Conformationally-locked N -glycosides with selective β-glucosidase inhibitory activity: Identification of a new non-iminosugar-type pharmacological chaperone for gaucher disease

Article abstract of DOI:10.1021/jm3006178

A series of conformationally locked N-glycosides having a cis-1,2-fused pyranose-1,3-oxazoline-2-thione structure and bearing different substituents at the exocyclic sulfur has been prepared. The polyhydroxylated bicyclic system was built in only three steps by treatment of the corresponding readily available 1,2-anhydrosugar with KSCN using TiO(TFA)₂ as catalyst, followed by S-alkylation and acetyl deprotection. In vitro screening against several glycosidase enzymes showed highly specific inhibition of mammalian β-glucosidase with a marked dependence of the potency upon the nature of the exocyclic substituent. The most potent representative, bearing an S-(ω-hydroxyhexadecyl) substituent, was further assayed as inhibitor of the human lysosomal β-glucocerebrosidase and as pharmacological chaperone in Gaucher disease fibroblasts. Activity enhancements in N370S/N370S mutants analogous to those achieved with the reference compound ambroxol were attained with a more favorable chaperone/inhibitor balance.

Full text of DOI:10.1021/jm3006178

Article
pubs.acs.org/jmc
Conformationally-Locked N-Glycosides with Selective β-Glucosidase
Inhibitory Activity: Identification of a New Non-Iminosugar-Type
Pharmacological Chaperone for Gaucher Disease
Javier Castilla,† Rocío Rísquez,‡ Deysi Cruz,† Katsumi Higaki,§ Eiji Nanba,§ Kousaku Ohno,∥
Yoshiyuki Suzuki,⊥ Yolanda Díaz,*,† Carmen Ortiz Mellet,*,‡ Jose M. García Fernandez,#
́
́
and Sergio Castillon†
́
†Department de Química Analítica i Química Organ
Spain
‡Departamento de Química Organ
Spain
̀
ica, Universitat Rovira i Virgili, C/Marcel·lí Domingo s/n, 43007 Tarragona,
ica, Facultad de Química, Universidad de Sevilla, C/Profesor García Gonzalez 1, 41012 Sevilla,
́ ́
§Division of Functional Genomics, Research Center for Bioscience and Technology, Faculty of Medicine, Tottori University, 86
Nishi-cho, Yonago, 683-8503, Japan
∥Division of Child Neurology, Insitute of Neurological Sciences, Faculty of Medicine, Tottori University, Nishi-cho, Yonago,
683-8504, Japan
⊥International University of Health and Welfare Graduate School, 2600-1 Kita Kanemaru, Otawara 324-8501, Japan
#Instituto de Investigaciones Químicas (IIQ), CSIC, Universidad de Sevilla, C/Amer
́
ico Vespucio 49, Isla de la Cartuja, 41092 Sevilla,
Spain
S
* Supporting Information
ABSTRACT: A series of conformationally locked N-glycosides having a cis-1,2-
fused pyranose−1,3-oxazoline-2-thione structure and bearing different substituents
at the exocyclic sulfur has been prepared. The polyhydroxylated bicyclic system was
built in only three steps by treatment of the corresponding readily available 1,2-
anhydrosugar with KSCN using TiO(TFA)2 as catalyst, followed by S-alkylation
and acetyl deprotection. In vitro screening against several glycosidase enzymes
showed highly specific inhibition of mammalian β-glucosidase with a marked
dependence of the potency upon the nature of the exocyclic substituent. The most potent representative, bearing an S-(ω-
hydroxyhexadecyl) substituent, was further assayed as inhibitor of the human lysosomal β-glucocerebrosidase and as
pharmacological chaperone in Gaucher disease fibroblasts. Activity enhancements in N370S/N370S mutants analogous to those
achieved with the reference compound ambroxol were attained with a more favorable chaperone/inhibitor balance.
dine-type iminosugar (+)-castanospermine (CS), with an
identical hydroxylation profile at the six-membered ring,
exhibits higher enzyme specificity compared with DNJ, which
is ascribed to the conformational restriction imposed by the
rigid bicyclic structure.22 In any case both DNJ and CS can
simultaneously inhibit several α- as well as β-glucosidases in
humans, which is a serious drawback for clinical application
(Figure 1).23
In connection with the design of more fine-tuned inhibitors,
numerous syntheses of DNJ and CS analogues have been
reported.24−34 Incorporation of alkyl substituents at the
nitrogen atom or at its vicinity in monocyclic iminosugar
frameworks, e.g., as in N-(n-nonyl)-1-deoxynojirimycin
(NNDNJ)35,36 or α-1-C-nonyl-1,5-dideoxy-1,5-iminoxylitol
(α-1-C-nonyl-DIX),37 has been shown to improve the affinity
toward certain glycosidases (Figure 1). Further chemical and
INTRODUCTION
■
Given the broad range of biological and pathological processes
in which glycosidases are involved, from the catabolism of
sugars to the biosynthesis of the complex oligosaccharide chains
in glycoproteins and glycolipids, specific inhibitors of these
enzymes bear strong potential for the development of new
pharmaceuticals. Examples include the treatment of viral
infections,1,2 such as human immunodeficiency virus (HIV),
human hepatitis C (HCV) or dengue virus, cancer,3−5
diabetes,6−8 tuberculosis,9,10 and lysosomal storage diseases
(LSDs),11−16 which has strongly stimulated research in this area
of glycobiology.17,18 With few exceptions,19,20 the glycosidase
inhibitors under study as drug candidates mimic the glycone
moiety of the putative substrate, which is shared within a series
of isoenzymes and enzymes acting on anomeric substrates. For
instance, the piperidine-type iminosugar 1-deoxynojirimycin
(DNJ) can be regarded as a stereochemical mimic of D-glucose,
which is consistent with its behavior as a potent inhibitor of
mammalian and plant α- and β-glucosidases.21 The indolizi-
Received: May 3, 2012
Published: July 4, 2012
© 2012 American Chemical Society
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prerequisite for strong enzyme affinity. Actually, protonation
under physiological conditions is probably responsible for the
broad range rather than selective activity of iminosugars.55,56 In
principle, the pyranose ring present in the natural glycoside
substrate could be directly incorporated in the molecular design
of bicyclic competitive inhibitors toward a target glycosidase to
account for the glycone specificity, which would greatly simplify
the synthetic scheme. Thus, thiamet G (Figure 1), a fused
pyranose−thiazoline derivative recently developed by Vocadlo
and co-workers,57 behaves as a potent inhibitor of O-linked 2-
acetamino-2-deoxy-β-D-glucopyranoside hydrolysis, being in-
vestigated as a potential therapeutic target that could hinder
progression of Alzheimer’s disease.58,59 To further test this
hypothesis, we have now prepared a new family of
glycomimetics having a cis-1,2-fused pyranose−2-alkylsulfanyl-
1,3-oxazoline structure (PSO). PSO derivatives share with
compound 1 the N-glycoside-type character (Figure 1).
Actually, they can be formally considered as conformationally
locked N-glycoside derivatives, which should warrant chemical
and enzymatic stability and at the same time impart
selectivity.60 On the other hand, similar to compounds 2 and
3 bearing an imine type nitrogen, the PSO structure is also very
well suited for the incorporation of a broad battery of
substituents on the exocyclic heteroatom. Here we report the
synthesis of the key synthetic precursor, the scope of the
approach, the assessment of the affinity and selectivity of the
final compounds against a panel of commercial glycosidases,
and the evaluation of a selected candidate as pharmacological
chaperone in human Gaucher disease fibroblasts.
Figure 1. Structures of representative iminosugar (DNJ, CS, NNDNJ,
α-1-C-nonyl-DIX), sp2-iminosugar (1 and 2), and bicyclic non-
iminosugar (3 and thiamet G) glycosidase inhibitors and general
structures of the pyranose−sulfanyloxazoline (PSO) glycomimetics
reported in this work.
structural evidence indicated that once the azaheterocycle ring
occupies the glycone site of the enzyme, the substituent can
favorably interact with other regions of the protein.38−40 This
result strongly suggests that exploiting non-glycone interactions
has the potential to become a general strategy to elaborate
selective glycosidase inhibitors. Implementing molecular
diversity-oriented strategies in conformationally locked bicyclic
glycomimetics is particularly attractive toward these channels.
Several polyhydroxylated bicyclic cores armed with anchoring
functionalities compatible with library generation schemes have
been proposed, among which the so-called sp2 iminosugars
have proven particularly useful (Figure 1).41−47 This family of
sugar mimics incorporates a pseudoamide group in the
structure that facilitates the installation of substituents either
at the pseudoanomeric position (e.g., in the carbamate-type
bicyclic nojirimycin derivative 1)48,49 or at an exocyclic nitrogen
(e.g., in the isourea-type bicyclic nojirimycin derivative 2).50,51
Total discrimination between α- and β-glucosidase enzymes
and even between closely related α-glucosidase isoenzymes
could be achieved in this manner, which translated into
interesting lead compounds in view of developing drug
candidates for the treatment of breast cancer49 and Gaucher
disease, the LSD with the highest prevalence.52,53 In the first
case, the biological activity was ascribed to selective inhibition
of the neutral α-glucosidases at the endoplasmic reticulum
(ER). In the second case, binding of the glycomimetic to the
active site of mutant β-glucocerebrosidase (GCase) in the ER
restored trafficking to the lysosome, acting as pharmacological
chaperone. Similarly, N′-alkylated bicyclic isoureas derived
from aminocyclitol scaffolds (e.g., 3) were also shown to behave
as very selective GCase inhibitors with strong pharmacological
chaperone potential (Figure 1).54
RESULTS
■
Synthesis. The initial synthetic objective of this research
was the preparation of S-alkyl cis-1,2-fused pyranose−(2-
alkylsulfany-1,3-oxazoline) carbohydrate derivatives bearing
different substituents at the exocyclic sulfur atom. A new
methodology has been developed for the construction of the
heteroatomic bicycle system that exploits the reactivity of sugar
epoxides. The reaction sequence started with the conventional
epoxidation of commercial tri-O-acetyl-D-glucal (4) to afford a
mixture of the corresponding tri-O-acetyl-1,2-anhydrosugars (5,
D-gluco/D-manno ratio of 7:1) in 90% yield.61 Treatment of 5
with potassium thiocyanate and catalytic amounts of TiO-
(CF3CO2)2 led to the key thionocarbamate 6 in 87% yield
(Scheme 1).62 The 1H NMR coupling constants of the
Scheme 1. Synthesis of 3,4,6-Tri-O-acetyl-1,2-dideoxy-α-D-
glucopyranoside[1,2-d]-1,3-oxazolidine-2-thione (6)
pyranose ring suggested a skew-boat conformation close to
0S2, instead of the 4C1 chair conformation typical of monocyclic
D-glucosyl derivatives, which is in agreement with previously
reported data for structurally related bicyclic cis-1,2-fused
glucopyranose structures in solution.63,64
Thionocarbamates display ambident functionality, offering
diverse reactivity.65,66 The different properties of both the N
and S electron-rich centers are explained by Pearson’s hard−
soft acid−base (HSAB) theory,67 where the nitrogen atom acts
The above examples illustrate the suitability of six-
membered−five-membered bicyclic glycomimetics to achieve
strong and selective glycosidase inhibition after incorporation
of substituents susceptible to participating in non-glycone
interactions. They also demonstrate that the presence of a basic
amine-type nitrogen in the six-membered ring is not a
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Table 1. Synthesis of 2-S-Alkylsulfanyl-1,3-oxazoline D-glucopyranose Derivatives
a
b
Carried out at rt with 1.0 equiv of substrate, 3.0 equiv of alkyl bromide, 3.0 equiv of Et3N, and 2 mol % DMAP in CH2Cl2. Carried out at rt with
1.0 equiv of of S-alkylated substrate and 5 mol % MeONa in MeOH.
a
Table 2. Ki Values (μM) against Bovine Liver (Cytosolic) β-Glucosidase and Inhibition of Human GCase Relative to Control
(%) for Compounds 16−25
enzyme
16
17
18
19
20
21
22
23
24
25
b
β-glucosidase (bovine liver)
GCase (Homo sapiens)
NI
99
220
97
580
92
310
91
280
91
73
87
52
86
12
36
210
88
230
91
a
Inhibition was competitive in all cases. No inhibition was observed for any of the compounds at 2 mM on almonds β-glucosidase, yeast α-
glucosidase, Jack bean α-mannosidase, Helix pomatia β-mannosidase, pig kidney trehalase, Aspergillus niger amyloglucosidase, Penicillium decumbens
b
naringinase, green coffee α-galactosidase, E. coli β-galactosidase, or yeast isomaltase. NI: no inhibition observed at 2 mM.
as a hard basic center while the sulfur atom shows a soft base
character. This reactivity has been extensively investigated by
Rollin et al.68−72 Reagents of R-X type are mostly considered as
soft electrophilic species, providing high-yielding S-alkyla-
tion.73−75
Reaction of 6 with a series of different alkyl bromides in basic
medium led to the expected 2-alkylsulfanyl-1,3-oxazoline
derivatives 7−15 in 69−91% yield (Table 1). Structures of
the S-alkylated compounds were ascertained by 13C NMR
spectroscopy by comparison with previously synthesized
thionocarbamates.68−72 The chemical shifts for the quaternary
sp2 carbon atom at position 2 of the five-membered heterocycle
varied from roughly 190 ppm (−N−CS in 1,3-oxazolidine-2-
thione 6) to approximately 170 ppm (−NC−SR in 2-
alkylsulfanyl-1,3-oxazoline- derivatives 7−15). Final removal of
the acetyl protecting groups using methanol under standard
NaOMe-catalyzed conditions provided the requested PSO
glucomimetics 16−25 in 89−99% yields (Table 1). Both the
alkylation and the deprotection steps requested longer reaction
times for the bulkier alkyl groups of the series (entries 6−8 and
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10, Table 1). The unprotected S-alkylated derivatives 17−25
maintain the skewed boat conformation already observed for
their corresponding acetylated precursors 7−15 and the parent
compound 6.
associated with Gaucher disease. The significant inhibitory
potential and total selectivity encountered for compound 23
against the commercial enzyme warranted further evaluation in
this sense. Determination of the inhibition activity on human
GCase, relative to control, for the whole set of compounds at
100 μM confirmed this point; only compound 23 within the
PSO derivatives surpassed 50% inhibition at this concentration
(Table 2). In further studies, ambroxol (ABX), a non-
glycomimetic-type GCase inhibitor under investigation as a
pharmacological chaperone for Gaucher disease,78 was assayed
in parallel. Inhibition studies in cell lysates from healthy
fibroblasts (Figure 2) indicated that 23 inhibited lysosomal
Evaluation of the Glycosidase Inhibitory Activity and
Chaperone Effect. All the new cis-1,2-fused D-glucopyr-
anose−(2-alkylsulfanyl-1,3-oxazoline) derivatives were first
screened as inhibitors against a panel of commercial
glycosidases including α-glucosidase (yeast), β-glucosidase
(almonds and bovine liver, cytosolic), α-mannosidase (Jack
bean), β-mannosidase (Helix pomatia), trehalase (pig kidney),
amyloglucosidase (Aspergillus niger), naringinase (β-glucosi-
dase/α-L-rhamnosidase, Penicillium decumbens), α-galactosidase
(green coffee beans), β-galactosidase (E. coli), and isomaltase
(yeast). The corresponding inhibition constants (Ki) are
collected in Table 2.
The S-unsubstituted PSO derivative 16 did not inhibit any of
the assayed glycosidases at concentrations up to 2 mM.
Interestingly, the S-substituted derivatives 17−25 behaved as
specific, though modest, inhibitors of the bovine liver β-
glucosidase among the 11 glycosidases tested. The capacity to
discriminate between the mammalian and the plant β-
glucosidases is particularly remarkable. Both the enzyme from
almonds and the enzyme from bovine liver belong to the same
glycosyl hydrolase family GH1 in the CAZy classification,76
meaning that they bear considerable similarities within their
active sites. The results support that subtle differences must
exist in areas close to the active site and further substantiate
that non-glycone interactions are better suited than glycone
interactions to attain high levels of selectivity among
isoenzymes.
Figure 2. Effects of ambroxol (ABX) and PSO 23 on lysosomal
enzyme activities in lysate from human normal fibroblasts. Enzyme
activity in normal cell lysates was determined in the absence or
presence of increasing concentrations of chaperones. Each point
represents the mean of triplicate determinations obtained in a single
experiment. Values were expressed relative to the activity in the
absence of compounds (100%). 4-Methylumbelliferyl β-D-glucopyr-
anoside was used as substrate.
A structure−activity relationship analysis within the PSO
series 17−25 indicated a notable influence of the nature of the
exocyclic substituent at the sulfur functionality on the inhibitory
potency. For linear alkyl substituents, a progressive decrease of
the corresponding Ki values, indicative of increased binding
affinity, with the chain length was observed, going from 584 μM
for the n-butyl derivative 18 to 52 μM for the n-hexadecyl
derivative 22, which is compatible with accommodation of the
aliphatic chain into a hydrophobic pocket of the protein. No
significant improvement was observed when aromatic (24) or
adamantyl residues (25) were present. Noteworthy, installation
of a terminal hydroxyl group at the hexadecyl chain (23)
further improved binding by a factor of 4.3-fold (Ki = 12.1 μM),
probably due to adventitious hydrogen-bonding interactions.
The molecular basis for the unprecedented specificity of the
PSO family toward the mammalian β-glucosidase is still
unknown. The presence of the glycosidic nitrogen atom
anchored in the α-configuration might seem to mismatch the
β-anomeric selectivity of this enzyme. However, recent X-ray
evidence on human GCase-inhibitor complexes has shown that
α-configured glycomimetics can be accommodated at the active
site in a skew-boat conformation in which the pseudoanomeric
substituent adopts a pseudoequatorial disposition.77 PSO
derivatives are preorganized in such a binding conformation,
which could be the origin of the observed specificity. In any
case, the results reported here represent a proof of concept of
the utmost importance for implementing non-glycone inter-
actions in the design of potent and specific glycosidase
inhibitors.
GCase slightly less potently compared to ABX (IC50 of 11.4
versus 4.1 μM). No inhibition of other lysosomal enzymes,
such as α-glucosidase, α-galactosidase, β-galactosidase, and β-
hexosaminidase, was observed, reproducing the selectivity
pattern already found in commercial enzymes.
Further comparative enzyme activity enhancement assays
were conducted in healthy and Gaucher fibroblasts from
patients having the N370S/N370S or the L444P/L444P
mutations. The first one, the most common mutation among
Gaucher patients, is located in the catalytic domain of the
enzyme, while the second one is located in a noncatalytic
domain. The cells were cultured for 5 days in the absence and
in the presence of various concentrations of 23 or ABX, then
lysed and the β-glucocerebrosidase activity determined using 4-
methylumbelliferyl β-D-glucopyranoside as substrate. In normal
cells, 23 had no effect on GCase activity, whereas ABX induced
a statistically significant activity enhancement compared to the
control at 10 and 30 μM. In N370S/N370S Gaucher fibroblasts
both 23 and ABX significantly up-regulated the activity of the
mutant enzyme. In the case of ABX a decrease in the relative
activity increase occurs from 30 μM, however, meaning that the
inhibitory activity overcomes the chaperone effect (Figure 3).
In contrast, the mutant enzyme activity enhancement induced
by PSO 23 steadily increased in a dose dependent manner in
the range 30−90 μM, indicating a more favorable chaperone/
inhibitor balance. Neither 23 nor ABX was effective at
increasing the activity in the case of the L444P/L444P mutant
GCase, nor did they exhibit toxic effect on any of the normal or
mutant cell lines assayed for 5 days of incubation.
Inhibition of bovine liver β-glucosidase is often used as a
preliminary parameter to select candidates as pharmacological
chaperones for mutant human β-glucocerebrosidase (GCase)
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appropriate alkyl bromide (3.00 mmol), Et3N (3.00 mmol), and
DMAP (0.2 mmol) followed by stirring at room temperature. After
completion of the reaction, the mixture was washed with saturated
NaHCO3 and brine, dried, and concentrated. Chromatographic
purification afforded the S-alkylated compounds in the yields shown
in Table 1 (see Supporting Information for details).
General Procedure for Acetyl Deprotection of Cis-1,2-Fused
D-Glucopyranose−2-Alkylsulfanyl-1,3-oxazoline Derivatives.
Sodium methoxide (0.05 mmol) was added to a solution of protected
1,3-oxazoline carbohydrate (1.00 mmol) in methanol (20 mL),
followed by stirring at room temperature. Upon completion of the
reaction, the solvent was removed in vacuo, and the crude product was
purified by flash chromatography on silica gel to afford the deprotected
compounds in the yields shown in Table 1.
Figure 3. Effect of ABX and PSO 23 in GCase activity in normal and
N370S/N370S fibroblasts. Fibroblasts from patients were cultured in
the absence or presence of the indicated concentrations of the
chaperone for 96 h, and the GCase activities in lysates were measured
using 4-methylumbelliferyl β-D-glucopyranoside as substrate. Each bar
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]allylsulfanyl-1,3-ox-
azoline (17). The title compound was prepared following the general
procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 7 (100 mg, 0.26 mmol),
MeONa (8 mg, 14 μmol), and MeOH (5.2 mL). The reaction mixture
was stirred at room temperature for 30 min. After standard workup,
the crude was purified by flash chromatography (1:9 MeOH/CH2Cl2)
to afford the desired compound (66.0 mg, 98% yield) as colorless
syrup. Rf (1:9 MeOH/CH2Cl2): 0.45. [α]D +18 (c 1.0, MeOH). FT-IR
(neat, cm−1): 3361, 1703, 1638, 1582, 1477, 1362, 1296, 1229, 1114,
represents the mean
SEM of three determinations each done in
triplicate. The asterisks indicate highly significant statistical difference
(p < 0.01) from the values in the absence of the compound (t test).
Altogether, the above data support that compound 23,
similar to the reference compound ABX, behaves as an active
site-directed pharmacological chaperone, although the observed
activity increase (1.8-fold) does not surpass the values reported
for the most effective iminosugar-type chaperones. For
instance, NNDNJ elicits up to 2.3-fold activity enhancement
in the N370S mutant and α-1-C-nonyl-DIX, developed by
Compain and co-workers, already reaches 1.8-fold increase at
10 nM.37 They also provide a proof of concept that non-
glycone interactions can be advantageously exploited to endow
a rigid pyranoid glycone moiety with high binding affinity and
selectivity toward a given glycosidase. Most importantly, the
synthetic methodology developed is very well adapted to
molecular-diversity-oriented strategies and compatible with lead
identification and optimization of pharmacological chaperones
for LSDs.
1
1046, 992, 949. H NMR (400 MHz, CD3OD) δ in ppm: 5.90 (ddt,
1H, J = 17.2, 10.4 Hz, J = 6.8 Hz), 5.78 (d, 1H, J = 7.2 Hz), 5.28 (dd,
1H, J = 17.2, 0.8 Hz), 5.19 (dd, 1H, J = 10.4, 0.8 Hz), 4.48 (dd, 1H, J =
7.2, 5.2 Hz), 3.74 (dd, 1H, J = 12.0, 2.8 Hz), 3.69 (dd, 1H, J = 12.0, 5.2
Hz), 5.20 (t, 1H, J = 6.8 Hz), 3.46 (dd, 1H, J = 8.8, 6.8 Hz), 3.51−3.30
(m, 3H). 13C NMR (100.6 MHz, CD3OD) δ in ppm: 171.8, 134.5,
119.6, 94.4, 84.4, 75.9, 75.6, 69.9, 63.3, 35.5. +TOF MS calcd for
C10H15NO5S m/z [M − H]+: 262.0749. Found: 262.0737.
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]butylsulfanyl-1,3-
oxazoline (18). The title compound was prepared following the
general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 8 (50.1 mg, 0.12 mmol),
MeONa (3.8 mg, 6 μmol), and MeOH (2.5 mL). The reaction
mixture was stirred at room temperature for 30 min. After standard
workup, the crude was purified by flash chromatography (1:9 MeOH/
CH2Cl2) to afford the desired compound (31.0 mg, 90% yield) as
colorless syrup. Rf (1:9 MeOH/CH2Cl2): 0.16. [α]D + 92 (c 2.5,
MeOH). FT-IR (neat, cm−1): 3342, 2930, 2872, 1579, 1455, 1294,
EXPERIMENTAL SECTION
■
General Methods. All chemicals were reagent grade and used as
supplied unless otherwise specified. TiO(CF3COO)2 catalyst was
prepared following a previously reported procedure.79 1H and 13C
NMR spectra were recorded on a Varian Mercury VX 400 (400 and
100.6 MHz, respectively) and Varian 400-MR spectrometer in CDCl3
or CD3OD as solvent, with the solvent resonance (δ) as the internal
standard (CDCl3 δ 7.26 ppm for 1H, δ 77.23 ppm for 13C; CD3OD δ
1
1144, 1114, 952. H NMR (400 MHz, CD3OD) δ in ppm: 5.77 (d,
1H, J = 7.2 Hz), 4.47 (dd, 1H, J = 7.2, 5.2 Hz), 3.78 (dd, 1H, J = 12.0,
2.8 Hz), 3.72 (dd, 1H, J = 12.0, 5.2 Hz), 3.68 (dd, 1H, J = 6.8, 5.2 Hz),
3.47 (dd, 1H, J = 9.2, 6.8 Hz), 3.34−3.29 (m, 1H), 3.03 (t, 2H, J = 7.2
Hz), 1.71−1.65 (m, 2H), 1.47−1.41 (m, 2H), 0.92 (t, 3H, J = 7.2 Hz).
13C NMR (100.6 MHz, CD3OD) δ in ppm: 170.8, 92.4, 82.3, 74.0,
1
3.31 ppm for H, δ 49.14 ppm for 13C) or using Me4Si as an internal
1
reference (δ 0.00 ppm for H and 13C). The 2D correlation spectra
73.7, 68.0, 61.4, 31.4, 30.7, 21.3; 12.5. +TOF MS calcd for
C11H19NO5S m/z [M − H]+: 278.1062. Found: 278.1054.
(gCOSY, NOESY, gHSQC, gHMBC) were visualized using the
VNMR program (Varian). ESI-MS was run on an Agilent 1100 series
LC/MSD instrument. Melting points (Mp) were measured on a
Griffin melting point apparatus and are uncorrected. Optical rotations
were measured at 598 nm at room temperature in a Perkin-Elmer 241
MC apparatus with 10 cm cells. IR spectra were recorded on a JASCO
FT/IR-600 plus Fourier transform infrared spectrometer ATR Specac
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]heptylsulfanyl-1,3-
oxazoline (19). The title compound was prepared following the
general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 9 (51.6 mg, 0.12 mmol),
MeONa (3.8 mg, 6 μmol), and MeOH (2.5 mL). The reaction
mixture was stirred at room temperature for 30 min. After standard
workup, the crude was purified by flash chromatography (1:9 MeOH/
CH2Cl2) to afford the desired compound (33.0 mg, 89% yield) as a
white solid. Rf (0.5:9.5 MeOH/CH2Cl2): 0.09. Mp: 48−50 °C. [α]D
+80.2 (c 5.5, MeOH). FT-IR (neat, cm−1): 3372, 2925, 2856, 1582,
1456, 1295, 1149, 1117, 954. 1H NMR (400 MHz, CD3OD) δ in ppm:
5.75 (d, 1H, J = 7.2 Hz), 4.44 (dd, 1H, J = 7.2, 5.2 Hz), 3.75 (dd, 1H, J
= 12.0, 2.8 Hz), 3.66 (dd, 1H, J = 12.0, 5.2 Hz), 3.66 (dd, 1H, J = 6.8,
5.2 Hz), 3.44 (dd, 1H, J = 9.2, 6.8 Hz), 3.32−3.27 (m, 1H), 3.0 (t, 2H,
J = 7.2 Hz), 1.72−1.65 (m, 2H), 1.40−1.25 (m, 8H), 0.87 (t, 3H, J =
7.2 Hz). 13C NMR (100.6 MHz, CD3OD) δ in ppm: 172.7, 94.2, 84.2;
75.9, 75.6, 69.9, 63.4, 33.4, 32.9, 31.2, 30.4, 30.1, 24.1, 14.9. +TOF MS
calcd for C14H25NO5S m/z [M − H]+: 320.1532. Found: 320.1519.
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]octylsulfanyl-1,3-
oxazoline (20). The title compound was prepared following the
́
Golden Gate in the Servei de Recursos Cientifics (SRCiT-URV).
Reactions were monitored by TLC carried out on 0.25 mm E. Merck
silica gel 60 F254 glass or aluminum plates. The plates were visualized
under a short-wave UV lamp (250 nm) or after dipping in a suitable
developing solution. Flash column chromatography was carried out
using forced flow of the indicated solvent on Fluka or Merck silica gel
60 (230−400 mesh). Radial chromatography was performed on 1 or 2
mm plates of Kieselgel 60 PF254 silica gel, depending on the amount
of product. Compounds 561 (as a mixture of the D-gluco and D-manno
epimers), 6,62 and 1662 were synthesized as previously reported. Purity
of all compounds was confirmed to be >95% by LC/MS and 1H NMR.
General Procedure for the S-Alkylation of Cis-1,2-Fused D-
Glucopyranose−1,3-Oxazolidine-2-thione Derivatives. To a
solution of 6 (1.00 mmol) in CH2Cl2 (3.5 mL) were added the
6861
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Journal of Medicinal Chemistry
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general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 10 (73.2 mg, 0.16 mmol),
MeONa (5.1 mg, 8 μmol), and MeOH (3.3 mL). The reaction
mixture was stirred at room temperature for 30 min. After standard
workup, the crude was purified by flash chromatography (1:9 MeOH/
CH2Cl2) to afford the desired compound (51.0 mg, 96% yield) as a
white solid. Rf (5:95 MeOH/CH2Cl2): 0.16. Mp: 56−58 °C. [α]D +44
(c 5.6, MeOH). FT-IR (neat, cm−1): 3259, 2923, 2854, 1578, 1467,
(q, 2H, J = 7.6 Hz), 1.53 (q, 2H, J = 6.8 Hz), 1.42−1.23 (m, 26H). 13C
NMR (100.6 MHz, CDCl3) δ in ppm: 172.4, 94.0, 83.9, 75.6, 75.3,
69.6, 63.2, 63.1, 33.8, 32.6, 30.9, 30.9, 30.8, 30.8, 30.7, 30.4, 29.8, 27.1.
+TOF MS calcd for C23H43NO6S m/z [M − H]+: 462.2884. Found:
463.2888. Calcd [M − Na]+: 484.2703. Found: 484.2684.
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]benzylsulfanyl-1,3-
oxazoline (24). The title compound was prepared following the
general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 14 (100 mg, 0.23 mmol),
MeONa (6.8 mg, 12 μmol), and MeOH (4.6 mL). The reaction
mixture was stirred at room temperature for 30 min. After standard
workup, the crude was purified by flash chromatography (1:9 MeOH/
CH2Cl2) to afford the desired compound (70.0 mg, 98% yield) as a
colorless syrup. Rf (1:9 MeOH/CH2Cl2): 0.16. [α]D +68 (c 1.0,
MeOH). FT-IR (neat, cm−1): 3345, 2923, 1579, 1495, 1453, 1296,
1144, 1113, 1045, 993, 949. 1H NMR (400 MHz, CD3OD) δ in ppm:
7.39−7.22 (stack, 5H), 5.78 (d, 1H, J = 7.2 Hz), 4.47 (dd, 1H, J = 7.2,
5.2 Hz), 4.29 (d, 1H, J = 13.4 Hz), 4.26 (d, 1H, J = 13.4 Hz), 3.74 (dd,
1H, J = 12.0, 2.8 Hz), 3.69 (dd, 1H, J = 12.0, 5.2 Hz), 3.65 (dd, 1H, J =
6.8, 5.2 Hz), 3.45 (dd, 1H, J = 8.8, 6.8 Hz), 3.30−3.26 (m, 1H). 13C
NMR (100.6 MHz, CD3OD) δ in ppm: 171.6, 138.1, 130.2, 129.9,
128.9, 94.1, 84.2, 75.6, 75.3, 69.6, 63.0, 36.8. +TOF MS calcd for
C14H17NO5S m/z [M − H]+: 312.0906. Found: 312.0895.
1,2-Dideoxy-α-D -glucopyranoside[1,2-d]-(6-(1-
adamantanecarboxamido)hexyl)sulfanyl-1,3-oxazoline (25).
The title compound was prepared following the general procedure
for acetyl deprotection of cis-1,2-fused 1,3-oxazoline carbohydrate
derivatives starting from 15 (36.9 mg, 0.06 mmol), MeONa (0.2 mg, 3
μmol), and MeOH (1.2 mL). The reaction mixture was stirred at
room temperature for 5 h. After standard workup, the crude was
purified by flash chromatography (2:98 MeOH/CH2Cl2) to afford the
desired compound (29.0 mg, 99% yield) as colorless syrup. Rf (1:1
AcOEt/hexane): 0. [α]D +0.54 (c 1.3, CD3OD). FT-IR (neat, cm−1):
3334, 2904, 2850, 1630, 1587, 1530, 1451, 1367, 1289, 1097. 1H NMR
(400 MHz, CD3OD) δ in ppm: 5.78 (d, 1H, J = 7.2 Hz), 4.47 (dd, 1H,
J = 7.2, 5.2 Hz), 3.78 (dd, 1H, J = 12.0, 2.8 Hz), 3.73 (dd, 1H, J = 12.0,
5.2 Hz), 3.69 (dd, 1H, J = 6.8, 5.2 Hz), 3.47 (dd, 1H, J = 9.2, 6.8 Hz),
3.32 (m, 1H, H-5), 3.15 (t, 2H, J = 7.0 Hz), 3.03 (t, 2H, J = 7.4 Hz),
1.98 (m, 3H), 1.81 (m, 6H), 1.76−1.65 (m, 8H), 1.50−1.24 (m, 6H).
13C NMR (100.6 MHz, CDCl3) δ in ppm: 181.0, 172.3, 94.0, 83.9,
75.6, 75.2, 69.6, 63.1, 41.9, 40.4, 40.3, 37.8, 32.5, 30.8, 30.5, 29.8, 29.4,
27.5. +TOF MS calcd for C24H38NO6S m/z [M − H]+: 483.2523.
Found: 483.2515. Calcd [M − Na]+: 505.2343. Found: 505.2335.
Inhibition Studies with Commercial Enzymes. Inhibition
constant (Ki) values were determined by spectrophotometrically
measuring the residual hydrolytic activities of the glycosidases against
the respective p-nitrophenyl α- or β-D-glycopyranoside, o-nitrophenyl
β-D-galactopyranoside (for β-galactosidases), or α,α′-trehalose (for
trehalase) in the presence of compounds 16−25. Each essay was
performed in phosphate buffer or phosphate−citrate buffer (for α- or
β-mannosidase and amyloglucosidase) at the optimal pH of each
enzyme. The reactions were initiated by addition of enzyme to a
solution of the substrate in the absence or presence of various
concentrations of inhibitor. The mixture was incubated for 10−30 min
at 37 or 55 °C (for amyloglucosidase), and the reaction was quenched
by addition of 1 M Na2CO3 or by heating and subsequent addition of a
solution of GLC-Trinder (Sigma, for trehalase). Reaction times were
appropiate to obtain 10−20% conversion of the substrate in order to
achieve linear rates. The absorbance of the resulting mixture was
determined at 405 or 492 nm (for trehalase). Approximate values of Ki
were determined using a fixed concentration of substrate (around the
Km value for the different glycosidases) and various concentrations of
inhibitor. Full Ki determinations and enzyme inhibition mode were
determined from the slope of Lineweaver−Burk plots and double
reciprocal analysis.
1
1352, 1289, 1163, 1099, 1046, 957. H NMR (400 MHz, CD3OD) δ
in ppm: 5.75 (d, 1H, J = 7.2 Hz), 4.44 (dd, 1H, J = 7.2, 5.2 Hz), 3.75
(dd, 1H, J = 12.0, 2.8 Hz), 3.70 (dd, 1H, J = 12.0, 5.2 Hz), 3.66 (dd,
1H, J = 6.8, 5.2 Hz), 3.44 (dd, 1H, J = 9.2, 6.8 Hz), 3.31−3.26 (m, 1H,
H-5), 2.99 (t, 2H, J = 7.6 Hz), 1.71−1.64 (m, 2H), 1.40−1.27 (m, 10
H), 0.87 (t, 3H, J = 7.2 Hz). 13C NMR (100.6 MHz, CD3OD) δ in
ppm: 171.7, 93.3; 83.2, 74.7, 73.6; 68.9, 62.4, 32.5, 32.0, 30.2, 29.8,
29.7, 29.2, 23.2, 13.9. +TOF MS calcd for C15H27NO5S m/z [M −
H]+: 334.1688. Found: 334.1672.
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]dodecylsulfanyl-
1,3-oxazoline (21). The title compound was prepared following the
general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 11 (49.17 mg, 0.10 mmol),
MeONa (2.7 mg, 4 μmol), and MeOH (1.7 mL). The reaction
mixture was stirred at room temperature for 30 min. After standard
workup, the crude was purified by flash chromatography (1:9 MeOH/
CH2Cl2) to afford the desired compound (36.0 mg, 97% yield) as
colorless syrup. Rf (5:95 MeOH/CH2Cl2): 0.26. [α]D +50 (c 1.0,
MeOH). FT-IR (neat, cm−1): 3300, 2919, 2850, 1573, 1468, 1349,
1292, 1163, 959. 1H NMR (400 MHz, CD3OD) δ in ppm: 5.8 (d, 1H,
J = 7.2 Hz), 4.47 (dd, 1H, J = 7.2, 5.2 Hz), 3.74 (dd, 1H, J = 12.0, 2.8
Hz), 3.69 (dd, 1H, J = 12.0 Hz, 5.2 Hz), 3.65 (dd, 1H, J = 6.8, 5.2 Hz),
3.43 (dd, 1H, J = 8.8, 6.8 Hz), 3.3−3.26 (m, 1H), 3.03 (t, 2H, J = 7.6
Hz), 1.71−1.63 (m, 2H), 1.39−1.25 (m, 18H), 0.92 (t, 3H, J = 7.6
Hz). 13C NMR (100.6 MHz, CD3OD) δ in ppm: 172.7, 94.3, 84.3,
75.9, 75.6, 69.9, 63.4, 33.6, 33.0, 31.3, 31.2, 31.1, 31.0, 30.7, 30.2, 24.2,
15.0. +TOF MS calcd for C19H35NO5S m/z [M − H]+: 390.2314.
Found: 390.2298.
1,2-Dideoxy-α-D-glucopyranoside[1,2-d]hexadecylsulfanyl-
1,3-oxazoline (22). The title compound was prepared following the
general procedure for acetyl deprotection of cis-1,2-fused 1,3-oxazoline
carbohydrate derivatives starting from 12 (83.7 mg, 0.15 mmol),
MeONa (0.5 mg, 9 μmol), and MeOH (3.0 mL). The reaction
mixture was stirred at room temperature for 20 h. After standard
workup, the crude was purified by flash chromatography (2:98
MeOH/CH2Cl2) to afford the desired compound (60.1 mg, 92%
yield) as a white solid. Rf (1:1 AcOEt/hexane): 0. Mp: 81−82 °C. [α]D
+0.58 (c 1.2, CD3OD). FT-IR (neat, cm−1): 3352, 2916, 2848, 1745,
1592, 1369, 1241, 1219, 1166, 1041. 1H NMR (400 MHz, CD3OD) δ
in ppm: 5.79 (d, 1H, J = 7.6 Hz), 4.47 (dd, 1H, J = 7.2, 5.2 Hz), 3.78
(dd, 1H, J = 12.0, 2.8 Hz), 3.73 (dd, 1H, J = 12.0, 5.2 Hz), 3.69 (dd,
1H, J = 6.8, 5.2 Hz), 3.48 (dd, 1H, J = 9.0, 6.8 Hz), 3.32 (m, 1H); 3.03
(t, 2H, J = 7.2 Hz), 1.72 (q, 2H, J = 7.6 Hz), 1.44−1.22 (m, 26H), 0.90
(t, 3H, J = 7.0 Hz). 13C NMR (100.6 MHz, CDCl3) δ in ppm: 172.7,
94.3; 84.3, 75.9, 75.6, 69.9, 63.4, 33.6, 33.0, 31.3, 31.2, 31.1, 31.0, 30.7,
30.2, 24.3, 15.0. +TOF MS calcd for C23H43NO5S m/z [M − H]+:
446.2940. Found: 446.2920.
1 , 2 - D i d e o x y - α - D - g l u c o p y r a n o s i d e [ 1 , 2 - d ] - ( 1 6 -
hydroxyhexadecyl)sulfanyl-1,3-oxazoline (23). The title com-
pound was prepared following the general procedure for acetyl
deprotection of cis-1,2-fused 1,3-oxazoline carbohydrate derivatives
starting from 13 (95.5 mg, 0.16 mmol), MeONa (0.6 mg, 10 μmol),
and MeOH (3.2 mL). The reaction mixture was stirred at room
temperature for 20 h. After standard workup, the crude was purified by
flash chromatography (2:98 MeOH/CH2Cl2) to afford the desired
compound (66.8 mg, 89% yield) as colorless syrup. Rf (1:1 AcOEt/
hexane): 0. [α]D +0.67 (c 1.2, CD3OD). FT-IR (neat, cm−1): 3397,
1
2918, 2849, 1745, 1587, 1464, 1158, 1219, 1039, 986. H NMR (400
Lysosomal Enzyme Activity Assay. Lysosomal enzyme activities
in cell lysates were determined as described previously.79,80 Briefly,
cells were scraped in ice-cold 0.1% Triton X-100 in water. After
centrifugation (6000 rpm for 15 min at 4 °C) to remove insoluble
materials, protein concentrations were determined using protein assay
MHz, CD3OD) δ in ppm: 5.78 (d, 1H, J = 7.0 Hz), 4.47 (dd, 1H, J =
7.0, 5.0 Hz), 3.77 (dd, 1H, J = 11.6, 2.8 Hz), 3.72 (dd, 1H, J = 11.6, 5.4
Hz), 3.69 (dd, 1H, J = 6.4, 5.2 Hz), 3.53 (t, 2H, J = 6.6 Hz), 3.48 (dd,
1H, J = 8.8, 6.4 Hz), 3.31 (m, 1H, H-5), 3.03 (t, 2H, J = 7.4 Hz), 1.71
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Journal of Medicinal Chemistry
Article
rapid kit (Wako, Tokyo, Japan). The lysates were incubated at 37 °C
with the corresponding 4-methylumbelliferyl β-D-glycopyranoside
solution in 0.1 M citrate buffer (pH 4). The librated 4-
methylumbelliferone was measured with a fluorescence plate reader
(exitation 340 nm; emission 460 nm; Infinite F500, TECAN Japan,
Kawasaki, Japan). For enzyme inhibition assay, cell lysates from
normal skin fibroblasts were mixed with the 4-methylumbelliferyl β-D-
glycopyranoside substrates in the absence or presence of increasing
concentrations of ABX or PSO 23.
Cell Culture and GCase Activity Enhancement Assay. Human
skin fibroblasts from a healthy and two Gaucher disease patients (with
N370S/N370S and L444P/L444P mutations) were maintained in our
laboratory with DMEM supplemented with 10% FBS as the culture
medium. For enzyme activity enhancement assay, cells were cultured
in the presence of different concentrations of ABX or PSO 23 or
DMSO alone (as a control) for 5 days and harvested by scraping.80,81
Cytotoxicity of the compounds was monitored by measuring the
lactate dehydrogenase activities in the cultured supernatants (LDH
assay kit, Wako, Tokyo, Japan).
(3) Wrodnigg, T. M.; Steiner, A. J.; Ueberbacher, B. J. Natural and
synthetic iminosugars as carbohydrate processing enzyme inhibitors
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ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental methods, experimental procedures,
compound characterization data, and NMR spectra for all
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(10) Wrodnigg, T. M.; Sprenger, F. K. Bioactive carbohydrates and
recently discovered analogues as chemotherapeutics. Mini-Rev. Med.
Chem. 2004, 4, 437−459.
́
(11) Benito, J. M.; García Fernandez, J. M.; Ortiz Mellet, C.
AUTHOR INFORMATION
Pharmacological chaperone therapy for Gaucher disease: a patent
review. Expert Opin. Ther. Pat. 2011, 21, 885−903.
■
Corresponding Author
(12) Parenti, G. Treating lysosomal storage diseases with
pharmacological chaperones: from concept to clinics. EMBO Mol.
Med. 2009, 1, 268−279.
*For Y.D.: phone, +34-977-559-556; e-mail, yolanda.diaz@urv.
cat. For C.O.M.: phone, +34-954-559-806; e-mail, mellet@us.
es.
(13) Wennekes, T.; van den Berg, R. J. B. H. N.; Boot, R. G.; van der
Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. F. G. Glycosphingolipids
nature, function, and pharmacological modulation. Angew. Chem., Int.
Ed. 2009, 8848−8869.
Notes
The authors declare no competing financial interest.
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storage disorders. Nat. Rev. Mol. Cell Biol. 2004, 5, 554−565.
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storage disorders: current techniques and future directions. Expert. Rev.
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(17) Alonzi, D. S.; Butters, T. D. Therapeutic targets for inhibitors of
glycosylation. Chimia 2011, 65, 35−39.
(18) Butters, T. D.; Dwek, R. A.; Platt, F. M. Inhibition of
glycosphingolipid biosynthesis: application to lysosomal storage
disorders. Chem. Rev. 2000, 100, 4683−4696.
(19) Zheng, W.; Padia, J.; Urban, D. J.; Jadhav, A.; Goker-Alpan, O.;
Simeonov, A.; Goldin, E.; Auld, D.; LaMarca, M. E.; Inglese, J.; Austin,
C. P.; Sidransky, E. Three classes of glucocerebrosidase inhibitors
identified by quantitative high-throughput screening are chaperone
leads for Gaucher disease. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
13192−13197.
(20) Marugan, J. J.; Zheng, W.; Motabar, O.; Southall, N.; Goldin, E.;
Westbroek, W.; Stubblefield, B. K.; Sidransky, E.; Aungst, R. A.; Lea,
W. A.; Simeonov, A.; Leister, W.; Austin, C. P. Evaluation of
quinazoline analogues as glucocerebrosidase inhibitors with chaperone
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(21) Asano, N.; Oseki, K.; Tomioka, E.; Kizu, H.; Matsui, K. N-
Containing sugars from Morus alba and their glycosidase inhibitory
activities. Carbohydr. Res. 1994, 259, 243−255.
ACKNOWLEDGMENTS
■
́
The Spanish Ministerio de Economia y Competitividad
(Contracts CTQ-2008-01569, CTQ2010-15848, and
SAF2010-15670), the Ministry of Health, Labour and Welfare
of Japan (H19-Nanji-Ippan-002, H22-Nanji-Ippan-002), and
the Japan Science and Technology Agency (AS232Z00009G)
are acknowledged. Cofinancing from the Fondo Europeo de
Desarrollo Regional (FEDER), the Fundacion
́
Ramon
and the Junta de Andalucia (P08-FQM-03711) are also
thanked. J.C. and R.R. thank the Ministerio de Educacion for
predoctoral fellowships. D.C. thanks CONACYT (Mexico) for
a grant.
́
Areces,
́
́
́
ABBREVIATIONS USED
■
LSD, lysosomal storage disorder; DNJ, 1-deoxynojirimycin; CS,
(+)-castanospermine; NNDNJ, N-(n-nonyl)-1-deoxynojirimy-
cin; DIX, 1,5-dideoxy-1,5-iminoxylitol; ER, endoplasmic
reticulum; GCase, β-glucocerebrosidase; PSO, cis-1,2-fused
pyranose−2-alkylsulfanyl-1,3-oxazoline; HSAB theory, hard−
soft acid−base theory; ABX, ambroxol
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