Synthesis of (-)-isofagomine

Article abstract of DOI:10.1080/07328300601039336

1,3-Dipolar cycloaddition of N-benzyl nitrone 2 to D-threo δ-lactone 15 proceeded with excellent stereoselectivity to provide only one adduct 16. Cycloadduct 16 was subsequently subjected to a sequence of reactions involving rearrangement to γ-lactone, gl

Full text of DOI:10.1080/07328300601039336

Journal of Carbohydrate Chemistry, 25:673–684, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300601039336
Synthesis of (2)-Isofagomine
Irma Panfil
Institute of Organic Chemistry of the Polish Academy of Sciences, Warsaw, Poland
Jolanta Solecka
National Institute of Hygiene, Warsaw, Poland
Marek Chmielewski
Institute of Organic Chemistry of the Polish Academy of Sciences, Warsaw, Poland
1,3-Dipolar cycloaddition of N-benzyl nitrone 2 to D-threo d-lactone 15 proceeded with
excellent stereoselectivity to provide only one adduct 16. Cycloadduct 16 was sub-
sequently subjected to a sequence of reactions involving rearrangement to g-lactone,
glycolic cleavage/reduction, protection of the terminal hydroxymethyl group, reduction
of the lactone, desilylation/mesylation, and hydrogenolysis of the N-O bond providing
(2)-isofagomine and its N-substituted derivatives. The biologic activity of N-substituted
(2)-isofagomines toward commercially available a- and b-glucosidases, a-D-mannosi-
dase, a-L-fucosidase, b-D-glucuronidase, and b-D-galactosidase was tested.
Keywords (2)-Isofagomine, Iminosugars, 1,3-Dipolar cycloaddition, Nitrones,
Unsaturated aldonolactones
INTRODUCTION
Glycosidases and glycosyl transferases play an important role as carbohydrate-
modifying enzymes. Carbohydrate mimics, such as iminosugars, carbasugars,
or C-glycosides, display properties as specific and competitive inhibitors of
these enzymes.[1] Consequently, they display many interesting biologic and
therapeutic activities such as antiviral, anticancer, antidiabetic, and antihy-
perglycemic.[1–7] The discovery of isofagomine (1) by a Danish group[8] as one
of the most potent b-glucosidase inhibitors has propelled, during the past
years, a growing interest of many laboratories in the synthesis of iminosugars
with nitrogen at the anomeric position.[9–21]
Received August 28, 2006; accepted November 9, 2006.
Address correspondence to Marek Chmielewski, Institute of Organic Chemistry of the
Polish Academy of Sciences, 01-224, Warsaw, Poland. E-mail: chmiel@icho.edu.pl
673

I. Panfil et al.
Recently we reported on 1,3-dipolar cycloaddition of N-benzyl nitrone 2 to
674
D-glycero g-lactone 3, L-erythro d-lactone 4, and D-threo d-lactone 5.[22] The
cycloaddition performed with 4 afforded two stereoisomers 7 and 8 in a ratio
of about 2.5:1. Cycloaddition of the same nitrone 2 to lactones 3 and 5
yielded, in each case as a sole product, 6 and 9, respectively (Chart 1).
Cycloadducts 6–9 were subsequently subjected to a sequence of reactions
involving hydrogenolysis of the N-O bond and intramolecular alkylation of
the nitrogen atom by the C-4 or C-5 carbon atom of the sugar backbone to
afford iminosugars 10–14 with a hydroxymethyl group at the C-2 carbon
atom.[23] This strategy proceeded with inversion of configuration at the second-
ary carbon atom (C-4 or C-5) of the starting sugar. In addition, the interchange
of the C-1 carbon atom by the aminomethyl group caused inversion of configur-
ation (according to the carbohydrate nomenclature) at the tertiary carbon atom
(C-2).[16]
RESULTS AND DISCUSSION
In all cases, the high preference for the anti addition of the nitrone to the
terminal methyl or substituted methyl groups in lactones was observed.[22–25]
Low stereoselectivity of cycloadditions of simple nitrones to erythro lactones
Chart 1

Synthesis of (2)-Isofagomine
675
compared to threo congeners caused by the 4-O-benzyloxy group diminished the
attractiveness of the former in the target-oriented synthesis. On the other hand,
cycloadducts involving threo lactones provide a wide range of synthetic possibi-
lities, particularly as the d-lactone fragment can be rearranged into the g one
and the terminal carbon atom of the adduct can be easily removed (Scheme 1).
By a such strategy, (2)-isofagomine 1-ent, the enantiomer of the natural
compound 1, can be synthesized. Adduct 16, obtained by the cycloaddition of
the acetylated lactone 15 and the nitrone 2, was rearranged to the g-lactone
by deacetylation to afford diol 17. Subsequent cleavage of the terminal diol in
17 followed by the reduction of the aldehyde group gave compound 18, which
was silylated yielding 19.
Subsequently, compound 19 was reduced to the diol 20, and both hydroxyl
groups were protected by the isopropylidene residue and provided bicyclic
compound 21, which was subsequently desilylated to afford the alcohol 22.
The primary hydroxy group in 22 was mesylated to give compound 23. Sub-
sequent deprotection of hydroxy groups caused immediate intramolecular
alkylation of the nitrogen atom. The crude ammonium salt 24 was subjected
to hydrogenolysis of the N-O bond to provide iminosugar 25, which was charac-
terized as its triacetate 26. The relative configuration of 26 was proven by trans
diaxial coupling constants between H-2, H-3, H-4, H-5, and H-6 protons equal
to 9.7, 8.9, 10.1, and 11.0 Hz. The hydrogenolysis of 26 followed by acetylation/
deacetylation sequence provided (2)-N-acetyl-isofagomine (28). Deacetylation
Scheme 1

I. Panfil et al.
676
of 26 followed by hydrogenolysis of the N-benzyl substituent afforded (2)-iso-
fagomine (1-ent). Compound 1-ent and its N-benzyl derivative 25 have been
synthesized before from 1-tert-butoxycarbonyl-1,2,3,6-tetrahydropyridyl-3-
methanol[13] and (þ)-L-tartaric acid (Scheme 1).[15b]
The biologic activity of compounds 1-ent, 25 and 28, toward commercially
available a- and b-glucosidases, a-D-mannosidase, a-L-fucosidase, b-D-glucur-
onidase, and b-D-galactosidase were tested. (2)-N-Acetyl-isofagomine (28)
was found to be a better inhibitor of tested enzymes than (2)-isofagomine
(1-ent). It showed moderate/weak activity against bovine kidney a-L-fucosi-
dase (50% inhibition required 3.5 mM), bovine liver b-D-galactosidase (50%
inhibition required at 6.6 mM), and bovine liver b-D-glucuronidase (50% inhi-
bition required at 9.1 mM), and little or no activity against rice a-, almond
b-D-glucosidases, and jack bean a-D-mannosidase. (2)-Isofagomine (1-ent)
showed moderate/weak activity against a-L-fucosidase, b-D-galactosidase,
and b-D-glucuronidase, and a little or no activity against a-D-glucosidases
and a-D-mannosidase. (2)-N-Benzyl-isofagomine (25) showed only inhibition
of a-L-fucosidase (50% inhibition at 5.1 mM).
CONCLUSION
It was demonstrated that 1,3-dipolar cycloaddition of the simple N-benzyl
nitrone to unsaturated threo 1,5-lactone 15 proceeded exclusively anti to
the substituents in the sugar. The adduct 16 after rearrangement to the
g-lactone 17 and removal of the terminal hydroxymethyl group could be
the attractive substrate for the synthesis of enantiomer of natural isofago-
mine (1-ent). Examination of the glycosidases’ inhibitory activity showed
that (2)-N-acetyl-isofagomine (28) was a better inhibitor of tested enzymes
than (2)-isofagomine (1-ent).
EXPERIMENTAL
1
Melting points were determined on a Koefler hot-stage apparatus. H NMR
spectra were recorded using Bruker Avance 500 and Varian Mercury 400
instruments. Absorptions of aromatic protons were not reported. IR spectra
were recorded on a Perkin-Elmer FTIR Spectrum 200 spectrophotometer.
Mass spectra were recorded using AMD-604 Inectra GmbH and HPLC-MS
with Mariner and API 356 detectors. Optical rotations were measured using
a JASCO P 3010 polarimeter at 22 + 38C. Column chromatography was per-
formed using E. Merck Kiesel Gel (230–400 mesh).
(3aR, 6R, 7R, 7aS)-7-Acetoxy-6-acetoxymethyl-2-benzyl-4-oxo-tetrahy-
dropyrano[3,4-d]isoxazolidine (16). Compound 16 was obtained according
to the procedure described earlier.[22] Syrup; [a]D þ 44.2 (c 1.5, CH2Cl2); IR

Synthesis of (2)-Isofagomine
677
1
(film): 1748 cm21; NMR (500 MHz, toluene-d8, 1008C) d: 5.09 (ddt, 1H, J 1.6,
3.3 Hz, H-7), 4.88 (dt, 1H, J 1.6, 6.3 Hz, H-6), 4.11 (dd, 1H, J 3.3,
8.0 Hz, H-7a), 4.11 (dd, 1H, J 6.3, 11.4 Hz, CHHOAc), 4.09 (dd, 1H, J 6.3,
11.4 Hz, CHHOAc), 3.69, 3.63 (2d, 2H, J 13.3 Hz, NBn), 3.22 (dt, 1H, J 5.4,
8.0 Hz, H-3a), 2.89 (bs, 1H, H-2), 2.83 (bt, 1H, H-20); HRMS (ESI) m/z
[M þ Na]þ, Calcd. for C18H21NO7Na: 386.1210. Found: 386.1230.
(3aR, 6R, 6aS, 10R)-2-Benzyl-6-(10,20-dihydroxyethyl)-4-oxo-isoxazoli-
dino[4,5-c]tetrahydrofurane (17). Adduct 16 (0.36 g, 1 mmol) was dissolved
in methanol (10 mL) containing 1.5% of ammonia and left at rt for 24 h. Sub-
sequently, solvent was evaporated and the crude product was purified by
chromatography using AcOEt as an eluent to afford 17 (0.26, 95%). m.p. 95–
978C; [a]D þ 25.2 (c 0.5, CH2Cl2); IR (film): 3588, 1777 cm21
;
1H NMR
(500 MHz, CDCl3) d: 4.99 (bt, 1H, H-6a), 4.65 (dd, 1H, J 3.7, 6.5 Hz, H-6),
4.10, 3.89 (2d, 2H, J 12.4 Hz, NBn), 4.06 (dd, 1H, J 5.1, 9.6 Hz, CHHOH),
3.74–3.56 (m, 4H, CHOH, CHHOH, H-3, H-3a), 2.82 (bm, 1H, H-30); HRMS
(LSIMS) m/z [M þ H]þ, Calcd. for C14H18NO5: 280.1185. Found: 280.1197.
(3aR, 6R, 6aS, 10R)-6-(10,20-Diacetoxyethyl)-2-benzyl-4-oxo-isoxazolidino
[4,5-c]tetrahydrofurane (17Ac). Syrup, [a]D þ 1.15 (c 0.8, CH2Cl2); IR (film):
1
1779, 1744 cm21; H NMR (500 MHz, toluene-d8) d: 5.50 (ddd, 1H, J 3.1, 4.2,
8.9 Hz, CHOAc), 4.22 (dd, 1H, J 3.1, 12.4 Hz, CHHOAc), 4.20 (dd, 1H, J 5.4,
7.1 Hz, H-6a), 4.16 (dd, 1H, J 5.4, 8.9 Hz, H-6), 4.10 (dd, 1H, J 4.2, 12.4 Hz,
CHHOAc), 3.63, 3.58 (2d, 2H, J 13.2 Hz, NBn), 3.17 (bd, 1H, J 9.3 Hz, H-3),
2.74 (t, 1H, J 7.1 Hz, H-3a), 2.33 (bt, 1H, H-30) 1.76, 1.70 (2s, 6H, 2Ac); HRMS
(ESI) m/z [M þ Na]þ, Calcd. for C18H21NO7Na: 386.1210. Found: 386.1232.
(3aR, 6R, 6aR)-2-Benzyl-6-hydroxymethyl-4-oxo-isoxazolidino[4,5-c]
tetrahydrofurane (18). Compound 17 (0.36 g, 1.0 mmol) was dissolved in
methanol/water 2:1 v/v (15 mL) and treated with sodium metaperiodate at
rt (0.24 g, 1.1 mmol). After 1.5 h the reaction was completed. After evaporation
of the solvent, water was added into the flask and the resulting solution was
extracted with AcOEt. Subsequently, the extract was evaporated and the
residue was dissolved in dry CH2Cl2(15 mL) and reduced with 1.5 molar
equiv. of NaBH(OAc)3(0.29 g). Standard workup provided the product, which
was purified on a silica gel column using AcOEt as an eluent to afford 18
(0.16 g, 60%). Syrup; [a]D þ 31.0 (c 0.84, CH2Cl2); IR (film): 3564, 1778 cm21
;
1H NMR (500 MHz, CDCl3) d: 4.94 (bt, 1H, H-6a), 4.22 (dt, 1H, J 5.1, 5.8,
5.8 Hz, H-6), 3.99, 3.95 (2bd, 2H, J 12.9 Hz, Bn), 3.89 (dd, 1H J 4.7, 12.3 Hz,
CHHOH), 3.83 (dd, 1H J 5.6, 12.3 Hz, CHHOH), 3.66–3.54 (m, 2H, H-3,
H-30), 2.80 (bs, 1H, H-3a); HRMS (EI) m/z Mþ, Calcd. for C13H15NO4:
249.1001. Found: 249.1007.
(3aR, 6R, 6aR)-6-Acetoxymethyl-2-benzyl-4-oxo-isoxazolidino[4,5-c]
tetrahydrofurane (18Ac). m.p. 108–1108C; [a]D 2 5.14 (c 1.0, CH2Cl2); IR

I. Panfil et al.
678
(film): 1778, 1741 cm21; 1H NMR (500 MHz, toluene-d8, 1008C) d: 4.32 (dd, 1H,
J 4.1, 12.2 Hz, CHHOAc), 4.24 (dd, 1H, J 7.6, 12.2 Hz, CHHOAc), 4.15 (ddd,
1H, J 4.1, 5.5, 7.6 Hz, H-6), 4.11 (dd, 1H, J 5.5, 7.2 Hz, H-6a), 3.66, 3.58 (2d,
2H, J 13.2 Hz, NBn), 3.14 (bd, 1H, J 9.3 Hz, H-3), 2.71 (dt, 1H, J 1.6 7.2 Hþz,
H-3a), 2.31 (bt, 1H, H-30), 1.69 (s, 3H, Ac); HRMS (LSIMS) m/z [M þ H] ,
Calcd. for C15H18NO5: 292.1185. Found: 292.1192.
(3aR, 6R, 6aR)-2-Benzyl-6-t-butyldiphenylsiloxymethyl-4-oxo-isoxazo-
lidino[4,5-c]tetrahydrofurane (19). Compound 18 (0.25 g, 1 mmol) dis-
solved in CH2Cl2(15 mL) was treated with t-butyldiphenylchlorosilane
(0.302, 1.1 mmol) and DMAP (1.1 mmol). The mixture was left overnight. Sub-
sequently, the solvent was evaporated and the residue was purified by chrom-
atography using hexane/AcOEt 4:1 v/v as an eluent to give 19 (0.35 g, 73%).
1
Syrup, [a]D þ 26.3 (c 0.9, CH2Cl2); IR (film): 1772 cm21; H NMR (500 MHz,
toluene-d8, 1008C) d: 4.22–4.16 (m, 2H, H-6, H-6a), 4.03 (dd, 1H, J 6.4,
11.1 Hz, CHHOTBDPS), 3.98 (dd, 1H, J 5.1, 11.1 Hz, CHHOTBDPS), 3.57,
3.54 (2d, 2H, J 13.3 Hz, NBn), 3.13 (bd, 1H, J 9.3 Hz, H-3), 2.73 (dt, 1H,
J 1.7, 7.0 Hz, H-3a), 2.33 (bt, 1H, H-30), 1.14 (s, 9H, t-Bu); HRMS (ESI) m/z
[M þ H]þ, Calcd. for C29H33NO4Si: 488.2252. Found: 488.2251.
(3aR, 8R, 8aR)-2-Benzyl-8-tert-butyldiphenylsiloxymethyl-6,6-dimethyl-
5,7-dioxa-isoxazolidino[4,5]cycloheptane (21). Compound 18 (0.49 g,
1 mmol) was dissolved in dry THF and treated with LiAlH4(0.04 g, 1 mmol).
The mixture was stirred for 0.5 h and then, consecutively, AcOEt (1 mL) and
water (1 mL) were added. Subsequently, the mixture was filtered through
Celite and solvents were evaporated. The residue was dissolved in brine
(5 mL) and extracted with AcOEt. The extract was dried and evaporated.
The residue (crude 20) was dissolved in dimethoxypropane (10 mL), treated
with catalytic amount of p-TsOH, and refluxed for 2 h. Subsequently, the
mixture was neutralized with Na2CO3 and evaporated. The crude product
was purified by chromatography using hexane/AcOEt 7:3 v/v as an eluent to
1
afford 21 (0.38 g, 71%). m.p. 84–868C; [a]D þ 24.5 (c 1.0, CH2Cl2); H NMR
(500 MHz, toluene-d8, 1008C) d: 4.17–4.12 (m, 2H, H-8, H-8a), 3.95 (d, 2H,
CH2OTBDPS), 3.87 (t, 1H, J 10.4, 11.9 Hz, H-4), 3.84, 3.82 (2d, 2H,
J 13.1 Hz, NBn), 3.20 (dd, 1H, J 5.2, 11.9 Hz, H-40), 2.76 (bt, 1H, J 7.8,
9.1 Hz, H-3), 2.38 (m, 1H, H-3a), 2.19 (bdd, 1H, H-30), 1.31, 1.29 (2s, 6H,
2CH3), 1.14 (s, 9H, t-Bu); HRMS (ESI) m/z [M þ H]þ, Calcd. for
C32H42NO4Si: 532.2878. Found: 532.2860.
(3S,4S,5S) N-Benzyl-3,4-diacetoxy-5-(acetoxymethyl)piperidine [(2)-
N-Benzyl-tri-O-acetyl-isofagomine] (26). Compound 21 (0.266 g,
0.5 mmol) was dissolved in THF (10 mL), treated with TBAF (0.16 g,
0.5 mmol), and stirred at rt for 3 h. The reaction mixture was evaporated and
crude product was passed through a silica gel column using AcOEt as an

Synthesis of (2)-Isofagomine
679
eluent. Subsequently, the solvent was evaporated and the product (22, 0.13 g,
90%) was dissolved in CH2Cl2(10 mL), treated with Et3N (0.06 g) and mesyl
chloride (0.06 g, 0.5 mmol), and left for 1 h at rt. The mixture was then
washed with water and evaporated. The product was dissolved in methanol
(10 mL) and acetic acid (2 mL) was added, the resulting solution was
refluxed for 10 min and evaporated, and the residue acetylated with acetic
anhydride/pyridine 1:1 mixture (2 mL). After standard workup and evapor-
ation, the crude mixture was dissolved in methanol (15 mL), treated with
K2CO3(20 mg), and hydrogenated using Degussa 10% Pd/C at rt for 24 h.
The mixture was filtered through Celite, evaporated, and acetylated using
1:1 acetic anhydride/pyridine mixture. After standard workup, the crude
product was purified by chromatography using hexane/AcOEt 1:1 v/v
mixture to afford 26 (0.07 g, 50%). Syrup, [a]D 2 10.5 (c 0.9, CH2Cl2); IR
1
(CHCl3): 1738 cm21; H NMR (500 MHz, toluene-d8, 1008C) d: 5.07 (ddd, 1H,
J 5.0, 8.9, 9.7 Hz, H-3), 4.91 (dd, 1H, J 8.9, 10.1 Hz, H-4) 3.98 (dd, 1H, J 6.2,
11.4 Hz, CHHOAc), 3.83 (dd, 1H, J 3.8, 11.4 Hz, CHHOAc), 2.96 (ddd, 1H, J
2.1, 5.0, 10.9 Hz, H-2), 2.68 (ddd, 1H, J 2.1, 4.1, 11.5 Hz, H-6), 2.02 (m, 1H,
H-5), 1.97 (dd, 1H, J 9.7, 10.9 Hz, H-20), 1.87 (t, 1H, J 11.1 Hz, H-60), 1.74,
1.68, 1.67 (3s, 12H, 3Ac); HRMS (ESI) m/z [M þ H]þ, Calcd. for C19H26NO6:
364.1755. Found: 364.1748.
(2)-N-Benzyl-isofagomine (25). Deacetylation of 26 with 1% of ammonia in
methanol gave 25 (90%). Colorless crystals; m.p. 147–1508C; [a]D 2 16.5 (c 0.4,
CH2Cl2), lit.[15b] [a]D 2 13.2 (c 1.1, ethanol); 1H NMR (400 MHz, methanol-d4) d:
3.78 (dd, 1H, J 3.7, 11.0 Hz, CHHOH), 3.58, 3.53 (2d, 2H, J 12.8 Hz, Bn), 3.51
(m, 1H, H-3), 3.49 (dd, 1H, J 6.9 11.0 Hz, CHHOH), 3.07 (dd, 1H, J 8.8, 10.3 Hz,
H-4), 3.01 (ddd, 1H, J 2.3, 3.8, 11.4 Hz, H-2), 2.96 (ddd, 1H, J 2.3, 4.8, 10.8 Hz,
H-3), 1.89 (t, 1H, J 11.4 Hz, H-20), 1.86 (t, 1H, J 10.8 Hz, H-30), 1.76 (m, 1H,
H-5); HRMS (ESI) m/z [M þ H]þ, Calcd. for C13H20NO3: 238.1438. Found:
238.1428.
(2)-N-Acetyl-tri-O-acetyl-isofagomine (27). Compound 26 was hydrogen-
ated in methanol under standard condition. After evaporation of the solvent,
the residue was acetylated with acetic anhydride pyridine mixture, which
after evaporation was purified on a silica gel column using AcOEt/MeOH 4:1
v/v as an eluent to afford 27 (90%) as a mixture of two rotamers in a ratio of
about 2:1. Colorless crystals; m.p. 89–908C; [a]D 2 14.7 (c 0.9, CH2Cl2); IR
(film): 1774, 1649 cm21 1H NMR (500 MHz, CDCl3) d: major rotamer 5.03
;
(bm, 1H, H-3), 4.73 (bm, 1H, H-4), 4.49 (bd, 1H, CHHOAc), 4.09 (m, 1H,
CHHAc), 4.03 (bm, 1H, H-2), 4.01 (bm, 2H, H-2, H-6), 3.11 (m, 1H, H-20), 2.81
(bt, 1H, H-60), 2.05 (m, 1H, H-5), 2.13, 2.11, 2.06, 2.05 (4s, 12H, 4Ac); minor
rotamer 5.02 (bs, 1H, H-3), 4.84 (bm, 1H, H-4), 4.38 (bm, 1H, CHHOAc), 4.11
(m, 1H, CHHOAc), 3.77 (bm, 1H, H-3), 3.21 (m, 1H, H-6), 3.08 (bm, 1H,
H-30), 3.05 (m, 1H, H-60), 1.56 (bm, 1H, H-5), 2.10, 2.09, 2.08, 2.03 (4s, 12H,

I. Panfil et al.
680
4Ac); HRMS (ESI), for the mixture of rotamers, m/z [M þ Na]þ, Calcd. for
C14H21NO7Na: 338.1210. Found: 338.1226.
(2)-N-Acetyl-isofagomine (28). Deacetylation of compound 27 with 1% of
ammonia in methanol gave 28 (90%) as a mixture of two rotamers in a ratio
of about 1:1. Colorless crystals; m.p. 60–628C; [a]D 2 8.7 (c 0.7, methanol); IR
(film): 1637 cm21 1H NMR (500 MHz, methanol-d4) d: 4.53, 4.50 (2 m, 1H,
;
H-4a and b), 3.95, 3.89 (2ddd, 1H, J 2.4, 4.4, 13.8 Hz, H-2a and b), 3.86, 3.78
(2dd, 1H, J 3.7, 11.1 Hz, CHHOHa and b), 3.60 (2dd, 0.5H, J 6.7, 11.1 Hz,
CHHOHa), 3.55 (2dd, 0.5H, J 7.5, 11.0 Hz, CHHOHb), 3.39 (ddd, 0.5H, J 4.8,
8.3, 10.1 Hz, H-6a), 3.35–3.24 (m, 1.5H, H-4a and b, H-6b), 2.96 (dd, 0.5H,
J 10.3, 13.3 Hz, H-20a), 2.93 (2dd, 0.5H, J 11.5, 13.1 Hz, H-20b), 2.56 (dd,
0.5H, J 11.5, 13.1 Hz, H-60a), 2.47 (dd, 0.5H, J 10.3, 12.7 Hz, H-60b), 2.11,
2.10 (2s, 3H, Ac a and b), 1.64, 1.55 (2 m, 1H, H-5a and b); HRMS (ESI), for
the mixture of rotamers, m/z [M þ Na]þ, Calcd. for C8H15NO4Na: 212.0893.
Found: 212.0890.
(2)-Isofagomine (1-ent). Compound 1-ent was obtained from 25 by hydro-
genation in methanol in the presence of 10% Pd/C (Degussa). Standard
workup and purification on a silica gel column using i-PrOH/NH3aq 3:1 v/v
as an eluent afforded 1-ent in 90% yield. Syrup; [a]D 2 17.8 (c 0.5, methanol),
lit.[15b] [a]D 2 15.7 (c 0.19, ethanol); IR (film): 3631, 3533, 3415 cm21 1H
;
NMR (500 MHz, methanol-d4) d: 3.78 (dd, 1H, J 3.8, 11.0 Hz, CHHOH), 3.55
(dd, 1H, J 6.8, 11.0 Hz, CHHOH), 3.40 (ddd, 1H, J 5.0, 8.7, 10.6 Hz, H-3),
3.20 (dd, 1H, J 8.8, 10.2 Hz, H-4), 3.08 (m, 2H, H-2, H-6), 2.39 (dd, 1H,
J 11.8, 12.8 Hz, H-20), 2.37 (dd, 1H, J 10.7, 12.4 Hz, H-60), 1.64 (m, 1H, H-5);
HRMS (EI) m/z Mþ, Calcd. for C6H13NO3: 147.0895. Found: 147.0892.
Measurements of Enzymes Inhibition
The following hydrolases were used: a-L-glucosidase from rice (type V,
63.43 U/mg, 1.34 mg/mL, Sigma), b-D-glucosidase from almonds (25.8 U/mg,
95.4% protein, Sigma), a-D-mannosidase from jack bean (6.2 mg prot./mL,
22 U/mg, Sigma), a-L-fucosidase from bovine kidney (28.0 U/mg, 0.55 mg
prot./mL, Sigma), b-D-galactosidase from bovine liver (0.148 U/mg, Sigma)
solution (0.562 U/mL) and b-D-glucuronidase from bovin liver (2630 U/mg,
Sigma) solution (4909 U/mL) (Table 1).
These enzymes were assayed with appropriate p-nitrophenyl glycoside
substrates (phenolphthalein b-glucuronide for b-glucuronidase), which were
purchased from Sigma. Hydrolase activities were measured by modification
of the procedures described previously.[24–29] The reaction mixtures (for each
enzyme) consisted of 25 mL of 16.5 mM p-nitrophenyl a-D-glucopyranoside,
403 mL of 0.1 M acetate buffer (pH 4.0), 50 mL of inhibitor solution (water or
methanol), and 22 mL of a-L-glucosidase solution (10 ꢀ diluted) for

Synthesis of (2)-Isofagomine
681
Table 1: Effect of (2)-isofagomine (1-ent) and (2)-N-acetyl-isofagomine (28) on
various glycosidases.
(2)-N-Accetyl-isofagomine
(2)-Isofagomine (1-ent)
(28)
Concentration
of inhibitor
[mM]
Enzyme
inhibition
[%]
Concentration
of inhibitor
[mM]
Enzyme
inhibition
[%]
Enzyme tested
a-L-Fucosidase
12.0
8.5
15.0
10.0
9.0
50
50
50
lack
50
lack
3.5
6.6
9.1
10.0
50.0
6.9
50
50
50
b-D-Galactosidase
b-D-Glucuronidase
a-D-Glucosidase
b-D-Glucosidase
a-D-Mannosidase
Lack
8.5
11.0
16
a-glucosidase activity; 100 mL of 14 mM p-nitrophenyl b-D-glucopyranoside,
250 mL of 0.2 M acetate buffer (pH 4.6), 50 mL of inhibitor solution, and
100 mL of b-D-glucosidase solution (30 mg/mL) for b-D-glucosidase activity;
5 mL of 20 mM p-nitrophenyl a-D-mannopyranoside, 200 mL of 0.1 M acetate
buffer (pH 4.5), 25 mL of inhibitor solution, and 20 mL of a-D-mannosidase
solution (100 ꢀ diluted) for a-D-mannosidase activity; 20 mL of a-L-fucosidase
solution (100 ꢀ diluted), 25 mL of inhibitor solution, 155 mL of 0.1 M acetate
buffer (pH 5.0), and 50 mL of 20 mM 4-nitrophenyl-a-L-fucopyranoside for a-L-
fucosidase activity; 90 mL of 4 mM phenolphthalein b-D-glucuronide, 95 mL of
0.1 M acetate buffer (pH 5.0), 25 mL of inhibitor solution, and 40 mL of b-D-glu-
curonidase solution for b-D-glucuronidase activity; 25 mL of 20 mM 4-nitrophe-
nyl b-D-galactopyranoside, 195 mL of 0.1 M acetate buffer (pH 5.0), 25 mL of
inhibitor solution, and 5 mL of b-D-galactosidase solution for b-D-galactosidase
activity. Generally, enzyme and inhibitor were mixed in buffer and reaction
was started by the addition of the substrate. Each reaction mixture was incu-
bated for 30 min (a- and b-glucosidases for 15 min) at 378C and was terminated
by adding 0.250 mL of 2% sodium carbonate. The absorbance of liberated
p-nitrophenol was measured at 405 nm (liberated phenolphthalein at 540 nm).
(2)-N-Benzyl-isofagomine (25) showed 50% inhibition of a-L-fucosidase
activity in concentration 5.1 mM, trace inhibition of b-D-glucosidase activity
in concentration 18 mM, and lack of inhibition of a-D-glucosidase, b-D-galacto-
sidase, b-D-glucuronidase, and a-D-mannosidase.
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