Synthesis and evaluation of novel phosphate ester analogs as neutral sphingomyelinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.042

A novel sphingomyelin inhibitor RY221B-a, which contains a bipyridyl moiety as a metal coordination site was designed based upon the mechanism of phosphate ester hydrolysis. RY221B-a was synthesized from N-Boc-sphingosine in three steps via selective etherification using stannyl acetal. Synthesized RY221B-a exhibited relatively-strong inhibitory activity against Bc-SMase (IC ₅₀ = 1.2 μM).

Full text of DOI:10.1016/j.bmcl.2010.05.042

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3868–3871
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel phosphate ester analogs as neutral
sphingomyelinase inhibitors
Hiroshi Imagawa a, , Masataka Oda , Takayuki Takemoto , Rieko Yamauchi , Tomomi Yoshikawa ,
a
a
a
a
*
a
a,
b
a
a
*
Hirofumi Yamamoto , Mugio Nishizawa , Hironobu Takahashi , Manabu Hashimoto , Kenta Yabiku ,
a
a,
*
Masahiro Nagahama , Jun Sakurai
a
Faculty of Pharmaceutical of Sciences, Tokushima Bunri University, Tokushima, Japan
Institute of Pharmacognosy, Tokushima Bunri University, Tokushima, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 March 2010
Revised 8 May 2010
Accepted 12 May 2010
Available online 15 May 2010
A novel sphingomyelin inhibitor RY221B-a, which contains a bipyridyl moiety as a metal coordination
site was designed based upon the mechanism of phosphate ester hydrolysis. RY221B-a was synthesized
from N-Boc-sphingosine in three steps via selective etherification using stannyl acetal. Synthesized
RY221B-a exhibited relatively-strong inhibitory activity against Bc-SMase (IC50 = 1.2 lM).
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Sphingomyelinase
SMase
Inhibitor
Sphingomyelin
Hydrolysis
Hydrolase-mediated hydrolysis of phosphate esters are
fundamental reactions in a variety of important biological processes
such as signal transduction, molecularrecognition, energy transduc-
tion, regulation of protein function and metabolism. Sphingomyeli-
coordinated with oxygen atoms in the phosphate ester of sphingo-
myelin, which controls the location of the substrate (Fig. 2). We fo-
cused on the metal ion mediated-coordination between the enzyme
7
1
nase (SMase , EC 3.1.4.12) hydrolyzes the phosphate ester-bond of
O
P
O
OH
O
sphingomyelin to generate phosphocholine and ceramide. This
hydrolysis reaction is important for lipid metabolism, regulation of
the immune mechanism and differentiation of cells, including angi-
N
O
O
HN
2
ogenesis (Fig. 1).
sphingomyelin
Therefore, an inhibitor of SMase might be expected to modulate a
wide variety of biological responses. So far, a number of naturally
occurring products showing SMase inhibition have been reported.
sphingomyelinase
OH
3
HO
Several synthetic inhibitors mimicking the phosphate ester moiety
by substitution with stable functional groups such as a carbamate
analog, carbon-analog, and a fluorinated carbon-analog have also
HN
O
P
O
+
N
O
O
OH
ceramide
4
been developed. A detailed mechanism for the hydrolysis reaction
mediated by SMase based on mutagenesis studies have been eluci-
phosphocholine
5
,6
dated. Obama et al. reported that Glu-53 of Bc-SMase has a crucial
2+
5d
function for a ligand for Mg and SMase activity. In 2006, the Sak-
OH
O
HO P O
O
urai group elucidated the three-dimensional structure of Bc-SMase
7
from Bacillus cereus. Analysis of the structure suggested that the io-
NH2
S-1-P
nic bond between Glu-53 and the divalent ion such as Mg2+ plays an
cellular growth
differentiation of cells
2
+
important role in hydrolysis of sphingomyelin, and the Mg ion is
regulation of the immune mechanism
angiogenesis
*
Corresponding authors. Tel.: +81 88 602 8448; fax: +81 88 655 3051 (H.I.).
E-mail address: imagawa@ph.bunri-u.ac.jp (H. Imagawa).
Figure 1. The role of SMase during lipid metabolism.
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.042

H. Imagawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3868–3871
3869
active site
N
O
H
O
P
O
N
O
O
O
O
H
H
Mg2+
H
H
O H
O
N
HO
Glu-53
NH
His-296
R
R
Coordination of 1 with metal ions
Bc-SMase (Bacillus
cereus)
N
O
O P
O
OH
Figure 3. Inhibitory effect of 6 (trigona), 10 (square) and 11 (argyle) for the
degradation of sphingomyeline liposome induced by Bc-SMase.
O
+HN
Mg2
O
Mg2+ complex of sphingomyelin (1)
OH
O
N
N
HN
Mg2+
O
Mg2+ complex of RY221B-a (6)
Figure 2. Design of a novel SMase inhibitor based upon substrate coordination in
the active site of the enzyme.
and sphingomyelin. If the phosphate ester moiety acts as a ligand for
2
+
the Mg ion, the functional group having high coordinating ability
with divalent metal ions will behave as an analog for the phosphate
ester of sphingomyelin during the hydrolysis reaction. From these
structural and mechanistic considerations, we designed a novel
sphingomyelin analog RY221B-a (6) having a bipyridine moiety that
acts as an efficient coordination site (Fig. 2).
The synthesis of 6 commenced with the preparation of N-Boc-
sphingosine (7) from serine methyl ester in five steps according
to the Suzuki protocol.8 Deprotection of 7 was achieved by using
Figure 4. Inhibitory effect of 6 (argyle) and 10 (square) for the hydrolysis of 14C-
sphingomyeline mediated by Bc-SMase.
1
M HCl. The resulting sphingosine (8) was treated with hexanoyl
chloride in the presence of Et N in CH Cl to provide ceramide 9
3
2
2
in 68% yield. The introduction of bipyridyl methyl ether into cera-
mide 9 was achieved by the etherification method via stannyl ace-
9
,10
tal
to give RY221B-a (6) along with isomer (RY221B-b, 10) after
HPLC separation in 53% and 18%, yield respectively (Scheme 1).
OH
OH
HO
C13H27
a
HN
O
HO
C13H27
NH2
O
7
8
OH
O
b
HO
C13H27
c, d, e
HN
Figure 5. Lineweaver–Burk plot analysis for 6. trigona: only Bc-SMase with 10 lM
of 6. Square: Bc-SMase with 5
lM of 6, argyle: Bc-SMase. In all cases, 40 ng/mL of
9
Bc-SMase was used.
N
N
O
The sphingomyelin liposome-disruption assay was used to eval-
6
+
HO
uate the synthetic compound 6 and its isomer 10 as potential SMase
7
,11
HN
inhibitors.
itory effect against Bc-SMase in a concentration-dependent manner
Fig. 3). By contrast, 10 showed only weak inhibitory activity and
Compound 6 was shown to display a significant inhib-
1
0
O
(
Scheme 1. Preparation of the novel sphingomyelin analog 6. Reagents and
conditions: (a) HCl, THF, 50 °C, 4 h, 96%; (b) hexanoyl chloride (1.0 equiv), DMAP,
, 0 °C, 15 min, 68%; (c) Bu
bromomethyl)-2,2 -bipyridine (1.2 equiv), n-Bu
bipyridine (11) displayed no detectable inhibitory action.
The detailed inhibitory activity of 6 and 10 against Bc-SMase
Et
(
3
N, CH
2
Cl
2
2
SnO (1.0 equiv), MeOH, reflux, 6 h; (d) 6-
1
4
7
0
were elucidated using [ C]-labeled SM. The IC50 of 6 was deter-
mined to be 1.2 M, which is classified as one of the most potent
4
NF (1.2 equiv), DMF, rt, 18 h; (e)
HPLC separation. Compound 6/10 (3:1, total 71% yield).
l

3
870
H. Imagawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3868–3871
The complex of 6 with Bc-SMase
The complex of 10 with Bc-SMase
A
2
+
2+
B The interaction of 6 with Mg in the active pocket
The interaction of 10 with Mg in the active pocket
N
N
O
Tyr-25
N
OH
N
H
N
Mg
O
OH
O
O
O
O
H
HN
Mg
O
Glu-53
Asn-197
O
NH2
HO
HO
Asp-195
Figure 6. Simulation analysis of the docking of RY221B-a (6) and RY221B-b (10) for Bc-SMase. For simplification of calculations, the aliphatic side chains in 6 and 10 were
truncated. (A) Simulation analysis of the docking of 6 and 10 for Bc-SMase. Left panel: docking between 6 and Bc-SMase. Right panel: docking between 10 and Bc-SMase. The
most stable structure of 6 and 10 on the molecular electrostatic potential surface were shown. The red and blue surfaces show the negative and positive electrostatic potential
surface, respectively. The gray, white, red, purple in the two compounds indicate carbon, hydrogen, oxygen, nitrogen, respectively. Mg2+ is represented in green. (B) The
interactions between the Bc-SMase and 6 or 10 in the active pocket.
revealed higher values for 6 compared with 10 (Table 1), further
highlighting the importance of the position of bipyridyl moiety
for more stable binding to Bc-SMase.
Table 1
Summary of docking energy measurement
6
10
In conclusion, we created a novel and potent Bc-SMase inhibi-
tor, 6 by molecular design based upon the phosphate-ligand analog
hypothesis. The simulated binding of 6 with Bc-SMase supported
the rationality of the molecular design process. We are currently
attempting to prepare more hydrolase inhibitors by an extension
of this approach.
Docking energy (kCal/mol)
ꢀ117
ꢀ58
Lower numbers indicate more stable interactions.
SMase inhibitors yet identified (Fig. 4). In this assay, a notable dif-
ference in the inhibitory effect between 6 and 10 was also observed
(
Fig. 4). Lineweaver–Burk plot analysis revealed that the inhibitory
Acknowledgments
effect of 6 is competitive with a K value of 5.2 M, showing that
i
l
the compound competed with sphingomyelin for binding to the
catalytic site (Fig. 5).
This study was financially supported by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technology of
the Japanese Government, and the Open Research Center program
of MEXT.
Next, we investigated the mode of binding of 6 and 10 to the ac-
tive pocket of Bc-SMase. Thus, we performed in silico docking sim-
ulation analyses of this interaction at the tertiary structure level
using Molecular Operating Environment (MOE) software. Our re-
sults showed that 6 almost perfectly fit into the active pocket of
Bc-SMase, moreover, the two nitrogen atoms of bipyridyl moiety,
carbonyl oxygen of the amide and ethereal oxygen in 6 coordinate
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.042.
2
+
with Mg in Bc-SMase (Fig. 6 A and B, left). By contrast, binding of
0 to the active pocket of Bc-SMase appears to be unstable (Fig. 6A,
right). Moreover, the bipyridyl moiety in 10 was not chelated with
1
References and notes
2
+
Mg in Bc-SMase (Fig. 6B, right). The structural difference between
these two compounds is the position of the bipyridyl group, sug-
gesting the importance of this moiety for binding to the Bc-SMase.
Calculation of the binding energy using docking simulations
1
2
.
.
Tomita, M.; Taguchi, R.; Ikezawa, H. Arch. Biochem. Biophys. 1983, 223, 202.
(a) Bradshaw, R. A.; Dennis, E. A. Handbook of Cell Signaling; Academic Press:
London, 2003; (b) Okamoto, Y.; Hannun, Y. A. In Recent Research Developments
in Biological Chemistry; Research Signpost: Kerala, 2002; Vol. 1. pp 209–222; (c)

H. Imagawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3868–3871
3871
Aug e´ , N.; Karakashian, M. N.; Carpentier, S.; Parthasarathy, S.; Salvayre, A. N.;
Salvayre, R.; Merrill, A. H.; Levade, T., Jr. J. Biol. Chem. 1999, 274, 21533; (d)
Johns, D. G.; Osborn, H.; Webb, R. C. Biochem. Biophys. Res. Commun. 1997, 237,
Shimeno, H. Bioorg. Med. Chem. Lett. 2001, 11, 1277; (c) Hakogi, T.; Monden, Y.;
Taichi, M.; Iwama, S.; Fujii, S.; Ikeda, K.; Katsumura, S. J. Org. Chem. 2002, 67,
4839; (d) Hakogi, T.; Taichi, M.; Katsumura, S. Org. Lett. 2003, 5, 2801; (e)
Taguchi, M.; Goda, K.; Sugimoto, K.; Akama, T.; Yamamoto, K.; Suzuki, T.;
Tomishima, Y.; Nishiguchi, M.; Arai, K.; Takahashia, K.; Kobori, T. Bioorg. Med.
Chem. Lett. 2003, 13, 3681; (f) Hakogi, T.; Yamamoto, T.; Fujii, S.; Ikeda, K.;
Katsumura, S. Tetrahedron Lett. 2006, 47, 2627.
9
5; (e) Spiegel, S.; Foster, D.; Kolesnick, R. Curr. Opin. Cell Biol. 1996, 8, 159; (f)
Merrill, A. H.; Schmelz, E. M., Jr.; Dillehay, D. L.; Spiegel, S.; Shayman, J. A.;
Schroeder, J. J.; Riley, R. T.; Voss, K. A.; Wang, E. Toxicol. Appl. Pharmacol. 1997,
1
42, 208; (g) Hannun, Y. A. J. Biol. Chem. 1994, 269, 3125.
(a) Nara, F.; Tanaka, M.; Hosoya, T.; Suzuki-Konagai, K.; Ogita, T. J. Antibiot.
999, 52, 525; (b) Nara, F.; Tanaka, M.; Masuda-Inoue, S.; Yamasato, Y.; Doi-
Yoshioka, H.; Suzuki-Konagai, K.; Kumakura, S.; Ogita, T. J. Antibiot. 1999, 52,
31; (c) Uchida, R.; Tomoda, H.; Dong, Y.; Omura, S. J. Antibiot. 1999, 52, 572;
d) Arenz, C.; Giannis, A. Angew. Chem., Int. Ed. 2000, 39, 1440; (e) Uchida, R.;
3
.
5. Mechanistic studies of SMase: (a) Ikezawa, H.; Tameishi, K.; Yamada, A.;
Tamura, H.; Tsukamoto, K.; Matsuo, Y.; Nishikawa, K. Amino Acids 1995, 9, 293;
(b) Fujii, S.; Inoue, B.; Yamamoto, H.; Ogata, K.; Shinki, T.; Inoue, S.; Tomita, M.;
Tamura, H.; Tsukamoto, K.; Ikezawa, H.; Ikeda, K. J. Biochem. 1998, 124, 1178;
(c) Tamura, H.; Tsukamoto, K.; Ikezawa, H.; Ikeda, K. J. Biochem. 1999, 126, 90;
(d) Obama, T.; Kan, Y.; Ikezawa, H.; Imagawa, M.; Tsukamoto, K. J. Biochem.
2003, 133, 279.
6. Mechanisms for general hydrolysis of phosphate ester by enzymes. (a)
Westheimer, F. H. Chem. Rev. 1981, 81, 313; (b) Knofel, T.; Strater, N. J. Mol.
Biol. 2001, 309, 239; (c) Kamerlin, S. C. L.; Wilkie, J. Org. Biomol. Chem. 2007, 5,
2098; (d) Rosta, E.; Kamerlin, S. C. L.; Warshel, A. Biochemistry 2008, 47, 3725.
7. Ago, H.; Oda, M.; Takahashi, M.; Tsuge, H.; Ochi, S.; Katunuma, N.; Miyano, M.;
Sakurai, J. J. Biol. Chem. 2006, 281, 16157.
1
5
(
Tomoda, H.; Arai, M.; Omura, S. J. Antibiot. 2001, 54, 882; (f) Pitsinos, E. N.;
Wascholowski, V.; Karaliota, S.; Rigou, C.; Couladouros, E. A.; Giannis, A.
ChemBioChem 2003, 4, 1223; (g) Hamada, M.; Iikubo, K.; Ishikawa, Y.; Ikeda, A.;
Umezawa, K.; Nishiyama, S. Bioorg. Med. Chem. Lett. 2003, 13, 3151; (h) Inoue,
M.; Yokota, W.; Murugesh, M. G.; Izuhara, T.; Katoh, T. Angew. Chem., Int. Ed.
2
004, 43, 4207; (i) Claus, R. A.; Wüstholz, A.; Müller, S.; Bockmeyer, C. L.;
Riedel, N. H.; Kinscherf, R.; Deigner, H.-P. ChemBioChem 2005, 6, 726; (j) Katoh,
T.; Izuhara, T.; Yokota, W.; Inoue, M.; Watanabe, K.; Nobeyama, A.; Suzuki, T.
Tetrahedron 2006, 62, 1590; (k) Wascholowski, V.; Giannis, A. Angew. Chem., Int.
Ed. 2006, 45, 827; (l) Inoue, M.; Yokota, W.; Katoh, T. Synthesis 2007, 622; (m)
Fujioka, H.; Sawama, Y.; Kotoku, N.; Ohnaka, T.; Okitsu, T.; Murata, N.; Kubo, O.;
Li, R.; Kita, T. Chem. Eur. J. 2007, 13, 10225.
8. Suzuki, H.; Mori, M.; Shibakami, M. Synlett 2003, 14, 2163.
9. Arena, G.; Contino, A.; Longo, E.; Sciotto, D.; Sgarlata, C.; Spoto, G. Tetrahedron
Lett. 2003, 44, 5415.
10. Nagashima, N.; Ohno, M. Chem. Lett. 1987, 141.
4
.
(a) Hakogi, T.; Monden, Y.; Iwama, S.; Katsumura, S. Org. Lett. 2000, 2, 2627; (b)
Yokomatsu, T.; Takechi, H.; Akiyama, T.; Shibuya, S.; Kominato, T.; Soeda, S.;
11. (a) Tomota, T.; Watanabe, M.; Yasuda, T. J. Biol. Chem. 1992, 267, 13391; (b)
Tomota, T.; Watanabe, M.; Yasuda, T. Biochim. Biophys. Acta 1992, 1104, 325.