Synthesis and biological evaluation of N-(2-fluorophenyl)-2β- deoxyfuconojirimycin acetamide as a potent inhibitor for α-L-fucosidases

Article abstract of DOI:10.1016/j.bmc.2013.08.028

In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of α-l-fucosidases. A structure-activity relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selectivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Consequently, N-(2-fluorophenyl)-2β-DFJ acetamide (18j) was found to display very potent and selective inhibition of bovine kidney, rat epididymis, and human lysosome α-l-fucosidases, with IC₅₀ value of 0.012, 0.044, and 0.0079 μM respectively. It is noteworthy that our designed N-phenyl-2β-DFJ acetamide derivative exhibited about 18-fold stronger effects on human lysosomal α-l-fucosidase than original DFJ and it occupied the active-site of this enzyme. We therefore expect that this compound may find applications in new therapeutic trials against genetic deficiency disorders.

Full text of DOI:10.1016/j.bmc.2013.08.028

Bioorganic & Medicinal Chemistry 21 (2013) 6565–6573
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of N-(2-fluorophenyl)-2b-deoxy-
fuconojirimycin acetamide as a potent inhibitor for
a-L-fucosidases
Atsushi Kato a, , Toru Okaki , Syohei Ifuku , Kasumi Sato , Yuki Hirokami , Ren Iwaki , Akiko Kamori ,
⇑
b
b
a
a
a
a
a
a
c
c
d
d
Shinpei Nakagawa , Isao Adachi , Peter G. Kiria , Osamu Onomura , Daishiro Minato , Kenji Sugimoto ,
Yuji Matsuya , Naoki Toyooka
d
b,e,
⇑
a
Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan
b
c
Graduate School of Science and Technology for Research, University of Toyama, Toyama 930-8555, Japan
Graduate School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8521, Japan
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-
Received 24 July 2013
Revised 13 August 2013
Accepted 14 August 2013
Available online 24 August 2013
deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of
a-L-fucosidases. A structure–activity
relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selec-
tivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Conse-
quently, N-(2-fluorophenyl)-2b-DFJ acetamide (18j) was found to display very potent and selective
inhibition of bovine kidney, rat epididymis, and human lysosome
.012, 0.044, and 0.0079 M respectively. It is noteworthy that our designed N-pheny
derivative exhibited about 18-fold stronger effects on human lysosomal -fucosidase than original DFJ
and it occupied the active-site of this enzyme. We therefore expect that this compound may find appli-
cations in new therapeutic trials against genetic deficiency disorders.
Ó 2013 Elsevier Ltd. All rights reserved.
a
-
L
-fucosidases, with IC50 value of
Keywords:
0
l
L-2b-DFJ acetamide
a-
L
-Fucosidase
a-L
1
-Deoxyfuconojirimycin
N-(2-Fluorophenyl)-2b-DFJ acetamide
Iminosugar
Fucosidosis
1
. Introduction
Iminosugars are sugar mimics with a nitrogen atom in place of
chronic, multisystem disease arising from mutations in the gene
encoding the CF transmembrane regulator (CFTR). Previous re-
views suggested that CF patients have increased terminal fucosyla-
tion and decreased sialylation, which is a hallmark of the CF
the ring oxygen of monosaccharides and inhibit various glycosi-
dases in a reversible and competitive manner.1 These inhibitors
are currently of great interest as potential therapeutic agents.
–3
7,8
glycosylation phenotype. More recently, competitive lysosomal
a-L-fucosidase inhibitors have been suggested to have therapeutic
a
-
L
-Fucosidase (EC 3.2.1.51) is involved in the cleavage of ter-
minal fucose linked 1–2 to a galactose residue and of fucose
linked 1–3, 1–4 or 1–6 to an N-acetylglucosamine residue on
glycoproteins and glycolipids. It has been reported that -fucosi-
potential for fucosidosis, which is an autosomal recessive lyso-
somal storage disease. Furthermore, a recent study demonstrated
the important connection between a-L-fucosidase and the adhe-
sion, growth, and pathogenicity of Helicobactor pylori and indicated
a
a
a
a
a-
L
dase activities and fucose-residue levels are closely related with
many disorders including inflammation, cancer, cystic fibrosis,
and fucosidosis. In silayl-Lewis X (sLe ), for instance, terminal fu-
inhibition of this enzyme is a potential target for clinical diagnosis
and therapeutic intervention of H. pylori related diseases.9
a-L-
x
Fucosidase is also known to be implicated in sperm-egg binding
cose is an essential feature recognized by cell adhesion molecules
and it is one of the most active glycosidases in epididymal sperm.10
4
such as E-selectins present on endothelial cells. In this capacity,
Previous studies suggested that
molecules inhibit human sperm-egg interactions.
cific -fucosidase inhibitors are expected to be useful tools in elu-
cidating the mechanisms of sterility.
We have previously reported the inhibitory activity towards
fucosidase of 15 piperidine- and pyrrolidine-type iminosugars from
L-fucose and L-fucose-containing
x
11,12
sLe enables homing of leukocytes to sites of inflammation and
Thus, spe-
mediates tumor metastasis. Indeed, many types of carcinomas dis-
a-L
x
play increased levels of sLe and fucose often correlated with
5,6
malignancy and patient survival rates. Cystic fibrosis (CF) is a
a-L-
1
3
the bark of the tree Angylocalyx pynaertii. Among them, 2,5-imino-
1,2,5-trideoxy- -glucitol and 6-O- -rhamnopyranosyl-1-deoxym-
annojirimycin showed potent inhibition against bovine kidney
⇑
Corresponding authors.
E-mail addresses: kato@med.u-toyama.ac.jp (A. Kato), toyooka@eng.u-toya-
L
a-L
ma.ac.jp (N. Toyooka).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.028

6
566
A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
a
-
L
-fucosidase, with IC50 values 1.0 and 0.92
l
M, respectively. 1-
tors, we turned our attention to the design of
inhibitors. An additional aim was to elucidate the inhibition profile
against different sources of -fucosidases. Here, we report design,
synthesis, evaluation and structure-activity relationship of novel
N-(2-fluorophenyl)-2b-DFJ acetamide as powerful and selective
a-L-fucosidase
Deoxymannojirimycin (DMJ) mimicking
be an inhibitor of class I
I and Golgi -mannosidase I but not of class II
However, DMJ is a better inhibitor of
D
-mannose is known to
a-mannosidases such as ER
a
-mannosidase
a-L
1
4
a
a
-mannosidases.
a-
L
-fucosidase than
nosidase. Furthermore, 1-deoxygalactonojirimycin (DGJ) mimick-
ing -galactose is known as an extremely powerful inhibitor of
human -galactosidase A, whereas its enantiomer -DGJ showed po-
tent inhibition against bovine epididymis -fucosidase, with IC50
A previous study suggested that sugar-mimic
a
-D
-man-
1
5
inhibitors of
a
-
L
-fucosidase.
. Results and discussion
.1. Chemistry
The synthesis began with
D
a
L
2
2
a
-L
1
5,16
value 0.63
-fucosidase inhibitors have a common structural feature; the
stereochemistry of their hydroxyl groups on the piperidine ring cor-
respond to C2–C4 of -fucose. Fleet et al. synthesized 1-deoxyfuc-
onojirimycin (DFJ) from methyl -glucopyranoside and reported
that it is a very powerful competitive inhibitor of bovine epididymis
-fucosidase with a K
value of 4.8 nM18 and some other potent
-fucosidase inhibitors have been designed and synthesized to
lM.
1
7
a-L
L
-alanine, which was converted to the
2
5
a-L
known alcohol (3) by the literature procedure. Treatment of 3
with MOMCl and Hünig base, and then the cross-metathesis reac-
tion of the resulting ether (4) with ethyl acryrate in the presence of
Hoveyda–Grubbs catalyst 2nd (5 mol %) provided the unsaturated
ester (5). Reduction of 5 followed by protection of the resulting
alcohol as the TBS ether afforded 7. Dihydroxylation of 7 gave rise
a
-D
a-
L
i
a-
L
1
9–22
date.
However, almost all these previous studies used a limited
2
6
range of enzyme sources such as commercially available bovine kid-
ney -fucosidase.
As part of our research program aimed at developing new po-
tent glycosidase inhibitors, we reported that -1-C-alkylated deriv-
atives of 1-deoxynojirimycin (DNJ) are more active and selective
towards b-glucocerebrosidase than DNJ or its N-alkylated deriva-
tives. Furthermore,
itol ( -1-C-butyl-LAB) dramatically improved its inhibition against
intestinal maltase and sucrase. This is a remarkable increase in
activity related to an alkyl chain at the C-1 position and, thus, in
connection with our recent work concerning the design and syn-
thesis of 1-C-alkylated iminosugars as potent glucosidase inhibi-
to the single diol (8), which was converted to the mesylate (11)
via the ether (9) and alcohol (10) in good overall yield. A cycliza-
tion reaction was performed by treatment of 11 with NaH to afford
the desired piperidine (12). Electronic oxidation of 12 gave the
methoxy derivative (13), which was treated with allytrimethylsi-
a
-L
a
lane in the presence of BF
3
ꢀEt
2
O to provide the allyl-substituted
2
3
2
7
a-1-C-butyl-1,4-dideoxy-1,4-imino-L-arabin-
piperidine (14) as a single isomer. The stereochemistry of 14
was determined by the NOE experiment of 18d as shown in
Scheme 1. The Lemieux–Johnson oxidation of 14 followed by the
Pinnick oxidation gave rise to the carboxylic acid (15). With the
key intermediate (15) in hand, the stage was now set for the syn-
a
24
thesis of
a variety of iminosugar derivatives. Thus, the
Scheme 1. Reagents and conditions: (a) SOCl
78 to 0 °C (45%); (d) MOMCl, DIPEA, CH Cl , reflux (86%); (e) ethyl acrylate, Hoveyda–Grubbs catalyst 2nd (5 mol %), CH
TBSCl, DMAP, Et N, CH Cl (86%); (h) OsO , NMO, acetone/H O (8:1) (91%); (i) MOMCl, DIPEA, CH Cl , reflux (91%); (j) TBAF, THF (86%); (k) MsCl, Et
NaI, THF (89%); (m) ꢁ2e, Et NBF , 100 mA, MeOH/MeCN (4:1), ꢁ15 °C (62%); (n) allyltrimethylsilane, BF ꢀEt O, CH Cl , ꢁ78 °C (69%); (o) OsO , NaIO
dioxane/H O (3:1); (p) NaH PO O, 2-methyl-2-butene, NaClO , t-BuOH/H O (3:1) (73%, two steps); (q) R-NH , EDC, CH Cl ; (r) 10% HCl (aq), THF, 40 °C; (s) H
ꢀ2H
Pd(OH) , MeOH.
2
, MeOH, reflux; (b) CbzCl, NaHCO
3
, EtOAc/H
2
O (1:1) (92%, two steps); (c) DIBAL, CH
Cl , reflux (98%); (f) DIBAL, CH
N, CH
2
Cl
2
, ꢁ78 °C; then vinylmagnesium chloride,
Cl
, ꢁ78 °C (77%); (g)
Cl (94%); (l) NaH,
, 2,6-lutidine, 1,4-
, 20%
ꢁ
2
2
2
2
2
2
3
2
2
4
2
2
2
3
2
2
4
4
3
2
2
2
4
4
2
2
4
2
2
2
2
2
2
2
2

A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
6567
OH
OH
OH
HO
OH
HO
OH
CH
HO
OH
CH
3
4
5
2
1
6
1
N
H
CH3
N
H
3
N
H
3
1
-deoxyfuconijirimycin
DFJ)
2
1
(
a: R = Phenyl
b: R = Benzyl
c: R = p-Methoxyphenyl
d: R = Butyl
OH
e: R = 0-Tolyl
HO
OH
f: R = m-Tolyl
O
g: R = p-Tolyl
R
h: R = 2,4-Dimethylphenyl
N
N
H
CH3
H
i : R = 2,4,6-Trimethylphenyl
j : R = 0-Fluorophenyl
1
8a-n
k: R = m-Fluorophenyl
l : R = p-Fluorophenyl
m: R = 2,4-Difluorophenyl
n: R = 2,4,6-Trifluorophenyl
Figure 1. Structure of DFJ, 1, 2, and 18a–n.
Table 1
Concentration of iminosugars giving 50% inhibition of
a
-L-fucosidases
IC50 (lM)
Bovine kidney
Rat epididymis
Human lysosome
DFJ
1
0.11
1.4
0.34
4.1
0.14
0.89
2
0.19
0.047
0.17
0.043
0.16
0.047
0.040
0.026
0.021
0.22
0.012
0.028
0.030
0.042
0.23
0.43
0.34
1.3
0.17
0.78
0.39
0.17
0.081
0.19
1.5
0.044
0.28
0.10
0.35
3.3
0.086
0.030
0.19
0.026
0.13
0.058
0.030
0.015
0.019
0.20
0.0079
0.031
0.014
0.035
0.18
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8a (R = phenyl)
8b (R = benzyl)
8c (R = p-methoxyphenyl)
8d (R = butyl)
8e (R = O-tolyl)
8f (R = m-tolyl)
8g (R = p-tolyl)
8h (R = 2,4-dimethylphenyl)
8i (R = 2,4,6-trimethylphenyl)
8j (R = O-fluorophenyl)
8k (R = m-fluorophenyl)
8l (R = p-fluorophenyl)
8m (R = 2,4-difluorophenyl)
8n (R = 2,4,6-trifluorophenyl)
condensation reaction of 15 with amine using EDC as the coupling
agent afforded the amides (16a–n) (Fig. 1). Finally, deprotection of
the MOM protecting group by acid (17a–n), and the Cbz group by
hydrogenolysis furnished the desired iminosugars (18a–n) as
shown in Scheme 1.
b-1-C-hexyl-DFJ (1), with IC50 values 0.19, 0.43, and 0.086 lM,
respectively but its inhibition potency was almost the same as par-
ent DFJ. These behaviors were obviously different from our previ-
2
3,24
ous results with
a
-1-C-alkyl-DNJ and
a
-1-C-alkyl-LAB.
0
Moreover, we were intrigued by the fact that b-1-C-(3 -propylphe-
nyl)-DFJ (2) showed a broader inhibition spectrum with an IC50 va-
2
2
.2. Biological results and discussion
lue of 883 lM towards bovine liver b-galactosidase (Table 2). Thus,
we turned out our strategy from introducing a simple n-alkyl chain
at the C-1 position directly to starting from 2b-DFJ acetic acid as a
lead compound to design more flexible variations of substitution
pattern. Consequently, N-phenyl-2b-DFJ acetamide (18a) was
.2.1. Comparison of b-1-C-alkyl-DFJ and N-alkyl-2b-DFJ
acetamide as a-L-fucosidase inhibitors
We first focused on the C-1 position and whether adding the al-
kyl chain at this position could influence the inhibition activities of
found to display more potent inhibition of
the original DFJ, with IC50 values of 0.047, 0.34, and 0.03 l
a
-
L
-fucosidases than
M (Ta-
1
-deoxyfuconojirimycin (DFJ). As shown in Table 1, the basic struc-
ture, DFJ showed potent inhibition towards bovine kidney, rat epi-
didymis, and human lysosome -fucosidases, with IC50 values of
.11, 0.34, and 0.14 M, respectively. Our preliminary results
showed that the introduction of a simple 1-C-alkyl substituent into
DFJ did not enhance their effectiveness as -fucosidase inhibitors
unpublished data). For example, b-1-C-hexyl-DFJ (1) was about 10
times weaker inhibitor than the parent DFJ against these enzymes
IC50 = 1.4, 4.1, and 0.89 M, respectively). Furthermore, b-1-C-(3-
propylphenyl)-DFJ (2) was found to be a much better inhibitor than
ble 1). For comparison, N-butyl-2b-DFJ acetamide (18d) was also
synthesized by the same step from 2b-DFJ acetic acid as shown
in Scheme 1. However, this compound was found to display less
activity than N-phenyl-2b-DFJ acetamide (18a). This result sug-
gested that the terminal phenyl ring changed the inhibition po-
tency. This behavior was similar to that observed with b-1-C-
a-L
0
l
a
-L
(
0
hexyl-DFJ (1) and b-1-C-(3 -propylphenyl)-DFJ (2). In order to
(
l
check if the change from phenyl to benzyl in the amide part had
an influence on the activity towards a-L-fucosidases, we prepared

6
568
A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
Table 2
Concentration of DFJ and derivatives giving 50% inhibition of various glycosidases
Enzyme
IC50 (lM)
DFJ
1
18d
2
18a
a-Glucosidase rat intestinal
NIa (5.7%)b
NI (8.3%)
NI (21.0%)
NI (1.9%)
NI (0%)
maltase
b-Glucosidase bovine liver
-Glucosidase coffee beans
b-Glucosidase bovine liver
-Mannosidase Jack beans
b-Mannosidase snail
NI (2.6%)
NI (5.2%)
NI (3.7%)
NI (4.9%)
NI (2.3%)
NI (37.8%)
NI (9.8%)
NI (49.7%)
NI (11.3%)
NI (0%)
NI (28.6%)
NI (6.6%)
NI (45.1%)
NI (0%)
NI (0%)
0.16
NI (41.3%)
NI (3.7%)
883
NI (9.6%)
NI (0%)
0.19
754
NI (6.7%)
299
NI (9.2%)
NI (0%)
0.047
a
a
a-
L
-Fucosidase bovine kidney 0.11
1.4
b-Glucuronidase E. coli NI (7.4%)
NI (19.8%)
NI (19.4%)
NI (26.8%)
NI (20.9%)
a
NI: no inhibition (less than 50% inhibition at 1000 lM).
b
(
): Inhibition % at 1000 lM.
N-benzyl-2b-DFJ acetamide (18b). This comparison revealed that
the inhibitory activity of this compound against -fucosidases
This compound inhibited bovine kidney
a
-
L
-fucosidase in a com-
a-L
petitive manner, with K value of 0.0021 lM. It is noteworthy that
i
was even weaker than that of N-phenyl-2b-DFJ acetamide. For
example, N-benzyl-2b-DFJ acetamide (18b) is 3.6-, 3.8- and 6.3-
fold less active on bovine kidney, rat epididymis, and human lyso-
some fucosidases than the corresponding N-phenyl-2b-DFJ acet-
amide (18a) (Table 1). On the basis of our results with the DFJ
series, it clearly appeared that the addition of the N-phenylacet-
amide unit to the C-1 position of DFJ might lead to highly potent
this compound 18j dramatically changed not only the inhibition
potency but also the inhibition profile changed. The compound
without fluoride 18a showed a broader inhibition spectrum includ-
ing activity towards bovine liver b-glucosidase and b-galactosi-
dase, with IC50 values of 754 and 299 lM (Table 2). In sharp
contrast, N-(2-fluorophenyl)-2b-DFJ acetamide (18j) exhibited less
than a 50% inhibition of bovine liver b-glucosidase and b-galactosi-
inhibitors of
a
-
L
-fucosidases.
dase even at concentrations as high as 1000
to clarify the structural basis of the interaction of N-(2-fluoro-
phenyl)-2b-DFJ acetamide (18j) with -fucosidase, N-(3-fluoro-
lM (Table 3). In order
2
.2.2. Effect of fluoro and methyl groups on the phenyl ring of
a-L
N-phenyl-2b-DFJ acetamide on the -fucosidase inhibition
For comparison, we next investigated whether the introduction
of fluoro or methyl groups on the phenyl ring had an influence on
a
-
L
phenyl)-2b-DFJ acetamide (18k) and N-(4-fluorophenyl)-2b-DFJ
acetamide (18l) were also tested with the hope of finding more po-
tent inhibitors. However, we found that the inhibitory potency of
the inhibition of
a
-L
-fucosidases and various glycosidases (Tables 1
compounds 18k and 18l against all a-L-fucosidase were much
and 3). N-(2-Fluorophenyl)-2b-DFJ acetamide (18j) showed an in-
weaker than that for compound 18j. Especially, the 3-fluoro deriv-
creased affinity for the bovine kidney, rat epididymis, and human
ative (18k) clearly showed decreased inhibition potency against rat
a-
L-fucosidases, with IC50 values of 0.012, 0.044, and 0.0079
l
M.
epididymis a-L-fucosidase (Table 1). It should be noted that the
Table 3
Concentration of N-phenyl-2b-DFJ derivatives giving 50% inhibition of various glycosidases
Enzyme
IC50 (lM)
18j
18e
18m
18h
18n
18i
a
-Glucosidase rat
intestinal maltase
NIa (21.7%)b
NI (34.3%)
NI (0.3%)
NI (49.5%)
NI (0%)
NI (0.3%)
NI (0%)
NI (0%)
NI (19.8%)
NI (0%)
798
NI (0%)
NI (9.0%)
b-Glucosidase bovine
7.1
NI (48.7%)
NI (0%)
346
NI (7.4%)
NI (0%)
NI (0%)
liver
a-Glucosidase coffee
NI (0.8%)
8.2
NI (0%)
beans
b-Glucosidase bovine
NI (42.6%)
NI (0%)
NI (29.1%)
NI (11.3%)
liver
a-Mannosidase Jack
NI (6.9%)
NI (11.0%)
NI (0%)
beans
b-Mannosidase snail
NI (4.8%)
0.012
NI (11.4%)
0.047
NI (24.3%)
0.042
NI (12.5%)
0.021
NI (0%)
0.23
588
0.22
a-L-Fucosidase bovine
kidney
b-Glucuronidase E. coli
NI (39.6%)
796
NI (30.9%)
NI (44.4%)
NI (0%)
NI (21.5%)
a
NI: no inhibition (less than 50% inhibition at 1000 lM).
b
(
): Inhibition % at 1000 lM.

A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
6569
suitable position of methyl groups was obviously different from
addition of fluoride. The most effective derivative was N-(4-to-
lyl)-2b-DFJ acetamide (18g) and it showed potent inhibition
for the enzyme than the parent DFJ. These results indicate that N-
(2-fluorophenyl)-2b-DFJ acetamide (18j) has the potential to be a
specific pharmacological chaperone of fucosidosis.
against bovine kidney, rat epididymis, and human lysosome
a-L-
fucosidases, with IC50 values 0.026, 0.081, and 0.015 M, respec-
tively. It clearly appeared that the addition of mono-fluoro or
mono-methyl groups on the phenyl ring of N-phenyl-2b-DFJ acet-
amide (18a) might lead to highly potent inhibitors of a-L-fucosid-
ases. Thus, we next focused on the number of fluoro or methyl
groups on the phenylacetamide and whether increasing the num-
ber could influence the inhibition activities. As shown in Table 3,
the inhibitory potency of N-phenyl-2b-DFJ acetamide (18a) deriv-
l
4
4
4
. Experimental
.1. Chemistry
.1.1. General information
Flash chromatography was performed with Kanto Kagaku silica
gel 60 N (63–210 mm). NMR spectra were recorded on a JEOL JNM-
A400 or JEOL JNM-ECX500 spectrometer in the solvent indicated.
Chemical shifts (d) are given in ppm downfield from TMS and ref-
atives against
a-L-fucosidase obviously decreased with increasing
the number of fluoro groups. For example, the tri-fluoro derivative
compound 18n was 19-times weaker inhibitor than mono-fluoro
3
erenced with CHCl (7.26 ppm) as an internal standard. Peak mul-
tiplicities are designated by the following abbreviations: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad and cou-
pling constants are given in (J) Hz. High resolution mass spectral
data was obtained on a JEOL MStation JMS-700. All commercial re-
agents were used as received unless otherwise noted.
derivative compound 18j against bovine kidney
Moreover, compound 18m showed activity on bovine liver b-galac-
tosidase, with an IC50 value of 346 M. On the other hand, the rela-
a-L-fucosidase.
l
tionship of the number of methyl groups and inhibition potency
was more complex. The di-methyl compound 18h was a 2.2-fold
better inhibitor than the mono-methyl compound 18e against bo-
vine kidney a-L-fucosidase, whereas the tri-methyl compound 18i
was a 4.7-times weaker inhibitor than compound 18e. It should be
4
1
.1.2. (3S,4S)-4-Benzyloxycarbonylamino-3-methoxymethoxy-
-pentene (4)
N-Ethyldiisopropylamine (3.00 mL, 17.4 mmol) and chloro-
noted that the introduction of two or three methyl groups drasti-
methyl methyl ether (1.09 mL, 13.2 mmol) were added to a solu-
tion of 3 (1.35 g, 5.74 mmol) in CH Cl (10 mL) at 0 °C. The
stirred reaction mixture was refluxed for 15 h. After cooling, the
volatiles were removed in vacuo to give a yellow oil, which was
chromatographed on silica gel (20 g, n-hexane/acetone 15:1) to
give 4 (1.391 g, 4.98 mmol, 87%) as a pale yellow oil. 1H NMR
cally improves their specificity as
pound 18e was a potent inhibitor of bovine liver b-glucosidase
and b-galactosidase, with IC50 values of 7.1 and 8.2 M. In contrast,
a-L-fucosidase inhibitors. Com-
2
2
l
the di-methyl compound 18h showed over 100-fold weaker inhibi-
tion than compound 18e against these b-glycosidases. Further-
more, the tri-methyl compound 18i completely lost the inhibition.
(
500 MHz, CDCl
0.1, 6.7 Hz), 5.27 (1H, dd, J = 13.7, 0.7 Hz), 5.26 (1H, dd, J = 10.1,
0.7 Hz), 5.10 (2H, s), 4.94 (1H, br), 4.53, 4.68 (2H, ABq, J = 6.6 Hz),
3
) d: 7.38–7.29 (5H, m), 5.72 (1H, ddd, J = 13.7,
1
3
. Conclusion
3
.98 (1H, dd, J = 6.7, 3.1 Hz), 3.89 (1H, br), 3.36 (3H, s), 1.22 (3H,
1
3
In conclusion, we report on the design and synthesis of a series
d, J = 6.8 Hz);
3
C NMR (125 MHz, CDCl ) d 155.99, 137.18,
of N-phenyl-2b-DFJ acetamide derivatives by starting from 2b-DFJ
acetic acid as a lead compound to design more flexible variations of
substitution pattern. Among the synthesized compounds, N-(2-
fluorophenyl)-2b-DFJ acetamide (18j) showed potent inhibitory
activity toward bovine kidney, rat epididymis, and human lyso-
137.03, 128.46, 128.02, 118.15, 117.84, 92.30, 76.74, 66.55, 55.64,
ꢁ1
+
53.29, 16.33; IR (neat): 3334, 1712 cm ; MS (EI): m/z 279 (M );
2
D
6
HRMS (EI) Calcd for C15
+45.6 (c 1.00, CHCl ).
H
21
O
4
N 279.1471; Found 279.1508; ½ ꢂ
a
3
some
.0079
show any significant inhibition activity against other glycosidases.
This study revealed that the catalytic site of -fucosidases strictly
a
-
L
-fucosidases, with IC50 values of 0.012, 0.044, and
4.1.3. Ethyl (2E,4S,5S)-5-benzyloxycarbonylamino-4-
methoxymethoxy-2-hexenoate (5)
Hoveyda–Grubbs catalyst, 2nd generation (7.0 mg, 10.5 lmol)
and ethyl acrylate (0.190 mL, 1.79 mmol) were added to a solution
0
lM, respectively. Furthermore, this compound did not
a
-L
recognized the fluoro or methyl groups on the phenylacetamide.
of 4 (100 mg, 0.356 mmol) in CH Cl (7 mL) at room temperature,
2
2
Consequently, the number and position influence on the inhibition
and the stirred reaction mixture was refluxed for 15 h. After cool-
ing, the volatiles were removed in vacuo to give a black oil, which
was chromatographed on silica gel (10 g, n-hexane/acetone 10:1)
to give 5 (122 mg, 0.347 mmol, 98%) as a pale yellow oil. H NMR
3
(400 MHz, CDCl ) d: 7.37–7.32 (5H, m), 6.81 (1H, dd, J = 15.8,
activity and selectivity against
associated with many diseases. However, almost all previous re-
ported studies on -fucosidase inhibitor design were using very
limited enzyme sources such as only commercially available bo-
vine kidney -fucosidase. In this study, therefore, we prepared
also -fucosidase from rat epididymis and human lysosome.
Our designed compounds especially inhibited human lysosome
-fucosidase. This enzyme is closely related with fucosidosis.
a-L-fucosidases. a-L-Fucosidases are
1
a-
L
a-
L
6.4 Hz), 6.01 (1H, d, J = 15.8 Hz), 5.09 (2H, s), 4.89 (1H, br), 4.59,
4.63 (2H, ABq, J = 7.0 Hz), 4.20 (2H, q, J = 7.1 Hz), 3.95 (1H, br),
3.75 (1H, dd, J = 6.6, 6.4 Hz), 3.37 (3H, s), 1.29 (3H, t, J = 7.1 Hz),
a-L
1
3
a-
L
3
1.22 (3H, d, J = 6.6 Hz); C NMR (100 MHz, CDCl ) d: 165.57,
Fucosidosis is a lysosomal storage disorder caused by the FUCA1
gene mutations that reduce or cause a deficiency of activity of acid
155.63, 144.03, 136.31, 128.33, 127.91, 127.87, 123.74, 94.79,
77.12, 66.50, 60.38, 55.72, 49.59, 17.20, 14.01; IR (neat): 3337,
ꢁ1
+
a-
L-fucosidase. Deficiency of the enzyme activity results in the pro-
1718, 1700 cm ; MS (EI): m/z 351 (M ); HRMS (EI) Calcd for
2
1
gressive accumulation of a variety of glycoproteins, glycolipids,
and oligosaccharides that contain fucose residue in the lysosomes
of vascular endothelial cells. Previous reports suggested that mu-
C
18
25
H O
6
N 351.1682; Found 351.1665; ½
aꢂ
D
3
+19.4 (c 1.00, CHCl ).
4.1.4. (2E,4S,5S)-5-Benzyloxycarbonylamino-4-
tated
with normal enzyme, except for thermostability. As in previous
pharmacological chaperone studies, potent and selective -fuco-
sidase inhibitors might also stabilize a mutant -fucosidase in
cells of fucosidosis patients. This study revealed that our designed
N-(2-fluorophenyl)-2b-DFJ acetamide had a much stronger affinity
a
-
L
-fucosidases of fucosidosis had similar kinetic properties
methoxymethoxy-2-hexen-1-ol (6)
A solution of DIBAL (1.0 M in n-hexane, 20.5 mL, 20.5 mmol,) at
ꢁ78 °C was added to a solution of 5 (1.60 g, 4.56 mmol) in CH Cl
(16 mL), and the reaction mixture was stirred at the same temper-
ature for 2 h. The reaction was quenched with sat. NH Cl (aq) and
then the insoluble materials were filtered off with a Celite pad. The
a-
L
2
2
a
-L
4

6
570
A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
organic layer was separated, dried over anhydrous Na
evaporated to give a pale yellow oil, which was chromatographed
2
SO
4
and
66.15, 62.63, 55.86, 55.42, 46.72, 25.60, 18.53, 17.92, 13.86,
ꢁ1
+
ꢁ5.74; IR (neat): 3338, 1719 cm ; MS (EI): m/z 545 (M ); HRMS
22
on silica gel (20 g, n-hexane/acetone 3:1) to give 6 (1.09 g,
(EI) Calcd for C26
(c 1.00, CHCl ).
H
47
O
9
NSi 545.3020; Found 545.3014; ½
aꢂ
+3.0
D
3
7
.52 mmol, 77%) as a colorless oil. 1H NMR (400 MHz, CDCl
3
) d:
.39–7.30 (5H, m), 5.87 (1H, dt, J = 14.7, 5.2 Hz), 5.61 (1H, dd,
3
J = 14.7, 6.8 Hz), 5.09 (2H, s), 4.96 (1H, br), 4.52, 4.67 (2H, ABq,
J = 6.8 Hz), 4.13 (2H, d, J = 5.2 Hz), 4.03 (1H, br), 3.88 (1H, br),
4.1.8. (2R,3R,4R,5S)-5-Benzyloxycarbonylamino-2,3,4-metho-
xymethoxyhexan-1-ol (10)
3
1
7
1
C
.36 (3H, s), 1.23 (3H, d, J = 6.8 Hz); 13C NMR (100 MHz, CDCl
3
) d:
A solution of tetrabutylammonium fluoride (1.0 M in THF,
5.70 mL, 5.70 mmol) was added to a solution of 9 (1.55 g,
3.39 mmol) in THF (10 mL) at 0 °C, and the reaction mixture was
stirred at room temperature for 1 h. After the reaction was com-
pleted, brine (4 mL) was added, and then the aqueous mixture
56.09, 136.42, 134.35, 128.38, 127.98, 127.92, 127.14, 93.64,
8.07, 66.55, 62.27, 55.53, 50.25, 17.69; IR (neat): 3339,
ꢁ
1
+
702 cm
; MS (EI): m/z 309 (M ); HRMS (EI) Calcd for
21
16
H
23
O
5
N 309.1576; Found 309.1614; ½
a
ꢂ
D
3
+44.7 (c 1.00, CHCl ).
was extracted with CH
combined, dried over anhydrous Na
2
Cl
2
(10 mL ꢃ 3). The organic layers were
4
.1.5. (2E,4S,5S)-5-Benzyloxycarbonylamino-1-tert-
2
SO and evaporated to give
4
butyldimethylsilyloxy-4-methoxymethoxy-2-hexene (7)
tert-Butyldimethylchlorosilane (0.935 g, 6.20 mmol), DMAP
0.126 g, 1.03 mmol) and Et N (1.10 mL, 7.83 mmol) were added
to a solution of 6 (1.07 g, 3.46 mmol) in CH Cl (15 mL) at 0 °C,
and the reaction mixture was stirred at room temperature for
5 h. The volatiles were removed in vacuo to give a yellow oil,
which was chromatographed on silica gel (20 g, n-hexane/acetone
10 as a yellow oil, which was chromatographed on silica gel
(20 g, n-hexane/acetone 3:1) to give 10 (1.38 g, 3.20 mmol, 94%)
1
(
3
as a colorless oil. H NMR (400 MHz, CDCl
3
) d: 7.36–7.31 (5H, m),
2
2
5.09 (2H, s), 4.75–4.65 (6H, m), 4.06 (1H, dd, J = 7.6, 7.5 Hz), 3.83
(2H, br), 3.75–3.66 (3H, m), 3.53 (1H, br), 3.45 (3H, s), 3.43 (3H,
1
3
1
3
s), 3.40 (3H, s), 1.24 (3H, d, J = 6.8 Hz); C NMR (100 MHz, CDCl )
d: 155.62, 136.37, 128.32, 127.94, 127.94, 98.62, 98.43, 97.56,
1
2
0:1) to give 7 (1.43 g, 3.38 mmol, 98%) as a pale yellow oil.
NMR (400 MHz, CDCl ) d: 7.36–7.30 (5H, m), 5.79 (1H, dt,
J = 14.4, 3.4 Hz), 5.58 (1H, dd, J = 14.4, 7.2 Hz), 5.09 (2H, s), 4.93
1H, br), 4.50, 4.68 (2H, ABq, J = 6.4 Hz), 4.17 (2H, d, J = 3.4 Hz),
H
80.96, 80.17, 78.05, 66.46, 62.75, 56.28, 56.14, 55.70, 46.30,
ꢁ
1
+
3
19.10; IR (neat): 3436, 1715 cm ; MS (FAB): m/z 432 (M +1);
HRMS (FAB) Calcd for C20 N 432.2234; Found 432.2234;
34 9
H O
2
3
(
½
aꢂ
ꢁ4.4 (c 1.00, CHCl
3
).
D
4
0
1
6
3
H
.00 (1H, br), 3.85 (1H, br), 3.35 (3H, s), 1.21 (3H, d, J = 6.6 Hz),
.91 (9H,s), 0.05 (6H, s); 13C NMR (100 MHz, CDCl
) d: 155.83,
4.1.9. (2R,3R,4R,5S)-5-Benzyloxycarbonylamino-1-methy-
lsulfonyloxy-2,3,4-methoxymethoxyhexane (11)
3
36.57, 134.89, 128.40, 128.01, 127.96, 125.74, 93.57, 78.13,
6.48, 62.76, 55.54, 50.26, 25.81, 18.27, 17.76, ꢁ5.33; IR (neat):
3
Methanesulfonyl chloride (0.40 mL, 5.15 mmol) and Et N
ꢁ
1
+
337, 1718 cm ; MS (EI): m/z 423 (M ); HRMS (EI) Calcd for C22-
(0.98 mL, 6.97 mmol) were added to a solution of 10 (1.14 g,
2.65 mmol) in CH Cl (15 mL) at 0 °C, and the reaction mixture
was stirred at room temperature for 15 h. After the reaction was
completed, sat. NaHCO (aq) (4 mL) was added, and then the aque-
ous mixture was extracted with CH Cl
(10 mL ꢃ 3). The organic
layers were combined, dried over anhydrous Na SO and evapo-
21
37
O
5
NSi 423.2411; Found 423.2435; ½
a
ꢂ
D
+36.6 (c 1.00, CHCl
3
).
2
2
4
.1.6. (2R,3S,4R,5S)-5-Benzyloxycarbonylamino-1-tert-
3
butyldimethylsilyloxy-4-methoxymethoxyhexane-2,3-diol (8)
NMO (0.782 g, 6.68 mmol) and OsO (2.0 mL, 0.171 mmol, 2% in
O) were added to a solution of 7 (1.41 g, 3.33 mmol) in acetone
16 mL) at 0 °C, and the reaction mixture was stirred at room tem-
perature for 15 h. After the reaction was completed, 10% Na
aq) (4 mL) was added, and then the aqueous mixture was ex-
tracted with CH Cl
(10 mL ꢃ 3). The organic layers were com-
bined, dried over anhydrous Na SO and evaporated to give a
2
2
4
2
4
H
2
rated to give 11 as a yellow oil, which was chromatographed on sil-
(
ica gel (20 g, n-hexane/acetone 3:1) to give 11 (1.27 g, 2.50 mmol,
1
2
2
S O
3
94%) as a pale yellow oil. H NMR (400 MHz, CDCl
3
) d: 7.37–7.31
(
(5H, m), 5.09 (2H, s), 4.79–4.67 (6H, m), 4.49–4.40 (2H, m), 4.08
(2H, br), 3.65 (2H, br), 3.42 (3H, s), 3.40 (3H, s), 3.39 (3H, s), 3.03
2
2
1
3
2
4
3
(3H, s), 1.24 (3H, d, J = 7.0 Hz); C NMR (100 MHz, CDCl ) d:
yellow oil, which was chromatographed on silica gel (20 g, n-hex-
155.69, 136.45, 128.40, 128.03, 128.02, 98.59, 98.45, 97.64, 80.95,
77.62, 75.27, 68.93, 66.54, 56.25, 56.13, 55.92, 46.59, 37.16,
ane/acetone 10:1) to give 8 (1.39 g, 3.04 mmol, 91%) as a colorless
1
ꢁ1
+
oil. H NMR (400 MHz, CDCl
3
) d: 7.37–7.31 (5H, m), 5.05, 5.13 (2H,
19.02; IR (neat): 3397, 1707 cm ; MS (FAB): m/z 510 (M +1);
HRMS (FAB) Calcd for C21 11NS 510.2009; Found 510.2012;
ABq, J = 12 Hz), 4.70, 4.74 (2H, ABq, J = 6.4 Hz), 4.18 (1H, br), 3.79
36
H O
2
D
2
(
(
3H, br), 3.62 (2H, br), 3.41 (3H, s), 1.25 (3H, d, J = 6.8 Hz), 0.90
½
aꢂ
ꢁ10.4 (c 1.00, CHCl
3
).
13
9H, s), 0.09 (6H, s);
3
C NMR (100 MHz, CDCl ) d: 156.45,
1
6
36.25, 128.29, 127.94, 127.92, 97.96, 79.96, 70.19, 69.02, 66.64,
4.1.10. (2S,3R,4S,5R)-2-Methyl-3,4,5-trimethoxymethoxy-
piperidine-1-carboxylic acid benzyl ester (12)
NaH (0.497 g, 12.4 mmol, 60% dispersion in paraffin liquid) and
NaI (0.746 g, 4.97 mmol) were added to a solution of 11 (1.27 g,
5.33, 56.12, 46.91, 25.69, 18.03, 17.25, ꢁ5.61; IR (neat): 3417,
ꢁ1
+
1
700 cm ; MS (EI): m/z 457 (M ); HRMS (EI) Calcd for C22
H
39
O
7-
2
D
2
NSi 457.2496; Found 457.2468; ½
a
ꢂ
ꢁ22.6 (c 1.00, CHCl
3
).
2
.50 mmol) in THF (20 mL) at 0 °C, and the reaction mixture was
stirred at room temperature for 20 h. After the reaction was com-
pleted, sat. NaHCO (aq) (6 mL) was added, and then the aqueous
mixture was extracted with CH Cl
(10 mL ꢃ 3). The organic layers
were combined, dried over anhydrous Na SO and evaporated to
4
.1.7. (2R,3R,4R,5S)-5-Benzyloxycarbonylamino-1-tert-
butyldimethylsilyloxy-2,3,4-methoxymethoxyhexane (9)
3
N-Ethyldiisopropylamine (2.10 mL, 12.2 mmol) and chloro-
2
2
methyl methyl ether (0.80 mL, 9.69 mmol) were added to a solu-
2
4
tion of 8 (1.37 g, 3.00 mmol) in CH
2
Cl
2
(15 mL) at 0 °C. The
give 12 as a yellow oil, which was chromatographed on silica gel
stirred reaction mixture was refluxed for 15 h. After cooling, the
volatiles were removed in vacuo to give a yellow oil, which was
chromatographed on silica gel (20 g, n-hexane/acetone 15:1) to
give 9 (1.57 g, 2.88 mmol, 96%) as a pale yellow oil. 1H NMR
(20 g, n-hexane/acetone 15:1) to give 12 (0.940 g, 2.28 mmol,
1
91%) as a pale yellow oil. H NMR (400 MHz, CDCl
3
) d: 7.35–7.27
(5H, m), 5.19, 5.09 (2H, ABq, J = 12.4 Hz), 4.80–4.67 (6H, m), 4.56
(2H, br), 4.09 (1H, br), 3.97–3.91 (2H, m), 3.83 (1H, br), 3.40 (3H,
s), 3.39 (3H, s), 3.38 (3H, s), 1.29 (3H, d, J = 7.1 Hz); 13C NMR
(
(
(
3
400 MHz, CDCl ) d: 7.35–7.30 (5H, m), 5.09 (2H, s), 4.79–4.66
6H, m), 4.09 (1H, br), 3.82–3.76 (3H, m), 3.74–3.68 (2H, m), 3.39
3
(100 MHz, CDCl ) d: 155.67, 136.74, 128.36, 127.84, 127.68,
3H, s), 3.38 (3H, s), 3.37 (3H, s), 1.25 (3H, d, J = 6.3 Hz), 0.89 (9H,
97.10, 95.43, 95.11, 75.74, 72.98, 71.52, 67.05, 55.61, 55.53,
1
3
ꢁ1
s), 0.06 (6H, s); C NMR (100 MHz, CDCl
3
) d: 155.56, 136.60,
55.52, 49.37, 37.74, 12.41; IR (neat): 1700 cm ; MS (EI): m/z
1
27.76, 127.70,128.17, 98.34, 97.60, 97.30, 80.03, 77.90, 77.25,

A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
6571
+
4
4
13 (M ); HRMS (EI) Calcd for C20
H
31
O
8
N 413.2050; Found
10.2 Hz), 2.70 (1H, dd, J = 14.2, 4.6 Hz), 1.35 (3H, d, J = 7.1 Hz);
2
2
13
13.2023; ½
a
ꢂ
ꢁ34.6 (c 1.00, CHCl
3
).
C NMR (100 MHz, CDCl
27.87, 127.59, 96.74, 95.60, 95.50, 76.74, 75.38, 71.15, 67.37,
55.68, 55.61, 55.56, 51.35, 37.72, 29.10, 15.96; IR (neat): 3485,
3
) d: 175.56, 155.70, 136.44, 128.38,
D
1
4
.1.11. (3S,4R,5R,6S)-2-Methoxy-6-methyl-3,4,5-
trimethoxymethoxypiperidine-1-carboxylic acid benzyl ester
ꢁ
1
+
1732, 1695 cm ; MS (EI): m/z 471 (M ); HRMS (EI) Calcd for
2
4
(
13)
Et
0.300 g, 0.73 mmol) in a 4:1 MeCN/MeOH mixture (20 mL) and
C
22
33
H O
10N 471.2104; Found 471.2105; ½
aꢂ
ꢁ16.4 (c 0.50, CHCl
3
).
D
4
4
NBF (0.400 g, 1.84 mmol) was added to a solution of 12
(
4.1.14. General procedure for the synthesis of amides (16a–n)
the reaction mixture was stirred at ꢁ15 °C for 4.5 h with 100 mA
electricity passed through by use of graphite anode and cathode
electrodes. Then the reaction solvent was removed to give 13 as
a yellow oil, which was chromatographed on silica gel (10 g, n-hex-
EDC (2 equiv), DMAP (0.1 equiv) and amine (1 equiv) were
added to a solution of 15 in CH Cl at room temperature, and the
reaction mixture was stirred at the same temperature for 15 h.
After the reaction was completed, the volatiles were removed in
vacuo, and the residue was chromatographed on silica gel to give
the amide (16).
2
2
ane/acetone 10:1) to give 13 (0.200 g, 0.45 mmol, 62%) as a pale
1
yellow oil. H NMR (400 MHz, CDCl
3
) d: 7.39–7.33 (5H, m), 5.19
(
(
2H, s), 4.78–4.66 (6H, m), 4.52 (1H, br), 4.09 (1H, br), 3.99-3.90
2H, m), 3.83 (1H, br), 3.40–3.38 (9H, m), 3.32 (3H, s), 1.43 (3H,
4.1.15. (2R,3R,4R,5R,6S)-2-[(2-Fluorophenylcarbamoyl)methyl]-
d, J = 7.0 Hz).
6-methyl-3,4,5-trimethoxymethoxypiperidine-1-carboxylic
acid benzyl ester (R = o-fluorophenyl, 16j)
1
4
.1.12. (2R,3R,4R,5R,6S)-2-Allyl-6-methyl-3,4,5-
trimethoxymethoxypiperidine-1-carboxylic acid benzyl ester
14)
Allyltrimethylsilane (0.46 mL, 2.9 mmol) and BF
.80 mmol) were added to a solution of 13 (0.335 g, 0.76 mmol) in
CH Cl
(4 mL) at ꢁ78 °C, and the reaction mixture was stirred at
the same temperature for 5 h. After the reaction was completed,
sat. NaHCO (aq) (6 mL) was added, and then the aqueous mixture
was extracted with CH Cl
(5 mL ꢃ 3). The organic layers were
combined, dried over anhydrous Na SO and evaporated to give
4 as a yellow oil, which was chromatographed on silica gel
10 g, n-hexane/acetone 15:1) to give 14 (0.238 g, 0.53 mmol,
0%) as a pale yellow oil. 1H NMR (400 MHz, CDCl
) d: 7.39–7.30
Yield 63%; H NMR (500 MHz, CDCl
3
) d: 8.23 (1H, d, J = 8.0 Hz),
7.35–7.28 (5H, m), 7.13–7.04 (3H, m), 5.21, 5.13 (2H, ABq,
J = 12.5 Hz), 4.79–4.65 (7H, m), 4.53 (1H, br), 4.08–4.01 (3H, m),
3.41 (3H, s), 3.40 (3H, s), 3.32 (3H, s), 3.20 (1H, dd, J = 14.5,
10.0 Hz), 2.75–2.72 (1H, br), 1.39 (3H, d, J = 7.5 Hz); 13C NMR
(
3
ꢀEt
2
O (0.10 mL,
0
2
2
3
(125 MHz, CDCl ) d: 168.88, 155.79, 152.52 (d, J = 241.0 Hz),
136.35, 128.34, 127.85, 127.64, 126.10, 126.02, 124.38, 122.21,
114.72 (d, J = 19.38 Hz), 96.77, 96.26, 95.50, 75.59, 75.29, 71.13,
67.36, 55.67, 55.62, 55.61, 52.54, 49.31, 16.06; IR (neat): 3315,
3
2
2
ꢁ
1
+
2
4
1695, 1684 cm ; MS (EI): m/z 564 (M ); HRMS (EI) Calcd for C28-
2
1
1
(
7
H
37
9
O N
2
F 564.2483; Found 564.2470; ½
aꢂ
D
3
+5.5 (c 0.50, CHCl ).
3
4.1.16. General procedure for the synthesis of triols (17a–n)
10% HCl (aq) was added to a solution of 16 in THF at room tem-
perature, and the reaction mixture was stirred at 40 °C for 48 h.
(
(
5H, m), 5.78 (1H, br), 5.17–5.02 (4H, m), 4.77–4.64 (6H, m), 4.51
1H, br), 4.38 (1H, br), 4.03 (1H, dd, J = 6.8, 2.3 Hz), 3.92 (1H, br),
3
.87 (1H, br), 3.40 (9H, s), 2.72–2.51 (2H, m), 1.37 (3H, d,
After the reaction was completed, NaHCO
then the aqueous mixture was extracted with AcOEt (5 mL ꢃ 3).
The organic layers were combined, dried over anhydrous Na SO
and evaporated to give the residue, which was chromatographed
on silica gel to give 17.
3
(aq) was added, and
1
3
J = 6.8 Hz); C NMR (100 MHz, CDCl
1
7
3
) d: 155.81, 136.71, 135.54,
28.35, 127.80, 127.65, 117.33, 96.48, 95.90, 95.67, 75.56, 75.54,
1.75, 67.11, 55.69, 55.56, 55.54, 49.63, 38.30, 29.19, 16.02; IR
2
4
,
ꢁ
1
+
(
neat): 1696 cm ; MS (EI): m/z 453 (M ); HRMS (EI) Calcd for
2
D
3
C
23
H
35
O
8
N 453.2363; Found 453.2358; ½
a
ꢂ
ꢁ8.7 (c 0.50, CHCl
3
).
4
.1.17. (2R,3R,4R,5R,6S)-2-[(2-Fluorophenylcarbamoyl)methyl]-
4
.1.13. (2R,3R,4R,5R,6S)-2-Carboxymethyl-6-methyl-3,4,5-
6-methyl-3,4,5-trihydroxypiperidine-1-carboxylic acid benzyl
trimethoxymethoxy-piperidine-1-carboxylic acid benzyl ester
ester (R = o-fluorophenyl, 17j)
1
(
15)
Yield 82%; H NMR (500 MHz, CDCl
3
) d: 8.19 (1H, t, J = 8.0 Hz),
2
,6-Lutidine (0.030 mL, 0.26 mmol), NaIO
(0.20 mL, 0.017 mmol, 2% in H O) were added to a solu-
tion of 14 (0.070 g, 0.15 mmol) in a 3:1 1,4-dioxane/H O mixture
4 mL) at 0 °C, and the resulting mixture was stirred at room tem-
perature for 2 h. After the reaction was completed, 10% Na
aq) (1 mL) was added, and then the aqueous mixture was ex-
tracted with AcOEt (5 mL ꢃ 3). The organic layers were combined,
washed with 10% HCl (aq), dried over anhydrous Na SO , and evap-
4
(0.132 g, 0.62 mmol)
7.35–7.29 (5H, m), 7.13–7.05 (3H, m), 5.15 (2H, s), 4.62 (1H, dd,
J = 7.3,3.0 Hz), 4.36 (1H, t, J = 7.3 Hz), 4.16 (1H, br), 4.07 (1H, br),
3.78 (1H, br), 3.22 (1H, dd, J = 15.5, 11.0 Hz), 2.73 (1H, dd,
and OsO
4
2
2
1
3
(
3
J = 15.5, 3.5 Hz), 1.32 (3H, d, J = 7.0 Hz); C NMR (125 MHz, CDCl )
2
2
S O
3
d: 170.31, 156.13, 152.88 (d, J = 243.0 Hz), 136.20, 128.72, 128.39,
127.91, 127.63, 124.78, 122.67, 114.73 (d, J = 19.5 Hz), 72.77,
72.21, 69, 51, 67.50, 53.72, 51.34, 41.55, 16.21; IR (neat): 3375,
(
ꢁ
1
+
2
4
1697, 1682 cm ; MS (EI): m/z 432 (M ); HRMS (EI) Calcd for C22-
2
1
orated to give the aldehyde as a colorless oil, which was used di-
rectly in the next step. 2-Methyl-2-butene (0.65 mL, 6.12 mmol),
H
25
6
O N
2
F 432.1697; Found 432.1694; ½
a
ꢂ
D
3
+10.0 (c 0.50, CHCl ).
NaH
0
3
2
PO
.91 mmol) were added to a solution of the above aldehyde in a
:1 t-BuOH/H O mixture (4 mL) at 0 °C. The reaction mixture was
4
ꢀ2H
2
O
(0.238 g, 1.53 mmol), and NaClO
2
(0.118 g,
4.1.18. General procedure for the synthesis of iminosugars
(18a–n)
2
2
20% Pd(OH) /C was added to a solution of 17 in MeOH, and the
stirred at room temperature for 2 h. After the reaction was com-
pleted, sat. NaHSO (aq) and 10% HCl (aq) were added, and then
the aqueous mixture was extracted with AcOEt (5 mL ꢃ 3). The or-
ganic layers were combined, dried over anhydrous Na SO , and
resulting suspension was hydrogenated at 1 atm under hydrogen
atmosphere for 2 days. The catalyst was filtered off with a Celite
pad, and the filtrate was evaporated to give a hydrogenated prod-
3
+
2
4
uct, which was purified using Dowex 50 W resin (X-8, H form, elu-
evaporated to give 15 as a yellow oil, which was chromatographed
on silica gel (10 g, n-hexane/acetone 15:1) to give 15 (0.053 g,
3
ent: 0.5 N aqueous NH ) to give 18.
0
7
.11 mmol, 73%) as a colorless oil. 1H NMR (400 MHz, CDCl
3
) d:
.36–7.30 (5H, m), 5.20, 5.13 (2H, ABq, J = 12.4 Hz), 4.77–4.63
4.1.19. N-Phenyl-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-
(phenylcarbamoylmethyl)deoxyfuconojirimycin] (18a)
1
(
3
7H, m), 4.51 (1H, br), 4.04 (1H, dd, J = 6.4, 2.4 Hz), 3.96 (2H, br),
.40 (3H, s), 3.39 (3H, s), 3.38 (3H, s), 3.21 (1H, dd, J = 14.2,
Yield 90%; H NMR (500 MHz, DMSO-d
6
) d: 7.56 (2H, d,
J = 7.6 Hz), 7.28 (2H, t, J = 7.6 Hz), 7.01 (1H, t, J = 7.6 Hz), 4.64–

6
572
A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
4
2
.33 (3H, br), 3.51 (1H, br), 3.18–3.12 (1H, m), 2.71–2.61 (1H, m),
.27–2.20 (1H, m), 0.99 (3H, d, J = 6.4 Hz); 13C NMR (125 MHz,
) d: 171.18, 130.19, 129.15, 120.40, 119.41, 75.86, 72.73,
4.1.25. N-(4-Tolyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-(4-
methylphenylcarbamoylmethyl)deoxyfuconojirimycin] (18g)
1
DMSO-d
0.09, 58.76, 53.30, 40.00, 15.92; IR (KBr): 3421, 1662 cm ; MS
6
Yield 68%; H NMR (500 MHz, DMSO-d
6
) d: 7.45 (2H, d,
ꢁ1
7
J = 8.5 Hz), 7.08 (2H, d, J = 8.5 Hz), 4.64–4.33 (3H, br), 3.46 (1H,
+
(
EI): m/z 280 (M ); HRMS (EI) Calcd for C14
H
20
O
4
N
2
280.1423;
br), 3.12–3.24 (1H, m), 2.69–2.61 (1H, m), 2.23–2.14 (4H, m),
2
D
5
13
Found 280.1421; ½
a
ꢂ
+7.5 (c 0.10, MeOH); mp 221–223 °C.
0.99 (3H, d, J = 6.5 Hz);
6
C NMR (125 MHz, DMSO-d ) d:
1
70.71, 137.32, 132.15, 129.51, 119.39, 76.32, 72.86, 71.98,
ꢁ
1
4
.1.20. N-Benzyl-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-
58.15, 53.24, 40.43, 20.93, 18.41; IR (KBr): 3411, 1656 cm
MS (EI): m/z 294 (M+); HRMS (EI) Calcd for
;
(
benzylcarbamoylmethyl)deoxyfuconojirimycin] (18b)
C
15
H
22
O
4
N
2
1
25
D
Yield 87%; H NMR (500 MHz, DMSO-d
6
) d: 7.30–7.21 (5H, m),
294.1580; Found 294.1585; ½
aꢂ
+4.8 (c 0.10, MeOH); mp 241–
4
.68–4.42 (3H, m), 4.35–4.21 (2H, m), 3.48 (1H, br), 3.21–3.14
242 °C.
(
1H, m), 2.71–2.54 (1H, m), 2.12 (1H, br), 1.01 (3H, d, J = 6.8 Hz);
1
3
C NMR (125 MHz, DMSO-d
6
) d: 171.84, 128.63, 127.72, 127.55,
4.1.26. N-(2,4-Dimethyphenyl)-2b-DFJ acetamide [(1R,2R,3R,-
4R,5S)-1-(2,4-dimethylphenylcarbamoylmethyl)deoxyfuconoji-
rimycin] (18h)
1
27.09, 74.69, 72.19, 71.71, 61.46, 52.93, 52.84, 40.89, 13.41; IR
ꢁ1
+
(
KBr): 3420, 1662 cm ; MS (EI): m/z 294 (M ); HRMS (EI) Calcd
25
Yield 90%; 1H NMR (500 MHz, DMSO-d
for C15
MeOH); mp 231–232 °C.
H
22
O
4
N
2
294.1580; Found 294.1583; ½
a
ꢂ
+5.7 (c 0.10,
6
) d: 7.65 (1H, d,
J = 8.0 Hz), 6.98 (1H, s), 6.93 (1H, d, J = 8.0 Hz), 4.70–4.46 (3H, br),
D
3
.49 (1H, br), 3.22–3.12 (1H, m), 2.74–2.58 (1H, m), 2.34–2.26
13
4
.1.21. N-(4-Methoxyphenyl)-2b-DFJ acetamide
(1R,2R,3R,4R,5S)-1-(4-
methoxyphenylcarbamoylmethyl)deoxyfuconojirimycin] (18c)
(1H, m), 2.22 (3H, s), 2.18 (3H, s), 1.03 (3H, d, J = 6.5 Hz);
C
[
6
NMR (125 MHz, DMSO-d ) d: 170.69, 134.95, 133.14, 131.13,
129.17, 126.88, 122.91, 76.09, 72.86, 71.84, 58.09, 53.26, 39.02,
1
ꢁ1
Yield 93%; H NMR (500 MHz, DMSO-d
J = 8.8 Hz), 6.86 (2H, d, J = 8.8 Hz), 4.69–4.41 (3H, br), 3.71 (3H, s),
.48 (1H, br), 3.20 (1H, br), 2.69 (1H, m), 2.24 (1H, m), 1.01 (3H,
6
) d: 7.47 (2H, d,
20.92, 18.36, 18.21; IR (KBr): 3412, 1653 cm ; MS (EI): m/z 308
+
(M ); HRMS (EI) Calcd for
C
16
H
24
O
4
N
2
308.1736; Found
2
5
3
308.1747; ½ ꢂ
a
+9.8 (c 0.10, MeOH); mp 215–216 °C.
D
1
3
d, J = 5.5 Hz); C NMR (125 MHz, DMSO-d
6
) d: 173.24, 136.27,
1
4
31.19, 128.92, 120.96, 74.02, 72.44, 72.04, 62.47, 58.65, 55,61,
4.1.27. N-(2,4,6-Trimethyphenyl)-2b-DFJ acetamide
[(1R,2R,3R,4R,5S)-1-(2,4,6-
ꢁ1
+
4.21, 16.74; IR (KBr): 3410, 1653 cm ; MS (EI): m/z 310 (M );
2
D
5
HRMS (EI) Calcd for C15
H
22
O
5
N
2
310.1529; Found 310.1527; ½
aꢂ
trimethylphenylcarbamoylmethyl)deoxyfuconojirimycin] (18i)
1
+9.4 (c 0.25, MeOH); mp 233–234 °C.
Yield 70%; H NMR (500 MHz, DMSO-d
6
) d: 6.84 (2H, s), 4.87–
4
.69 (3H, br), 3.53 (1H, br), 3.27 (1H, br), 2.85–2.57 (1H, m), 2.21
13
4
.1.22. N-Butyl-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-
(3H, s), 2.14–2.07 (7H, m), 1.05 (3H, d, J = 5.5 Hz); C NMR
(125 MHz, DMSO-d ) d: 169.56, 139.28, 135.17, 128.63, 122.48,
79.13, 72.32, 72.18, 57.97, 53.57, 40.59, 20.98, 18.63, 16.36; IR
(KBr): 3418, 1653 cm ; MS (EI): m/z 322 (M ); HRMS (EI) Calcd
for C17
(
butylcarbamoylmethyl)deoxyfuconojirimycin] (18d)
6
1
Yield 71%; H NMR (500 MHz, DMSO-d
6
) d: 4.57–4.20 (3H, br),
ꢁ1
+
3
.49 (1H, br), 3.16–3.14 (1H, br), 3.08–3.00 (2H, m), 2.61 (1H, dd,
2
D
5
J = 13.0, 6.5 Hz), 2.00–1.96 (1H, m), 1.38–1.25 (4H, m), 0.96 (3H,
H
26
O
4
N
2
322.1893; Found 322.1883; ½
aꢂ
+5.0 (c 0.10,
d, J = 6.5 Hz), 0.86 (3H, t, J = 7.0 Hz); 13C NMR (125 MHz, DMSO-
MeOH); mp 237–240 °C.
6
d ) d: 171.82, 76.40, 72.84, 72.14, 58.25, 53.32, 39.54, 38.48,
ꢁ
1
3
1.72, 20.07, 18.37, 14.21; IR (KBr): 3419, 1652 cm ; MS (EI):
4.1.28. N-(2-Fluorophenyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-
1-(2-fluorophenylcarbamoylmethyl)deoxyfuconojirimycin]
+
m/z 260 (M ); HRMS (EI) Calcd for C12
2
H
24
O
4
N
2
260.1736; Found
+13.0 (c 0.40, MeOH); mp 230–231 °C.
2
1
60.1737; ½
aꢂ
(18j)
D
1
Yield 65%; H NMR (500 MHz, DMSO-d
6
) d: 7.60 (1H, dd, J = 8.5,
4
.1.23. N-(2-Tolyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-(2-
2.0 Hz), 7.34–7.23 (2H, m), 6.84 (1H, td, J = 8.5, 2.0 Hz), 4.73–4.49
methylphenylcarbamoylmethyl)deoxyfuconojirimycin] (18e)
(3H, br), 3.48 (1H, br), 3.20–3.16 (1H, m), 2.88–2.58 (1H, m),
2.31–2.24 (1H, m), 1.01 (3H, d, J = 6.5 Hz); C NMR (125 MHz,
DMSO-d ) d: 171.18, 160.86 (d, J = 216.0 Hz), 141.76, 141.53,
6
130.83 (d, J = 35.1 Hz), 115.38 (d, J = 37.8 Hz), 109.97, 76.07,
1
13
Yield 64%; H NMR (500 MHz, DMSO-d
J = 7.5 Hz), 7.19 (1H, d, J = 7.5 Hz), 7.14 (1H, t, J = 7.5 Hz), 7.04
1H, t, J = 7.5 Hz), 5.22–4.88 (3H, br), 3.60 (1H, br), 3.11–2.95 (1H,
br), 2.84 (1H, br), 2.22 (3H, s), 2.21–2.09 (1H, m), 1.15 (3H, d,
6
) d: 7.30 (1H, d,
(
72.69, 71.67, 57.87, 53.40, 37.80, 18.54; IR (KBr): 3412,
1
3
ꢁ1
+
J = 6.0 Hz);
6
C NMR (125 MHz, DMSO-d ) d: 168.63, 131.26,
219 4-
1653 cm ; MS (EI): m/z 298 (M ); HRMS (EI) Calcd for C14H O
2
3
1
5
30.71, 126.54, 126.40, 121.74, 119.92, 74.83, 71.51, 71.27, 58.03,
N
2
F 298.1329; Found 298.1334; ½
a
ꢂ
+13.5 (c 0.10, MeOH); mp
D
ꢁ1
3.90, 18.43, 15.40; IR (KBr): 3402, 1654 cm ; MS (EI): m/z 294
208–209 °C.
+
(
M ); HRMS (EI) Calcd for
C
15
H
22
O
4
N
2
294.1580; Found
2
D
6
2
94.1583; ½
aꢂ
+2.8 (c 0.20, MeOH); mp 217–218 °C.
4.1.29. N-(3-Fluorophenyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-
-(3-fluorophenylcarbamoylmethyl)deoxyfuconojirimycin]
(18k)
6
Yield 85%; H NMR (500 MHz, DMSO-d ) d: 8.20 (1H, td, J = 8.0,
1
4
.1.24. N-(3-Tolyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-1-(3-
1
methylphenylcarbamoylmethyl)deoxyfuconojirimycin] (18f)
1
Yield 87%; H NMR (500 MHz, DMSO-d
1H, d, J = 7.5 Hz), 7.15 (1H, t, J = 7.5 Hz), 6.83 (1H, d, J = 7.5 Hz),
.68–4.39 (3H, br), 3.47 (1H, br), 3.21–3.14 (1H, m), 2.69–2.62
1H, m), 2.26 (3H, s), 2.21–2.12 (1H, m), 1.00 (3H, d, J = 6.5 Hz);
6
) d: 7.42 (1H, s), 7.33
1.5 Hz), 7.25–7.03 (3H, m), 4.71–4.45 (3H, br), 3.50 (1H, br), 3.22–
3.11 (1H, m), 2.59–2.53 (1H, m), 2.40–2.35 (1H, m), 1.05 (3H, d,
(
4
1
3
6
J = 6.5 Hz); C NMR (125 MHz, DMSO-d ) d: 171.50, 152.93 (d,
(
J = 243.0 Hz), 127.49, 125.05, 122.37, 115.69 (d, J = 18.3 Hz),
108.42 (d, J = 12.5 Hz), 76.17, 72.92, 71.96, 58.01, 53.31, 18.16; IR
1
3
C NMR (125 MHz, DMSO-d
6
) d: 170.87, 139.74, 138.31, 129.00,
ꢁ
1
+
1
2
24.08, 119.95, 116.62, 76.26, 72.81, 71.89, 58.12, 53.31, 39.83,
1.71, 18.34; IR (KBr): 3397, 1655 cm ; MS (EI): m/z 294 (M );
(KBr): 3406, 1654 cm ; MS (EI): m/z 298 (M ); HRMS (EI) Calcd
ꢁ1
+
25
for C14H
219
O
4
N
2
F 298.1329; Found 298.1313; ½
aꢂ
+3.3 (c 0.10,
D
2
D
6
HRMS (EI) Calcd for C15
H
22
O
4
N
2
294.1580; Found 294.1578; ½ ꢂ
a
MeOH); mp 197–198 °C.
+13.5 (c 0.20, MeOH); mp 200–201 °C.

A. Kato et al. / Bioorg. Med. Chem. 21 (2013) 6565–6573
6573
4
1
.1.30. N-(4-Fluorophenyl)-2b-DFJ acetamide [(1R,2R,3R,4R,5S)-
-(4-fluorophenylcarbamoylmethyl)deoxyfuconojirimycin]
using an appropriate p-nitrophenyl glycoside as substrate at the
optimum pH of each enzyme. The reaction mixture (0.2 mL) con-
tained 2 mM of the substrate and the appropriate amount of en-
zyme. The reaction was stopped by adding 0.4 mL of 400 mM
(
18l)
Yield 94%; H NMR (500 MHz, DMSO-d
1
6
) d: 7.59 (2H, dd, J = 8.8,
5
3
.0 Hz), 7.12 (2H, t, J = 8.8 Hz), 4.75–4.51 (3H, br), 3.49 (1H, br),
2 3
Na CO . The released p-nitrophenol was measured spectrometri-
cally at 400 nm.
.21 (1H, br), 2.72–2.63 (1H, m), 2.30–2.26 (1H, m), 1.02 (3H, d,
1
3
J = 7.0 Hz); C NMR (125 MHz, DMSO-d
6
) d: 170.72, 158.25 (d,
J = 236.9 Hz), 136.33, 121.15, 115.71 (d, J = 23.1 Hz), 76.15, 72.72,
Acknowledgments
ꢁ1
7
1.76, 57.99, 53.34, 18.20; IR (KBr): 3409, 1661 cm ; MS (EI):
+
m/z 298 (M ); HRMS (EI) Calcd for C14
H
19
O
4
N
2
F 298.1329; Found
The authors wish to thank Dr. Fumihiro Ishikawa (FUSHIMI
pharma. Co., Ltd.) and Professor Robert J. Nash (IBER/Phytoquest.
Ltd.) for valuable discussion. This work was supported in part by
a Grant-in-Aid for Scientific Research (C) from Japan Society for
the Promotion of Science (JSPS).
2
D
4
2
98.1352; ½
aꢂ
+3.2 (c 0.10, MeOH); mp 204–205 °C.
4
.1.31. N-(2,4-Difluorophenyl)-2b-DFJ acetamide
[
(1R,2R,3R,4R,5S)-1-(2,4-
difluorophenylcarbamoylmethyl)deoxyfuconojirimycin] (18m)
1
Yield 77%; H NMR (500 MHz, DMSO-d
.33–7.28 (1H, m), 7.05–7.02 (1H, m), 4.72–4.46 (3H, br), 3.49
1H, br), 3.17–3.11 (1H, m), 2.70–2.57 (1H, m), 2.32–2.22 (1H,
6
) d: 8.17–8.13 (1H, m),
Supplementary data
7
(
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.08.028.
13
m), 1.03 (3H, d, J = 6.5 Hz); C NMR (125 MHz, DMSO-d
6
) d:
71.30, 157.89 (d, J = 374 Hz), 155.62 (d, J = 193.3 Hz), 123.41,
20.33, 111.60 (d, J = 47.0 Hz), 103.44 (t, J = 8.5 Hz), 79.67, 72.79,
1
1
7
References and notes
ꢁ
1
1.86, 57.93, 53.22, 18.10; IR (KBr): 3419, 1654 cm ; MS (EI):
+
m/z 316 (M ); HRMS (EI) Calcd for C14
H
18
O
4
N
2
F
4
316.1235; Found
1. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Tetrahedron: Asymmetry
2000, 11, 1645.
2
4
D
3
16.1224; ½
a
ꢂ
+1.7 (c 0.10, MeOH); mp 224–226 °C.
2
3
.
.
Asano, N. Glycobiology 2003, 13, 93R.
Nash, R. J.; Kato, A.; Yu, C.-Y.; Fleet, G. W. J. Future Med. Chem. 2011, 3, 1513.
4
.1.32. N-(2,4,6-Trimethyphenyl)-2b-DFJ acetamide
4. Rinnbauer, M.; Ernst, B.; Wagner, B.; Magnani, J.; Benie, A. J.; Petera, T.
Glycobiology 2003, 13, 435.
[
(1R,2R,3R,4R,5S)-1-(2,4,6-
5
.
Wang, J. W.; Ambros, R. A.; Weber, P. B.; Rosano, T. G. Cancer Res. 1995, 55,
654.
6. Shah, M.; Telang, S.; Raval, G.; Shah, P.; Patel, P. S. Cancer 2008, 113, 336.
trifluorophenylcarbamoylmethyl)deoxyfuconojirimycin] (18n)
3
1
Yield 85%; H NMR (500 MHz, DMSO-d
6
) d: 7.25 (2H, t,
7
8
.
.
Scanlin, T. F.; Glick, M. C. Biochim. Biophys. Acta 1999, 1455, 241.
Rhim, A. D.; Stoykova, L.; Glick, M. C.; Scanlin, T. F. Glycoconjugate J. 2001, 18,
J = 8.5 Hz), 4.70–4.26 (3H, br), 3.52 (1H, br), 2.90–2.85 (1H, m),
1
3
2
.73–2.57 (1H, m), 2.33–2.26 (1H, m), 1.05 (3H, d, J = 6.5 Hz);
C
649.
NMR (125 MHz, DMSO-d
6
) d: 170.1, 160.0 (d, J = 234.6 Hz), 157.9
9. Liu, T. W.; Ho, C. W.; Huang, H. H.; Chang, S. M.; Popat, S. D.; Wang, Y. T.; Wu,
M. S.; Chen, Y. J.; Lin, C. H. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 14581.
10. Rao, V. D. N.; Smith, M. O.; Ellinwood, N. M. J. Androl. 1998, 19, 444.
(
d, J = 232.1 Hz), 114.7 (t, J = 6.3 Hz), 100.0 (t, J = 6.1 Hz), 71.58,
ꢁ1
7
1.04, 70.43, 53.24, 51.44, 17.91; IR (KBr): 3408, 1653 cm ; MS
11. Lucas, H.; Bercegeay, S.; Pendu, J.; Jean, M.; Mirallie, S.; Barriere, P. Hum.
+
(
EI): m/z 334 (M ); HRMS (EI) Calcd for C14
H
17
O
4
N
2
F
3
334.1140;
Reprod. 1994, 9, 1532.
2
5
1
1
2. De Cerezo, J. M.; Marquinez, A. C.; Sarchi, M. I.; Cerezo, A. S. Biocell 1996, 20, 11.
3. Asano, N.; Yasuda, K.; Kizu, H.; Kato, A.; Fan, J.-Q.; Nash, R. J.; Fleet, G. W. J.;
Molyneux, R. J. Eur. J. Biochem. 2001, 268, 35.
Found 334.1127; ½ ꢂ
a
+4.0 (c 0.10, MeOH); mp 267–270 °C.
D
4
.2. Biological assays
14. Kato, A.; Wamg, L.; Ishii, K.; Asano, N.; Suzuki, T. J. Biochem. 2011, 149, 415.
15. Kato, A.; Kato, N.; Kano, E.; Adachi, I.; Ikeda, K.; Yu, L.; Okamoto, T.; Banba, Y.;
Ouchi, H.; Takahata, H.; Asano, N. J. Med. Chem. 2005, 48, 2036.
The enzymes b-glucosidase (from bovine liver),
from coffee beans), b-galactosidase (from bovine liver),
nosidase (from jack bean), b-mannosidase (from snail),
nosidase (from Penicillium decumbens), -fucosidase (from
a
-galactosidase
-man-
-rham-
16. Jenkinson, S. F.; Fleet, G. W. J.; Nash, R. J.; Koike, Y.; Adachi, I.; Yoshihara, A.;
Morimoto, K.; Izumori, K.; Kato, A. Org. Lett. 2011, 13, 4064.
(
a
L
a
-
17. Winchester, B.; Barker, C.; Baines, S.; Jacob, G. S.; Namgoong, S. K.; Fleet, G. W. J.
Biochem. J. 1990, 265, 277.
a-L
18. Fleet, G. W. J.; Shaw, A. N.; Evans, S. V.; Fellows, L. E. J. Chem. Soc., Chem.
bovine kidney), b-glucuronidases (from bovine liver; from Esche-
richia coli), p-nitrophenyl glycosides, and various disaccharides
were purchased from Sigma–Aldrich Co. Brush border membranes
were prepared from the rat small intestine according to the meth-
Commun. 1985, 841.
19. Pearson, M. S. M.; Floquet, N.; Bello, C.; Vogel, P.; Plantier-Royon, R.;
Szymoniak, J.; Bertus, P.; Behr, J.-B. Bioorg. Med. Chem. 2009, 17, 8020.
20. Moreno-Clavijo, E.; Carmona, A. T.; Moreno-Vargas, A. J.; Rodríguez-Carvajal,
M. A.; Robina, I. Bioorg. Med. Chem. 2010, 18, 4648.
2
8
od of Kessler et al. and were assayed at pH 6.8 for rat intestinal
maltase using maltose. Rat epididymis -fucosidase was partially
prepared from epididymis according to the method of Skudlared
21. Ak, A.; Prudent, S.; Lenouën, D.; Defoin, A.; Tarnus, C. Bioorg. Med. Chem. Lett.
010, 20, 7410.
2. Wu, C.-Y.; Chang, C.-F.; Chen, J. S.; Wong, C.-H.; Lin, C.-H. Angew. Chem., Int. Ed.
003, 42, 4661.
2
a
-L
2
2
2
9
et al. Human lysosome
a-
L
-fucosidase was prepared according
to our previous methods and was assayed using 4-methylumbel-
liferyl- -fucopyranoside as substrate. For rat intestinal maltase
23. Yu, L.; Ikeda, K.; Kato, A.; Adachi, I.; Godin, G.; Compain, P.; Martin, O. R.; Asano,
2
7
N. Bioorg. Med. Chem. 2006, 14, 7736.
2
4. Kato, A.; Hayashi, E.; Miyauchi, S.; Adachi, I.; Imahori, T.; Natori, Y.;
Yoshimutra, Y.; Nash, R. J.; Shimaoka, H.; Nakagome, I.; Koseki, J.; Hirono, S.;
Takahata, H. J. Med. Chem. 2012, 55, 10347.
a
-L
activities, the reaction mixture (0.2 mL) contained 25 mM maltose
and the appropriate amount of enzyme, and the incubations were
performed for 10 min at 37 °C. The reaction was stopped by heat-
ing at 100 °C for 3 min. After centrifugation (600 g; 10 min),
25. Gryko, D.; Urba n´ czyk-Lipkowska, Z.; Jurczak, J. Tetrahedron: Asymmetry 1997, 8,
4059.
2
2
6. Cha, J. K.; Christ, W. J.; Kishi, Y. Tetrahedron Lett. 1983, 24, 3943.
7. Deslongchamps, P. Stereoelectronic Effects in Organic Chemistry; Pergamon: New
York, 1983. p 209.
0
.035 mL of the resulting reaction mixture were added to 2.1 mL
2
2
8. Kessler, M.; Acuto, O.; Strelli, C.; Murer, H.; Semenza, G. A. Biochem. Biophys.
Acta 1978, 506, 136.
9. Skudlarek, M. D.; Tulsiani, D. R. P.; Orgebin-Crist, M.-C. Biochem. J. 1992, 286,
907.
of the Glucose CII-test Wako (Wako Pure Chemical Ind., Osaka, Ja-
pan). The absorbance at 505 nm was measured to determine the
amount of the released
D-glucose. Other glycosidase activities
(
including the rat epididymis
a-L-fucosidase) were determined