Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors

Article abstract of DOI:10.1021/jm501181z

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of d-gluco and l-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (d-gluco and l-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted l-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their d-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

Full text of DOI:10.1021/jm501181z

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Article
Identification and development of biphenyl substituted
iminosugars as improved dual glucosylceramide
synthase/neutral glucosylceramidase inhibitors
Herman S. Overkleeft, Amar Ghisaidoobe, Richard J.B.H.N van den Berg, Saleem
Butt, Wilma E Donker-Koopman, Saskia Scheij, Adrianus M.C.H. van den Nieuwendijk,
Gerrit-Jan Koomen, Mark Leemhuis, Arnold van Loevezijn, Tom Wennekes, Mario van
der Stelt, Gijsbert A. van der Marel, Stan van Boeckel, and Johannes M.F.G. Aerts
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501181z • Publication Date (Web): 24 Sep 2014
Downloaded from http://pubs.acs.org on October 5, 2014
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Identification and development of biphenyl substituted iminosugars as improved
dual glucosylceramide synthase/neutral glucosylceramidase inhibitors
1#
1#
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Amar T. Ghisaidoobe , Richard J. B. H. N. van den Berg , Saleem S. Butt , Anneke
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Strijland , Wilma E. DonkerꢀKoopman , Saskia Scheij , Adrianus M. C. H. van den
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3$
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Nieuwendijk , GerritꢀJan Koomen , Arnold van Loevezijn , Mark Leemhuis , Tom
1&
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Wennekes , Mario van der Stelt , Gijsbert A. van der Marel , Constant A. A. van Boeckel,
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Johannes M. F. G. Aerts * and Herman S. Overkleeft *
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Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55,
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300 RA Leiden, The Netherlands, Department of Medical Biochemistry, Academic Medical
Center, University of Amsterdam, Biochemistry, Academic Medical Center, Meibergdreef 15,
3
1
105 AZ Amsterdam, University of Amsterdam, van ‘t Hoff Institute for Molecular Sciences,
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P.O. Box 94157, 1090 GD Amsterdam, The Netherlands and Pivot Park Screening Centre,
Molenstraat 110, 5342 CC Oss, The Netherlands
*
Corresponding
authors.
Eꢀmail:
j.m.aerts@amc.uva.nl
or
h.s.overkleeft@chem.leidenuniv.nl
Keywords
Glucosylceramide synthase, glucosylceramide metabolism, neutral glucosylceramidase,
deoxynojirimycin, Gaucher disease, type 2 diabetes
ABSTRACT
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This work details the evaluation of a number of Nꢀalkylated deoxynojirimycin derivatives on
their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.
Building on our previous work we synthesised a series of Dꢀgluco and Lꢀidoꢀconfigured
iminosugars Nꢀmodified with a variety of hydrophobic functional groups. We found that
iminosugars featuring Nꢀpentyloxymethylaryl substituents are considerably more potent
inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next
optimization round we explored a series of biphenylꢀsubstituted iminosugars of both
configurations (Dꢀgluco and Lꢀido) with the aim to introduce structural features known to
confer metabolic stability to drugꢀlike molecules. From these series two sets of molecules
emerge as lead series for further profiling. Biphenylꢀsubstituted Lꢀidoꢀconfigured
deoxynojirimycin derivatives are selective for glucosylceramidase and the nonꢀlysosomal
glucosylceramidase and we consider these as leads for the treatment of neuropathological
lysosomal storage disorders. Their Dꢀglucoꢀcounterparts are also potent inhibitors of
intestinal glycosidases and by this virtue we regard these as leads for type 2 diabetes
therapeutics.
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INTRODUCTION
NꢀSubstituted deoxynojirimycin derivatives are an important class of molecules in medicinal
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chemistry and drug discovery. Nꢀ(Hydroxyethyl)ꢀdeoxynojirimycin 1 (Figure 1B) is
marketed as Miglitol as an antiꢀdiabetic agent (type 2 diabetes) and acts as a broadꢀ
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spectrum inhibitor of several intestinal glycosidases (maltase, sucrase, lactase). Nꢀbutylꢀ
deoxynojirimycin (Zavesca, 2) was developed as a glucosylceramide synthase inhibitor and
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is used in the clinic to treat the lysosomal storage disorder, Gaucher disease. The
therapeutic success of these molecules serves as an inspiration for many researchers and
several reports on deoxynojirimycin derivatives bearing various Nꢀsubstituents have
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appeared in recent years.
Figure 1. A) Glucosylceramide metabolism (GCS = glucosylceramide synthase, GBA1 = acid glucosylceramidase,
GBA2
= neutral glucosylceramidase). B) Clinical drugs miglitol (1) and miglustat (Zavesca, 2) based on
deoxynojirimycin and lead structures 3ꢀ6 that form the basis of the here presented studies.
Our research on deoxynojirimycin derivatives focuses on compounds able to interfere with
5
glucosylceramide metabolism (Figure 1A). Glucosylceramide is the condensation product of
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UDPꢀglucose and ceramide and its formation is catalysed by glucosylceramide synthase
GCS). Glucosylceramide is the starting point of a considerable part of all human
(
glycosphingolipids and pharmacological interference with glucosylceramide metabolism also
affects these glycoconjugates. Under normal circumstances glucosylceramide is degraded in
lysosomes by acid glucosylceramidase (glucocerebrosidase, GBA1), however the neutral
glucosylceramidase (GBA2) residing in the cytoplasm is also capable of processing
glucosylceramide. Although its physiological role remains unclear, GBA2 is involved in
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neuropathological effects observed in several lysosomal storage disorders. In Gaucher
disease and NiemannꢀPick type
B disease (caused by inherited deficiency in acid
sphingomyelinase), elevated lysosomal glucosylceramide levels lead to partial leakage to
the cytosol, where it is processed by GBA2 to produce cytosolic ceramide. It may well be
that the thus produced cytosolic ceramide itself in neuronal cells is at the basis of the
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8a
observed neuropathology. We have however indications that GBA2, like GBA1 acts also as
a retaining transglucosylase by transferring glucose from ceramide to cholesterol, and that
the thus formed unusual (in man) glycolipid, cholesterolꢀbetaꢀglucoside, might be causative
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in neuropathological lysosomal storage disorders (LSDs).
In recent years the realisation has grown that each of the enzymes involved in
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glucosylceramide metabolism are relevant targets for drug development. Moreover, and in
contrast to the clinical dogma of compound selectivity, it may well be that the most effective
compounds should inhibit a combination of glucosylceramide processing enzymes, or
alternatively one of these in combination with unrelated glycoprocessing enzymes. The
clinical drug used in substrate reduction therapy for type 2 Gaucher patients, Miglustat 2,
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inhibits GCS at relatively high concentrations, but is a rather potent GBA2 inhibitor. The
observed clinical effect of this drug may therefore be based on this dual inhibition effect. It
should be noted that the beneficial effect of Miglustat is accompanied by gastrointestinalꢀ
and metabolic side effects as a result of its strong inhibitory activity on intestinal
glycosidases. In the same vein, a pharmacological chaperone for GBA1 – pharmacological
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chaperone therapy being a new yet clinically unproven concept aimed at stabilizing the
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proper fold of mutant GBA1 in the endoplasmic reticulum (ER) – that acts by active site
binding may turn out to be a GCS inhibitor as well. In such a strategy, abnormal
glucosylceramide levels may be restored by, at once reducing its biosynthesis (through GCS
inhibition) and enhancing its turnover (by increasing lysosomal GBA1 activity). Finally we
demonstrated recently that dual inhibition of GCS and intestinal glycosidases results in
restoration of insulin sensitivity as well as reduction of glucose levels in type 2 diabetes
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animal models.
In this concept, AMPꢀDNM 3 emulates (and does so at lower
concentrations) the action of the established drug Miglitol 1 – known to act by reducing
blood glucose levels by blocking intestinal glycosidases – and complements this by reduction
of cell surface glycolipids. Of these, ganglioside GM3 can interfere with insulin signalling
through binding at the outer cell surface to the insulin receptor, thereby preventing insulin
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receptor dimerisation – a prerequisite for insulin signalling.
Ganglioside GM3 is a
downstream metabolite of glucosylceramide and GCS inhibition therefore also affects the
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production of this glycosphingolipid.
Our work on Nꢀalkylated iminosugars in the past years has focused on Nꢀ
(
(
adamantanemethyloxypentyl)ꢀdeoxynojirimycin (AMPꢀDNM, 3, Figure 1) and its C5ꢀepimer,
adamantanemethyloxypentyl)ꢀLꢀidoꢀdeoxynojirimycin (idoꢀAMPꢀDNM, 4). In our first
studies, we found AMPꢀDNM 3 to be a superior GCS inhibitor compared to Zavesca 2 (2:
9
IC50 = 25 ꢀM, 3: IC50 = 200 nM). Compound 3 also strongly inhibits GBA1, GBA2 as well as
intestinal lactase, sucrase and isomaltase. Though not a selective compound we could show
that coꢀinhibition of GCS and intestinal glycosidases through application of 3 has a beneficial
11
effect in the aforementioned studies on type 2 diabetes models. Inversion of configuration
at C5 gave idoꢀAMPꢀDNM 4, which inhibits GCS and GBA2 with about equal potency as 3 but
with little to no activity against the intestinal glycosidases, and we proposed this compound
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as a suitable lead for further development towards LSD therapeutics.
Therapeutics
directed against type 2 diabetes and Gaucher disease should ideally not interfere with GBA1
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activity. Importantly, Lꢀido congener 4 is comparatively the weaker GBA1 inhibitor (3: IC50
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=
200 nM, 4: IC50 = 2 ꢀM). In an initial study we found that replacing the adamantyl
group in 4, but not in 3, with linear alkyl substituents (propyl up to nonyl, exemplified by
structures 5 and 6, respectively) led to a considerably improved selectivity. Whereas GCS
activity of both Dꢀgluco and Lꢀido congeners remained essentially the same (with the Lꢀidoꢀ
based inhibitors being consistently slightly more potent), activity against GBA1 dropped
significantly for the Lꢀidoꢀcompounds, but not in the Dꢀglucoꢀseries. We here report an inꢀ
depth study based on these initial findings. In a first set of compounds we explore a
comprehensive series of aliphatic, unsaturated and aromatic alkyl side chains grafted onto
the nitrogen of both deoxynojirimycin and Lꢀidoꢀdeoxynojirimycin. Replacement of the
adamantane moiety with any of a series of aromatic substituents gives rise to compounds
with remarkably improved GCS inhibitory potency. Of these series, we selected those
compounds equipped with a methylbiphenyl substituent for further profiling since this
particular moiety has proven its merits as a bioisostere for fatty acids with clear guidelines
for structural modification to deliver drugꢀlike compounds, which not only elicit strong
inhibitory activity, but for which we may expect enhanced metabolic stability, bioavailability
and reduced toxicity compared to the adamantyl functionality. In a second series of
compounds we demonstrate that such alterations can be introduced without hampering
enzyme inhibitory activity and selectivity, yielding a number of improved leads for
development towards neuropathological LSDs and type 2 diabetes.
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RESULTS
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R
N
R
N
HO
HO
HO
OH
HO
OH
OH
OH
D-gluco-series
L-ido-series
O
O
O
O
O
O
3
IC50 GCS 200 nM
IC50 GBA1 100 nM
IC50 GBA2 2 nM
13 IC50 GCS 300 nM
IC50 GBA1 400 nM
IC50 GBA2 1 nM
4
IC50 GCS 100 nM
IC50 GBA1
IC50 GBA2 1 nM
26 IC50 GCS 80 nM
IC50 GBA1 10
IC50 GBA2 2 nM
2
ꢀ
M
ꢀM
O
O
O
7
IC50 GCS 500 nM
14 IC50 GCS NA
20 IC50 GCS 70 nM
IC50 GBA1 60
27 IC50 GCS 2.5
ꢀM
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IC50 GBA1 1.5
ꢀ
M
IC50 GBA1 2.5
ꢀ
M
ꢀ
M
IC50 GBA1 50 ꢀM
IC50 GBA2 100 nM
IC50 GBA2 4 nM
IC50 GBA2 100 nM
IC50 GBA2 6 nM
O
O
O
NH
NH
8
9
IC50 GCS
IC50 GBA1 500 nM
IC50 GBA2 9 nM
1
ꢀ
M
15 IC50 GCS 750 nM
IC50 GBA1 300 nM
IC50 GBA2 1 nM
21 IC50 GCS 400 nM
28 IC50 GCS 200 nM
IC50 GBA1 10 ꢀM
IC50 GBA2 3 nM
IC50 GBA1 40
ꢀ
M
IC50 GBA2 15 nM
O
O
O
O
IC50 GCS NA
IC50 GBA1 500 nM
IC50 GBA2 5 nM
16 IC50 GCS 3 nM
22 IC50 GCS NA
29 IC50 GCS 3 nM
IC50 GBA1 50 ꢀM
IC50 GBA2 1 nM
IC50 GBA1
1
ꢀ
M
IC50 GBA1 12
ꢀ
M
IC50 GBA2 1 nM
IC50 GBA2 8 nM
O
O
10 IC50 GCS 300 nM
IC50 GBA1 100 nM
IC50 GBA2 2 nM
17 IC50 GCS NA
23 IC50 GCS 250 nM
30 IC50 GCS NA
IC50 GBA1 600 ꢀM
IC50 GBA2 200 nM
IC50 GBA1 10
ꢀ
M
IC50 GBA1
5
ꢀ
M
IC50 GBA2 100 nM
IC50 GBA2 2.5 nM
O
O
O
O
11
IC50 GCS NA
IC50 GBA1 60 nM
IC50 GBA2 10 nM
18 IC50 GCS 3 nM
IC50 GBA1 500 nM
IC50 GBA2 3 nM
24 IC50 GCS NA
31 IC50 GCS 3 nM
IC50 GBA1 15 ꢀM
IC50 GBA2 1 nM
IC50 GBA1 25
ꢀ
M
IC50 GBA2 20 nM
H
H
1
9
IC50 GCS 50 nM
IC50 GBA1 400 nM
IC50 GBA2 2 nM
32 IC50 GCS 8 nM
IC50 GBA1 20
ꢀM
IC50 GBA2 3 nM
12
IC50 GCS
IC50 GBA1 500 nM
IC50 GBA2 5 nM
1
ꢀ
M
25 IC50 GCS
IC50 GBA1 15
IC50 GBA2 20 nM
1 ꢀM
O
O
ꢀM
Figure 2. Inhibition of GCS, GBA1 and GBA2 by Dꢀglucoꢀdeoxynojirimycin and Lꢀidoꢀdeoxynojirimycin derivatives
featuring a variety of hydrophobic Nꢀsubstituents. NA: IC50 > 20 ꢀM.
Figure 2 depicts the inhibitory potency of 26 Nꢀalkylated iminosugars (7ꢀ32) towards the
three enzymes involved in glucosylceramide metabolism: GCS, GBA1 and GBA2, in
comparison to the lead structures, AMPꢀDNM 3 and idoꢀAMPꢀDNM 4. The compounds were
synthesised following established procedures. Briefly, we prepared gram quantities of
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deoxynojirimycin and Lꢀidoꢀdeoxynojirimycin. These were Nꢀalkylated with the halide of
the respective Nꢀsubstituents, which in turn were prepared by standard chemical
transformations (see Supporting Information). In this fashion, we prepared a series of close
analogues of the two lead compounds in which the nature of the adamantane moiety is
altered, as well as compounds bearing comparatively more different apolar substituents
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ranging from farnesyl over cholesteryl to a number of aromatic moieties. Comparison of the
inhibition data of this first set of compounds allows for several interesting observations.
Within the series of adamantane analogues, it appears that the nature of the linkage
between the spacer moiety and the bulky head group is important for GCS inhibition.
Whereas replacement of the ether linkage in Dꢀgluco/Lꢀido derivatives 3/4 by an amide, as
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in 8 /21, has limited influence on GCS (IC fourꢀfive fold lower), elimination of the oxygen
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(11/24, also 9/22) has a drastic negative effect on GCS inhibition. Interestingly, the effect
on GBA1 with a 20 to 50ꢀfold decrease is much more pronounced. Linking the adamantane
through a secondary carbon, instead of a tertiary carbon, yields compounds with an
inhibitory profile essentially the same as that of the parent compounds (compare 13/26
with 3/4). The same holds true when substituting the adamantane for either the more
moderately sized hexyl or bulky cholesteryl moiety (compare 13/26 and 12/25,
respectively, with 3/4). Compounds 14/27, having farnesyl ether as a branched and
partially unsaturated bulky head group, are poor GCS inhibitors. Substitution of the aliphatic
head group in 3/4 by aromatic moieties however provided drastically improved GCS
inhibitors. Benzyl derivatives 15/28 inhibit GCS in the range also observed for adamantane
leads 3/4. In contrast, enlarging the size of the aromatic moiety to probe the size of the
hydrophobic binding cavity, as in the Dꢀgluco/Lꢀido pairs 16/29, 18/31 and 19/32,
afforded a series of low nanomolar GCS inhibitors. Interestingly, whereas naphthyl
derivatives 16/29 and pyrenyl derivatives 18/31 proved to be single digit nanomolar GCS
inhibitors, the corresponding phenanthryl derivatives 17/30 did not inhibit GCS at the
measured concentrations (up to 10 ꢀM). Apparently, branched hydrophobic groups at this
side are not well accommodated in the GCS active site (it should be noted that no GCS
structure is available for structureꢀactivity studies). When comparing the Dꢀgluco series with
the Lꢀido series in general on GCS inhibition potency we observe a trend we have witnessed
before, namely that the inhibition potency of a Dꢀglucoꢀdeoxynojirimycin derivative is at
least matched (for instance, 18 and 31) and often surpassed (for instance, compare 13
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with 26) by that of the corresponding Lꢀidoꢀcongener. With respect to GBA1 inhibition
without exception the Dꢀgluco compounds are the stronger inhibitors. Therefore, and as we
observed before, the Lꢀido configuration is preferable when aiming for correcting for
glucosylceramide levels in Gaucher disease (in which as said GBA1 activity is already
impaired) in substrate reduction therapy strategies by inhibition of GCS. We finally observe
that, with IC50 values ranging from 1 to 200 nM, all compounds are potent GBA2 inhibitors,
and that the most potent GCS inhibitors without exception also belong to the most potent
GBA2 inhibitors.
10
11
12
13
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15
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17
18
19
20
21
22
23
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29
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32
33
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Arguably and based on these initial studies, the most attractive selective dual GCS/GBA2
inhibitors are compounds 29, 31 and 32, which are single digit nanomolar GCS/GBA2
inhibitors that inhibit GBA1 only in the micromolar range. Of the three aromatic moieties we
reasoned that the biphenyl moiety, present in 19/32, would be the most suitable for further
development for several reasons. Naphthylꢀ and pyrenyl moieties are prone to metabolic
oxidation to deliver reactive aromatic epoxides that are mutagenic. Biphenyl moieties are
less prone to the formation of such toxic metabolites, have better drugꢀlike properties and
are more attractive from a medicinal chemistry point of view to make various structural
modifications. We therefore selected the biphenylꢀmethyl moiety for further optimization
and identified a number of positions for chemical modification to arrive at a series
comprising leads with not only different selectivity and potency profiles, but also improved
DMPK properties. Chemical modifications are both in the external and internal ring, in the
benzylic CH connecting the biphenyl to the spacer, and in the spacer pentane itself. In our
2
optimization program we decided on Dꢀglucoꢀderivative 19 as our starting point. This for
practical reasons since the starting iminosugar, deoxynojirimycin, is comparatively easier to
access on a suitable scale and also gives better yields in the Nꢀalkylation step than the
corresponding Lꢀido congener. This also for intrinsic reasons since 19 can be considered as
an improved lead structure compared to 3 for development towards type 2 diabetes
therapeutics.
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1
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R
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
OH
OH
37 IC50 GCS 150 nM
IC50 GBA1 2.5
IC50 GBA2 20 nM
43 IC50 GCS 25 nM
IC50 GBA1 250 nM
IC50 GBA2 1 nM
49 IC50 GCS 40 nM
IC50 GBA1 250 nM
IC50 GBA2 30 nM
55 IC50 GCS 750 nM
IC50 GBA1
IC50 GBA2 5 nM
61 IC50 GCS 70 nM
IC50 GBA1 400
IC50 GBA2 3 nM
D-gluco-series
ꢀ
M
3
ꢀ
M
ꢀM
N
OMe
OMe
F
F
O
O
F
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
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8
9
0
1
2
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8
9
0
1
3
9
IC50 GCS 50 nM
IC50 GBA1 400 nM
IC50 GBA2 2 nM
38 IC50 GCS 50 nM
IC50 GBA1 600 nM
IC50 GBA2 3 nM
44 IC50 GCS 75 nM
IC50 GBA1 600 nM
IC50 GBA2 2 nM
50 IC50 GCS 25 nM
56 IC50 GCS 50 nM
62 IC50 GCS 60 nM
IC50 GBA1 300 nM
IC50 GBA2 3 nM
IC50 GBA1
3
ꢀ
M
IC50 GBA1 1.25
ꢀM
IC50 GBA2 15 nM
IC50 GBA2 3 nM
O
O
O
F
3
IC50 GCS 100 nM
IC50 GBA1 250 nM
IC50 GBA2 3 nM
39 IC50 GCS 150 nM
IC50 GBA1 350 nM
IC50 GBA2 3 nM
45 IC50 GCS 25 nM
IC50 GBA1 500 nM
IC50 GBA2 2 nM
51 IC50 GCS
2
2
ꢀ
M
57 IC50 GCS 75 nM
IC50 GBA1 300 nM
IC50 GBA2 2 nM
63 IC50 GCS 25 nM
IC50 GBA1 250 nM
IC50 GBA2 0.08 nM
IC50 GBA1
ꢀ
M
IC50 GBA2 10 nM
N
CN
Cl
O
O
O
O
O
O
N
3
3
4
5
IC50 GCS 100 nM
IC50 GBA1 250 nM
IC50 GBA2 2 nM
40 IC50 GCS 50 nM
IC50 GBA1 700 nM
IC50 GBA2 2 nM
46 IC50 GCS 20 nM
IC50 GBA1 800 nM
IC50 GBA2 10 nM
52 IC50 GCS
IC50 GBA1
2
3
ꢀ
M
58 IC50 GCS 200 nM
64 IC50 GCS 25 nM
IC50 GBA1 200 nM
IC50 GBA2 0.08 nM
F
ꢀ
M
IC50 GBA1 1.5
ꢀM
IC50 GBA2 10 nM
IC50 GBA2 3 nM
N
CN
CN
CF
3
F
F
O
O
O
O
O
O
CF
3
IC50 GCS 25 nM
IC50 GBA1 70 nM
IC50 GBA2 0.5 nM
41 IC50 GCS 150 nM
IC50 GBA1 600 nM
IC50 GBA2 5 nM
47 IC50 GCS 20 nM
IC50 GBA1 800 nM
IC50 GBA2 15 nM
53 IC50 GCS
2
ꢀ
M
59 IC50 GCS 1.25
ꢀ
M
65 IC50 GCS 25 nM
IC50 GBA1 200 nM
IC50 GBA2 0.12 nM
IC50 GBA1 10
ꢀ
M
IC50 GBA1 125 nM
IC50 GBA2 10 nM
IC50 GBA2 25 nM
N
O
N
F
3
C
CF
3
F
F
O
O
O
O
O
3
6
IC50 GCS 25 nM
IC50 GBA1 2.5
IC50 GBA2 10 nM
42 IC50 GCS 25 nM
IC50 GBA1 400 nM
IC50 GBA2 1 nM
48 IC50 GCS 25 nM
IC50 GBA1 500 nM
IC50 GBA2 2 nM
54 IC50 GCS 150 nM
60 IC50 GCS 60 nM
IC50 GBA1 300 nM
IC50 GBA2 1 nM
66 IC50 GCS 25 nM
IC50 GBA1 100 nM
IC50 GBA2 0.08 nM
CF3
ꢀ
M
IC50 GBA1
2 ꢀM
IC50 GBA2 4 nM
O
N
F
OMe
F
O
Figure 3. Inhibition of GCS, GBA1 and GBA2 by a variety of biphenylꢀsubstituted Dꢀglucoꢀdeoxynojirimycin
derivatives.
We thus assembled a comprehensive number of analogues of biphenylꢀDNM derivative 19
and assessed their inhibitory potency on GCS, GBA1 and GBA2. The most attractive
modifications were transferred to the Lꢀidoꢀdeoxynojirimycin scaffold to afford a list of
compounds that were again tested on GCS, GBA1 and GBA2. Finally we selected from both
series a number of compounds that we tested on a panel of intestinal glycosidases. Figure 3
depicts our focused library of biphenylꢀsubstituted deoxynojirimycin derivatives and their
inhibitory potency towards GCS, GBA1 and GBA2. Compounds 33ꢀ50 are biphenyl
analogues with the terminal phenyl substituted at the ortho, metaꢀ or para position, and
carrying varying substitutions on the terminal phenyl ring. We selected both carbon
substituents and heteroatom substituents and included both electronꢀdonating and electronꢀ
withdrawing functional groups in our studies. As can be seen, the effect of these
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modifications on GCS inhibition is modest, and all compounds inhibit GCS with an IC50
between 20 nM and 150 nM. Essentially the same holds true for GBA2 (IC50 0.7ꢀ10 nM)
whereas the nature of the functional group appears to have a somewhat more profound
effect on GBA1 inhibition. Orthoꢀmethyl derivative 35 inhibits GBA1 with an IC50 of 70 nM
and is the most potent GBA1 inhibitor of this series. In contrast, the weakest inhibitor of
this series is dioxetane derivative 50 with an IC50 of 3 ꢀM. Overall however the effect of the
substituents in 33ꢀ50 is limited when looking at inhibitory potency and selectivity. All
modifications are tolerated, which holds promise for stabilizing the external phenyl ring at
for instance the orthoꢀ or para position, positions susceptible to oxidation by cytochrome
P450 enzymes. Substitution of the terminal phenyl for pyridine (51, 52) or pyrazine (53)
has a more pronounced effect on GCS inhibition, with a drop in IC50 from 40 nM (19) to 2
10
11
12
13
14
15
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17
18
19
20
21
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23
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28
29
30
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32
33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
M. Apparently, a weak basic character at this position is poorly tolerated by the GCS active
site, an observation underscored by the finding that inhibition is moderately (55) to largely
54) restored by decreasing the basicity through instalment of a fluorine substituent onto
(
the pyridine moiety. Essentially the same trend within this series is observed for GBA1
whereas as before the nature of the modification appears to have limited influence on GBA2
inhibition. Orthoꢀbiphenyl derivative 56 and metaꢀbiphenyl derivative 57 resemble paraꢀ
biphenyl derivative 19 where it comes to GCS and GBA2 inhibition, but interestingly the
metaꢀanalogue appears to fit better within the GBA1 active site. Substitution of the internal
phenyl for pyridyl, as in 58, has some detrimental effect on GCS inhibition, but not on
GBA1/2 inhibition. The same holds true for analogue 59, with the hydrogens on the spacer
carbon beta to the deoxynojirimycin nitrogen substituted for fluorine. Arguably, this
modification, designed to prohibit metabolic oxidation at this site, lowers the basic character
of the ring nitrogen such that the molecule is accommodated slightly more poorly within the
GCS active site. Substitution of the spacer methylene beta to the ether oxygen (for the
same reason to increase metabolic stability) as in 60 has the opposite effect: stronger GCS
inhibitor and weaker GBA1 inhibitor. Introduction of a branching methyl group at the
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benzylic carbon (enantiomeric pair 61 and 62) – a modification known to confer stability to
this oxidationꢀprone position – yields compounds with an attractive GCS/GBA1 selectivity
window: good GCS inhibitors and weak GBA1 inhibitors. Interestingly, diastereomers 61
and 62 appear equally potent inhibitors for all three enzymes and thus no enantiospecificity
is at play with respect to the chiral benzyllic positions. Although we have no explanation for
this finding, it might be that the benzyllic position is not directly interacting with the enzyme
active sites but rather embedded within a membrane (both GCS and GBA2 being
membraneꢀassociated enzymes). Compounds 63ꢀ66 finally bear either a fluorine or
trifluormethyl substituent at either the orthoꢀ or metaꢀposition of the inner phenyl ring.
These compounds are GCS inhibitors (IC50 of 25 nM) and are highly potent GBA2 inhibitors
0
1
2
3
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5
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7
8
9
0
1
2
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8
9
0
1
2
3
4
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9
0
(
sub nanomolar activity). They are also amongst the strongest GBA1 inhibitors of this
series.
Figure 4 depicts a small focused library of Lꢀidoꢀderivatives 67ꢀ76 containing biphenyl
modifications we selected from the Dꢀglucoꢀseries. We made this selection based on both
inhibitor potency and selectivity and putative stabilizing effect on metabolic enzymes. In
general and as before (Figure 2) the Lꢀidoꢀisomers when compared with their respective Dꢀ
glucoꢀcongeners are more potent GCS inhibitors, are (considerably) less potent GBA1
inhibitors and inhibit GBA2 in about equal potency. We consider compounds 73ꢀ76
especially interesting from these series, as they emulate the beneficial properties of parent
compound 32 (single digit nanomolar GCS inhibitor, subnanomolar GBA2 inhibitor offset by
only micromolar GBA1 inhibition) and combine this with stabilizing substituents at the inner
phenyl ring. We note that these compounds may not yet contain the most optimal
combination of substitutions, and for instance including the distinguishing features in
compounds 42 and 60 (Figure 3) may result in even more optimal compounds. We also
conclude that, for optimal GCS inhibition, paraꢀconfigured biphenyl moieties are the more
attractive substituents (compare 32 with 69 and 70).
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R
N
F
F
O
O
O
O
O
HO
HO
CF
3
F
OH
OH
67 IC50 GCS 40 nM
IC50 GBA1 10
IC50 GBA2 2 nM
69 IC50 GCS 25 nM
IC50 GBA1 15
IC50 GBA2 3 nM
71 IC50 GCS 20 nM
IC50 GBA1 500
IC50 GBA2 2 nM
73 IC50 GCS 3 nM
IC50 GBA1
IC50 GBA2 0.1 nM
75 IC50 GCS 5 nM
IC50 GBA1
IC50 GBA2 0.4 nM
L-ido-series
ꢀ
M
ꢀ
M
ꢀ
M
5
ꢀ
M
8 ꢀM
F
F
O
O
O
O
O
O
3
2
IC50 GCS 8 nM
IC50 GBA1 20
IC50 GBA2 3 nM
68 IC50 GCS 30 nM
70 IC50 GCS 70 nM
72 IC50 GCS 15 nM
IC50 GBA1 12.5
IC50 GBA2 2 nM
74 IC50 GCS 3 nM
CF3
76 IC50 GCS 2.5 nM
F
ꢀ
M
IC50 GBA1 10
ꢀ
M
IC50 GBA1 20
ꢀ
M
ꢀ
M
IC50 GBA1
6
ꢀ
M
IC50 GBA1
6 ꢀM
IC50 GBA2 1 nM
IC50 GBA2 2 nM
IC50 GBA2 0.1 nM
IC50 GBA2 0.6 nM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Inhibition of GCS, GBA1 and GBA2 by
a variety of biphenylꢀsubstituted Lꢀidoꢀdeoxynojirimycin
derivatives.
From the set of Lꢀidoꢀdeoxynojirimycin derivatives depicted in Figure 4 we consider
compounds 73ꢀ76 as the most suitable leads for further profiling as dual GCS/GBA2
inhibitors for the treatment of neuropathological lysosomal storage disorders. For this the
compounds preferably should not display inhibition of intestinal glycosidases. For this
purpose, but also to establish whether the analogous Dꢀglucoꢀdeoxynojirimycin derivatives
would be viable leads for development towards antiꢀtype 2 diabetics (coꢀinhibition of GCS
and intestinal glycosidases) we assessed inhibition of intestinal sucrase, maltase and lactase
with a selected number of compounds: all biphenyl modified Lꢀidoꢀdeoxynojirimycin
derivatives alongside their Dꢀglucoꢀcongeners (Table 1). Without exception the hydrophobic
Dꢀglucoꢀdeoxynojirimycin derivatives inhibit all three enzymes measured, whereas the Lꢀ
idoꢀdeoxynojirimycin derivatives do not. In this sense both series emulate the properties of
their respective parent compounds, AMPꢀDNM 3 and LꢀidoꢀAMPꢀDNM 4, respectively, and
reflect the general trend that the intestinal glycosidases tested prefer glucose as the
(
natural) substrate over the nonꢀnatural (in mammals) Lꢀidose configuration.
D-gluco-series
L-ido-series
sucrase
lactase
35
maltase
sucrase
lactase
200
Maltase
>500
3
0.6
3
2
4
>100
70
1
9
0.25
20
32
80
>100
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1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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5
5
5
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5
5
5
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6
1
0.25
0.5
0.2
0.2
35
9
35
2.5
4
67
68
69
70
71
72
73
74
75
76
>100
>100
95
>100
45
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
62
5
5
6
7
17
1.5
1.5
>100
9
25
20
100
60
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
59
>100
27
>100
>100
>100
>100
>100
>100
>100
80
6
6
6
0
5
6
0.4
1.5
1.25
0.25
0.4
50
8
125
100
60
45
7
63
30
2.5
3
6
4
20
50
Table 1. Inhibition of intestinal glycosidases by a selection of Dꢀglucoꢀ and Lꢀidoꢀdeoxynojirimycin derivatives
bearing biphenyl substituents. Inhibition values are given as IC50 (ꢁM) values.
DISCUSSION
In this paper we have described an inꢀdepth study on 72 deoxynojirimycin derivatives
differing in both configuration at C5 and the nature of the hydrophobic moiety attached to
the ringꢀnitrogen, and their inhibitory profile towards the enzymes involved in
glucosylceramide metabolism; GCS, GBA1 and GBA2. At the onset of our studies we set out
to identify compounds with improved properties with respect to inhibition profile (potency,
selectivity, compounds that would target two enzymes/enzyme classes simultaneously).
Addition of hydrophobic moieties to the iminosugar in general increases potency, however,
it may also lead to a deterioration of the physicoꢀchemical properties required for oral
bioavailability.
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41
42
43
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49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Lipophilic ligand efficiency (LipE) values of matched molecular pairs from the Lꢀido and Dꢀgluco series for
GCS activity (square = Lꢀido series; circle = Dꢀgluco series; triangle = log P).
To gain insight in these issues, the lipophilic ligand efficiency index (LipE), which is defined
as pIC50ꢀLogP, is often used as a composite parameter to guide the optimization of
compounds. A high LipE index indicates that the affinity of a compound for a target is likely
driven by favourable inhibitorꢀprotein interactions, rather than an aspecific entropyꢀdriven
18
effect. In Figure 5, we have plotted the LipE values of matched molecular pairs from the Lꢀ
ido and Dꢀgluco series (for this purpose, LogP values were calculated using the ChemDraw
version 13 software). It should be noted that most iminosugars have high LipE values (>
1
0.000ꢀfold selectivity for GCS than for a generic hydrophobic surrounding). The Lꢀido
series appear to outperform the Dꢀgluco series in this evaluation, indicating that they
exhibit a more specific interaction with GCS. In general, the LipE value of both compound
series decreases with more hydrophobic substituents indicative of an entropyꢀdriven binding
to GCS.
Based on previous results we consider both neuropathological lysosomal storage disorders
and type 2 diabetes as therapeutic application areas. GCS is an attractive target for both
(established for Gaucher disease and indicated for type 2 diabetes) but according to our
previous results is ideally targeted in combination with either GBA2 (LSDs) or intestinal
glycosidases (type 2 diabetes). Our results reveal that subtle modification of the biphenyl
core influences inhibitory potency towards GCS and GBA1, while having less effect on GBA2
inhibition. Our results also confirm our previous finding that Dꢀglucoꢀconfigured
deoxynojirimycin derivatives bearing a hydrophobic Nꢀsubstituent are relatively good
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6
inhibitors of the intestinal glycosidases maltase, sucrase and lactase, whereas the
corresponding Lꢀidoꢀconfigured iminosugars are inactive towards these enzymes. This work
thus presents a number of improved lead structures for further development. We propose
biphenylꢀsubstituted Nꢀalkylated D-gluco-deoxynojirimycin derivatives as type 2 diabetes
leads based on their dual GCS/intestinal glycosidase inhibitory capacity. We note that these
compounds also target GBA1 and GBA2. Our focused libraries delivered leads such as
compound 61 with low GBA1 inhibitory activity. On the other hand it is difficult to steer
away from GBA2 inhibitory activity. Interestingly though, a recent paper by Summers and
coꢀworkers indicate that the culprit (glyco)sphingolipid in type 2 diabetes varies between
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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5
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9
0
1
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0
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0
1
9
tissues. They indicate that in adipocytes elevated levels of ganglioside GM3 are causative
of insulin resistance. This confirms our previous study showing that downregulation of
ganglioside GM3 through GCS inhibition restores insulin signalling in obese diabetic mice
and rats. They however also show compelling evidence that in myocetes ceramide is the
actual diseaseꢀinducing lipid. If true, then coꢀinhibition of GCS and GBA2, alongside
inhibition of intestinal glycosidases (prevention of glucose assimilation) may actually turn
out to be beneficial. Our Lꢀidoꢀconfigured, Nꢀalkylated deoxynojirimycin derivatives we
propose as leads for neuronopathic lysosomal storage disorders based on their dual
GCS/GBA2 inhibitory activity. Compounds 73ꢀ76 are single digit nanomolar GCS inhibitors
and as such equal or even surpass the activity of nonꢀiminosugar GCS inhibitors reported in
20
recent years. The latter compounds are based on ceramide and as far as we know do not
target GBA2.
Finally,
next
to
Dꢀglucoꢀ
and
Lꢀidoꢀconfigured
iminosugars,
Nꢀalkylated
deoxygalactonojirimycin derivatives have been studied as compounds able to interfere with
21
glucosylceramide metabolism. Originally
studied
as GCS
inhibitors, Nꢀalkylꢀ
22
deoxygalactonojirimycin derivatives were recently put forward as inhibitors selective for
14
GBA2 over GBA1. We have previously reported a headꢀtoꢀhead study comparing, amongst
others, Nꢀbutyl and Nꢀadamantanemethyloxypentyl substituted iminosugars differing in
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configuration at C4 and C5. The data obtained for the Dꢀgluco, Lꢀido and Dꢀgalactoꢀ
configured iminosugars bearing these substituents are summarised in Figure 6 and to
complete the list we prepared and evaluated biphenylꢀmodified galactonojirimycin 80.
Though our values deviate from those reported by van der Spoel and coꢀworkers for
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compound 78 , the trend as observed from the Nꢀbutyl series (2, 77, 78) that GBA2 is
considerably more sensitive to inhibitors of the deoxynojirimycin class is confirmed in the
adamantaneꢀsubstituted (3, 4, 79) and biphenylꢀsubstituted (19, 32, 80) series. The Dꢀ
galactoꢀconfigured compounds indeed favour GBA2 over GBA1 when compared to the Dꢀ
glucoꢀconfigured compounds, though the differences are less pronounced when comparing
with the Lꢀidoꢀconfigured compounds.
D-gluco
L-ido
D-galacto
2
3
IC50 GCS 50
IC50 GBA1 400
IC50 GBA2 230 nM
ꢀ
ꢀ
Ma
77 IC50 GCS 10
ꢀ
Ma
78 IC50 GCS 10
IC50 GBA1 320
ꢀ
Ma
N
N
N
HO
HO
a
a
HO
HO
IC50 GBA1 NAa
HO
HO
a
a
(non detectedb)
M
ꢀ
M
IC50 GBA2 250 nMa
IC50 GBA2 300 nM (1-6 ꢀΜb
)
OH
OH
OH
OH
OH
OH
O
O
O
a
N
IC50 GCS 200 nM
IC50 GBA1 100 nM
IC50 GBA2 2 nM
N
4
IC50 GCS 100 nM
IC50 GBA1
IC50 GBA2 1 nM
N
79 IC50 GCS 500 nM
HO
HO
HO
HO
2
ꢀ
M
HO
HO
a
IC50 GBA1 200 nM
a
IC50 GBA2 1 nM
OH
OH
OH
OH
OH
OH
OH
O
O
O
N
19 IC50 GCS 50 nM
IC50 GBA1 400 nM
IC50 GBA2 2 nM
N
32 IC50 GCS 8 nM
IC50 GBA1 20
IC50 GBA2 1 nM
N
80 IC50 GCS 100 nM
IC50 GBA1 200
IC50 GBA2 2 nM
HO
HO
HO
HO
ꢀ
M
HO
HO
ꢀM
OH
OH
OH
OH
OH
Figure 6. Comparison of inhibitory potency differently substituted Dꢀgluco, Lꢀido and Dꢀgalactoꢀconfigured
a
b
deoxynojirimycins on GCS, GBA1 and GBA2 ( values taken from reference 14, values taken from reference 22).
In conclusion, our inꢀdepth study on modifications on the biphenyl core, how to prepare
these (as shown in detail in the experimental section) and their effect on enzyme inhibition
potency and selectivity, will help the identification, after further studies on in vivo efficacy
and pharmacochemical properties, of lead compounds for development in the direction of
both human disease areas.
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EXPERIMENTAL SECTION
1
13
Synthetic procedures. The H and CꢀNMR spectra were recorded on a 200/50, 300/75,
4
00/100, 500/125, or 600/150 MHz spectrometer. Chemical shifts are given in ppm (δ)
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relative to tetramethylsilane as internal standard for all H NMR measurements in CDCl and
3
the deuterated solvent signal for all other NMR measurements. Coupling constants (J) are
given in Hz. High resolution mass spectra were recorded on a mass spectrometer (Thermo
Finnigan LTQ Orbitrap) equipped with an electrospray ion source in positive mode with
resolution R = 60 000 at m/z 400 (mass range m/z = 150ꢀ2000). All tested iminosugars
were analyzed via a combination of HPLC and LC/MS that showed a purity of >95%. All
iminosugars were tested as their trifluoroacetic acid salts. AMPꢀDNM 3 and LꢀidoꢀDNM 4
14
were prepared as described previously. Compounds 7ꢀ76 were prepared following either
of two general schemes as demonstrated in the experimental scheme for two representative
examples.
Experimental Scheme. Reagents and conditions: ROUTE A: (a) TrCl, Et N, CH Cl , 48h,
3
2
2
9
4%; (b) OsO , NMO, H O, acetone, tBuOH, 85%; (c) Bu SnO, toluene, reflux (Deanꢀ
4 2 2
Stark), 18h, then 4ꢀbromomethylbiphenyl, TBABr, 90 °C, 3h, 69%; (d) DMSO, oxalyl
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chloride, CH Cl , ꢀ78 °C, 10 min, then add 80, ꢀ70 °C, 10 min, then Et N, ꢀ60 °C to rt,
2
2
3
9
2%; (e) DAST, CH Cl , 54 h, 94 %; (f) BF ꢂOEt , MeOH/toluene 1:1, 6h, 99%; (g) CBr ,
2 2 3 2 4
PPh , CH Cl , 0 °C, 73%; (h) K CO , DMF, 80 °C, 24h, 47%; ROUTE B: (i) 1) TrCl, TEA,
3
2
2
2
3
EtOAc, 85 °C, 99% 2) TsCl, TEA, CH Cl , 20h, 96%; (j) NaH, DMF, 88, 4h, 80%; (k)
2
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BF ꢂOEt , MeOH/toluene 1:1, 6h, 84%; (l) CBr , PPh , CH Cl , 0 °C, 99%; (m) K CO , DMF,
3
2
4
3
2
2
2
3
8
0 °C, 24h, 67%; (n) NaOMe, 92, 93, Pd(PPh ) , EtOH, 65 °C, 31%.
3 4
In ROUTE A, key step is the direct alkylation of the unprotected iminosugar to the
functionalized alkyl substituent under alkaline conditions. The route is exemplified for the
16
synthesis of fluorinated derivative 60. Alkylation of deoxynojirimycin 85
with alkyl
bromide 84 is accomplished in DMF at 0.2 M substrate concentration. Amine 85 and
bromide 84 are reacted in stoichiometric amounts in the presence of a threeꢀfold excess of
potassium carbonate. The requisite alkyl bromides are prepared individually following
established procedures, as is outlined here fore 84. In ROUTE B, terminally modified
phenyl substituents are attached to benzyl bromide core 92 using Suzuki crossꢀcoupling
chemistry. The synthesis of the required building block 92 follows chemistry related tot that
described in ROUTE A, and as an individual example the crossꢀcoupling of 92 with 93 to
give library member 55 is depicted. The library members are prepared using the following
general strategies: ROUTE A starting from deoxynojirimycin 85: compounds 7ꢀ19 and 56ꢀ
66. ROUTE A starting from Lꢀidoꢀdeoxynojirimycin: compounds 20ꢀ32 and 67ꢀ76. ROUTE
B starting from deoxynojirimycin 85: compounds 33ꢀ55. All compounds are purified to
>
95% purity by preparative HPLCꢀMS. See for full experimental and analytical details on all
intermediates and endꢀproducts the Supplementary Information.
Enzyme inhibition assays
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The enzyme assays used for determining the inhibition of activity of glucosylceramide
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synthase (GCS) , glucocerebrosidase (GBA1) , βꢀglucosidase 2 (GBA2) , sucrase, lactase
16b
and maltase
were carried out as described previously.
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ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Authors
*
Phone:
+31ꢀ71ꢀ5274342;
email
j.m.aerts@amc.uva.nl
or
h.s.overkleeft@chem.leidenuniv.nl
Present Address
&
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB
$
Wageningen, The Netherlands; Albemarle Catalysts Company BV, Analytical Research and
Quality Department, Nieuwendammerkade 1ꢀ3, 1022 AB Amsterdam, The Netherlands.
Author Contributions
#
A.T.G. and R.J.B.H.N.v.d.B. contributed equally to the work
Notes
The authors declare no competing financial interest
ACKNOWLEDGMENTS
The authors are grateful for financial support from the Netherlands Organization for
Scientific Research (NWOꢀCW, TOP Grant to HSO and JMFGA) and the European Research
Council (Advanced Grant, to HSO).
ABBREVIATIONS USED
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GCS, glucosylceramide synthase; GBA1, acid glucosylceramidase, GBA2; neutral
glucosylceramidase
Supporting information: details on the synthesis and analysis of all new compounds
described in this work. This information is available free of charge via the Internet at
http://pubs.acs.org.
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ganglioside GM3. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3445ꢀ3449. (c)Kabayama,
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(
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14) Wennekes, T.; Meijer, A. J.; Groen, A. K.; Boot, R. G.; Groener, J. E.; van Eijk, M.;
Ottenhoff, R.;Bijl, N.; Ghauharali, K.; Song, H.; O'Shea, T. J.; Liu, H.; Yew, N.;
Copeland, D.; van den Berg, R. J.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J.
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(
15) Ghisaidoobe, A.; Bikker, P.; de Bruijn, A. C. J.; Godschalk, F. D.; Rogaar, E.; Guijt,
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S.; Wijzenbroek, M.; Zweegers, T.; DonkerꢀKoopman, W. E.; Strijland, A.; Boot, R.;
van der Marel, G.; Overkleeft, H. S.; Aerts, J. M. F. G.; van den Berg, R. J. B. H. N.
Identification of potent and selective glucosylceramide synthase inhibitors from a
library of Nꢀalkylated iminosugars, ACS Med. Chem. Lett. 2011, 2, 119ꢀ123.
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van Wiltenburg, J.; Wolberg, M.; Aerts, J. M. F. G.; Overkleeft, H. S. Largeꢀscale
synthesis of the glucosylceramide synthase inhibitor Nꢀ[5ꢀ(adamantanꢀ1ylꢀ
methoxy)ꢀpentyl]ꢀ1ꢀdeoxynojirimycin. Org. Process Res. Dev. 2008, 12, 414ꢀ423.
(
(
b) Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.;
Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. Development of adamantanꢀ1ꢀylꢀ
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methoxyꢀfunctionalized 1ꢀdeoxynojirimycin derivatives as selective inhibitors of
glucosylceramide metabolism in man. J. Org. Chem. 2007, 72, 1088ꢀ1097.
(
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