Conformationally-locked N -glycosides: Exploiting long-range non-glycone interactions in the design of pharmacological chaperones for Gaucher disease

Article abstract of DOI:10.1016/j.ejmech.2014.11.002

Pyranoid-type glycomimetics having a cis-1,2-fused glucopyranose-2-alkylsulfanyl-1,3-oxazoline (Glc-PSO) structure exhibit an unprecedented specificity as inhibitors of mammalian β-glucosidase. Notably, their inhibitory potency against human β-glucocerebrosidase (GCase) was found to be strongly dependent on the nature of aglycone-type moieties attached at the sulfur atom. In the particular case of υ-substituted hexadecyl chains, an amazing influence of the terminal group was observed. A comparative study on a series of Glc-PSO derivatives suggests that hydrogen bond acceptor functionalities, e.g. fluoro or methyloxycarbonyl, significantly stabilize the Glc-PSO:GCase complex. The S-(16-fluorohexadecyl)-PSO glycomimetic turned out to be a more potent GCase competitive inhibitor than ambroxol, a non glycomimetic drug currently in pilot trials as a pharmacological chaperone for Gaucher disease. Moreover, the inhibition constant increased by one order of magnitude when shifting from neutral (pH 7) to acidic (pH 5) media, a favorable characteristic for a chaperone candidate. Indeed, the fluoro-PSO derivative also proved superior to ambroxol in mutant GCase activity enhancement assays in N370S/N370S Gaucher fibroblasts. The results presented here represent a proof of concept of the potential of exploiting long-range non-glycone interactions for the optimization of glycosidase inhibitors with chaperone activity.

Full text of DOI:10.1016/j.ejmech.2014.11.002

European Journal of Medicinal Chemistry 90 (2015) 258e266
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Conformationally-locked N-glycosides: Exploiting long-range non-
glycone interactions in the design of pharmacological chaperones for
Gaucher disease
Javier Castilla a, Rocío Rísquez b, Katsumi Higaki c, Eiji Nanba c, Kousaku Ohno d,
,
,
*
f
Yoshiyuki Suzuki e, Yolanda Díaz a *, Carmen Ortiz Mellet b , Jose M. García Fernandez ,
ꢀ
ꢀ
a
ꢀ
Sergio Castillon
a
ꢁ
Department de Química Analítica i Química Organica, Universitat Rovira i Virgili, C/ Marcel·lí Domingo s/n, 43007 Tarragona, Spain
b
ꢀ
ꢀ
Departamento de Química Organica, Facultad de Química, Universidad de Sevilla, C/ Profesor García Gonzalez 1, 41012 Sevilla, Spain
c Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-cho, Yonago 683-8503, Japan
d Sanin Rosai Hospital, Yonago 683-8605, Japan
e Tokyo Metropolitan Institute of Medical Science, Tokyo 204-8588, Japan
f
ꢀ
Instituto de Investigaciones Químicas (IIQ), CSIC e Universidad de Sevilla, C/ Americo Vespucio 49, Isla de la Cartuja, 41092 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyranoid-type glycomimetics having a cis-1,2-fused glucopyranosee2-alkylsulfanyl-1,3-oxazoline (Glc-
Received 29 August 2014
Received in revised form
13 October 2014
Accepted 1 November 2014
Available online 4 November 2014
PSO) structure exhibit an unprecedented specificity as inhibitors of mammalian
their inhibitory potency against human -glucocerebrosidase (GCase) was found to be strongly depen-
dent on the nature of aglycone-type moieties attached at the sulfur atom. In the particular case of
b-glucosidase. Notably,
b
u
-
substituted hexadecyl chains, an amazing influence of the terminal group was observed. A comparative
study on a series of Glc-PSO derivatives suggests that hydrogen bond acceptor functionalities, e.g. fluoro
or methyloxycarbonyl, significantly stabilize the Glc-PSO:GCase complex. The S-(16-fluorohexadecyl)-
PSO glycomimetic turned out to be a more potent GCase competitive inhibitor than ambroxol, a non
glycomimetic drug currently in pilot trials as a pharmacological chaperone for Gaucher disease. More-
over, the inhibition constant increased by one order of magnitude when shifting from neutral (pH 7) to
acidic (pH 5) media, a favorable characteristic for a chaperone candidate. Indeed, the fluoro-PSO de-
rivative also proved superior to ambroxol in mutant GCase activity enhancement assays in N370S/N370S
Gaucher fibroblasts. The results presented here represent a proof of concept of the potential of exploiting
long-range non-glycone interactions for the optimization of glycosidase inhibitors with chaperone
activity.
Keywords:
Glycomimetic
Glycosidase inhibitor
Pharmacological chaperone
Gaucher disease
Glucocerebrosidase
Lysosomal storage disorders
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
neurodegenerative diseases [15,16]. On the other hand, compounds
stabilizing the proper folding of trafficking-incompetent mutant
The search for compounds capable of modulating the activity of
glycosidases, the enzymes that catalyze the hydrolysis of the
glycosidic bond in polysaccharides and glycoconjugates, represents
one of the more active research fields in glycobiology [1]. Glycosi-
dase inhibitors are fundamental tools to interrogate biological
processes involving biosynthesis, metabolism and recognition of
carbohydrates [2] and bear strong potential for the development of
drugs against associated pathologies [3], including cancer [4e8],
glycosidases at the endoplasmic reticulum (ER), thereby rescuing
them from degradation by the quality control system of the cell,
show high promise as pharmacological chaperones for the treat-
ment of lysosomal storage disorders [17e21] such as Gaucher
[22e27], Fabry [28e30] or GM1 gangliosidosis [31e34], formally
acting as effectors of the corresponding dysfunctional enzyme.
Someway counterintuitively, the glycosidase inhibitory and
chaperoning activities often coexist, the balance between them
being a function of concentration and relative binding affinities at
neutral (ER) and acidic pH (lysosome) [35].
diabetes
[9,10],
infection
[11e13],
ischemia
[14]
or
With few exceptions [36e40], most naturally occurring or de
* Corresponding authors.
E-mail addresses: yolanda.diaz@urv.cat (Y. Díaz), mellet@us.es (C. Ortiz Mellet).
novo
synthesized
glycosidase
inhibitors/chaperones
are
http://dx.doi.org/10.1016/j.ejmech.2014.11.002
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
259
carbohydrate-like derivatives (glycomimetics) in which the acetal
2. Results
group characteristic of glycosides has been modified while pre-
serving a hydroxylation pattern of stereochemical complemen-
tarity with the aglycone moiety of the putative glycosidase
substrate. Yet, recent work has shown the importance of imple-
menting non-glycone interactions to achieve glycosidase selectivity
levels within isoenzymes compatible with clinical applications
[41e47]. Most of the work in this sense has focused on nitrogen-
(iminosugars [48,49], sp2-iminosugars [50e58], azasugars [59,60])
and carbon-in-the-ring (carbasugars [61,62], cyclitols [63,64]) car-
bohydrate mimics. Surprisingly, aglycon effects for glycomimetics
keeping the pyranose core intact have been much less studied
[65e68], even though modifications at the glycosidic region has the
potential to be compatible with molecular diversity-oriented stra-
tegies with a relatively low synthetic cost [69].
2.1. Synthesis
ThepreparationofPSOglycomimeticswithahydroxylationprofile
matching that of -glucose and differing in the S-linked aglycon
D
portion relies on the S-alkylation reaction of the pivotal
glucopyranoso-based 1,3-oxazolidine-2-thione intermediate 1. Tak-
ing into consideration that the target enzyme GCase has been shown
to exhibit low discriminating capabilities between active site-binding
ligands differing in the configuration at C-4 for some glycomimetic
families [74], the corresponding
also initially considered. The methodology used for the synthesis of 1
and 2 started from tri-O-acetyl- -glucal (3) or - -galactal (5),
respectively, and involved epoxidation of the double bond with in situ
generated dimethyldioxirane. In the first case, the reaction afforded a
mixture of the
anhydrosugars 4 (
Epoxidation of 5 provided instead exclusively the
D
-galacto configured epimer 2 was
D
D
In a previous report [70], we developed a new family of
pyranoid-type glycomimetics having a glucopyranoso-based 2-
alkylsulfanyl-1,3-oxazoline structure (Glc-PSO, Fig. 1) behaving as
a-D
-gluco-and
-gluco/ -manno ratio 7:1) in 90% yield [75].
-epoxide 6 (
b-D-manno-configured tri-O-acetyl-1,2-
D
D
selective
b
-glucosidase inhibitors. PSO derivatives can be formally
a
a-D-
considered as conformationally locked N-glycosides, which war-
rant chemical and enzymatic stability. Their fused six-member-
eddfive-membered bicyclic skeleton, analogous to that of the
potent O-(N-acetylglucosaminidase) inhibitors NAG-thiazoline,
NButGT and thiamet-G [71] (Fig. 1), imposes a skew-boat confor-
mation to the pyranose ring, which has been found to impart
glycosidase transition state mimic character [72]. Structure-
chaperone activity relationship studies on fibroblasts from
Gaucher disease patients evidenced a strong impact of the nature of
exocyclic S-substituents of Glc-PSO glycomimetics on the mutant
galacto configuration) in an almost quantitative yield [75]. Reaction of
4 and 6 with potassium thiocyanate and catalytic amounts of
TiO(CH3CO2)2 led to the requested thionocarbamates 1 and 2 in 87
and 79% yield, respectively [76,77] (Scheme 1).
In view of the good GCase chaperon properties observed for the
D-
gluco-PSO derivative bearing an S-( -hydroxyhexadecyl) aglycon
u
moiety Glc-PSO-HHD (Fig. 1) and the significant contribution of the
terminal hydroxyl to this behavior [70], in this work we have chosen
to examine the incorporation of a series of u-substituted hexadecyl
chains bearing different terminal groups, including ester, carboxylic
acid, iodo and fluoro. These compounds were prepared using the
corresponding distal iodo derivatives as the alkylating agents. Methyl
16-iodohexadecanoate and 16-iodohexadecanoic acid were prepared
from the corresponding commerciallyavailable 16-bromo derivatives
by reaction with sodium iodide in acetone (see Supporting
Information). Given the ambident character of the thionocarbamate
functionality [78], the reaction conditions for the alkylation step had
to be carefully adjusted in order to warrant the alkylation reaction
regioselectively at the sulfur atom. Thus, compound 1 was treated
with a series of different alkyl iodides in dichloromethane in the
presence of triethylamine and catalytic amounts of N,N-dimethyla-
minopyridine (DMAP) or triazabicyclodecene (TBD) following the
work by Rollin et al. [79e82]. Reactions carried out under these soft
conditions afforded the expected acetyl-protected PSO derivatives
7e10 in52e87% yields (Table 1). Interestingly, reaction of1 with 1,16-
diiodohexadecane not only afforded the expected S-(16-
iodohexadecyl)sulfanyl derivative 8, but also the dialkylation prod-
uct 9, isolated in 32% yield (Table 1, entry 2). We also prepared the
lysosomal
b-glucosidase (b-glucocerebrosidase; GCase) effector
abilities. Notably, the S-(16-hydroxyhexadecyl) derivative (Glc-
PSO-HHD) was as efficient as the non-glycomimetic chaperone
candidate ambroxol (ABX), currently in pilot trials in humans [73],
for the N370S homozygous GCase mutation, the most prevalent for
this lysosomal storage disorder. The possibility of hydrogen
bonding involvement of the terminal hydroxyl group, once the
pyranoid ring sits in the active site of the enzyme, with an amino
acid residue located at an appropriate distance was advanced. To
test this hypothesis, we have now expanded the PSO family with
the preparation of a series of analogues keeping the hexadecyl
chain in the aglycone but modifying the terminal group or the
configuration of the core. The synthetic strategy and the evaluation
of the new compounds as glycosidase inhibitors and chaperone
candidates are reported.
corresponding
D-galacto-PSO derivative 11, bearing the S-(16-
hydroxyhexadecyl) antenna, to test the effect of the configurational
change on the inihibitory/chaperone properties.
The S-alkyl character of compounds 7e11 was confirmed by 13
C
NMR spectroscopy: the chemical shift for the quaternary sp2 carbon
atom at position 2 in the five-membered heterocycle varied from
roughly 190 ppm (eNeC]S in 1,3-oxazolidine-2-thiones 1 and 2)
to approximately 170 ppm (eN]CeSR in PSO derivatives 7e11).
This data is in accordance with analogous thionocarbamates
already reported in the literature [70].
The target fully-unprotected PSOA-glycomimetics 12e16 were
obtained in 81e100 % yield by final removal of the acetyl protecting
groups using methanol under standard NaOMe-catalyzed condi-
tions. In order to obtain derivative 17, precursor 7 was treated with
a solution of sodium hydroxide in methanol under similar reactions
conditions (Table 1, entry 2). The vicinal protoneproton coupling
constants about the pyranose ring both for the acetylated (7e11)
and the unprotected (12e17) PSO derivatives were in agreement
Fig. 1. General structure of Glc-PSO glycomimetics, structures of the related glycosi-
dase inhibitors NAG-thiazoline, NBuGT and thiamet-G and structure of the previously
reported Glc-PSO derivative Glc-PSO-HHD, which exhibited pharmacological chap-
erone abilities towards human glucocerebrosidase in Gaucher disease fibroblasts.

260
J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
Scheme 1. Synthesis of glycopyranoso-based 1,3-oxazolidine-2-thione derivatives 1 and 2.
with a skew-boat conformation close to 0S2 as previously observed
by us [70] and others [83,84] in structurally related bicyclic cis-1,2-
fused glucopyranose structures in solution.
the fluoro (15) and methoxycarbonyl (12) derivatives exhibit
comparable binding affinities, much higher that the iodo (13) or
carboxylic acid (17) partners. It is interesting to speculate that the
terminal group is acting as hydrogen bond acceptor and that opti-
mizing this long-range hydrogen bonding interaction may provide
a general strategy for the design of potent and specific GCase in-
hibitors/chaperones. The IC50 value for 15 is even lower as
compared to that encountered for the reference chaperone drug
2.2. Biological evaluation
The new PSO derivatives 12e17 were first screened as inhibitors
against a panel of commercial glycosidases including
(yeast), -glucosidase ( -Glcase; almonds and bovine liver, cyto-
solic), -mannosidase (Jack bean), -mannosidase (Helix pomatia),
trehalase (pig kidney), amyloglucosidase (Aspergillus niger), nar-
inginase ( -glucosidase/ -L-rhamnosidase, Penicillium decumbens),
-galactosidase (green coffee beans), -galactosidase (E. coli), and
amyloglucosidase (A. niger). All compounds behaved as competitive
inhibitors of the mammalian -Glcase and exhibited total selec-
a-glucosidase
ambroxol against GCase in a similar assay (4.1 mM) [70].
b
b
Compounds 12 and 15 were selected for further enzyme activity
enhancement assays in healthy and Gaucher fibroblasts from pa-
tients 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
a
b
b
a
a
b
b
tivity within the series of enzymes assayed; a unique signature of
the PSO family whose molecular basis is still unknown. The cor-
12 or 15, then lysed and the b-glucocerebrosidase activity deter-
mined using 4- methylumbelliferyl
substrate.
b-D-glucopyranoside as
responding inhibition constants (Ki) at the optimal pH of
(7.3) ranged from 5.5 to 26.4 M (Tables 2 and 3), similar to that
previously encountered for Glc-PSO-HHD (12 M) [70]. Interest-
b-Glcase
m
In normal cells, 12 and 15 had no effect on GCase activity. In
N370S/N370S Gaucher fibroblasts (Fig. 4) compound 12 had only a
m
ingly, the inhibition potency decreased from 2 to 11-fold when
moving from pH 7.3 to 5.5, suggesting that the neutral form of the
compounds is the most active species.
marginal effect (12% activity increase at 30
whereas 15 up-regulated the activity of the mutant enzyme in a
very significant manner (62% at 30 M), actually more efficiently
than the hydroxyl-bearing analogue Glc-PSO-HHD (55% at 90 M).
In a parallel assay, ABX led to a maximum activity enhancement of
33% at 10 M [70], with a decrease in the chaperone efficiency
when increasing the concentration to 30 M, meaning that the
inhibitory activity overcomes the chaperone effect. In the case of 15,
this situation occurred only at 60 M, indicating a more favorable
mM concentration),
m
Inhibition of bovine liver b-glucosidase is often used as a pre-
m
liminary parameter to select candidates as pharmacological chap-
erones for mutant human GCase associated with Gaucher disease.
Yet, the predictive character of the data must be taken with care:
although both enzymes are members of the GHA clan in the CAzY
classification [85], meaning that they share three-dimensional
structural similarities, they belong to different glycosyl hydrolase
families, namely GH1 and GH30, with only limited sequence sim-
ilarity [85]. Indeed, determination of the inhibition activity against
m
m
m
chaperone/inhibitor balance. Neither 12 or 15 nor ABX or Glc-PSO-
HHD were 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.
The ensemble of results supports the potential of the PSO scaf-
human GCase (pH 7.0) revealed that the D-Gal-PSO representative
16 was inactive, whereas a strong influence of the terminal func-
tional group at the hexadecyl aglycone chain was observed in the
Glc-PSO series, the inhibitory potency decreasing in the sense F (15;
fold for the design of selective
b-Glc inhibitors and active site-
directed pharmacological chaperones for Gaucher disease.
Although the observed activity enhancements do not surpass the
values reported for the most efficient iminosugar-type chaperones
[86], they are in principle medically useful. Most importantly, they
also provide a proof of concept that long-range non-glycone in-
teractions can be advantageously exploited to endow a rigid pyr-
anoid glycone moiety with high binding affinity and selectivity
toward a given glycosidase and suggest a key structural element for
the design of GCase chaperones. The current data also validate the
synthetic methodology for the purpose of structureeactivity rela-
tionship studies and pharmacological chaperone optimization.
IC50 3.9
230 M) > COOH (17; IC50 420
turned out to be inactive (Table 2). No inhibition of other lysosomal
enzymes, such as -glucosidase, -galactosidase, -galactosidase,
and -hexosaminidase, was observed, reproducing the selectivity
pattern already found in commercial enzymes. Most interestingly,
an about one-order-of-magnitude decrease in the GCase inhibition
strength was also observed at pH 5 (Table 2; see also Fig. 2 for the
most active compounds 15 and 12), a favorable feature for chap-
erone candidates.
m
M)
>
COOMe (12; IC50 33
mM) > I (13; IC50
m
mM). The dimeric derivative 14
a
a
b
b
The dramatic effect of the
u-substituent in the hexadecyl chain
on the ability of PSO derivatives to inhibit GCase, above two orders
of magnitude when comparing the fluoro and the carboxylic acid
groups, reinforces our initial hypothesis that PSO:GCase complexes
might be stabilized by a long-range interaction involving this ter-
minal group and an amino acid residue located at a maximum
distance of 22 Å from the catalytic site (Fig. 3). Comparing with Glc-
3. Experimental section
3.1. General methods
All chemicals were reagent grade and used as supplied unless
PSO-HHD (IC50 11.2
mM) [70], where the
u
-substituent is OH, only
otherwise specified. TiO(CF3COO)2 catalyst was prepared following

J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
261
Table 1
Synthesis of glycopyranoso-based 2-alkylsulfanyl-1,3-oxazoline derivatives.
Entry
1
Starting M.
Alkyl halide
S-alkylationa
Product
O-deprotectionb
Product
Yield (%)
52
Yield (%)
1
95
81
2
1
64
32
100
94
3
4
1
2
87
89
70
90
a
Carried out at rt during 24 h with 1.0 eq. substrate, 3.0 eq. alkyl halide, 3.0 eq. Et3N, and 2 mol% DMAP (or TBD) in CH2Cl2.
Carried out at rt during 12 h with 1.0 eq. of S-alkylated substrate and 5 mol% MeONa in MeOH.
b
a previously reported procedure [87]. 1H and 13C NMR spectra were
recorded on a Varian Mercury VX 400 (400 MHz and 100.6 MHz
respectively) and Varian 400-MR spectrometer in CDCl3 or CD3OD
0.00 ppm for 1H and 13C). 2D correlation spectra (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 melting point apparatus
Griffin and are uncorrected. Optical rotations were measured at
598 nm at 20 ꢀC in a PerkineElmer 241 MC apparatus with 10 cm
cells. IR spectra were recorded on a JASCO FT/IR-600 plus Fourier
Transform Infrared Spectrometer ATR Specac Golden Gate in the
Servei de Recursos Científics (SRCiT-URV). Reactions were moni-
tored by TLC carried out on 0.25 mm E. Merck Silica gel 60 F254
as solvents, with the solvent resonance (
(CDCl3 d 7.26 ppm for 1H, 77.23 ppm 13C; CD3OD
49.14 ppm for 13C) or using Me4Si as an internal reference (
d
) as the internal standard
d
d
3.31 ppm for 1H,
d
d
Table 2
Ki Values (
12e17.
mM) against Bovine Liver (Cytosolic) b
-Glucosidasea for Compounds
Enzyme
pH
12
13
14
11.8
15
16
5.5
17
Table 3
b
b
-Glcase (bovine liver)
-Glcase (bovine liver)
7.3
5.5
26.4
45
19.3
98
11.1
66
22.6
61
IC50 values (
mM) against human GCase for compounds 12e17.
139
62
a
Enzyme
pH
12
33
13
14
15
3.9
16
17
Inhibition was competitive in all cases. No inhibition was observed for any of the
-glucosidase, yeast -glucosidase, jack bean
-mannosidase, pig kidney trehalase, Aspergillus niger
amyloglucosidase, Penicillium decumbens naringinase, green coffee -galactosidase,
E. coli -galactosidase, or yeast isomaltase.
compounds at 2 mM on almonds
mannosidase, Helix pomatia
b
a
a-
GCase (Homo sapiens)
GCase (Homo sapiens)
7.0
5.0
230
>1000
NIa
NI
NI
NI
420
>1000
b
300
27
a
a
b
NI: no inhibition observed at 1 mM.

262
J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
completion of the reaction, the mixture was washed with saturated
NaHCO3 and brine, dried, and concentrated. Chromatographic pu-
rification afforded the S-alkylated compounds in the yields shown.
3.5. General procedure for acetyl deprotection of D-glycopyranoso-
based 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 comple-
tion 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.
Fig. 2. Effects of the PSO derivatives 15 and 12 on GCase activity in lysate from human
normal fibroblasts as a function of pH. Enzyme activity in normal cell lysates was
determined in the absence or presence of increasing concentrations of the 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 com-
3.6. 4,5-Dihydro-(3,4,6-tri-O-acetyl-1,2-dideoxy-a-D-
galactopyranoso)[1,2-d]-1,3-oxazol-2-thione (2)
pounds (100%). 4-Methylumbelliferyl b-D-glucopyranoside was used as substrate.
The title compound was prepared following the general proce-
dure for the synthesis of cis-1,2-fused 1,3-oxazolidine-2-thione
carbohydrate derivatives starting from 6 (810 mg, 2.81 mmol),
KSCN (642 mg, 8.43 mmol), TiO(CF3COO)2 (16.6 mg, 0.06 mmol) in
dry CH3CN (14 mL). The reaction mixture was stirred under reflux
for 2,5 h. Standard workup, followed by flash chromatography on
silica gel (2:1 AcOEt/hexane), afforded 2 (776 mg, 79% yield) as a
white solid. Data: Rf (4:1 AcOEt/hexane): 0.56. Mp: 52e55 ꢀC.
glass or aluminium 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 (230e400 mesh). Radial chromatography was performed on
1 or 2 mm plates of Kieselgel 60 PF254 silica gel, depending on the
amount of product.
[a
]
þ46 (c 1.38, CHCl3). FT-IR (neat)
y
in cmꢂ1: 3301, 2923, 2853,
1743, 1490, 1368, 1225, 1174, 1033. 1H NMR (400 MHz, CDCl3)
in
D
3.2. General procedure for the preparation of the 1,2-
anhydrosugars 4 and 6 [75]
d
ppm: 8.41 (bs,1H, ¼NH); 5.75 (d,1H, J1,2 ¼ 6.0 Hz, H-1); 5.56 (pt,1H,
J4,3 ¼ 3.2 Hz, J4,5 ¼ 3.6 Hz, H-4); 5.17 (dd, 1H, J3,2 ¼ 6.6 Hz,
J3,4 ¼ 3.2 Hz, H-3); 4.85 (pt, 1H, J2,1 ¼ 6.0 Hz, J2,3 ¼ 6.6 Hz, H-2); 4.37
The corresponding glycal 3 or 5 (1.00 mmol) was dissolved in an
ice bath cooled biphasic solution of CH2Cl2 (4 mL), acetone (0.4 mL)
and saturated aqueous NaHCO3 (6.5 mL). The mixture was vigor-
ously stirred and a solution of Oxone® (1.23 g, 2.00 mmol) in H2O
(5 mL) was added dropwise over 15 min. The crude reaction was
vigorously stirred at 0 ꢀC for 30 min and then allowed to warm to
room temperature until complete consumption of the glycal (TLC
monitoring). The organic phase was separated and the aqueous
phase was extracted with CH2Cl2 (2 ꢁ 4 mL). The combined organic
phases were dried over MgSO4, filtered and concentrated to afford
0
(ddd, J4,5 ¼ 3.6 Hz, J5,6 ¼ 7.4 Hz, J5,6 ¼ 5.8 Hz, 1H, H-5); 4.25 (dd,
0
0
J6,5 ¼ 7.4 Hz, J6,6 ¼ 11.6 Hz, H-6); 3.99 (dd, J6 ,5 ¼ 5.8 Hz,
J6 ,6 ¼ 11.6 Hz, H-60); 2.11 (s, 3H, AcO); 2.08 (s, 3H, AcO); 2.05 (s, 3H,
0
AcO). 13C NMR (100.6 MHz, CDCl3)
d in ppm: 189.8 (C]S); 170.9,
169.9, 168.7 (C(O), AcO); 82.1 (C-1); 81.0 (C-2); 70.7 (C-3); 70.4 (C-
5); 66.2 (C-4); 60.6 (C-6); 20.8, 20.7, 20.6 (CH3, AcO). þTOF MS Calcd
for
C
13H17NO8S m/z [MꢂNa]þ: 370.0567, found: 370.0584;
[2MꢂNa]þ: 717.1242, found: 717.1273.
the 1,2-anhydro-pyranoses 4 (90%, mixture
D-gluco/
D-manno 7:1) or
3.7. 2-(16-(Methoxycarbonyl)hexadecyl)sulfanyl-4,5-dihydro-
6 (98%, only -epoxide). The crude could not be purified due the
a
(3,4,6-tri-O-acetyl-1,2-dideoxy-a-D-glucopyranoso)[1,2-d]-1,3-
inherent instability of 1,2-anhydrosugars.
oxazole (7)
3.3. General procedure for the synthesis of D-glycopyranoso-based
1,3-oxazolidine-2-thione precursors 1 and 2 [76,77]
The title compound was prepared following the general proce-
dure for the S-alkylation of glycopyranoso-based 1,3-oxazolidine-
2-thione derivatives starting from 1 (41.3 mg, 0.12 mmol), methyl
To a stirred solution of the crude 1,2-anhydro-sugar 4 or 6
(1.00 mmol) and KSCN (3.00 mmol) in dry CH3CN (5 mL), finely
powdered TiO(CF3COO)2 (0.02 mmol) was added under an Ar at-
mosphere, and the mixture was heated to boiling for appropriate
time. After completion of the reaction (TLC monitoring), the
mixture was cooled to room temperature, H2O (10 mL) was added
and the resultant mixture was extracted with EtOAc (2 ꢁ 20 mL).
The combined organic extracts were dried over MgSO4, filtered and
concentrated. Flash chromatography on silica gel (ethyl acetate/
hexanes 2:1) gave the bycyclic derivatives 1 or 2 (87% and 79%,
respectively).
16-iodo hexadecanoate (141.4 mg, 0.36 mmol), Et3N (52
mL,
0.36 mmol), TBD (0.4 mg, 3 mol) and CH2Cl2 (1.5 mL). The reaction
m
mixture was stirred at rt for 24 h. After standard workup, the crude
was purified by flash chromatography (from 1:10 to 2:1 AcOEt/
hexane) followed by recrystallization from AcOEt-Hexane to afford
compound 7 (38.1 mg, 52% yield) as a white solid. Data: Rf (1:1
AcOEt/hexane): 0.74. Mp: 67e68 ꢀC. [
(neat)
1012, 883. 1H NMR (400 MHz, CDCl3)
a
]
D þ45 (c 3.13, CHCl3). FT-IR
in cmꢂ1: 2917, 2851,1734,1602,1372,1218,1110,1045,1034,
in ppm: 5.86 (d, 1H,
y
d
J1,2 ¼ 7.5 Hz, H-1); 5.18 (t, 1H, J3,2 ¼ J3,4 ¼ 4.1 Hz, H-3); 4.90 (dd, 1H,
J4,3 ¼ 4.1 Hz, J4,5 ¼ 8.1 Hz, H-4); 4.50 (ddd, 1H, J2,1 ¼ 7.5 Hz,
J2,3 ¼ 4.0 Hz, J2,4 ¼ 0.8 Hz, H-2); 4.26 (dd, 1H, J6,5 ¼ 5.5 Hz,
J6,6 ¼ 12.2 Hz, H-6); 4.15 (dd, 1H, J6 ,5 ¼ 3.1 Hz, J6 ,6 ¼ 12.2 Hz, H-60);
0
0
0
3.4. General procedure for the S-alkylation of D-glycopyranoso-
based 1,3-oxazolidine-2-thione derivatives
0
3.67 (ddd, J5,4 ¼ 8.1 Hz, J5,6 ¼ 5.5 Hz, J5,6 ¼ 3.1 Hz, 1H, H-5); 3.64 (s,
3H, MeO); 3.04 (t, 2H, J ¼ 7.4 Hz, CH2eS aliph); 2.28 (t, 2H,
J ¼ 7.8 Hz, CH2eC(O) aliph); 2.10 (s, 3H, AcO); 2.06 (s, 3H, AcO); 2.05
(s, 3H, AcO); 1.69 (quint, 2H, J ¼ 7.5 Hz, CH2 aliph.); 1.59 (quint, 2H,
J ¼ 7.4 Hz, CH2 aliph.); 1.38 (quint, 2H, J ¼ 7.3 Hz, CH2 aliph.);
To
a solution of the 1,3-oxazolidine-2-thione derivative
(1.00 mmol) in CH2Cl2 (3.5 mL), the corresponding alkyl iodide
(3.00 mmol), Et3N (3.00 mmol) and either DMAP or TBD (0.2 mmol)
were added followed by stirring at room temperature. After
1.35e1.22 (20H, CH2 aliph.). 13C NMR (100.6 MHz, CDCl3)
d in ppm:

J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
263
J ¼ 7.4 Hz, CH2 aliph); 1.41e1.20 (24H, CH2 aliph). 13C NMR
(100.6 MHz, CDCl3) d in ppm: 171.2 (C]N); 170.9,169.8,169.6 (C(O),
AcO); 92.5 (C-1); 77.1 (C-2); 70.2 (C-3); 68.0 (C-4); 67.4 (C-5); 63.5
(C-6); 32.4 (CH2eS); 30.7, 29.9, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3,
28.9, 28.8, 27.0 (CH2 aliph); 21.1, 21.0, 21.0 (CH3, AcO); 7.7
(CH2eI). þTOF MS Calcd for C29H48INO8S m/z [MꢂNa]þ: 720.2038,
Fig. 3. 3D Molecular model of Glc-PSO-HDD (carbons in grey, oxygens in red, nitrogen
in light blue, sulfur in yellow; hydrogens have been omitted for the sake of clarity). The
pyranose ring is in a 0S2 conformation and the hexadecyl chain has been set to the
more stable extended conformation. The distance between the anomeric nitrogen
atom and the terminal substituent in the chain, which remains identical in all the new
PSO derivatives prepared in this work, is indicated. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
found: 720.2049. Data for 9: Rf (1:1 AcOEt/hexane): 0.26. [
a
]
D þ1 (c
in cmꢂ1: 3460, 2923, 2852, 1741, 1590,
1368, 1224, 1147, 1111, 1038, 752. 1H NMR (400 MHz, CDCl3)
in
3.98, CHCl3). FT-IR (neat)
y
d
ppm: 5.88 (d, 2H, J1,2 ¼ 7.4 Hz, 2ꢁ H-1); 5.2 (pt, 2H, J3,2 ¼ 4.0,
J3,4 ¼ 4.2 Hz, 2ꢁ H-3); 4.93 (ddd, 2H, J4,2 ¼ 0.8 Hz, J4,3 ¼ 4.2 Hz,
J4,5 ¼ 8.8 Hz, 2ꢁ H-4); 4.52 (ddd, 2H, J2,1 ¼ 7.4 Hz, J2,3 ¼ 4.0 Hz,
0
J2,4 ¼ 0.8 Hz, 2ꢁ H-2); 4.29 (dd, 2H, J6,5 ¼ 5.4 Hz, J6,6 ¼ 12.2 Hz, 2ꢁ
H-6); 4.18 (dd, 2H, J6 ,5 ¼ 2.8 Hz, J6 ,6 ¼ 12.2 Hz, 2ꢁ H-60); 3.69 (m,
2H, 2ꢁ H-5); 3.06 (t, 4H, J ¼ 7.4 Hz, 2ꢁ CH2eS); 2.12 (s, 6H, 2ꢁ AcO);
2.09 (s, 6H, 2ꢁ AcO); 2.07 (s, 6H, 2ꢁ AcO); 1.72 (quint, 4H, J ¼ 7.4 Hz,
2ꢁ CH2 aliph); 1.43e1.20 (24H, CH2 aliph). 13C NMR (100.6 MHz,
0
0
CDCl3)
d
in ppm: 172.1 (2ꢁ C]N); 170.9, 169.8, 169.5 (2ꢁ C(O),
AcO); 92.5 (2ꢁ C-1); 77.1 (2ꢁ C-2); 70.2 (2ꢁ C-3); 68.0 (2ꢁ C-4);
67.4 (C-5); 63.5 (2ꢁ C-6); 32.4 (2ꢁ CH2eS); 29.8, 29.8, 29.8, 29.7,
29.4, 29.3, 28.9 (2ꢁ CH2 aliph); 21.0, 21.0, 21.0 (2ꢁ CH3, AcO). þTOF
MS Calcd for C42H64N2O16S2 m/z [MꢂNa]þ: 939.3589, found:
939.3565.
3.9. 2-(16-Fluorohexadecyl)sulfanyl-4,5-dihydro-(3,4,6-tri-O-
acetyl-1,2-dideoxy-a-D-glucopyranoso)[1,2-d]-1,3-oxazole (10)
The title compound was prepared following the general proce-
dure for the S-alkylation of glycopyranoso-based 1,3-oxazolidine-
2-thione derivatives starting from 1 (50.7 mg, 0.15 mmol), 1-fluoro-
Fig. 4. Effect of PSO glycomimetics 12 and 15 in GCase activity in N370S/N370S fi-
broblasts. 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
b-D-glucopyranoside as substrate. Each
16-iodohexadecane (138 mg, 0.37 mmol), Et3N (52 mL, 0.37 mmol),
bar 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).
TBD (0.4 mg, 0.03 mmol) and CH2Cl2 (0.5 mL). The reaction mixture
was stirred at rt for 24 h. After standard workup, the crude was
purified by flash chromatography (from 1:9 to 1:1 AcOEt/hexane)
to afford compound 10 (74.7 mg, 87% yield) as an off-white solid.
174.6 (C]N); 171.2, 170.9, 169.8, 169.5 (C(O), AcO); 92.5 (C-1); 77.1
(C-2); 70.2 (C-3); 68.1 (C-4); 67.5 (C-5); 63.5 (C-6); 51.6 (MeO); 34.3
(CH2eC(O)); 32.4 (CH2eS); 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3,
29.3, 28.9, 25.1 (CH2 aliph); 21.0, 21.0, 20.9 (CH3, AcO). þTOF MS
Calcd for C30H49NO10S m/z [MꢂNa]þ: 638.2969, found: 638.2976.
Data: Rf (1:1 AcOEt/hexane): 0.66. Mp: 51e52 ꢀC. [
CHCl3). FT-IR (neat)
1213, 1141, 1109, 1049. 1H NMR (400 MHz, CDCl3)
a
]
D þ42 (c 3.62,
in cmꢂ1: 2918, 2850, 1742, 1600, 1464, 1371,
in ppm: 5.88 (d,
y
d
1H, J1,2 ¼ 7.4 Hz, H-1); 5.20 (pt, 1H, J3,2 ¼ 3.8 Hz, J3,4 ¼ 4.2 Hz, H-3);
4.92 (dd, 1H, J4,3 ¼ 4.2 Hz, J4,5 ¼ 8.8 Hz, H-4); 4.52 (dd, 1H,
J2,1 ¼ 7.4 Hz, J2,3 ¼ 3.8 Hz, H-2); 4.44 (dt, 2H, J ¼ 6.0 Hz, JHF ¼ 47.3,
0
3.8. 2-(16-Iodohexadecyl)sulfanyl-4,5-dihydro-(3,4,6-tri-O-acetyl-
CH2eF aliph); 4.29 (dd, 1H, J6,5 ¼ 5.0 Hz, J6,6 ¼ 12.0 Hz, H-6); 4.17
0
0
1,2-dideoxy-a-D-glucopyranoso)[1,2-d]-1,3-oxazole (8) and 1,16-
(dd, 1H, J6 ,5 ¼ 2.8 Hz, J6 ,6 ¼ 12.0 Hz, H-60); 3.69 (ddd, J5,4 ¼ 8.8 Hz,
0
bis((4,5-dihydro-(3,4,6-tri-O-acetyl-1,2-dideoxy-
a-D-
J5,6 ¼ 5.0 Hz, J5,6 ¼ 2.8 Hz, 1H, H-5); 3.06 (t, 2H, J ¼ 7.4 Hz, CH2eS
glucopyranoso)[1,2-d]-1,3-oxazolyl)thio)hexadecane (9)
aliph); 2.12 (s, 3H, AcO); 2.09 (s, 3H, AcO); 2.07 (s, 3H, AcO);
1.77e1.60 (4H, CH2 aliph); 1.40e1.20 (24H, CH2 aliph). 13C NMR
The title compounds were prepared following the general pro-
cedure for the S-alkylation of glycopyranoso-based 1,3-
(100.6 MHz, CDCl3) d in ppm: 171.1 (C]N); 170.8, 169.8, 169.5 (C(O),
AcO); 92.5 (C-1); 84.4 (d, JCF ¼ 164.6 Hz, CH2eF); 77.1 (C-2); 70.2 (C-
3); 68.0 (C-4); 67.4 (C-5); 63.5 (C-6); 32.4 (CH2eS); 30.7, 30.5, 29.8,
29.8, 29.7, 29.7, 29.7, 29.7, 29.4, 29.3, 28.9, 25.3, 25.3 (CH2 aliph);
oxazolidine-2-thione derivatives starting from
0.10 mmol), 1,16-diiodohexadecane (130 mg, 0.27 mmol), Et3N
(38 L, 0.27 mmol), TBD (0.3 mg, 2 mol) and CH2Cl2 (0.5 mL). The
1 (37.3 mg,
m
m
21.0, 21.0, 20.9 (CH3, AcO). 19F NMR (376 MHz, CDCl3)
d in
0
reaction mixture was stirred at rt for 24 h. After standard workup,
the crude was purified by flash chromatography (from 1:9 to 1:2
AcOEt/hexane) to afford 8 (48.4 mg, 64% yield) as a colorless syrup
and 9 (31.6 mg, 32% yield) as an off-white syrup. Data for 8: Rf (1:1
ppm: ꢂ218.0 (tt, JFH ¼ 47.3 Hz, JFH ¼ 24.8, CH2eF Hz). þTOF MS
Calcd for C29H48FNO8S m/z [MꢂNa]þ: 612.2977, found: 612.2986.
3.10. 2-(16-Hydroxyhexadecyl)sulfanyl-4,5-dihydro-(3,4,6-tri-O-
AcOEt/hexane): 0.78. [
cmꢂ1: 2922, 2852, 1745, 1592, 1461, 1367, 1222, 1147, 1110, 1038. 1H
NMR (400 MHz, CDCl3)
a
]
þ42 (c 0.82, CHCl3). FT-IR (neat)
y
in
acetyl-1,2-dideoxy-a-D-galactopyranoso)[1,2-d]-1,3-oxazole (11)
D
d
in ppm: 5.89 (d, 1H, J1,2 ¼ 7.4 Hz, H-1); 5.2
The title compound was prepared following the general proce-
dure for the S-alkylation of glycopyranoso-based 1,3-oxazolidine-
2-thione derivatives starting from 2 (54.0 mg, 0.16 mmol), 16-
(pt, 1H, J3,2 ¼ 3.8, J3,4 ¼ 4.2 Hz, H-3); 4.93 (ddd, 1H, J4,2 ¼ 0.8 Hz,
J4,3 ¼ 4.2 Hz, J4,5 ¼ 8.8 Hz, H-4); 4.52 (ddd, 1H, J2,1 ¼ 7.4 Hz,
J2,3 ¼ 3.8 Hz, J2,4 ¼ 0.8 Hz, H-2); 4.29 (dd, 1H, J6,5 ¼ 5.2 Hz,
iodohexadecanol (172 mg, 0.47 mmol), Et3N (66 mL, 0.47 mmol),
J6,6 ¼ 12.2 Hz, H-6); 4.18 (dd, 1H, J6 ,5 ¼ 2.8 Hz, J6 ,6 ¼ 12.2 Hz, H-60);
3.70 (m, 1H, H-5); 3.19 (t, 2H, J ¼ 7.0 Hz, CH2eI); 3.06 (t, 2H,
J ¼ 7.4 Hz, CH2eS); 2.12 (s, 3H, AcO); 2.09 (s, 3H, AcO); 2.07 (s, 3H,
AcO); 1.82 (quint, 2H, J ¼ 7.2 Hz, CH2 aliph); 1.72 (quint, 2H,
TBD (0.4 mg, 0.03 mmol) and CH2Cl2 (0.5 mL). The reaction mixture
was stirred at rt for 24 h. After standard workup, the crude was
purified by flash chromatography (from 1:10 to 1:3 AcOEt/hexane)
to afford compound 11 (89 mg, 91% yield) as a colorless syrup. Data:
0
0
0

264
J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
Rf (1:1 AcOEt/hexane): 0.46. Mp: 32e33 ꢀC. [
CHCl3). FT-IR (neat)
in cmꢂ1: 3410, 2921, 2851, 1747, 1590, 1467,
1369, 1215, 1162, 1117, 1051, 753. 1H NMR (400 MHz, CDCl3)
in
a]
þ86 (c 2.50,
C-2); 74.8 (2ꢁ C-5); 74.4 (2ꢁ C-3); 68.9 (2ꢁ C-4); 62.4 (2ꢁ C-6);
32.4 (2ꢁ CH2eS); 30.5, 30.5, 30.4, 30.4, 30.3, 30.0, 29.5 (2ꢁ CH2
aliph). þTOF MS Calcd for C30H52N2O10S2 m/z [MꢂNa]þ: 687.2956,
found: 687.2904.9.
D
y
d
ppm: 5.85 (d, 1H, J1,2 ¼ 7.1 Hz, H-1); 5.43 (pt, 1H, J4,3 ¼ 3.0 Hz,
J4,5 ¼ 2.2 Hz, H-4); 4.96 (dd, 1H, J3,2 ¼ 7.3 Hz, J3,4 ¼ 3.0 Hz, H-3); 4.56
(pt, 1H, J2,1 ¼ 7.1 Hz, J2,3 ¼ 7.3 Hz, H-2); 4.22 (pdt, J5,4 ¼ 2.2 Hz,
3.13. 2-(16-Fluorohexadecyl)sulfanyl-4,5-dihydro-(1,2-dideoxy-a-
D-glucopyranoso)[1,2-d]-1,3-oxazole (15)
0
0
J5,6 ¼ 7.0 Hz, J5,6 ¼ 6.6 Hz, 1H, H-5); 4.15 (dt, 1H, J6 ,5 ¼ 6.6 Hz,
J6 ,6 ¼ 11.0 Hz, H-60); 4.12 (dd, 1H, J6,5 ¼ 7.0 Hz, J6,6 ¼ 11.0 Hz, H-60);
3.61 (t, 2H, J ¼ 6.6 Hz, CH2eOH aliph); 3.03 (m, 2H, CH2eS aliph);
2.11 (s, 3H, AcO); 2.05 (s, 3H, AcO); 2.03 (s, 3H, AcO); 1.67 (quint, 2H,
J ¼ 7.4 Hz, CH2 aliph); 1.61 (bs, 1H, OH); 1.54 (quint, 2H, J ¼ 6.9 Hz,
CH2 aliph); 1.40e1.20 (24H, CH2 aliph). 13C NMR (100.6 MHz, CDCl3)
0
0
The title compound was prepared following the general proce-
dure for acetyl deprotection of D-glycopyranoso-based 2-
alkylsulfanyl-1,3-oxazoline derivatives starting from 10 (44.3 mg,
0.08 mmol), MeONa (0.2 mg, 5 mmol) and MeOH (1 mL). The re-
d
in ppm: 170.7 (C]N); 170.7, 170.2, 170.1 (C(O), AcO); 93.9 (C-1);
action mixture was stirred at rt for 12 h. After standard workup, the
crude was purified by flash chromatography (from 0:1 to 1:9
MeOH/CH2Cl2) to afford compound 15 (24.3 mg, 70% yield) as a
white solid. Data: Rf (1:9 MeOH/CH2Cl2): 0.53. Mp: 72e73 ꢀC.
77.9 (C-2); 71.7 (C-3); 69.3 (C-5); 66.2 (C-4); 63.1 (CH2eOH); 61.4
(C-6); 32.9 (CH2 aliph); 32.4 (CH2eS); 29.8, 29.8, 29.7, 29.7, 29.6,
29.6, 29.2, 28.8, 25.9 (CH2 aliph); 20.9, 20.8, 20.8 (CH3, AcO). þTOF
MS Calcd for
610.3039.
C
29H49NO9S m/z [MꢂNa]þ: 610.3020, found:
[
a
]
þ21 (c 2.25, 2:1 CH3OH/CHCl3). FT-IR (neat)
y
in cmꢂ1: 3327,
D
2915, 2850, 1579, 1469, 1162, 1115, 720. 1H NMR (400 MHz, 2:1
CD3OD/CDCl3)
in ppm: 5.76 (d, 1H, J1,2 ¼ 7.2 Hz, H-1); 4.43 (dd, 1H,
d
3.11. 2-(16-Iodohexadecyl)sulfanyl-4,5-dihydro-(1,2-dideoxy-
glucopyranoso[1,2-d]-1,3-oxazole (13)
a
-D-
J2,1 ¼ 7.2 Hz, J2,3 ¼ 5.3 Hz, H-2); 4.39 (dt, 2H, J ¼ 6.4 Hz, JHF ¼ 41.0,
CH2eF aliph); 3.78 (dd, 1H, J6 ,5 ¼ 3.2 Hz, J6 ,6 ¼ 12.5 Hz, H-60); 3.74
(dd, 1H, J6,5 ¼ 4.6 Hz, J6,6 ¼ 12.4 Hz, H-6); 3.70 (dd, 1H, J3,2 ¼ 5.3 Hz,
J3,4 ¼ 7.3, H-3); 3.49 (dd, 1H, J4,3 ¼ 7.3 Hz, J4,5 ¼ 9.2 Hz, H-4); 3.35
0
0
The title compound was prepared following the general proce-
dure for acetyl deprotection of cis-1,2-fused D-glucopyranosed2-
alkylsulfanyl-1,3-oxazoline derivatives starting from 8 (30.3 mg,
0
(ddd, 1H, J5,4 ¼ 9.2 Hz, J5,6 ¼ 4.6 Hz, J5,6 ¼ 3.2 Hz, H-5); 2.79 (m, 2H,
CH2eS); 1.715e1.57 (4H, CH2 aliph); 1.40e1.20 (24H, CH2 aliph). 13
C
0.04 mmol), MeONa (0.2 mg, 3
mmol) and MeOH (1 mL). The re-
NMR (100.6 MHz, 2:1 CD3OD/CDCl3) d in ppm: 170.8 (C]N); 92.5
action mixture was stirred at rt for 12 h. After standard workup, the
crude was purified by flash chromatography (1:9 MeOH/CH2Cl2) to
afford compound 13 (24.8 mg, 100% yield) as a white solid. Data: Rf
(C-1); 84.0 (d, JCF ¼ 163.0 Hz, CH2eF); 82.1 (C-2); 73.8 (C-5); 73.6 (C-
3); 67.9 (C-4); 61.5 (C-6); 31.5 (CH2eS); 30.3, 30.1 29.4, 29.0, 28.9,
28.4, 24.9 (CH2 aliph). 19F NMR (376 MHz, CDCl3)
d
in ppm: ꢂ215.1
(1:9 MeOH/CH2Cl2): 0.53. Mp: 64e66 ꢀC. [
FT-IR (neat)
1163, 1117, 1073, 964. 1H NMR (400 MHz, 2:1 CD3OD/CDCl3)
a
]
D þ15 (c 1.35, CH3OH).
in cmꢂ1: 3304, 2915, 2859, 1579, 1469, 1350, 1292,
in
(tt, JFH ¼ 41.0 Hz, JFH ¼ 24.8, CH2eF Hz). þTOF MS Calcd for
0
y
C23H42FNO5S m/z [MꢂNa]þ: 486.2660, found: 486.2661.
d
ppm: 5.78 (d, 1H, J1,2 ¼ 7.0 Hz, H-1); 4.47 (dd, 1H, J2,1 ¼ 7.0 Hz,
3.14. 2-(16-Hydroxyhexadecyl)sulfanyl-4,5-dihydro-(1,2-dideoxy-
J2,3 ¼ 5.3 Hz, H-2); 3.78 (dd, 1H, J6 ,5 ¼ 2.8 Hz, J6 ,6 ¼ 12.0 Hz, H-60);
a-D-galactopyranoso)[1,2-d]- 1,3-oxazole (16)
0
0
0
0
3.72 (dd, 1H, J6 ,5 ¼ 5.2 Hz, J6 ,6 ¼ 12.0 Hz, H-6); 3.69 (dd, 1H,
J3,2 ¼ 5.3 Hz, J3,4 ¼ 7.0, H-3); 3.47 (dd, 1H, J4,3 ¼ 7.0 Hz, J4,5 ¼ 8.8 Hz,
H-4); 3.32 (m, 1H, H-5); 3.22 (t, 2H, J ¼ 7.0 Hz, CH2eI); 3.03 (t, 2H,
J ¼ 7.4 Hz, CH2eS); 1.79 (quint, 2H, J ¼ 7.2 Hz, CH2 aliph); 1.71
(quint, 2H, J ¼ 7.4 Hz, CH2 aliph). 1.45e1.28 (24H, CH2 aliph). 13C
The title compound was prepared following the general proce-
dure for acetyl deprotection of D-glycopyranoso-based 2-
alkylsulfanyl-1,3-oxazoline starting from 11 (45.2 mg, 0.08 mmol),
MeONa (0.3 mg, 5 mmol) and MeOH (1 mL). The reaction mixture
NMR (100.6 MHz, 2:1 CD3OD/CDCl3)
d
in ppm: 172.1 (C]N); 93.6
was stirred at rt for 20 h. After standard workup, the crude was
purified by recrystallization from MeOH to afford compound 16
(31.9 mg, 90% yield) as white crystals. Data: Rf (1:1 AcOEt/hexane):
(C-1); 83.4 (C-2); 75.0 (C-5); 74.9 (C-3); 69.1 (C-4); 62.7 (C-6); 34.5
(CH2 aliph); 32.4 (CH2eS); 31.3, 30.6, 30.5, 30.5, 30.4, 30.0, 29.5,
29.4 (CH2 aliph); 7.2 (CH2eI). þTOF MS Calcd for C23H42INO5S m/z
[MꢂNa]þ: 594.1721, found: 594.1701.
0.00. Mp: 90e92 ꢀC. [
(neat)
1300, 1161, 1119, 1057, 984. 1H NMR (400 MHz, 1:2 CD3OD/CDCl3)
a
]
þ32 (c 2.73, CH3OH/CHCl3 2:1). FT-IR
D
y
in cmꢂ1: 3487, 3299, 2919, 2851, 1744, 1590, 1468, 1374,
3.12. 1,16-Bis((4,5-dihydro-(1,2-dideoxy-
d]-1,3-oxazolyl)thio)hexadecane (14)
a
-D-glucopyranoso)[1,2-
d
in ppm: 5.60 (d, 1H, J1,2 ¼ 7.0 Hz, H-1); 4.34 (pt, 1H, J2,1 ¼ 7.0 Hz,
J2,3 ¼ 6.6 Hz, H-2); 3.75 (dd, 1H, J4,3 ¼ 3.2 Hz, J4,5 ¼ 2.0 Hz, H-4);
3.65e3.49 (stack, 3H, H-5, H-6, and H-60); 3.47 (dd, 1H, J3,2 ¼ 6.6 Hz,
J3,4 ¼ 3.2, H-3); 3.35 (t, 2H, J ¼ 7.0 Hz, CH2eOH aliph); 2.80 (m, 2H,
CH2eS aliph); 1.50 (quint, 2H, J ¼ 7.6 Hz, CH2 aliph); 1.33 (quint, 2H,
J ¼ 7.0 Hz, CH2 aliph); 1.25e1.00 (24H, CH2 aliph). 13C NMR
The title compound was prepared following the general proce-
dure for acetyl deprotection of D-glycopyranoso-based 2-
alkylsulfanyl-1,3-oxazoline derivatives starting from 9 (31.6 mg,
0.03 mmol), MeONa (0.1 mg, 2
mmol) and MeOH (1 mL). The re-
(100.6 MHz, 1:2 CD3OD/CDCl3) d in ppm: 170.7 (C]N); 92.8 (C-1);
action mixture was stirred at rt for 12 h. After standard workup, the
crude was purified by crystallization from MeOH to afford com-
pound 14 (21.6 mg, 94% yield) as a white solid. Data: Rf (1:9 MeOH/
82.1 (C-2); 73.0 (C-5); 71.3 (C-3); 67.4 (C-4); 62.1 (CH2eOH); 61.1 (C-
6); 32.3 (CH2eS); 31.5, 29.4, 29.4, 29.3, 29.3, 29.3, 29.0, 28.9, 28.4,
25.6 (CH2 aliph). þTOF MS Calcd for C23H43NO6S m/z [MꢂNa]þ:
484.2703, found: 484.2704.
CH2Cl2): 0.53. Mp: 125e126 ꢀC. [
FT-IR (neat)
1155, 1094, 997. 1H NMR (400 MHz, 2:1 CD3OD/CD3Cl)
a
]
D þ19 (c 3.10, 2:1 CH3OH/CHCl3).
in cmꢂ1: 3330, 2919, 2850, 1743, 1590, 1462, 1352,
in ppm:
y
d
3.15. 2-(15-Carboxyhexadecyl)sulfanyl-4,5-dihydro-(1,2-dideoxy-
5.78 (d, 2H, J1,2 ¼ 7.3 Hz, 2ꢁ H-1); 4.47 (dd, 2H, J2,1 ¼ 7.3 Hz,
a
-D-glucopyranoso)[1,2-d]- 1,3-oxazole (17)
0
0
J2,3 ¼ 5.4 Hz, 2ꢁ H-2); 3.79 (dd, 2H, J6 ,5 ¼ 3.2 Hz, J6 ,6 ¼ 12.0 Hz, 2ꢁ
H-60); 3.78e3.71 (stack, 3H, 2ꢁ H-3 and 2ꢁ H-6); 3.52 (dd, 2H,
J4,3 ¼ 7.0 Hz, J4,5 ¼ 9.0 Hz, 2ꢁ H-4); 3.35 (m, 2H, 2ꢁ H-5); 3.01 (td,
4H, J ¼ 7.0 Hz, J0 ¼ 1.9 Hz, CH2eS); 1.69 (quint, 4H, J ¼ 7.5 Hz, 2ꢁ CH2
aliph); 1.45e1.23 (24H, CH2 aliph). 13C NMR (100.6 MHz, 2:1
The title compound was prepared following the general proce-
dure for acetyl deprotection of D-glycopyranoso-based 2-
alkylsulfanyl-1,3-oxazoline derivatives starting from 7 (31.3 mg,
0.05 mmol), NaOH (2.4 mg, 0.06 mmol) and MeOH (1 mL). The
reaction mixture was stirred at rt for 12 h. After standard workup,
CD3OD/CD3Cl)
d
in ppm: 172.2 (2ꢁ C]N); 93.5 (2ꢁ C-1); 83.0 (2ꢁ

J. Castilla et al. / European Journal of Medicinal Chemistry 90 (2015) 258e266
265
the crude was purified by flash chromatography (5:95 MeOH/
CH2Cl2) to afford compound 17 (19.6 mg, 81% yield) as a white solid.
FBS as the culture medium. For enzyme activity enhancement
assay, cells were cultured in the presence of different concentra-
tions of Glc-PSO derivatives 12 and 15 or DMSO alone (as a control)
for 5 days and harvested by scraping [88,89]. Cytotoxicity of the
compounds was monitored by measuring the lactate dehydroge-
nase activities in the cultured supernatants (LDH assay kit, Wako,
Tokyo, Japan).
Data: Rf (1:9 MeOH/CH2Cl2): 0.94. Mp: 77e75 ꢀC. [
a
]
D þ44 (c 0.86,
in cmꢂ1: 3320, 2918, 2849, 1736,
1579, 1468, 1379, 1229, 1161, 1030. 1H NMR (400 MHz, CD3OD)
in
MeOH/CHCl3 2:1). FT-IR (neat)
y
d
ppm: 5.79 (d, 1H, J1,2 ¼ 7.4 Hz, H-1); 4.48 (dd, 1H, J2,1 ¼ 7.4 Hz,
J2,3 ¼ 5.2 Hz, H-2); 3.78 (dd, 1H, J6 ,5 ¼ 2.8 Hz, J6 ,6 ¼ 12.0 Hz, H-60);
0
0
0
0
3.73 (dd, 1H, J6 ,5 ¼ 5.2 Hz, J6 ,6 ¼ 12.0 Hz, H-6); 3.71 (dd, 1H,
J3,2 ¼ 5.2 Hz, J3,4 ¼ 6.8, H-3); 3.48 (dd, 1H, J4,3 ¼ 6.8 Hz, J4,5 ¼ 9.0 Hz,
H-4); 3.30 (m, 1H, H-5); 3.03 (t, 2H, J ¼ 7.6 Hz, CH2eS); 2.31 (t, 2H,
J ¼ 7.4 Hz, CH2eCO2Me); 1.72 (quint, 2H, J ¼ 7.4 Hz, CH2 aliph); 1.59
(quint, 2H, J ¼ 7.2 Hz, CH2 aliph). 1.45e1.28 (stack, 22H, CH2 aliph).
Acknowledgments
ꢀ
The Spanish Ministerio de Ciencia e Innovacion (contract
numbers DGI CTQ2011-22872-BQU) and SAF2013-44021-R), the
Ministry of Education, Culture, Science, Sports and Technology of
Japan (22390207 and 23591498) and the Ministry of Health, Labour
and Welfare of Japan (H17-Kokoro-019, H20-Kokoro-022) are
acknowledged. Cofinancing from the Fondo Europeo de Desarrollo
Regional (FEDER) and the Fondo Social Europeo (FSE), the
13C NMR (100.6 MHz, CD3OD)
d in ppm: 176.1 (C]O); 172.2 (C]N);
93.8 (C-1); 83.7 (C-2); 75.4 (C-5); 75.0 (C-3); 69.4 (C-4); 62.9 (C-6);
34.8 (CH2eCO2H); 32.4 (CH2eS); 30.7, 30.7, 30.7, 30.6, 30.6, 30.4,
30.2, 30.2, 29.6, 26.0 (CH2 aliph). þTOF MS Calcd for C23H41NO7S m/
z [MꢂNa]þ: 498.2496, found: 498.2509.
ꢀ
Fundacion Mahuer, and the Junta de Andalucía are also thanked.
3.16. Inhibition studies with commercial enzymes
K.H. was supported by Takeda Science Foundation. J.C. and R.R.
ꢀ
thank the Ministerio de Educacion for predoctoral fellowships.
Inhibition constant (Ki) values were determined by spectro-
photometrically measuring the residual hydrolytic activities of the
Appendix A. Supplementary data
glycosidases against the respective p-nitrophenyl
pyranoside, o-nitrophenyl -galactopyranoside (for
dases) or ’-trehalose (for trehalase) in the presence of
compounds 12e17. Each assay was performed in phosphate buffer
or phosphate-citrate buffer (for - or -mannosidase and amylo-
a- or
b-D
-glyco-
b
-D
b-galactosi-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.11.002.
a,a
a
b
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