An Efficient Synthesis of the Glycosidase Inhibitor 1,6-Dideoxy-6,6- difluoronojirimycin

Article abstract of DOI:10.1515/znb-2011-0809

1,6-Dideoxy-6,6-difluoronojirimycin (1) was prepared from an easily accessible and commercially available starting material, methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside. Key steps of this synthesis are a difluorination reaction using DAST and a reductiv

Full text of DOI:10.1515/znb-2011-0809

An Efficient Synthesis of the Glycosidase Inhibitor
1,6-Dideoxy-6,6-difluoronojirimycin
Rene´ Csuk, Erik Prell, and Claudia Korb
Martin-Luther-Universita¨t Halle-Wittenberg, Bereich Organische Chemie, Kurt-Mothes-Straße 2,
06120 Halle (Saale), Germany
Reprint requests to Prof. Dr. Rene´ Csuk. Fax: 0049 345 5527030.
E-mail: rene.csuk@chemie.uni-halle.de
Z. Naturforsch. 2011, 66b, 837 – 842; received June 21, 2011
1,6-Dideoxy-6,6-difluoronojirimycin (1) was prepared from an easily accessible and commercially
available starting material, methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside. Key steps of this syn-
thesis are a difluorination reaction using DAST and a reductive amination. The overall yield of the
synthesis is 44 %. Compound 1 has been tested for its activity as an inhibitor of various glucosidases
and galactosidases.
Key words: Deoxynojirimycin, Fluorination, Reductive Amination, Glycosidase Inhibitor
Introduction
Several polyhydroxylated iminosugars are strong
and/or specific inhibitors of glycosidases and glyco-
syltransferases [1, 2]. 1-Deoxynojirimycin (2, Fig. 1)
is a natural iminosugar, and it has been shown to be an
effective inhibitor for various glycosidases [3, 4]. Gly-
cosidases are critical for the normal cellular develop-
ment of all organisms. Several iminosugar-based com-
pounds have been discussed for the therapeutic treat-
ment of diabetes mellitus, type 1 Gaucher disease and
lysosomal storage disorders [5, 6].
Modification of a known iminosugar inhibitor is a
promising strategy for obtaining better or more se-
lective inhibitors. Fluorine’s special properties (high
electronegativity, small size) contribute to its impor-
tance in medicinal chemistry. Thus, the effects of flu-
orine substitution on the biological behavior of bioac-
tive molecules have been used effectively in the de-
velopment of new drugs. The germinal difluoromethyl
group is a strong electron-withdrawing group, and it is
an isopolar and isosteric substituent for oxygen [7, 8].
Fig. 1. Structure of deoxynojirimycin (2) and its 6,6-difluoro-
analog 1.
compound since 1 is a competitive inhibitor [9] for
an α-glucosidase (from yeast). To obtain significant
amounts of 1 a new strategy for its synthesis had to
be developed. Retrosynthetic analysis of 1 revealed a
suitably protected gluco-configurated pyranoside hav-
ing an unprotected primary hydroxyl group at C-6 as
an ideal starting material for the synthesis of 1.
Thus, Swern oxidation [10, 11] of commercially
available methyl 2,3,4-tri-O-benzyl-α-D-glucopyr-
anoside (3, Scheme 1) gave aldehyde 4. React◦ion of
crude 4 with DAST in dichloromethane at 25 C for
4 d yielded 85.5% of the 6,6-difluoro compound 5. As
a by-product of this fluorination reaction generated in
an elimination reaction, 6 was obtained in 6 % yield.
Compound 5 is characterized in its 19F NMR spec-
trum by the presence of two signals centered at δ =
−132.46 and −134.97 ppm showing 2JF,F = 283.4 Hz.
From this spectrum as well as from the corresponding
Results and Discussion
Several years ago, Szarek et al. [9] were able to syn-
thesize 1,6-dideoxy-6,6-difluoro-nojirimycin (1,5,6-
trideoxy-6,6-difluoro-1,5-imino-D-glucitol) (1) start-
ing from L-sorbose; the overall yield of this syn-
thetic approach was 8 %. We became interested in this
1H NMR spectra the coupling constants 2JF
=
=
A
,H-6
54.5, 2JF ,H-6 = 54.4 Hz, 3JF ,H-5 = 18.3, and 3JF ,H-5
B
A
B
c
0932–0776 / 11 / 0800–0837 $ 06.00 ꢀ 2011 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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R. Csuk et al. · The Glycosidase Inhibitor 1,6-Dideoxy-6,6-difluoronojirimycin
Scheme 1. a) DMSO, TFAA, −78 ◦C, 1 h, then NEt3, −78 → 25 ◦C, 24 h, quant. (crude product); b) DAST, dichloromethane,
25 ◦C, 4 d, 85.5 % of 5 and 6 % of 6; c) H2SO4 (4 N), HOAc, 85 ◦C, 9 d, 79.5 %; d) LiAlH4, THF, 25 ◦C, 4 h, 99.6 %; e) DMSO,
TFAA, −78 ◦C, 1 h, then NEt3, −78 ◦C → 25 ◦C, 1 h, 75.6 %; f) BnNH2, AcOH, MeOH, −78 ◦C, then NaBH3CN, 88 % of 10
and 6.9 % of 11; g) H2, Pd/C, 35 ◦C, 4.64 atm, 48 h, 98.9 %.
Table 1. Inhibitory activity of 1. IC50 values (in mM) using
several representative glycosidases in a p-nitrophenolate as-
say.
8.8 Hz were determined. Carbon atom C-6 exhibits
1JC,F = 247.3 and 243.9 Hz couplings, whereas for
C-5 2JC,F = 19.9 and 20.2 Hz were measured.
Enzyme
Organism
IC50 (mM)
1.46
4.36
3.07
3.33
Reacting 5 for 9 d with a mixture of 4 N sulfuric
acid/acetic acid at 85 ◦C gave 79.5% of the glucopyra-
nose 7. During the progress of the reaction the forma-
tion of both anomers could be detected by TLC. Af-
ter work-up, however, only the α-anomer was isolated
showing 3JH-1,H-2 = 3.5 Hz in its 1H NMR spectrum.
Reduction of 7 with LiAlH4 in THF gave an al-
most quantitative yield of the diol 8 whose Swern
oxidation [10, 11] using DMSO/TFAA/NEt3 gave a
75.6 % yield of the hex-5-ulose 9. This compound was
immediatly used as a starting material for a reductive
amination using benzylamine and sodium cyanoboro-
hydride [12]. From this reaction, a mixture of the
epimeric compounds 10 and 11 was obtained. The
main epimer 10 was hydrogenated in the presence of
Pd/C to yield target compound 1 in an almost quantita-
tive yield.
α-Glucosidase
α-Glucosidase
β-Glucosidase
α-Galactosidase
β-Galactosidase
B. stearothermophilus
baker’s yeast
almonds
green coffee beans
E. coli
0.90
Whereas for the α-glucosidases and the galactosi-
dases a competitive inhibition is found, inhibition of
the β-galactosidase from almonds takes place by a
mixed competitive inhibition.
Experimental Section
General methods
Melting points are uncorrected (Leica hot stage micro-
scope). Optical rotations were obtained using a Perkin-Elmer
341 polarimeter (1 cm micro cell, 20 ◦C). NMR spectra were
recorded using the Varian spectrometers Gemini 200, Gem-
ini 2000 or Unity 500 (δ given in ppm, J in Hz, internal
Me4Si or internal CCl3F), IR spectra (film or KBr pellet)
on a Perkin-Elmer FT-IR spectrometer Spectrum 1000. MS
spectra were taken on an Intectra GmbH AMD 402 (electron
impact, 70 eV) or on a Thermo Electron Finnigan LCQ (elec-
trospray, voltage 4.5 kV, sheath gas nitrogen) instrument. For
Compound 1 exhibited no significant cytotoxicity as
determined in colorimetric sulforhodamine B assays
using 9 different human cancer cell lines. The results
of an in vitro evaluation of 1 to act as an enzyme in-
hibitor using commercially avaliable glycosidases are
compiled in Table 1.
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R. Csuk et al. · The Glycosidase Inhibitor 1,6-Dideoxy-6,6-difluoronojirimycin
839
elemental analysis a Foss-Heraeus Vario EL instrument was Methyl 2,3,4-tri-O-benzyl-6-deoxy-6,6-difluoro-α-D-gluco-
used. TLC was performed on silica gel (Merck 5554, detec- pyranoside (5) and methyl 2,3-di-O-benzyl-4,6-dideoxy-6,6-
tion by UV absorption or by treatment with a solution of difluoro-β-L-threo-hex-4-enopyranside (6)
10 % sulfuric acid, ammonium molybdate and cerium(IV)
sulfate) followed by gentle heating. The solvents were dried
according to usual procedures.
To a solution of 4 (1.00 g, 2.16 mmol) in dry
dichloromethane (40 mL), DAST (446 µL, 3.24 mmol) was
added, and the mixture was stirred at 25 ◦C for 4 d. Methanol
(5.0 mL) was carefully added, and the solvents were removed
under reduced pressure. The oily residue was dissolved in
Biological testing: p-nitrophenolate assay [13]
dichloromethane (100 mL) and washed with water (50 mL).
The p-nitrophenolate assay was performed as a mi-
The aq. phase was reextracted with dichloromethane (3 ×
100 mL); the organic phases were combined, and the sol-
vent was evaporated under reduced pressure. The remain-
ing residue was subjected to chromatography (silica gel,
hexane-ethyl acetate, 5 : 3) to afford 5 (890 mg, 85.5 %) and 6
(48 mg, 6 %).
crotiter plate assay using a Tecan instrument. The α-gluco-
sidase (from Bacillus stearothermophilus) was obtained from
Sigma (96 U mg−1; Sigma-Aldrich, Steinheim, Germany),
β-glucosidase (almonds) from Fluka (8.92 U mg−1; Sigma-
Aldrich), α-galactosidase (green coffee beans) from Sigma
(45.6 U mg−1), and the β-galactosidase (165 U mg−1) from
Fluka. For all experiments, 0.06 M phosphate buffer (pH =
6.0) was used except for the β-glucosidase where an ac-
etate buffer (0.05 M, pH = 5.0) was applied. Dilution factors
of 6.0, 4.0, 3.0, 2.0, 1.0, 0.03, and 0.01 mg mL−1 were ap-
plied for the inhibitor and 1.0, 0.5, 0.1, 0.05, and 0.025 mM
for the 4-nitrophenol. UV measurements were performed at
λ = 415 nm at least in triplicate, resulting in an SD of 0.0188
and R2 = 0.99988.
Data for 5: colorless oil. – [α]D = +10.46◦ (c = 0.45,
CHCl3) (lit.: +9.5◦ (c = 0.1, CHCl3) [14]). – Rf = 0.80
(hexane-ethyl acetate, 5 : 3). – IR (film): ν = 3388m, 3089m,
3064s, 3032s, 3005m, 2919s, 1954m, 1877w, 1811w, 1732m,
1606m, 1586m, 1497s, 1454s, 1404s, 1361s, 1328s, 1196s,
1156s, 1051s, 918m, 821m, 788m, 737s, 697s, 631m, 554m,
531m, 463m cm−1. – 1H NMR (500 MHz, CDCl3): δ =
7.36 – 7.23 (m, 15 H, Ph), 5.89 (dd, 1 H, 2J6,F = 54.4, 2J6,F
=
2
54.5 Hz, 6-H), 4.99 (d, 1 H, J = 10.9 Hz, CH2OBn), 4.88
(d, 1 H, 2J = 10.9 Hz, CH2OBn), 4.81 (d, 1 H, 2J = 10.8 Hz,
Methyl 2,3,4-tri-O-benzyl-α-D-gluco-hexodialdo-1,5-pyran-
oside (4)
CH2OBn), 4.79 (d, 1 H, 2J = 12.0 Hz, CH2OBn), 4.64
3
(d, 1 H, 2J = 12.0 Hz, CH2OBn), 4.63 (d, 1 H, J1,2
=
3.5 Hz, 1-H), 4.59 (d, 1 H, 2J = 10.8 Hz, CH2OBn), 4.00
To a solution of dry DMSO (0.93 g, 107.64 mmol) in
3
3
◦
(dd, 1 H, J4,3 = 9.2, J4,5 = 9.2 Hz, 4-H), 3.84 (ddd, 1 H,
dry dichloromethane (8.0 mL) at −78 C, a solution of tri-
3J5,F = 8.8, 3J5,4 = 9.2, 3J5,F = 18.8 Hz, 5-H), 3.57 (dd, 1 H,
fluoroacetic acid anhydride (1.99 mL, 8.49 mmol) in dry
dichloromethane (2.0 mL) was added dropwise, and the
mixture was stirred at this temperature for 45 min. A so-
lution of 3 (1.00 g, 2.15 mmol) in dry dichloromethane
(8.0 mL) was added dropwise maintaining the tempera-
ture at −78 ◦C. The mixture was stirred for 2 h, then
a solution of triethylamine (2.2 mL, 15.78 mmol) in dry
dichloromethane (8.0 mL) was added dropwise, and the mix-
3J3,2 = 9.7, J3,4 = 9.2 Hz, 3-H), 3.53 (dd, 1 H, J2,1 = 3.5,
3J2,3 = 9.7 Hz, 2-H), 3.38 (s, 3 H, OCH3) ppm. – 13C NMR
(125 MHz, CDCl3): δ = 138.4, 137.8, 137.6, 128.8, 128.6,
128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.9, 127.7 (each
3
3
1
1
Car), 113.7 (dd, J6,F = 247.3, J6,F = 243.9 Hz, C–6), 98.2
(C-1), 81.7 (C-4), 79.5 (C-2), 77.2 (C-3), 75.8, 75.1, 73.5
2
2
(each CH2OBn), 68.8 (dd, J5,F = 19.9, J5,F = 20.2 Hz,
C–5), 55.4 (OCH3) ppm. – 19F NMR (188 MHz, CDCl3):
◦
◦
ture was allowed to warm to 25 C. After stirring at 25 C
for 12 h, dichloromethane (200 mL) and water (100 mL)
were added, the phases were separated, and the organic
phase was extracted with water (3 × 100 mL). The com-
bined organic phases were evaporated under reduced pres-
sure, and the remaining residue was subjected to chro-
matography (silica gel, hexaneethyl acetate 5 : 3) to afford 4
(quant.) as a mixture of aldehyde and its corresponding hy-
3
2
2
δ = −132.46 (ddd, 1 F, JF,5 = 8.81, JF,6 = 54.4, JF,F
=
=
283.4 Hz, FA), −134.97 (ddd, 1 F, JF,5 = 18.3, JF,6
3
2
54.5, JF,F = 283.4 Hz, FB) ppm. – MS (ESI): m/z (%) =
502.1 (200) [M+NH4]+, 507.2 (55) [M+Na]+, 990.5 (20)
[M2+Na]+. – C28H30O5F2 (484.53): calcd. C 69.41, H 6.24;
found C 69.28, H 6.36.
2
Data for 6: colorless oil. – [α]D = +118.89◦ (c =
drate; colorless oil. – [α]D = +55.97◦ (c = 0.34, CHCl3). – 0.51, CHCl3). – Rf = 0.80 (hexane-ethyl acetate, 5 : 3). –
Rf = 0.45 (hexane-ethyl acetate, 5 : 3). – IR (film): ν = IR (film): ν = 3442m, 3065m, 3032s, 2934s, 1724w,
3031m, 2932s, 1704s, 1640w, 1497w, 1454s, 1383m, 1096s, 1694s, 1600w, 1585m, 1497m, 1454s, 1385s, 1346s, 1278s,
739m, 698m cm−1. – MS (ESI): m/z (%) = 498.1 (5) 1197s, 1158s, 1106s, 1049s, 925m, 811m, 739s, 698s,
[M+NH4, H2O]+, 503.3 (15) [M+Na, H2O]+, 512.1 (70) 650m, 608m, 536m, 462m cm−1. – 1H NMR (500 MHz,
([M+NH4, MeOH]+), 517.3 (100) [M+Na, MeOH]+, 1010.8 CDCl3): δ = 7.37 – 7.27 (m, 10 H, Ph), 5.90 (t, 1 H,
(80) ([M2+Na, (MeOH)2]+. – C28H30O6 (462.53): calcd. 2J6,F = 54.4 Hz, 6-H), 5.34 (m, 1 H, 4-H), 4.89 (d,
3
C 72.71, H 6.54; found C 72.55, H 6.80.
1 H, J1,2 = 2.5 Hz, 1-H), 4.80 (d, 1 H, 2J = 12.2 Hz,
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840
R. Csuk et al. · The Glycosidase Inhibitor 1,6-Dideoxy-6,6-difluoronojirimycin
CH2OBn), 4.73 (d, 1 H, 2J = 12.2 Hz, CH2OBn), 4.65 493.3 (23) [M+Na]+, 962.8 (52) [M2+Na]+. – C27H28O5F2
(m, 2 H, 1 H, CH2OBn), 4.27 (m, 1 H, 3-H), 3.79 (dd, (470.51): calcd. 68.92, 5.99; found 68.79,
C
H
C
3
3
1 H, J2,1 = 2.5, J2,3 = 7.3 Hz, 2-H), 3.48 (s, 3 H, H 6.12
OCH3) ppm. – 13C NMR (125 MHz, CDCl3): δ = 143.9
(dd, J5,F = 23.5, J5,F = 24.0 Hz, C-5), 138.0, 137.9,
137.9, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7
(each Car), 110.4 (dd, J6,F = 239.4, J6,F = 239.3 Hz,
2
2
2,3,4-Tri-O-benzyl-6-deoxy-6,6-difluoro-D-glucitol (8)
1
1
To an ice-cold solution of 7 (370 mg, 0.79 mmol) in
abs THF (20 mL), lithium aluminum hydride (118 mg,
3.15 mmol) was added in several portions and the sus-
pension was stirred for 20 min at this temperature and
for 4 h at 25 ◦C. Methanol (1 mL) was carefully added
and stirring was continued for another 30 min. The sol-
vents were evaporated under reduced pressure. The remain-
ing residue was dissolved in dichloromethane (100 mL) and
was washed with water (30 mL). The aqueous phase was
reextracted with dichloromethane (4 × 100 mL), the com-
bined organic phases were dried (Na2SO4) and the solvent
was evaporated under reduced pressure. The residue was
subjected to chromatography (silica gel, hexane-ethyl ac-
etate, 5 : 3) to afford 8 (370 mg, 99.6 %) as a colorless oil. –
[α]D = +5.23◦ (c = 0.74, CHCl3). – Rf = 0.12 (hexane-
ethyl acetate, 5 : 3). – IR (KBr): ν = 3418m, 3064m, 3032m,
2929m, 1704m, 1604m, 1497m, 1454m, 1398m, 1211m,
1059s, 1028s, 915w, 737m, 699s, 606w, 462m cm−1. –
1H NMR (500 MHz, CDCl3): δ = 7.37 – 7.26 (m, 15 H,
3
3
C-6), 102.6 (dd, J6,F = 6.0, J6,F = 6.0 Hz, C-4), 99.9
(C-1), 76.0 (C-2), 73.3 (CH2(OBn)), 72.7 (C-3), 71.8 (d,
5JCH (OBn),F = 10.6 Hz, (CH2(OBn)), 56.7 (OCH3) ppm. –
2
19F NMR (188 MHz, CDCl3): δ = −122.94 (d, 2 F, 2JF,6
=
54.4 Hz, F) ppm. – MS (ESI): m/z (%) = 394.3 (100)
[M+NH4]+, 399.3 (88) [M+Na]+, 774.7 (64) [M2+Na]+. –
C21H22O4F2 (376.39): calcd. C 67.01, H 5.89; found
C 66.82, H 6.01.
2,3,4-Tri-O-benzyl-6-deoxy-6,6-difluoro-α-D-glucopyranose
(7)
A mixture of 5 (350 mg, 7.22 mmol), acetic acid
(2.56 mL) and 4 N H2SO4 (1.44 mL) was heated to 85 C
and the mixture was stirred for 9 d. After cooling to 25 C
◦
◦
the mixture was slowly poured into an ice-cold aqueous so-
lution of sodium hydrogencarbonate (200 mL). The mix-
ture was extracted with chloroform (4 × 100 mL), the com-
bined organic phases were dried (Na2SO4), and the sol-
vent was evaporated under reduced presure. The remain-
ing residue was subjected to chromatography (silica gel,
hexane-ethyl acetate, 5 : 3) to afford 7 (270 mg, 79.5 %) as
3
2
2
Ph), 5.84 (ddd, 1 H, J6,5 = 2.7, J6,F = 54.9, J6,F
54.9 Hz, 6-H), 4.70 (d, 1 H, J = 11.3 Hz, CH2OBn), 4.65
(s, 2 H, CH2OBn), 4.65 (d, 1 H, J = 11.3 Hz, CH2OBn),
=
2
2
3
4.57 (s, 2 H, CH2OBn), 4.01 (dddd, 1 H, J5,6 = 2.7,
a colorless solid. M. p. 118 – 120 C. – [α]D = +3.32◦ (c =
◦
3J5,4 = 7.1, J5,F = 9.0, J5,F = 16.3 Hz, 5-H), 3.90 (dd,
3
3
3
3
0.35, CHCl3); Rf = 0.68 (hexane-ethyl acetate, 5 : 3). – IR
(KBr): ν = 3425s, 3064m, 3031m, 2918m, 1748w, 1607w,
1498m, 1454m, 1364m, 1328m, 1218m, 1157m, 1132m,
1098m, 1040s, 910w, 792w, 728m, 694m, 632m, 552w,
528w, 458w cm−1. – 1H NMR (500 MHz, CDCl3): δ =
1 H, J3,2 = 7.1, J3,4 = 4.2 Hz, 3-H), 3.85 (dd, 1 H,
3J4,3 = 4.2, J4,5 = 7.1 Hz, 4-H), 3.81 (ddd, 1 H, J2,1A
=
3
3
3
3
4.5, J2,1B = 4.7, J2,3 = 7.1 Hz, 2-H), 3.79 (dd, 1 H,
3J1B,2 = 4.7, J1B,1A = 11.9 Hz, 1-HA), 3.64 (dd, 1 H,
2
3J1B,2 = 4.5, J1B,1A = 11.9 Hz, 1-HA) ppm. – 13C NMR
2
7.29 – 7.17 (m, 15 H, Ph), 5.84 (dd, 1 H, 2J6,F = 54.1, 2J6,F
=
(125 MHz, CDCl3): δ = 137.7, 137.2, 137.1, 128.8, 128.7,
128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9,
3
54.1 Hz, 6-H), 5.17 (d, 1 H, J1,2 = 3.5 Hz, 1-H), 4.89 (d,
1 H, J = 10.9 Hz, CH2OBn), 4.81 (d, 1 H, J = 10.8 Hz,
CH2OBn), 4.77 (d, 1 H, J = 10.9 Hz, CH2OBn), 4.71 (d,
1 H, J = 11.8 Hz, CH2OBn), 4.61 (d, 1 H, J = 11.8 Hz,
2
2
1
127.8, 127.7, 127.6 (each Car), 115.1 (dd, J6,F = 243.3,
2
1J6,F = 243.7 Hz, C-6), 78.7 (C-3), 78.6 (C-4), 75.9 (C-2),
2
2
74.4 (CH2(OBn)), 73.2 (CH2(OBn)), 73.2 (CH2(OBn)), 70.8
2
2
2
CH2OBn), 4.55 (d, 1 H, J = 10.8 Hz, CH2OBn), 4.07 (dd,
(dd, J5,F = 21.6, J5,F = 21.5 Hz, C-5), 61.6 (C-1) ppm. –
1 H, 3J3,2 = 9.4, 3J3,4 = 9.2 Hz, 3-H), 3.94 (dd, 1 H, 3J4,3
9.2, 3J4,5 = 9.2 Hz, 4-H), 3.54 (ddd, 1 H, 3J5,4 = 9.2, 3J5,F
9.9, 3J5,F = 16.8 Hz, 5-H), 3.50 (dd, 1 H, 3J2,1 = 3.5, 3J2,3
=
=
=
19F NMR (188 MHz, CDCl3): δ = −130.80 (ddd, 1 F,
3JF,5 = 9.0, JF,6 = 54.9, JF,F = 286.5 Hz, FA), −133.28
2
2
3
2
2
(ddd, 1 F, JF,5 = 16.3, JF,6 = 55.9, JF,F = 286.5 Hz,
FB) ppm. – MS (ESI): m/z (%) = 473.2 (5) [M+H]+,
490.3 (16) [M+NH4]+, 495.4 (38) [M+Na]+, 966.8 (100)
[M2+Na]+. – C27H30O5F2 (472.52): calcd. C 68.63, H 6.40;
found C 68.50, H 6.61.
9.4 Hz, 2-H) ppm. – 13C NMR (100 MHz, CDCl3): δ =
138.4, 137.6, 137.6, 130.8, 128.8, 128.6, 128.5, 128.4, 128.2,
128.1, 128.0, 127.9, 127.8, 127.7, 127.6 (each Car), 113.9
1
1
(dd, J6,F = 242.8, J6,F = 242.8 Hz, C-6), 91.3 (C-1), 81.2
(C-2), 79.7 (C-4), 75.8, 75.1, 73.5 (each CH2OBn), 71.8
2
2
(C-3), 69.3 (dd, J5,F = 20.3, J5,F = 20.3 Hz, C-5) ppm. –
2,3,4-Tri-O-benzyl-6-deoxy-6,6-difluoro-D-xylo-hexos-5-
19F NMR (188 MHz, CDCl3): δ = −132.23 (ddd, 1 F,
ulose (9)
3JF,5 = 9.9, JF,6 = 54.1, JF,F = 283.1 Hz, FA), −134.20
2
2
3
2
2
(ddd, 1 F, JF,5 = 16.8, JF,6 = 54.1, JF,F = 283.1 Hz,
To a mixture of DMSO (1.05 g, 13.53 mmol) and
FB) ppm. – MS (ESI): m/z (%) = 488.3 (100) [M+NH4]+, dry dichloromethane (9.0 mL) at −78 ◦C a solution of
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R. Csuk et al. · The Glycosidase Inhibitor 1,6-Dideoxy-6,6-difluoronojirimycin
841
trifluoroacetic anhydride (1.91 mL, 9.36 mmol) in dry (film): ν = 3081w, 2883w, 2225w, 1919s, 1879s, 1653w,
dichloromethane (2.2 mL) was added, and the mixture was 1511w, 1323w, 1285m, 1229w, 969m, 735w, 617w, 577w,
stirred for 45 min at −78 ◦C. A solution of 8 (560 mg, 564w, 509m, 451s cm−1. – 1H NMR (500 MHz, CDCl3):
3
1.19 mmol) in dry dichloromethane (9.0 mL) was slowly δ = 7.37 – 7.22 (m, 20 H, Ph), 6.22 (dt, 1 H, J6,5 = 2.0,
added, and stirring was continued at −78 ◦C for 2 h. A 2J6,F = 54.3 Hz, 6-H), 4.89 (d, 1 H, 2J = 10.9 Hz, CH2OBn),
solution of triethylamine (2.46 mL, 1.81 mmol) in dry 4.86 (d, 1 H, 2J = 10.9 Hz, CH2OBn), 4.71 (d, 1 H, 2J =
2
dichloromethane (9.0 mL) was added, and stirring was 11.5 Hz, CH2OBn), 4.63 (d, 1 H, J = 11.5 Hz, CH2OBn),
continued for another 1 h. Dichloromethane (50 mL) and 4.61 (d, 1 H, 2J = 11.5 Hz, CH2OBn), 4.55 (d, 1 H, 2J =
2
water (20 mL) were added, and the aqueous phase was 11.5 Hz, CH22OBn), 4.02 (d, 1 H, J = 14.2 Hz, CH2NBn),
extracted with dichloromethane (3 × 50 mL), the com- 3.92 (d, 1 H, J = 14.2 Hz, CH2NBn), 3.84 – 3.77 (m, 2 H,
bined organic phases were dried (Na2SO4) and the sol- 4-H, 3-H), 3.58 (ddd, 1 H, 3J2,1A = 7.0, 3J2,1B = 11.1, 3J2,3
=
=
=
3
vents were evaporated under reduced pressure. The remain- 7.2 Hz, 2-H), 3.27 (m, 1 H, 5-H), 2.95 (dd, 1 H, J1B,2
2
3
ing residue was subjected to chromatography (silica gel, 11.1, J1B,1A = 11.4 Hz, 1-HB), 2.87 (dd, 1 H, J1A,2
hexane-ethyl acetate, 5 : 3) to afford 9 (787 mg, 75.6 %) 7.0, 2J1A,1B = 11.4 Hz, 1-HA) ppm. – 13C NMR (125 MHz,
as a colorless oil that was used immediately for the next CDCl3): δ = 139.0 (Ph (NBn)), 138.9, 138.4, 138.4, 128.5,
reaction step. – [α]D = +31.15◦ (c = 0.27, MeOH). – 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5,
1
Rf = 0.55 (hexane-ethyl acetate, 5 : 3). – IR (film): ν = 127.4, 127.3, 127.2 (each Car), 119.1 (dd, J6,F = 245.7,
3033w, 2923w, 1770m, 1498w, 1455m, 1362w, 1211m, 1J6,F = 248.1 Hz, C–6), 83.0 (C-3), 78.8 (d, JC,F = 5.3 Hz,
1092s, 739m, 699w, 605w cm−1. – MS (ESI, MeOH): m/z C-4), 78.5 (C-2), 75.5 (CH2OBn), 73.6 (CH2OBn), 72.7
(%) = 486.3 (12) [M+NH4]+, 491.4 (18) [M+Na]+, 504.2 (CH2OBn), 58.9 (t, 1J5,F = 18.2 Hz, C–5), 59.2 (CH2NBn),
(14) [M+NH4, H2O]+, 509.4 (24) [M+Na, H2O]+, 518.3 49.4 (d, J1,F = 3.4 Hz, C–1) ppm. – 19F NMR (188 MHz,
(38) [M+NH4, MeOH]+, 523.4 (68) [M+Na, MeOH]+, CDCl3): δ = −119.93 (dt, 2 F, 3JF,5 = 16.0, 2JF,6 = 54.3 Hz,
958.7 (22) [M2+Na]+, 990.0 (20) [M2+NH4, (H2O)2]+, F) ppm. – MS (ESI): m/z (%) = 544.3 (100) [M+H]+,
1008.8 (34) [M2+NH4, (H2O)2]+, 1022.7 (100) [M2+Na, 566.3 (18) [M+Na]+, 1086.1 (1) [M2+H]+. – C34H35O3F2N
(MeOH)2]+. – MS (ESI, EtOH): m/z (%) = 491.4 (25) (543.64): calcd. C 75.12, H 6.49, N 2.58; found C 75.98,
[M+Na]+, 531.5 (33) [M+NH4, EtOH]+, 537.5 (53) [M+Na, H 6.57, N 2.43.
EtOH]+, 553.3 (30) [M+K, EtOH]+, 599.4 (100) [M+K,
(EtOH)2]+.
Data for 11: colorless oil. – [α]D = +0.06◦ (c = 0.30,
MeOH). – Rf = 0.52 (hexane-ethyl acetate, 85 : 15). – IR
(film): ν = 2999w, 2887w, 2014w, 1649w, 1519w, 1407w,
1363m, 1272w, 1165w, 1109w, 956s, 920s, 800w, 692w,
617s, 578s cm−1. – 1H NMR (500 MHz, CDCl3): δ = 7.34 –
7.19 (m, 20 H, Ph), 6.04 (ddd, 1 H, 3J6,5 = 1.8, 2J6,F = 55.5,
2J6,F = 55.3 Hz, 6-H), 4.87 (d, 1 H, 2J = 10.7 Hz, CH2OBn),
2,3,4-Tri-O-benzyl-1,6-dideoxy-6,6-difluoro-nojirimycin
(10) and 2,3,4-tri-O-benzyl-1,6-dideoxy-6,6-difluoro-L-
idonojirimycin (11)
To a solution of 9 (250 mg, 0.53 mmol) in dry methanol 4.85 (d, 1 H, 2J = 10.7 Hz, CH2OBn), 4.74 (d, 1 H, 2J =
2
(5.0 mL), sodium sulfate (6◦50 mg) was added, and the so- 11.1 Hz, CH2OBn), 4.58 (d, 1 H, J = 11.1 Hz, CH2OBn),
lution was cooled to −78 C. A solution of benzylamine 4.50 (d, 1 H, 2J = 11.7 Hz, CH2OBn), 4.45 (d, 1 H, 2J =
2
(70 µL, 0.65 mmol) and acetic acid (111 µL, 1.92 mmol) 11.7 Hz, CH2OBn), 4.14 (d, 1 H, J = 13.6 Hz, CH2NBn),
in dry methanol (5.0 mL) was added, and the mixture was 3.69 – 3.56 (m, 3 H, 4-H, 3-H, 2-H), 3.56 (d, 1 H, 2J =
3
2
stirred for 2 h at this temperature. Sodium cyanoborohydride 13.6 Hz, CH2NBn), 2.98 (dd, 1 H, J1B,2 = 4.6, J1B,1A
=
(74 mg, 1.17 mmol) was added in small porti◦ons, and the sus- 12.1, 1-HB), 2.93 (m, 1 H, 5-H), 2.23 (dd, 1 H, 3J1A,2 = 9.5,
pension was stirred for another 2 h at −78 C. The solution 2J1A,1B = 12.1, 1-HA) ppm. – 13C NMR (125 MHz, CDCl3):
was allowed to warm to 25 ◦C, and stirring was continued for δ = 138.4 (Ph (NBn)), 138.4, 128.1, 137.8, 128.8, 128.6,
additional 24 h followed by 48 h at 50 ◦C. Methanol (20 mL) 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6, 127.5,
1
was added, and the suspension was filtered through a thin 127.4, 127.3, 127.2, 127.1 (each Car), 115.8 (dd, J6,F
layer of silica gel. The solvents were evaporated, the remain- 245.7, J6,F = 245.2 Hz, C–6), 84.9 (C–3), 77.7 (C-2), 76.8
ing residue was dissolved in dichloromethane (50 mL) and (d, JC,F = 5.3 Hz, C-4), 74.6, 74.4, 72.1 (each CH2OBn), 65.9
=
1
1
1
washed with an aqueous solution of sodium hydrogen car- (dd, J5,F = 19.2, J5,F = 19.2 Hz, C–5), 58.0 (CH2NBn),
bonate and sodium chloride (satd., 20 mL each). The organic 51.4 (C-1) ppm. – 19F NMR (188 MHz, CDCl3): δ = −119.9
3
2
2
layer was dried (Na2SO4), the solvents were evaporated, and (ddd, 1 F, JF,5 = 9.3, JF,6 = 55.5, JF,F = 280.7 Hz,
the remaining residue was subjected to chromatography (sil- FA), −129.4 (ddd, 1 F, JF,5 = 18.1, JF,6 = 55.3, JF,F
=
3
2
2
ica gel, hexane-ethyl acetate, 85 : 15) to afford 10 (220 mg, 280.7 Hz, FB) ppm. – MS (ESI): m/z (%) = 544.3 (100)
88.0 %) and 11 (20 mg, 6.9 %).
[M+H]+, 566.3 (20) [M+Na]+. – C34H35O3F2N (543.64):
Data for 10: colorless oil. – [α]D = +85.56◦ (c = 0.29, calcd. C 75.12, H 6.49, N 2.58; found C 74.96, H 6.70,
MeOH). – Rf = 0.56 (hexane-ethyl acetate, 85 : 15). – IR N 2.27
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842
R. Csuk et al. · The Glycosidase Inhibitor 1,6-Dideoxy-6,6-difluoronojirimycin
1,6-Dideoxy-6,6-difluoro-nojirimycin (1,5,6-trideoxy-6,6-di- 2J6,F = 56.6 Hz, 6-H), 3.89 (m, 1 H, 2-H), 3.85 (m, 1 H,
fluoro-1,5-imino-D-glucitol) (1)
3-H), 3.77 (m, 1 H, 4-H), 3.34 (m, 1 H, 5-H), 3.20 (dd,
3
2
1 H, J1B,2 = 1.9, J1B,1A = 13.0 Hz, 1-HB), 3.07 (dd, 1 H,
2
3J1A,2 = 2.0, J1A,1B = 13.1 Hz, 1-HA) ppm. – 13C NMR
A solution of 10 (180 mg, 0.34 mmol) in dry methanol
(25.0 mL) containing palladium on charcoal (10 %, 210 mg)
was hydrogenated (35 ◦C, 4.64 atm, 48 h). The solution was
filtered through a Celite pad, and this pad was washed with
methanol (3 × 25 mL). The combined organic phases were
dried (MgSO4), the solvent was removed under reduced pres-
sure, and the residue was subjected to chromatography (sil-
ica gel, methanol-ethyl acetate, 20 : 80) to afford 1 (60 mg,
98.9 %) as a colorless foam. – [α]D = +29.48◦ (c = 0.3,
MeOH); (lit.: [α]D = +31.3◦ (c = 0.5, MeOH) [9]); Rf =
0.38 (methanol-ethyl acetate = 20 : 80). – IR (film): ν =
3457w, 2449w, 2171w, 2015w, 1563w, 1325s, 1245s, 1010m,
939w, 861m, 705m, 628w cm−1. – 1H NMR (500 MHz,
1
1
(125 MHz, CD3OD): δ = 116.6 (dd, J6,F = 240.9, J6,F
=
240.4 Hz, C-6), 69.6 (d, JC,F = 4.9 Hz, C-4), 69.1 (C-3), 68.2
2
2
(C-2), 58.8 (dd, J5,F = 20.6, J5,F = 20.6 Hz, C-5), 46.7
(C-1) ppm. – 19F NMR (188 MHz, CD3OD): δ = −128.77
(dd, 1 F, JF,6 = 55.2, JF,F = 297.7 Hz, FA), −131.59 (dd,
1 F, 2JF,6 = 57.1, 2JF,F = 298.5 Hz, FB) ppm. – MS (ESI): m/z
(%) = 184.3 (100) [M+H]+. – C6H11O3F2N (183.15): calcd.
C 39.35, H 6.05, N 7.65; found C 39.27, H 6.18, N 7.56.
2
2
Acknowledgements
We thank Dr. D. Stro¨ hl for recording the NMR spectra,
Dr. R. Kluge for numerous ESI-MS spectra, and St. Schwarz
and B. Siewert for the cell tests.
3
2
CD3OD): δ = 6.02 (ddd, 1 H, J6,5 = 6.6, J6,F = 55.9,
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