Sphingomyelin analogues as inhibitors of sphingomyelinase

Article abstract of DOI:10.1016/S0960-894X(03)00360-3

To search for neutral sphingomyelinase inhibitors we designed and synthesized hydrolytically stable analogues of sphingomyelin. The novel compounds 8 and 9 which were replaced the phosphodiester moiety of sphingomyelin with the carbamate moiety showed inhibitory activity with an IC₅₀ value of μM on neutral sphingomyelinase in rat brain microsomes. Compound 8i showed a selective neutral sphingomyelinase inhibitory activity.

Full text of DOI:10.1016/S0960-894X(03)00360-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1963–1966
Sphingomyelin Analogues as Inhibitors of Sphingomyelinase
Minoru Taguchi,a,* Kikuo Sugimoto,b Ken-ichi Goda,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 14 February 2003; accepted 9 April 2003
Abstract—To search for neutral sphingomyelinase inhibitors we designed and synthesized hydrolytically stable analogues of
sphingomyelin. The novel compounds 8 and 9 which were replaced the phosphodiester moiety of sphingomyelin with the carbamate
moiety showed inhibitory activity with an IC50 value of mM on neutral sphingomyelinase in rat brain microsomes. Compound 8i
showed a selective neutral sphingomyelinase inhibitory activity.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
cells.8 Therefore the inhibition of N-SMase warrants
study as a possible therapy for subjects with the neuro-
nal diseases. Additionally, selective N-SMase inhibitors
will be useful to clarify the physiological functions of N-
SMase.
Recent studies suggested that apoptosis is involved in
neuronal cell death which accompanies stroke and neu-
rodegenerative diseases.1 Additionally, it was suggested
that ceramide, an intracellular lipid messenger, induces
apoptotic neuronal cell death.2 Ceramide is the product
of sphingomyelinase (SMase)-catalysed hydrolysis of
sphingomyelin (SM), one of the most abundant sphin-
golipid species in plasma membranes of cells in mam-
malians (Fig. 1).
Nara et al. reported that Scyphostatin, a constituent of
Dasyscyphus mollissimus inhibits N-SMase of rat brain
microsomes with an IC50 value of 1 mM9 and Omura et
al. reported N-SMase inhibitors of microbial origin,
KF-1040A
(IC50=5.4
mM)10
and
Alutenusin
(IC50=40mM).11 On the other hand, Yokomatsu et al.
reported a SM difluoromethylene analogue which
inhibited N-SMase of bovine brain microsomes with an
IC50 value of 3.3 mM12 and Katsumura et al. reported
SM carbon analogues.13
Stress conditions such as ischemia, stimuli by cytokines
or irradiation,3 activate SMase, increase the amount of
ceramide and can initiate apoptosis.2 Many different
SMase activities have been noted in mammalians,
including lysosomal acidic SMase (A-SMase),4 the
membrane neutral magnesium-dependent SMase (N-
SMase),5 the cytosolic magnesium-independent SMase,6
and others. Although the relevance of each activity on
specific cellular reactions and diseases has not been
clarified, N-SMase distributes mainly in the brain7 and
may be the major activity participating in the patho-
genesis of stroke and neurodegenerative diseases. It was
reported that ceramide generated by activation of N-
SMase mediated hypoxic cell death in neuronal PC12
We have searched for N-SMase inhibitors among the
analogues of SM. In designing the analogues, we con-
sidered simplification of synthesis and improvement of
the poor solubility of SM and we converted the phos-
phodiester moiety the hydrolysis position in SMase. For
improvement of the solubility, we studied shortening of
the 2-N-acyl group and replacement of the trimethyl-
ammonium group with the dialkylamino or pyridyl
group which was recognized as the surrogate for the
choline moiety in investigation of the muscarinic
antagonist. We also studied ester or carbamate analo-
gues for conversion of the phosphodiester moiety.
Therefore we found that novel carbamate compound, as
*Corresponding author. Tel.: +81-4866-93029; fax: +81-4865-27254;
e-mail: minoru.taguchi@po.rd.taisho.co.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00360-3

1964
M. Taguchi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1963–1966
into the 1-hydroxy group of 2. Thus the 2-N-Boc group
of 2 was deprotected under acidic conditions and N-
acylation with pivaloyl chloride yielded 2-N-pivaloyl
sphingosine 3. Pivaloylation of the 1-hydroxyl group of
3, followed by silylation of the 3-hydroxyl group with
t-butyldimethylsilyl chloride, yielded 5. Deprotection of
the 1-pivaloyloxy group of 5 with DBU gave 2-N-piva-
loyl-3-O-t-butyldimethylsilyl-d-erythro-sphingosine
6
that was condensed with 4-(dimethylamino)butyric acid.
Subsequent desilylation afforded the desired ester 7. On
the other hand, 6 was treated with trichloromethyl
chloroformate and the resulting compound was reacted
with amines corresponding to the analogues 8. Subse-
quently desilylation gave the desired carbamate analo-
gues 8. In transformation of the 2-N-acyl group, the
2-amino group of sphingosine was coupled with car-
boxy acids and the resulting compounds were converted
to the 2-N-isobutyryl analogue 9, the 2-N-acetyl analo-
gue 10 and the 2-N-steroyl analogue 11, respectively,
using the method described above.
Figure 1. Metabolism of Sphingomyelin (SM) by Sphingomyelinase
(SMase).
analogues of SM, showed a selective inhibitory activity
on N-SMase. We describe here the syntheses and struc-
ture activity relationships of N-SMase inhibitors we
investigated.
Preparation of Compounds
The synthetic method we used is shown Scheme 1. The
desired intermediate, 2-N-Boc-protected d-erythro-
sphingosine 2 [a]D20 À1.1 (c 1.1,CHCl3); lit.[a]2D5 À1.4 (c
1.1, CHCl3), was synthesized from the Garner aldehyde
1 according to the synthesis method of Herold et al.14
The ester analogue 7 and the carbamate analogues 8
were obtained by introducing the desired substitution
The 2-N-Boc-1-carbamate analogue 14 was prepared in
the following manner, as shown in Scheme 2. The
1-hydroxyl group of 2-N-Boc sphingosine 2 protected
by the pivaloyl group was followed by t-butyldi-
methylsilylation of the 3-hydroxyl group, gave 12.
Hydrolysis of the 1-pivaloyl group of 12, the resulting
alcohol 13 was treated in a manner similar to that
described for the preparation of 8 from 6, to give the
desired carbamate analogue 14. Furthermore, the 2-
amino-1-carbamate analogue 15 was obtained by
deprotection of the 2-N-Boc on 14.
Biological Effects
The inhibitory activity of our synthesized analogues on
N-SMase was examined using rat brain microsomes as
the enzyme source. To measure N-SMase activity,
Scheme 1. (a) Lit. 14; (b) TFA, 0 ꢀC then tBuCOCl, Et3N, THF, rt
(50%); (c) tBuCOCl, Py, À10 ꢀC (82%); (d) TBDMSCl, imidazole,
DMF, rt (95%); (e) DBU, MeOH, rt (97%); (f) Me2N(CH2)3CO2H, 1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide, DMAP, CHCl3, rt
(80%); (g) 2% HF, Py, CH3CN, rt (42%); (h) ClCO2CCl3, Py,
CH2Cl2, À78 ꢀC to À15 ꢀC, then R1NH2, À30 ꢀC (40–90%); (i) 2%
HF, Py, CH3CN, rt (59–97%).
Scheme 2. (a) tBuCOCl, Py, À20 ꢀC (91%); (b) TBDMSCl, imidazole,
DMF, rt (quant); (c) DBU, MeOH, rt (93%); (d) ClCO2CCl3, Py,
CH2Cl2, À78 ꢀC to À15 ꢀC, then 4-PyCH2NH2, À30 ꢀC (63%); (e)
Bu4NF, THF, 0 ꢀC (95%); (f) TFA, 0 ꢀC (quant).

M. Taguchi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1963–1966
Table 2. Inhibitor capacity of carbamate analogues
1965
microsomes (50mg of protein) was incubated for 25 min
at 37 ꢀC in 80mL of buffer containing 0.3mM 14C-label-
led SM, 100 mM Tris–HCl (pH 7.5), 20 mM MgCl2,
0.25% TritonX-100 and various concentrations of test
compounds. Radioactive phosphorylcholine produced
from 14C-labelled SM was extracted with chloroform/
methanol (2/1,v/v). The amount of phosphorylcholine
in the aqueous phase was determined by scintillation
counting. Results are shown in Tables 1and 2 . The IC50
value of compound 7 transformed to an ester moiety
with dimethylaminobutylate was 31mM. This transfor-
Compd
R2
IC50 mM
8i
9
10
11
14
15
COBut
COPri
COMe
COC17H35
CO2But
H
2.8
1.8
>100
>100
>100
>100
Table 1. Inhibitor capacity of ester and carbamate analogues
mation of the ester moiety to the carbamate moiety
increased the inhibitory activity to 4.1mM (compound
8a). Transformation from the methyl group of the
amine moiety to the bulky i-propyl group led to a 4-fold
less potent activity (compound 8d). Replacement of a
nitrogen atom of the amino group with a carbon atom
resulted in a significant loss of the inhibitory activity
(compound 8e). Compounds that were replaced the
amino group with the pyridyl group were somewhat
more potent than compound 8a. In the 2-pyridyl 8g,
3-pyridyl 8h or 4-pyridyl analogue 8i,15 the activity was
almost the same (IC50=2 to 5mM). The inhibitory
activity of compounds 8h, 8i was almost equal com-
pared with Scyphostatin.9 Regarding the distance
between carbamate linkage and the pyridyl group, the
one-carbon tether was most active and a direct bond
and the two-carbon tether were less active. Activity of
the long 2-N-fatty acyl compound, the 2-N-steroyl ana-
logue 11 was not found, perhaps solubility was low. The
2-N-isobutyryl analogue 9 showed activity (IC50=1.8
mM) almost equivalent to the 2-N-pivaloyl analogue 8i.
The short 2-N-acyl chain compound, the 2-N-acetyl
analogue 10, was less active. On the other hand, the 2-
N-Boc-1-carbamate analogue 14 and the 2-amino-1-
carbamate analogue 15 did not show activity.
Compd
R1
IC50 mM
8a
4.1
8b
8c
8d
8e
8f
4.9
8.7
18
To examine the specificity of compound 8i for N-SMase,
we investigated effects of this compound against
A-SMase from rat brain lysosomes, bacterial SMase
(Bacillus cereus SMase; Sigma, S-9396) and phospholi-
pase C. No significant inhibition of each lipase occurred
at 10 and 100 mM. Additionally, we found no significant
activity, that is, IC50>10mM, of 8i on 29 receptors and 10
enzymes (data not shown). These results are interpreted
to mean that 8i is a selective inhibitor of N-SMase.
77
7.9
5.0
2.1
2.8
8.4
8.2
8g
8h
8i
From this study, we confirmed that the carbamate ana-
logues are selective N-SMase inhibitors. The carbamate
moiety is available as a surrogate for the phosphodiester
moiety of SM, which is not hydrolyzed by SMase.
Additionally, the 2-N-pivaloyl or 2-N-isobutyryl analo-
gues are more advantageous in comparison with long 2-
N-fatty acyl compounds with regard to solubility during
synthesis and for evaluation of biological activity. We
are examining the possibility of using these novel
N-SMase inhibitors to treat subjects with ceramide-
induced cell death.
8j
8k

1966
M. Taguchi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1963–1966
8. Yoshimura, S.; Banno, Y.; Nakanishi, Y.; Takenaka, K.;
Acknowledgements
Sakai, H.; Nishimura, Y.; Sakai, N.; Shimizu, S.; Eguchi, Y.;
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Ogita, T. J. Antibiot. 1999, 52, 525. (b) Nara, F.; Tanaka, M.;
Masuda-Inoue, S.; Yamasato, Y.; Doi-Yoshioka, H.; Suzuki-
Konagai, K.; Ogita, T. J. Antibiot. 1999, 52, 531.
We thank Dr. Y. Hirabayashi (RIKEN Brain Science
Institute) for providing useful advice regarding this
research.
10. Omura, S.; Tomoda, H.; Masuma, R. PCT Patent
WO9941265, 1999.
11. Uchida, R.; Tomoda, H.; Dong, Y.; Omura, S. J. Antibiot.
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References and Notes
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15. Spectral data for compound 8i: [a]2D0 À1.3(c 1.1,CHCl3);
1H NMR(CDCl3, 200MHz) d 0.88(t, J=6.5 Hz, 3H), 1.15 (s,
9H), 1.20–1.44 (m, 22H), 2.02 (m, 2H), 3.72 (m, 1H), 4.13–4.17
(m, 3H), 4.34–4.48 (m, 3H), 5.45 (dd, J=6.6, 15.4 Hz, 1H),
5.74 (dt, J=15.3, 6.6 Hz, 1H), 6.29 (d, J=7.4 Hz, 1H), 7.20
(m, 2H), 8.57 (m, 2H); IR (KBr): 3320, 2959, 2919, 2852, 1705,
1635, 1537, 1471, 1420, 1368, 1271, 1154, 1095, 1043, 1007,
971, 905, 796, 718cmÀ1
;
MS(SIMS) m/e: 518 (MH+);
HRMS(ESI) m/e: calcd for C30H51N3O4: 518.3958 (MH+).
Found: 518.3963.