Glucosylceramide mimics: Highly potent GCase inhibitors and selective pharmacological chaperones for mutations associated with types 1 and 2 gaucher disease

Article abstract of DOI:10.1002/cmdc.201300327

A series of iminoxylitol derivatives carrying a C-linked di-O-acyl or di-O-alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal b-glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di-O-hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di-Odecanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nm); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat-stress studies, the diether was found to be much more effective in stabilizing the wildtype enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di-C10-ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.

Full text of DOI:10.1002/cmdc.201300327

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DOI: 10.1002/cmdc.201300327
Glucosylceramide Mimics: Highly Potent GCase Inhibitors
and Selective Pharmacological Chaperones for Mutations
Associated with Types 1 and 2 Gaucher Disease
Wojciech Schçnemann,[a, b] Estelle Gallienne,[a] Kyoko Ikeda-Obatake,[c] Naoki Asano,[c]
Shinpei Nakagawa,[d] Atsushi Kato,[d] Isao Adachi,[d] Marcin Gꢀrecki,[e] Jadwiga Frelek,[e] and
Olivier R. Martin*[a]
A series of iminoxylitol derivatives carrying a C-linked di-O-acyl
or di-O-alkyl glyceryl substituent were prepared and character-
ized. All of these compounds, which were designed as gluco-
sylceramide (GlcCer) mimics, were nanomolar inhibitors of lyso-
somal b-glucosidase (glucocerebrosidase, GCase). Two of these
pseudoglycolipids were further evaluated for their ability to
enhance the activity of mutant GCase in human Gaucher cells.
Although the di-O-hexyl ether was surprisingly devoid of chap-
eroning activity on both N370S and L444P GCases, the di-O-
decanoyl ester was a potent chaperone of the L444P hydro-
lase, capable of increasing the residual activity of the enzyme
by a factor of two at a very low concentration (50 nm); such
a significant effect on the L444P mutation in human fibroblasts
has not yet been observed. In heat-stress studies, the diether
was found to be much more effective in stabilizing the wild-
type enzyme than the diester. Four representative pseudogly-
colipids were also assayed as inhibitors of GlcCer synthase, be-
cause such compounds could find use in the substrate reduc-
tion therapy approach to treat lysosomal storage diseases, but
these compounds revealed only moderate activity. As efficient
pharmacological chaperones, new structures such as the
di-C10-ester constitute leads for the development of therapeu-
tic agents for types 2 and 3 Gaucher disease, the most severe
neuronopathic forms of this lysosomal disease.
Introduction
Pharmacological chaperone therapy (PCT) is gaining increasing
attention as a promising approach to treat diseases due to
protein misfolding.[1] The potential of using small organic “cor-
rectors” for the treatment of such genetic diseases is extremely
attractive and justifies the current intense research activity
toward new molecules with chaperoning activity. Protein mis-
folding causes a number of severe diseases including, for ex-
ample, cystic fibrosis (CF), Gaucher disease (GD), and other ly-
sosomal disorders (LSDs), as well as certain types of cancers.[1]
Although finding chemical chaperones for such proteins as the
CF transmembrane conductance regulator (CFTR) transporter
in CF is a difficult endeavor,[2] the search for chaperones for the
deficient enzymes responsible for LSDs is guided by a knowl-
edge of the substrate and inhibitor structures.[3] Such mole-
cules intrinsically have a high affinity for the protein and have
already met with significant success: one drug candidate, 1-de-
oxygalactonojirimycin (Migalastat), is undergoing phase III clini-
cal trials for Fabry disease,[4] an LSD that is caused by a deficien-
cy of a-galactosidase A.
A great deal of effort has been dedicated to the search for
pharmacological chaperones (PCs) for Gaucher disease in
recent years.[3,5] GD, which is the most prevalent lysosomal dis-
ease, is caused by a mutation in the gene encoding for b-glu-
cocerebrosidase (GCase; EC 3.2.1.45).[6,7] This enzyme is respon-
sible for the hydrolysis of b-glucosylceramide (GlcCer) into glu-
cose and ceramide. Mutations in the GCase gene lead to aber-
rant folding in the endoplasmic reticulum (ER), which results in
impaired trafficking and degradation of the defective protein
by the ER quality-control system.[8] Although a large number of
mutations have been identified, those involving the N370S or
L444P single amino acid exchange are by far the most
common.[9] Both of these defective enzymes exhibit residual
hydrolytic activity (30 and 10–12%, respectively),[10] which is
a requirement for PCT to be effective. Gaucher disease is char-
acterized by three clinical subtypes. Type 1, in which patients
suffer from bone pain, skeletal lesions, anemia, and enlarged
liver or spleen, is due largely to the N370S mutation. Types 2
[a] W. Schçnemann, Dr. E. Gallienne, Prof. O. R. Martin
Institute of Organic & Analytical Chemistry (ICOA)
University of Orlꢀans, CNRS-UMR7311
Rue de Chartres, 45067 Orlꢀans (France)
E-mail: olivier.martin@univ-orleans.fr
[b] W. Schçnemann
Faculty of Chemistry, Jagellonian University
Ingardena 3, 30-060 Krakow (Poland)
[c] Dr. K. Ikeda-Obatake, Dr. N. Asano
Faculty of Pharmaceutical Sciences, Hokuriku University
Ho-3 Kanagawa-machi, Kanazawa 920-1181 (Japan)
[d] S. Nakagawa, Dr. A. Kato, Dr. I. Adachi
Department of Hospital Pharmacy, University of Toyama
2630 Sugitani, Toyama 930-0194 (Japan)
[e] Dr. M. Gꢁrecki, Prof. J. Frelek
Institute of Organic Chemistry, Polish Academy of Sciences
ul. M. Kasprzaka 44/52, 01-224 Warsaw (Poland)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300327.
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and 3 are neuronopathic forms of the disease: type 2 is an
the residual activity of the b-glucosidase in fibroblasts from
Gaucher patients at nanomolar concentrations. Most impor-
tantly, unlike the more common deoxynojirimycin (DNJ) deriva-
tives, this compound does not inhibit a-glucosidases, including
lysosomal and intestinal a-glucosidases. This favorable selectiv-
ity is related to the absence of the 5-CH2OH group in the glu-
coside mimic, because it is known that GCase has xylosidase
activity.[21]
acute, infantile form with
a median patient lifespan of
9 months, whereas type 3 is more viable. The L444P allele is
most frequently associated with these neuronopathic forms.
Gaucher disease is a rare, autosomal recessive disease, which
affects about one in 40000–60000 live births, but it has
a much higher prevalence in certain populations (Ashkenazi
Jews: 1:800 live births).[11] Currently approved treatments in-
clude enzyme replacement therapy (ERT; with Imiglucerase)[12]
and substrate reduction therapy (SRT; with Miglustat).[13] Al-
though these treatments can decrease the symptoms signifi-
cantly and improve the quality of life of type 1 Gaucher pa-
tients, they are not as effective on types 2 and 3.[14] The search
for therapeutic agents for this disease is further supported by
an increased awareness that GCase mutations represent a sig-
nificant genetic risk factor for Parkinson’s disease: Gaucher pa-
tients have an estimated 10–20-fold increased risk of develop-
ing Parkinson’s disease, because GCase is involved in the ca-
tabolism of a-synuclein in the brain.[15]
To further improve the properties of these compounds and
search for more efficient PCs, we decided to investigate
a series of 1-C-substituted iminoxylitol derivatives carrying an
“aglycone” mimicking more closely the lipidic component of
GlcCer (Figure 2). Herein we report the synthesis and the char-
The diversity of molecules that have been investigated as
potential PCs for Gaucher disease was summarized in a recent,
thorough review by Boyd et al.[3d] Most of the PCs identified so
far are carbohydrate mimics that are also good inhibitors of
GCase, such as monocyclic and bicyclic iminosugar derivatives,
aminocyclitols, and hydroxylated lactams, many of which also
carry a lipophilic group. The most extensively studied com-
pound is isofagomine (IFG; Figure 1),[16] which was developed
Figure 2. Structure of GlcCer and of a target compound.
acterization of a series of novel iminoglycolipids, constituted
by an iminoxylitol unit linked, as a glucose surrogate, by a CÀC
bond to a di-O-acyl or di-O-alkyl glyceryl group. We also report
the activity of these compounds as GCase inhibitors. The effect
as pharmacological chaperones on the most common mutant
GCases was determined for selected compounds. These studies
revealed some of the most important enhancements of L444P
GCase in Gaucher cells detected so far. The activity of selected
compounds as glucosylceramide synthase (GlcCer synthase,
GCS) inhibitors was also determined, because inhibitors of this
transferase are of interest as potential therapeutic agents for
LSDs by the SRT strategy.
Figure 1. Typical iminosugars investigated as pharmacological chaperones
for GCase.
by Amicus Therapeutics as a candidate (Plicera) for the treat-
ment of GD but was eventually discontinued in phase II trials
because it failed to meet efficacy expectations.[1a,3d] It is inter-
esting to note that, among the large diversity of compounds
exhibiting significant chaperoning effect on N370S GCase, the Results and Discussion
activity enhancement very rarely exceeds 2–2.5-fold (whereas
Synthesis of target compounds
the effect can be much higher on other mutations). In addi-
tion, enhancement effects on homozygous L444P patient cell
lines are generally nonexistent or very weak. Enhancements
greater than 50% on this mutation have been observed for
isofagomine[17] under specific conditions and for the bicyclic
l-idonojirimycin derivatives recently reported by Giraldo,
Garcia-Fernandez and co-workers.[18]
Glucosylceramide mimics of type 1 are readily available by way
of a 1-C-allyl-iminopentitol derivative, such as 5a. According to
the pioneering studies of Nicotra and co-workers on the syn-
thesis of 1-C-substituted iminosugars[22] and our experience in
this area,[23] such compounds can be made by the chain elon-
gation of a glycosyl amine, followed by a piperidine-forming
ring closure. Tri-O-benzyl-d-xylopyranose 2,[24] prepared in
three steps from d-xylose, was converted into N-benzylglyco-
sylamine 3 by reaction with BnNH2 (Scheme 1). Glycosylamine
3 was then treated with allylmagnesium bromide to give the
corresponding chain-extended aminopentitols 4,[25] in a diaste-
reomeric ratio of ~6:1. Cyclization was performed from the
In the course of our own program on iminosugars as poten-
tial pharmacological chaperones for LSDs,[19] we have identified
a family of highly potent and selective GCase inhibitors.[20] The
lead compound of this series is a-1-C-nonyl-iminoxylitol (a-1-C-
nonyl-DIX; Figure 1), a nanomolar inhibitor of GCase (Ki =
2.0 nm) that acts as a chaperone, being able to nearly double
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and 37% yields by flash chromatography and then cleaved by
methanolysis to give 7a and 7b, respectively.
The configuration at the C7 atom in epimers 7a/b was de-
termined by the so-called in situ methodology of electronic cir-
cular dichroism (ECD) spectroscopy. As we have recently
shown,[23] this technique is applicable to compounds contain-
ing multiple stereogenic centers, with the chiral diol moiety
being converted into an appropriate system for ECD by mixing
with dimolybdenum tetraacetate, which acts as an auxiliary
chromophore.[26] The resulting CD spectra are suitable for the
assignment of the absolute configuration, because the deter-
mining factor of the signs of Cotton effects (CEs) arising within
the d–d absorption bands of the metal cluster is the sense of
twist within the diol moiety.[26,27]
Based on such CD results, the molecular stereostructure of
vicinal diols can be determined with confidence through the
helicity rule.[27,28] In the case of compound 7a, the positive
sign of the CE at 304 nm points to a positive sign of the tor-
sion angle in the (HO)ÀCÀCÀ(OH) unit, whereas the negative
sign of the CE occurring in 7b at 303 nm corresponds to a neg-
ative torsion angle in the diol moiety (Scheme 3). With the as-
Scheme 1. Synthesis of precursors 5a and 5b and of 1-C-propyl-iminoxyli-
tols 6a and 6b. Reagents and conditions: a) MeOH, HCl (from SOCl2), reflux;
b) NaH, BnBr, TBAI, anhydrous DMF; c) 1m H2SO4, AcOH, dioxane, reflux,
64% (3 steps); d) BnNH2, pTsOH, CH2Cl2, 97%; e) AllylMgBr, THF, then H2O,
67%; f) MsCl, py, 1008C, 5a: 67%; 5b: 8%; g) H2, Pd/C, CH2Cl2/iPrOH/1n
HCl, 97%. Bn: benzyl; TBAI: tetrabutylammonium iodide; DMF: N,N-dime-
thylformamide; pTs: toluene-4-sulfonyl; THF: tetrahydrofuran; Ms: methane-
sulfonyl; py: pyridine.
mixture of epimers, by reaction with mesyl chloride in pyridine
at 1008C. The two epimeric products, compounds 5a and 5b,
that resulted from this reaction could be separated by column
chromatography.
The two 1-C-allyl epimers were submitted to hydrogenation
in order to obtain the reference 1-C-propyl-iminoxylitols 6a
and 6b, the structures of which were unambiguously estab-
lished from their NMR data. Dihydroxylation of 5a by using
catalytic OsO4 in the presence of 4-methylmorpholine N-oxide
(NMO) gave an approximate 1:1 mixture of diols 7a and 7b in
81% yield (Scheme 2). These diols could not be separated at
this stage, so derivatives were prepared in order to obtain
pure samples of each isomer. The mixture was acylated with
p-tert-butylbenzoyl chloride, to form the corresponding ben-
zoates in good yields. Esters 8a and 8b were isolated in 33
Scheme 3. Top: Dimolybdenum tetraacetate (left) and the diol ligand (right).
Bottom left: CD spectra of in situ formed Mo2 complexes of diols 7a (S) and
7b (R). Bottom right: preferred antiperiplanar conformation of the investi-
gated diols after complexation to the Mo2 dimer with the signs of the deci-
sive Cotton effects.
sumption that the preferred conformation for these com-
pounds is an antiperiplanar arrangement of the hydroxy group
at the C8 atom with respect to the bulky substituent at the C7
atom, the absolute configuration of compound 7a can be de-
termined as C7(S) on the basis of its positive (HO)ÀCÀCÀ(OH)
torsional angle. In contrast, from the negative sign of the
(HO)ÀCÀCÀ(OH) torsional angle, the absolute configuration of
the stereogenic center at the C7 atom in 7b is R. It has to be
added that 7a measured in dimethyl sulfoxide (DMSO) without
stock dimolybdenum complex was transparent in the spectral
range that is decisive for chiral Mo complexes with vicinal
Scheme 2. Synthesis and separation of epimers 7a and 7b. Reagents and
conditions: a) OsO4, NMO, THF/tBuOH/H2O, 81%; b) 4-tBuC6H4COCl, py, then
separation by flash chromatography (PhMe/CH2Cl2, 85:15), 8a: 33%; 8b:
37%; c) MeONa/MeOH/CH2Cl2, 85–89%.
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Biological investigations
Activity as GCase inhibitors
All of the new iminoglycolipids were submitted to an evalua-
tion of their activity as GCase inhibitors. The results are report-
ed in Table 1. Most of the compounds were found to be very
Table 1. Inhibition of GCase by GlcCer analogues 10, 13, and 14.
Compd
Structure
IC50 [nm][a]
Ki [nm][a]
Scheme 4. Synthesis of diesters. Reagents and conditions: a) RCOCl (R: C5H11,
C9H19, or C13H27), py, 22–71%; b) H2, Pd/C, EtOH/HCl, 67–100%.
10aS
24
–
diols, that is, 280–650 nm. Thus, the configuration at the C7
atom is established to be S in 7a and 8a and R in 7b and 8b.
Compound 7a (or 7S) and 7b (or 7R) were then di-O-acylat-
ed by using acyl chlorides with C6, C10, and C14 chain lengths,
to afford compounds 9a–cR and 9a,bS (Scheme 4). The result-
ing diesters were then debenzylated by hydrogenolysis over
palladium on charcoal in acidic ethanol, which thus provided
the glucosylceramide mimics carrying two acyl chains, 10a–cR
and 10a,bS, all as hydrochlorides.
Diols 7S and 7R were also converted into mono- and di-O-
hexyl ethers. The reaction of 7S and 7R with an excess of
1-bromohexane in the presence of NaH gave a mixture of
mono-O-hexyl (8-O-alkylated) and di-O-hexyl derivatives, com-
pounds 11R or S and 12R or S, respectively (Scheme 5); the
10aR
10bS
10bR
5
7
5
–
1.8
–
10cR
14S
64
4
–
2.2
14R
13S
13R
3
50
16
–
–
–
Scheme 5. Synthesis of mono- and diethers 13 and 14. Reagents and condi-
tions: a) NaH, excess C6H13Br, DMF, 11R: 45%; 12R: 8%; 11S: 52%; 12S: 41%;
b) H2, Pd/C, EtOH/HCl, 88–100%.
[a] Data represent the mean of triplicate experiments.
monoethers could be readily separated from the diethers by
flash chromatography. O- and N-debenzylation of the resulting
compounds were achieved by hydrogenolysis under the same
conditions as those used for the diesters, which thus gave glu-
cosylceramide mimics carrying one O-hexyl chain (13R or S) or
two hexyl chains (14R or S), all as hydrochlorides.
powerful GCase inhibitors, with IC50 values in the nm range. In
the esters, the effect of the configuration at the C7 atom,
which mimics the possible configuration of the ceramide
carbon atom carrying the amido group, played a minor role in
the inhibitory activity; in the decanoyl series, both epimers
have essentially the same activity. The effect of the chain
length was found to be unfavorable if the chain length is very
long (more than 10 carbon atoms). In the ether series, the re-
sults clearly showed that two chains are better than one, with
the OH group apparently having a detrimental effect, but the
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configuration at the C7 atom is unimportant. The Ki values
were measured for two representative compounds, diester
10bS and diether 14S; both of them exhibited a purely com-
petitive inhibition with Ki values in the low nm range (details
are given in the Supporting Information).
Table 2. Enzyme enhancement (EE) effects of glucosylceramide mimics
10bS and 14S on N370S and L444P GCase (see Figure 4).
Compd
10bS
14S
Structure
EE [fold increase]
N370S
L444P
ꢂ1.5
(50 nm)
ꢂ2
(50 nm)
Activity as pharmacological chaperones
The ability of the two representative glucosylceramide mimics
to stabilize the wild-type glycosidase against heat stress was
first determined (Figure 3). The diether 14S had a much stron-
ꢂ1.0
ꢂ0.9–1.0
exhibit a potent chaperone effect on L444P GCase: the activity
was enhanced twofold at a concentration of 50 nm, which is
the strongest effect observed so far on Gaucher patient L444P
fibroblasts (Figure 4). As this mutation is associated with neu-
Figure 3. Effect of a) diether 14S and b) diester 10bS on the in vitro thermo-
stability of human lysosomal b-glucocerebrosidase. The enzyme was incu-
bated at 508C in 100 mm sodium citrate buffer (pH 5.2) containing 0.25%
(w/v) sodium taurocholate and 0.1% (v/v) Triton X-100 for various times
Figure 4. Effect of GlcCer mimics 10bS and 14S on L444P GCase in
GM00877 Gaucher cells treated with increasing concentrations of a) 10bS or
b) 14S. Mean values Æ SD are shown for triplicate experiments.
*
^
with or without 14S and 10bS. Concentrations of 14S were: 0 ( ), 0.025 ( ),
^
*
^
^
and 0.1 mm ( ). Concentrations of 10bS were 0 ( ), 20 ( ), and 100 mm ( ).
ronopathic forms of the disease, compounds such as 10bS
hold great promise as new leads for the development of a PC
treatment for the most severe form of Gaucher disease. By
comparison, isofagomine has an enhancement effect of 1.3-
fold in the same cellular assay, which could be increased to
close to twofold by a special treatment that was designed to
maximize GCase activity.[17] Diether 14S was also assayed under
the same conditions: this compound was found, however, to
be devoid of chaperoning effect on both GCase mutations (for
L444P, see Figure 4).
ger stabilizing effect on the human enzyme submitted to heat
stress than the corresponding diester did. Whereas a concentra-
tion of 0.1 mm diether 14S was sufficient to protect the
enzyme from degradation after 1 h, a concentration of 100 mm
was necessary to achieve the same effect with the diester
10bS. The two representative compounds were then investi-
gated as pharmacological chaperones on Gaucher cells carry-
ing either the N370S or L444P mutations. The results are gath-
ered in Table 2. The diester 10bS promoted a 1.5-fold increase
in the activity of the N370S GCase, which is significant but not
remarkable given that many other compounds have an effect
in the order of twofold. However, this compound was found to
These unexpected results demonstrate that an enzyme en-
hancement effect is not directly linked to the activity of a com-
pound as an enzyme inhibitor. Several studies have already re-
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vealed that weak GCase inhibitors can have very good chaper-
oning effects, notably in the work of Ye, Butters, and co-work-
ers.[29] Whereas 10bS and 14S are both very powerful inhibitors
of GCase, only one of them acts as a pharmacological chaper-
one for the two most common mutant forms of this enzyme.
The difference of activity between these two compounds
must, however, be interpreted with caution: the diester is
labile in the cell and may undergo processing, such as partial
or total deacylation, so the observed effects may be due to
species derived from 10bS and not to 10bS itself. The effect
of a substrate analogue combining both an iminosugar and
a di-O-acyl glyceryl unit as a mimic of the highly lipophilic
aglycone of GlcCer was investigated here for the first time, and
this combination appears to be quite favorable in promoting
a very significant enhancement of the L444P enzyme activity,
which is highly desirable in the search for a treatment for
type 2 and 3 Gaucher diseases. It appears that this effect is
sensitive to subtle structure–activity relationships, because the
replacement of the two esters by ether functions completely
abolishes the chaperoning effect, even on the N370S enzyme
(note: the activity of N370S GCase is enhanced 1.8-fold by the
more simple a-1-C-nonyl-DIX).
Table 3. Inhibition of GlcCer synthase by selected GlcCer mimics.
Compd
Structure
IC50 [mm][a]
10bS
106
14S
89
13S
13R
NI[b]
NI[b]
[a] Data represent the mean of triplicate experiments. [b] No inhibition at
1000 mm.
All of these compounds exhibited a powerful activity as GCase
inhibitors, with IC50 values in the nm range. Although the pres-
ence of the ceramide mimic does not significantly increase the
inhibitory activity relative to the parent iminoxylitol carrying
a single C9 chain (a-1-C-nonyl-DIX), it lends some of these com-
pounds remarkable properties as pharmacological chaperones.
The diester was indeed found to be the best pharmacological
chaperone identified so far for L444P GCase in Gaucher pa-
tients fibroblasts, which is a very desirable property because
this mutation is generally associated with neurological forms
of Gaucher disease. In contrast, the corresponding diether was
found to be inactive, which indicates subtle structure–activity
relationships for the chaperone effect at the molecular level. It
appears that enzyme enhancement can be fine-tuned by ad-
justing the structure of the aglycone in the glycoside mimics,
as also shown by the recent work of Withers and co-workers
on the same iminoxylitol scaffold.[33] Although the chaperone
effect has usually been considered as a protective effect from
the small molecules in the course of protein biosynthesis in
the ER, new hypotheses are emerging, such as the reduced
degradation and longer lifetime of the mutant protein in the
lysosome.[34] Further work is required to better understand the
chaperoning effect at the molecular level, but our results, as
well as those of a number of other groups, bring to light the
very promising nature of pharmacological chaperones as thera-
peutic agents to treat lysosomal diseases and other protein-
folding diseases.
Finally, the observation that the diether 14S is capable of
stabilizing human GCase much more efficiently at 608C than
the diester 10bS is quite remarkable, but it cannot be correlat-
ed with the potential chaperone effect of each compound
owing to the possible transformation of 10bS in the cell, as
mentioned above. The stabilizing property of diether 14S sug-
gests that it could be used in improving the effectiveness of
ERT for Gaucher disease, because it might increase the stability
of the therapeutic enzyme and increase lysosomal delivery.[30]
Activity as inhibitors of GlcCer synthase
Selected compounds were also assayed as inhibitors of the
glucosyltransferase responsible for the synthesis of GlcCer
(GlcCer synthase, GCS, EC 2.4.1.80). The GlcCer analogues pos-
sess a bisubstrate-type structure, so they may behave as inhibi-
tors of this enzyme. A decrease in the production of GlcCer-
based glycolipids by the action of an inhibitor of this transfer-
ase is the basis of the SRT strategy for the treatment of GD.[31]
N-butyl-DNJ (NB-DNJ, Zavesca) is now an approved drug for
type 1 GD and remains the reference compound in this area,
although a number of other iminosugars have been reported
to be good inhibitors of GCS.[32] The results are summarized in
Table 3. The compounds tested were found to be fairly weak
inhibitors of GCS, with IC50 values in the 100 mm range for
GlcCer mimics with two lipophilic chains (10bS and 14S), al-
though they were not much weaker than NB-DNJ (IC50 =
20.4 mm),[31] or to have no inhibition effect at all for the mono-
ethers (13S and 13R).
Experimental Section
General procedures: All reactions requiring anhydrous conditions
were carried out by using oven-dried glassware under an atmos-
phere of dry Ar. THF was distilled from sodium benzophenone. Di-
chloromethane was distilled from calcium hydride. All reagent-
grade chemicals were obtained from commercial suppliers and
were used as received. Infrared spectra were recorded on a Nicolet
Conclusions
We have prepared a series of glucosylceramide mimics based
on an iminoxylitol core and carrying a di-O-acyl or di-O-alkyl
glyceryl group as a mimic of the lipidic component of GlcCer.
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iS10 FTIR spectrometer. Low-resolution mass spectra were recorded
on a Perkin–Elmer Sciex API 3000 instrument. High-resolution mass
spectra were recorded on a Waters Q-TOF microspectrometer by
the Centre Rꢃgional de Mesures Physiques in Clermont-Ferrand or
4H; PhCH2O), 4.94–5.10 (m, 2H; H8), 5.54–5.71 (m, 0.85H; H7A),
5.75–5.89 (m, 0.15H; H7B), 7.16–7.28 ppm (m, 20H; HAr); HRMS
(ESI): m/z calcd for C36H42NO4: 552.310835 [M+H]+; found:
552.310775.
1
on a Bruker Q-TOF MaXis spectrometer in Orlꢃans. H and 13C NMR
(1R)- and (1S)-1-C-Allyl-N-benzyl-2,3,4-tri-O-benzyl-1,5-dideoxy-
1,5-imino-d-xylitol (5a and 5b): Compound 4 (1.58 g, 2.86 mmol)
was dissolved in dry pyridine (30 mL) in the presence of activated
4 ꢄ molecular sieves. After the reaction mixture had been stirred
for 10 min at room temperature, MsCl (0.55 mL, 7.11 mmol,
2.5 equiv) was added. The reaction mixture was stirred for 4 h at
1008C and then cooled down and filtered through Celite. The
crude product was diluted with EtOAc (100 mL). The organic phase
was washed three times with H2O (200 mL), dried over MgSO4, con-
centrated under vacuum, and co-evaporated with toluene. The
crude product was purified by flash chromatography on silica gel
(petroleum ether/EtOAc, 20:1) to afford two separable diastereo-
mers 5a (1.03 g, 67%) and 5b (0.12 g, 8%) as yellow oils.
spectra were recorded on Bruker Avance DPX 250 or Bruker
Avance 400 spectrometers. Chemical shifts are given in ppm and
are referenced to the residual solvent signal or to tetramethylsilane
(TMS) as an internal standard. Carbon multiplicities were assigned
by distortionless enhancement by polarization transfer (DEPT) ex-
1
periments. H and 13C signals were attributed on the basis of H–H
and C–H correlations. Specific rotations were measured at 208C in
a 1 dm cell with a Perkin–Elmer 341 polarimeter. The CD spectra
were acquired at room temperature in DMSO on a Jasco J-815
spectropolarimeter, and step scans were collected at 0.5 nmstepÀ1
with an integration time of 1 s in the 10 mm and 2 mm cells over
the spectral range of 250–650 nm. For the standard CD measure-
ments, the chiral vicinal diol (5–5.5 mg, ~0.003mLÀ1) was dissolved
in a stock solution of [Mo2(OAc)4] (2.7–2.8 mg, ~0.002mLÀ1) in
DMSO (3 mL), so that the molar ratio of the stock complex to
ligand was ~1:1.5. The real complex structure, as well as the con-
centration of the chiral complex formed in solution, is not known,
so the CD data are presented as the De’ values. These De’ values
are calculated in the usual way as De’=DA/cꢂd, in which c is the
molar concentration of the chiral ligand, with the assumption of
100% complexation, A is the absorption, and d is the path length
of the cell). De’ is expressed in [mÀ1 ꢂcmÀ1] units. [Mo2(OAc)4] and
DMSO (Uvasol) were commercially available from Fluka AG and
Merck, respectively, and were used without further purification. An-
alytical thin-layer chromatography was performed by using silica
gel 60 F254 precoated plates (Merck) with visualization by ultraviolet
light and phosphomolybdic acid solution (2% in H2SO4/EtOH, 1:7).
Flash chromatography was performed on silica gel 60 (230–
5a: [a]D = +25.1 (c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3): d=
2.36–2.49 (m, 2H; H6), 2.55–2.61 (m, 1H; H5a), 2.78–2.82 (m, 1H;
H5b), 3.14–3.19 (m, 1H; H1), 3.58–3.65 (m, 2H; H3, H4), 3.67 (d, 1H,
J=13.8 Hz; PhCH2N), 3.72–3.75 (m, 1H; H2), 3.82 (d, 1H, J=
13.8 Hz; PhCH2N), 4.55–4.66 (m, 4H; PhCH2O), 4.84 (d, 1H, J=
10.8 Hz; PhCH2O), 4.90 (d, 1H, J=10.8 Hz; PhCH2O), 4.97–5.09 (m,
2H; H8), 5.81–5.91 (m, 1H; H7), 7.21–7.36 ppm (m, 20H; HAr);
13C NMR (100 MHz, CDCl3): d=28.01 (C6), 48.04 (C5), 59.01
(PhCH2N), 60.09 (C1), 72.60, 73.07, 75.67 (PhCH2O), 78.57, 80.65,
82.88 (C2, C3, C4), 115.67 (C8), 127.10–128.45 (CHAr), 138.39 (C7),
138.63–139.45 ppm (CAr); IR (neat): n˜ =3063, 3029, 2903, 2868,
1495, 1453, 1089, 1069, 734, 696 cmÀ1; MS (ESI+): m/z 534.5 [M+
H]+.
5b: 1H NMR (400 MHz, CDCl3): d=1.80–1.85 (m, 1H; H5a), 2.36–
2.38 (m, 1H; H1), 2.60 (brs, 2H; H6), 2.90 (d, 1H, J=11.4 Hz; H5b),
3.08 (d, 1H, J=13.3 Hz; PhCH2N), 3.38–3.46 (m, 3H; H2, H3, H4),
4.02 (d, 1H, J=13.3 Hz; PhCH2N), 4.41 (d, 1H, J=11.7 Hz; PhCH2O),
4.47 (d, 1H, J=11.7 Hz; PhCH2O), 4.58 (d, 1H, J=10.9 Hz; PhCH2O),
4.73 (d, 1H, J=10.9 Hz; PhCH2O), 4.88–4.92 (m, 2H; PhCH2O), 5.04–
5.08 (m, 2H; H8), 5.86–5.96 (m, 1H; H7), 7.14–7.25 ppm (m, 20H;
HAr); 13C NMR (100 MHz, CDCl3): d=32.11 (C6), 53.87 (C5), 55.95
(PhCH2N), 64.35 (C1), 72.56, 75.26, 75.46 (PhCH2O), 78.32, 80.12,
87.60 (C2, C3, C4), 117.59 (C8), 127.14–128.47 (CHAr), 134.30 (C7),
138.52–139.06 ppm (CAr).
1
400 mesh). Copies of H and 13C NMR spectra of final products are
provided in the Supporting Information.
N-Benzyl-2,3,4-tri-O-benzyl-d-xylopyranosylamine (3): 2,3,4-Tri-O-
benzyl-d-xylopyranose (2; 1.50 g, 3.57 mmol) was dissolved in dry
CH2Cl2 (3.6 mL). p-Toluenesulfonic acid (0.67 g, 3.52 mmol, 1 equiv)
and BnNH2 (780 mL, 7.14 mmol, 2 equiv) were added slowly. The re-
action mixture was stirred for 20 h at room temperature, and then
CH2Cl2 (100 mL) was added. The organic phase was separated,
washed twice with saturated Na2CO3 (200 mL), dried over MgSO4,
and concentrated under vacuum. Most of the residual amine was
co-evaporated with toluene to afford crude compound 3 as
a white solid (1.76 g, 97%), which was used in the next step with-
out further purification.
(1R)- and (1S)-1-C-Propyl-1,5-dideoxy-1,5-imino-d-xylitol (6a and
6b): The protected compound (5a or 5b) was dissolved in a mix-
ture of CH2Cl2 (1 mL), iPrOH (5 mL), and 1n HCl (1:5:1; 0.03m).
10% Pd/C and Pd black (1:1, 500 mgmmolÀ1) were added to the
mixture, which was stirred overnight under an H2 atmosphere. The
reaction mixture was filtered through a membrane, and the cata-
lyst was washed with iPrOH. The filtrate was introduced onto an
ion-exchange resin column (H+, Dowex 50WX8). The column was
washed with MeOH and H2O, and then the product was eluted by
using 0.5n NH4OH and concentrated under vacuum.
(1R,1S)-1-C-Allyl-2,3,4-tri-O-benzyl-1-benzylamino-1-deoxy-d-xy-
litol (4): Crude xylosylamine 3 (2.17 g, 4.26 mmol) was dissolved in
dry THF (200 mL) and treated with allylmagnesium bromide (1m in
Et2O, 20 mL, 20 mmol, 4.7 equiv) at 08C. The reaction mixture was
stirred overnight at room temperature and then carefully hydro-
lyzed (H2O, 100 mL). The aqueous phase was extracted three times
with EtOAc (100 mL). The organic phases were combined, dried
over MgSO4, and concentrated under vacuum. The crude product
was purified by flash chromatography on silica gel (petroleum
ether/EtOAc, 4:1) to afford 4 as a colorless oil and as an insepara-
ble 85:15 mixture of diastereomers (1.58 g, 67%): 1H NMR
(250 MHz, CDCl3): d=2.18–2.47 (m, 2H; H6), 2.58–2.64 (m, 0.85H;
H1A), 2.87–2.94 (m, 0.15H; H1B), 3.34–3.39 (m, 0.85H; H4A), 3.58 (d,
1H, J=13.0 Hz; PhCH2N), 3.59–3.79 (m, 3.15H; H2, H5, H4B), 3.87
(d, 1H, J=13.0 Hz; PhCH2N), 4.05–4.10 (m, 1H; H3), 4.38 (d, 1H, J=
11.7 Hz; PhCH2O), 4.55 (d, 1H, J=11.7 Hz; PhCH2O), 4.63–4.84 (m,
6a (64 mg, 97%) was obtained from 5a (200 mg, 0.375 mmol) as
a colorless oil: [a]D =À17.9 (c=1.0, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.95 (t, 3H, J=7.0 Hz; H8), 1.34–1.48 (m, 3H; H6a, H7),
1.50–1.60 (m, 1H; H6b), 2.79 (d, 1H, J=12.8 Hz; H5a), 2.89–2.91 (m,
1H; H1), 3.04 (d, 1H, J=12.8 Hz; H5b), 3.54 (brs, 2H; H2, H4),
3.77 ppm (brs, 1H; H3); 13C NMR (100 MHz, CD3OD): d=14.51 (C8),
20.15 (C7), 33.00 (C6), 47.43 (C5), 55.27 (C1), 70.46 (C2 or C4), 71.12
(C3), 71.65 ppm (C2 or C4); MS (ESI+): m/z 176.5 [M+H]+; HRMS
(ESI): m/z calcd for C8H18NO3: 176.128120 [M+H]+; found:
176.128682.
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6b (38 mg, 97%) was obtained from 5b (119 mg, 0.223 mmol) as
a white solid: [a]D =À44.6 (c=0.97, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.94 (t, 3H, J=7.1 Hz; H8), 1.23–1.40 (m, 2H; H6a,
H7a), 1.46–1.58 (m, 1H; H7b), 1.78–1.85 (m, 1H; H6b), 2.31–2.36
(m, 1H; H1), 2.39 (dd, 1H, J=10.8, 12.1 Hz; H5a), 2.97 (t, 1H, J=
9.1 Hz; H2), 3.05 (dd, 1H, J=5.1, 12.1 Hz; H5b), 3.15 (t, 1H, J=
9.1 Hz; H3), 3.41 ppm (ddd, 1H, J=5.1, 9.1, 10.8 Hz; H4); 13C NMR
(100 MHz, CD3OD): d=14.64 (C8), 19.86 (C7), 35.24 (C6), 51.41 (C5),
61.33 (C1), 72.59 (C4), 76.67 (C2), 80.63 ppm (C3); IR (neat): n˜ =
3258, 2957, 2908, 2872, 1455, 1362, 1078, 1047, 998 cmÀ1; MS (ESI+
): m/z 176.5 [M+H]+; HRMS (ESI): m/z calcd for C8H18NO3:
176.128120 [M+H]+; found: 176.128591.
2.39 (m, 1H; H6b), 2.62 (dd, 1H, J=10.7, 13.7 Hz; H5a), 2.82 (dd,
1H, J=5.0, 13.7 Hz; H5b), 3.26–3.29 (m, 1H; H1), 3.63 (d, 1H, J=
13.0 Hz; PhCH2N), 3.63–3.74 (m, 2H; H3, H4), 3.78 (d, 1H, J=
13.0 Hz; PhCH2N), 3.84 (dd, 1H, J=5.6, 9.4 Hz; H2), 4.44 (dd, 1H,
J=5.7, 11.8 Hz; H8a), 4.55–4.67 (m, 5H, PhCH2O; H8b), 4.87 (d, 1H,
J=10.8 Hz; PhCH2O), 4.91 (d, 1H, J=10.8 Hz; PhCH2O), 5.62–5.68
(m, 1H; H7), 7.06–7.42 (m, 24H; HAr), 7.89 (d, 2H, J=8.5 Hz; HAr),
7.95 ppm (d, 2H, J=8.5 Hz; HAr); 13C NMR (100 MHz, CDCl3): d=
25.93 (C6), 31.23 (CH3), 35.17, 35.19 (C(CH3)3), 46.67 (C5), 56.45 (C1),
59.07 (PhCH2N), 66.13 (C8), 70.30 (C7), 75.84, 73.09, 72.79 (PhCH2O),
76.77 (C4), 78.05 (C2), 83.43 (C3), 125.37, 125.48, 127.23 (CHAr),
127.27, 127.53 (CAr), 127.63–129.72 (CHAr), 138.51–139.09 (CAr),
156.67, 156.70 (CAr), 166.03, 166.38 ppm (C=O); IR (neat): n˜ =2961,
1718, 1609, 1455, 1363, 1264, 1096, 1069, 1017, 734, 698 cmÀ1; MS
(ESI+): m/z 889.0 [M+H]+; HRMS (ESI): m/z calcd for C58H66NO7:
888.4839 [M+H]+; found: 888.4841.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-(2,3-dihydroxypropyl)-1,5-di-
deoxy-1,5-imino-d-xylitol (7): Compound 5a (1.20 g, 2.25 mmol)
was dissolved in a mixture of THF (7 mL), tBuOH (20 mL), and H2O
(4 mL). Subsequently, N-methylmorpholine-N-oxide (317 mg,
2.71 mmol, 1.2 equiv) was added. After the reaction mixture had
been stirred for 5 min, a 2.5% (w/v) solution of OsO4 in tBuOH
(1.55 mL, 0.118 mmol, 0.05 equiv) was added. The reaction mixture
was stirred at room temperature for 40 h. The mixture was then
treated with a saturated Na2S2O5 solution (10 mL) and stirred for
30 min. It was diluted with CH2Cl2 (40 mL) and H2O (20 mL). The
aqueous phase was separated and extracted with CH2Cl2 (40 mL).
The combined organic phases were dried over MgSO4 and concen-
trated under vacuum. The crude product was purified by flash
chromatography on silica gel (petroleum ether/EtOAc, 1:4) to
afford 7 as a colorless oil and as an inseparable 1:1 mixture of dia-
stereomers 7a and 7b (1.03 g, 81%).
General procedure for methanolysis: MeONa (1 equiv) was added
to a solution of 8a or 8b in a mixture of MeOH/CH2Cl2 (1:6;
0.06m). The reaction mixture was stirred at room temperature for
2 d and then diluted with CH2Cl2. The organic phase was washed
with 5% aqueous HCl, saturated NaHCO3, and H2O, dried over
MgSO4, and concentrated under vacuum. The crude product was
purified by flash chromatography on silica gel (petroleum ether/
EtOAc, 1:4).
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2S)-2,3-dihydroxypropyl)-
1,5-dideoxy-1,5-imino-d-xylitol (7a): Compound 7a (640 mg,
85%) was obtained from 8a (1.18 g, 1.33 mmol) as a colorless oil:
[a]D =À8.1 (c=0.98, CHCl3); 1H NMR (400 MHz, CDCl3): d=1.71–
1.82 (m, 2H; H6), 2.75–2.86 (m, 2H; H5), 3.15–3.20 (m, 1H; H1),
3.42 (dd, 1H, J=5.3, 11.2 Hz; H8a), 3.56 (dd, 1H, J=3.4, 11.2 Hz;
H8b), 3.66–3.83 (m, 6H; H2, H3, H4, H7, PhCH2N), 4.58 (d, 1H, J=
11.8 Hz; PhCH2O), 4.59 (d, 1H, J=11.8 Hz; PhCH2O), 4.69 (d, 1H, J=
11.8 Hz; PhCH2O), 4.73 (d, 1H, J=11.8 Hz; PhCH2O), 4.87 (s, 2H;
PhCH2O), 7.05–7.08 (m, 2H; HAr), 7.24–7.39 ppm (m, 18H; HAr);
13C NMR (100 MHz, CDCl3): d=25.91 (C6), 46.42 (C5), 58.63
(PhCH2N), 60.61 (C1), 66.94 (C8), 73.23, 73.47 (PhCH2O), 73.55 (C3 or
C7), 75.51 (C2 or C4), 75.96 (PhCH2O), 76.43 (C2 or C4), 83.38 (C3 or
C7), 127.76–129.03 (CHAr), 137.39–138.84 ppm (CAr); IR (neat): n˜ =
3393, 3029, 2867, 1495, 1454, 1363, 1067, 1027, 735, 697 cmÀ1; MS
(ESI+): m/z 568.5 [M+H]+; HRMS (ESI): m/z calcd for C36H42NO5:
568.3063 [M+H]+; found: 568.3067.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-(2,3-di-(4-tert-butylbenzoyl-
oxy)propyl)-1,5-dideoxy-1,5-imino-d-xylitol (8a and 8b): Diol 7
(530 mg, 0.934 mmol) was dissolved in dry pyridine (8 mL) and
treated with 4-tert-butylbenzoyl chloride (513 mL, 2.63 mmol,
2.8 equiv). After the reaction mixture had been stirred for 20 h at
room temperature, another equivalent of acid chloride (171 mL,
0.876 mmol) was added. The reaction mixture was stirred at room
temperature for 24 h and then diluted with CH2Cl2 (20 mL). The
mixture was washed three times with H2O (20 mL), dried over
MgSO4, concentrated under vacuum, and co-evaporated with tolu-
ene. The crude product was purified by flash chromatography on
silica gel (toluene/CH2Cl2, 85:15) to afford the separable diastereo-
mers 8a (276 mg, 33%) and 8b (306 mg, 37%) as yellow oils.
1
8a: [a]D =À10.1 (c=1.0, CHCl3); H NMR (400 MHz, CDCl3): d=1.31
(s, 9H; (CCH3)3), 1.32 (s, 9H; (CCH3)3), 2.07–2.14 (m, 1H; H6a), 2.19–
2.25 (m, 1H; H6b), 2.72 (dd, 1H, J=10.4, 13.1 Hz; H5a), 2.86 (dd,
1H, J=3.7, 13.1 Hz; H5b), 3.20–3.24 (m, 1H; H1), 3.66–3.69 (m, 3H;
H3, H4, PhCH2N), 3.78 (d, 1H, J=13.4 Hz; PhCH2N), 3.85 (dd, 1H,
J=5.6, 9.2 Hz; H2), 4.29 (dd, 1H, J=5.5, 12.0 Hz; H8a), 4.37 (dd,
1H, J=3.0, 12.0 Hz; H8b), 4.57 (d, 1H, J=11.6 Hz; PhCH2O), 4.63–
4.71 (m, 3H; PhCH2O), 4.88 (s, 2H; PhCH2O), 5.59–5.65 (m, 1H; H7),
7.12–7.43 (m, 24H; HAr), 7.93 (d, 2H, J=8.4 Hz; HAr), 7.94 ppm (d,
2H, J=8.4 Hz; HAr); 13C NMR (100 MHz, CDCl3): d=25.70 (C6), 31.20
(CH3), 35.15 (C(CH3)3), 47.82 (C5), 56.01 (C1), 58.57 (PhCH2N), 64.88
(C8), 71.42 (C7), 73.00, 73.29, 75.81 (PhCH2O), 77.61 (C4), 78.85 (C2),
83.29 (C3), 125.42, 125.44 (CHAr), 127.25 (CAr), 127.31 (CHAr), 127.52
(CAr), 127.59–129.67 (CHAr), 138.40–139.09 (CAr), 156.68 (CAr), 166.14,
166.29 ppm (C=O); IR (neat): n˜ =2961, 1717, 1610, 1545, 1454,
1344, 1264, 1095, 1069, 1026, 732, 698 cmÀ1; MS (ESI+): m/z 889.0
[M+H]+; HRMS (ESI): m/z calcd for C58H66NO7: 888.4839 [M+H]+;
found: 888.4838.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-2,3-dihydroxypropyl)-
1,5-dideoxy-1,5-imino-d-xylitol (7b): Compound 7b (665 mg,
89%) was obtained from 8b (1.17 g, 1.32 mmol) as a colorless oil:
[a]D =À2.6 (c=0.92, CHCl3); H NMR (400 MHz, CDCl3): d=1.79 (t,
1
2H, J=5.9 Hz; H6), 2.66 (dd, 1H, J=10.4, 13.3 Hz; H5a), 2.81 (dd,
1H, J=4.2, 13.3 Hz; H5b), 3.17 (q, 1H, J=5.9 Hz; H1), 3.33 (dd, 1H,
J=7.0, 11.0 Hz; H8a), 3.43 (dd, 1H, J=4.0, 11.0 Hz; H8b), 3.61 (d,
1H, J=13.1 Hz; PhCH2N), 3.66–3.73 (m, 4H; H3, H4, H7, PhCH2N),
3.79 (dd, 1H, J=5.9, 9.5 Hz; H2), 4.58 (d, 1H, J=11.6 Hz; PhCH2O),
4.63 (d, 1H, J=10.9 Hz; PhCH2O), 4.66 (d, 1H, J=10.9 Hz; PhCH2O),
4.74 (d, 1H, J=11.6 Hz; PhCH2O), 4.86 (d, 1H, J=10.9 Hz; PhCH2O),
4.90 (d, 1H, J=10.9 Hz; PhCH2O), 7.10–7.12 (m, 2H; HAr), 7.26–
7.38 ppm (m, 18H; HAr); 13C NMR (100 MHz, CDCl3): d=26.91 (C6),
47.93 (C5), 57.09 (C1), 58.39 (PhCH2N), 66.71 (C8), 71.10 (C3, C4, or
C7), 73.07, 73.79, 75.79 (PhCH2O), 77.38 (C3, C4, or C7), 78.07 (C2),
83.22 (C3, C4, or C7), 127.65–128.77 (CHAr), 137.93–138.92 ppm
(CAr); IR (neat): n˜ =3394, 3029, 2869, 1495, 1453, 1363, 1068, 1027,
735, 696 cmÀ1; MS (ESI+): m/z 568.5 [M+H]+; HRMS (ESI): m/z
calcd for C36H42NO5: 568.3063 [M+H]+; found: 568.3053.
1
8b: [a]D = +5.5 (c=1.2, CHCl3); H NMR (400 MHz, CDCl3): d=1.32
(brs, 18H; (CCH3)3), 2.00 (ddd, 1H, J=3.4, 11.0, 14.5 Hz; H6a), 2.32–
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General procedure for esterification: Diol 7a or 7b was dissolved
in dry pyridine (0.08m) and treated with the corresponding acid
chloride. The reaction mixture was stirred at room temperature for
several days and then diluted with EtOAc. The organic phase was
washed with H2O, dried over MgSO4, concentrated under vacuum,
and co-evaporated with toluene.
signals; 13C NMR (100 MHz, CDCl3): d=14.24 (CH3), 22.81, 25.06,
25.08 (CH2), 25.54 (C6), 29.30–29.60, 32.01 (CH2), 34.30, 34.55
(CH2CO), 47.85 (C5), 56.01 (C1), 58.47 (PhCH2N), 64.44 (C8), 70.71
(C7), 73.07, 73.30, 75.82 (PhCH2O), 77.78 (C4), 79.04 (C2), 83.26 (C3),
127.35–128.52 (CHAr), 138.45–139.11 (CAr), 173.41, 173.51 ppm (C=
O); IR (neat): n˜ =2924, 2854, 1736, 1454, 1362, 1244, 1156, 1099,
1067, 1027, 734, 697 cmÀ1; MS (ESI+): m/z 876.5 [M+H]+; HRMS
(ESI): m/z calcd for C56H78NO7: 876.5778 [M+H]+; found: 876.5756.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2S)-2,3-di(hexanoyloxy)-
propyl)-1,5-dideoxy-1,5-imino-d-xylitol (9aS): Isomer 7a (129 mg,
0.227 mmol) was treated with hexanoyl chloride (3 equiv) for 1 d.
Purification was performed by flash chromatography on silica gel
(petroleum ether/EtOAc, 9:1) to afford 9aS (112 mg, 65%) as a col-
orless oil: [a]D = +6.4 (c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.86–0.91 (m, 6H; CH3), 1.23–1.36 (m, 8H; CH2), 1.54–1.63 (m,
4H; CH2), 1.81–1.88 (m, 1H; H6a), 1.91–1.97 (m, 1H; H6b), 2.13–
2.28 (m, 4H; CH2CO), 2.60 (dd, 1H, J=10.1, 13.1 Hz; H5a), 2.80 (dd,
1H, J=4.7, 13.1 Hz; H5b), 3.03–3.07 (m, 1H; H1), 3.61 (d, 1H, J=
13.5 Hz; PhCH2N), 3.62–3.69 (m, 2H; H3, H4), 3.74 (d, 1H, J=
13.5 Hz; PhCH2N), 3.79 (dd, 1H, J=5.6, 9.1 Hz; H2), 3.88 (dd, 1H,
J=6.3, 12.0 Hz; H8a), 4.09 (dd, 1H, J=3.0, 12.0 Hz; H8b), 4.56 (d,
1H, J=11.5 Hz; PhCH2O), 4.63–4.65 (m, 3H; PhCH2O), 4.87 (s, 2H;
PhCH2O), 5.21 (qd, 1H, J=3.0, 3ꢂ6.3 Hz; H7), 7.16–7.18 (m, 2H;
HAr), 7.23–7.37 ppm (m, 18H; HAr); 13C NMR (100 MHz, CDCl3): d=
14.04 (CH3), 22.45, 24.71, 24.73 (CH2), 25.55 (C6), 31.41 (CH2), 34.24,
34.49 (CH2CO), 47.86 (C5), 55.99 (C1), 58.46 (PhCH2N), 64.44 (C8),
70.71 (C7), 73.07, 73.30, 75.81 (PhCH2O), 77.77 (C4), 79.02 (C2),
83.25 (C3), 127.34–128.52 (CHAr), 138.45–139.11 (CAr), 173.40,
173.50 ppm (C=O); IR (neat): n˜ =3030, 2929, 2869, 1735, 1454,
1362, 1163, 1095, 1066, 1028, 735, 697 cmÀ1; MS (ESI+): m/z 765.0
[M+H]+; HRMS (ESI): m/z calcd for C48H62NO7: 764.4526 [M+H]+;
found: 764.4515.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-2,3-di(decanoyloxy)-
propyl)-1,5-dideoxy-1,5-imino-d-xylitol
(9bR):
Isomer
7b
(183 mg, 0.322 mmol) was treated with decanoyl chloride (6 equiv)
for 3 d. Purification was performed by flash chromatography on
silica gel (petroleum ether/EtOAc, 95:5) to afford 9bR (165 mg,
59%) as
a
yellow oil: [a]D = +13.2 (c=1.2, CHCl3); 1H NMR
(400 MHz, CDCl3): very similar to 9aR, except for the integration of
the ester chain signals; 13C NMR (100 MHz, CDCl3): d=14.25 (CH3),
22.81, 25.06, 25.08 (CH2), 25.54 (C6), 29.30–29.60, 32.01 (CH2), 34.30,
34.55 (CH2CO), 47.85 (C5), 56.01 (C1), 58.47 (PhCH2N), 64.44 (C8),
70.71 (C7), 73.07, 73.30, 75.82 (PhCH2O), 77.78 (C4), 79.04 (C2),
83.26 (C3), 127.34–128.50 (CHAr), 138.45–139.11 (CAr), 173.41,
173.51 ppm (C=O); IR (neat): n˜ =3030, 2924, 2854, 1737, 1455,
1360, 1246, 1205, 1157, 1102, 1069, 1027, 735, 697 cmÀ1; MS
(ESI+): m/z 877.0 [M+H]+; HRMS (ESI): m/z calcd for C56H78NO7:
876.5778 [M+H]+; found: 876.5760.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-2,3-di(tetradecanoyl-
oxy)propyl)-1,5-dideoxy-1,5-imino-d-xylitol (9cR): Isomer 7b
(64 mg, 0.113 mmol) was treated with myristoyl chloride (6 equiv)
for 2 d. Purification was performed by flash chromatography on
silica gel (petroleum ether/EtOAc, 9:1) to afford 9cR (25 mg, 22%)
as a yellow oil: [a]D = +6.0 (c=1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): very similar to 9aR, except for the integration of the ester
chain signals; 13C NMR (100 MHz, CDCl3): d=14.27 (CH3), 22.84,
25.08 (CH2), 25.72 (C6), 29.33–29.85, 32.01 (CH2), 34.34, 34.52
(CH2CO), 46.96 (C5), 55.88 (C1), 59.00 (PhCH2N), 65.42 (C8), 69.61
(C7), 72.79, 73.21, 75.89 (PhCH2O), 77.38 (C4), 77.88 (C2), 83.44 (C3),
127.35–128.76 (CHAr), 138.50–139.10 (CAr), 173.21, 173.63 ppm (C=
O); IR (neat): n˜ =3030, 2922, 2852, 1738, 1455, 1361, 1246, 1161,
1107, 1071, 1028, 734, 697 cmÀ1; MS (ESI+): m/z 989.0 [M+H]+;
HRMS (ESI): m/z calcd for C64H94NO7: 988.7030 [M+H]+; found:
988.7038.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-2,3-di(hexanoyloxy)-
propyl)-1,5-dideoxy-1,5-imino-d-xylitol (9aR): Isomer 7b (131 mg,
0.231 mmol) was treated with hexanoyl chloride (3 equiv) for 1 d.
Purification was performed by flash chromatography on silica gel
(petroleum ether/EtOAc, 9:1) to afford 9aR (126 mg, 71%) as a col-
orless oil: [a]D = +7.5 (c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.85–0.94 (m, 6H; CH3), 1.21–1.34 (m, 8H; CH2), 1.50–1.57 (m,
2H; CH2), 1.58–1.66 (m, 2H; CH2), 1.78 (ddd, 1H, J=3.5, 10.9,
14.6 Hz; H6a), 2.02–2.20 (m, 3H; H6b, CH2CO), 2.31 (t, 2H, J=
7.5 Hz; CH2CO), 2.57 (dd, 1H, J=10.9, 13.9 Hz; H5a), 2.79 (dd, 1H,
J=5.4, 13.9 Hz; H5b), 3.07–3.12 (m, 1H; H1), 3.57–3.63 (m, 2H; H3,
PhCH2N), 3.67–3.73 (m, 1H; H4), 3.76 (d, 1H, J=13.1 Hz; PhCH2N),
3.82 (dd, 1H, J=5.6, 9.7 Hz; H2), 4.03 (dd, 1H, J=5.6, 11.8 Hz; H8a),
4.29 (dd, 1H, J=3.7, 11.8 Hz; H8b), 4.56 (d, 1H, J=11.6 Hz;
PhCH2O), 4.64–4.67 (m, 3H; PhCH2O), 4.85 (d, 1H, J=10.8 Hz;
PhCH2O), 4.89 (d, 1H, J=10.8 Hz; PhCH2O), 5.19–5.25 (m, 1H; H7),
7.12–7.14 (m, 2H; HAr), 7.21–7.38 ppm (m, 18H; HAr); 13C NMR
(100 MHz, CDCl3): d=14.01, 14.03 (CH3), 22.42, 24.68, 24.70 (CH2),
25.70 (C6), 31.39 (CH2), 34.24, 34.41 (CH2CO), 46.97 (C5), 55.81 (C1),
58.96 (PhCH2N), 65.37 (C8), 69.59 (C7), 72.75, 73.17, 75.84 (PhCH2O),
76.74 (C4), 77.81 (C2), 83.40 (C3), 127.31–128.73 (CHAr), 138.47–
139.08 (CAr), 173.15, 173.57 ppm (C=O); IR (neat): n˜ =3030, 2929,
2869, 1735, 1454, 1360, 1244, 1163, 1094, 1070, 1028, 735,
697 cmÀ1; MS (ESI+): m/z 765.0 [M+H]+; HRMS (ESI): m/z calcd for
C48H62NO7: 764.4526 [M+H]+; found: 764.4530.
General procedure for etherification: NaH (60% suspension in
mineral oil) was added to a solution of 7a or 7b in anhydrous
DMF (0.02m). After a few minutes, 1-bromohexane was added. The
reaction mixture was stirred at room temperature for the time indi-
cated and then quenched with MeOH. The mixture was diluted
with EtOAc and washed with saturated NaHCO3 and H2O. The or-
ganic phase was dried over MgSO4 and concentrated under
vacuum.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2S)-(3-hexyloxy-2-hydroxy)-
propyl)-1,5-dideoxy-1,5-imino-d-xylitol (11S) and (1R)-N-benzyl-
2,3,4-tri-O-benzyl-1-C-((2S)-(2,3-dihexyloxy)propyl)-1,5-dideoxy-
1,5-imino-d-xylitol (12S): Isomer 7a (132 mg, 0.233 mmol) was
treated with 60% NaH (5 equiv) and 1-bromohexane (4 equiv) for
16 h. Purification was performed by flash chromatography on silica
gel (petroleum ether/EtOAc, 95:5) to afford 12S (71 mg, 41%) as
a colorless oil and 11S (79 mg, 52%) as a yellow oil.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2S)-2,3-di(decanoyloxy)-
propyl)-1,5-dideoxy-1,5-imino-d-xylitol (9bS): Isomer 7a (124 mg,
0.218 mmol) was treated with decanoyl chloride (6 equiv) for 4 d.
Purification was performed by flash chromatography on silica gel
(petroleum ether/EtOAc, 9:1) to afford 9bS (66 mg, 35%) as
1
11S: [a]D =À3.0 (c=1.1, CHCl3); H NMR (400 MHz, CDCl3): d=0.88
(t, 3H, J=6.8 Hz; CH3), 1.26–1.37 (m, 6H; CH2), 1.54–1.61 (m, 2H;
CH2), 1.64–1.71 (m, 1H; H6a), 1.92 (dt, 1H, J=2ꢂ2.8, 14.6 Hz; H6b),
2.74–2.85 (m, 2H; H5), 3.17–3.22 (m, 1H; H1), 3.30 (dd, 1H, J=4.9,
9.7 Hz; H8a), 3.36 (dd, 1H, J=5.6, 9.7 Hz; H8b), 3.43 (t, J=6.8 Hz,
1
a yellow oil: [a]D = +8.7 (c=1.2, CHCl3); H NMR (400 MHz, CDCl3):
very similar to 9aS, except for the integration of the ester chain
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1805 – 1817 1813

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2H; CH2O), 3.65–3.69 (m, 2H, H3; PhCH2N), 3.74–3.83 (m, 4H; H2,
H4, H7, PhCH2N), 4.56–4.72 (m, 4H; PhCH2O), 4.88 (s, 2H; PhCH2O),
5.58 (brs, 1H; OH), 7.09–7.11 (m, 2H; HAr), 7.24–7.38 ppm (m, 18H;
HAr); 13C NMR (100 MHz, CDCl3): d=14.19 (CH3), 22.74, 25.92 (CH2),
26.82 (C6), 29.73, 31.83 (CH2), 46.50 (C5), 58.65 (PhCH2N), 60.57
(C1), 71.85 (CH2O), 72.33 (C7), 73.17, 73.30 (PhCH2O), 75.30 (C8),
75.88 (C4), 75.93 (PhCH2O), 76.94 (C2), 83.41 (C3), 127.71–128.97
(CHAr), 137.70–138.97 ppm (CAr); IR (neat): n˜ =3029, 2928, 2858,
1454, 1363, 1099, 1068, 1027, 734, 698 cmÀ1; MS (ESI+): m/z 652.5
[M+H]+; HRMS (ESI): m/z calcd for C42H54NO5: 652.4002 [M+H]+;
found: 652.3984.
H8b), 3.52–3.70 (m, 5H; H3, H4, H7, CH2O, PhCH2N), 3.79–3.84 (m,
2H; H2, PhCH2N), 4.55 (d, 1H, J=11.6 Hz; PhCH2O), 4.57 (d, 1H, J=
11.6 Hz; PhCH2O), 4.66 (d, 1H, J=11.5 Hz; PhCH2O), 4.70 (d, 1H, J=
11.5 Hz; PhCH2O), 4.82 (d, 1H, J=10.8 Hz; PhCH2O), 4.90 (d, 1H, J=
10.8 Hz; PhCH2O), 7.19–7.37 ppm (m, 20H; HAr); 13C NMR (100 MHz,
CDCl3): d=14.19, 14.20 (CH3), 22.79, 26.04 (CH2), 27.19 (C6), 29.86,
30.50, 31.87, 31.90 (CH2), 47.41 (C5), 55.81 (C1), 59.01 (PhCH2N),
70.57 (CH2O), 71.78 (CH2O), 72.12, 73.17 (PhCH2O), 74.22 (C8), 75.76
(PhCH2O), 76.35 (C7), 77.38 (C4), 78.68 (C2), 83.43 (C3), 127.22–
128.58 (CHAr), 138.74–139.70 ppm (CAr); IR (neat): n˜ =3030, 2928,
2857, 1454, 1361, 1092, 1070, 1028, 734, 697 cmÀ1; MS (ESI+): m/z
737.0 [M+H]+; HRMS (ESI): m/z calcd for C48H66NO5: 736.4941 [M+
H]+; found: 736.4960.
12S: [a]D = +10.9 (c=1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d=
0.85–0.89 (m, 6H; CH3), 1.22–1.34 (m, 12H; CH2), 1.45–1.56 (m, 4H;
CH2), 1.68–1.75 (m, 1H; H6a), 1.86 (dt, 1H, J=2ꢂ5.7, 14.4 Hz; H6b),
2.51 (dd, 1H, J=10.1, 12.6 Hz; H5a), 2.76 (dd, 1H, J=4.8, 12.6 Hz;
H5b), 3.23 (q, 1H, J=5.7 Hz; H1), 3.27–3.38 (m, 5H; H8, CH2O),
3.50–3.70 (m, 5H; H3, H4, H7, CH2O, PhCH2N), 3.79–3.80 (m, 1H;
H2), 3.80 (d, 1H, J=13.8 Hz; PhCH2N), 4.55 (d, 1H, J=11.6 Hz;
PhCH2O), 4.64 (d, 1H, J=11.6 Hz; PhCH2O), 4.67 (s, 2H; PhCH2O),
4.86 (d, 1H, J=10.8 Hz; PhCH2O), 4.88 (d, 1H, J=10.8 Hz; PhCH2O),
7.21–7.36 ppm (m, 20H; HAr); 13C NMR (100 MHz, CDCl3): d=14.19
(CH3), 22.76, 25.98, 26.00 (CH2), 26.67 (C6), 29.83, 30.35, 31.86 (CH2),
47.93 (C5), 56.63 (C1), 58.42 (PhCH2N), 70.36 (CH2O), 71.57 (C8 or
CH2O), 72.93, 73.00 (PhCH2O), 73.32 (C8 or CH2O), 75.71 (PhCH2O),
77.35 (C7), 78.33 (C4), 80.39 (C2), 83.29 (C3), 127.01–128.45 (CHAr),
138.66–139.70 ppm (CAr); IR (neat): n˜ =3063, 3029, 2927, 2857,
1454, 1363, 1091, 1068, 1028, 733, 696 cmÀ1; MS (ESI+): m/z 737.0
[M+H]+; HRMS (ESI): m/z calcd for C48H66NO5: 736.4941 [M+H]+;
found: 736.4933.
General procedure for hydrogenolysis: The protected compound
was dissolved in EtOH (0.01m) in the presence of 1m HCl (5 equiv).
Pd/C (10%) was added and the reaction mixture was stirred under
an H2 atmosphere at room temperature. More catalyst was added
if needed. The mixture was filtered through a membrane and the
catalyst was washed with MeOH. The filtrate was concentrated
under vacuum to afford the pure compounds as hydrochlorides.
(1R)-1-C-((2S)-2,3-Di(hexanoyloxy)propyl)-1,5-dideoxy-1,5-imino-
d-xylitol (10aS): Compound 9aS (50 mg, 0.065 mmol) was stirred
under an H2 atmosphere for 42 h to afford 10aS (30 mg, 100%) as
a
yellow oil: [a]D =À11.0 (c=1.0, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.91 (t, 6H, J=6.8 Hz; CH3), 1.30–1.37 (m, 8H; CH2),
1.58–1.66 (m, 4H; CH2), 1.99 (ddd, 1H, J=4.0, 10.4, 14.0 Hz; H6a),
2.22 (ddd, 1H, J=3.1, 10.7, 14.0 Hz; H6a), 2.29–2.38 (m, 4H;
CH2CO), 3.23 (d, 1H, J=13.0 Hz; H5a), 3.41–3.51 (m, 2H; H1, H5b),
3.85–3.87 (m, 1H; H2), 3.92–3.96 (m, 2H; H3, H4), 4.04–4.09 (m,
1H; H8a), 4.39 (dd, 1H, J=3.2, 12.0 Hz; H8b), 5.22–5.27 ppm (m,
1H; H7); 13C NMR (100 MHz, CD3OD): d=14.24 (CH3), 23.36, 23.38,
25.62, 25.65 (CH2), 30.51 (C6), 32.34, 32.37 (CH2), 34.80, 35.00
(CH2CO), 47.43 (C5), 53.20 (C1), 65.79 (C8), 67.51, 67.98 (C3, C4),
68.22 (C7), 68.78 (C2), 174.68, 174.83 ppm (C=O); IR (neat): n˜ =
3308, 2956, 2930, 2860, 1737, 1588, 1453, 1378, 1245, 1169, 1102,
1063, 1027 cmÀ1; MS (ESI+): m/z 404.0 [M+H]+; HRMS (ESI): m/z
calcd for C20H38NO7: 404.2648 [M+H]+; found: 404.2649.
(1R)-N-Benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-(3-hexyloxy-2-hy-
droxy)propyl)-1,5-dideoxy-1,5-imino-d-xylitol (11R) and (1R)-N-
benzyl-2,3,4-tri-O-benzyl-1-C-((2R)-(2,3-dihexyloxy)propyl)-1,5-di-
deoxy-1,5-imino-d-xylitol (12R): Isomer 7b (142 mg, 0.250 mmol)
was treated with 60% NaH (2 equiv) and 1-bromohexane (5 equiv)
for 2 d. Purification was performed by flash chromatography on
silica gel (petroleum ether/EtOAc, 85:15) to afford 12R (14 mg, 8%)
as a yellow oil and 11R (73 mg, 45%) as a colorless oil.
1
11R: [a]D = +5.6 (c=1.1, CHCl3); H NMR (400 MHz, CDCl3): d=0.88
(1R)-1-C-((2R)-2,3-Di(hexanoyloxy)propyl)-1,5-dideoxy-1,5-imino-
d-xylitol (10aR): Compound 9aR (68 mg, 0.089 mmol) was stirred
under an H2 atmosphere for 27 h to afford 10aR (38 mg, 97%) as
(t, 3H, J=6.7 Hz; CH3), 1.26–1.37 (m, 6H; CH2), 1.52–1.59 (m, 2H;
CH2), 1.77–1.90 (m, 2H; H6), 2.66 (dd, 1H, J=10.3, 13.2 Hz; H5a),
2.79 (dd, 1H, J=4.4, 13.2 Hz; H5b), 3.22 (dd, 1H, J=5.7, 9.6 Hz;
H8a), 3.29–3.35 (m, 2H; H1, H8b), 3.38–3.42 (m, 2H; CH2O), 3.65 (d,
1H, J=13.3 Hz; PhCH2N), 3.65–3.71 (m, 2H; H3, H4), 3.74 (d, 1H,
J=13.3 Hz; PhCH2N), 3.80 (dd, 1H, J=5.4, 9.4 Hz; H2), 3.84–3.90
(m, 1H; H7), 4.57 (d, 1H, J=11.6 Hz; PhCH2O), 4.65–4.68 (m, 3H;
PhCH2O), 4.88 (s, 2H; PhCH2O), 7.13–7.15 (m, 2H; HAr), 7.24–
7.37 ppm (m, 18H; HAr); 13C NMR (100 MHz, CDCl3): d=14.18 (CH3),
22.75, 25.97 (CH2), 26.91 (C6), 29.81, 31.84 (CH2),47.69 (C5), 56.73
(C1), 58.61 (PhCH2N), 69.26 (C7), 71.73 (CH2O), 73.12, 73.16
(PhCH2O), 74.70 (C8), 75.81 (PhCH2O), 77.40 (C4), 78.29 (C2), 83.35
(C3), 127.50–128.81 (CHAr), 138.27–139.12 ppm (CAr); IR (neat): n˜ =
2928, 2858, 1454, 1363, 1093, 1070, 1027, 734, 697 cmÀ1; MS
(ESI+): m/z 653.0 [M+H]+; HRMS (ESI): m/z calcd for C42H54NO5:
652.4002 [M+H]+; found: 652.4001.
a
white wax: [a]D =À7.0 (c=1.1, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.91 (t, 6H, J=6.7 Hz; CH3), 1.28–1.38 (m, 8H; CH2),
1.60–1.65 (m, 4H; CH2), 2.04 (ddd, 1H, J=4.4, 6.6, 15.0 Hz; H6a),
2.17 (ddd, 1H, J=6.6, 9.0, 15.0 Hz; H6a), 2.32–2.38 (m, 4H; CH2CO),
3.26 (d, 1H, J=12.9 Hz; H5a), 3.40 (d, 1H, J=12.9 Hz; H5b), 3.48 (t,
1H, J=6.6 Hz; H1), 3.80 (brs, 1H; H2), 3.93–3.96 (m, 2H; H3, H4),
4.11 (dd, 1H, J=5.9, 12.0 Hz; H8a), 4.39 (dd, 1H, J=3.3, 12.0 Hz;
H8b), 5.21–5.25 ppm (m, 1H; H7); 13C NMR (100 MHz, CD3OD): d=
14.24 (CH3), 23.35, 25.61 (CH2), 31.78 (C6), 32.34 (CH2), 34.78, 35.00
(CH2CO), 47.56 (C5), 53.84 (C1), 65.53 (C8), 67.81, 67.84 (C3, C4),
69.75 (C7), 70.98 (C2), 174.82, 175.19 ppm (C=O); IR (neat): n˜ =
3331, 2956, 2931, 2861, 1736, 1628, 1453, 1378, 1245, 1168, 1100,
1060 cmÀ1; MS (ESI+): m/z 404.5 [M+H]+; HRMS (ESI): m/z calcd
for C20H38NO7: 404.2648 [M+H]+; found: 404.2639.
12R: [a]D = +12.7 (c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3): d=
0.84 (t, 3H, J=6.9 Hz; CH3), 0.88 (t, 3H, J=6.9 Hz; CH3), 1.22–1.41
(m, 12H; CH2), 1.53–1.62 (m, 4H; CH2), 1.71 (ddd, 1H, J=3.4, 9.6,
14.5 Hz; H6a), 1.80 (ddd, 1H, J=3.2, 9.4, 14.5 Hz; H6b), 2.61 (dd,
1H, J=10.6, 13.5 Hz; H5a), 2.77 (dd, 1H, J=5.4, 13.5 Hz; H5b), 3.13
(dt, 1H, J=2ꢂ6.8, 9.2 Hz; CH2O), 3.34 (dd, 1H, J=5.0, 10.0 Hz;
H8a), 3.35–3.43 (m, 3H; H1, CH2O), 3.46 (dd, 1H, J=5.6, 10.0 Hz;
(1R)-1-C-((2S)-2,3-Di(decanoyloxy)propyl)-1,5-dideoxy-1,5-imino-
d-xylitol (10bS): Compound 9bS (63 mg, 0.072 mmol) was stirred
under an H2 atmosphere for 24 h to afford 10bS (31 mg, 78%) as
a white solid: m.p. 106–1098C (decomp.); [a]D =À9.6 (c=1.0,
1
MeOH); H NMR (400 MHz, CD3OD): very similar to 10aS, except for
the integration of the ester chains; 13C NMR (100 MHz, CD3OD): d=
14.44 (CH3), 23.72, 25.96, 26.00, 30.43–30.58, 33.05 (CH2, C6), 34.87,
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35.05 (CH2CO), 47.42 (C5), 53.21 (C1), 65.80 (C8), 67.51, 67.97 (C3,
C4), 68.24 (C7), 68.80 (C2), 174.66, 174.80 ppm (C=O); IR (neat): n˜ =
3309, 2956, 2923, 2852, 1737, 1575, 1455, 1369, 1276, 1253, 1214,
1166, 1110, 1059, 1026 cmÀ1; MS (ESI+): m/z 516.5 [M+H]+; HRMS
(ESI): m/z calcd for C28H54NO7: 516.3900 [M+H]+; found: 516.3881.
a white wax: [a]D = +5.2 (c=1.2, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.89–0.93 (m, 6H; CH3), 1.29–1.41 (m, 12H; CH2), 1.55–
1.61 (m, 4H; CH2), 1.97 (t, 2H, J=6.0 Hz; H6), 3.22 (d, 1H, J=
13.0 Hz; H5a), 3.39 (d, 1H, J=13.0 Hz; H5b), 3.47–3.55 (m, 5H; H8,
CH2O), 3.64–3.71 (m, 3H; H1, H7, CH2O), 3.82 (brs, 1H; H2), 3.91
(brs, 1H; H4), 3.94–3.95 ppm (m, 1H; H3); 13C NMR (100 MHz,
CD3OD): d=14.39 (CH3), 23.70, 26.98, 26.99, 30.75, 31.10, 32.85,
32.87 (CH2), 33.32 (C6), 47.51 (C5), 54.49 (C1), 68.04, 68.11 (C3, C4),
70.91 (CH2O), 71.49 (C2), 72.75, 72.90 (C8, CH2O), 76.34 ppm (C7);
IR (neat): n˜ =3327, 2955, 2928, 2858, 1593, 1454, 1377, 1265, 1102,
1066 cmÀ1; MS (ESI+): m/z 376.0 [M+H]+; HRMS (ESI): m/z calcd
for C20H42NO5: 376.3063 [M+H]+; found: 376.3061.
(1R)-1-C-((2R)-2,3-Di(decanoyloxy)propyl)-1,5-dideoxy-1,5-imino-
d-xylitol (10bR): Compound 9bR (50 mg, 0.057 mmol) was stirred
under an H2 atmosphere for 18 h to afford 10bR (21 mg, 67%) as
a beige solid: [a]D =À10.0 (c=0.95, MeOH); 1H NMR (400 MHz,
CD3OD): very similar to 10aR, except for the integration of the
ester chains; 13C NMR (100 MHz, CD3OD): d=14.44 (CH3), 23.73,
25.98, 30.20–30.59 (CH2), 31.87 (C6), 33.05 (CH2), 34.86, 35.07
(CH2CO), 47.61 (C5), 53.85 (C1), 65.56 (C8), 67.85, 67.89 (C3, C4),
69.71 (C7), 71.06 (C2), 174.81, 175.26 ppm (C=O); IR (neat): n˜ =
3219, 2922, 2852, 1734, 1599, 1467, 1376, 1249, 1213, 1166, 1110,
1056, 1031 cmÀ1; HRMS (ESI): m/z calcd for C28H54NO7: 516.3900
[M+H]+; found: 516.3917.
(1R)-1-C-((2S)-(2,3-Dihexyloxy)propyl)-1,5-dideoxy-1,5-imino-d-
xylitol (14S): Compound 12S (71 mg, 0.096 mmol) was stirred
under an H2 atmosphere for 48 h to afford 14S (39 mg, 98%) as
a yellow oil: [a]D =À25.6 (c=1.2, MeOH); 1H NMR (400 MHz,
CD3OD): d=0.91 (t, 6H, J=6.7 Hz; CH3), 1.26–1.41 (m, 12H; CH2),
1.53–1.60 (m, 4H; CH2), 1.81–1.90 (m, 1H; H6a), 2.05 (ddd, 1H, J=
3.2, 8.6, 14.0 Hz; H6b), 3.22 (d, 1H, J=12.8 Hz; H5a), 3.40–3.56 (m,
6H; H5b, H8, CH2O), 3.62–3.74 (m, 3H; H1, H7, CH2O), 3.87 (brs,
1H; H2), 3.93 (brs, 1H; H4), 3.96–3.98 ppm (m, 1H; H3); 13C NMR
(100 MHz, CD3OD): d=14.38, 14.39 (CH3), 23.69, 26.95, 26.99, 30.74,
31.17 (CH2), 32.40 (C6), 32.83, 32.85 (CH2), 47.34 (C5), 55.26 (C1),
67.88 (C3), 68.13 (C4), 69.76 (C2), 71.07 (CH2O), 72.67, 73.46 (C8,
CH2O), 76.00 ppm (C7); IR (neat): n˜ =3312, 2928, 2858, 1586, 1455,
1377, 1261, 1115, 1068 cmÀ1; MS (ESI+): m/z 376.0 [M+H]+; HRMS
(ESI): m/z calcd for C20H42NO5: 376.3063 [M+H]+; found: 376.3074.
(1R)-1-C-((2R)-2,3-Di(tetradecanoyloxy)propyl)-1,5-dideoxy-1,5-
imino-d-xylitol (10cR): Compound 9cR (24 mg, 0.024 mmol) was
stirred under an H2 atmosphere for 20 h to afford 10cR (13 mg,
82%) as a white wax: 1H NMR (400 MHz, CD3OD): very similar to
10aR, except for the integration of the ester chains; 13C NMR
(100 MHz, CD3OD): d=14.42, 14.44 (CH3), 23.69, 25.94, 25.96,
30.16–30.7 (CH2), 31.82 (C6), 33.02 (CH2), 34.85, 35.06 (CH2CO),
47.54 (C5), 53.74 (C1), 65.55 (C8), 67.86, 67.89 (C3, C4), 69.66 (C7),
71.06 (C2), 174.79, 175.25 ppm (C=O); MS (ESI+): m/z 629.0 [M+
H]+; HRMS (ESI): m/z calcd for C36H70NO7: 628.5152 [M+H]+;
found: 628.5131.
(1R)-1-C-((2R)-(3-Hexyloxy-2-hydroxy)propyl)-1,5-dideoxy-1,5-
imino-d-xylitol (13R): Compound 11R (50 mg, 0.077 mmol) was
stirred under an H2 atmosphere for 4 h to afford 13R (25 mg,
100%) as a colorless oil: [a]D = +1.7 (c=1.2, MeOH); 1H NMR
(400 MHz, CD3OD): d=0.91 (t, 3H, J=6.8 Hz; CH3), 1.29–1.41 (m,
6H; CH2), 1.55–1.62 (m, 2H; CH2), 1.82 (ddd, 1H, J=4.8, 8.2,
14.9 Hz; H6a), 2.04 (ddd, 1H, J=3.9, 8.7, 14.9 Hz; H6b), 3.22 (d, 1H,
J=12.8 Hz; H5a), 3.39–3.50 (m, 5H; H5b, H8, CH2O), 3.72–3.75 (m,
1H; H1), 3.83 (brs, 1H; H2), 3.92 (brs, 1H; H4), 3.94–4.00 ppm (m,
2H; H7, H3); 13C NMR (100 MHz, CD3OD): d=14.38 (CH3), 23.68,
26.92, 30.71, 32.86 (CH2), 34.24 (C6), 47.38 (C5), 54.16 (C1), 67.72,
68.05, 68.07 (C3, C4, C7), 71.70 (C2), 72.72, 75.79 ppm (C8, CH2O);
IR (neat): n˜ =3300, 2928, 2858, 1586, 1451, 1378, 1260, 1096,
1058 cmÀ1; MS (ESI+): m/z 292.0 [M+H]+; HRMS (ESI): m/z calcd
for C14H30NO5: 292.2124 [M+H]+; found: 292.2110.
Biological investigations
Inhibition of GCase: The inhibitory activity toward GCase was
measured with recombinant human b-glucocerebrosidase (Cere-
zyme) from Genzyme (Boston, MA) as the enzyme source and p-ni-
trophenyl-b-d-glucopyranoside (Sigma Chemical Co.) as the sub-
strate. The reaction mixture consisted 0.15m citrate–phosphate
buffer (pH 5.5; 75 mL), 1% sodium taurocholate/Triton X-100
(50 mL; Sigma Chemical Co.), the enzyme solution (50 mL), and an
inhibitor solution or H2O (25 mL). The reaction mixture was pre-in-
cubated at 08C for 10 min, and the reaction was started by the ad-
dition of a 10 mm substrate solution (50 mL). This was followed by
incubation at 378C for 10 min. The reaction was stopped by the
addition of a 0.4m Na2CO3 solution (2 mL). Liberated p-nitropheno-
late was measured (400 nm) with a F-4500 fluorescence spectro-
photometer (Hitachi, Tokyo, Japan). Enzyme inhibition modes and
Ki values of inhibitors were determined from the slope of Linewea-
ver–Burk plots by using increasing concentrations of substrate and
two inhibitor concentrations.
(1R)-1-C-((2S)-(3-Hexyloxy-2-hydroxy)propyl)-1,5-dideoxy-1,5-
imino-d-xylitol (13S): Compound 11S (75 mg, 0.115 mmol) was
stirred under an H2 atmosphere for 14 h to afford 13S (38 mg,
100%) as a yellow oil: [a]D =À19.4 (c=1.0, MeOH); 1H NMR
(400 MHz, CD3OD): d=0.90 (t, 3H, J=6.7 Hz; CH3), 1.29–1.40 (m,
6H; CH2), 1.54–1.61 (m, 2H; CH2), 1.82 (ddd, 1H, J=6.7, 10.6,
14.4 Hz; H6a), 1.96 (ddd, 1H, J=2.9, 6.7, 14.4 Hz; H6b), 3.23 (d, 1H,
J=12.0 Hz; H5a), 3.39–3.50 (m, 5H; H5b, H8, CH2O), 3.53 (t, 1H, J=
6.7 Hz; H1), 3.88 (brs, 1H; H2), 3.92–3.97 ppm (m, 3H; H3, H4, H7);
13C NMR (100 MHz, CD3OD): d=14.37 (CH3), 23.65, 26.86, 30.67,
32.83 (CH2), 33.26 (C6), 47.22 (C5), 55.48 (C1), 68.08, 68.10, 68.87
(C3, C4, C7), 70.42 (C2), 72.64 (CH2O), 76.22 ppm (C8); IR (neat): n˜ =
3287, 2929, 2858, 1586, 1451, 1378, 1260, 1109, 1058 cmÀ1; MS
(ESI+): m/z 292.0 [M+H]+; HRMS (ESI): m/z calcd for C14H30NO5:
292.2124 [M+H]+; found: 292.2120.
Effect of compounds 14S and 10bS on GCase thermostability:
Human recombinant b-glucocerebrosidase (Genzyme) was incubat-
ed at 508C in 100 mm sodium citrate buffer (pH 5.2) containing
0.25% (w/v) sodium taurocholate and 0.1% (v/v) Triton X-100 for
various incubation time (20, 40, or 60 min) in the absence or pres-
ence of 14S (concentration: 0.025 or 0.1 mm) or 10bS (concentra-
tion: 20 or 100 mm). After incubation, an aliquot was diluted with
a fivefold volume of 100 mm sodium citrate buffer (pH 5.2) con-
taining 0.25% (w/v) sodium taurocholate and 0.1% (v/v) Triton X-
100. The remaining b-glucocerebrosidase activity was then assayed
immediately by using 4-methylumbelliferyl-b-d-glucopyranoside as
the substrate. Liberated 4-methylumbelliferone was measured
(lEx =362 nm, lEm =450 nm) with a RF-5300PC fluorescence spec-
trophotometer (Shimadzu, Tokyo, Japan).
(1R)-1-C-((2R)-(2,3-Dihexyloxy)propyl)-1,5-dideoxy-1,5-imino-d-
xylitol (14R): Compound 12R (24 mg, 0.033 mmol) was stirred
under an H2 atmosphere for 5 h to afford 14R (12 mg, 88%) as
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D. L. Marsden, P. B. Boudes, J. Inherited Metab. Dis. 2010, 33 (Suppl. 1),
S148 (abstract); d) Amicus Therapeutics: http://www.amicusrx.com/fab-
ry.aspx (accessed September 5, 2013).
Chaperone activity: The Gaucher cell line GM00372 (Coriell Cell
Repositories, Camden, NJ) with an N370S mutation (Gaucher dis-
ease type I) was cultured with 10bS or 14S at 0–10 mm for 4 days
before being collected for the enzyme assay. After being washed
twice with phosphate buffer, the cell pellets were homogenized in
a citrate buffer (pH 5.2) containing 0.25% sodium taurocholate and
0.1% Triton X-100 by using a microhomogenizer (Physcotron, Niti-
on Inc., Chiba, Japan). The supernatant obtained from the homo-
genate after centrifugation at 10000 g for 5 min was subjected to
enzyme assays and protein determination. Intracellular glycosidase
activities were determined with 4-methylumbelliferyl-b-d-glucoside
as the substrate at pH 5.5. Mean values Æ standard deviation (SD)
are shown for triplicate experiments. The same procedure was ap-
plied to Gaucher cell line GM00877 (Coriell Cell Repositories,
Camden, NJ) with an L444P mutation (Gaucher disease type 2) to
determine the chaperone effect of compounds 10bS or 14S on
L444P GCase.
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Acknowledgements
The authors from ICOA thank Sabrina Schmidt (University of
Aachen, Germany) for her experimental contribution. Support of
this study by grants from the French National Research Agency
(ANR) (07MRAR-015-01), the French National Center for Scientific
Research (CNRS), and the French Department of Research is
gratefully acknowledged. The authors also thank LABEX SynOrg
(ANR-11-LABX-0029) for financial support.
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Keywords: Gaucher disease
inhibitors · iminosugars · pharmacological chaperones
·
glycolipids
·
glycosidase
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Received: July 30, 2013
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Published online on October 2, 2013
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