1-N-Iminosugars: Potent and selective inhibitors of β-glycosidases

Article abstract of DOI:10.1021/ja973443k

A series of 1-N-iminosugars were synthesized to supply the need for glycosidase inhibitors that are both highly potent and selective for β- glycosidases. Designed on the basis of the transition-state model of the β- glucosidase reaction, these iminosugar

Full text of DOI:10.1021/ja973443k

J. Am. Chem. Soc. 1998, 120, 3007-3018
3007
1-N-Iminosugars: Potent and Selective Inhibitors of â-Glycosidases
Yoshitaka Ichikawa,* Yasuhiro Igarashi, Mie Ichikawa, and Yoshitomo Suhara
Contribution from the Department of Pharmacology and Molecular Sciences, Johns Hopkins UniVersity
School of Medicine, Baltimore, Maryland 21205
ReceiVed October 2, 1997
Abstract: A series of 1-N-iminosugars were synthesized to supply the need for glycosidase inhibitors that are
both highly potent and selective for â-glycosidases. Designed on the basis of the transition-state model of the
â-glucosidase reaction, these iminosugar inhibitors differ from the currently available inhibitors in possessing
a nitrogen atom at the anomeric position of the pyranose ring, thereby generating a positive charge on the
anomeric position rather than on the ring oxygen of the sugar. Their syntheses, starting with a readily available
carbohydrate derivative, involve (i) introduction of an amino functionality as an azido group, (ii) formation of
a 1-N-iminopyranose ring with reductive amination, and (iii) stereoselective introduction of a hydroxymethyl
or methyl group and were accomplished in a highly stereoselective and efficient manner. The inhibitory
potencies of the 1-N-iminosugars were evaluated against several R- and â-glycosidases, and they were found
to be extremely potent and highly specific against the corresponding â-glycosidases, with Ki values in the
nanomolar range.
Inhibitors of glycosidases have been attractive target com-
pounds for synthetic chemists and biochemists, not only because
they serve as useful biological tools for studying the biological
functions of oligosaccharides1 but also because they may have
great potential as drugs to treat a variety of carbohydrate-
mediated diseases.2 This group of inhibitors, which are deriva-
tives of naturally occurring azasugars, are now finding clinical
application as anti-HIV,3 anticancer,4 and antidiabetic agents,5
and their effectiveness has made the development of additional
glycosidase inhibitors a matter of considerable interest to
synthetic chemists.6,7
R-Glycosidases are generally believed to go through an E2-
type elimination mechanism (2 f 3 in Figure 1) during which
a positively charged aglycon (leaving group) and the lone pair
of the ring oxygen are positioned antiperiplanar and coopera-
tively facilitate the glycosidic bond-cleaving reaction.8 In the
case of the â-glycosidase reaction, if the enzyme proceeds via
an E2-type mechanism similar to that of the R-glycosidases,
the protonated substrate 5 has to go through a highly strained
intermediate 68 that may not favor further reaction. It is,
therefore, quite natural to assume that the â-glycosidase reaction
may follow different reaction steps than those of the R-glycosi-
dase reaction in order to reach the presumed reaction intermedi-
ate 3.
(1) (a) Albein, A. D. Annu. ReV. Biochem. 1987, 56, 497-534. (b) Dwek,
R. A. Chem. ReV. 1996, 96, 683-720.
When we used a molecular mechanics program to study the
reaction intermediate of the â-glucosidase reaction in terms of
conformation and charge distribution, we found that the positive
charge was generated at the anomeric position rather than at
the position of the ring oxygen.9 In addition, extensive work
by the Withers group using 2-fluoro10 or 5-fluoro11 carbohydrate
derivatives has made it clear that retaining â-glycosidases carry
out the glycoside-cleaving reaction via a covalent glycosyl-
enzyme intermediate.12 On the basis of these two findings, we
have designed a new class of iminosugars, 1-N-iminosugars 9,
(2) Hughs, A. B.; Rudge, A. J. Nat. Prod. Rep. 1994, 35-162.
(3) (a) 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. U.S.A. 1988, 85, 9229-9233. (b) Fleet, G. W. J.; Karpas,
A.; Dwek, R. A.; Fellows, L. E.; Tyms, A. S.; Petursson, S.; Namgoong,
S. K.; Ramsden, N. G.; Smith, P. W.; Son, J. C.; Wilson, F.; Witty, D. R.;
Jacob, G. S.; Rademacher, T. W. FEBS Lett. 1988, 237, 128-132. (c)
Wikler, D. A.; Holan, G. J. Med. Chem. 1989, 32, 2084-2089. (d) Ratner,
L.; Heyden, N. V.; Dedera, D. Virology 1991, 181, 180-192.
(4) For review on glycosidase inhibitor as anticancer agents, see: Gross,
P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W. Clin. Cancer Res. 1995,
1, 935-944.
(5) (a) Joubert, P. H.; Venter, C. P.; Joubert, H. F.; Hillebrand, I. Eur.
J. Pharmacol. 1985, 28, 705-708. (b) Anzeveno, P. B.; Creemer, L. J.;
Daniel, J. K.; King, C.-H.; Liu, P. S. J. Org. Chem. 1989, 54, 2539-2542.
(c) Balfour, J. A.; McTavish, D. Drugs 1993, 46, 1025-1054. (d) Truscheit,
E.; Frommer, W.; Junge, B.; Mu¨ller, L.; Schmidt, D. D.; Wingender, W.
Angew. Chem., Int. Ed. Engl. 1981, 20, 744-761.
(6) For reviews on the synthesis of DNJ-based iminosugar inhibitor,
see: (a) Paulsen, H.; Todt, K. AdV. Carbohydr. Chem. Biochem. 1968, 23,
115-232. (b) Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res.
1993, 26, 182-190. (c) Ganem. B. B. Acc. Chem. Res. 1996, 29, 340-
347. (d) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. ReV.
1996, 96, 443-473. (e) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.;
Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 412-432 and 521-
546.
(8) For reviews on the reaction mechanims of glycosidase reactions,
see: (a) Kirby, A. J. Acc. Chem. Res. 1984, 17, 305-311. (b) Gorenstein,
D. G. Chem. ReV. 1987, 87, 1047-1077. (c) Legler, G. AdV. Carbohydr.
Chem. Biochem. 1990, 48, 319-384. (d) Sinnott, M. L. Chem. ReV. 1990,
90, 1171-1202.
(9) Kajimoto, T.; Liu, K. K.-C.; Pederson, R. L.; Zhong, Z.; Ichikawa,
Y.; Porco, J. A., Jr.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 6187-
6196.
(10) (a) Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. J. Am.
Chem. Soc. 1991, 113, 7530-7531. (b) Withers, S. G.; Rupitz, K.; Street,
I. P. J. Biol. Chem. 1988, 263, 7929-7932. (c) McCarter, J.; Adam, M.;
Braun, C.; Namchuk, M.; Tull, D.; Withers, S. G. Carbohydr. Res. 1993,
249, 77-90.
(11) McCarter, J. D.; Withers, S. G. J. Am. Chem. Soc. 1996, 118, 241-
242.
(12) (a) McCarter, J. D.; Withers, S. G. Curr. Opin. Struct. Biol. 1994,
4, 885-892. (b) Davies, G. Structure 1995, 3, 853-859. (c) Stu¨tz, A. E.
Angew. Chem., Int. Ed. Engl. 1996, 35, 1926-1928.
(7) For some potent designed glycosidase inhibitors, see: (a) Heightman,
T. D.; Ermert, P.; Klein, D.; Vasella, A. HelV. Chim. Acta 1995, 78, 514-
532. (b) Pan, Y.-T.; Kanshal, G. P.; Papandreou, G.; Ganem, B.; Elbein,
A. D. J. Biol. Chem. 1992, 267, 8313-8318.
S0002-7863(97)03443-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/17/1998

3008 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.
Figure 1. Well-accepted R- and â-galactosidase reaction mechanism.
Figure 2. General design strategy of glycosidase inhibitor.
in which a nitrogen atom is at the anomeric position of a
monosaccharide; these new iminosugars are different from the
conventional deoxynojirimycin (DNJ)-type iminosugars 10
where a nitrogen atom replaces the ring oxygen of a monosac-
charide. We have synthesized a variety of 1-N-iminosugars,
including D-glucose-type 11,13,14 D-galactose-type 12,15 L-fucose-
type 13,16,17 D-glucuronic acid-type 14,18 and D-xylose-type 15
(Figure 2). We describe herein the detailed synthesis of these
1-N-iminosugars and the evaluation of their inhibitory activities
against a variety of glycosidases.
Synthesis of D-Glucose-Type 1-N-Iminosugar 11 (Scheme
1). The synthesis of D-glucose-type 1-N-iminosugar 11 was
based on our previous synthesis of another D-glucose-type 1-N-
iminosugar with a 5-OH group.13 Bols et al. have prepared
D-glucose-type 1-N-iminosugar 11, but their synthesis was rather
complicated.14 We therefore sought to synthesize 11 in a simple
and practical manner. D-Lyxose was sequentially treated with
(1) concentrated H2SO4/acetone;19 (2) TsCl/pyr; and (3) BzCl/
pyr to give a 5-tosyl-1-O-benzoate 17 in high yield. Azido
substitution of the 5-tosyloxy group of 17 gave a 5-azido
derivative 18. Treatment of 18 with methanolic sodium
methoxide followed by Ho’s aldol reaction20 (K2CO3/HCHO/
aqueous MeOH) afforded 19 with a hydroxymethyl group at
the C-2 position which subsequently became the C-5 position
of the iminosugar. Hydrogenation of 19 over Pd(OH)2 under-
went an intramolecular cyclization to give a free glucose-type
1-N-iminosugar 20 with an additional OH group at the C-5
position. Sequential treatment of 20 with Boc2O in MeOH and
BzCl in pyridine gave 21 in high yield and left only the tertiary
5-OH free. Attempts (thiocarbonyldiimidazole, phenoxythio-
carbonyl chloride-DMAP) to remove the 5-OH group at the
C-5 failed because of its low reactivity.
Methyloxalyl chloride-tributyltin hydride has been used to
deoxygenate a tertiary OH group in steroids21 and nucleoside22
derivatives, and we employed this system for our synthesis.
When 21 was treated with methyloxalyl chloride in acetonitrile
in the presence of DMAP,22 the reaction proceeded quickly (∼30
min) to convert 21 to a faster moving compound in TLC.
(13) Ichikawa, M.; Igarashi, Y.; Ichikawa, Y. Tetrahedron Lett. 1995,
36, 1767-1770.
(14) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup, T.; Jundt,
I.; Bols, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1778-1779.
(15) Ichikawa, Y.; Igarashi, Y. Tetrahedron Lett. 1995, 36, 4585-4586.
(16) Igarashi, Y.; Ichikawa, M.; Ichikawa, Y. Bioorg. Med. Chem. Lett.
1996, 6, 553-558.
(17) Hansen, A.; Tagmose, T. M.; Bols, M. J. Chem. Soc., Chem.
Commun. 1996, 2649-2650.
(18) Igarashi, Y.; Ichikawa, M.; Ichikawa, Y. Tetrahedron Lett. 1996,
37, 2707-2708.
(19) (a) Hughes, N. A.; Speakman, P. R. H. Carbohydr. Res. 1965, 1,
171. (b) Kaskar, B.; Heise, G. L.; Michalak, R. S.; Vishnuvajjala, R.
Synthesis 1990, 1031-1032.
(20) (a) Ho, P.-T. Tetrahedron Lett. 1978, 1623-1626. (b) Ho, P.-T.
Can. J. Chem. 1979, 57, 381-383.
(21) Dolan, S. C.; MacMillan, J. J. Chem. Soc., Chem. Commun. 1985,
1588-1589.
(22) Matsuda, A.; Takenuki, K.; Sasaki, T.; Ueda, T. J. Med. Chem.
1991, 34, 234-239.

1-N-Iminosugars
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3009
Scheme 1a
a Reagents and conditions: (a) (i) acetone-concentrated H2SO4/rt/overnight, (ii) TsCl/pyridine/0 °C to r.t./overnight, (iii) BzCl/pyridine/0 °C to
r.t./overnight (41% overall yield); (b) NaN3/DMSO/100 °C/overnight (74%); (c) (i) NaOMe/MeOH/r.t. (ii) K2CO3/30% HCHO-MeOH/85 °C/
overnight (80-90%); (d) (i) H2/Pd(OH)2/MeOH/r.t./overnight, (ii) 1 N HCl; (e) (i) Boc2O/Et3N/MeOH/r.t./overnight, (ii) BzCl/pyridine/0 °C to
r.t./overnight; (f) (i) MeOCOCOCl/DMAP/CH3CN, (ii) Bu3SnH/VAZO/toluene/100 °C, (iii) SiO2 chromatography with iPrOH/H2O/NH4OH (7:2:
1).
Scheme 2a
a Reagents and conditions: (a) (i) NaOMe/MeOH, (ii) H2/Pd(OH)2/MeOH/r.t./overnight, (b) Boc2O/Et3N/MeOH; (c) DMSO/(COCl)2/CH2Cl2/
-78 °C then Et3N/-78 to 0 °C; (d) n-BuLi/CH3PPh3+Br-/DME/0 °C to r.t./overnight; (e) (i) 9-BBN/THF/0 °C to r.t./3 h, (ii) NaOH/30% H2O2/
r.t./overnight; (f) 1 N HCl/r.t./overnight.
Purification of the product was troublesome because of the
instability of the product which was easily hydrolyzed back to
the starting material as has been noted by the Matsuda group.22
For the workup, we passed the ethyl acetate extract through a
short column of dry silica gel and eluted the column with ethyl
acetate in order to dry the oxalate 22. Reduction of 22 with
tributyltin hydride in toluene in the presence of 1,1′-azobis-
(cyclohexanecarbonitrile) (VAZO) gave a moderate conversion
of the oxalate 22 into a mixture of the deoxygenated products,
D-glucose and L-idose derivatives. After stepwise removal of
the protective groups (i) NaOMe and (ii) 1 N HCl, each of the
isomers was completely separated by silica gel chromatography
with 2-propanol-water-NH4OH (7:2:1, v/v) to afford the faster
moving D-glucose-type 11 and the slower moving L-idose-type
23 iminosugars in a ratio of 2:1. These structures were
and the subsequent Witting methylenation gave an exo-
methylene derivative 27. As expected, hydroboration of 27 with
a sterically bulky 9-BBN followed by oxidative treatment gave
a single isomer 28, with a desired galacto configuration. Acid
treatment of 28 gave the galactose-type 1-N-iminosugar 12.
Synthesis of L-Fucose-Type 1-N-Iminosugar 13 (Scheme
3). Synthesis of L-fucose-type 1-N-iminosugar was carried out
using a strategy similar to that used for synthesis of the
D-galactose-type iminosugar.13 D-Ribose 29 was converted to
a 5-azido-1-O-benzoyl-2,3-O-isopropylidene derivative 31 in a
manner similar to that for synthesizing 18 from 16. Removal
of the 1-O-benzoate group of 31 followed by reductive amina-
tion afforded a piperidine derivative 32, which was then treated
with Boc2O to give 33. By a strategy similar to that used for
preparing the galactose-type iminosugar 12, the piperidine
derivative 33 was oxidized, giving a 5-keto derivative 34, which
was subsequently methylenated to give 35. Hydrogenation of
35 on Pd-C occurred from the less hindered side (convex side)
to give only a single isomer 36 with a desired L-fuco config-
uration. Acidic treatment of 36 furnished the synthesis of
L-fucose-type 1-N-iminosugar 13.
Synthesis of D-Glucuronic Acid-Type 1-N-Iminosugar 14
(Scheme 4). D-Glucuronic acid-type 1-N-iminosugar 14 was
synthesized in a strategy similar to that for the D-galactose-
type iminosugar:18 (i) introduction of a C-5 exo-methylene
group, (ii) hydroboration, and (iii) subsequent oxidation of the
hydroxymethyl group to the corresponding carboxylate. D-
Arabinose 37 was converted to a 5-azidofuranose derivative 38
according to the published procedure of Legler et al.24 Treat-
ment of 38 with 4-methoxyphenol in the presence of TMSOTf
gave a single isomer of 39. Exchange of the O-protective groups
1
confirmed with their H NMR spectra.
Synthesis of D-Galactose-Type 1-N-Iminosugar 12 (Scheme
2). For the synthesis of D-galactose-type 1-N-iminosugar, we
decided to introduce the 5-hydroxymethyl group in a stepwise
manner: (i) introduction of a C-5 exo-methylene group and (ii)
subsequent hydroboration reaction.15 In this strategy, we
anticipated that the stereoselectivity of the hydroboration could
be controlled by the bicyclic structure of the substrate in favor
of the formation of the desired D-galacto configuration.
O-Debenzoylation of the synthetic intermediate of the D-
glucose-type iminosugar 18 followed by hydrogenation led to
intramolecular reductive amination, giving a cyclic imino
derivative 24. This step was followed by the N-protection with
a Boc group, giving a 3,4-O-isopropylidenepiperidine derivative
25 with a free OH group at the C-5 position. Swern oxidation23
of 25 with DMSO-(COCl)2 afforded a 5-keto derivative 26,
(23) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43,
2480-2482.
(24) Legler, G.; Stu¨z, A. E.; Immich, H. Carbohydr. Res. 1995, 272,
17-30.

3010 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.
Scheme 3a
a Reagents and conditions: (a) (i) acetone-concentrated H2SO4/r.t./overnight, (ii) TsCl/pyridine/0 °C to r.t./overnight, (iii) BzCl/pyridine/0 °C to
r.t./3 h (48% overall); (b) NaN3/DMSO/60-65 °C/overnight (91%); (c) (i) NaOMe/MeOH/r.t. (ii) H2/Pd(OH)2/MeOH/r.t./overnight (75% overall);
(d) Boc2O/Et3N/MeOH/r.t./overnight (73%); (e) (i) DMSO/(COCl)2/CH2Cl2/-60 °C then Et3N/-60 to 0 °C (80%); (f) n-BuLi/CH3PPh3+Br-/DME/
0 °C to r.t./overnight (77%); (g) H2/Pd-C/MeOH/r.t./5 h (82%); (h) 1 N HCl/r.t./overnight (90-95%).
Scheme 4a
a Reagents and conditions: (a) ref; (b) 4-MeO-phenol/TMSOTf/CH2Cl2/r.t./4 h; (c) (i) NaOMe/MeOH, (ii) NaH/BnBr/DMF/0 °C to r.t./overnight;
(d) (NH4)2Ce(NO3)6/CH3CN-H2O/0 °C/5 min; (e) (i) H2/Lindlar catalyst/MeOH/r.t./overnight, (ii) Boc2O/Et3N/MeOH/r.t./4 h; (f) DMSO/(COCl)2/
CH2Cl2/-78 °C then Et3N/-78 to 0 °C; (g) LiN(TMS)2/CH3PPH3+Br-/THF/0 °C to r.t./overnight; (h) 9-BBN/THF/0 °C to r.t./overnight then
NaOH/35% H2O2/0 °C to r.t./overnight; (i) DMSO/(COCl)2/CH2Cl2/-78 °C then Et3N/-78 °C to r.t./overnight; (j) NaClO2/35% H2O2/CH3CN-
phosphate buffer/0 °C to r.t./1 h; (k) (i) H2/Pd(OH)2/MeOH/r.t./overnight, (ii) 1 N HCl, (iii) SiO2 chromatography.
Scheme 5a
a Reagents and conditions: (a) thiocarbonyldiimidazole/(CH2Cl)2/80 °C/overnight; (b) Bu3SnH/AlCN/toluene/100 °C/overnight; (c) (i) H2/Pd(OH)2/
MeOH/room temperature/overnight, (ii) 1 N HCl.
of 39 from the acetyl to the more stable benzyl groups afforded
40, which was then treated with (NH4)2Ce(NO3)6 to remove
the C-1 4-methoxyphenol group and give an anomeric free 41.
Reductive amination of 41 over a Lindlar catalysis25 (Pd-
BaSO4) afforded a piperidine derivative 42 after N-butoxycar-
bonylation. Oxidation of 42 with DMSO-(COCl)223 to give a
5-keto derivative 43 followed by a Wittig methylenation gave
44. Hydroboration of 44 with 9-BBN gave a mixture of 45
with a D-gluco configuration (R1 ) CH2OH, R2 ) H) and the
other isomer of L-idose 45′. Conversion of 45 to the corre-
sponding uronic acid derivative was performed in a stepwise
removal of the benzyl group of 47 followed by acidic treatment
furnished the D-glucuronic acid-type 1-N-iminosugar 14 and an
easily separable L-iduronic acid derivative 48.
Synthesis of D-Xylose-Type Iminosugar 15 (Scheme 5).
The D-xylose-type 1-N-iminosugar 15 was easily prepared from
a synthetic intermediate 42 during the synthesis of the D-
glucuronic acid-type iminosugar 14. Treatment of 43 with
thiocarbonyldiimidazole gave a thiocarbonylimidate derivative
49, which was subsequently subjected to radical deoxygenation
with Bu3SnH to give a deoxygenated derivative 50. Hydro-
genolysis of 50 followed by acidic treatment afforded the
D-xylose-type 1-N-iminosugar 15.
23
manner. First, oxidation of 45 with DMSO-(COCl)2 gave
N-Alkylation of 1-N-Iminosugars. N-Alkylation of 1-N-
iminosugars was performed by reductive amination of n-butanal
and borane-pyridine complex in MeOH-phosphate buffer27
to give the corresponding N-n-butyl 1-N-iminosugars almost
quantitatively.
an aldehyde 46, which was subsequently further oxidized by
H2O2 and NaClO2 in a CH3CN-phosphate buffer system26 to
produce a D-glucuronic acid derivative 47. Hydrogenolytic
(25) Corey, E. J.; Nicolaou, K. C.; Balanson, R. D.; Machida, Y. Synthesis
1975, 590-591.
(26) Dalcanale, E.; Montanari, F. J. Org. Chem. 1986, 51, 567-569.
(27) Yoshida, T.; Lee, Y. C. Carbohydr. Res. 1994, 251, 175-186.

1-N-Iminosugars
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3011
Evaluation of the Inhibitory Potencies of the 1-N-Imino-
sugar Derivatives. We evaluated28 the ability of each of the
synthesized 1-N-iminosugars to inhibit a series commercially
available glycosidases: R-glucosidase (yeast), â-glucosidase
(almond), R-galactosidase (green coffee beans), â-galactosidase
(Aspergillus orizae), R-mannosidase (jack bean), R-fucosidase
(bovine kidney), and â-glucuronidase (bovine liver), all obtained
from Sigma Chemical Co., (St. Louis, MO). The IC50 values
obtained are summarized in Table 1.
in 5530 and 56,30 significantly abolished the inhibitory activity
of the iminosugar (entries 8 and 9).
Fuconojirimycin, a DNJ-type iminosugar, is a potent inhibitor
of R-fucosidase, with a Ki of 4.8 nM;32 however, L-fucose-type
1-N-iminosugar 13 (entry 10) was, as expected, a moderate
inhibitor against R-fucosidase, with an IC50 of 26 µM, because
the 1-N-iminosugar was designed as an inhibitor for â-glycosi-
dase and not for R-glycosidase.16 It is noteworthy that the
N-butyl derivative 57 (Entry 11) was found to inhibit â-glu-
cosidase effectively, with an IC50 of 80 µM.
The conventional type iminosugar deoxynojirimycin (DNJ)
inhibits R-glucosidase more potently (Ki ) 12.6 µM) than
â-glucosidase (Ki ) 47 µM).8c As expected, the glucose-type
1-N-iminosugar 11 (entry 1) was found to be a very potent
inhibitor of â-glucosidase with an IC50 value of 0.1 µM; in
contrast, it did not effectively inhibit R-glucosidase (IC50, 150
µM). The structure of the 1-N-iminosugar does not include a
C-2 OH group; however, the IC50 value of this iminosugar
against R-mannosidase was determined to be 250 µM, which
is similar to that for R-glucosidase, and it may be concluded
that the glucose-type 1-N-iminosugar 11 did not discriminate
between R-glucosidase and R-mannosidase. Even though 11
did not inhibit either R-galactosidase and R-fucosidase, it
showed a modest inhibition of â-galactosidase. When the
nitrogen group was butylated, the N-butylglucose-type 1-N-
iminosugar 51 (entry 2) inhibited â-glucosidase 380-fold less
potent than did the N-unmodified iminosugar 11. N-Modifica-
tion (alkylation) has been reported to not alter the inhibitory
activity of the DNJ-type iminosugar very much;29 however, the
inhibitory potencies of 1-N-iminosugars were found to be
significantly affected. It is interesting that the inhibitory potency
against R-glucosidase remained the same upon N-alkylation.
With an additional OH group at the C-5 position of 11,
compound 2011 (entry 3) was also a potent inhibitor of
â-glucosidase but was not as potent as 11, which lacks this OH
group. The N-butyl derivative 52 showed an inhibition pattern
similar to that of 51.
DNJ-type iminosugar galactonojirimycin is a very potent
inhibitor of R-galactosidase with a Ki of 1.6 nM but is not very
effective for R-galactosidase (Ki ) 160 nM).8c,31 The galactose-
type 1-N-iminosugar 12 (entry 5) was found to be an extremely
potent inhibitor of â-galactosidase, with an IC50 value of 12
nM (Ki ) 4 nM).15 It was also a potent inhibitor of â-glucosi-
dase (IC50 of 190 nM), although the stereochemistry at the C-4
OH group is different. These data suggested that the most
critical factor required for potent inhibition may be the charge
distribution. N-Butylation of 12 (entry 6) dramatically weak-
ened the inhibitory potency against â-galactosidase (by ∼400-
fold); the IC50 of 53 was determined to be 5 µM. However, 53
showed a slightly higher (about 4-fold) inhibitory activity against
R-galactosidase. The N-oxide derivative 54 (entry 7) showed
moderate inhibitory activity similar to that of 53, and it may
indicate that the oxidation of the nitrogen atom did not help
produce a clear positive charge on the nitrogen atom. The
presence of the an additional OH group at the C-5 position, as
A conventional glucuronic acid-type iminosugar is a weak
inhibitor of â-D-glucuronidase, with a Ki of 80 µM,33 whereas,
as expected, the glucuronic acid-type 1-N-iminosugar 15 (Entry
13) was a very potent inhibitor of â-D-glucuronidase with a Ki
of 79 nM, and this iminosugar did not inhibit other glycosidases
at concentrations below 1 mM.18 L-Iduronic acid-type imino-
sugar 48 (Entry 14) inhibits â-D-glucuronidase only modestly
(IC50 ) 1.3 µM).
We have also examined the inhibitory potencies of other 1-N-
iminosugars that were easily prepared from the synthetic
intermediates of the syntheses of 11-14. A C-2 symmetrical
iminosugar 58 (Entry 15) which was prepared from 32 by acidic
treatment was found to be a potent inhibitor of â-glucosidase,
with an IC50 of 8.8 µM. It also inhibited â-galactosidase only
weakly (IC50 ) 40 µM). The N-butyl derivative 59 (Entry 16)
had a 60-fold lower inhibitory activity against â-glucosidase.
The L-galactose-type 1-N-iminosugar 60, which was prepared
by hydroboration of 36, and its N-butyl derivative 61 inhibited
â-glucosidase and â-galactosidase only weakly (entries 17 and
18). Another iminosugar 6224,34a (Entry 19), prepared from the
synthetic intermediate 24, was found to inhibit R- and â-ga-
lactosidases equally, with IC50 values of 50-70 µM. It is
interesting that the L-idose-type 1-N-iminosugar 23 (Entry 21)
inhibited â-glucosidase quite well, with an IC50 of 8 µM.
Discussion. We have designed and synthesized a new class
of glycosidase inhibitors, 1-N-iminosugars. All the syntheses
were straightforward and highly stereoselective and were carried
out using readily available monosaccharides. There was a
remarkable difference between our 1-N-iminosugars 9 and the
conventional DNJ-type iminosugars 10. Iminosugars with a
nitrogen atom replacing the ring oxygen position 10 tend to
inhibit the corresponding R-glycosidases more potently than the
â-glycosidases.8c In contrast, we have found that our 1-N-
iminosugars 9 with a nitrogen atom at the anomeric position
are extremely potent inhibitors of the corresponding â-glycosi-
dases, and show only modest inhibitory potencies against
R-glycosidases.
In the case of the R-galactosidase reaction (A in Figure 3),
(1) a substrate R-galactoside as a ground-state structure is bound
by the enzyme active site, (2) the aglycon of the enzyme-bound
substrate is protonated by a general acid, and (3) an E2-like
elimination reaction takes place between the lone electron pair
of the ring oxygen and the protonated aglycon shown in 64.8
Because these two components in the E2 elimination are
positioned antiperiplanar to each other, this elimination process
proceeds rapidly without a noticeable intermediate to lead to a
(28) (a) Halvorson, H. Methods Enzymol. 1966, 8, 559-562. (b) Dale,
M. P.; Ensley, H. E.; Kern, K.; Sastry, K. A. R.; Byers, L. D. Biochemistry
1985, 24, 3530-3539.
(29) (a) For study of N-alkylated iminosugars with isolated enzymes,
see: Al Daher, S.; Fleet, G.; Namgoong, S. K.; Winchester, B. Biochem. J.
1989, 258, 613-615. (b) For study of N-alkylated iminosugars with cells,
see: Tan, A.; van den Broek, L.; van Boeckel, S.; Ploegh, H.; Bolscher, J.
J. Biol. Chem. 1991, 266, 14504-14510.
(30) Ichikawa, M.; Ichikawa, Y. Biorg. Med. Chem. 1995, 3, 161-165.
(31) (a) Paulsen, H.; Hayauchi, Y.; Sinnwell, V. Chem. Ber. 1980, 113,
2601-2608. (b) Legler, G.; Pohl, S. Carbohyr. Res. 1986, 155, 119-129.
(c) Bernotas, R. C.; Pezzone, M. A.; Ganem, B. Carbohydr. Res. 1987,
167, 305-311.
(32) Fleet, G. W.; Shaw, A. N.; Evans, S. V.; Fellows, L. E. J. Chem.
Soc., Chem. Commun. 1985, 841-842.
(33) Cenci di Bello, I.; Dorling, P.; Fellows, L.; Winchester, B. FEBS
Lett. 1984, 176, 61-64.
(34) We synthesized the iminosugars 62 and 63 from 24 which was a
synthetic intermediate for the galactose-type 1-N-iminosugar 12. Their
syntheses have been reported. For 62: (a) Bernotas, R. C.; Papandreous,
G.; Urbach, J.; Ganem, B. Tetrahedron Lett. 1990, 31, 3393-3396. (b)
See ref 24. For 63: McCaig, A. E.; Chomier, B.; Wightman, R. H. J.
Carbohydr. Chem. 1994, 13, 397-407.

3012 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.

1-N-Iminosugars
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3013
Figure 3. Proposed reaction mechanism of R- and â-galactosidases.
well-accepted transition-state structure 65 in which the galactose
has a half-chair conformation and a positive charge developing
at the position of the ring oxygen. A naturally occurring
deoxygalactonojirimycin (DGJ) 68, a conventional DNJ-type
iminosugar, is an extremely potent inhibitor of R-galactosidase
(Ki ) 1.6 nM)8c,31 presumably because it has a ground-state
conformation and has a positive charge that is close enough to
be able to interact with the negative charge of the general base.
A galactosylamine derivative 69 has a positive charge on the
glycosyl amino group that is a little too far to interact with the
general base, and therefore it shows a modest inhibitory potency
against R-galactosidase.8c Ganem’s amidine derivative 7035 has
both characteristics, making it a potential potent inhibitor of
glycosidase: a half-chair conformation and a positive charge
at the ring oxygen position mimicking the transition-state
structure 65; however, its Ki value was reported to be 8.3 µM
for R-galactosidase.6c,35 This result was rather unexpected
because it has been thought that a transition-state mimetics
would be a more potent inhibitor than would a compound that
is a ground-state analogue. It is possible to assume that a
conformational difference between the ground-state 64 and the
amidine derivative 70 could disrupt the binding of the inhibitor
to an enzyme active site that binds to a substrate with a ground-
state conformation. A five-membered iminosugar 71 prepared
by the Wong group,24 which shares some of the characteristics
of both DGJ and Ganem’s amidine-type compound, also showed
an inhibitory potency that was similar to that of the amidine
derivative 70, probably for the reasons already cited for the
amidine 70. Our 1-N-iminosugar inhibitor 12 showed only a
weak inhibition of R-galactosidase (IC50 ) 200 µM) because
the positive charge on the C-1 nitrogen atom is too far away
for interacting with the general base.
As in the case of the â-galactosidase reaction mechanism (B
in Figure 3), a protonated aglycon of the enzyme-bound substrate
â-galactoside 66 departs from the sugar ring with the aid of the
general base. This leaves (albeit transiently) a galactose
derivative having a positive charge at the anomeric position 67.
We gave the name “First Intermediate” to this derivative 67, a
relatively stable reaction intermediate of the â-galactosidase
reaction that is stabilized by the general base group. The
complex of First Intermediate-general base is similar in
structure to the glycosyl enzyme complex described by the
Withers group.12,37 First Intermediate is then collapsed, with
the aid of the lone electron pair of the ring oxygen, shown as
67, to yield the common reaction intermediate 65. DGJ 68 was
found to be a less potent inhibitor of â-galactosidase than of
R-galactosidase8c presumably because positive charge may be
located far from the general base, even though it has a ground-
state conformation which is favorable for binding to the enzyme
active site. The galactosylamine derivative 69 is still a moderate
inhibitor of â-galactosidase,8c again because its positive charge
is located far from the general base. Because Ganem’s amidine
70 was prepared to target, with respect to conformational and
charge characters, the well-accepted transition-state structure 65,
which is common to both R- and â-galactosidases, the com-
pound shows similar inhibitory potencies against both R- (Ki
) 8.3 µM) and â-galactosidases (Ki ) 6.5 µM).6c,35 This finding
may indicate that a compound which mimics the transition state
of the glycosidase reaction could be a potent inhibitor but rather
nonspecific. Wong’s five-membered iminosugar 71 shows a
similar decrease in inhibitory potency when compared to that
(35) (a) Tong, M. K.; Papandreou, G.; Ganem. B J. Am. Chem. Soc.
1990, 112, 6137-6139. (b) An unsuccessful approach to a similar amidine
derivative was reported: Bird, P.; Dolphin, D. H.; Withers, S. G. Can. J.
Chem. 1990, 68, 317-322.
(36) Wang, Y.-F.; Takaoka, Y.; Wong, C.-H. Angew. Chem., Int. Ed.
Engl. 1994, 33, 1242-1244.
(37) Withers, S. G.; Street, I. P. J. Am. Chem. Soc. 1988, 110, 8551-
8553.

3014 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.
of DGJ.36 Our 1-N-iminosugar 12 has both characteristics
required for targeting First Intermediate 67: a ground-state-
like conformation and correct positioning of the positive charge
to allow it to interact with the general base; consistent with
these features, it was found to be an extremely potent inhibitor
of â-galactosidase, with a Ki value of 4.1 nM. Further study
is necessary to determine whether First Intermediate is formed
via an SN1-type process followed by subsequent charge stabi-
lization or whether it is formed via an assisted internal concerted
process involving the carboxylate ion as a nucleophile as
suggested by the Wither’s group.10-12 In any cases, our work
has not only further confirmed the structure of the intermediate
of glycosidase reaction by the Wither’s group but has also
suggested a strategy for designing a potent and specific inhibitor
of â-glycosidase: (i) a proper charge distribution (positive
charge at the anomeric position) and (ii) a ground-state-like
conformation (a chair conformation).
Summary. We have described the synthesis, from readily
available monosaccharides, of a new class of 1-N-iminosugars
with a nitrogen atom at the anomeric position. These syntheses
were carried out in a highly stereoselective and efficient manner
and produced 1-N-iminosugars that are highly potent and
specific inhibitors of â-glycosidases. The concept of imino-
sugars that we demonstrated herein clearly indicates a design
strategy for development of selective inhibitors of glycosi-
dases: We could develop a potent and specific inhibitor of a
â-glycosidase by synthesizing an analogue of our 1-N-imino-
sugars 9; in contrast, an analogue of the conventional DNJ-
type iminosugars 10 would be expected to be a potent inhibitor
of R-glycosidases.
H-5), 4.83 (d, 1H, J ) 5.8 Hz, H-2), 4.87 (dd, 1H, J ) 3.2 and 5.8 Hz,
H-3), 6.35 (s, 1H, H-1), 7.32 (d, 2H, J ) 8.0 Hz, o-aromatic H of
benzoyl group), 7.45 (t, 2H, J ) 7.7 Hz, m-aromatic H of benzoyl
group), 7.59 (tt, 1H, J ) 1.4 and 7.4 Hz, p-aromatic H of benzoyl
group), 7.81 (d, 2H, J ) 8.3 Hz, aromatic H of tosyl group), 7.98 (d,
2H, J ) 8.4 Hz, aromatic H of tosyl group). Anal. Calcd for C22H24-
SO8: C, 58.92; H, 5.39. Found: C, 58.52; H, 5.52.
5-Azido-5-deoxy-2-(hydroxymethyl)-2,3-O-isopropylidene-r/â-D-
lyxofuranose (19). A mixture of 17 (21.0 g, 46.8 mmol) and NaN3
(13.2 g, 203 mmol) in DMF (150 mL) was heated overnight at 120
°C, and the reaction mixture was poured into ice-water and extracted
with EtOAc. The combined extracts were successively washed with
water, aqueous NaHCO3 solution, and brine, dried, and concentrated
to give an azido derivative 18 as a colorless solid. This was employed
for the next step without further purification.
A solution of the above 18 in MeOH (250 mL) and 25% methanolic
NaOMe (2.5 mL) was stirred for 30 min at room temperature, and the
solution was neutralized by the addition of AcOH. The mixture was
concentrated, and the residue was passed through silica gel with
toluene-EtOAc (20:1) to give the crude O-debenzoylated derivative
as a colorless oil. This was employed for the next step without further
purification.
A solution of the above anomeric free derivative, 30% HCHO
solution (125 mL), and K2CO3 (12.5 g, 90.4 mmol) in MeOH (200
mL) was heated for 24 h at 85 °C. After cooling, the reaction mixture
was neutralized by the addition of 10% H2SO4 solution, concentrated,
diluted with water, and extracted with EtOAc. The combined extracts
were successively washed with water, aqueous NaHCO3 solution, and
brine, dried, and concentrated. The residue was chromatographed on
silica gel with hexanes-EtOAc (20:1 to 2:1) to give 19 (9.2 g, 80%)
as a colorless oil, which crystallized on standing. A small portion was
recrystallized from hexanes-EtOAc to give an analytical sample as
colorless needles: mp 79-81 °C (hexanes-EtOAc); 1H NMR (CDCl3)
δ 1.48 and 1.57 (each s, each 3H, CH3 of the isopropylidene group),
3.56 (dd, 1H, J ) 6.43 and 13.04 Hz, H-5a), 3.59 (dd, 1H, J ) 6.47
and 13.04 Hz, H-5b), 3.74 (ddd, 1H, J ) 2.86 and 6.45 Hz, H-4), 3.80
(d, 1H, J ) 2.89 Hz, H-3), 3.85 (d, 1H, J ) 11.7 Hz, H-2′a), 4.60 (d,
1H, J ) 2.97 Hz, H-1), 4.92 (d, 1H, J ) 11.7 Hz, H-2′b).
Experimental Section
General Methods. All melting points are uncorrected. The reagents
used were purchased from Aldrich, Sigma, or Acros, and the solvents
were reagent grade and used as supplied. Organic extracts were dried
over anhydrous MgSO4 and concentrated in vacuo. 1H NMR spectra
were recorded at 300 MHz, and 13C NMR spectra were recorded at 75
MHz on a Bruker AXM 300 spectrometer. Internal standards used in
1H NMR spectra were TMS (δ 0.00) for CDCl3, HOD (δ 4.78) for
D2O, and in 13C NMR were CDCl3 (δ 75.0) for CDCl3 and CH3CN (δ
1.30) for D2O. Elemental analysis were performed by Galbraith
Laboratories (Knoxville, TN), and mass spectral data were analyzed
by Mass Spectrometry Laboratory at University of Illinois (Urbana-
Champaign, IL).
Synthesis of D-Glucose-Type 1-N-Iminosugar. 1-O-Benzoyl-2,3-
O-isopropylidene-5-O-p-toluenesulfonyl-r-D-lyxofuranose (17). A
mixture of D-lyxose (12.6 g, 83.9 mmol) and concentrated H2SO4 (0.74
mL) in dry acetone (300 mL) was stirred overnight at room temperature,
and the reaction mixture was neutralized by stirring with solid
anhydrous Na2CO3 (30 g) for 3 h. The suspension was filtered and
concentrated to give crude 2,3-O-isopropylidene-D-lyxofuranose as a
colorless syrup. This was employed for the next step without further
purification.
p-Toluenesulfonyl chloride (17.6 g, 92.3 mmol) was added portion-
wise to a cooled solution of the above isopropylidene derivative in
pyridine (300 mL) at 0-5 °C, and the mixture was stirred overnight at
room temperature. The reaction mixture was then cooled to 0-5 °C,
and benzoyl chloride (14.2 g, 101 mmol) was added dropwise to the
mixture. After 1 h, the reaction mixture was poured into ice-water
and extracted with EtOAc. The combined extracts were successively
washed with diluted HCl solution, aqueous NaHCO3 solution, and brine,
dried, and concentrated to give crude 17 (29.3 g, 78% from D-lyxose)
as a colorless solid. A small portion was recrystallized from hexanes-
EtOAc to give an analytical sample as colorless needles: mp 115-
118 °C (hexanes-EtOAc); 1H NMR (CDCl3) δ 1.30 and 1.39 (each s,
each 3H, CH3 of isopropylidene group), 2.42 (s, 3H, CH3 of tosyl
group), 4.21 (ddd, 1H, J ) 3.2 and 8.9 Hz, H-4), 4.36-4.44 (m, 2H,
tert-Butyl (3S,4S,5R)-4,5-Di-O-benzoyl-3-((benzoyloxy)methyl)-
3,4,5-trihydroxypiperidine-1-carboxylate (21). A mixture of 19 (2.0
g, 8.16 mmol) and Pd(OH)2 (450 mg) in MeOH (100 mL) was
vigorously stirred under an atmosphere of H2 at room temperature for
12 h, the reaction mixture was filtered through a Celite pad, and the
filter cake was washed with aqueous 50% MeOH. The combined
filtrates were concentrated to give a crude piperidine derivative. This
was employed for the next step without further purification.
A solution of the above piperidine derivative in 1 N HCl (30 mL)
was stirred overnight at room temperature, and the mixture was
concentrated. The residue was coevaporated with MeOH and 1,4-
dioxane several times to give crude 20. This was employed for the
next step without further purification.
A mixture of the above 20, Et3N (1.79 mL, 12.9 mmol), and (Boc)2O
(3.74 g, 17.2 mmol) in MeOH (100 mL) was stirred overnight at room
temperature, and the reaction mixture was concentrated to give an
N-Boc derivative. This was employed for the next step without further
purification.
Benzoyl chloride (6.03 g, 42.9 mmol) was added dropwise to a
solution of the above N-Boc derivative in pyridine (50 mL) at 0-5
°C, and the reaction mixture was stirred overnight at room temperature.
The reaction mixture was poured into ice-water and extracted with
EtOAc. The combined extracts were successively washed with water,
diluted CuSO4 solution, aqueous NaHCO3 solution, and brine, dried,
and concentrated. The residue was chromatographed on silica gel with
hexanes-EtOAc (20:1 to 2:1) to give 21 (2.3 g, 49% overall) as a
colorless solid. A small portion was recrystallized from hexanes-
EtOAc to give an analytical sample as colorless cubes: mp 174-176
1
°C (hexanes-EtOAc); H NMR (CDCl3) δ 1.45 (s, 9H, tBu of Boc
group), 3.35 (m, 2H), 4.18 (m, 1H), 4.38 (m, 1H), 4.40 (d, 1H, J )
11.7 Hz), 4.50 (d, 1H, J ) 11.7 Hz), 5.52 (m, 1H, H-3), 5.68 (d, 1H,
J ) 8.2 Hz, H-4), 7.32-7.58 (m, 9H, aromatic H of benzoyl group),
7.91-8.02 (m, 6H, aromatic H of benzoyl group).

1-N-Iminosugars
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3015
D-Glucose-Type 1-N-Iminosugar: (3R,4R,5R)-5-(Hydroxymethyl)-
piperidine-3,4-diol Hydrochloride Salt (11). MeOCOCOCl (0.19 mL,
255 mg, 2.08 mmol) was added dropwise to a cooled solution of 21
(700 mg, 1.22 mmol) and DMAP (340 mg, 2.78 mmol) in CH3CN (10
mL) at 0-5 °C, and the reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was poured into ice-water and
extracted with EtOAc, and the combined extracts were successively
washed with water, aqueous NaHCO3 solution, and brine, dried, and
passed through a short column of dry silica gel with EtOAc. Due to
the liability of the compound, the combined filtrates were concentrated
and the compound was employed for the next step immediately.
A solution of Bu3SnH (809 mg, 2.78 mmol) and VAZO (30 mg,
catalytic amount) in toluene (30 mL) was added dropwise to a solution
of the above oxalyl ester in toluene (10 mL), and the mixture was heated
at 100 °C for 1 h. The reaction mixture was concentrated, and the
residue was passed through silica gel with toluene-EtOAc (40:1) to
give crude deoxygenated derivatives.
A solution of the above deoxygenated derivatives and 25% metha-
nolic NaOMe (0.20 mL) in MeOH (5 mL) was stirred for 2 h at room
temperature. Next, 1 N HCl (5 mL) was added to the mixture, and the
reaction mixture was stirred overnight at room temperature. The
mixture was diluted with water (20 mL) and applied onto a column of
Dowex 50W-X8 [H+] resin (20 mL). The column was washed with
water (30 mL), and the product was eluted out with 3% NH4OH (50
mL). The fractions containing the mixture of 11 and 23 were pooled
and concentrated. The residue was chromatographed on silica gel with
2-propanol-H2O-NH4OH (7:2:1) to give chromatographically pure
11 (Rf 0.37 in 2-propanol-H2O-NH4OH, 7:2:1) and 23 (Rf 0.26 in
2-propanol-H2O-NH4OH, 7:2:1). Water (10 mL) was added to the
residue, and the solution was concentrated in order to remove the
residual NH4OH. Water (10 mL) and 1 N HCl (5 mL) were added to
residue, and the solution was concentrated in order to form a
hydrochloride salt of the iminosugar. The residue was applied onto a
column of Sephadex G-25 (1.0 × 25 cm) and eluted with water. The
fractions containing the product were pooled and concentrated. The
residue was lyophilized from water (5 mL) to afford 11 (80 mg, 36%)
as a colorless amorphous powder (HCl salt). In a similar manner, 23
(40 mg, 18%) was also obtained as a colorless amorphous powder (HCl
salt). For 11 (D-glucose-type): 1H NMR (D2O) δ 1.91-2.02 (m, 1H,
H-5), 2.88 (t, 1H, J ) 11.92 Hz), 2.97 (t, 1H, J ) 12.68 Hz), 3.48-
3.56 (m, 2H), 3.71-3.85 (m, 3H). The data were in good agreement
with those reported by Bols et al.14 For L-idose-type 1-N-iminosugar
(3R,4R,5S)-5-hydroxymethyl-piperidine-3,4-diol hydrochloride salt
(23): 1H NMR (D2O) δ 2.37-2.45 (m, 1H, H-5), 3.00 (t, 1H, J )
12.52 Hz), 3.22-3.38 (m, 3H), 3.59 (dd, 1H, J ) 7.19, 11.29 Hz, H-7a),
3.70 (dd, 1H, J ) 6.56, 11.29 Hz, H-7b), 3.98-4.01 (m, 1H), 4.04-
4.07 (m, 1H).
N-Butyl D-Glucose-Type 1-N-Iminosugar: (3R,4R,5R)-N-Butyl-
5-(hydroxymethyl)piperidine-3,4-diol Hydrochloride Salt (51). BH3‚
pyridine (8 M; 50 mL, 0.40 mmol) was added to a cooled mixture of
11 (24.7 mg, 0.13 mmol) and n-PrCHO (19 mg, 0.27 mmol; 25 mL)
in MeOH (2 mL) and phosphate buffer (100 mM, pH 6.8; 5 mL) at
0-5 °C, and the reaction mixture was stirred overnight at room
temperature. The mixture was diluted with water (30 mL), and Dowex
50W-X8 [H+] resin (30 mL) was carefully added to the mixture
(hydrogen bubble was evolved). After the bubbling ceased, the resin
was packed in a column and washed with water (50 mL). The product
was eluted out with 3% NH4OH, and the fractions containing the
product were pooled and concentrated. The residue was diluted with
water and 1 N HCl (until the solution became acidic), and the resulting
mixture was concentrated. The residue was purified with Sephadex
G-25, with water, and the fractions were concentrated. The product
was lyophilized from water to give 51 (25 mg, 80%): 1H NMR (D2O)
δ 0.94 (t, 3H, J ) 7.38 Hz, CH3 of Bu), 1.39 (q, 2H, J ) 7.43 Hz,
CH3CH2CH2CH2N), 1.68-1.79 (m, 2H, CH3CH2CH2CH2N), 1.92-2.07
(m, 1H, H-5), 2.85-3.04 (m, 1H), 3.18-3.25 (m, 2H), 3.53 (t, 1H, J
) 10.13 Hz), 3.61-3.66 (m, 1H), 3.72-3.86 (m, 3H).
and the reaction mixture was neutralized by the addition of AcOH.
The mixture was concentrated, and the residue was passed through silica
gel with toluene-EtOAc (20:1) to give crude anomeric free product.
This was employed for the next step without further purification.
A mixture of the above product and Pd(OH)2 (250 mg) in MeOH
(200 mL) was vigorously stirred under 1 atm of H2 for 12 h at room
temperature, the reaction mixture was filtered through a Celite pad,
and the filter cake was washed with aqueous MeOH. The combined
filtrates were concentrated to give a piperidine derivative 24. This was
employed for the next step without further purification.
A solution of the above 24, (Boc)2O (1.02 g, 4.70 mmol), and Et3N
(0.48 g, 4.70 mmol) in MeOH (20 mL) was stirred overnight at room
temperature, and the reaction mixture was concentrated. The residue
was diluted with EtOAc, and the organic layer was successively washed
with water and brine, dried, and concentrated to give 25. This was
employed for the next step without further purification.
A solution of DMSO (1.18 g, 15.1 mmol) in CH2Cl2 (5 mL) was
added dropwise to a cooled solution of (COCl)2 (3.77 mL, 7.6 mmol,
2.0 M solution) in CH2Cl2 (15 mL) at -78 °C. After 30 min, to the
solution was added dropwise a solution of the above 25 in CH2Cl2 (7
mL) at -78 °C, and the mixture was stirred for another 40 min at -78
°C. A solution of Et3N (1.90 g, 18.8 mmol) in CH2Cl2 (5 mL) was
then added dropwise to the reaction mixture at -78 °C, and the reaction
temperature was gradually raised to 0-5 °C in 30 min. The reaction
mixture was poured into ice-water and extracted with EtOAc. The
combined extracts were successively washed with water, aqueous
NaHCO3 solution, and brine, dried, and concentrated. The residue was
chromatographed on silica gel with toluene-EtOAc (10:1 to 4:1) to
give 26 (365 mg, 43% overall) as a colorless needle: mp 116.0-116.5
°C (hexanes-EtOAc); 1H NMR (CDCl3) δ 1.40 (s, 3H, CH3 of
isopropylidene group), 1.45 (s, 9H, t-Bu of Boc group), 1.47 (s, 3H,
CH3 of isopropylidene group), 3.51 (m, 1H, H-6), 3.60 (m, 1H, H-6),
4.65 (m, 1H, H-2), 4.83 (m, 2H, H-4 and -5), 4.95 (m, 1H, H-2); 13C
NMR (CDCl3) δ 24.9, 26.3, 80.5, 111.2, 154.2, 202.8.
tert-Butyl (3R,4S)-3,4-O-Isopropylidene-5-methylene-3,4-dihy-
droxypiperidine-1-carboxylate (27). A solution of 1.6 M n-BuLi (3.47
mL, 5.17 mmol) in hexane was added dropwise to a cooled suspension
of methyltriphenylphosphonium bromide (2.19 g, 5.77 mmol) in DME
(35 mL) at 0-5 °C, and the mixture was stirred for 1 h at 0-5 °C. A
solution of 26 (320 mg, 1.18 mmol) in DME (2 mL) was added
dropwise to the solution at 0-5 °C, and the mixture was stirred for 2
h at 0-5 °C. The reaction mixture was then stirred for another 8 h at
the ambient temperature. The reaction mixture was poured into ice-
water and extracted with EtOAc. The combined extracts were
successively washed with water, aqueous NaHCO3 solution, and brine,
dried, and concentrated. The residue was chromatographed on silica
gel with toluene-EtOAc (30:1 to 10:1) to give 27 (246 mg, 77%) as
a colorless oil: 1H NMR (CD3OD) δ 1.34 (s, 3H, CH3 of isopropylidene
group), 1.40 (s, 3H, CH3 of isopropylidene group), 1.46 (s, 9H, tBu of
Boc group), 2.94 (m, 1H, H-2), 3.74 (m, 1H, H-6), 3.89 (dd, J ) 2.4
and 14.4 Hz, H-2), 4.24-4.36 (m, 2H, H-3 and H-6), 4.71 (d, J ) 7.5
Hz, 1H, H-4), 5.20 (s, 1H, CdCH2), 5.27 (s, 1H, CdCH2); 13C NMR
(CD3OD) δ 24.94, 27.05, 28.76, 76.05, 77.33, 80.87, 110.25, 141.24,
156.91.
tert-Butyl (3R,4S,5R)-3-(Hydroxymethyl)-4,5-O-isopropylidene-
4,5-dihydroxypiperidine-1-carboxylate (28). To a stirred solution of
27 (200 mg, 0.743 mmol) in THF (6 mL) was added dropwise a 0.5 M
solution of 9-BBN (7.43 mL, 3.72 mmol) in THF at 0-5 °C, and the
reaction mixture was stirred for 15 h at room temperature and then
cooled to 0-5 °C. To the cooled mixture were successively added
water (0.5 mL), 1 N NaOH solution (0.5 mL), and 30% H2O2 solution
(0.5 mL) at 0-5 °C, and the reaction mixture was stirred for 12 h at
room temperature. The reaction mixture was diluted with water and
extracted with EtOAc. The combined extracts were successively
washed with water and brine, dried, and concentrated. The residue
was chromatographed on silica gel with toluene-EtOAc (20:1 to 3:1)
to give 28 (121 mg, 56%) as a colorless oil: 1H NMR (CDCl3) δ 1.35
and 1.45 (each s, each 3H, CH3 of isopropylidene group), 1.46 (s, 9H,
tBu of Boc group), 2.01 (s, 1H), 3.22 (m, 2H), 3.50 (m, 1H), 3.75 (m,
4H), 4.46 (dd, 1H, J ) 2.67 and 7.02 Hz); HRMS (CI) calcd for C14H25-
NO5 (M+) 287.1733, found 287.1738.
Synthesis of D-Galactose-Type 1-N-iminosugar. tert-Butyl (4R,5R)-
4,5-O-Isopropylidene-4,5-dihydroxy-3-piperidone-1-carboxylate (26).
A solution of 18 (1.0 g, 3.13 mmol) and 25% methanolic NaOMe (0.2
mL) in MeOH (20 mL) was stirred for 30 min at room temperature,

3016 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.
D-Galactose-Type 1-N-Iminosugar: (3R,4S,5R)-5-(Hydroxymeth-
yl)piperidine-3,4-diol Hydrochloride Salt (12). A solution of 28 (103
mg, 0.36 mmol) in 1 N HCl (10 mL) was stirred overnight at room
temperature, and the reaction mixture was concentrated. The residue
was coevaporated with water (10 mL) and purified with a column of
Sephadex G-25 with water. The fractions containing iminosugar were
pooled and concentrated, and the residue was lyophilized from water
(5 mL) to give 12 (62 mg, 94%) as a colorless amorphous powder: 1H
NMR (D2O) δ 1.89-1.99 (m, 1H, H-5), 2.73 (t, 1H, J ) 12.6 Hz,
H-2ax), 2.86 (t, 1H, J ) 11.9 Hz, H-6ax), 3.05 (dd, 1H, J ) 4.29 and
12.21 Hz, H-6eq), 3.09 (dd, 1H, J ) 7.07 and 11.89 Hz, H-2eq), 3.40
(dd, 1H, J ) 7.24 and 11.29 Hz, H-7a), 3.40 (dd, 1H, J ) 6.70 and
11.26 Hz, H-7b), 3.77 (ddd, 1H, J ) 2.69, 7.05, and 11.55 Hz, H-3),
3.93 (br s, 1H, H-4); 13C NMR (D2O) δ 41.54, 42.31, 44.65, 62.71,
68.32, 68.70; HRMS (CI) calcd for C6H13NO3 (M + H+) 148.0974,
found 148.0976.
3.51 (m, 1H, H-6), 3.60 (m, 1H, H-6), 4.65 (m, 1H, H-2), 4.83 (m,
2H, H-4 and -5), 4.95 (m, 1H, H-2); 13C NMR (CDCl3) δ 24.9, 26.3,
80.5, 111.2, 154.2, 202.8; FABMS calcd for C13H24N2O5 (M + NH3)+
288.1, found 288.1.
tert-Butyl (3S,4R)-3,4-O-Isopropylidene-5-methylene-3,4-dihy-
droxypiperidine-1-carboxylate (35). In a manner similar to the
preparation of the methylene derivative 27 from the ketone derivative
26, 34 (935 mg, 3.45 mmol) was converted to 35 (610 mg, 66%) as a
colorless oil: 1H NMR (CD3OD) δ 1.34 (s, 3H, CH3 of isopropylidene
group), 1.40 (s, 3H, CH3 of isopropylidene group), 1.46 (s, 9H, tBu of
Boc group), 2.94 (m, 1H, H-2), 3.74 (m, 1H, H-6), 3.89 (dd, J ) 2.4
and 14.4 Hz, H-2), 4.24-4.36 (m, 2H, H-3 and H-6), 4.71 (d, J ) 7.5
Hz, 1H, H-4), 5.20 (s, 1H, CdCH2), 5.27 (s, 1H, CdCH2); 13C NMR
(CD3OD) δ 24.94, 27.05, 28.76, 76.05, 77.33, 80.87, 110.25, 141.24,
156.91; HRMS (CI) calcd for C14H23NO4 (M + H+) 270.1705, found
270.1710.
Synthesis of L-Fucose-Type 1-N-Iminosugar. 1-O-Benzoyl-2,3-
O-isopropylidene-5-O-p-toluenesulfonyl-â-D-ribose (30). In a manner
similar to the preparation of the tosyl derivative 17 from D-lyxose 16,
D-ribose 29 (10 g) was converted to the tosyl derivative 30 (20.0 g,
48% from D-ribose) as colorless needles: mp 82.5-83.0 °C (hexanes-
tert-Butyl (3S,4R,5R)-3,4-O-Isopropylidene-5-methyl-3,4-dihy-
droxypiperidine-1-carboxylate (36). A suspension of 35 (493 mg,
1.83 mmol) and 10% Pd/C (50 mg) in EtOAc-EtOH (30 mL-20 mL)
was vigorously stirred under an atmosphere of H2 for 10 h at room
temperature, the reaction mixture was filtered through a Celite pad,
and the filter cake was washed with EtOAc. The combined filtrates
were concentrated, and the residue was chromatographed on silica gel
with hexanes-EtOAc (30:1 to 10:1) to give 36 (408 mg, 82%) as a
colorless oil: 1H NMR (CDCl3) δ 1.05 (d, 3H, J ) 6.9 Hz, CH3 at
C-5), 1.34 and 1.44 (each s, each 3H, CH3 of isopropylidene group),
1.46 (s, 9H, tBu of Boc group), 1.90 (m, 1H, H-5), 2.99 (t, 1H, J )
12.3 Hz), 3.34 (m, 2H), 3.68 (m, 1H), 4.20 (dd, 1H, J ) 2.5 and 6.9
Hz), 4.27 (br s, 1H); 13C NMR (CDCl3) δ 24.7, 26.5, 28.4, 30.7, 72.7,
74.9, 108.2, 164.1; HRMS (CI) calcd for C14H25NO4 (M + H+)
272.1862, found 272.1860.
L-Fucose-Type 1-N-Iminosugar: (3S,4R,5R)-5-methylpiperidine-
3,4-diol Hydrochloride Salt (13). In a manner similar to the
preparation of 12 from the protected derivative 28, 36 (278 mg, 1.02
mmol) was converted to 13 (154 mg, 90%) as a colorless amorphous
powder (HCl salt): 1H NMR (D2O) δ 0.93 (d, 3H, J ) 6.75 Hz, C-5
CH3), 1.96 (m, 1H, H-5), 2.73 (dd, 1H, J ) 12.6 Hz, H-2ax or -6ax),
2.91 (dd, 1H, J ) 11.7 Hz, H-6ax or -2ax), 3.01 (dd, 1H, J ) 3.65 and
12.6 Hz, H-2eq or -6eq), 3.17 (dd, 1H, J ) 4.22 and 11.3 Hz, H-6eq
or -2eq), 3.85 (s, 1H, H-4), 3.87 (dm, 1H, J ) 12 Hz, H-3); 13C NMR
(D2O) δ 13.89, 31.88, 41.85, 43.22, 66.25, 69.70; HRMS (CI) calcd
for C6H13NO2 (M + H)+ 132.1025, found 132.1023.
Synthesis of D-Glucuronic Acid-Type 1-N-Iminosugar 14. p-
Methoxyphenyl 5-Azido-5-deoxy-2,3-di-O-acetyl-r-D-arabinofura-
noside (39). TMSOTf (2.06 g, 1.79 mL, 9.27 mmol) was added
dropwise to a cooled solution of 38 (9.3 g, 30.9 mmol) and p-
methoxyphenol (5.75 g, 46.4 mmol) in CH2Cl2 (200 mL) at 0-5 °C,
and the mixture was stirred for 3 h at room temperature. The reaction
mixture was diluted with CH2Cl2, washed successively with saturated
NaHCO3 solution and brine, dried, and concentrated. The residue was
chromatographed on silica gel with hexanes-EtOAc (20:1 to 4:1) to
give 39 (9.5 g, 84%) as a colorless oil: 1H NMR (CDCl3) δ 2.14 (s,
3H, CH3 of acetyl group), 2.15 (s, 3H, CH3 of acetyl group), 3.47 (dd,
1H, J ) 4.8 and 13.3 Hz, H-5a), 3.69 (dd, 1H, J ) 3.0 and 13.3 Hz,
H-5b), 3.77 (s, 3H, OCH3), 4.35 (ddd, 1H, J ) 3.0, 4.8, and 4.9 Hz,
H-4), 5.08 (dd, 1H, J ) 1.4 and 4.9 Hz, H-3), 5.36 (d, 1H, J ) 1.4 Hz,
H-2), 5.61 (s, 1H, H-1), 6.83 (dm, 2H, J ) 9.1 Hz, aromatic H), 7.01
(dm, 2H, J ) 9.1 Hz, aromatic H); 13C NMR (CDCl3) δ 20.76, 51.34,
55.65, 77.68, 81.45, 82.86, 104.91, 114.63, 118.34, 149.93, 155.33,
169.75, 170.40. Anal. Calcd for C16H19N3O7: C, 52.60; H, 5.24; N,
11.50. Found: C, 52.80; H, 5.32; N, 11.29.
p-Methoxyphenyl 5-Azido-5-deoxy-2,3-di-O-benzyl-r-D-arabino-
furanoside (40). A mixture of 39 (8.0 g, 21.9 mmol) and 25%
methanolic NaOMe (1.6 mL) in MeOH (160 mL) was stirred for 30
min at room temperature, and the reaction mixture was neutralized with
Dowex 50W-X8 [H+], filtered to remove the resins, and concentrated.
The residual oil was diluted with Et2O, dried, and concentrated.
NaH (60% mineral oil dispersion; 2.10 g, 52.6 mmol) was added
portionwise to a cooled solution of the above residue in dry DMF (150
mL) at 0-5 °C, and the suspension was stirred for 30 min at room
temperature. The mixture was cooled again at 0-5 °C, benzyl bromide
1
EtOAc); H NMR (CDCl3) δ 1.35 (s, 3H, CH3), 1.57 (s, 3H, CH3),
2.39 (s, 3H, ArCH3), 4.05 (dd, 1H, J ) 6.9, 10.3 Hz, H-5), 4.10 (dd,
1H, J ) 5.1, 10.3 Hz, H-5), 4.48 (dd, 1H, J ) 5.1, 6.9 Hz, H-4), 4.87
(s, 2H, H-2,3), 6.43 (s, 1H, H-1), 7.22 (d, 2H, J ) 8.2 Hz, aromatic H
of the tosyl group), 7.48 (dd, 2H, J ) 7.7 Hz, aromatic H of the benzoyl
group), 7.62 (dd, 1H, J ) 7.3 Hz, aromatic H of the benzoyl group),
7.66 (d, 2H, J ) 8.2 Hz, aromatic H of the tosyl group), 8.00 (d, 2H,
J ) 7.3 Hz, aromatic H of the benzoyl group); 13C NMR (CDCl3) δ
21.47, 24.87, 26.33, 68.84, 81.09, 84.92, 85.09, 102.73, 113.32, 127.78,
128.45, 129.73, 129.80, 133.48, 145.04, 164.61. Anal. Calcd for
C22H22O8S: C, 59.19; H, 4.96. Found: C, 55.53; H, 5.50.
5-Azido-1-O-benzoyl-5-deoxy-2,3-O-isopropylidene-â-D-ribose (31).
In a manner similar to the preparation of the azido derivative 18 from
the tosyl derivative 17, 30 (18.7 g, 41.7 mmol) was converted to 31
(12.2 g, 92%) as colorless needles: mp 80.0-80.5 °C (EtOH-hexane);
1H NMR (CDCl3) δ 1.37 (s, 3H, CH3), 1.59 (s, 3H, CH3), 3.31 (dd,
1H, J ) 6.8 and 12.7 Hz, H-5), 3.55 (dd, 1H, J ) 7.0 and 12.7 Hz,
H-5), 4.48 (dt, 1H, J ) 0.93 and 6.9 Hz, H-4), 4.76 (dd, 1H, J ) 0.93
and 6.0 Hz, H-3), 4.90 (d, 1H, J ) 6.0 Hz, H-2), 7.46 (dd, 2H, J ) 7.4
and 7.9 Hz, m-aromatic H), 7.60 (tt, 1H, J ) 1.3 and 7.4 Hz, p-aromatic
H), 8.01 (dd, 2H, J ) 1.3 and 7.9 Hz, o-aromatic H); 13C NMR (CDCl3)
δ 24.9, 26.4, 53.0, 81.7, 85.2, 86.6, 103.1, 113.4, 128.5, 129.6, 133.5,
164.7. Anal. Calcd for C15H17N3O5: C, 56.42; H, 5.36; N, 13.16.
Found: C, 56.76; H, 5.43; N, 12.98.
(3S,4R,5R)-3,4-O-Isopropylidenepiperidine-3,4,5-triol (32). In a
manner similar to the preparation of the piperidine derivative 24 from
the azido derivative 18, 31 (4.62 g, 14.5 mmol) was converted to 32
(1.88 g, 95% overall) as a colorless amorphous powder: mp 92-95
°C; 1H NMR (CDCl3) δ 1.38 and 1.55 (each s, each 3H, CH3 of
isopropylidene group), 2.74-3.15 (m, 6H, H-2, -6, OH and NH), 3.79
(m, 1H), 4.15 (m, 1H), 4.27 (m, 1H); 13C NMR (CDCl3) δ 25.5, 26.9,
46.6, 47.2, 64.9, 71.8, 74.2, 108.7; HRMS (CI) calcd for C8H16NO3
(M + H)+ 174.1130, found 174.1129.
tert-Butyl (3S,4R,5R)-2,3-O-Isopropylidene-3,4,5-trihydroxypip-
eridine-1-carboxylate (33). In a manner similar to the preparation of
the N-Boc derivative 25 from the piperidine derivative 24, 32 (4.50 g,
26.0 mmol) was converted to 33 (5.10 g, 73%) as colorless needles:
1
mp 82.5-83.5 °C (from hexanes-EtOAc); H NMR (CDCl3) δ 1.38
and 1.51 (each s, each 3H, CH3 of isopropylidene group), 1.46 (s, 9H,
tBu of Boc group), 2.79 (m, 1H), 3.18 (dd, 1H, J ) 9.9 and 12.2 Hz),
3.45 (dd, 1H, J ) 4.0 and 14.0 Hz), 3.56 (m, 2H), 3.90 (m, 1H), 4.38
(m, 2H); 13C NMR (CDCl3) δ 24.7, 26.6, 28.3, 65.6, 72.2, 79.8, 109.3.
Anal. Calcd for C13H23NO5: C, 57.13; H, 8.48; N, 5.12. Found: C,
57.08; H, 8.74; N, 5.21.
tert-Butyl (4S,5S)-4,5-O-Isopropylidene-4,5-dihydroxy-3-piperi-
done-1-carboxylate (34). In a manner similar to the preparation of
the ketone derivative 26 from the hydroxyl derivative 25, 33 (2.06 g,
7.54 mmol) was converted to 34 (1.63 g, 80%) as a colorless oil: 1H
NMR (CDCl3) δ 1.40 (s, 3H, CH3 of isopropylidene group), 1.45 (s,
9H, t-Bu of Boc group), 1.47 (s, 3H, CH3 of isopropylidene group),

1-N-Iminosugars
J. Am. Chem. Soc., Vol. 120, No. 13, 1998 3017
(8.99 g, 6.25 mL, 52.6 mmol) was added dropwise to this solution,
and the reaction mixture was stirred overnight at room temperature.
The reaction mixture was poured onto ice-water and extracted with
EtOAc. The combined extracts were successively washed with water
and brine, dried, and concentrated. The residue was chromatographed
on silica gel with hexanes-EtOAc (1:0 to 10:1) to give 40 (9.5 g, 99%)
as a colorless oil: 1H NMR (CDCl3) δ 3.31 (dd, 1H, J ) 5.0 and 13.2
Hz, H-5a), 3.51 (dd, 1H, J ) 2.8 and 13.2 Hz, H-5b), 3.77 (s, 3H,
OCH3), 4.00 (dd, 1H, J ) 3.3 and 6.7 Hz, H-3), 4.30 (dd, 1H, J ) 1.1
and 3.5 Hz, H-2), 4.32 (m, 1H, H-4), 4.54 (d, 2H, J ) 11.8 Hz, benzylic
H), 4.64 (d, 2H, J ) 11.8 Hz, benzylic H), 5.60 (s, 1H, H-1), 6.83 (d,
2H, J ) 9.1 Hz, aromatic H of p-MeOPh group), 6.98 (d, 2H, J ) 9.1
Hz, aromatic H of p-MeOPh group), 7.31-7.35 (m, 10H, aromatic H
of benzyl groups); 13C NMR (CDCl3) δ 51.64, 55.68, 72.34, 72.50,
80.77, 83.61, 88.21, 105.37, 114.61, 118.04, 127.88, 128.01, 128.04,
128.09, 128.51, 128.55, 137.17, 137.55, 150.46, 155.03. Anal. Calcd
for C26H27N3O5: C, 67.67; H, 5.89; N, 9.10. Found: C, 68.00; H,
6.00; N, 8.87.
(3R,4R,5R)-3,4-Di-O-benzyl-3,4,5-trihydroxypiperidine (42). (NH4)2-
Ce(NO3)6 (18.8 g, 34.3 mmol) was added to a cooled solution of 40
(5.0 g, 11.4 mmol) in CH3CN (90 mL) and H2O (30 mL) at 0-5 °C,
and the mixture was stirred for 5 min at this temperature. The reaction
mixture was diluted with Et2O, washed successively with water, diluted
Na2SO3 solution, saturated NaHCO3 solution and brine, dried, and
concentrated. The residue was chromatographed on silica gel with
hexanes-EtOAc (20:1 to 4:1) to give crude 41 as a colorless oil.
A suspended mixture of the above 41 and Pd-CaCO3 (1.50 g) in
MeOH (600 mL) was vigorously stirred under the atmosphere of H2
for 24 h at room temperature. The reaction mixture was filtered through
a Celite pad, and the filtrate was concentrated. The residue was
chromatographed on silica gel with CHCl3-MeOH (1:0 to 10:1) to
give the imine (2.14 g, 60%) as a colorless oil: 1H NMR (CDCl3) δ
2.49 (dd, 1H, J ) 8.7 and 13.4 Hz, H-2ax), 2.65 (dd, 1H, J ) 2.4 and
13.8 Hz, H-6ax), 3.00 (dd, 1H, J ) 4.6 and 13.8 Hz, H-6eq), 3.14 (dd,
1H, J ) 4.3 and 13.4 Hz, H-2eq), 3.51 (dd, 1H, J ) 3.1 and 7.8 Hz,
H-4), 3.65 (ddd, 1H, J ) 4.4, 7.8, and 8.7 Hz, H-3), 3.95 (ddd, 1H, J
) 2.4, 3.1, and 4.6 Hz, H-5), 4.63 (d, 1H, J ) 11.8 Hz, benzylic H),
4.672 (d, 1H, J ) 11.7 Hz, benzylic H), 4.674 (d, 1H, J ) 11.8 Hz,
benzylic H), 4.73 (d, 1H, J ) 11.7 Hz, benzylic H), 7.28-7.36 (m,
10H, aromatic H); 13C NMR (CDCl3) δ 48.00, 49.19, 67.20, 72.35,
72.54, 76.02, 81.28, 127.66, 127.79, 127.88, 128.40, 128.50, 138.18,
138.56; HRMS calcd for C19H24NO3 (M + H)+ 314.1756, found
314.1755.
was stirred for 18 h at ambient temperature. The reaction mixture was
evaporated, and the residue was dissolved in water and EtOAc. The
organic layer was successively washed with water and brine, dried,
and concentrated. The residue was chromatographed on silica gel with
hexanes-EtOAc (30:1 to 10:1) to give 44 (596 mg, 81%) as a colorless
oil: 1H NMR (CDCl3) δ 1.44 (s, 9H, t-Bu of Boc group), 3.56 (br s,
2H), 3.75 (dd, 1H, J ) 5.3 and 14.2 Hz), 3.91 (m, 2H), 4.15 (m, 1H),
4.45 (d, 1H, J ) 11.8 Hz, benzylic H), 4.57 (d, 1H, J ) 11.5 Hz,
benzylic H), 4.61 (d, 1H, J ) 11.5 Hz, benzylic H), 4.71 (d, 1H, J )
11.8 Hz), 5.13 (s, 1H, CdCH), 5.23 (br s, 1H, CdCH), 7.28-7.36
(m, 10H, aromatic H). Anal. Calcd for C24H31NO4: C, 72.52; H, 7.86;
N, 3.52. Found: C, 72.60; H, 8.14; N, 3.48.
tert-Butyl (3R,4R,5RS)-3,4-Di-O-benzyl-3,4-dihydroxy-5-(hy-
droxymethyl)piperidine-1-carboxylate (45). In a manner similar to
the hydroboration reaction of the methylene derivative 27 to the
hydroxyl derivative 28, 44 (339 mg, 0.853 mmol) was converted to 45
(concomitant with 45′) (404 mg, quantitative) as a colorless oil: 1H
NMR (CDCl3) δ 1.43 (s, 9H, t-Bu of Boc group), 1.84 (m, 1H, H-5),
3.47-3.84 (m, 8H), 4.58 (d, 1H, J ) 11.6 Hz), 4.66 (d, 1H, J ) 11.4
Hz), 4.72 (dm, 1H, J ) 11.6 Hz), 4.84 (m, 1H), 7.30-7.35 (m, 10H,
aromatic H of benzyl group); FABMS calcd for C25H34NO5 (M + H)+
427.2, found 427.3.
tert-Butyl (3R,4R,5RS)-3,4-Di-O-benzyl-5-formyl-3,4-dihydrox-
ypiperidine-1-carboxylate (46). In a manner similar to the oxidation
of the hydroxyl derivative 25 to the ketone derivative 26, 45 (300 mg,
0.724 mmol) was converted to 46 (251 mg, 84%) as a colorless oil:
1H NMR (CDCl3) δ 1.45 (s, 9H, tBu of Boc group), 2.90-3.12 (m,
2H), 3.60-4.66 (m, 9H), 7.20-7.36 (m, 10H, aromatic H of benzyl
group); FABMS calcd for C25H32NO5 (M + H)+ 426.2, found 426.3.
D-Glucuronic Acid-Type 1-N-Iminosugar: (3R,4R,5S)-5-Carboxy-
lpiperidine-3,4-diol Hydrochloride Salt (14). To a mixture of 46 (220
mg, 0.53 mmol) and NaH2PO4‚H2O (736 mg, 5.3 mmol) in CH3CN-
H2O (10 mL-2 mL) were successively added a 35% H2O2 solution
(0.062 mL, 0.64 mmol) and a solution of NaClO2 (80%, 72 mg, 0.64
mmol) in water (10 mL) at 0-5 °C, and the reaction mixture was stirred
for 1 h at room temperature. Na2SO3 (100 mg) was added to the
mixture to quench the oxidants, and the resulting mixture was extracted
with EtOAc. The combined extracts were successively washed with
water and brine, dried, and concentrated. The residue was passed
through silica gel with CHCl3-MeOH (9:1) to give crude 47 as a
colorless oil. This was employed for the next step without further
purification.
A mixture of the above 47 and Pd(OH)2 (50 mg) in MeOH (12 mL)
was vigorously stirred under the atmosphere of H2 for 18 h at room
temperature. The mixture was filtered through a Celite pad, and the
filtrate was concentrated. A mixture of the residue in 1 N HCl (3 mL)
was stirred for 3 h at room temperature, and the mixture was
concentrated. The residue was chromatographed on silica gel with
2-propanol-H2O-30% NH4OH (7:2:1; v/v) to give a chromatographi-
cally pure 14, which was dissolved in 0.1 N HCl and concentrated.
The residue was diluted with water (1 mL) and applied onto a column
of Sephadex G-25 (1.0 × 25 cm) and eluted with water. The fractions
containing the product were pooled and concentrated. The residue was
lyophilized from water to give 14 (27.5 mg, 26% overall) as a colorless
amorphous powder (HCl salt): 1H NMR (300 MHz, D2O) δ 2.90 (ddd,
1H, J ) 4.3, 7.4, 7.8 Hz, H-5), 3.07 (dd, 1H, J ) 7.7, 12.9 Hz, H-2ax),
3.38 (dd, 1H, J ) 7.8, 13.2 Hz, H-6ax), 3.50 (dd, 1H, J ) 3.6, 12.9
Hz, H-2eq), 3.51 (dd, 1H, J ) 4.3, 13.2 Hz, H-6eq), 3.90 (ddd, 1H, J
) 3.6, 7.2, 7.7 Hz, H-3), 4.08 (t, 1H, J ) 7.0 Hz, H-4); HRMS calcd
for C6H12NO4 (M + H)+ 162.0766, found 162.0766.
D-Xylose-Type 1-N-Iminosugar: (3R,4R)-Piperidine-3,4-diol Hy-
drochloride Salt (15). A mixture of 42 (900 mg, 2.18 mmol) and
thiocarbonyldiimidazole (1.55 g, 8.7 mmol) in ClCH2CH2Cl (50 mL)
was heated under gentle reflux overnight, and the reaction mixture was
concentrated. The residue was chromatographed on silica gel, with
hexanes-EtOAc (20:1), to give 49 (940 mg, 83%). Its 1H NMR
spectrum (in CDCl3) showed the presence of two components probably
due to the isomers around the N-Boc group: 1H NMR (300 MHz,
CDCl3) δ 1.31 and 1.39 (br s each, Boc group), 5.72 and 5.84 (br s
each, H-5), 7.03 (br s, 1H, imidazole), 7.54 (br s, 1H, imidazole).
tert-Butyl (3R,4R,5R)-3,4-Di-O-benzyl-3,4,5-trihydroxypiperidine-
1-carboxylate (42). A solution of 41 (1.61 g, 5.14 mmol), (Boc)2O
(1.68 g, 7.71 mmol), and Et3N (0.78 g, 7.71 mmol) in MeOH (30 mL)
was stirred for 4 h at room temperature, and the reaction mixture was
concentrated. The residue was chromatographed on silica gel with
hexanes-EtOAc (20:1 to 4:1) to give 42 (1.55 g, 75%) as a colorless
oil: 1H NMR (CDCl3) δ 1.44 (s, 9H, tBu of Boc group), 3.40-3.50
(m, 3H), 3.60 (dd, 1H, J ) 3.2 and 6.0 Hz, H-4), 3.70 (m, 2H), 3.96
(br s, 1H, H-5), 4.53 (d, 1H, J ) 11.6 Hz, benzylic H), 4.62 (d, 1H, J
) 11.8 Hz, benzylic H), 4.68 (d, 1H, J ) 11.8 Hz, benzylic H), 4.70
(d, 1H, J ) 11.6 Hz, benzylic H), 7.30-7.36 (m, 10H, aromatic H);
FABMS calcd for C24H31NO5 412.2, found 412.2.
tert-Butyl (4S,5R)-4,5-O-Dibenzyl-4,5-dihydroxy-3-piperidone-1-
carboxylate (43). In a manner similar to the preparation of the ketone
derivative 26 from the hydroxyl derivative 25, 42 (1.36 g, 3.39 mmol)
was converted to 43 (1.06 g, 78%) as a colorless oil; 1H NMR (CDCl3)
δ 1.44 (s, 9H, tBu of Boc group), 3.67 (dd, 1H, J ) 6.0 and 13.8 Hz),
3.83 (m, 1H), 3.93-4.00 (m, 3H), 4.14 (d, 1H, J ) 17.0 Hz), 4.56 (d,
1H, J ) 11.6 Hz, benzylic H), 4.62 (d, 1H, J ) 11.9 Hz, benzylic H),
4.68 (d, 1H, J ) 11.9 Hz, benzylic H), 4.79 (d, 1H, J ) 11.6 Hz,
benzylic H), 7.28-7.37 (m, 10H, aromatic H). Anal. Calcd for C23H29-
NO5: C, 69.16; H, 7.31; N, 3.51. Found: C, 69.22; H, 6.95; N, 3.27.
tert-Butyl (3R,4R)-3,4-Di-O-benzyl-5-methylene-3,4-dihydroxypi-
peridine-1-carboxylate (44). (TMS)2NLi (1M solution; 15.7 mL, 15.7
mmol) was added dropwise to a cooled suspension of CH3PPh3Br (6.68
g, 18.7 mmol) in DME (80 mL), and the mixture was stirred for 1 h at
0-5 °C. A solution of 43 (740 mg, 1.85 mmol) in DME (10 mL) was
then added dropwise to the mixture at 0-5 °C, and the reaction mixture

3018 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Ichikawa et al.
A solution of 49 (940 mg, 1.80 mmol) and 1,1′-azobis(cyclohex-
anecarbonitrile) (30 mg) in toluene (20 mL) was added dropwise to a
preheated mixture of n-Bu3SnH (1.31 g, 4.49 mmol; 1.21 mL) in toluene
(50 mL) at 90 °C, and the reaction mixture was heated overnight at 90
°C. The mixture was concentrated, and the residue was chromato-
graphed on silica gel, with hexanes-EtOAc (50:1), to give 50 (213
mg, 36%) contaminated with a trace amount of tin compound.
A mixture of 50 (213 mg, 0.64 mmol) and 20% Pd(OH)2 (220 mg)
in MeOH (5 mL) and 1 N HCl (20 mL) was stirred at 1 atm of H2 for
18 h at room temperature. The reaction mixture was filtered through
a Celite pad, and the filtrate was concentrated. The residue was
dissolved in water (30 mL), applied on a column of Dowex 50W-X8
[H+] resin, and eluted with 3% NH4OH. The fractions containing the
product were pooled, concentrated, and sequentially coevaporated with
1 N HCl and water. The residue was chromatographed on Sephadex
G-20, with water, and lyophilized from water to give 15 (65 mg,
66%): 1H NMR (300 MHz, D2O) δ 1.69-1.82 (m, 1H, H-5ax), 2.24
(tdd, 1H, J ) 3.70,3.80,14.84 Hz, H-5eq), 2.97-3.05 (m, 1H), 3.13
(ddd, 1H, J ) 3.94,8.49, 12.96 Hz), 3.36 (ddd, 1H, J ) 3.96,7.61,
13.20 Hz), 3.45 (dd, 1H, J ) 2.45,12.60 Hz, H-2ax), 3.78-3.87 (m,
2H, H-3,4); HRMS calcd for C5H12NO2 (M + H)+ 118.0868, found
118.0866.
General Procedure for Inhibition Assay. Inhibitory potencies of
the 1-N-iminosugars were determined by measuring the residual
hydrolytic activities of the glycosidases of the corresponding p- or
o-nitrophenyl glycosides in the presence of iminosugars spectropho-
tometrically on a Beckman Model DU 650. Glycosidases used in this
study were purchased from Sigma (St. Louis), and they were R-glu-
cosidase (yeast; G 5003), â-glucosidase (almond; G 4511), R-galac-
tosidase (green coffee beans; G8507), â-galactosidase (Aspergillus
orizae; G7256), R-mannosidase (Jack bean; M 7257), R-fucosidase
(bovine kidney; F 5884), and â-glucuronidase (bovine liver; G 0501).
Each assay was performed in either phosphate buffer (25 mM; pH
6.8) or acetate buffer (25 mM; pH 5.0) with 4-nitrophenyl (p-
nitrophenyl) glycoside derivatives of the sugars except for â-galac-
tosidase assay in which 2-nitrophenyl (o-nitrophenyl) â-galactoside was
used. The reactions were initiated by addition of enzyme to a cooled
solution of the substrate (0.4-5 mM) at 0-5 °C in the presence or
absence of a various concentration of iminosugars in buffer which had
been thermodynamically equilibrated. After the mixture was incubated
for 10-15 min at 37 °C, the reaction was quenched by addition of 200
mM Na2CO3 solution. The absorbance of the resulting mixture was
read at 400 nm (for 4-nitrophenol) or 420 nm (for 2-nitrophenol). IC50
values were determined as a concentration of the iminosugars that
inhibits 50% of the enzyme activity. Ki values were determined with
a Sigma Plot program (version 4.1, Jandel Scientific).
Acknowledgment. The NMR studies was performed in the
Biochemistry NMR Facility at Johns Hopkins University, which
was established by a grant from the National Institutes of Health
(GM 27512) and a Biomedical Shared Instrumentation Grant
(1S10-RR06262-0). Support from the American Cancer Society
(JFA-515), the National Institutes of Health (GM 52324), and
the Mizutani Foundation of Glycobiology were greatly appreci-
ated. We also appreciated valuable suggestions from the
reviewers.
Supporting Information Available: Experimental data for
compounds 53 and 57-63 (3 pages, print/PDF). See any current
masthead page for ordering information and Web access
instructions.
JA973443K