Sphingolactones: Selective and irreversible inhibitors of neutral sphingomyelinase

Article abstract of DOI:10.1002/anie.200501983

Ceramide analogues which act as potent inhibitors of the neutral sphingomyelinase (N-SMase) without needing to contain reactive epoxy groups are the sphingolactones (see picture for an example). Sphingolactones are useful chemical tools for exploring the

Full text of DOI:10.1002/anie.200501983

Angewandte
Chemie
Sphingolactones
phosphate (3), more complex glycosphingolipids (4), and
sphingosine-1-phosphate (5).
[
1]
DOI: 10.1002/anie.200501983
It has been assumed for a few years that the hydrolysis of
sphingomyelin is the major process for the formation of
Sphingolactones: Selective and Irreversible
Inhibitors of Neutral Sphingomyelinase
biological active ceramide. In the tightly regulated sphingo-
[1–3,6]
myelin cycle
(Scheme 1) ceramide is generated through
the action of either an acid sphingomyelinase (A-SMase) or a
membrane-bound neutral sphingomyelinase (N-SMase). Dif-
ferent cytokines, such as TNF-a, interleukin-1b, interferon-g,
Veit Wascholowski and Athanassios Giannis*
as well as radiation, heat, oxidative agents, vitamin D , and
3
[
3,4]
Sphingolipids are important components of the plasma
membranes of eukaryotic cells. In the last few years increasing
attention has been focussed on sphingolipid metabolites,
because of their numerous biological effects and particularly
because of their possible function as second messengers.
The primary catabolite of sphingomyelin (1) is ceramide
NO are all able to activate sphingomyelinases.
However, various aspects of ceramide-mediated signal
transduction, particularly its role in apoptosis, are controver-
[
3–7]
sial.
Additionally, the question as to which of the
sphingomyelinases is important for stimulus-induced ceram-
ide production is still a point of controversy.
[
3,8–11]
The
(
2), which is generated through the enzymatic activity of
membrane-located neutral sphingomyelinase, which under-
lies physiological regulation through glutathione and arach-
idonic acid, is believed to play a relevant role in signal
sphingomyelinases and is assumed to be involved in cell
regulation, modulation of inflammatory processes, and also in
programmed cell death (apoptosis).
to activate different signal-transduction cascades, both
directly and indirectly. Control of these biological effects
is reached through regulation of the intracellular ceramide
concentration by enzymes of the ceramide biosynthesis and
by the formation of other sphingolipids, such as ceramide-1-
[
1–5]
[1,3]
Ceramide is also able
transduction.
Potent, and above all, selective inhibitors are necessary to
understand the biological function of ceramide and the
sphingomyelinases. Although some inhibitors of N-SMase
[1]
[
12,13]
[12b,c,g]
are known,
7,
only scyphostatin (6),
spiroepoxide
[
13a,b]
[12a]
and manumycin A (8),
as well as its simple
Scheme 1. The sphingomyelin cycle. Ceramide 2 is generated from spingomyelin (1) through the action ofsphingomyelinases. This competes in a
metabolic equilibrium with ceramide-1-phosphate (3), more complex glycosphingolipids (e.g., 4, Glc=glucose), and sphingosine-1-phosphate (5).
analogues have been used to examine the precise roles of
these enzymes (Scheme 2). However, all these compounds
[
*] Dipl.-Chem. V. Wascholowski, Prof. Dr. A. Giannis
University ofLeipzig
Institute ofOrganic Chemistry
[14]
contain reactive epoxy groups, which are able to modify
different cellular proteins through covalent interactions.
Therefore, it is difficult to interpret the results of biological
cell experiments. Here, we present the development of a new
class of potent, selective and epoxy-free inhibitors of neutral
sphingomyelinase, which we term sphingolactones.
04103 Leipzig (Germany)
Fax : (+49)341-973-6599
E-mail: giannis@chemie.uni-leipzig.de
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 2. Scyphostatin (6), spiroepoxide 7, and manumycin (8).
The conception of the inhibitors is based on the following
facts: Both, scyphostatin (6) as well as manumycin A (8)
contain a polyunsaturated fatty acid residue as a common
structural element. Furthermore, synthetic manumycin ana-
logues with unsaturated carboxylic acids in the amide moiety
possess a higher affinity to N-SMase relative to those with
[
12a]
saturated fatty acid chains.
own research, that the primary hydroxy group of 7 is essential
Moreover, it is known from our
[
13c]
for the inhibitory activity.
As a replacement for the
Scheme 3. Synthesis ofsphingolactones. a) 1.2 equiv LiCl, 1.2 equiv
(PhO) P(O)CH((CH CH )COOtBu (10), 1.1 equiv DBU, THF, 67%;
b) 1. 1.5 equiv Py, 1.3 equiv TfO , CH Cl , 2. 4 equiv NaN , DMF, 62%;
reactive epoxy groups we chose the g-butyrolactone scaffold,
a privileged structure, which is a recurrent structural motif
present in many biological active molecules. Figure 1shows
the structural features of the desired inhibitor.
2
2
)
5
3
[
15]
2
2
2
3
c) 1.6 equiv Ph P, THF, H O, 608C, 94%; d) 1.5 equiv NEt , 1.1 equiv
3
2
3
RCOCl, THF, 87–98%; e) cat. p-TsOH, DMF, 1,2-ethandiol, 76–85%;
f) cat. Pd/C, H , ethanol, quant.; g) 1.5 equiv NEt , 1.1 equiv RCOCl,
2
3
THF, 94–97%; h) cat. p-TsOH, DMF, 1,2-ethandiol, 72–79%.
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, Py=pyridine, Tf=trifluoro-
methanesulfonyl, Ts=toluene-4-sulfonyl.
fonic acid and in situ lactonization yielded the desired
lactones 18–21. The syntheses of 24 and 25 were completed
by catalytic hydrogenolysis of compound 12 with Pd on
charcoal (Scheme 3). The reaction results in an inseparable
mixture of diastereomers in a ratio of 4:1. Further conversion
into the desired compounds 24 and 25 was carried out in
analogy to the described synthesis above.
Figure 1. Structural features of the desired inhibitor.
The synthesis of the targeted inhibitor (Scheme 3) starts
with the reaction of 2,4-O-benzylidene-d-threose (9), which is
readily accessible by periodate cleavage of 4,6-O-benzyli-
The synthesized compounds were then evaluated regard-
ing their inhibitory effect against N-SMase, for which a
[
16,17]
2+
dene-d-galactose,
phosphonate 10
with the appropriate modified aryl
microsome preparation containing Mg -dependent N-SMase
[
18]
[19]
in an Ando variant
of the Horner–
at À788C. The
from rat brain was used. Indeed, lactone 18, as well as 24 and
25 proved to be time-dependent (irreversible) inhibitors of
the N-SMase (Figure 2). Derivatives 19–21 also proved to be
effective (irreversible inhibition, data not shown).
Subsequently, we investigated the inhibitors to determine
which functional group is crucial for irreversible inhibition. In
this context, the reduced derivative 24, which contains a more
[
20]
Wadsworth–Emmons (HWE) reaction
desired Z isomer 11 was obtained together with the corre-
sponding E isomer in a ratio of 20:1. After azide exchange
and reduction, amine 13 was modified with different carbox-
ylic acid chlorides to obtain the amides 14–17. Cleavage of the
protecting groups with catalytic amounts of para-toluenesul-
828
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Figure 2. Time-dependence ofthe inhibition ofthe neutral sphingo-
myelinase by 18 (^), 24 (!), and 25 (*) (final concentration 350 mm).
Hypothetical curve through a reversible competitive inhibition by 25:
Figure 4. Effect of 24 (*) on A-SMase (final concentration 350 mm).
control:
&
.
~
; control:
time.
&
; A=activity; cpm=counts per minute; t=preincubation
The selective N-SMase inhibitors describe, which we term
sphingolactones, are characterized by their high potency, with
compound 24 possessing the highest inhibitory activity. In
contrast to the previously described N-SMase inhibitors such
as scyphostatin (6), spiroepoxide 7, and manumycin A (8), the
sphingolactones are stable and contain no epoxy function.
Therefore, they represent useful chemical tools to explore the
biological significance of ceramide and N-SMase in apoptosis,
inflammation responses, malignant processes, and neuro-
degenerative diseases, such as Alzheimerꢀs disease or multiple
electrophilic lactone group, was identified as a better inhibitor
than compound 18. Therefore, it seems apparent that the
inhibitory activity correlates directly with the lactone and is
not accomplished by a 1,4-addition to the Michael system of
compound 18. This hypothesis is supported by the fact that N-
SMase is also irreversibly inhibited by the saturated lactone
2
5, as well as by derivative 21. Moreover, increasing concen-
[
1,21]
trations of sphingomyelin in the assay attenuate the inhibitory
sclerosis.
effect of 24 (Figure 3). This result indicates that inhibitor 24
Received: June 8, 2005
Published online: December 19, 2005
Keywords: ceramide · inhibitors · lactones · signal transduction ·
.
sphingomyelinase
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