Synthesis and biological evaluation of glycosidase inhibitors: gem-difluoromethylenated nojirimycin analogues

Article abstract of DOI:10.1021/jm060066q

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel gem-difluoromethylenated glycosidase inhibitors, gem-4,4-difluoromethylenated iminosugars (5-9) were synthesized. The biological evaluation of these synthetic iminos

Full text of DOI:10.1021/jm060066q

J. Med. Chem. 2006, 49, 2989-2997
2989
Synthesis and Biological Evaluation of Glycosidase Inhibitors: gem-Difluoromethylenated
Nojirimycin Analogues
Ruo-Wen Wang,† Xiao-Long Qiu,† Mikael Bols,‡ Fernando Ortega-Caballero,‡ and Feng-Ling Qing*,†,§
Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu,
Shanghai 200032, China, Institute of Biological Sciences and Biotechnology, Donghua UniVersity, 2999 North Renmin Lu, Shanghai 201620,
China, and Department of Chemistry, UniVersity of Aarhus, DK-8000 Aarhus C, Denmark
ReceiVed January 19, 2006
In our ongoing program aimed at the design, synthesis, and biological evaluation of novel gem-
difluoromethylenated glycosidase inhibitors, gem-4,4-difluoromethylenated iminosugars (5-9) were syn-
thesized. The biological evaluation of these synthetic iminosugars showed that the gem-difluoromethylenyl
group generally reduced the inhibition of glycosidases. However, this was not the case at pH 5.0, where the
gem-difluoromethylenated iminosugar 6 was a stronger inhibitor than comparable iminosugars 1 and 36,
suggesting that the influence of this group is mainly through its effect on the amine. It is proposed that the
unprotonated iminosugar is the species preferably bound by â-glucosidase, due to the lower pKa value of
iminosugar 6 than of 1 or 36, leaving iminosugars 1 and 36 mostly protonated at pH 5.0, while iminosugar
6 is not. Iminosugar 6 also displayed good and selective inhibition of â-glucosidase at pH 6.8.
Introduction
bioactivity, especially when electron-withdrawing groups are
located in the piperidine ring. As a strong electron-withdrawing
group and suggested as an isopolar and isosteric substituent for
oxygen, the gem-difluoromethylene has been successfully used
in modifying nucleosides into anticancer drugs.8 In view of the
aforementioned and the awakening comprehension of structure-
activity relationships for iminosugars,6,9 we designed and
synthesized 4,4-difluoromethylenated iminosugars. The design
of our target molecules was based on the following two
considerations: First, the C2-OH and the C3-OH groups in
an iminosugar typically are critical for a good binding to the
enzymes, whereas the C4-OH group in some cases is non-
essential. Second, the strongly electron-withdrawing gem-
difluoromethylene group would greatly affect the pKa of an
ammonium salt, which could have some interesting conse-
quences, such as improved selectivity. Consequently, we
wondered how the presence of a CF2 group in the C4 position
of the piperidine ring in nojirimycin analogues would affect
the biological activity and selectivity of target molecules D-1,4-
dideoxy-4,4-difluoronojirimycin 5, D-1,4-dideoxy-4,4-difluoro-
mannonojirimycin 6, L-1,4-dideoxy-4,4-difluorogulonojirimycin
7, D-1,4,6-trideoxy-4,4-difluoronojirimycin 8, and L-1,4,6-
trideoxy-4,4-difluoronojirimycin 9 (Figure 2).
Polyhydroxylated piperidines and iminosugars play an im-
portant role in acting as strong and specific inhibitors of
carbohydrate-processing enzymes (i.e. glycosidases and gly-
cotransferases).1 Perhaps, the best known iminosugars are the
naturally occurring 1-deoxynojirimycin (DNJ) 1 and L-1-
deoxyfuconojirimycin 2 (Figure 1), both of which have been
demonstrated to be excellent inhibitors of glucosidase and
fucosidase, respectively.2 These iminosugars have a tremendous
potential as leads for therapeutic reagents for a number of
diseases,3 due to the simple fact that glycosidases are critical
for the normal cellular development of all organisms. Especially
noteworthy are the biologically highly active iminosugars
N-butyl-1-DNJ (Zavesca) 3 and N-hydroxyethyl-DNJ (miglitol)
4 (Figure 1), which successfully have completed clinical trials
for type 1 Gaucher disease and lysosomal storage disorder.4
However, notwithstanding extensive synthesis and investigation
of highly bioactive iminosugars, a remaining drawback associ-
ated with the use of many iminosugars is their lack of selectivity
for R- and â-glycosidase inhibitors, and this has been shown to
cause problems and side effects in therapeutic applications.1a
For example, the clinical trials with N-butyl-DNJ have high-
lighted some important side effects mediated by the inhibition
of glucosidases.
Results and Discussion
Modification of a known iminosugar inhibitor is a promising
strategy for obtaining stronger and more selective inhibitors
toward a certain glycosidase of therapeutic interest. In general,
there are two main strategies for modification of iminosugars:
(a) alterations of the ring hydroxyl residues or (b) introduction
of different alkyl groups on the amino group. In the past decade,
tremendous efforts have been devoted to the alteration of the
ring hydroxyl groups, which involved protection of the hydroxyl
groups,5 substitution with other groups for the hydroxyl groups,6
or changing the stereoconfiguration.7 However, few studies have
dealt with the effect of electron-withdrawing groups on the
Chemistry. The synthesis of D-1,4-dideoxy-4,4-difluoro-
mannonojirimycin 6, which is presented in Scheme 1, features
an efficient intramolecular cyclization to construct the piperidine
ring skeleton. Starting from (R)-glyceraldehyde acetonide and
3-bromo-3,3-difluoropropene, diols 11 and 17 were conveniently
prepared using our reported methodology.10 Selective benzoy-
lation of the primary hydroxyl group in diol 11 gave benzoate
12 in 90% yield. Then, treatment of compound 12 with
trifluoromethanesulfonic anhydride in dichloromethane at -25
°C afforded the corresponding triflate intermediate, which
directly reacted with NaN3 in DMF at room temperature to give
azide 13 in 89% yield. Removal of isopropylidene ketal in azide
13 with 75% acetic acid at 50 °C smoothly gave diol 14 in
96% yield. Selective mesylation of the primary hydroxyl group
in diol 14 proceeded well, and desirable methanesulfonate 15
* Corresponding author. Office: 86-21-5492-5187. Fax: 86-21-6416-
6128. E-mail: flq@mail.sioc.ac.cn.
† Shanghai Institute of Organic Chemistry.
§ Donghua University.
‡ Aarhus University.
10.1021/jm060066q CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/13/2006

2990 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Wang et al.
Figure 1. Some highly bioactive iminosugars 1-4.
C5 in mesylate 24 via SN2 nucleophilic substitution, that basic
conditions should be avoided due to the strong electron-
withdrawing property of the difluoromethylenyl group. It renders
the neighboring hydrogen somewhat acidic and might result in
racemization if basic conditions were utilized. Thus, the
somewhat acidic conditions of NaOAc/Ac2O was adopted in
the SN2 nucleophilic reaction of compound 24, and desired
diacetate 26 was obtained in 53% yield after treatment with an
excess of NaOAc in Ac2O for 36 h at 140 °C. In addition,
diacetate 26 could also be provided in 74% yield by removal
of the TBS protecting group of 24 with AcOH/H2O/THF and
subsequent treatment of resulting alcohol 25 with excess sodium
acetate in Ac2O for 24 h. Then, removal of the acetyl groups in
compound 26 with HCl/MeOH proceeded well to give the diol
27 in 97% yield. Finally, using the procedure as that described
for synthesis of iminosugar 6 from diol 14, compound 27 was
converted into D-1,4-dideoxy-4,4-difluoronojirimycin 5.
Figure 2. Design of gem-difluoromethylenated iminosugars 5-8.
was obtained in 81% yield. Reduction of azide group in
compound 15 with triphenylphosphine successfully afforded the
corresponding piperidine, which was then directly treated with
benzyloxycarbonyl chloride to give carbamate 16 in 82% overall
yield. Finally, one-step removal of the benzyl group and
N-benzyloxycarbonyl group of carbamate 16 via hydrogenation
followed by deprotection of the benzoyl group with a saturated
solution of ammonia in methanol gave the expected iminosugar
6 in 91% yield. Following the same procedure, iminosugar 7
was also easily prepared starting from diol 17.
It was evident that the reversion of the center C2 in compound
6 would result in accessing iminosugar 5. However, initial
attempts to carry out the chiral inversion via the Mitsunobu
reaction of piperidine 16 failed to afford the desired compound.
Fortunately, we were surprised to find that the inverse config-
uration could be conveniently achieved prior to cyclization
(Scheme 2). Selective protection of the primary hydroxyl group
in diol 14 as the tert-butyldimethylsiyl ether 23 progressed well
in 86% yield. Then, exposure of the alcohol 23 to MsCl/Et3N
in CH2Cl2 at room temperature gave the mesylate 24 in 95%
yield. It should be noted, about inversing the configuration at
Iminosugars 8 and 9 were prepared according to our
developed synthetic strategy (Scheme 3).11 That is, Alcohol 30,
prepared from 2,2,2-trifluoroethanol,12 was first treated with
MsCl/Et3N to give mesylate 31 in 96% yield. Then, Pd(0)-
catalyzed regioselective allylic substitution of 31 with NaN3
afforded the desired azide 32 in 96% yield. Conversion of the
azide 32 into the N-Cbz-amine 33 was accomplished by
treatment with PPh3 in dry THF followed by hydrolysis of the
intermediate phosphoryl imine and addition of CbzCl. The
asymmetric AD reaction of compound 33 was carried out, and
diols 34 and 35 were obtained with good ee values (82-84%)
and in moderate yield using (DHQ)2PHAL and (DHQD)2PHAL
as the ligand, respectively. Finally, deprotection of diols 34 and
35, followed by a highly diastereoselective hydrogenation of
intermediates, gave the desired iminosugars 8 and 9, respec-
tively.
Scheme 1. Synthesis of D-1,4-Dideoxy-4,4-difluoromannonojirimycin 6 and l-1,4-Dideoxy-4,4-difluorogulonojirimycin 7 from
(R)-Glyceraldehyde Acetonidea
a Reagents and conditions: (a) BzCl, pyridine, CH2Cl2, -78 °C, 2 h; (b) (i) Tf2O, pyridine, CH2Cl2, -25 °C, 3 h; (ii) NaN3, DMF, rt, 10 h; (c) 75%
AcOH, 50 °C, 2 h; (d) MsCl, collidine, CH2Cl2, 0 °C, 12 h; (e) (i) PPh3, THF, rt, 20 h; (ii) saturated NaHCO3, 65 °C, 12 h; (iii) CbzCl, rt, 3 h; (f) (i) H2,
Pd(OH)2/C, MeOH, 1 atm, rt, 10 h; (ii) saturated NH3/MeOH, rt, 36 h.

gem-Difluoromethylenated Nojirimycin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2991
Scheme 2. Synthesis of D-1,4-Dideoxy-4,4-difluoronojirimycin 5 via the Inversion of Mesylate 25a
a Reagents and conditions: (a) TBSCl, imidazole, DMF, rt, 2 h; (b) MsCl, Et3N, DMAP, CH2Cl2, rt, 12 h; (c) THF/H2O/AcOH (1:1:3), rt, 48 h; (d)
AcOK, Ac2O, 140 °C, 24 h; (e) HCl, MeOH, CH2Cl2, rt, 8 h; (f) MsCl, collidine, CH2Cl2, 0 °C, 12 h; (g) (i) PPh3, THF, rt, 20 h; (ii) saturated NaHCO3,
65 °C, 12 h; (iii) CbzCl, rt, 3 h; (h) (i) H2, Pd(OH)2/C, MeOH, 1 atm, rt, 10 h; (ii) saturated NH3/MeOH, rt, 36 h.
Scheme 3. Synthesis of d-1,4,6-Trideoxy-4,4-difluoronojirimycin 8 and L-1,4,6-Trideoxy-4,4-difluoronojirimycin 9 from
Trifluoroethanola
a Reagents and conditions: (a) MsCl, Et3N, DMAP, CH2Cl2, rt, 12 h; (b) NaN3, Pd(PPh3)4, THF/H2O, rt, 6 h; (c) (i) PPh3, THF, rt, 20 h; (ii) H2O, 65 °C,
12 h; (iii) NaHCO3, CbzCl, rt, 4 h; (d) AD-mix-R, MeSO2NH2, rt, 48 h; (e) AD-mix-â, MeSO2NH2, rt, 48 h; (f) (i) SOCl2, MeOH, rt, 10 h; (ii) H2, Pd/C,
80 psi, rt, 16 h.
Enzymology. Difluoromethylenylated iminosugars 5-9 were
used to investigate their inhibition activities toward 10 different
glycosidases, which included the â-glucosidase from almonds,
R-glucosidase from yeast, â-galactosidases from Saccharomyces
fragilis and Aspergillus orizae, R-galactosidase from green
coffee beans, R-mannosidases from jack bean and almonds,
â-mannosidase from snail, and the R-fucosidases from bovine
kidney and human placenta. Using the corresponding nitrophenyl
glycoside substrates, all assays were performed at 25 °C and
pH 6.8 except the snail â-mannosidase assay, where pH was
4.0. Iminosugars 5, 7, and 8 showed no or negligible inhibition
activities against all 10 enzymes at concentrations, which meant
that the Ki was larger than 1 mM (Table 1). Similarly compound
6 displayed no or negligible inhibition (Ki was larger than 1
mM) against all the enzymes except almond â-glucosidase,
while 9 had Ki > 1 mM for all enzymes except the two
fucosidases (Table 2). In addition, the inhibition of almond
â-glucosidase by 6 and the inhibition of bovine kidney and
human placenta R-fucosidase by 9 was competitive.
iminosugar 7, which resembles the unnatural L-allose or
L-gulose, could be expected to be the bad inhibitor of the 10
enzymes, as it was found to be. Somewhat surprising may be
the observation that compound 8, which is a 6-deoxy-D-glucose
or galactose analogue, did not display any inhibition of any of
glucosidases or galactosidases, but as 1,6-dideoxynojirimycin
9 has been found to be a relatively poor glycosidase inhibitor
(Table 1),6a it is not. Even more surprising was the observation
that compound 5 essentially is not an inhibitor of glucosidase
or galactosidases and 6 is not an inhibitor of galactosidases.
Iminosugars 6 and 9 are analogues of d-mannose and l-fucose,
respectively, so in these cases inhibition of mannosidases or
fucosidases could be expected and be used to evaluate the
influence of displacing the 4-OH with a gem-difluoro group.
The fluoro atom is normally expected to be able to mimic the
hydrogen-bond-accepting properties of an OH, but at the same
time the gem-difluoro group is strongly electron-withdrawing
and will affect the pKa of the iminosugar profoundly. Therefore,
analysis of the influence of the difluoro group is therefore
complicated by these various effects. The pKa of 6 was measured
to 5.3, while the pKa of 1-deoxymannonojirimycin is 7.6,14
meaning an electron withdrawing effect of 2.3 pH units of the
difluoro group. Using our pKa prediction methodology,13,15 the
pKa of compound 6 is predicted to be 5.1, which is quite close
to the measured value.
The inhibition profile of these five compounds 5-9 is not
entirely surprising on the basis of their stereochemistry. Previous
experience has shown that glycosidase inhibitors in most cases
are crucially dependent on their configuration and that epimer-
ization of a hydroxyl group away from the stereochemistry of
the substrate usually decreases inhibition.13 Therefore, the

2992 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Wang et al.
Table 1. Inhibition Constants at 25 °C of gem-Difluorosugars 5, 6, and 8 in Comparison with Iminosugars 36-42a
a Data for compounds 36-42 are taken from refs 6a (1, 37, 38), 16 (1, 36), 17 (1, 36), 18 (39-41), and 19 (42). -, Not available. Ki in µM.
Table 2. Inhibition Constants at pH 6.8 and 25 °C of
OH is unimportant can it be replaced with fluorine and activity
be retained. Since the 4-OH is not crucial for binding to
gem-Difluorosugar 9 in Comparison with Fuconojirimycin 2a
â-glucosidase, it is interesting that the fluorination increased
binding to the enzyme, as this increase must be caused by the
electron-withdrawing influence of the fluorine atoms on the pKa
of the nitrogen. As 1 (pKa ) 6.7) and 36 (pKa ) 7.6) are largely
protonated at pH 5.0 while 6 is not, the good inhibition by 6
and poor binding of 1 and 36 at this pH strongly suggest that
the unprotonated iminosugar is the binding species. Furthermore,
if the protonated iminosugars where the binding species, one
would not expect 6 to be a stronger inhibitor than 36 at pH 6.8,
a Data for compound 2 are taken from refs 21 and 22.
because at this pH 6 contains much less protonated form and
there is no reason why the protonated form of 6 should bind
From the comparison of 6 with its 4-hydroxylated parent 36
stronger than the protonated form of 36. We can therefore
it is seen that generally the introduction of the 4,4′-gem-difluoro
conclude that the unprotonated iminosugar is the species
functionality reduces binding (Table 1). Thus, while 36 is a fair
preferably bound by â-glucosidase.
to good mannosidase and R-glucosidase inhibitor, compound 6
displays no inhibition. On the other hand, 6 is, compared to 36,
The binding constants of difluoroiminosugar 9 to fucosidases
is shown in Table 2 and compared to the parent iminosugar 2.
a very good inhibitor of almond â-glucosidase. In fact, 6 is,
While 36 is a fair inhibitor, the drop in binding when compared
despite having the wrong configuration at the 2-position, as
to the very potent inhibitor 2 is so remarkable that the 4-OH
potent as the gluco-configured 1-deoxynojirimycin 1 at pH 6.8
must have an important interaction with the enzymes that the
and 9-fold more potent at pH 5.0.
As is seen from the data of previously made fluorinated
gem-difluoro group does not have.
In summary, we designed and synthesized nojirimycin
analogues gem-4,4-difluoromethylenated iminosugars 5-9. The
biological evaluation of these synthesized iminosugars showed
that the strongly electron-withdrawing gem-difluoromethylenyl
group had an important influence on the inhibition of glycosi-
dase, which was attributed to the great change of pKa value of
iminosugars resulting from the gem-difluoromethylenyl sub-
analogues of 1 and 36 (Table 1), it is clear that introduction of
the fluorine atom at positions 2, 3, and 6 has reduced binding
to â-glucosidase. The hydroxyl groups in the 2, 3, and 6
positions are known to be important for binding to almond
â-glucosidase, while the 4-OH is unimportant.18,20 Therefore,
it appears, in general, that the fluorine atom cannot emulate an
OH in the interaction with the enzyme, since only when the

gem-Difluoromethylenated Nojirimycin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2993
1
colorless oil: [R]20 0.9 (c 1.350, CHCl3); H NMR (300 MHz,
stituent. In addition, this research has also demonstrated that,
although the gem-difluoromethylenyl group could not substitute
for the key hydroxyl groups in iminosugar without resulting in
decreased affinity, the replacement of a hydroxyl group in an
iminosugar with gem-difluoromethylenyl group would be ad-
vantageous provided the unprotonated iminosugar is the pre-
ferred binding species.
D
CDCl3) δ 8.04-8.07 (d, J ) 7.8 Hz, 2H), 7.58-7.63 (t, J ) 7.5
Hz, 1H), 7.44-7.49 (t, J ) 7.8 Hz, 2H), 7.33-7.40 (m, 5H), 4.78-
4.89 (m, 3H), 4.57-4.64 (m, 1H), 4.07-4.18 (m, 3H), 3.85-3.86
(d, J ) 4.5 Hz, 1H), 1.49 (s, 1H), 1.40 (s, 3H); 19F NMR (282
MHz, CDCl3) δ -114.35 (ddd, J ) 5.9, 18.9, 264.0 Hz, 1F),
-115.93 (ddd, J ) 6.2, 18.6 H, 263.7 Hz, 1F); IR (thin film) 3431,
2113, 1728, 1603, 1454, 1275, 711 cm-1; MS m/z (ESI) 439 (M +
NH4+). Anal. Calcd for C20H21F2N3O5: C, 57.01; H, 5.02; N, 9.97.
Found: C, 57.18; H, 5.22; N, 9.89.
Experimental Section
(2R,4R,5R)-2-Azido-4-O-benzyl-3,3-difluoro-5-hydroxy-6-
(methylsulfonyloxy)hex-1-yl Benzoate (15). Collidine (4.0 mL,
30 mmol) was added to a solution of the diol 14 (1.26 g, 3 mmol)
in CH2Cl2 (60 mL) at room temperature. The mixture was cooled
to 0 °C and MsCl (360 mg, 3.15 mmol) in CH2Cl2 (5.0 mL) was
added dropwise. The reaction mixture was stirred for 20 h at 0 °C
and quenched with aq NaHCO3 (6 mL). The organic layer was
separated and the aqueous layer was extracted with CH2Cl2 (8 mL).
The combined organic layer was dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by column chromatog-
raphy to give compound 15 (1.21 g, 81% yield) as colorless oil:
[R]20D -1.5 (c 0.900, CHCl3); 1H NMR (300 MHz, CDCl3) δ 8.04-
8.07 (d, J ) 7.8 Hz, 2H), 7.56-7.66 (t, J ) 7.5 Hz, 1H), 7.43-
7.48 (t, J ) 7.8 Hz, 2H), 7.33-7.40 (m, 5H), 4.71-4.92 (m, 3H),
4.14-4.62 (m, 6H), 3.03 (s, 3H); 19F NMR (282 MHz, CDCl3) δ
-112.4 (dm, J ) 264.8 Hz, 1F), -114.54 (d m, J ) 265.6 Hz,
1F); IR (thin film) 3511, 2115, 1725, 1603, 1452, 1275, 1177, 1115,
712 cm-1; MS m/z (ESI) 522 (M + Na+). Anal. (C21H23 F2N3O7S)
C, H, N.
Chemistry. All reactions were performed under an argon
atmosphere. Glassware was dried by heating in an oven at a
temperature above 125 °C for at least 6 h. Rotary evaporation was
performed under reduced pressure and at maximum temperature
40 °C. High vacuum refers to a pressure of approximate 2 mmHg.
Petrol refers to the fraction of light petroleum ether with bp 60-
90 °C. Dichloromethane (DCM) was distilled from calcium hydride,
and methanol was distilled from magnesium methoxide. All other
reagents and solvents were purified by standard procedures or used
as obtained from the supplier. All reactions were monitored by thin-
layer chromatography (TLC). Optical rotations were measured using
1
a Perkin-Elmer 241 polarimeter. H and 13C NMR spectra were
recorded on a Bruker AM 300 or a Varian Mercury 300 instrument
at room temperature. 19F NMR spectra were recorded on a Bruker
AM300 spectrometer (CFCl3 as outside standard and low field is
positive). The following abbreviations were used for multiplici-
ties: br ) broad, s ) singlet, d ) doublet, t ) triplet, and q )
quartet, p ) pentet. The signal of the solvent was used as an internal
reference. Asterisk-marked shifts may be interchanged. COSY,
NOESY, HMBC, and HMQC spectra were used for assignment of
the NMR signals.
4,4-Difluoro-6-O-benzoyl-3-O-benzyl-1,5-[(benzyloxycarbon-
y)imino]-1,4,5-trideoxy-D-mannitol (16). A solution of PPh3 (635
mg, 2.65 mmol) in THF (10 mL) was dropwise added to a solution
of compound 15 (1.10 g, 2.20 mmol) in THF (50 mL) at room
temperature. After the starting material was consumed, saturated
aq NaHCO3 (10 mL) was added and the reaction mixture was stirred
for 24 h at reflux. The reaction mixture was cooled to the room
temperature and CbzCl (412.85 mg, 2.42 mmol) was added. The
reaction mixture was stirred for further 4 h, the phases were
separated, and the aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with brine, dried over Na2-
SO4, and concentrated in vacuo. The residue was purified by silica
gel column chromatography to afford compound 16 (922 mg, 82%
(2R,4R)-2-Azido-4-O-benzyl-4-((R)-2,2-dimethyl-1,3-dioxolan-
4-yl)-3,3-difluorobut-1-yl Benzoate (13). Compound 12 (3.15 g,
7.19 mmol) was dissolved in dry CH2Cl2 (55 mL) and fresh distilled
pyridine (4.6 mL) was added. After the resulted mixture was cooled
to -35 °C, trifluoromethanesulfonic anhydride (2.23 g, 8.63 mmol)
in CH2Cl2 (15 mL) was dropwise added to the mixture, and then
the reaction mixture was stirred for about 2 h at about -25 °C.
Water and NaHCO3 aqueous solution (10 mL) were added after
the mixture was warmed to the room temperature. The mixture was
extracted with CH2Cl2 (15 mL) and the organic phase was dried
over anhydrous Na2SO4. After filtration and removal of the solvent
in vacuo, the residue was purified by silica gel chromatograpy to
afford crude trifluoromethanesulfonic ester, which was directly
dissolved in DMF (60 mL), and resulting solution was cooled to 0
°C by ice bath. Then, sodium azide (560 mg, 8.6 mmol) was added
carefully with stirring. The reaction mixture was stirred overnight
at room temperature. After that, water was added to quench the
reaction. The reaction mixture was extracted with CH2Cl2 and the
combined organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography to afford compound
yield) as a white solid: mp 84 °C; [R]20 23.2 (c 1.400, CHCl3);
D
1H NMR (300 MHz, CDCl3) δ 7.89-8.00 (dd, J ) 7.8, 25.8 Hz,
2H), 7.51-7.56 (t, J ) 7.4 Hz, 1H), 7.17-7.41 (m, 12H), 4.83-
5.08 (m, 5H), 4.48-4.64 (m, 2H), 3.84-4.33 (dd, J ) 11.1 Hz,
48.0 Hz, 1H), 3.97 (s, 1H), 3.84 (s, 1H), 3.02-3.12 (m, 1H), 2.21
(s, 1H); 19F NMR (282 MHz, CDCl3) δ -102.76 (ddd, J ) 15.8,
55.3, 267.6 Hz, 1F), -111.18 (dd, J ) 93.6, 266.7 Hz, 1F); IR
(thin film) 3431, 1723, 1603, 1498, 1271, 1124, 1067, 713, 699
cm-1; MS m/z (ESI) 512 (M + H+). Anal. (C28H27 F2NO6) C, H,
N.
13 (2.66 g, 81% yield) as a colorless oil: [R]20 -4.4 (c 1.300,
D-4,4-Difluoro-1,4-dideoxymannonojirimycin (6). A solution
of 16 (664 mg, 1.3 mmol) in MeOH (20 mL) was hydrogenated at
atmospheric pressure and at 25 °C, using 10% Pd(OH)2/C (300
mg) as the catalyst. After stirring for 24 h, the reaction mixture
was filtered through Celite, and the solvent was evaporated. The
residue was dissolved in a saturated solution of ammonia in
methanol (25 mL) and stirred for 48 h. After removal of the solvent,
the residue was purified by silica gel column chromatography to
afford compound 6 (211 mg, 89% yield) as a white solid, which
D
CHCl3); 1H NMR (300 MHz, CDCl3) δ 8.04-8.07 (d, J ) 7.8 Hz,
2H), 7.58-7.63 (t, J ) 7.5 Hz, 1H), 7.45-7.50 (t, J ) 7.8 Hz,
2H), 7.33-7.40 (m, 5H), 5.03-5.07 (d, J ) 10.8, 1H), 4.74-4.83
(m, 2H), 4.50-4.62 (m, 2H), 4.05-4.39 (m, 4H), 1.49 (s, 3H),
1.40 (s, 3H); 19F NMR (282 MHz, CDCl3) δ -116.39 (dd, J )
27.6, 260.9 Hz, 1F), -117.62 (ddd, J ) 5.6, 18.6, 263.7 Hz); IR
(thin film) 2989, 2113, 1730, 1603, 1454, 1270, 1115, 853, 711
cm-1; MS m/z (ESI) 484 (M + Na+). Anal. (C23H25 F2N3O5) C, H,
N.
deliquated soon if placed in air: [R]20 -6.5 (c 1.250, CH3OH);
D
1H NMR (400 MHz, MeOD) δ 3.88-3.90(m, 1H), 3.68-3.84 (m,
3H), 2.98-3.04 (dd, J ) 3.9, 13.8 Hz, 1H), 2.74-2.91 (m, 2H);
13C NMR (75.5 MHz, CDCl3) δ 123.52, 72.24, 70.55, 62.42, 59.68,
50.15; 19F NMR (282 MHz, CD3OD) δ -112.99 (d, J ) 236.6
Hz, 1F), -128.05 (br, 1F); IR (thin film) 3435, 1107, 844, 807,
619 cm-1; HRMS found 184.07798, C6H12NO3F2 requires 184.07797.
(2S,4R)-2-Azido-4-O-benzyl-4-((R)-2,2-dimethyl-1,3-dioxolan-
4-yl)-3,3-difluorobut-1-yl benzoate (19) was prepared by the same
(2R,4R,5R)-2-Azido-4-O-benzyl-3,3-difluoro-5,6-dihydroxyhex-
1-yl Benzoate (14). A mixture of compound 13 (1.98 g, 4.3 mmol)
and 75% aqueous AcOH (20 mL) was stirred at 50 °C for 3 h.
Then, the solvent was removed in vacuo. The resulted residue was
dissolved in EtOAc (30 mL) and washed with aq NaHCO3 (10 mL).
The aqueous layer was extracted with EtOAc (20 mL). The
combined organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was quickly purified by silica gel column
chromatography to afford compound 14 (1.72 g, 95% yield) as a
procedure as described for 13 from 18 as a colorless oil: [R]20
D

2994 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Wang et al.
1
15.5 (c 1.050, CHCl3); H NMR (300 MHz, CDCl3) δ 8.05-8.08
(dm, J ) 263.7 Hz, 1F), -116.73 (dm, J ) 261.1 Hz, 1F); IR
(thin film) 3511, 2115, 1726, 1603, 1455, 1358, 1276, 830, 713
cm-1; MS m/z (ESI) 558 (M + Na+). Anal. (C26H35 F2N3O5Si) C,
H, N.
(d, J ) 8.1 Hz, 2H), 7.58-7.62 (t, J ) 7.5 Hz, 1H), 7.45-7.50 (t,
J ) 7.8 Hz, 2H), 7.30-7.37 (m, 5H), 4.71-4.87 (m, 3H), 4.45-
4.52 (t, J ) 9.3 Hz, 1H), 4.33-4.37 (m, 2H), 4.05-4.14 (m, 2H),
3.92-3.97 (t, J ) 12.0 Hz, 1H), 1.58 (s, 3H), 1.47 (s, 3H); 19F
NMR (282 MHz, CDCl3) δ -114.41 (ddd, J ) 6.5, 13.8, 262.5
Hz, 1F), -116.54 (ddd, J ) 11.8, 16.1, 264.2 Hz, 1F); IR (thin
(2R,4R,5R)-2-Azido-4-O-benzyl-6-(tert-butyldimethylsilyloxy)-
3,3-difluoro-5-(methylsulfonyloxy)hex-1-yl Benzoate (24). To a
stirred solution of alcohol 23 (1.070 g, 2 mmol) in CH2Cl2 (15
mL) were added triethylamine (0.70 mL, 5 mmol), DMAP (10 mg,
0.08 mmol), and methanesulfonyl chloride (0.18 mL, 2.3 mmol) at
0 °C. The mixture was stirred for 12 h at room temperature and
then the reaction was quenched with H2O (4 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 5 mL), and the combined
organic layers were washed with brine and then dried over MgSO4.
After concentration in vacuo, the residue was purified by column
chromatography to give compound 24 (1.14 g, 93% yield) as
colorless oil: [R]20D -6.3° (c 0.700, CHCl3); 1H NMR (300 MHz,
CDCl3) δ 8.03-8.07 (m, 2H), 7.57-7.63 (m, 1H), 7.34-7.49 (m,
7H), 5.01-5.05 (m, 1H), 4.93-4.97 (d, J ) 11.4 Hz, 1H), 4.84-
4.87 (m, 1H), 4.70-4.74 (d, J ) 11.4 Hz, 1H), 4.53-4.60 (m,
1H), 4.35-4.44 (m, 1H), 4.12-4.26 (m, 1H), 3.94-4.07 (m, 2H),
3.10 (s, 3H), 0.92 (s, 1H), 0.10 (s, 3H), -0.11 (s, 3H); 19F NMR
(282 MHz, CDCl3) δ -116.42 (dm, J ) 264.5 Hz, 1F), -117.93
(dm, J ) 260.0 Hz, 1F); IR (thin film) 2956, 2115, 1730, 1603,
1455, 1363, 1272, 1179, 1114, 838, 712 cm-1; MS m/z (ESI) 631
(M + NH4+). Anal. (C27H37 F2N3O7SSi) C. H. N.
film) 2990, 2114, 1729, 1603, 1454, 1271, 1116, 850, 712 cm-1
;
MS m/z (ESI) 484 (M + Na+). Anal. (C23H25 F2N3O5) C, H, N.
(2S,4R,5R)-2-Azido-4-O-benzyl-3,3-difluoro-5,6-dihydroxyhex-
1-yl benzoate (20) was prepared by the same procedure as described
for 14 from 19 as a colorless oil: [R]20D 19.6 (c 1.300, CHCl3); 1H
NMR (300 MHz, CDCl3) δ 8.05-8.08 (d, J ) 7.8 Hz, 2H), 7.57-
7.62 (t, J ) 7.5 Hz, 1H), 7.44-7.49 (t, J ) 7.2 Hz, 2H), 7.35-
7.38 (m, 5H), 4.67-4.89 (m, 3H), 4.40-4.55 (m, 2H), 3.98-4.07
(m, 2H), 3.73-3.84 (m, 2H); 19F NMR (282 MHz, CDCl3) δ
-113.28 (ddd, J ) 6.8, 14.4, 267.1 Hz, 1F), -114.75 (ddd, J )
8.5, 18.9, 265.1 Hz, 1H); IR (thin film) 3433, 2115, 1727, 1603,
1454, 1275, 1116, 712 cm-1; MS m/z (ESI) 439 (M + NH4+). Anal.
(C20H21F2N3O5) C, H, N.
(2S,4R,5R)-2-Azido-4-O-benzyl-3,3-difluoro-5-hydroxy-6-
(methylsulfonyloxy)hex-1-yl benzoate (21) was prepared by the
same procedure as described for 15 from 20 as a colorless oil:
[R]20 -1.5° (c 0.900, CHCl3); H NMR (300 MHz, CDCl3) δ
1
D
8.07-8.10 (d, J ) 7.8 Hz, 2H), 7.58-7.63 (t, J ) 6.9 Hz, 1H),
7.45-7.50 (t, J ) 7.5 Hz, 2H), 7.33-7.39 (m, 5H), 4.72-4.90 (m,
3H), 4.47-4.57 (m, 3H), 4.35-4.40 (m, 1H), 4.26 (s, 1H), 4.02-
4.16 (m, 1H), 3.48 (s, 1H), 3.03 (s, 3H); 13C NMR (75.5 MHz,
CDCl3) δ 166.3, 136.6, 133.7, 129.9, 129.2, 128.8, 128.7, 128.6,
128.5, 121.5 (t, J ) 252.2 Hz), 77.27 (t, J ) 22.4 Hz), 75.84, 68.67,
62.54, 61.92 (t, J ) 23.4 Hz), 37.38; 19F NMR (282 MHz, CDCl3)
δ -112.71 (dd, J ) 9.0 Hz, J ) 263.1 Hz, 1F), -114.83 (dm, J )
267.6 Hz, 1F); IR (thin film) 3516, 3067, 2115, 1726, 1603, 1454,
1657, 1274, 1177, 1116, 713 cm-1; HRMS found 522.1119,
C21H23N3O7F2SNa requires 522.1117 (M +Na+).
(2R,4R,5R)-2-Azido-4-O-benzyl-3,3-difluoro-6-hydroxy-5-
(methylsulfonyloxy)hex-1-yl Benzoate (25). A solution of 24 (1.10
g, 1.80 mmol) in the THF/H2O/AcOH (1:1:3) mixture (35 mL) was
stirred at room temperature for 48 h. Then, the solvent was then
removed in vacuo. The residue was dissolved in EtOAc (30 mL)
and washed with aq NaHCO3 (10 mL). The aqueous layer was
extracted with EtOAc (20 mL). The combined organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography to afford compound
25 (799 mg, 89% yield) as a colorless oil: [R]20D -7.99 (c 1.500,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 8.03-8.06 (d, J ) 7.8 Hz,
2H), 7.3-7.62 (m, 8H), 5.11-5.13 (t, J ) 2.2 Hz, 1H), 4.94-4.97
(d, J ) 11.1 Hz, 1H), 4.83-4.87 (m, 1H), 4.72-4.75 (d, J ) 10.8
Hz, 1H), 4.54-4.61 (dd, J ) 9.0, 11.7 Hz, 1H), 4.35-4.45 (m,
1H), 4.17-4.26 (m, 1H), 3.97-4.11 (m, 2H), 3.12 (s, 3H), 2.72
(br, 1H); 13C NMR (75.5 MHz, CDCl3) δ 166.0, 136.2, 133.6, 129.9,
129.1, 128.8, 128.7, 128.6, 128.4, 120.8 (t, J ) 254.5 Hz), 81.4,
77.1 (dd, J ) 23.7, 28.8 Hz), 75.4, 61.8, 61.5, 59.4 (dd, J ) 23.2,
30.3 Hz), 38.5; 19F NMR (282 MHz, CDCl3) δ -116.68 (dm, J )
263.4 Hz, 1F), -117.86 (dm, J ) 260.9 Hz, 1F); IR (thin film)
4,4-Difluoro-6-O-benzoyl-3-O-benzyl-1,5-[(benzyloxycarbon-
y)imino]-1,4,5-trideoxy-L-gulositol (22) was prepared by the same
procedure as described for 16 from 21 as a white solid: bp 88 °C;
[R]20D -1.1 (c 11.00, CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.85-
7.88 (d, J ) 7.8 Hz, 2H), 7.55-7.61 (m, 1H), 7.27-7.42 (m, 12H),
5.05-5.17 (m, 3H), 4.89-4.93 (d, J ) 12.0 Hz, 1H), 4.74-4.78
(d, J ) 12.0 Hz, 1H), 4.47-4.50 (m, 1H), 4.43-4.46 (m, 1H),
4.09 (s, 1H), 3.75-3.84 (dm, J ) 20 Hz, 1H), 3.21-3.26 (d, J )
15 Hz, 1H), 2.56 (s, 1H); 19F NMR (282 MHz, CDCl3) δ -106.53
(d, J ) 263.1 Hz, 1F), -108.68 (d, J ) 255.5 Hz, 1F); IR (thin
film) 3512, 1722, 1602, 1440, 1276, 1118, 708 cm-1; MS m/z (ESI)
512 (M + H+). Anal. (C28H27 F2NO6) C, H, N.
3531, 2115, 1727, 1603, 1454, 1359, 1274, 1176, 810, 712 cm-1
HRMS found 522.1128, C21H23N3O7F2SNa requires 522.1117.
;
L-4,4-Difluoro-1,4-dideoxygulonojirimycin (7) was prepared by
(2S,3R,5R)-5-Azido-6-O-benzoyl-3-O-benzyl-4,4-difluorohex-
ane-1,2-diyl Diacetate (26). A mixture of 25 (744 mg, 1.49 mmol),
potassium acetate (1.46 g, 10 mol equiv), and Ac2O (60 mL) was
stirred for 24 h at 140 °C. After the mixture was cooled to room
temperature, the solvent was removed in vacuo. The residue was
dissolved in EtOAc (30 mL) and washed with aq NaHCO3 (10 mL).
The aqueous layer was extracted with EtOAc (20 mL). The
combined organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column chroma-
tography to afford 26 (564 mg, 75% yield) as a white solid: mp
63 °C; [R]20D -18.3 (c 1.000, CHCl3); 1H NMR (300 MHz, CDCl3)
δ 8.03-8.06 (dd, J ) 8.4, 1.2 Hz, 2H), 7.57-7.62 (t, J ) 7.5 Hz,
1H), 7.34-7.49 (t, J ) 7.5 Hz, 2H), 7.33-7.44 (m, 5H), 5.49-
5.55 (m, 1H), 4.76-4.86 (m, 3H), 4.55-4.61 (dd, J ) 12.3, 9.0
Hz, 1H), 4.40-4.44 (dd, J ) 3.6, 12.0 Hz, 1H), 4.11-4.27 (m,
3H), 2.07 (s, 6H); 19F NMR (282 MHz, CDCl3) δ -115.52 (dm, J
) 266.2 Hz, 1F), -116.67 (dm, J ) 264.5 Hz, 1F); IR (thin film)
the same procedure as described for 16 from 21 as a white solid:
[R]20 -16.7° (c 3.750, CH3OH); H NMR (400 MHz, MeOD) δ
1
D
3.83-3.93 (m, 3H), 3.70-3.78 (m, 1H), 3.57-3.63 (dd, J ) 7.2,
11.4 Hz, 1H), 3.07-3.19 (dm, J ) 27.0 Hz, 1H), 2.75-2.89 (m,
2H); 13C NMR (75.5 MHz, CDCl3) δ 122.19, 72.22, 69.76, 60.20,
57.31, 50.35; 19F NMR (282 MHz, CD3OD) δ -115.52 (dd, J )
8.7, 251.8 Hz, 1F), -125.11 (dd, J ) 24.8, 251.2 Hz, 1F); IR (thin
film) 3440, 3310, 1078, 998, 669, 607 cm-1; HRMS found
184.07798, C6H12NO3F2 requires 184.07797.
(2R,4R,5R)-2-Azido-4-O-benzyl-6-(tert-butyldimethylsilyloxy)-
3,3-difluoro-5-hydroxyhex-1-yl Benzoate (23). To a solution of
14 (968 mg, 2.3 mmol) in dry DMF (13 mL) were added imidazole
(234 mg, 3.45 mmol) and TBSCl (380 mg, 2.5 mmol) at 0 °C, and
the mixture was stirred for 1 h at 0 °C. The reaction was quenched
with water (50 mL), and the mixture was extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by column chromatography
to give 23 (1.06 g, 86%) as a clear oil: [R]20D 0.6 (c 1.450, CHCl3);
1H NMR (300 MHz, CDCl3) δ 8.04-8.08 (m, 2H), 7.56-7.61 (t,
J ) 8.4 Hz, 1H), 7.44-7.47 (t, J ) 7.2 Hz, 1H), 7.30-7.37 (m,
5H), 4.77-4.88 (m, 3H), 4.54-4.61 (m, 1H), 4.24-4.36 (m, 1H),
4.05-4.14 (m, 2H), 3.72-3.87 (m, 2H), 2.80 (s, 1H), 0.92 (s, 9H),
0.09 (s, 3H), 0.08 (s, 3H); 19F NMR (282 MHz, CDCl3) δ -114.02
3034, 2125, 1747, 1732, 1456, 1242, 1224, 1114, 749, 709 cm-1
;
MS m/z (ESI) 528 (M + Na+). Anal. (C24H25 F2N3O7) C, H, N.
(2R,4R,5S)-2-Azido-4-O-benzyl-3,3-difluoro-5,6-dihydroxyhex-
1-yl Benzoate (27). A solution of HCl in MeOH [prepared at 0 °C
from acetyl chloride (2 mL) and methanol (50 mL)] was added to
a solution of compound 26 (560 mg, 1.1 mmol) in CH2Cl2 (15
mL). After stirring for 10 h at room temperature, the mixture was

gem-Difluoromethylenated Nojirimycin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2995
evaporated to dryness. The residue was dissolved in EtOAc (20
mL), washed with brine, and then dried over MgSO4. After
concentration in vacuo, the residue was purified by column
chromatography to give 27 (449 mg, 97% yield) as a white solid:
mp 81 °C; [R]20D 9.4 (c 0.850, CHCl3); 1H NMR (300 MHz, CDCl3)
δ 8.05-8.08 (m, 2H), 7.37-7.63 (m, 8H), 4.76-4.91(m, 3H),
4.58-4.65 (dd, J ) 12.3, 9.0 Hz, 1H), 4.26-4.38 (m, 1H), 3.94-
4.02 (m, 2H), 3.65-3.67 (d, J ) 4.8 Hz, 1H), 2.61 (br, 1H); 19F
NMR (282 MHz, CDCl3) δ -113.04 (dm, J ) 270.4 Hz, 1F),
-114.38 (dm, J ) 268.5 Hz, 1F); IR (thin film) 3440, 2121, 1731,
1603, 1456, 1319, 1270, 751, 711 cm-1; MS m/z (ESI) 444 (M +
Na+). Anal. (C20H21F2N3O5) C, H, N.
(2R,4R,5S)-2-Azido-4-O-benzyl-3,3-difluoro-5-hydroxy-6-
(methylsulfonyloxy)hex-1-yl benzoate (28) was prepared by the
same procedure as described for 15 from 27 as a colorless oil:
[R]20D 11.2 (c 0.900, CHCl3); 1H NMR (300 MHz, CDCl3) δ 8.05-
8.07 (dd, J ) 1.2, 7.5 Hz, 2H), 7.57-7.63 (m, 1H), 7.37-7.50 (t,
J ) 7.5 Hz, 7H), 4.48-4.92 (m, 3H), 4.58-4.65 (m, 1H), 4.24-
4.35 (m, 4H), 3.97-4.04 (m, 1H), 3.04 (s, 3H); 13C NMR (75.5
MHz, CDCl3) δ 165.9, 136.2, 133.5, 129.9, 129.2, 128.8, 128.6,
128.5, 128.4, 121.0, 76.7, 75.9, 69.7, 67.3, 61.9, 60.4, 37.6; 19F
NMR (282 MHz, CDCl3) δ -113.06 (dm, J ) 269.3 Hz, 1F),
-114.25 (dm, J ) 267.9 Hz, 1F); IR (thin film) 3513, 2115, 1726,
1603, 1454, 1357, 1275, 1177, 1117, 713 cm-1; HRMS found
522.1119, C21H23N3O7F2SNa requires 522.1117.
compound 32 (820 mg, 96%) as a clear oil: 1H NMR (300 MHz,
CDCl3) δ 6.20 (d, JH-Htrans ) 15.6 Hz, 1H), 5.96-6.04 (m, 1H),
5.12 (s, 2H), 4.80 (d, J ) 3.0 Hz, 1H), 4.66-4.68 (m, 1H), 3.92
(s, 2H), 3.74-3.77 (m, 2H), 3.54-3.57 (m, 2H), 3.39 (s, 3H); 19
F
NMR (282 MHz, CDCl3) δ -101.17 (d, J ) 10.4 Hz, 2F); IR (thin
film) 2931, 2886, 2109, 1653, 1081, 998, 853 cm-1; MS m/z (ESI)
281 (M + NH4+). Anal. (C10H13 F2 N3O3) C, H, N.
N-(Benzyloxycarbonyl)-4,4-difluoro-5-(2-methoxyethoxy-
methoxy)hexa-2,5-dienamine (33). Triphenylphosphine (470 mg,
1.79 mmol) was added to a solution of 32 (400 mg, 1.52 mmol) in
dry THF (24 mL). After the reaction mixture was stirred room
temperature for 16 h, H2O (1.8 mL) was added, and the reaction
was allowed to stir for another 16 h at 65 °C. When the reaction
was cooled to room temperature, K2CO3 (320 mg, 2.30 mmol) and
CbzCl (0.30 mL, 2.20 mmol) were added. After stirring at room
temperature for 8 h, the reaction was extracted with ether (30 mL
× 2), and the organic layers were washed with brine and dried
over MgSO4. After concentration in vacuo, the residue was purified
by flash column chromatography to give 33 (510 mg, 90%) as a
clear oil: 1H NMR (300 MHz, CDCl3) δ 7.35 (s, 5H), 6.17 (d,
JH-Htrans ) 17.3 Hz, 1H), 5.76-5.88 (m, 1H), 5.12 (s, 2H), 5.10 (s,
2H), 4.76 (d, J ) 3.3 Hz, 1H), 4.63-4.64 (m, 1H), 3.92 (s, 2H),
3.72-3.76 (m, 2H), 3.53-3.56 (m, 2H), 3.37 (s, 3H); 19F NMR
(282 MHz, CDCl3) δ -101.19 (d, J ) 10.7 Hz, 2F); IR (thin film)
3335, 2931, 1716, 1652, 1532, 1255, 852, 699 cm-1; MS m/z (ESI)
389 (M + NH4+). Anal. (C18H23 F2NO5) C, H, N.
(2R,3R)-1-(Benzyloxycarbonylamino)-4,4-difluoro-5-(2-meth-
oxyethoxymethoxy)hex-5-ene-2,3-diol (34). To a solution of 33
(370 mg, 1.0 mmol) in tert-butyl alcohol (5 mL) and water (5 mL)
at 0 °C were added K3[Fe(CN)6] (987 mg, 3 mmol), K2CO3 (414
mg, 3 mmol), (DHQ)2PHAL (47 mg, 0.06 mmol), K2OsO2(OH)4
(8.0 mg, 0.02 mmol), and CH3SO2NH2 (95 mg, 1 mmol). After
the mixture was stirred at 0 °C for 36 h, the reaction was quenched
by addition of saturated aqueous Na2S2O3. After stirring for 30 min,
the resulting mixture was extracted with ethyl acetate (3 × 10 mL).
The combined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified by
flash column chromatography to give a mixture of 33 and MeSO2-
NH2 (2:1) (260 mg, 55%) as a viscous oil in 82% ee (determined
by chiral HPLC analysis on Chiralcel AD, hexane/2-propanol (90:
10 v/v), 0.7 m/mL, tR (minor) ) 36.5 min, tR (major) ) 40.1 min):
1H NMR (300 MHz, CDCl3) δ 7.35 (s, 5H), 5.27 (s, 1H), 5.08-
5.16 (m, 4H), 4.86 (d, J ) 5.4 Hz, 1H), 4.74 (t, J ) 2.7 Hz, 1H),
4.09-4.12 (m, 1H), 3.85-3.93 (m, 1H), 3.75-3.78 (m, 2H), 3.54-
3.57 (m, 1H); 19F NMR (282 MHz, CDCl3) δ -110.65 (d, J )
256.6 Hz, F), -118.44 (dd, J ) 19.2, 256.3 Hz); IR (thin film)
3355, 2939, 1706, 1657, 1533, 1456, 1329, 1264, 1152, 1089, 996,
699 cm-1; MS m/z (ESI) 406 (M + H+). Anal. (2C18H25F2NO7‚CH5-
NO2S) C, H, N, S.
(2S,3S)-1-(Benzyloxycarbonylamino)-4,4-difluoro-5-(2-meth-
oxyethoxymethoxy)hex-5-ene-2,3-diol (35). To a solution of 33
(590 mg, 1.6 mmol) in tert-butyl alcohol (8 mL) and water (8 mL)
at 0 °C were added K3[Fe(CN)6] (1580 mg, 3 mmol), K2CO3 (662
mg, 3 mmol), (DHQD)2PHAL (75 mg, 0.06 mmol), K2OsO2(OH)4
(13.0 mg, 0.032 mmol), and CH3SO2NH2 (152 mg, 1.6 mmol). After
the mixture was stirred at 0 °C for 36 h, the reaction was quenched
by addition of saturated aqueous Na2S2O3. After stirring for 30 min,
the resulting mixture was extracted with ethyl acetate (3 × 15 mL).
The combined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified by
flash column chromatography to give a mixture of 35 and MeSO2-
NH2 (2:1) (430 mg, 57%) as a viscous oil in 84% ee (determined
by chiral HPLC analysis on Chiralcel OD, hexane/2-propanol (90:
10 v/v), 0.7m/ml, tR (major) ) 30.4 min, tR (minor) ) 33.5 min):
1H NMR (300 MHz, CDCl3) δ 7.36 (s, 5H), 5.28 (s, 1H), 5.08-
5.14 (m, 4H), 4.86 (d, J ) 3.3 Hz, 1H), 4.73 (t, J ) 2.7 Hz, 1H),
4.09-4.13 (m, 1H), 3.84-3.92 (m, 1H), 3.74-3.77 (m, 2H), 3.53-
3.55 (m, 2H), 3.44-3.51 (m, 2 H), 3.37 (s, 3 H), 3.26-3.35 (m, 1
H), 3.10 (s, 1.5 H), 3.00 (s, 1 H); 19F NMR (282 MHz, CDCl3) δ
-110.65 (d, J ) 256.6 Hz, 1F), -118.44 (dd, J ) 19.2, 256.3 Hz,
1F); IR (thin film) 3358, 2939, 1707, 1656, 1533, 1330, 1265, 1151,
4,4-Difluoro-6-O-benzoyl-3-O-benzyl-1,5-[(benzyloxycarbon-
y)imino]-1,4,5-trideoxy-D-glucitol (29) was prepared by the same
procedure as described for 15 from 27 as a white solid: mp 77 °C;
[R]20D 10.3 (c 1.400, CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.94
(br, 2H), 7.19-7.57 (m, 13H), 4.62-5.10 (m, 6H), 4.50-4.54 (d,
J ) 11.7 Hz, 1H), 4.22 (br, 1H), 3.96 (br, 1H), 3.75 (s, 1H), 3.53-
3.58 (d, J ) 15 Hz, 1H), 2.27 (br, 1H); 19F NMR (282 MHz, CDCl3)
δ -99.21 (d, J ) 267.2 Hz, 1F), -110.80 (dd, J ) 268.7 Hz, 1F);
IR (thin film) 3571, 1723, 1711, 1602, 1498, 1270, 1114, 703 cm-1
;
MS m/z (ESI) 512 (M + H+). Anal. (C28H27 F2NO6) C, H, N.
D-4,4-Difluorono-1,4-dideoxyjirimycin (5) was prepared by the
same procedure as described for 6 from 29 as a white solid, which
deliquated soon in air: [R]20D -39.2 (c 1.2250, CH3OH); 1H NMR
(300 MHz, MeOD) δ 3.88-3.93 (dd, J ) 3.9, 11.4 Hz, 1H), 3.46-
3.66 (m, 3H), 3.14-3.20 (m, 1H), 2.82-2.94 (dm, J ) 24.0 Hz,
1H), 2.47-2.51 (t, J ) 10.8 Hz, 1H); 13C NMR (75.5 MHz, CDCl3)
δ 121.42, 77.39, 72.00, 62.57, 60.02, 51.05; 19F NMR (282 MHz,
CD3OD) δ -117.59 (dd, J ) 244.8 Hz, 1F), -134.52 (dm, J )
243.9 Hz, 1F); IR (thin film) 3431, 1107, 843, 808, 619 cm-1
HRMS found 184.0776, C6H12NO3F2 requires 184.07798.
;
3,3-Difluoro-4-mesyloxy-2-(2-methoxyethoxymethoxy)hexa-
1,5-diene (31). To a stirred solution of alcohol 30 (2.02 g, 8.48
mmol) in CH2Cl2 (23 mL) were added triethylamine (3.0 mL, 21.5
mmol), DMAP (39 mg, 0.32 mmol), and methanesulfonyl chloride
(0.90 mL, 11.72 mmol) at 0 °C. The mixture was stirred for 12 h
at room temperature and then quenched with 10 mL of H2O. The
aqueous layer was extracted with CH2Cl2 (10 mL × 2), and the
combined organic layers were washed with brine and then dried
over MgSO4. After concentration in vacuo, the residue was purified
by flash column chromatography to give 31 (2.57 g, 96%) as a
colorless oil: 1H NMR (300 MHz, CDCl3) δ 5.87-5.98 (m, 1H),
5.64 (br d, JH-Htrans ) 17.4 Hz, 1H), 5.56 (br d, JH-Hcis ) 10.5 Hz,
1H), 5.44-5.34 (m, 1H), 5.12 (s, 2H), 4.86 (d, J ) 3.3 Hz, 1H),
4.81-4.82 (m, 1H), 3.76-3.80 (m, 2H), 3.56 (t, J ) 5.1 Hz, 2H),
3.39 (s, 3H), 3.06 (s, 3H); 19F NMR (282 MHz, CDCl3) δ -114.02
(d, J ) 10.5 Hz, 2F); IR (thin film) 2940, 1658, 1368, 1180, 1102,
998, 856 cm-1; MS m/z (ESI) 334 (M + NH4+). Anal. (C11H18
F2O6S) C, H.
6-Azido-3,3-difluoro-2-(2-methoxyethoxymethoxy)hexa-1,4-
diene (32). To a stirred solution of 31 (1.03 g, 3.26 mmol) in THF
(60 mL) and H2O (15 mL) were added Pd(PPh3)4 (200 mg, 5 mol
%) and sodium azide (268 mg, 4.12 mmol). After stirring for 10 h
at room temperature, the reaction mixture was extracted with ether
(100 mL × 2) and the combined organic layers were washed with
brine and then dried over MgSO4. After concentration in vacuo,
the residue was purified by flash column chromatography to give

2996 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Wang et al.
1094, 996, 699 cm-1; MS m/z (ESI) 406 (M + H+). Anal.
(2C18H25F2NO7‚CH5NO2S) C, H, N.
inhibition constant, Ki, was calculated. All assays were performed
at pH 6.8 and 25 °C except the â-mannosidase assay, which was
performed at pH 4.0. The inhibition constants (Ki) were obtained
from the formula Ki ) [I]/(KM′/KM - 1), where KM′ and KM are
Michaelis-Menten constants with and without inhibitor present.
4,4-Difluoro-1,4,6-trideoxynojirimycin (8). Thionyl chloride
(0.09 mL, 1.23 mmol) was added slowly to a cooled solution (0
°C) of 34 (190 mg, 0.60 mmol) in methanol (10 mL). The reaction
mixture was allowed to stir overnight at room temperature. The
methanol was removed in vacuo, and the residue was resolved in
ethyl acetate (20 mL) and washed with saturated aqueous K2CO3.
After concentration in vacuo, the residue was mixed with 10% Pd/C
(80 mg) in methanol (8 mL) and hydrogenated under 80 psi of H2
(12h). Then, the reaction mixture was filtered through Celite, and
the filtrate was evaporated to give a residue. The residue was
purified by flash column chromatography to give 8 (53 mg, 53%)
as a white solid. This product was recrystalized from CH3OH/
Acknowledgment. We thank the National Natural Science
Foundation of China, Ministry of Education of China and
Shanghai Municipal Scientific Committee for funding this work.
Supporting Information Available: Elemental analyses data
of compounds and 1H and 13C NMR spectra for all the compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AcOEt (1:10) to give optically pure 8: mp 178-180 °C; [R]20
D
References
1
+30.2 (c 0.350, CH3OH); H NMR (300 MHz, CD3OD) δ 3.50-
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3.64 (m, 2H), 3.38 (d, J ) 11.7 Hz, 1H), 3.06-3.13 (q, 1H), 2.83-
2.96 (m, 1H), 2.48-2.56 (q, 1H), 1.21 (d, J ) 6.2 Hz, 3H); 19F
NMR (282 MHz, CD3OD) δ -116.67 (dd, J ) 3.4, 241.7 Hz, 1F),
-138.70 (dm, 243.4 Hz, 1F); IR (thin film) 3327, 3254, 1232 1065,
1027, 820, 689 cm-1; MS m/z (ESI) 168 (M + H+). Anal. (C6H11F2-
NO2) C, H, N.
L-4,4-Difluoro-1,4,6-trideoxynojirimycin (9). Thionyl chloride
(0.06 mL, 0.86 mmol) was added slowly to a cool solution (0 °C)
of 35 (130 mg, 0.40 mmol) in methanol (8 mL). The reaction
mixture was allowed to stir overnight at room temperature.
Methanol was removed in vacuo, and the residue was resolved in
ethyl acetate (15 mL) and washed with saturated aqueous K2CO3.
After concentration in vacuo, the residue was mixed with 10% Pd/C
(60 mg) in methanol (6 mL) and hydrogenated under 80 psi of H2
(12 h). Then, the reaction mixture was filtered through Celite, and
the filtrate was evaporated to give a residue. The residue was
purified by flash column chromatography to give 9 (32 mg, 46%)
as a white solid. This product was recrystallized from CH3OH/
AcOEt (1:10) to give optically pure 9: mp 179-182 °C; [R]20
D
1
-30.2 (c 0.550, CH3OH); H NMR (300 MHz, CD3OD) δ 3.41-
3.58 (m, 2H), 3.26-3.31 (m, 1H), 2.96-3.04 (m, 1H), 2.74-2.88
(m, 1H), 2.37-2.46 (m, 1H), 1.21 (d, J ) 6.2 Hz, 3H); 13C NMR
(75.5 MHz, CD3OD) δ 119.8 (t, JC-F ) 247.9 Hz), 75.56 (t, JC-F
) 19.9 Hz), 70.53 (d, JC-F ) 7.9 Hz), 54.39 (t, JC-F ) 23.7 Hz),
49.28, 10.81 (d, JC-F ) 6.3 Hz); 19F NMR (282 MHz, CD3OD) δ
-117.69 (dd, J ) 3.7, 246.7 Hz, 1F), -138.69 (dm, J ) 244.8,
1F); IR (thin film) 3323, 3254, 1232, 1065, 1027, 821, 688 cm-1
HRMS found 190.0652, C6H11NO2F2Na requires 190.0650.
;
Enzyme Inhibition. Each glycosidase assay was performed by
preparing eight 2-mL samples in cuvettes, containing 1 mL of
sodium phosphate buffer (0.1 M) of pH 6.8 or acetate buffer of pH
4.0, along with 0.04-0.80 mL of different substrate. The concentra-
tion of the substrate was in the range from 0.25KM to 5KM. The
substrates used were 2-nitrophenyl-â-D-galactopyranoside, 4-nitro-
phenyl-R-D-galactopyranoside, 4-nitrophenyl-â-D-glucopyranoside,
4-nitrophenyl-R-D-glucopyranoside, 4-nitrophenyl-R-L-fucopyra-
noside, 4-nitrophenyl-R-D-mannopyranoside, or 4-nitrophenyl-â-
D-mannopyranoside. Also added was 0.02-0.1 mL of a solution
of either the inhibitor or water, and finally each cuvette was filled
up to a total volume of 1.9 mL with distilled water. Seven of the
samples contained the inhibitor at a fixed concentration but with
varying concentrations of nitrophenyl glycoside. The other seven
samples contained no inhibitor but also varying concentrations of
nitrophenyl glycoside. Finally, the reaction was started by adding
0.1 mL of a diluted solution of enzyme solution. The formation of
4- or 2-nitrophenol was monitored for 2 min at 25 °C by
measurement of the absorbance at 400 nm. In the case of the
â-mannosidase assay, the velocity of substrate hydrolysis was
measured by quenching 200 µL of solution with 1800 µL of borate
buffer (1.0 M, pH 9) every 30 s over 3 min and then measuring
absorbance at 400 nm. Initial velocities were calculated from the
slopes from each reaction and used to construct two Hanes plots
([S]/V vs [S]), one with and one without inhibitor, which also was
used to check whether inhibition was competitive. From the two
Michaelis-Menten constants, KM and KM′, thus obtained, the
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JM060066Q