIC) for the
growth is determined with an aid of formazan color develop-
ment of MTC (methyl tetrazolim chloride). The production of
biofilm is represented by the length of slime produced in each
(+)-(3R,4R,5R,6R)-N-(ter t-Bu tylcar bon yl)-2,3-dideh ydr o-
4,5-d i-O-(m eth oxym eth oxy)-6-tr iflu or oa ceta m id o-3-p ip -
er id in eca r boxylic Acid (42). Procedures used were similar
to those used for the preparation of 30 from 29; the yield was
87%: [R]23 +24.3° (c 1.0, CHCl3); NMR (CDCl3, 400 MHz) δ
D
1.48 (9H, s), 3.04 (1H, dt, J ) 4.0, 7.3 Hz), 3.40 and 3.42 (3H,
s each), 4.04 (1H, dd, J ) 4.0, 14.4 Hz), 3.78 (1H, dd, J ) 7.3,
14.4 Hz), 4.09 (1H, dd, J ) 4.0, 6.3 Hz), 4.14 (1H, dd, J ) 2.9
Hz), 4.66 and 4.72 (2H, ABq, J ) 6.6 Hz), 4.74 (2H, t, J ) 7.3
Hz), 5.86 (1H, dd, J ) 2.9, 5.9 Hz) and 8.25-8.50 (1H, br s).
Anal. Calcd for C17H27N2O9F3: C, 44.35; H, 5.91; N, 6.08.
Found: C, 44.47; H, 5.94; N, 6.05.
(-)-(3R,4R,5R,6S)-4,5-Dih yd r oxy-6-tr iflu or oa ceta m id o-
3-p ip er id in eca r boxylic Acid (11). Procedures used were

Uronic Acid-Type gem-Diamine 1-N-Iminosugars
J . Org. Chem., Vol. 65, No. 1, 2000 11
holes. When 48 plugs of polyethylene (2 mm diameter, 20 mm
length) arranged on the cover slip of the microplate are placed
in the holes of plate before the incubation, the amount and
stickiness of slime production after 18 h incubation can be
assayed by measuring the length of slime until the slime is
splitted, if the biofilm covered plugs is lifted up gradually from
the hole. Thus, as shown in Table 3, erythromycin as the
control showed full growth in holes of 4 µg/mL, but scarce
growth in 20 and 100 µg/mL. Despite the full growth, the slime
production on a plug in the holes of 4 µg/mL was inhibited
and splitted at 50 mm length of lifting (the slime production
in holes of 0.8 µg/mL or less was not inhibited and splitted at
>100 mm length; no slime was produced in 100 µg/mL).
Ack n ow led gm en t. The authors are grateful to Dr.
Shinichi Kondo for his helpful discussions and encour-
agement. We also thank Ms. Fukiko Kojima for the
biological evaluation of the derivatives, Ms. Hikaru
Nakamura for X-ray crystallographic analysis, and Ms.
Maya Yasuda for technical assistance.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra of 27, 28, 33, 34, 8, 9, 10, and 11 and an X-ray
crystallographic report for 25. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O982448C