Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase

Article abstract of DOI:10.1016/j.bmcl.2003.08.020

Seeking neutral sphingomyelinase inhibitors, we designed and synthesized hydrolytically stable analogues of sphingomyelin. These novel analogues replace the phosphodiester moiety of sphingomyelin with carbamate and urea moiety, resulting in inhibition of neutral sphingomyelinase. Compound 1 prevented ceramide generation and apoptotic neuronal cell death in a model of ischemia based on organotypic hippocampal slice cultures.

Full text of DOI:10.1016/j.bmcl.2003.08.020

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3681–3684
Biological Evaluation of Sphingomyelin Analogues as Inhibitors
of Sphingomyelinase
Minoru Taguchi,a,* Ken-ichi Goda,b Kikuo Sugimoto,b Tomoko Akama,a
Kyoko Yamamoto,a Taizo Suzuki,a Yasumitsu Tomishima,a Mariko Nishiguchi,a
Koshi Arai,a Kenzo Takahashia and Takeo Koborib
aMedicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Kita-ku, Saitama-shi,
Saitama 331-9530, Japan
bSagami Chemical Research Center, 2743-1 Hayakawa, Ayase-shi, Kanagawa 252-1193, Japan
Received 13 May 2003; revised 8 August 2003; accepted 11 August 2003
Abstract—Seeking neutral sphingomyelinase inhibitors, we designed and synthesized hydrolytically stable analogues of sphingo-
myelin. These novel analogues replace the phosphodiester moiety of sphingomyelin with carbamate and urea moiety, resulting in
inhibition of neutral sphingomyelinase. Compound 1 prevented ceramide generation and apoptotic neuronal cell death in a model
of ischemia based on organotypic hippocampal slice cultures.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
independent SMase, and so on. While it remains unclear
which specific SMase activity contributes most sig-
nificantly to neuronal apoptosis, N-SMase is distributed
primarily in the brain. Ceramide generated by activation
of N-SMase mediates hypoxic cell death in neural PC12
cells.6 Thus, N-SMase may be a major factor in the
pathogenesis of stroke and neurodegenerative disease,
making the inhibition of N-SMase a topic worthy of
further attention. We sought out selective N-SMase
inhibitors that presented potential for use in the treat-
ment of these diseases.
Sphingomyelinase (SMase), which generates ceramide
and phosphorylcholine by hydrolyzing the phosphodie-
ster linkage of sphingomyelin (SM), is activated by
stress conditions such as ischemia or cytokine stimula-
tion, including tumor necrosis factor a (TNF-a) and
interleukin 1b (IL-1b).1 It has become apparent that no
less than cell proliferation or cell differentiation, cer-
amide, a SMase product, plays an important role in the
signal transduction of extracellular stimuli to apoptotic
cell death as a lipid second messenger.2 On the other
hand, recent studies suggest the deep implication of
apoptosis in stroke-related neuronal cell death and
neurodegenerative diseases.3 Thus, ceramide may play a
key role in these neuronal diseases, in which case the
inhibition of SMase may emerge as an unique therapy.
The administration of exogenous ceramides induces
apoptotic neuronal cell death,4 while SMase inhibition
suppresses apoptotic cell death in neuronal PC12
cells.5,6 Various SMase activities have been noted in
mammalian tissues and cells, including lysosomal acidic
SMase (A-SMase), the membrane neutral magnesium-
dependent SMase (N-SMase), the cytosolic magnesium-
Previously, we reported that a non-phosphate analogue
1 that replaces the phosphodiester moiety of SM with a
carbamate moiety selectively inhibited N-SMase.7 Sub-
sequently, we synthesized various new derivatives of 1
and examined the anti-apoptotic effects of 1 on neuro-
nal cell death. In this paper, we describe herein structure
activity relationships of derivatives of 1 and an eval-
uation of the bioactivity of 1 (Fig. 1).
Preparation of compounds
The urea analogue 7 was synthesized according to
Scheme 1. The alcohol 3 prepared from 2-N-Boc-pro-
tected d-erythro-sphingosine 2 in five steps, as reported
in our previous paper,7 was oxidized to give 4. The
*Corresponding author. Tel.: +81-4866-93029; fax: +81-4865-27254;
e-mail: minoru.taguchi@po.rd.taisho.co.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.020

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M. Taguchi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3681–3684
Scheme 2. (a) ClC(S)OPh, Py, THF, À78 to À20 ꢀC (33%); (b)
PyCH2NH2, DMSO, rt (69%); (c) Bu4NF, THF, 0 ꢀC (73%).
Figure 1. Sphingomyelin (SM) and sphingomyelinase (SMase)
inhibitor 1.
Scheme 3. (a) ClCO2CCl3, Py, CH2Cl2, À78 to À20 ꢀC, then
PyCH2OH, À20 ꢀC (59%); (b) Bu4NF, THF, 0 ꢀC (72%).
Scheme 4. (a) ClCO2CCl3, DMAP, CH2Cl2, À78 to À20 ꢀC, then
PyCH2NHOTBDMS, À20 ꢀC (5%); (b) Bu4NF, THF, 0 ꢀC (87%).
give the protected derivative of 11, which was desily-
lated to give the desired analogue 11.
Compound 12 or 13, an aliphatic side chain modifi-
cation of 1, was synthesized in a manner similar to that
of 1, in which an acetylene derivative corresponding to
the side chain of 12 or 13 was used as a starting mate-
rial. The saturated compound 14 or 15 was prepared
from 1 or 12, respectively, by catalytic reduction (H2/
10% Pd/C/MeOH).
Scheme 1. (a) PySO3, Et3N, DMSO, THF, rt (90%); (b) HONH2/
HCl, i-Pr2NEt, THF, rt (98%); (c) Ac2O, Py, THF, 0 ꢀC (74%); (d)
NaBH4, EtOH, À20 ꢀC (58%); (e) Boc2O, DMAP, CH2Cl2 then 4-
PyCH2NH2, rt (74%); (f) Bu4NF, THF, 0 ꢀC (47%).
Biological effects
aldehyde 4 was treated with hydroxylamine, followed by
acylation of the oxime to give the acetoxyimine 5.
Reduction of 5 with sodium borohydride gave the
amine 6. t-Butoxy carbonylation of the amine 6, the
resulting compound was treated with 4-pyridine-
methylamine to give the protected urea derivative of 7.
Subsequent desilylation afforded the desired urea
analogue 7.
The inhibitory activity of synthesized novel analogues
on N-SMase was examined using rat brain microsomes,
previously described.7 The results are given in Tables 1
and 2. The inhibitory activity of the urea 7 (IC50=2.9
mM) was almost equal to that of carbamate 1. The
thiourea 9, the reverse carbamate 10, and the N-
hydroxy carbamate 11 were 10–20 times less potent than
compound 1.
The thiourea analogue 9, the carbamate analogue 10,
and the N-hydroxycarbamate analogue 11 were pre-
pared as shown in Schemes 2–4, respectively. Thiono-
On the other hand, compounds 12 or 13, which have
different carbon chains, showed somewhat weaker inhi-
bitory activity than compound 1. Saturated compounds
14 and 15 decreased the inhibitory activity.
carbonylation of the amine
6
with phenyl
chlorothionoformate gave the adduct 8. Treatment of 8
with 4-pyridylmethylamine, followed by desilylation
gave the desired thiourea 9. The carbamate analogue 10
was prepared from 6. The amine 6 was treated with tri-
chloromethyl chloroformate, followed by condensation
with 4-pyridylmethanol. Subsequent desilylation gave
the desired carbamate analogue 10. The N-hydroxy-
carbamate analogue 11 was prepared in a manner simi-
lar to that of 1. The alcohol 3 was treated with
trichloromethyl chloroformate, followed by condensa-
tion with (4-pyridylmethyl)hydoxylamine O-TBDMS to
For a functional evaluation of compound 1, we used a
model of ischemia based on organotypic hippocampal
slice cultures. Organotypic hippocampal slice cultures
were prepared from 7-day-old rats, as described by
Sakaguchi et al.,8 but with certain modifications. The
hippocampal slices were cultured for 7 days, then
exposed to ischemic stress with oxygen-glucose depriva-
tion for 24 h, followed by reperfusion for 24 h. Apop-
totic cell death was determined in each slice by the

M. Taguchi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3681–3684
Table 1. Inhibitor potential of the derivatives of 1
3683
Compd
R
IC50 mM
1
2.8
7
2.9
45
Figure 2. Effect of 1 on organotypic hippocampal slice after ischemia.
Results are means of nine different experiments. The SE is given.
#p<0.05, significantly different from control (Student’s t-test).
*p<0.05, significantly different from ischemia (Dunnett’s multiple
comparison test).
9
10
11
58
21
Table 2. Inhibitor potential of the derivatives of 1
Compd
R
IC50 mM
Figure 3. Compound 1 inhibits ceramide accumulation induced by in
vitro ischemia. Results are means of four different experiments. The
SE is given. # p<0.05, significantly different from control (Student’s t-
test). *p<0.05, significantly different from ischemia (Student’s t-test).
1
2.8
12
13
14
15
6.8
6.6
The results of this study appear to confirm that the
selective N-SMase inhibitor 1 attenuates neuronal cell
death and ceramide generation induced by ischemia.
These results suggest that N-SMase inhibitors should be
valuable in the treatment of stroke and other neurode-
generative diseases. The paper also proposes various
new surrogates for the phosphodiester moiety of SM
and demonstrates the possibility of conversion to low
molecular weight and non-phosphate compounds.
Based on these results, we may look forward to progress
toward the development of SMase inhibitors.
>100
>100
terminal deoxynucleotidyl transferase-mediated dUTP-
biotin nick-end labeling (TUNEL) method, a histo-
chemical assay of DNA fragmentation.9 A basal level of
neuronal cell death due to apoptosis occurred under
these culture conditions. Following ischemic stress, the
cultured hippocampal slices contained a large number
of TUNEL-positive neurons; 3 mM of compound 1 sig-
nificantly prevented this increase in apoptotic cell death
(Fig. 2).
Acknowledgements
We thank Dr. Y. Hirabayashi of the RIKEN Brain
Science Institute for helpful advice during our research.
We also examined ceramide levels in hippocampal slices
exposed to ischemic stress with and without compound
1. As shown in Figure 3, ceramide levels were increased
by ischemia. This increase was significantly attenuated
by compound 1, suggesting that compound 1 protects
neurons from apoptosis by inhibiting ceramide accu-
mulation, which has been reported to induce apoptosis.
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