Identification of selective inhibitors of sphingosine kinases 1 and 2 through a structure–activity relationship study of 4-epi-jaspine B

Article abstract of DOI:10.1016/j.bmc.2017.03.059

We recently reported that 4-epi-jaspine B exhibits potent inhibitory activity towards sphingosine kinases (SphKs). In this study, we investigated the effects of modifying the 2-alkyl group, as well as the functional groups on the THF ring of 4-epi-jaspine B using a diversity-oriented synthesis approach based on a late-stage cross metathesis reaction. The introduction of a p-phenylene tether to the alkyl group was favored in most cases, whereas the replacement of a carbon atom with an oxygen atom led to a decrease in the inhibitory activity. Furthermore, the introduction of a bulky alkyl group at the terminus led to a slight increase in the inhibitory activity of this series towards SphKs compared with 4-epi-jaspine B (the Q values of compound 13 for SphK1 and SphK2 were 0.2 and 0.4, respectively). Based on this study, we identified two isoform selective inhibitors, including the m-phenylene derivative 4 [IC₅₀ (SphK1) ≥30?μM; IC₅₀ (SphK2)?=?2.2?μM] and the methyl ether derivative 22 [IC₅₀ (SphK1)?=?4.0?μM; IC₅₀ (SphK2) ≥30?μM].

Full text of DOI:10.1016/j.bmc.2017.03.059

Accepted Manuscript
Identification of selective inhibitors of sphingosine kinases 1 and 2 through a
structure–activity relationship study of 4-epi-jaspine B
Hiroaki Ohno, Maho Honda, Naoka Hamada, Jun Miyagaki, Akira Iwata,
Kazuhiro Otsuki, Toru Maruyama, Shinya Nakamura, Isao Nakanishi, Shinsuke
Inuki, Nobutaka Fujii, Shinya Oishi
PII:
S0968-0896(17)30410-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.059
BMC 13657
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 February 2017
21 March 2017
24 March 2017
Please cite this article as: Ohno, H., Honda, M., Hamada, N., Miyagaki, J., Iwata, A., Otsuki, K., Maruyama, T.,
Nakamura, S., Nakanishi, I., Inuki, S., Fujii, N., Oishi, S., Identification of selective inhibitors of sphingosine kinases
1 and 2 through a structure–activity relationship study of 4-epi-jaspine B, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.03.059
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Bioorganic & Medicinal Chemistry
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Identification of selective inhibitors of sphingosine kinases 1 and 2 through a
structure–activity relationship study of 4-epi-jaspine B
Hiroaki Ohnoa,∗, Maho Hondaa, Naoka Hamadaa, Jun Miyagakia, Akira Iwataa, Kazuhiro Otsukib, Toru
Maruyamab, Shinya Nakamurac, Isao Nakanishic, Shinsuke Inukid, Nobutaka Fujiia, Shinya Oishia
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
bMedicinal Chemistry Research Laboratories, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
cFaculty of Pharmacy, Department of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
dDepartment of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagama 223-8522, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We recently reported that 4-epi-jaspine B exhibits potent inhibitory activity towards sphingosine
kinases (SphKs). In this study, we investigated the effects of modifying the 2-alkyl group, as well as
the functional groups on the THF ring of 4-epi-jaspine B using a diversity-oriented synthesis
approach based on a late-stage cross metathesis reaction. The introduction of a p-phenylene tether to
the alkyl group was favored in most cases, whereas the replacement of a carbon atom with an oxygen
atom led to a decrease in the inhibitory activity. Furthermore, the introduction of a bulky alkyl group
at the terminus led to a slight increase in the inhibitory activity of this series towards SphKs
compared with 4-epi-jaspine B (the Q values of compound 13 for SphK1 and SphK2 were 0.2 and
0.4, respectively). Based on this study, we identified two isoform selective inhibitors, including the
m-phenylene derivative 4 [IC50 (SphK1) = >30 µM; IC50 (SphK2) = 2.2 µM] and the methyl ether
derivative 22 [IC50 (SphK1) = 4.0 µM; IC50 (SphK2) = >30 µM].
Available online
Keywords:
Sphingosine kinase
Kinase inhibitor
Jaspine B
Structure–activity relationship
2009 Elsevier Ltd. All rights reserved.
1. Introduction
numerous diseases, such as cancer,13,14 asthma,15,16
atherosclerosis,17 inflammation,18 fibrosis19 and sickle cell
disease.20,21 The development of effective SphK inhibitors,
especially isoform-selective compounds, has therefore emerged
as an important area of research in drug discovery.2,22,23
Sphingosine kinases (SphKs) catalyze the conversion of
sphingosine (Sph) to sphingosine 1-phosphate (S1P), which plays
an important role in the regulation of numerous biological
processes, including cell growth, proliferation, survival, cell
death and migration.1–3 SphKs exist in two isoforms in mammals,
including SphK1 and SphK2. SphK1 is the smaller of these two
proteins, and lacks the N-terminal nuclear localization sequence
that is found in SphK2.4 SphK1 is located primarily in the cytosol
and overexpressed in many tumor tissues.5-7 The S1P generated
by SphK1 interacts with G-protein coupled sphingosine-1-
phosphate receptors (S1P1−5) to elicit prosurvival and
proliferative effects.8 The knockdown of SphK1 leads to a
reduction in the plasma levels of S1P, as well as a significant
increase in cellular ceramides.9 SphK2 in mainly located in the
nucleus, but can also be found in several other organelles,
including the endoplasmic reticulum and mitochondria. Notably,
the expression of SphK2 has been linked to an increase in gene
expression10 and apoptosis.11 Furthermore, it has been reported
that the siRNA knockdown of SphK2 expression inhibited the
proliferation of glioblastoma cells more effectively than the
knockdown of SphK1.12 S1P signaling is also involved in
Several different types of SphK inhibitors have been
developed during the last decade.3 N,N-Dimethylsphingosine
(DMS)24 and SKI-II25 inhibit both isoforms of the SphKs (Fig.
1).26 PF-543,27 oxadiazole A28 and CB546813926 are potent
SphK1 inhibitors that are more than 50-fold selective for this
particular isoform over SphK2. In contrast, there have been very
few reports to date pertaining to the development of SphK2-
selective inhibitors, and the activities and/or selectivities of those
inhibitors that have been reported are not high. For example,
ABC294640,29 which is currently in phase II clinical trials for the
treatment of pancreatic cancer and solid tumors, exhibits
moderate inhibitory activity and selectivity towards SphK2 [IC50
(SphK1) = >100 µM; IC50 (SphK2) = ~60 µM]. Several other
SphK2 selective inhibitors have been reported, including K145,30
(R)-FTY720-OMe31 and SLP120701,32 although these
compounds typically only demonstrate 2- to 10-fold selectivity
for SphK2 over SphK1. The difficulties associated with
∗Corresponding authors. Tel.: +81 75 753 4571; fax: +81 75 753 4570.
E-mail address: hohno@pharm.kyoto-u.ac.jp.

non-selective
SphK1 selective
O
O
CH3
Cl
N
OH
n-C12H25
OH OH
S
NH2
N
n-C13H27
O
N
H
N
O
NH·HCl
N
NMe2
DMS
O
SO2Ph
N
Cl
SKI-II
PF-543
A
CB5468139
OH
IC50 (SphK1): ~60 µM
IC50 (SphK2): 20 µM
IC50 (SphK1): 35 µM
IC50 (SphK2): 20 µM
IC50 (SphK1): 0.0027 µM
IC50 (SphK2): 0.36 µM
Ki (SphK1): 0.04 µM
Ki (SphK2): 14.1 µM
IC50 (SphK1): ~2 µM
IC50 (SphK2): >100 µM
SphK2 selective
N
O
n-C8H17
N
Cl
N
NH2
HN
N
n-C8H17
OH
NH2
NH
S
O
NH2
N
n-BuO
MeO
O
O
ABC294640
IC50 (SphK1): >100 µM
60
K145
IC50 (SphK1): >10 µM
4.3
(R)-FTY720-OMe
SLP120701
IC50 (SphK1): >50 µM
27
Ki (SphK1): >10 µM
M
µ
M
µ
M
µ
1.2 M
µ
IC50 (SphK2):
~
IC50 (SphK2):
IC50 (SphK2):
Ki (SphK2):
Figure 1. Representative SphK inhibitors.
development of highly potent SphK2-selective inhibitors can be
attributed in part to the lack of structural information for SphK2,
although homology models of this protein were reported in
201333 and 201434 based on the X-ray crystal structure of human
SphK1.
introduction of a phenylene/heteroatom tether and bulky alkyl
groups), as well as the effect of modifying the hydroxy and
amino groups on the THF ring (Fig. 2B). Notably, this work led
to the identification of several isoform selective inhibitors
towards SphK1 and SphK2.
(A) previous works
2. Results and discussion
2.1. Design and synthesis
H2N
OH
H2N
OH
H2N
OH
4
3
2
C14H29
C14H29
C14H29
O
O
O
We designed a new series of jaspine B derivatives 3–22 (Fig.
3) based on the SAR information obtained during our previous
studies, as well as the structures of the known inhibitors shown in
Fig. 1. Consideration of the structures of compound A, (R)-
FTY720-OMe (and its parent compound FTY720) and
SLP120701 suggested that the inclusion of a phenylene tether
was tolerated, having no significant impact on the interaction
between these inhibitors and the SphKs. These results also
indicated that the positioning of the substituents on the phenyl
ring could affect the isoform selectivity. With this in mind, we
prepared a series of derivatives 3–11 containing a phenylene
tether bearing an alkyl group (R) at the o-, m- or p-position. We
reasoned that the alkyl chain length of the R group should be C8
or C9, given that the benzene ring would correspond to a C3 or
C4 alkyl group. Bulky-alkyl derivatives 12–16 were designed to
determine whether the introduction of a branched alkyl group
would lead to an increase in the lipophilicity of the side chain
without significantly affecting the chain length. The oxygen atom
introduced in the alkyl chain of 12–16 was for ease of synthesis.
The two ether derivatives 17 and 18 were also designed to assess
the impact of incorporating an oxygen atom in the alkyl chain.
Notably, the alkyl chain in these two analogs was the same length
as that of jaspine B. The methoxy and guanidino groups in (R)-
FTY720-OMe and SLP120701, respectively, prompted us to
design derivatives 19–22 bearing guanidino, amidino, acetamido
and methoxy moieties as the polar head groups, respectively.
1
jaspine B ( )
2
4-epi-jaspine B ( )
other 6 isomers
µ
µ
IC50 (SphK1): 4.6 µM
IC50 (SphK2): 6.6 µM
IC50 (SphK1): 0.94 µM
IC50 (SphK2): 0.48 µM
IC50 (SphK1): 0.59–3.9
IC50 (SphK2): 1.8–15.8
M
M
(B) this work
modification
H2N
OH
branched alkyl group
O
phenylene or oxygen tether
Figure 2. Our works on SphK inhibitory activities of jaspine B
derivatives.
Our group has an ongoing interest in the development of
SphK inhibitors based on the structure of the naturally occurring
anhydrophytosphingosine derivative jaspine B (1) (Fig. 2A).35-37
In terms of its main structural features, this compound consists of
a functionalized THF moiety (polar head group) that is attached
to an alkyl side chain (lipophilic tail).38 Several synthetic39-43 and
structure–activity relationship studies44-46 have been conducted
around jaspine B and its derivatives. However, most of these
studies have focused on the polar head group and very little is
known about the impact of making changes to the lipophilic
tail.37,47,48 In our previous study, we synthesized all of the
possible stereoisomers of jaspine B and evaluated their biological
activities. The results revealed that all of the isomers exhibited
inhibitory activities toward SphKs and that 4-epi-jaspine B (2)
exhibited the most potent activities of all of the isomers tested
towards SphKs 1 and 2.36 The optimum length of the linear alkyl
chain in epimer 2 for SphK inhibition was determined to be C14,
which is the same length as the chain in natural jaspine B.37
However, we did not investigate the impact of modifications of
the alkyl side chain or the functional groups on the THF moiety.
Herein, we report the results of our recent structure–activity
relationship study on the alkyl side chain of jaspine B (i.e., the

H2N
OH
H2N
OH
phenylene derivatives. As anticipated, the use of too short or too
long an alkyl chain, as exemplified by compounds 7 and 11,
respectively, resulted in a decrease in the inhibitory activity.
O
( )n
O
O
R
3 11
–
12 16
–
(R = o-, m-, or p-alkyl group)
(n = 8–11)
Table 1
SphK inhibitory activities of the phenylene tether derivatives
H2N
OH
R1HN OR2
3–11.
4
3
O
( )m
( )n
C14H29
O
O
H2N
OH
R1
17, 18
(n + m = 12)
19–22
O
Figure 3. Design of SphK inhibitors 3–22.
IC50 (µM)a
Qb
Compd
R1
SphK1
SphK2 SphK1 SphK2
Compounds 3–22 were prepared according to our diversity-
oriented synthesis of jaspine B derivatives, involving the use of a
late-stage cross metathesis reaction.37,49-51 A representative
example of this process is shown in Scheme 1. The reduction of
the known ketone 23 with NaBH4, followed by the dehydration
of the resulting alcohol with TsOH gave alkene 25.52 The
subsequent cross metathesis of 25 with the known
tetrahydrofuran derivative 26 using the Hoveyda–Grubbs second
generation catalyst, followed by the hydrogenation of the
resulting C=C double bond and the deprotection of the Boc group
gave the desired phenylene derivative 9. All of the other 4-epi-
jaspine B derivatives in this series were readily prepared in a
similar manner (see Supplementary material).
2
3
C14H29
0.82–1.1 0.60–0.74 1.0
1.0
>30
>30
–
–
C8H17
R
4
5
6
(R = n-C7H15) >30
2.2
2.1
1.7
–
3.7
2.8
2.8
(R = n-C8H17)
(R = n-C9H19)
16
12
20
11
O
OH
NaBH4
TsOH·H2O
R
EtOH, rt
(85%)
toluene, reflux
(97%)
C10H21
7
8
(R = n-C6H13) >30
8.3
–
14
0.76
1.1
35
–
C10 H21
C10H21
23
24
25
(R = n-C8H17)
(R = n-C10H21)
(R = n-C12H25)
1.4
0.56
0.67
1.7
0.38
0.80
–
1) Hoveyda-Grubbs II
(10 mol %)
CH2Cl2, reflux
9
0.42
0.88
H2N
OH
BocHN
OH
10
21
>30
O
2) Pd/C, H2, EtOH, rt
3) TFA, CH2Cl2, 0 °C
(32% in 3 steps)
11
(R = n-C14H29) >30
O
C10H21
9
a
26
IC50 values (i.e., the concentration required to inhibit the
phosphorylation of sphingosine by SphK1 or SphK2 by 50%).
b
Scheme 1. Synthesis of 4-epi-jaspine B derivative 9.
The
Q
values were calculated as follows:
Q
=
IC50(compound)/IC50(4-epi-jaspine B).
2.2. SphK inhibitory activity
The in vitro inhibitory activities of all of the 4-epi-jaspine B
derivatives prepared in the current study were evaluated towards
SphK1 and SphK2. The results for derivatives 3–11 bearing a
phenylene tether are shown in Table 1. The o-substituted
derivative 3 exhibited no inhibitory activity towards SphK1 or
SphK2 at 30 µM. In contrast, the m-substituted derivatives 4–6
exhibited potent inhibitory activities toward SphK2 (IC50 = 1.7–
2.2 µM), although their activities toward SphK1 were generally
weak (>12 µM) and appeared to be dependent on the chain length
of the R group. It is noteworthy that the heptyl derivative 4 (R =
n-C7H15) exhibited 14-fold selectivity for SphK2 over SphK1. Of
all the p-phenylene tether-containing derivatives prepared in the
current study (7–11), compounds 8–10 bearing an alkyl group of
the appropriate chain length exhibited potent inhibitory activities,
especially towards SphK1 (0.42–1.4 µM). Furthermore, a slight
improvement over the activity of 4-epi-jaspine B (2) was
observed in some cases [Q (SphK1) for 9 = 0.38; Q (SphK2) for
8 = 0.76]. Interestingly, the inhibitory activities of 8–10 towards
SphK2 (0.56–21 µM) were more sensitive to the alkyl chain
length of the R group than their activities towards SphK1 (0.42–
1.4 µM), which contrasted with the results observed for the m-
The SphK inhibitory activities of the bulky alkyl derivatives
12–16 are shown in Table 2. Pleasingly, we found that the
introduction of a cyclohexyl group bearing an oxygen atom-
containing tether generally favored SphK inhibition. For example,
we observed slight improvement in the inhibitory activities of
compounds 13 [Q (SphK1) = 0.20; Q (SphK2) = 0.40] and 14 [Q
(SphK1) = 0.61; Q (SphK2) = 0.76] compared with 4-epi-jaspine.
However, the same tendency was not observed for compound 15
bearing a shorter alkyl chain (Q = >2.2 for both the isoforms). A
comparison of the cyclohexyl analog 12 (IC50 = 0.38–0.78 µM)
with the isopropyl analog 16 (IC50 = 0.71–0.84 µM) revealed that
the inclusion of an isopropyl group as the terminal moiety was
well tolerated.

SLP120701 and oxadiazole A, did not lead to any improvements
in the inhibitory activity [Q (SphK1) = 1.0–2.8; Q (SphK2) =
3.2–16]. The acetylation of the amino group (21) resulted in a
significant drop in the inhibitory activity towards both isoforms
(IC50 = >30 µM). Given that the degree of steric hindrance
observed in the modified amino group analogs 19–21 was almost
identical, these results highlighted the importance of the cationic
functional group in 4-epi-jaspine B (2). Pleasingly, methyl ether
22, which was designed to mimics (R)-FTY720-OMe, inhibited
the activity of SphK1 (IC50 = 4.0 µM) much more effectively
than that of SphK2 (IC50 = >30 µM).
Table 2
SphK inhibitory activities of the bulky-alkyl derivatives 12–
16
H2N
OH
R2
( )8
O
IC50 (µM)a
SphK1 SphK2 SphK1SphK2
0.82–1.1 0.60–0.74 1.0 1.0
Qb
Compd
R2
2
12
0.38
0.22
0.67
0.78
0.24
0.46
0.46 1.1
0.20 0.40
0.61 0.76
Table 4
SphK inhibitory activities of amino or hydroxy-modified
derivatives
O
O
13
14
15
R1HN
OR2
C14H29
O
O
IC50 (µM)a
SphK1 SphK2 SphK1SphK2
1.1–2.8 0.32–0.60 1.0 1.0
Qb
Compd
NHR1
OR2
OH
OH
O
2.8
1.3
2.5
2.2
NH2
2
16
0.71
0.84
0.65 1.4
O
NH
a
IC50 values (i.e., the concentration required to inhibit the
19
1.1
3.1
1.9
9.4
1.0 3.2
b
N
H
NH2
phosphorylation of sphingosine by SphK1 or SphK2 by 50%).
The Q values were calculated as Q = IC50(compound)/IC50(4-epi-
jaspine B).
NH
OH
20
21
2.8
16
N
H
NHAc
NH2
OH >30
OMe
>30
>30
–
–
–
The introduction of an oxygen tether had an adverse effect on
inhibitory activities of these compounds (Table 3). For example,
the ether derivative 18 did not exhibit any inhibitory activity at
30 µM. In contrast, the introduction of an oxygen atom at a
position further away from the polar head was better tolerated,
although a considerable decrease was observed in the inhibitory
activity (e.g., 17, Q = 6.5–8.7).
22
4.0
3.6
a
IC50 values (i.e., the concentration required to inhibit the
b
phosphorylation of sphingosine by SphK1 or SphK2 by 50%).
The Q values were calculated as Q = IC50(compound)/IC50(4-epi-
jaspine B).
2.3. Docking mode analysis
Table 3
SphK inhibitory activities of heteroatom tether derivatives
To develop a deeper understanding of the SAR information
and generate some new insights for further SAR studies, we
investigated the binding modes of some jaspine B derivatives.
Calculations were performed using MOE-Dock based on the
crystal structures of SphK1 and the Sph complex (PDBID:
3VZB). It is noteworthy that we experienced some difficulty in
quantifying these binding interactions because of the flexible
nature of the alkyl side chain, which did not form any meaningful
interactions. An RMSD value of 2.1 Å was obtained for the re-
docking of Sph, highlighting the flexibility of these ligands.
H2N
OH
X
( )4 ( )6
O
IC50 (µM)a
Qb
Compd
X
SphK1
SphK2 SphK1SphK2
2
1.1–2.2 0.60–1.1 1.0
1.0
6.5
–
17
9.6
>30
3.9
>30
8.7
–
O
As shown in Fig. 4A, all the stereoisomers of jaspine B bound
to the Sph binding site of SphK1 through several polar residues,
including Asp81, Asp178, Leu268 and Ser168, regardless of the
stereochemistries of the amino and hydroxy groups of the THF
ring. The relatively high inhibitory activity of 4-epi-jaspine B (2)
can be explained by the formation of favorable hydrophilic
interactions without the need to undergo any conformational
strain (i.e., the formation of a hydrogen bond from the hydroxy
group to the carbonyl group of Leu268, and the formation of
electrostatic interactions between the amino group and the
carboxy groups of Asp178 and Asp81) (Fig. 4B). The
corresponding interactions of jaspine B (1) are shown in Fig. 4C.
The hydrophobic pocket of SphK1 contains additional space
18
O
a
IC50 values (i.e., the concentration required to inhibit the
phosphorylation of sphingosine by SphK1 or SphK2 by 50%).
The Q values were calculated as Q = IC50(compound)/IC50(4-epi-
b
jaspine B).
We also investigated the effect of modifying the amino and
hydroxy groups on the THF ring (Table 4). Disappointingly, the
introduction of a guanidino or amidino group (19 and 20,
respectively); changes inspired by the substructures of

around the terminal region of 2 (C8–C14). Notably, the docking
simulation of the cyclohexyl analog 12 revealed the presence of a
reasonable interaction, as shown in Fig. 4D. These calculations
therefore provided a strong rationale for the slight improvements
observed in the inhibitory activities of compounds bearing a
branched alkyl group as their terminal moiety (Table 2). Taken
together with their inhibitory activities, the results of this docking
mode analysis suggest that further modifications could be made
to the alkyl chain, as well as the functional groups on the THF to
improve the isoform selectivity and inhibitory activity of these
compounds.
(A)
(D)
(B)
Figure 4. (A) Electrostatic interactions between 4-epi-jaspine B
stereoisomers and SphK1. Each amino group and hydroxy group is shown in
small sphere. (B) Predicted binding mode of 4-epi-jaspine B (2) (cyan). (C)
Predicted binding mode of jaspine B (1) (green). (D) Predicted binding mode
of 12 (yellow).
3. Conclusions
In this study, we investigated the effect of modifying the alkyl
side chain, as well as the hydroxy and amino groups of 4-epi-
jaspine B (2) on the inhibitory activity and isoform selectivity of
this compound towards SphK1 and SphK2. The incorporation of
a phenylene tether in the alkyl group resulted in the SphK2
selective inhibitor 4 with moderate activity and selectivity [IC50
(SphK1) = >30 µM; IC50 (SphK2) = 2.2 µM]. We also achieved a
slight improvement in the inhibitory activity of this compound
series following the introduction of a cyclohexyl group at the end
of the alkyl chain, as exemplified by the Q values for 13, which
were 0.20 (SphK1) and 0.40 (SphK2). The methyl etherification
of the hydroxy group in 2 led to the identification of 22, which
exhibited moderate selectivity [SphK1 (IC50 = 4.0 µM); SphK2
(IC50 = >30 µM)].
(C)

4. Experimental
catalyst (27 mg, 0.044 mmol; 10 mol %), and the resulting
mixture was heated under reflux for 1.5 h. The reaction was then
cooled to room temperature and filtered through a short NH2
silica gel column eluting with EtOAc. The solvent was removed
under reduced pressure to give the crude metathesis product as a
residue, which was dissolved in a 1:1 mixture of EtOH and THF
(15 mL). The solution was treated with 10 wt% Pd/C (23 mg,
0.022 mmol; 5 mol%) at room temperature, and the resulting
mixture was stirred at room temperature for 2 h under an
atmosphere of H2. The mixture was then filtrated through a short
pad of Celite. The solvent was removed under vacuum to give the
crude hydrogenation product, which was dissolved in CH2Cl2 (9
mL). This solution was cooled to 0 °C and treated with TFA (9
mL), and the resulting mixture was stirred at room temperature
for 30 min. The reaction was then concentrated under reduced
pressure to give an oily residue, which was purified by column
chromatography over silica gel eluting with CHCl3–MeOH–28%
1
General Methods. H NMR spectra were recorded on a 500
MHz JEOL AL-500 spectrometer using CDCl3 as a solvent.
Chemical shifts (δ) were reported in ppm relative to Me4Si,
which was used as an internal reference standard. 13C NMR
spectra were recorded on a JEOL AL-500 spectrometer using
CDCl3 as a solvent. The signals were reported relative to the
residual CHCl3 signal. IR spectra were recorded on a JASCO
FT/IR-4100 spectrometer. High resolution mass spectrometry
(HRMS) analyses were conducted on a JMS-HX/HX 110A mass
spectrometer or Shimadzu LC-ESI-IT-TOF-MS system.
Purification by column chromatography was conducted using
Wakogel C-300E (Wako), Chromatorex NH-DM1020 (Fuji
Silysia) or Aluminum oxide 90 (Merck-Millipore). The purities
of the compounds submitted for biological analysis were
determined to be >95% by HPLC.
NH4OH (94:5:1) to give 9 (48.4 mg, 32% yield in 3 steps) as a
General procedure for the ketone reduction: synthesis of
1-(4-decylphenyl)ethan-1-ol (24). NaBH4 (436 mg, 11.5 mmol)
was added to a stirred solution of ketone 23 (2.50 g, 9.60 mmol)
in EtOH (32 mL; 0.3 M) at 0 °C, and the resulting mixture was
allowed to warm to room temperature with stirring for 2 h. The
reaction was then cooled to 0 °C and quenched by the addition of
a saturated aqueous solution of NH4Cl. The solvent was removed
under reduced pressure, and the resulting residue was dissolved
in EtOAc. The resulting solution was washed sequentially with
water and brine, before being dried over MgSO4. The mixture
was then evaporated to dryness under reduced pressure to give an
oily residue, which was purified by flash column
chromatography over silica gel eluting with n-hexane and EtOAc
(10:1) to give 24 (2.14 g, 85% yield) as a colorless oil: 1H-NMR
(500 MHz, CDCl3) δ: 0.88 (t, J = 6.9 Hz, 3H), 1.22–1.34 (m,
14H), 1.49 (d, J = 6.3 Hz, 3H), 1.57–1.63 (m, 2H), 1.75 (d, J =
3.4 Hz, 1H), 2.59 (t, J = 7.7 Hz, 2H), 4.85–4.90 (m, 1H), 7.17 (d,
J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H); 13C-NMR (125 MHz,
CDCl3) δ: 14.1, 22.7, 25.0, 29.3 (2C), 29.5, 29.58, 29.60, 31.5,
31.9, 35.6, 70.2, 125.3 (2C), 128.5 (2C), 142.2, 143.0; HRMS
(FAB+) calcd for C18H30O [M+], 262.2297; found, 262.2294.
1
white solid: [α]29 –16.1 (c 1.08, CHCl3); H-NMR (500 MHz,
D
CDCl3) δ: 0.88 (t, J = 6.9 Hz, 3H), 1.20–1.37 (m, 14H), 1.58 (tt, J
= 7.4, 7.4 Hz, 2H), 1.65 (br s, 3H), 1.84–1.93 (m, 1H), 1.93-2.02
(m, 1H), 2.56 (t, J = 7.4 Hz, 2H), 2.61–2.69 (m, 1H), 2.79 (ddd, J
= 14.3, 9.2, 5.2 Hz, 1H), 3.39 (dd, J = 9.2, 3.4 Hz, 1H), 3.42–3.47
(m, 1H), 3.77–3.80 (m, 1H), 3.89–3.94 (m, 1H), 4.20 (dd, J = 9.2,
5.7 Hz, 1H), 7.09 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H);
13C-NMR (CDCl3) δ: 14.1, 22.7, 29.3, 29.4, 29.5, 29.58, 29.59,
30.4, 31.6, 31.9, 32.1, 35.5, 60.0, 73.6, 79.6, 80.2, 128.2 (2C),
128.4 (2C), 138.9, 140.5; HRMS (FAB+) calcd for C22H38NO2 [M
+ H]+, 348.2903; found, 348.2905.
Sphingosine kinase assay. SphK inhibitory activities were
evaluated by the off-chip mobility shift assay by the QuickScout®
service from Carna Bioscience (Kobe, Japan). SphK1(1-384) and
SphK2(1-618) were expressed as N-terminal GST-fusion proteins
using a baculovirus expression system. They were purified using
glutathione sepharose chromatography. Each chemical in DMSO
at different concentrations was diluted fourfold with reaction
buffer [20 mM HEPES (pH 7.5), 0.01% Triton X-100, 2 mM
DTT]. For SphK reactions, a combination of the compound, 1
µM Sph, 5 mM MgCl2, ATP (25 µM for SphK1; 600 µM for
SphK2) in reaction buffer (20 µL) were incubated with each
SphK in 384-well plates at room temperature for 1 h (n = 2). The
reaction was terminated by addition of 60 or 70 µL of termination
buffer (Carna Biosciences). Substrate and product were separated
by electrophoretic means using the LabChip system. The kinase
reaction was evaluated by the product ratio, which was calculated
from the peak heights of the substrate (S) and product (P):
[P/(P+S)]. Inhibition data were calculated by comparing with no-
enzyme controls for 100% inhibition and no-inhibitor reactions
for 0% inhibition. IC50 values were calculated using GraphPad
Prism 4 software (GraphPad Software, Incorporated, La Jolla,
CA, USA).
General procedure for the acid-catalyzed dehydration of
alcohols: synthesis of 1-decyl-4-vinylbenzene (25). TsOH·H2O
(140 mg, 0.74 mmol) was added to a stirred solution of alcohol
24 (1.94 g, 7.39 mmol) in toluene (150 mL), and the resulting
mixture was heated under reflux for 30 min. The mixture was
then cooled to room temperature and basified by the addition of a
saturated aqueous solution of NaHCO3. The organic phase was
separated and washed sequentially with water and brine, before
being dried over MgSO4. The solvent was removed under
reduced pressure to give a residue, which was purified by flash
column chromatography over silica gel eluting with n-hexane to
1
give 25 (1.74 g, 97% yield) as a colorless oil. The H and 13C
NMR spectra of 25 were consisted with those reported in the
1
literature52: H-NMR (500 MHz, CDCl3) δ: 0.88 (t, J = 6.9 Hz,
Acknowledgments
3H), 1.21–1.30 (m, 14H), 1.56–1.62 (m, 2H), 2.58 (t, J = 7.7 Hz,
2H), 5.18 (d, J = 10.9 Hz, 1H), 5.70 (d, J = 17.8 Hz, 1H), 6.69
(dd, J = 17.8, 10.9 Hz, 1H), 7.13 (d, J = 8.0 Hz, 2H), 7.32 (d, J =
8.0 Hz, 2H); 13C-NMR (125 MHz, CDCl3) δ: 14.1, 22.7, 29.30,
29.34, 29.5, 29.60, 29.62, 31.5, 31.9, 35.7, 112.8, 126.1 (2C),
128.6 (2C), 135.0, 136.7, 142.7.
This work was supported by the JSPS KAKENHI (Grant
Numbers JP15KT0061 and JP15H04654), the Uehara Memorial
Foundation, and the Platform for Drug Design, Discovery and
Development from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan.
General procedure for the synthesis of 4-epi-jaspine B
through
derivatives
sequential
cross-metathesis,
Supplementary data
hydrogenation and deprotection reactions: synthesis of
(2S,3S,4R)-4-amino-2-(4-decylphenethyl)tetrahydrofuran-3-ol
(9). To a stirred solution of 26 (100 mg, 0.436 mmol) in CH2Cl2
(9 mL) under argon at room temperature was added 25 (434 mg,
1.78 mmol), followed by the 2nd generation Hoveyda–Grubbs
Supplementary data associated with this article (provided by
the authors including experimental procedures and

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online version at http://dx.doi.org/10.1016/j.bmc
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