696 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Wennekes et al.
potency of inhibition of GCS, it seems warranted to investigate
its potential as a therapeutic agent for inherited glycosphingo-
lipidoses.
found 564.3090 [M þ H]þ, calculated for [C37H41O4N1 þ H]þ
564.3108.
2,3,4,6-Tetra-O-benzyl-L-ido-1-deoxynojirimycin (13). Potas-
sium tert-butoxide (259 mg, 2.3 mmol) was added to a solution
of 12 (2.598 g, 4.61 mmol) in DMSO (9.2 mL), and the resulting
brown reaction mixture was heated at 100 °C for 30 min. The
reaction mixture was charged with 1 M aqueous HCl (9 mL) and
stirred vigorously for 15 min. The mixture was poured into
saturated aqueous NaHCO3 (100 mL) and extracted with Et2O
(3 ꢀ 100 mL). The organic phase was isolated, dried (Na2SO4),
and concentrated. The residue was purified by flash silica gel
chromatography (2:1 f 1:2 PE/EtOAc) to furnish 13 (1.954 g,
3.73 mmol) in 81% yield as a yellow oil. Rf = 0.2 (1:1 PE/
In summary, lipophilic iminosugar 2 exerts beneficial effects
on glycemic control by virtue of its dual lowering of visceral
glycosphingolipids and buffering of carbohydrate assimilation.
This dual action is desirable for control of hyperglycemia,
a hallmark of type 2 diabetes. The L-ido analogue of 2 we
developed, 4, specifically inhibits glycosphingolipid biosynthesis
and may be of interest to intervene in inherited glycosphingo-
lipidoses. The outcome of this study also indicates that tailored
iminosugars can be developed for specific therapeutic indica-
tions by designing them to exclusively act on glycosphingolipid
metabolism or on other related glycoprocessing pathways.
1
EtOAc). H NMR (400 MHz, CDCl3) δ 7.38-7.20 (m, 20H),
4.67-4.50 (m, 8H), 3.67 (dd, J=9.5, 9.5, 1H), 3.64-3.59 (m,
2H), 3.55 (dd, J = 5.2, 9.5, 1H), 3.44 (dd, J = 6.3, 10.5, 1H),
3.41-3.35 (m, 1H), 3.00 (dd, J=4.1, 12.9, 1H), 2.86 (dd, J=6.7,
12.9, 1H), 2.00 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 138.8,
138.7, 138.6, 138.5, 128.6, 128.5, 128.0, 127.9, 127.8, 127.7,
78.1, 77.3, 77.1, 74.2, 73.5, 72.8, 72.2, 67.4, 54.8, 44.4. HRMS:
found 524.2776 [M þ H]þ, calculated for [C34H37O4N1 þ H]þ
524.2795.
Experimental Section
Chemistry. The 1H and 13C NMR experiments were recorded
on a 200/50, 300/75, 400/100, 500/125, or 600/150 MHz spectro-
meter. Chemical shifts are given in ppm (δ) relative to tetra-
methylsilane as internal standard for all 1H NMR measure-
ments in CDCl3 and the deuterated solvent signal for all other
NMR measurements. Coupling constants (J ) are given in Hz.
High resolution mass spectra were recorded on a mass spectro-
meter (Thermo Finnigan LTQ Orbitrap) equipped with an
electrospray ion source in positive mode with resolution R =
60 000 at m/z 400 (mass range m/z = 150-2000). All tested
iminosugars were analyzed via a combination of HPLC and
LC/MS that showed a purity of >95%. The L-tartaric acid salt
of 1 was obtained from Genzyme and used as received.13 The
N-[5-(Adamantan-1-ylmethoxy)pentyl]-L-ido-1-deoxynojiri-
mycin (4). A solution of 13 (1.954 g, 3.73 mmol) and 1621 (1.029
g, 4.11 mmol) in EtOH/AcOH (93 mL, 9/1, v/v) was purged of
oxygen by bubbling argon through the solution. Pd/C (10 wt %,
329 mg) was added to the solution, and the reaction mixture was
exposed to 4 bar of hydrogen for 20 h. Removal of Pd/C by
filtration over a glass microfiber filter and concentration of the
filtrate provided the crude N-alkylated intermediate (Rf = 0.6 in
4:1 PE/EtOAc) as a light-yellow oil. A solution of crude inter-
mediate in EtOH (50 mL) was acidified with 2 M aqueous HCl
(12 mL) and purged of oxygen by bubbling argon through the
solution. Pd/C (10 wt %, 500 mg) was added to the solution, and
the reaction mixture was exposed to 4 bar of hydrogen for 20 h.
After removal of Pd/C and concentration, the residue was
purified by flash silica gel chromatography (0 f 10% MeOH
in CHCl3 þ 1% NH4OH) to afford 4 (1.146 g, 2.88 mmol) in
77% yield over two steps as a white foam. Rf = 0.3 (3:1 CHCl3/
methanesulfonic acid salt (2 MSA) of lead compound 2 was
3
synthesized as previously described and used as such in the
enzyme assays and animal studies.21 Compounds 3 and 4 were
used in enzyme assays and animal studies as their hydrochloric
acid salts (3 HCl and 4 HCl). Compound 5 was obtained
3
3
commercially and used as received (Zavesca, Actelion). The
remaining iminosugars were tested in the enzyme assays as their
trifluoromethanesulfonic acid salt.
1
N-Allyl-2,3,4,6-tetra-O-benzyl-L-ido-1-deoxynojirimycin (12).
Methanesulfonyl chloride (1.45 mL, 18.75 mmol) was added
dropwise to a cooled (0 °C) solution of 10 (4.070 g, 7.50 mmol,
prepared from 6 via a previously reported procedure)16 in
pyridine (30 mL). After TLC analysis indicated complete con-
sumption of starting material (2 h; Rf(10) = 0.25 in 2:1 PE/
EtOAc), water (20 mL) was added and the reaction mixture was
concentrated. The residue was dissolved in EtOAc (100 mL) and
washed successively with 1 M aqueous HCl (2 ꢀ 100 mL),
saturated aqueous NaHCO3 (100 mL) and saturated aqueous
NaCl (100 mL). The organic phase was isolated, dried (Na2SO4),
and concentrated to yield crude 11 (4.790 g, 6.86 mmol) in
∼91% yield as a yellow oil. Rf = 0.55 (2:1 PE/EtOAc). Crude 11
(4.790 g, 6.86 mmol) was coevaporated with toluene, dissolved
in allylamine (34 mL) and refluxed for 20 h. The reaction
mixture was concentrated, dissolved in EtOAc (100 mL), and
washed successively with saturated aqueous NaHCO3 (2 ꢀ 100
mL) and saturated aqueous NaCl (100 mL). The organic phase
was isolated, dried (Na2SO4), and concentrated. The residue was
purified by flash silica gel chromatography (isocratic 6:1 PE/
EtOAc) to produce 12 (2.591 g, 4.60 mmol) in 67% yield as an
orange oil. Rf = 0.8 (2:1 PE/EtOAc). 1H NMR (400 MHz,
CDCl3) δ 7.38-7.15 (m, 20H), 5.84-5.70 (m, 1H), 5.12 (dd, J =
1.3, 17.2, 1H), 5.07 (d, J = 10.2, 1H), 4.85 (d, J = 11.1, 1H), 4.80
(d, J = 11.1, 1H), 4.68-4.55 (m, 4H), 4.46 (s, 2H), 3.83 (dd, J =
6.6, 10.2, 1H), 3.76-3.66 (m, 2H), 3.61-3.51 (m, 2H), 3.45-3.33
(m, 2H), 3.18 (dd, J = 6.9, 14.1, 1H), 2.92 (dd, J = 4.5, 11.8,
1H), 2.58-2.53 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 139.2,
138.7, 138.6, 138.5, 136.2, 129.6, 129.2, 128.6, 128.3, 128.2,
127.9, 127.7, 127.5, 127.4, 127.3, 127.1, 126.7, 117.1, 82.9,
80.1, 78.7, 75.3, 73.2, 72.8, 72.6, 64.6, 59.9, 57.9, 49.1. HRMS:
MeOH þ 1% NH4OH). H NMR (400 MHz, MeOD, major
conformation) δ 3.91 (d, J = 5.1, 2H), 3.85 (s, 1H), 3.74 (s, 1H),
3.63 (s, 1H), 3.40 (t, J = 6.2, 2H), 3.31-3.27 (m, 1H), 3.23-2.97
(m, 4H), 2.97 (s, 2H, OCH2), 1.95 (s, 3H), 1.80-1.59 (m, 10H),
1.56 (d, J = 2.0, 6H), 1.47-1.38 (m, 2H). 13C NMR (100 MHz,
MeOD) δ 83.2, 72.5, 72.5, 70.2, 64.1, 59.2, 55.4, 53.6, 41.0, 38.5,
35.3, 30.5, 29.9, 25.0. HRMS: found 398.2889 [M þ H]þ,
calculated for [C22H39O5N1 þ H]þ 398.2901.
Biology. Animals. Experimental procedures were all approved
by the appropriate Ethics Committee for Animal Experiments.
C57Bl/6J and ob/ob mice (C57Bl/6J background) were obtained
from Harlan (Horst, The Netherlands), and ZDF (ZDF/GMi-
fa/fa) rats and lean littermates were from Charles River La-
boratories (Wilmington, MA). Animals were housed in a light-
and temperature-controlled facility. Animals were fed a com-
mercially available lab chow (RMH-B, Hope Farms BV, Woer-
den, The Netherlands) containing about 6% fat and ∼0.01%
cholesterol (w/w). Iminosugars were mixed in the food. In the
case of experiments with ZDF rats, compound was administered
by oral gavage two times daily.
Plasma and Tissue Sampling. Blood samples were collected by
either tail vein or retro-orbital plexus puncture. Animals were
sacrificed under isoflurane anesthesia. A large blood sample was
collected by cardiac puncture. Tissues were quickly removed
and frozen for further analysis.
Analysis of Lipids and Measurement of Enzyme Activities.
Lipids were extracted according to Folch et al.34 Ceramide and
glucosylceramide collected from the chloroform phase were
determined by HPLC analysis of o-phthalaldehyde-conjugated
lipids according to a procedure described previously.35 The
chloroform layer was thoroughly dried, and deacylation of