(5aR)-5a-C-Pentyl-4-epi-isofagomine: A powerful inhibitor of lysosomal β-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B

Article abstract of DOI:10.1016/j.ejmech.2016.09.095

This report is about the identification, synthesis and initial biological characterization of derivatives of 4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal β-galactosidase. The two epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human β-galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the enzyme (IC₅₀?=?8?nM, 30× more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant β-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B.

Full text of DOI:10.1016/j.ejmech.2016.09.095

Accepted Manuscript
(5aR)-5a-C-pentyl-4-epi-isofagomine: A powerful inhibitor of lysosomal β-
galactosidase and a remarkable chaperone for mutations associated with GM1-
gangliosidosis and Morquio disease type B
Sophie Front, Anna Biela-Banaś, Patricie Burda, Diana Ballhausen, Katsumi Higaki,
Anna Caciotti, Amelia Morrone, Julie Charollais-Thoenig, Estelle Gallienne, Stéphane
Demotz, Olivier R. Martin
PII:
S0223-5234(16)30858-3
10.1016/j.ejmech.2016.09.095
EJMECH 8961
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2016
Revised Date: 26 September 2016
Accepted Date: 29 September 2016
Please cite this article as: S. Front, A. Biela-Banaś, P. Burda, D. Ballhausen, K. Higaki, A. Caciotti,
A. Morrone, J. Charollais-Thoenig, E. Gallienne, S. Demotz, O.R. Martin, (5aR)-5a-C-pentyl-4-epi-
isofagomine: A powerful inhibitor of lysosomal β-galactosidase and a remarkable chaperone for
mutations associated with GM1-gangliosidosis and Morquio disease type B, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.095.
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ACCEPTED MANUSCRIPT
Graphical abstract
R201C/H281Y mutant
16
14
12
10
8
1
6
4
2
0
A potent inhibitor of hGal (IC50 = 8 nM)
An effective chaperone for
0
0.016
0.08
0.4
2
10
several mutant hGal
Compound 1 (µM)
1

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(5aR)-5a-C-Pentyl-4-Epi-Isofagomine: a Powerful
Inhibitor of Lysosomal β-Galactosidase and a
Remarkable Chaperone for Mutations Associated with
GM1-Gangliosidosis and Morquio Disease Type B
Sophie Front a, Anna Biela-Banaś a, Patricie Burda b, Diana Ballhausen c Katsumi Higaki d
, ,
,f
Anna Caciotti e , Amelia Morrone e
,
Julie Charollais-Thoenig g
,
Estelle Gallienne a, Stéphane
Demotz g Olivier R. Martin a,*
,
a Université d’Orléans & CNRS, Institut de Chimie Organique et Analytique (ICOA), UMR 7311, Rue
de Chartres, 45067 Orléans, France
b Division of Metabolism and Children’s Research Center, University Children’s Hospital Zürich,
Zürich, Switzerland
c Center for Molecular Diseases, Lausanne University Hospital, 1005 Lausanne, Switzerland
d
Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori
University, Yonago, Japan
e
Neuroscience, Psychology, Pharmacology and Child Health Department, University of Florence,
Florence, 50139, Italy
f
Molecular and Cell Biology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience
Department, Meyer Children’s Hospital, Florence, 50139, Italy
1

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g DORPHAN, EPFL Innovation Park, 1015 Lausanne, Switzerland
* Corresponding author. E-mail address: olivier.martin@univ-orleans.fr (O. R. Martin)
ABSTRACT
This report is about the identification, synthesis and initial biological characterization of derivatives of
4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal β-galactosidase. The two
epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-
epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of
nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human β-
galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the
enzyme (IC50 = 8 nM, 30x more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for
this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1
showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant β-
galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell
lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-
pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-
gangliosidosis and Morquio disease type B.
Keywords: galactosidase inhibitors, GM1-gangliosidosis, Morquio disease type B, iminosugars,
pharmacological chaperones
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1. Introduction
GM1-gangliosidosis [1] and Morquio disease type B [2] are two clinically distinct lysosomal storage
disorders due to deficiency of the lysosomal enzyme β-galactosidase, which hydrolyzes terminal
galactose residues from cell constituents, including GM1-gangliosides, glycoproteins, oligosaccharides,
and the glycosaminoglycan keratan sulfate. Both diseases are inherited in an autosomal recessive
manner. A defective lysosomal enzyme results in the accumulation of its physiological substrates in
lysosomes, and more largely in cells, tissues, organs and body fluids. Depending on the mutations,
degradation of one or the other of the β-galactosidase substrates is more or less impaired. If
degradation of GM1-gangliosides is predominantly defective, the patients develop the symptomatology
of GM1-gangliosidosis, while accumulation of keratan sulfate is an indication for Morquio disease type
B. GM1-gangliosidosis is a neurodegenerative disorder with three clinical forms based on the age of
onset of symptoms, severity, and residual β-galactosidase activity. Morquio disease type B belongs to
the mucopolysaccharidoses, a group of inherited inborn errors of metabolism caused by defects in the
degradation of glycosaminoglycans. This disease presents clinical features overlapping those of
Morquio disease type A, but is the result of a different deficient enzyme (β-galactosidase vs. N-acetyl-
galactosamine-6-sulfatase).
Morquio disease type B is characterized by normal intelligence and skeletal dysplasia that results in
short stature and skeletal abnormalities. GM1-gangliosidosis and Morquio disease type B are very rare
diseases, with a prevalence in the range of 1 in 100’000 and 1 in 250’000 births, respectively [2]. For
GM1-gangliosidosis, this value is reported to be higher in some geographical regions, especially in
Brazil, with 6 new cases per 100’000 births [3]. As of today, both diseases remain orphan pathologies.
Patients are only offered palliative treatments, such as orthopedic surgery in the case of Morquio
disease type B.
Therapies relying on enzyme substitution, substrate reduction, gene replacement and
pharmacological chaperones constitute options for the treatment of lysosomal diseases [4].
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Pharmacological chaperones (PCs) offer the advantages that they can be designed to distribute into the
target organs, in particular the brain and the bones, and to be orally available [5]. In addition, as they
are low molecular weight compounds, they should be devoid of immunogenic properties and the cost of
manufacturing might be kept affordable. Finally, the recent development of 1-deoxygalactonojirimycin
(DGJ) as a pharmacological chaperone for α-galactosidase (Fabry disease) validates the clinical and
commercial viability of the pharmacological chaperone-based therapies for lysosomal storage diseases
[6].
Over the last decade, a few investigators reported the development of β-galactosidase PCs, the first
example being galactose [7]. One of the most investigated compounds, N-octyl-4-epi-β-valienamine
(NOEV), was already reported in 2003 by Suzuki and coworkers [8]. Importantly, their study showed
that this compound was therapeutically active in β-galactosidase-deficient transgenic mice with a
mutated form of the human β-galactosidase. This was the first in vivo evidence of the therapeutic
potential of PCs for the treatment of these two pathologies. Other β-galactosidase PCs were reported,
which were mainly exploiting the DGJ scaffold [9-12]. A bicyclic derivative of DGJ developed by
Garcia-Fernandez, Higaki and coworkers was also evaluated in the humanized GM1-gangliosidosis
mouse model. As observed with NOEV, this new compound was therapeutically active, albeit to a
lesser extent, which likely reflects the high concentrations required to restore β-galactosidase activity in
patient cells [12]. To our knowledge, none of the other β-galactosidase PCs was evaluated in disease
animal models, and none is currently undergoing further preclinical development.
Our report focus on the identification, synthesis and initial biological characterization of a derivative
of 4-epi-isofagomine (4-epi-IFG) as a potent and specific PC for the human lysosomal β-galactosidase
[13]. Previous studies by our group [14] and others [15] have shown that the substitution of an
iminosugar moiety with a group mimicking an aglycone, substantially improved the potency and the
specificity of iminosugar-based glycosidase inhibitors. Following this approach, 4-epi-IFG (Fig. 1) [16]
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was modified with a C5-alkyl chain at the C-5a position in either of the two possible configurations. In
one of them, the modification considerably increased the potency and the selectivity of this compound,
previously known as a moderate inhibitor of the human β-galactosidase, but lacking the desired
selectivity for development as a drug candidate. In addition, the resulting compound 1 (Fig. 1)
increased β-galactosidase enzyme activity in two-thirds of the tested GM1-gangliosidosis and Morquio
disease type B patient cell lines. Taken together, the results presented in this report indicate that
compound 1 constitutes a potential PC-based drug candidate for the treatment of GM1-gangliosidosis
and Morquio disease type B.
5-C-alkyl imino-L-ribitol
iminoribitol
5a
1
2
3
4
6
5
-galactoside
β
(5aR)-5a-C-pentyl-4-epi-IFG
(5aS)-5a-C-pentyl-4-epi-IFG
1
2
4-epi-IFG
Fig. 1. Structures of galactoside mimics
2. Results and discussion
2.1. Synthesis of iminosugars 1 and 2
We recently reported iminoribitol derivatives (Figure 1) as β-galactosidase inhibitors [17]. SAR
studies showed that adding an alkyl group next to the nitrogen atom in the imino- -ribitol structure
L
leads to a 400-fold improvement of its inhibitory activity on bovine galactosidase. No similar effect
was observed on the human enzyme. In these investigations we also observed that 4-epi-IFG was a
5

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much better inhibitor than iminoribitol. We therefore decided to investigate 4-epi-IFG derivatives
carrying a substituent at C-5a, hypothesizing that this modification would improve the activity of the
parent compound, as it was observed in the iminoribitol series. We therefore embarked on the synthesis
of (5aR)- and (5aS)-5a-C-pentyl-4-epi-IFG 1 and 2 (strictly (2R, 3R, 4S, 5R)- and (2S, 3R, 4S, 5R)-3-
hydroxymethyl-2-pentylpiperidine-4,5-diol). To our knowledge, there is only one example of a related
compound in the literature: 5a-C-methyl-4-epi-IFG was prepared from a bicyclic lactam in the course
of synthetic studies [18]. Very recently, preliminary results on 4-epi-IFG derivatives carrying a
functionalized alkyl group at C-5a were reported and the compounds were shown to be good β-
galactosidase inhibitors [19]. 5a-C-Alkyl-isofagomine derivatives (as glucoside mimics) have also been
synthesized and found to be potent inhibitors of β-glucocerebrosidase [20], the enzyme responsible for
Gaucher disease, and good candidates as PCs [21]. Our synthesis differs significantly from these
studies, as it is inspired from the concise synthesis of IFG developed by Andersch and Bols, which
used the addition of nitromethane to a pentopyranose-derived ketone as the key step [22]. Using a
longer chain nitroalkane, we planned to simultaneously introduce the nitrogen atom and the C-C5
fragment of the final molecule. A C5-chain was introduced as a good compromise to provide some
lipophilic character to the molecule without risking problems of toxicity that are common with longer
alkyl chains [23].
The synthesis started from D-lyxose (Scheme 1), which was converted to ketone 5 by adaptation of
known procedures [24,25]. The reaction of this ketone in neat nitrohexane in the presence of NEt3 gave
the branched-chain sugar derivative 6 in 52% yield; use of a co-solvent resulted in a significant drop in
yield. Interestingly the addition of nitrohexane was quite stereoselective, as it led to a 7:3 mixture of
the epimers at the nitromethine group, and a single configuration at the ring carbon atom. The C-4(R)
configuration was indeed favored by both kinetic (addition to the less hindered face of the ketone) and
thermodynamic factors (equatorial disposition of the nitrohexyl group). Two consecutive catalytic
hydrogenations promoted the reduction of the nitro function, the cleavage of the benzyl group, and an
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intramolecular reductive amination, leading to an inseparable mixture of two iminosugars, epimeric at
C-5a (8) and still bearing a hydroxyl group at position C-5.
a
b
c
d
D
-Lyxose
3
4
5
1
4
e
f
2
3
5a
5
6
6
7
8
Scheme 1. Reagents and conditions: (a) BnOH, PTSA, 60 °C, 78 h, 78%; (b) 2,2-dimethoxypropane,
acetone, PTSA, rt, 18 h, 64%; (c) Dess-Martin periodinane, CH2Cl2, rt, 16 h, 99%; (d) 1-nitrohexane,
NEt3, rt, 4 d, 52%; (e) H2, Raney Ni, AcOH, iPrOH, rt, 5 d, 85%; (f) H2, Pd(OH)2/C, AcOH, iPrOH, rt,
3 d, quant.
In order to remove this group, dehydration of branched nitroaldol 6 was attempted under various
conditions, including acetylation/elimination according to literature procedures [26], but these reactions
were unsuccessful. Finally, reductive cleavage of the tertiary alcohol function of 8 could be achieved
under radical conditions by way of a cyclic thiocarbonate (Scheme 2). Preliminary protection of the
nitrogen atom in 8 as a benzyl carbamate allowed the separation of the isomers 9 and 10. On both
isomers, the cyclic thiocarbonate was prepared efficiently and radical deoxygenation was realized with
Bu3SnH, which gave the expected product containing a tertiary C-H bond [27]. Interestingly, the
reaction of thiocarbonate 11 led to a single compound, 12, precursor of the desired (5aR)-5a-C-pentyl-
4-epi-IFG 1, whereas compound 14 gave a separable mixture of the two iminosugars 15 and 16, which
were later identified as the precursors of (5aS)-5a-C-pentyl-4-epi-IFG 2 and its epimer at C-5 (19).
Hydrogen transfer at C-5 anti with respect to the substituents at C-4 and C-5a appeared to be highly
favored in the case of 11, whereas the transfer became less stereoselective when the substituents at C-4
and C-5a were trans as in 14. Final deprotection steps were conducted on each epimer, providing thus
7

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the three isomeric products 1, 2 and 19. NMR analyses of the three compounds unambiguously
confirmed their configuration at C-5 and C-5a.
b
c
d
d
d
e
a
R
11
12
15
16
13
17
18
1
epimer 5a
9,
e
8
c
b
2
e
S
14
, epimer 5a
10
19
Scheme 2. Reagents and conditions: (a) benzyl chloroformate, DIPEA, EtOH, rt, 16 h, 9: 18%, 10:
31%; (b) thiocarbonyldiimidazole, THF, reflux, 4 h, 11: 86%, 14: 91%; (c) Bu3SnH, AIBN, toluene,
reflux, 16 h, 12: 60%, 15: 38%, 16: 43%; (d) H2, 10% Pd/C, iPrOH, rt, 13: 96%, 17: 49%, 18: 67%; (e)
Dowex 50WX8, dioxane/H2O (5:3), rt, 18h, 1: 84%, 2: 86%, 19: quant.
2.2. Inhibition studies on human β-galactosidase
Compound 1 was first evaluated for its capacity to inhibit the human β-galactosidase activity. Using
lysate of human peripheral blood mononuclear cells (PBMC) as a source of enzyme, it was found that
the IC50 of 1 was 8 nM at pH 7.3 (Table 1). By comparison, the parent compound, 4-epi-IFG, had an
IC50 of 240 nM, indicating that the introduction of a pentyl chain at position C-5a resulted in a 30-fold
increase in potency. Epimers 2 and 19 were also tested for their inhibitory capacity of the human β-
galactosidase. Remarkably, 5,5a-trans isomer 2 was found to be 1600-fold less active than all-cis
isomer 1 and the double epimer of 1 at C-5 and C-5a, compound 19, was only weakly active on the
8

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enzyme (IC50 >150 µM), illustrating the exquisite specificity of the human β-galactosidase for structure
1.
Table 1. Inhibitory activities of 4-epi-IFG derivatives towards human β-galactosidase
IC50 (µM)
H
N
OH
OH
OH
4-epi-IFG
0.24 ± 0.015
12 ± 2.8
1
2
19
>150
nda
pH 7.3
pH 4.3
0.008 ± 0.0004
0.18 ± 0.019
13 ± 4.7
nda
a not determined
The selectivity of 1 was next evaluated on a set of human glycosidases. As for the β-galactosidase,
assays were established using PBMC lysate, as a source of human enzyme, and a corresponding
chromogenic or fluorogenic substrate. A compound blocking one of the glycosidase activities was
included in each assay, as a validation of the different enzymatic tests. Five of the 6 glycosidases tested
were not inhibited by compound 1 up to 160 µM. The β-glucosidase was the only glycosidase weakly
blocked by this compound, with an IC50 of 100 µM, indicating that compound 1 was considerably less
active on the β-glucosidase than on the β-galactosidase (Table 2). Overall, compound 1 was found
exquisitely selective for human β-galactosidase, as it was not active on a set of closely related
glycosidases.
Table 2. Inhibitory activities towards a set of human glycosidases
IC50 (µM) IC50 (µM)
Structure
Human enzymesa
compound reference
reference compound
compound
1
9

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0.01
0.24
4-epi-IFG
DGJ
β-galactosidase
α-galactosidase
> 160
0.08
0.05
> 160
100
DNJ
α-glucosidase
β-glucosidase
30
Conduritol B epoxide
b
O H
O
H
N
> 160
> 160
0.1
15
α-hexosaminidase
β-hexosaminidase
HO
NHAc
O H
c
a Human PBMC lysates were used as the source of glycosidase activities.
b DORPHAN’s proprietary inhibitor [28].
c Ref. [29]
2.3. Heat denaturation of β-galactosidase
The capacity of a compound to protect an enzyme from denaturation by heat and chemical treatments is
regarded as predictive of its pharmacological chaperone activity [30]. Compound 1 was evaluated for
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its capacity to protect β-galactosidase from heat-induced inactivation. β-Galactosidase activity in
human PBMC lysates was reduced by half following heat exposition at 48 oC for 3 min. In the presence
of 20 and 200 nM of compound 1, the same effect was obtained after 4 and 17 min at 48oC,
respectively. At higher concentrations of compound 1, the level of β-galactosidase activity remained
o
above 50% over at least 40 min at 48 C, illustrating the remarkable capacity of this compound to
stabilize the native structure of the β-galactosidase protein (Fig. 2).
Fig. 2. Protection of β-galactosidase against heat denaturation by compound 1. The concentrations of
compound 1 were 0 (squares), 20 nM (triangles), 200 nM (diamonds) and 2 µM (circles).
2.4. Evaluation of chaperone activity in treated fibroblasts from GM1-gangliosidosis and
Morquio disease type B patients
It was next investigated whether compound 1 had the capacity to restore β-galactosidase activity in
cells from GM1-gangliosidosis and Morquio disease type B patients. Following an experimental design
applied in several studies [7,9,11,31], patient fibroblasts were first cultured for 5 days in the presence
of increasing concentrations of compound 1. β-Galactosidase and β-hexosaminidase activities were
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then determined in cell lysates. β-Hexosaminidase activity was used as a means to assess cell viability
and cell number between cultures. Figure 3 presents the results obtained with fibroblasts derived from a
GM1-gangliosidosis patient bearing the R201C/H281Y mutations. The β-galactosidase activity was
enhanced with increasing concentrations of compound 1; exposure to 0.08, 0.4 and 2 µM of compound
1 resulted in a 7, 12 and 15-fold activity increase of β-galactosidase respectively. By contrast, the β-
hexosaminidase activity remained stable over the range of concentrations tested (Fig. 3). These
observations indicated that compound 1 had the capacity to specifically induce the recovery of β-
galactosidase activity in patient cells.
Fig. 3. Chaperone activity on fibroblasts from a GM1-gangliosidosis patient. Results presented are
means of 4 values obtained from independent cultures.
These studies were then extended to a panel of 22 patient cell lines. As the chaperone effect was
observed in numerous tests over a broad concentration range, Table 3 presents a compilation of the β-
galactosidase activity values induced by concentrations of compound 1 ranging between 2 and 10 µM,
concentrations at which maximal fold enhancements were usually observed. Although the set of cell
lines represents only a limited fraction of the mutations reported thus far, our observations indicate that
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mutations associated with the different forms of GM1-gangliosidosis (infantile, juvenile and late onset),
as well as with Morquio disease type B, were responsive to compound 1. Overall, increased β-
galactosidase activity was detected in the cell lines carrying missense mutations while, as expected, no
rescue of activity was detected in the cell lines carrying a null allele at homozygous level (patients 1
and 17). The chaperone effect was heterogeneous across different genetic backgrounds, from a modest
1.5-fold up to a 35-fold enhancement of the β-galactosidase activity. As residual enzyme activity in the
patient cells is very variable, from less than 1% up to 5-10%, it was expected that the resulting induced
recovery of glycosidase activity would span a broad range, from a few percent to more than 50% of the
level found in cells from healthy individuals. Remarkably, in half of the cell lines, the recovery reached
at least 10% of the level found in normal cells, a value believed to represent a threshold above which a
therapeutic benefit might be obtained [32,33].
Table 3. Enhancement of β-galactosidase activity in fibroblasts of GM1-gangliosidosis and Morquio
disease type B patients following treatment with compound 1
Maximal fold Residual Restored
Patient Disease/Phenotype
Mutation
enhancementa activityb activityc
1
2
3
4
5
6
7
8
9
GM1-gangliosidosis/infantile
GM1-gangliosidosis/adult
Homozygous insertion 733+2T>C
I51T/I51T
1.1
1.6
1.4
1.1
1.1
1
11
15
5.4
18
3.5
6.7
4.7
4.4
20
35
1
0.25
4d
0.28
6.4
0.7
nd
0.9
0.4
11
75
11
nd
23
GM1-gangliosidosis/infantile
GM1-gangliosidosis/infantile
GM1-gangliosidosis/infantile
GM1-gangliosidosis/infantile
GM1-gangliosidosis/late infantile
GM1-gangliosidosis/juvenile
GM1-gangliosidosis/juvenile
R59H/R59H
R59H/R59H
C127Y/W161G
R148S/S532G/L305F
S191N/R351Term
R201C/R201C
R201C/R201C
R201C/H281Y
R201H/c.247dup1
R201H/G76E
R201H/H281Y
R208C/IVS10+1 G>A
Q255H/K578R
H281Y/splicing
R351Ter/R351Ter
G438E/G438E
0.5
nd
0.8
0.4
1
5
2d
10 GM1-gangliosidosis/juvenile
11 GM1-gangliosidosis/juvenile
12 GM1-gangliosidosis/juvenile
13 GM1-gangliosidosis/juvenile
14 GM1-gangliosidosis/infantile
15 GM1-gangliosidosis/infantile
16 GM1-gangliosidosis/infantile
17 GM1-gangliosidosis/infantile
18 GM1-gangliosidosis/juvenile/adult
19 GM1-gangliosidosis/adult
20 GM1-gangliosidosis/adult
nd
6.7
2.3
4.6
nd
2
2.6
0.3
3.4
3.2
4d
15
22
nd
40
91
0.3
6.8
4.8
29
2
1.5
7.3
R442Q/W92Term
R457Q/R457Q
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21 GM1-gangliosidosis/infantile
22 Morquio B
23 Morquio B
P549L/P549L
W273L/R482H
W273L/W509C
1.3
1.5
1.5
0.8
6.5
6.5
1
10
10
a Fold enhancement was determined following treatment of the cells for up to 5 days in the presence or
the absence of compound 1. The maximal enhancement obtained, up to the maximal concentration of
compound tested (10 µM), is presented.
b
The values indicate the β-galactosidase activity (in %) in fibroblasts of untreated patient cells as
compared with the activity in cells from healthy individuals
c The values are calculated by multiplying the fold enhancement factor with the residual activity
d Data from [31]
nd: not determined
2.5. pH dependence of β-galactosidase inhibition
Compound 1 was found to have significant chaperone activity on an array of β-galactosidase mutants.
The tight binding of compound 1 to β-galactosidase is believed to induce proper folding of the newly
synthesized protein in the endoplasmic reticulum, and to rescue it from early degradation before
trafficking to the lysosomes. Once the protein has reached the lysosome, it is desirable that the
inhibitory activity would be reduced in the acidic environment of this organelle. Compound 1 was
found indeed to have appropriate pH dependence as a galactosidase inhibitor (Table 1). At a pH = 7.3,
mimicking the endoplasmic reticulum medium, the compound was a strong inhibitor of human β-
galactosidase, with an IC50 = 8 nM. The IC50 value was found to increase progressively with decreasing
pH values, to reach an IC50 = 180 nM at pH 4.3, a value 22-fold higher than at pH 7.3 (Table 1); the
catalytic rate of the enzyme was 4-fold higher than at neutral pH. As a result of this pH dependence,
compound 1 dissociates readily from the enzyme in the acidic environment of the lysosomes and thus
does not prevent its action on lysosomal substrates.
3. Conclusions
(5aR)-5a-C-Pentyl-4-epi-IFG 1 and its (5aS)-epimer 2 were synthesized from D-lyxose and evaluated as
inhibitors of human lysosomal β-galactosidase. The all-cis, (5aR)-epimer was found to be a very potent
14

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inhibitor of this enzyme (IC50 = 8 nM), much more potent than the (5aS)-epimer and the parent 4-epi-
isofagomine (1600x and 30x respectively), thus revealing fine structure-activity relationship in this
family of 1-N-iminosugar derivatives. Compound 1 behaved as a remarkable PC, considering it is
capable of restoring β-galactosidase activity and expression in two-thirds of the tested GM1-
gangliosidosis and Morquio disease type B patient cell lines, with enhancement of residual activities up
to 35-fold, and restoration of enzyme activity up to 91%. In addition its capacity to dissociate readily
from the enzyme in an acidic environment indicated that compound 1 constitutes a promising PC-based
drug candidate for the treatment of these two severe lysosomal storage disorders. Further investigations
in this direction are in progress and will be reported in due course.
4. Experimental section
4.1. Chemistry.
Materials and methods: All reactions requiring anhydrous conditions were carried out using oven-
dried glassware under an atmosphere of dry Ar. THF was dried through a column containing activated
alumina under nitrogen pressure (Dry Solvent Station GT S100, GlassTechnology, Geneva,
Switzerland). CH2Cl2 was distilled from P2O5. All reagent-grade chemicals and anhydrous toluene were
obtained from commercial suppliers and were used as received. High resolution mass spectra were
1
13
recorded on a Bruker Q-TOF MaXis spectrometer. H and C NMR spectra were recorded on Bruker
Avance II (250 MHz), Bruker Avance III HD nanobay (400 MHz) or Bruker Avance III (600 MHz)
spectrometers. Chemical shifts are given in ppm and are referenced to the residual solvent signal or to
1
TMS as internal standard. Carbon multiplicities were assigned by DEPT experiments. H and 13C
signals were attributed on the basis of H-H and C-H correlations. Spectral splitting patterns are
designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; br, broad. The purity
of tested iminosugars was shown to be ≥96% by HPLC (VWR Hitachi LaChrom Elite system) using a
Nucleodur Sphinx RP column (Macherey-Nagel, 150 mm x 4.6 mm, particle size: 5 µm) and a SEDEX
15

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85 LT-ELSD detector (SEDERE). A gradient of solvent from a 98:2 mixture of H2O/MeOH containing
0.5% of formic acid to pure MeOH containing 0.5% of formic acid in 30 min was used (flow rate: 1
mL/min). Specific rotations were measured in a 1-dm cell with a Perkin-Elmer 341 polarimeter.
Melting points were determined in capillary tubes with a Büchi apparatus. Analytical thin layer
chromatography was performed using Silica Gel 60F254 precoated plates (Merck) with visualization by
UV light and ceric sulfate/ammonium molybdate solution (0.5 g/0.24 g in 500 mL of 1M aqueous
H2SO4). Flash chromatography was performed on Silica Gel 60 (40-63 µm, Merck).
Numbering schemes for NMR data:
7
1
5
5
1
2
4
2
4
3
3
6
6
7
Benzyl α-
D
-lyxopyranoside (3). To a solution of
D
-lyxose (20.0 g, 0.133 mol) in benzyl alcohol (37
mL) was added PTSA (210 mg, 1.10 mmol, 0.008 eq). The mixture was heated at 60 °C for 78 h.
Toluene (10 mL) was added and the resulting mixture was concentrated under vacuum and co-
evaporated with toluene (3 x 30 mL) to give a soft white solid, which was then suspended in a mixture
of hexane/CH2Cl2 (2:1) (180 mL). The solid was filtered and washed thoroughly with cold Et2O. The
filtrate was concentrated under vacuum and the procedure was repeated three times to give the desired
1
compound 3 (24.8 g, 78%) as a white solid. H NMR spectrum is in accordance with literature data
[24].
Benzyl 2,3-O-isopropylidene-α-D-lyxopyranoside (4). To a suspension of compound 3 (10.1 g, 0.042
mol) in acetone (140 mL) were added 2,2-dimethoxypropane (17.5 mL, 0.142 mol, 3.4 eq) and PTSA
(178 mg, 0.936 mmol, 0.02 eq). The mixture was stirred at rt for 18 h. A 1:1 mixture of hexane/Et2O
16

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(120 mL) was added and the solution was washed with saturated aqueous NaHCO3 (60 mL), water (2 x
60 mL) and dried over MgSO4. Concentration under vacuum gave crude 4 as a colorless oil, which was
purified by flash column chromatography on silica gel (petroleum ether/EtOAc 85:15, then 7:3, then
1
6:4) to afford compound 4 (7.55 g, 64%) as a white solid. H NMR spectrum is in accordance with
literature data [24].
Benzyl 2,3-O-isopropylidene-β-L-erythro-pent-4-uloside (5). To a solution of alcohol 4 (1.0 g, 3.57
mmol) in anhydrous CH2Cl2 (36 mL) under Ar was added Dess-Martin periodinane (1.82 g, 4.29
mmol, 1.2 eq). The mixture was stirred for 16 h at rt. The solvent was evaporated under vacuum. The
residue was suspended in cold Et2O and the solid was removed by filtration through Celite. The filtrate
was concentrated under vacuum to give the clean desired ketone 5 (0.98 g, 99%) as a pale yellow solid.
1H NMR spectrum is in accordance with literature data [25].
Benzyl 2,3-O-isopropylidene-4-C-[(1R,S)(1-nitrohex-1-yl)]-β-L-ribopyranosides (6a) and (6b). To
a solution of ketone 5 (1.98 g, 7.11 mmol) in 1-nitrohexane (5.5 mL) was added NEt3 (2.8 mL, 20.1
mmol, 2.8 eq). The mixture was stirred for 4 d at rt. The reaction was quenched with saturated aqueous
NH4Cl (40 mL). The aqueous phase was extracted with EtOAc (80 mL). The organic phase was then
washed with brine (40 mL), dried over MgSO4 and concentrated under vacuum to give the crude
product. Flash column chromatography on silica gel (petroleum ether/EtOAc, 92:8) afforded an
inseparable mixture of the two diastereoisomers 6a and 6b (1.5 g, 52%) in a 7:3 ratio as a colorless oil.
1H NMR (400 MHz, CDCl3): δ 7.47-7.28 (m, 5H, Harom), 5.05 (br s, 1H, H1), 4.75 (d, 1H, CH2Ph, J =
11.6 Hz), 4.60-4.51 (m, 1H, H6), 4.54 (d, 1H, CH2Ph, J = 11.6 Hz), 4.26 (d, 0.7H, H3MAJ, J = 6.4 Hz),
4.17-4.09 (m, 1.3H, H2MAJ, H3min, H2min), 3.88 (d, 0.3H, H5bmin, J = 12.4 Hz), 3.81 (d, 0.7H, H5bMAJ, J
= 12.0 Hz), 3.73 (d, 0.7H, H5aMAJ, J = 12.0 Hz), 3.58 (d, 0.3H, H5amin, J = 12.4 Hz), 3.04 (s, 0.7H,
OHMAJ), 2.96 (s, 0.3H, OHmin), 2.16-1.95 (m, 1.3H, H7bMAJ, 2H7min), 1.72-1.65 (m, 0.7H, H7aMAJ),
1.55 (s, 0.9H, CH3iPrmin), 1.54 (s, 2.1H, CH3iPrMAJ), 1.37 (s, 0.9H, CH3iPrmin), 1.36 (s, 2.1H,
17

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13
CH3iPrMAJ), 1.30-1.29 (m, 6H, CH2hex), 0.95-0.82 (m, 3H, CH3hex). C NMR (100 MHz, CDCl3):
δ 136.65 (Carom), 128.69-128.28 (CHarom), 110.24 (CiPrMAJ), 110.15 (CiPrmin), 96.60 (C1MAJ), 96.40
(C1min), 92.71 (C6min), 92.68 (C6MAJ), 74.35 (C2MAJ), 74.32 (C2min), 72.52 (C3min), 72.03 (C3MAJ),
69.74 (CH2PhMAJ), 69.66 (CH2Phmin), 69.49 (C4MAJ), 69.05 (C4min), 61.26 (C5min), 60.79 (C5MAJ),
31.12, 28.02, 25.99, 22.41 (CH2hexMAJ), 31.17, 27.23, 25.93, 22.45 (CH2hexmin), 26.17, 25.36
(CH3iPrMAJ), 26.13, 25.36 (CH3iPrmin), 14.00 (CH3hexmin), 13.98 (CH3hexMAJ). HRMS (ESI): m/z
[M+NH4]+ calcd for C21H35NO7: 427.2439, found: 427.2438; m/z [M+Na]+ calcd for C21H31NNaO7:
432.1993, found: 432.1992.
Benzyl 2,3-O-isopropylidene-4-C-[(1R,S)(1-aminohex-1-yl)]-β-L-ribopyranosides (7a) and (7b). To
a solution of the mixture of isomers 6a and 6b (289 mg, 0.706 mmol) in iPrOH (7.1 mL) were added
glacial AcOH (0.71 mL) and Raney Ni (excess, 2800, slurry in H2O). The mixture was stirred under a
H2 atmosphere for 5 d at rt. Then the catalyst was filtered through a membrane, washed with MeOH
and the filtrate was concentrated under vacuum to give the crude product as a green oil. Flash column
chromatography on silica gel (CH2Cl2/MeOH 9:1) gave an inseparable mixture of the two
1
diastereoisomers 7a and 7b (228 mg, 85%) in a 7:3 ratio as a colorless oil. H NMR (400 MHz,
CD3OD): δ 7.43-7.28 (m, 5H, Harom), 4.90 (d, 1H, H1, J = 3.3 Hz), 4.79 (d, 1H, CH2Ph, J = 11.9 Hz),
4.61 (d, 1H, CH2Ph, J = 11.9 Hz), 4.36 (d, 1H, H3, J = 5.8 Hz), 4.13 (dd, 1H, H2, J = 3.3, 5.8 Hz), 3.78
(d, 1H, H5b, J = 12.0 Hz), 3.57 (d, 1H, H5a, J = 12.0 Hz), 2.96 (dd, 0.7H, H6MAJ, J = 2.4, 9.6 Hz), 2.81-
2.79 (m, 0.3H, H6min), 1.69-1.26 (m, 14H, CH2hex, CH3iPr), 0.95 (t, 3H, CH3hex, J = 6.6 Hz). 13C
NMR (100 MHz, CD3OD): δ 138.63 (Carom), 129.41, 129.19, 128.90 (CHarom), 110.96 (CiPrMAJ),
110.63 (CiPrmin), 99.73 (C1min), 99.56 (C1MAJ), 76.33 (C2), 76.09 (C3), 71.20 (C4), 70.76 (CH2Ph),
64.84 (C5min), 63.52 (C5MAJ), 58.39 (C6min), 57.59 (C6MAJ), 32.95, 30.66, 27.24, 23.60 (CH2hexMAJ),
31.66, 27.48 (CH2hexmin), 26.92 (CH3iPrmin), 26.84, 25.96 (CH3iPrMAJ), 14.39 (CH3hex). HRMS (ESI):
m/z [M+H]+ calcd for C21H34NO5: 380.2432, found: 380.2433.
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(2R,S, 3S, 4R, 5R)-3-Hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperidine-3,4,5-triol (8a) and
(8b). To a solution of the mixture of isomers 7a and 7b (367 mg, 0.967 mmol) in iPrOH (10 mL) were
added glacial AcOH (1 mL) and 20% Pd(OH)2/C (150 mg). The mixture was stirred under a H2
atmosphere for 3 d at rt. Then the catalyst was filtered through a membrane, washed with iPrOH and
the filtrate was concentrated under vacuum to give the crude product as a clean mixture of epimers 8a
and 8b (264 mg, quant.) in a 7:3 ratio as a colorless oil. 1H NMR (600 MHz, CD3OD): δ 4.55 (dt, 0.7H,
H2MAJ, J = 3.9, 3.9, 7.2 Hz), 4.48 (dt, 0.3H, H2min, J = 4.8, 4.8, 6.6 Hz), 4.29 (d, 0.7H, H3MAJ, J = 7.2
Hz), 4.21 (dd, 0.3H, H3min, J = 1.2, 6.6 Hz), 3.72 (d, 0.3H, H6bmin, J = 11.4 Hz), 3.66 (d, 0.3H, H6amin
,
J = 11.4 Hz), 3.64 (d, 0.7H, H6bMAJ, J = 11.4 Hz), 3.61 (d, 0.7H, H6aMAJ, J = 11.4 Hz), 3.51 (dd, 0.7H,
H1bMAJ, J = 3.9, 13.5 Hz), 3.34 (dd, 0.3H, H1bmin, J = 5.4, 13.2 Hz), 3.23 (dd, 0.7H, H5MAJ, J = 3.6,
13.2 Hz), 3.21-3.18 (m, 0.3H, H5min), 3.18 (dd, 0.7H, H1aMAJ, J = 3.9, 13.5 Hz), 2.92 (dd, 0.3H,
H1amin, J = 6.0 Hz, 13.2 Hz), 1.93-1.83 (m, 1H, CH2pent), 1.76-1.60 (m, 2H, CH2pent), 1.51 (s, 3H,
CH3iPr), 1.40-1.27 (m, 5H, CH2pent), 1.38 (s, 3H, CH3iPr), 0.94 (t, 3H, CH3pent, J = 6.6 Hz). 13C
NMR (62.5 MHz, CD3OD): δ 111.20 (CiPrmin), 110.68 (CiPrMAJ), 75.52 (C3min), 75.20 (C3MAJ), 72.82
(C4min), 72.74 (C4MAJ), 70.82 (C2min), 70.79 (C2MAJ), 65.27 (C6min), 64.70 (C6MAJ), 60.01 (C5min),
54.83 (C5MAJ), 42.43 (C1MAJ), 41.61 (C1min), 32.84, 28.41, 26.53, 23.45 (CH2pentMAJ), 32.67, 28.60,
27.40 (CH2pentmin), 27.18, 24.86 (CH3iPrmin), 27.08, 24.58 (CH3iPrMAJ), 14.35 (CH3pent). HRMS
(ESI): m/z [M+H]+ calcd for C14H28NO4: 274.2013, found: 274.2015.
(2R, 3S, 4R, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperi-
dine-3,4,5-triol (9) and (2S, 3S, 4R, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopro-
pylidene-2-pentylpiperidine-3,4,5-triol (10). The mixture of epimers 8a and 8b (500 mg, 1.83 mmol)
was dissolved in EtOH (15 mL). At 0 °C, DIPEA (956 µL, 5.49 mmol, 3 eq) and benzyl chloroformate
(392 µL, 2.75 mmol, 1.5 eq) were added. The mixture was stirred for 30 min at 0 °C and then 16 h at rt.
The solvent was removed under vacuum and the residue was purified by flash column chromatography
19

ACCEPTED MANUSCRIPT
on silica gel (toluene/acetone 7:1) to give the major epimer 10 (228 mg, 31%) and the minor one 9 (132
mg, 18%), both as white solids and mixtures of rotamers (65:35).
20
1
9: mp = 140-143°C. [α]D = + 6.4 (c = 1.01, CHCl3). H NMR (400 MHz, CDCl3): δ 7.44-7.22 (m,
5H, Harom), 5.25-5.03 (m, 2H, CH2Ph), 4.40-4.23 (m, 2H, H1bmin, H2, H5MAJ), 4.22-4.16 (m, 0.35H,
H5min), 4.12 (dd, 0.65H, H1bMAJ, J = 7.2, 13.6 Hz), 4.04-3.97 (m, 1H, H3), 3.77-3.64 (m, 2H, H6), 3.22
(br s, 0.65H, OHMAJ), 2.81-2.58 (m, 1.65H, H1a, OHMAJ), 2.58 (s, 0.35H, OHmin), 2.17 (br s, 0.35H,
OHmin), 2.10-1.93 (m, 1H, H7b), 1.47 (s, 3H, CH3iPr), 1.31 (s, 3H, CH3iPr), 1.41-1.02 (m, 7H,
3CH2pent, H7a), 0.95-0.78 (m, 3H, CH3pent). 13C NMR (100 MHz, CDCl3): δ 157.29 (COMAJ), 156.64
(COmin), 136.67 (Carommin), 136.56 (CaromMAJ), 129.10-127.70 (CHarom), 110.04 (CiPrmin), 109.95
(CiPrMAJ), 76.34 (C3), 74.31 (C4MAJ), 73.57 (C4min), 70.03 (C2MAJ), 69.85 (C2min), 67.70 (CH2PhMAJ),
67.62 (CH2Phmin), 65.49 (C6MAJ), 65.06 (C6min), 57.45 (C5min), 57.10 (C5MAJ), 40.27 (C1MAJ), 39.87
(C1min), 31.59 (CH2pentmin), 31.54 (CH2pentMAJ), 28.53, 26.20 (CH3iPrMAJ), 28.48, 26.27 (CH3iPrmin),
25.99-25.68, 22.63 (CH2pent), 14.14 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C22H34NO6:
408.2381, found: 408.2377; m/z [M+Na]+ calcd for C22H33NNaO6: 430.2200, found: 430.2209.
20
1
10: mp = 84-86°C. [α]D = + 52.2 (c = 1.38, MeOH). H NMR (400 MHz, CDCl3): δ 7.42-7.22 (m,
5H, Harom), 5.21-5.04 (m, 2H, CH2Ph), 4.40-4.25 (m, 2H, H2, H3), 4.25-4.09 (m, 1H, H1b), 4.09-4.00
(m, 0.65H, H5MAJ), 3.96-3.87 (m, 0.35H, H5min), 3.78 (br d, 1H, H6b, J = 11.2 Hz), 3.57-3.44 (m, 1H,
H6a), 3.35-3.20 (m, 1H, H1a), 2.82-2.72 (m, 1H, OH), 2.58 (br s, 0.65H, OHMAJ), 2.41 (br s, 0.35H,
OHmin),1.69-1.41 (m, 2H, H7), 1.40-1.11 (m, 12H, 3CH2pent, 2CH3iPr), 0.97-0.76 (m, 3H, CH3pent).
13C NMR (100 MHz, CDCl3): δ 156.92 (COMAJ), 156.58 (COmin), 138.00 (Carommin), 136.92 (CaromMAJ),
129.17-127.90 (CHarom), 109.11 (CiPrmin), 109.00 (CiPrMAJ), 75.47, 73.46 (C2min, C3min), 75.27, 73.15
(C2MAJ, C3MAJ), 72.84 (C4MAJ), 72.42 (C4min), 67.58 (CH2Phmin), 67.24 (CH2PhMAJ), 65.59 (C6min),
65.22 (C6MAJ), 54.38 (C5min), 54.06 (C5MAJ), 41.90 (C1min), 41.80 (C1MAJ), 32.13 (CH2pent), 28.76
(C7min), 28.09 (C7MAJ), 26.24 (CH2pentmin), 26.08 (CH2pentMAJ), 26.01 (CH3iPrmin), 25.88 (CH3iPrMAJ),
20

ACCEPTED MANUSCRIPT
24.53 (CH3iPr), 22.72 (CH2pent), 14.19 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C22H34NO6:
408.2381, found: 408.2377; m/z [M+Na]+ calcd for C22H33NNaO6: 430.2200, found: 430.2204.
General procedure for the formation of cyclic thiocarbonate: The diol was dissolved in anhydrous
THF (0.1 M) under Ar. Thiocarbonyldiimidazole (1.5 eq) was added. The mixture was heated under
reflux for 4 h, then cooled to rt. The solvent was removed under vacuum and the residue was purified
by flash column chromatography on silica gel to give the cyclic thionocarbonate as a mixture of
rotamers (6:4).
(2R, 3S, 4R, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperi-
dine-3,4,5-triol 3,3’-thiocarbonate (11). Compound 11 (74 mg, 86%) as a colorless syrup was
obtained from diol 9 (78 mg, 0.191 mmol) following general procedure, after purification using
1
petroleum ether/EtOAc (7.5:1) as the eluent. H NMR (400 MHz, CDCl3): δ 7.46-7.28 (m, 5H, Harom),
5.25-5.07 (m, 2H, CH2Ph), 4.85-4.72 (m, 1H, H6b), 4.65-4.54 (m, 0.4H, H5min), 4.50-4.32 (m, 3.2H,
H6a, H3, H5MAJ, H1bMAJ), 4.24-4.14 (m, 1.4H, H2, H1bmin), 2.94-2.82 (m, 0.4H, H1amin), 2.80-2.70 (m,
0.6H, H1aMAJ), 2.05-1.91 (m, 1H, H7b), 1.51 (s, 3H, CH3iPr), 1.36 (s, 3H, CH3iPr), 1.32-1.10 (m, 7H,
13
3CH2pent, H7a), 0.95-0.80 (m, 3H, CH3pent). C NMR (100 MHz, CDCl3): δ 189.92 (CSmin), 189.78
(CSMAJ), 155.52 (COMAJ), 155.47 (COmin), 136.31 (Carommin), 136.20 (CaromMAJ), 128.70-127.92
(CHarom), 111.12 (CiPr), 87.71 (C4min), 87.52 (C4MAJ), 74.60 (C2MAJ), 74.35 (C2min), 72.95 (C6), 69.83
(C3min), 69.55 (C3MAJ), 68.08 (CH2PhMAJ), 67.89 (CH2Phmin), 55.62 (C5MAJ), 55.17 (C5min), 39.73
(C1min), 39.07 (C1MAJ), 31.45, 26.32 (CH2pentmin), 31.32, 25.96, 25.45 (CH2pentMAJ), 28.01
(CH3iPrMAJ), 27.81 (CH3iPrmin), 26.14 (CH3iPr), 22.48 (CH2pent), 14.03 (CH3pent). HRMS (ESI): m/z
[M+H]+ calcd for C23H32NO6S: 450.1945, found: 450.1943; m/z [M+NH4]+ calcd for C23H35N2O6S:
467.2210, found: 467.2210; m/z [M+Na]+ calcd for C23H31NNaO6S: 472.1764, found: 472.1763.
(2S, 3S, 4R, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperi-
dine-3,4,5-triol 3,3’-thiocarbonate (14). Compound 14 (200 mg, 91%) as a colorless syrup was
21

ACCEPTED MANUSCRIPT
obtained from diol 10 (199 mg, 0.488 mmol) following general procedure, after purification using
1
petroleum ether/EtOAc (6:1) as the eluent. H NMR (400 MHz, CDCl3): δ 7.49-7.26 (m, 5H, Harom),
5.23-5.05 (m, 2H, CH2Ph), 4.89-4.78 (m, 1H, H6b), 4.49-4.29 (m, 4H, H6a, H5MAJ, H2, H3, H1bmin),
4.28-4.16 (m, 1H, H1bMAJ, H5min), 3.29-3.13 (m, 1H, H1a), 1.65-1.51 (m, 2H, H7), 1.46-1.11 (m, 12H,
3CH2pent, 2CH3iPr), 0.95-0.78 (m, 3H, CH3pent). 13C NMR (100 MHz, CDCl3): δ 190.53 (CSMAJ),
190.40 (CSmin), 156.78 (COMAJ), 156.04 (COmin), 136.42 (CaromMAJ), 135.89 (Carommin), 128.97-128.10
(CHarom), 110.69 (CiPrMAJ), 110.54 (CiPrmin), 87.95 (C4min), 87.71 (C4MAJ), 75.53 (C6MAJ), 75.43
(C6min), 74.56, 72.79 (C2MAJ, C3MAJ), 74.47, 72.91 (C2min, C3min), 68.11 (CH2Phmin), 67.58
(CH2PhMAJ), 55.14 (C5min), 54.58 (C5MAJ), 42.07 (C1MAJ), 41.61 (C1min), 31.93, 25.37 (CH2pentMAJ),
31.86, 25.50 (CH2pentmin), 28.36 (C7MAJ), 28.08 (C7min), 25.68, 24.37 (CH3iPrmin), 25.54, 24.20
(CH3iPrMAJ), 22.55 (CH2pent), 14.08 (CH3pentMAJ), 14.05 (CH3pentmin). HRMS (ESI): m/z [M+H]+
calcd for C23H32NO6S: 450.1945, found: 450.1944; m/z [M+Na]+ calcd for C23H31NNaO6S: 472.1764,
found: 472.1765.
General procedure for deoxygenation: Half of a degassed solution of AIBN (0.12 eq, 4 mM) in
anhydrous toluene was added to a degassed solution of the cyclic thiocarbonate (0.05 M) and Bu3SnH
(2 eq) in anhydrous toluene. The mixture was stirred for 2 h at rt, and then the second half of the
solution of AIBN was added. The mixture was stirred for 16 h under reflux. After cooling to rt, the
reaction mixture was washed twice with 10% aqueous KF (v mL = 1.5 x total volume of toluene) and
saturated aqueous NaHCO3 (v mL). The combined aqueous phases were extracted 3 times with EtOAc
(3v mL). The combined organic phases were dried over MgSO4 and concentrated under vacuum. The
residue was purified by flash column chromatography on silica gel (petroleum ether/EtOAc 7:3) to give
the protected iminosugar as a mixture of rotamers (1:1) containing traces of impurities coming from
Bu3SnH.
22

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(2R, 3R, 4S, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperi-
dine-4,5-diol (12). Compound 12 (38 mg, 60%) as a colorless syrup was obtained from cyclic
1
thionocarbonate 11 (72 mg, 0.160 mmol) following general procedure. H NMR (400 MHz, CDCl3): δ
7.43-7.20 (m, 5H, Harom), 5.20 (d, 0.5H, CH2Ph, J = 12.4 Hz), 5.12 (s, 1H, CH2Ph), 5.05 (d, 0.5H,
CH2Ph, J = 12.4 Hz), 4.42-4.26 (m, 2H, H3, H5), 4.22 (dd, 0.5H, 0.5H1b, J = 7.2, 13.6 Hz), 4.17-4.00
(m, 1.5H, H2, 0.5H1b), 3.87-3.73 (m, 2H, 2H6), 2.82-2.64 (m, 1H, H1a), 2.26-2.13 (m, 1H, H4), 2.03-
1.81 (m, 2H, H7b, OH), 1.49 (s, 3H, CH3iPr), 1.33 (s, 3H, CH3iPr), 1.43-1.05 (m, 7H, 3CH2pent, H7a),
0.90-0.78 (m, 3H, CH3pent). 13C NMR (100 MHz, CDCl3): δ 155.76, 155.48 (CO), 136.78, 136.74
(Carom), 128.61-127.76 (CHarom), 109.54 (CiPr), 73.41, 73.31 (C3), 71.22, 70.98 (C2), 67.45, 67.31
(CH2Ph), 61.82, 61.75 (C6), 51.17, 50.99 (C5), 42.24, 41.86 (C4), 39.84, 39.49 (C1), 31.70, 25.90,
25.81, 22.66 (CH2pent), 28.77, 26.29, 26.24 (CH3iPr), 26.93, 26.78 (C7), 14.15, 14.14 (CH3pent).
HRMS (ESI): m/z [M+H]+ calcd for C22H34NO5: 392.2432, found: 392.2432; m/z [M+Na]+ calcd for
C22H33NNaO5: 414.2251, found: 414.2250.
(2S, 3R, 4S, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperi-
dine-4,5-diol (15) and (2S, 3S, 4S, 5R)-N-Benzyloxycarbonyl-3-hydroxymethyl-4,5-O-isopropyli-
dene-2-pentylpiperidine-4,5-diol (16). Compound 16 (71 mg, 43%) and compound 15 (63 mg, 38%)
as colorless syrups were obtained from cyclic thionocarbonate 14 (189 mg, 0.420 mmol) following
general procedure.
15: 1H NMR (400 MHz, CDCl3): δ 7.45-7.23 (m, 5H, Harom), 5.22-5.04 (m, 2H, CH2Ph), 4.61-4.51 (m,
1H, H3), 4.37-4.21 (m, 1.5H, H2, 0.5H1b), 4.21-4.08 (m, 1.5H, 0.5H1b, H5), 3.90-3.72 (m, 2H, 2H6),
2.91-2.78 (m, 1H, H1a), 2.54-2.29 (m, 1H, OH), 1.77-1.58 (m, 2H, H4, H7b), 1.48-1.09 (m, 13H, H7a,
3CH2pent, 2CH3iPr), 0.91-0.74 (m, 3H, CH3pent). 13C NMR (100 MHz, CDCl3): δ 156.99, 156.57
(CO), 137.07, 136.74 (Carom), 128.46, 128.02, 127.88 (CHarom), 109.01, 108.96 (CiPr), 74.37, 74.01,
73.64, 73.56 (C2, C3), 67.32, 66.93 (CH2Ph), 62.92, 62.78 (C6), 49.11, 48.82 (C5), 42.93, 42.79 (C1),
23

ACCEPTED MANUSCRIPT
40.85, 40.76 (C4), 33.50, 33.10 (C7), 32.20, 24.79, 22.70 (CH2pent), 25.97, 24.32 (CH3iPr), 14.15
(CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C22H34NO5: 392.2432, found: 392.2429; m/z
[M+NH4]+ calcd for C22H37N2O5: 409.2697, found: 409.2692; m/z [M+Na]+ calcd for C22H33NNaO5:
414.2251, found: 414.2249.
16: 1H NMR (400 MHz, CDCl3): δ 7.44-7.22 (m, 5H, Harom), 5.28-5.03 (m, 2H, CH2Ph), 4.61-4.45 (m,
1H, H1b), 4.45-4.26 (m, 1H, H5), 4.18-4.02 (m, 2H, H3, H2), 3.83 (dd, 1H, H6b, J = 7.2, 10.4 Hz),
3.61 (br s, 1H, H6a), 3.26-3.10 (m, 1H, H1a), 2.30 (br s, 1H, OH), 2.03-2.15 (m, 1H, H4), 1.46 (s, 3H,
CH3iPr), 1.42-1.10 (m, 11H, 4CH2pent, CH3iPr), 0.90-0.76 (m, 3H, CH3pent). 13C NMR (100 MHz,
CDCl3): δ 156.11 (CO), 137.05 (Carom), 128.49, 127.92, 127.46 (CHarom), 109.02 (CiPr), 74.58, 71.80
(C2, C3), 67.19 (CH2Ph), 63.62 (C6), 51.93 (C5), 44.14 (C4), 39.44 (C1), 31.59, 26.93, 25.51, 22.65
(CH2pent), 28.43, 26.49 (CH3iPr), 14.08 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C22H34NO5:
392.2432, found: 392.2430; m/z [M+NH4]+ calcd for C22H37N2O5: 409.2697, found: 409.2694; m/z
[M+Na]+ calcd for C22H33NNaO5: 414.2251, found: 414.2250.
General procedure for benzyloxycarbonyl deprotection: To a solution of the N-protected compound
in iPrOH (0.02 M) was added 10% Pd/C (500 mg/mmol). The mixture was stirred under a H2
atmosphere at rt until completion of the reaction (TLC). More catalyst was added if needed. Then the
catalyst was filtered through a membrane, washed with iPrOH and the filtrate was concentrated under
vacuum to give the deprotected compound free of Sn-containing impurities.
(2R, 3R, 4S, 5R)-3-Hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperidine-4,5-diol (13).
Compound 13 (24 mg, 96%) as a yellow syrup was obtained from N-protected compound 12 (38 mg,
20
1
0.097 mmol) following general procedure. [α]D = + 31.7 (c = 1.00, MeOH). H NMR (250 MHz,
CD3OD): δ 4.38 (dd, 1H, H3, J = 3.3, 5.8 Hz), 4.14 (dt, 1H, H2, J = 5.8, 5.8, 7.0 Hz), 3.68 (d, 2H, 2H6,
J = 7.5 Hz), 2.99 (ddd, 1H, H5, J = 2.9, 6.3, 10.9 Hz), 2.79 (dd, 1H, H1b, J = 5.8, 13.0 Hz), 2.68 (dd,
24

ACCEPTED MANUSCRIPT
1H, H1a, J = 7.0, 13.0 Hz), 2.16 (ddt, 1H, H4, J = 3.3, 6.3, 7.5, 7.5 Hz), 1.92-1.74 (m, 1H, H7b), 1.46
(s, 3H, CH3iPr), 1.31 (s, 3H, CH3iPr), 1.56-1.14 (m, 7H, 3CH2pent, H7a), 0.92 (t, 3H, CH3pent, J = 6.6
13
Hz). C NMR (100 MHz, CD3OD): δ 109.81 (CiPr), 73.90 (C3), 73.32 (C2), 61.76 (C6), 52.70 (C5),
42.84 (C1), 42.46 (C4), 33.09, 27.46, 23.62 (CH2pent), 29.26 (C7), 28.06, 25.45 (CH3iPr), 14.40
(CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C14H28NO3: 258.2064, found: 258.2066.
(2S, 3R, 4S, 5R)-3-Hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperidine-4,5-diol (17).
Compound 17 (17 mg, 49%) as a colorless syrup was obtained from N-protected compound 15 (54 mg,
0.138 mmol) following general procedure, after purification using CH2Cl2/MeOH (9:1) as the eluent.
[α]D20 = - 28.3 (c = 1.00, MeOH). 1H NMR (250 MHz, CD3OD): δ 4.47 (dd, 1H, H3, J = 3.5, 5.5 Hz),
4.11 (dt, 1H, H2, J = 5.5, 5.5, 7.9 Hz), 3.33-3.29 (m, 2H, 2H6), 2.93 (dd, 1H, H1b, J = 5.5, 12.6 Hz),
2.58 (dd, 1H, H1a, J = 7.9, 12.6 Hz), 2.64-2.51 (m, 1H, H5), 1.72 (dddd, 1H, H4, J = 3.5, 5.3, 7.5, 11.0
Hz), 1.66-1.58 (m, 1H, H7b), 1.47 (s, 3H, CH3iPr), 1.58-1.42 (m, 1H, H7a), 1.34 (s, 3H, CH3iPr), 1.42-
13
1.23 (m, 6H, 3CH2pent), 0.92 (t, 3H, CH3pent, J = 6.8 Hz). C NMR (100 MHz, CD3OD): δ 110.62
(CiPr), 73.90 (C3), 72.50 (C2), 61.64 (C6), 53.44 (C5), 46.08 (C1), 43.58 (C4), 33.57 (C7), 33.06,
26.02, 23.58 (CH2pent), 27.72, 25.46 (CH3iPr), 14.37 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for
C14H28NO3: 258.2064, found: 258.2064.
(2S, 3S, 4S, 5R)-3-Hydroxymethyl-4,5-O-isopropylidene-2-pentylpiperidine-4,5-diol (18).
Compound 18 (26 mg, 67%) as a yellow syrup was obtained from N-protected compound 16 (60 mg,
20
1
0.153 mmol) following general procedure. [α]D = + 22.4 (c = 1.00, MeOH). H NMR (250 MHz,
CD3OD): δ 4.33-4.22 (m, 2H, H2, H3), 3.90 (dd, 1H, H6b, J = 5.0, 11.1 Hz), 3.69 (dd, 1H, H6a, J =
4.3, 11.1 Hz), 3.15 (dd, 1H, H1b, J = 6.1, 12.9 Hz), 3.10 (dt, 1H, H5, J = 3.8, 7.4, 7.4 Hz), 2.82 (dd,
1H, H1a, J = 7.0, 12.9 Hz), 2.11-2.05 (m, 1H, H4), 1.64-1.52 (m, 2H, 2H7), 1.50 (s, 3H, CH3iPr), 1.46-
1.28 (m, 9H, 3CH2pent, CH3iPr), 0.98-0.87 (m, 3H, CH3pent). 13C NMR (100 MHz, CD3OD): δ 109.43
(CiPr), 76.72 (C3), 71.95 (C2), 61.56 (C6), 54.94 (C5), 47.02 (C1), 41.80 (C4), 32.94, 26.90, 23.56
25

ACCEPTED MANUSCRIPT
(CH2pent), 30.98 (C7), 28.53, 26.45 (CH3iPr), 14.36 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for
C14H28NO3: 258.2064, found: 258.2064.
General procedure for isopropylidene deprotection: The protected compound was dissolved in a
mixture of dioxane/H2O (5:3; 0.01 M) and Dowex 50WX8 ion-exchange resin (H+ form, 10
mL/mmol), previously washed with dioxane/H2O (1:1), was added. The mixture was stirred for 18 h at
rt, then poured into a column and washed with dioxane/H2O (1:1) and with H2O. Elution with aqueous
0.5 N NH4OH (800 mL/mmol), followed by concentration under vacuum gave the expected
iminosugar.
(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-pentylpiperidine-4,5-diol or (5aR)-5a-C-pentyl-4-epi-
isofagomine (1). Compound 1 (16 mg, 84%) as a colorless syrup was obtained from protected
1
iminosugar 13 (23 mg, 0.089 mmol) following general procedure. [α]D20 = - 9.5 (c = 1.00, MeOH). H
NMR (250 MHz, CD3OD): δ 3.90 (dd, 1H, H6b, J = 3.5, 11.5 Hz), 3.87-3.84 (m, 1H, H3), 3.80 (dd,
1H, H6a, J = 4.5, 11.5 Hz), 3.75-3.68 (m, 1H, H2), 3.00 (dd, 1H, H1b, J = 2.8, 13.4 Hz), 2.73 (d, 1H,
H1a, J = 13.4 Hz), 2.70-2.64 (m, 1H, H5), 1.89-1.82 (m, 1H, H4), 1.72-1.53 (m, 2H, 2H7), 1.49-1.26
13
(m, 6H, CH2pent), 0.97-0.87 (m, 3H, CH3pent). C NMR (100 MHz, CD3OD): δ 72.04 (C3), 68.92
(C2), 58.63 (C5), 58.15 (C6), 50.76 (C1), 44.08 (C4), 33.11, 27.39, 23.63 (CH2pent), 32.96 (C7), 14.39
(CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C11H24NO3: 218.1751, found: 218.1753.
(2S, 3R, 4S, 5R)-3-Hydroxymethyl-2-pentylpiperidine-4,5-diol or (5aS)-5a-C-pentyl-4-epi-
isofagomine (2). Compound 2 (12 mg, 86%) as a colorless syrup was obtained from protected
iminosugar 17 (17 mg, 0.067 mmol) following general procedure. [α]D20 = - 34.5 (c = 1.05, MeOH). 1H
NMR (250 MHz, CD3OD): δ 4.10 (t, 1H, H3, J = 2.5 Hz), 3.65 (d, 2H, 2H6, J = 6.0 Hz), 2.53 (ddd,
1H, H2, J = 2.5, 6.6, 9.3 Hz), 2.92-2.71 (m, 3H, H5, 2H1), 1.73-1.55 (m, 1H, H7b), 1.53-1.40 (m, 1H,
H4), 1.53-1.22 (m, 7H, H7a, 3CH2pent), 0.98-0.85 (m, 3H, CH3pent). 13C NMR (100 MHz, CD3OD): δ
26

ACCEPTED MANUSCRIPT
70.91 (C2), 70.69 (C3), 62.14 (C6), 52.06 (C5), 48.02 (C4), 46.91 (C1), 33.64 (C7), 33.32, 25.92,
23.63 (CH2pent), 14.40 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for C11H24NO3: 218.1751, found:
218.1752.
(2S, 3S, 4S, 5R)-3-Hydroxymethyl-2-pentylpiperidine-4,5-diol (19). Compound 19 (22 mg, quant.)
as a colorless syrup was obtained from protected iminosugar 18 (26 mg, 0.101 mmol) following
general procedure. [α]D20 = + 9.7 (c = 1.00, MeOH). 1H NMR (250 MHz, CD3OD): δ 4.02 (t, 1H, H3, J
= 3.2 Hz), 3.87 (dt, 1H, H2, J = 3.2, 7.4, 7.4 Hz), 3.78 (dd, 1H, H6b, J6B-4= 7.0, 11.3 Hz), 3.69 (dd, 1H,
H6a, J6A-4= 4.6, 11.3 Hz), 3.13 (dt, 1H, H5, J = 3.8, 7.0, 7.0 Hz), 2.87 (d, 2H, 2H1, J = 7.4 Hz), 2.02-
1.94 (m, 1H, H4), 1.58-1.44 (m, 2H, 2H7), 1.44-1.26 (m, 6H, 3CH2pent), 0.97-0.86 (m, 3H, CH3pent).
13C NMR (100 MHz, CD3OD): δ 71.15 (C3), 67.38 (C2), 60.54 (C6), 53.50 (C5), 46.84 (C1), 45.72
(C4), 33.01, 27.19, 23.59 (CH2pent), 31.60 (C7), 14.36 (CH3pent). HRMS (ESI): m/z [M+H]+ calcd for
C11H24NO3: 218.1751, found: 218.1752.
4.2. Enzymatic assays
Human PBMC lysates were used as a source of human glycosidases. Each assay was conducted using
lysate from the indicated number of cells with the appropriate buffer and substrate supplemented with
protease inhibitors (cOmpleteTM Protease Inhibitor Cocktail Tablets, Roche) and 1% Triton X-100.
Otherwise indicated, the β-galactosidase assays were conducted with the equivalent of 2 x 106 cells per
reaction in 50 mM sodium phosphate, pH 7.3, using, as substrate, 4-methylumbelliferyl β-D-
galactopyranoside at 0.8 mM. The α-galactosidase assays were conducted with the equivalent of 2 x
106 cells per reaction in 100 mM sodium citrate, pH 4.3, using, as substrate, 4-methylumbelliferyl α-
galactopyranoside at 1 mM. The α-glucosidase assays were conducted with the equivalent of 2 x 106
D
-
cells per reaction in 100 mM sodium citrate, pH 4.3, using, as substrate, 4-methylumbelliferyl α-D-
glucopyranoside at 1 mM. The β-glucosidase assays were conducted with the equivalent of 2 x 106
cells per reaction in 100 mM sodium citrate, pH 6.0, supplemented with 10 mM taurocholic acid and 5
27

ACCEPTED MANUSCRIPT
mM 2-mercaptoethanol, using, as substrate, 4-methylumbelliferyl β-D-glucopyranoside at 1 mM. The
N-acetyl-α-D-glucosaminidase assays were conducted with the equivalent of 2.5 x 106 cells per
reaction in 50 mM sodium citrate, pH 4.3, using, as substrate, 4-methylumbelliferyl 2-acetamido-2-
deoxy-α-D-glucopyranoside at 0.25 mM. The β-hexosaminidase assays were conducted with the
equivalent of 0.4 x 106 cells per reaction in 50 mM sodium citrate, pH 4.3, using, as substrate, 4-
nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside at 2 mM. The enzymatic reactions were run for
2 hours at 37oC, with the exception of the N-acetyl-α-
D-glucosaminidase assays, which were incubated
for 17 hours. The fluorogenic reactions were stopped by the addition of an equal volume of glycine
0.15 M, pH 10.2 and fluorescence emission was determined at 445 nm using an excitation wavelength
at 365 nm. The colorimetric reactions were stopped by the addition of an equal volume of NaOH 0.2 M
and absorbance was determined at 405 nm.
4.3. Heat denaturation assays
The tests were conducted essentially as described in the literature [34]. Samples of human PBMC
lysates (equivalent of 2 x 106 cells/reaction) in 50 mM sodium phosphate, pH 7.3, supplemented with
protease inhibitors and 1% Triton X-100 were incubated at 48 oC for 0, 5, 10, 20, and 40 minutes in the
presence of graded concentrations of compound 1. The samples were then diluted in citrate buffer, pH
o
4.3, and 0.25 mM of 4-methylumbelliferyl β-
D
-galactopyranoside was added. After 17 hours at 37 C,
the reactions were stopped by the addition of an equal volume of glycine 0.15 M, pH 10.2.
Fluorescence was then determined at 445 nm using an excitation wavelength at 365 nm.
4.4. Cell-based chaperone assays
Patient fibroblasts were purchased from the Coriell Institute (Camden, NJ) or were obtained directly
from patients according to the ethical committee requirements of the respective institutions in which
the cell lines were generated. The GM03348 fibroblast cell line, from a healthy donor, was from the
Coriell Institute. The chaperone assays were conducted following the method described in Rigat et al
28