Pachastrissamine (jaspine B) and its stereoisomers inhibit sphingosine kinases and atypical protein kinase C

Article abstract of DOI:10.1016/j.bmc.2011.07.061

Sphingosine kinases (SphKs) are oncogenic enzymes that regulate the critical balance between ceramide and sphingosine-1-phosphate. Much effort has been dedicated to develop inhibitors against these enzymes. Naturally occurring pachastrissamine (jaspine B)

Full text of DOI:10.1016/j.bmc.2011.07.061

Bioorganic & Medicinal Chemistry 19 (2011) 5402–5408
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pachastrissamine (jaspine B) and its stereoisomers inhibit sphingosine kinases
and atypical protein kinase C
⇑
⇑
Yuji Yoshimitsu, Shinya Oishi , Jun Miyagaki, Shinsuke Inuki, Hiroaki Ohno, Nobutaka Fujii
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine kinases (SphKs) are oncogenic enzymes that regulate the critical balance between ceramide
and sphingosine-1-phosphate. Much effort has been dedicated to develop inhibitors against these
enzymes. Naturally occurring pachastrissamine (jaspine B) and all its stereoisomers were prepared and
evaluated for their inhibitory effects against SphKs. All eight stereoisomers exhibited moderate to potent
inhibitory activity against SphK1 and SphK2. Inhibitory effects were profiled against protein kinase C
Received 24 June 2011
Revised 27 July 2011
Accepted 28 July 2011
Available online 4 August 2011
(PKC) isoforms by in vitro experiments. Atypical PKCs (PKCf and PKCi) were inhibited by several pachas-
Keywords:
Jaspine B
Pachastrissamine
Protein kinase C
Sphingosine kinase
Sphingolipid
trissamine stereoisomers. The improved activity over N,N-dimethylsphingosine suggests that the cyclic
scaffold in pachastrissamines facilitates potential favorable interactions with SphKs and PKCs.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
revealed that a selective inhibitor of SphK2 attenuates experimen-
tal osteoarthritis.12 Accordingly, SphKs could be promising drug
Sphingolipid metabolites such as ceramide (Cer), sphingosine
(Sph), and sphingosine-1-phosphate (S1P) play important parts in
diverse biological processes.1 It has been reported that Cer and
Sph promote apoptosis and inhibit proliferation, whereas S1P
mediates cell proliferation and angiogenesis.2,3 The binding of
S1P to a family of five specific G protein-coupled receptors termed
S1PR1–5 controls various biological functions. S1P can also regulate
intracellular processes, but molecular targets are not fully defined.4
Sphingosine kinases (SphKs), which catalyze the phosphorylation
of Sph to form S1P are important lipid kinases.5 S1P levels are
mainly regulated by SphKs, S1P lyase, and S1P phosphatases
(Fig. 1). Hitherto, two distinct isoforms of SphKs, SphK1, and SphK2,
have been reported.6,7 SphK1 is overexpressed in various human
tumors,8 thereby impairing the efficacy of chemotherapy.9 SphK1
inhibition by siRNA-mediated knockdown or pharmacologic inhi-
bition induces apoptosis with elevation of the Cer/S1P ratio,9 and
increases the effectiveness of docetaxel in prostate cancer cell
lines.10
targets for cancer chemotherapy and inflammatory diseases. So
far, number of sphingosine kinase inhibitors have been
reported.13
Pachastrissamine, a naturally occurring anhydrophytosphingo-
sine derivative, was isolated from the Okinawan marine sponge,
a
OH
OH
dihydroceramide
desaturase
HO
C13H27
HO
C13H27
HN
O
HN
O
R
R
dihydroceramide
ceramide (Cer)
ceramidase
ceramide
synthase
OH
HO
NH2
sphingosine
sphingosine
SphK2 is defferent in its amino terminus and central region
from SphK1. These two enzymes have different kinetic properties
and tissue expression, implying that they may have distinct phys-
iological roles. Indeed, in contrast to pro-survival SphK1, the SphK2
induces apoptosis by cytochrome c release.11 Recent investigations
kinase (SphK)
sphingosine-1-
phosphate
O
HO
P
phosphatase
O
P
OH
NH2
HO
HO
HO
O
sphingosine-1-
O
C13H27
phosphate lyase
phosphoethanolamine
NH2
OHC
C13H27
sphingosine-1-phosphate (S1P)
hexadecenal
⇑
Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-
u.ac.jp (N. Fujii).
Figure 1. Metabolism of sphingolipids in mammalian cells.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.061

Y. Yoshimitsu et al. / Bioorg. Med. Chem. 19 (2011) 5402–5408
5403
4S-isomers synthesized from
L
-serine
against isolated sphingolipid metabolizing enzymes has yet to be
investigated. In the present study, we investigated the inhibitory
activities of eight pachastrissamine stereoisomers 1–8 against
SphKs in vitro (Fig. 2).
H2N
OH
2
4
3
O
2. Results and discussion
pachastrissamine (jaspine B, 1)
H2N
OH OH
H2N
H2N
OH
2.1. Synthesis of pachastrissamine stereoisomers
C14H29
C14H29
C14H29
Previously, we developed a stereoselective synthesis of four
pachastrissamine diastereomers 1–4 with the 4S-configuration.18
This synthetic route divergently provides four pachastrissamine
diastereomers from S-Garner’s aldehyde as a sole chiral pool. We
prepared the other enantiomeric stereoisomers 5–8 using this suc-
cessful approach from R-Garner’s aldehyde (Scheme 1). Briefly, R-
Garner’s aldehyde was converted into 11 by treatment with a
phosphonium ylide followed by dihydroxylation with OsO4.19 The
diol 11 was converted into the corresponding bis-tosylate 12 with
TsCl, Et3N and Me3NꢀHCl. Treatment of 12 with TsOH successfully
constructed the expected tetrahydrofuran ring. Cleavage of the to-
syl group with Mg in MeOH gave the (2R,3R,4R)-isomer 5.
O
O
O
(2R,3S,4S)-isomer (2) (2S,3R,4S)-isomer (3) (2R,3R,4S)-isomer (4)
4R-isomers synthesized from
D
-serine
H2N H2N
OH
OH
C14H29
(2R,3R,4R)-isomer (5) (2S,3R,4R)-isomer (6)
OH OH
H2N H2N
C14H29
O
O
C14H29
C14H29
O
O
Regioselective tosylation of the primary hydroxy group of the D-
(2R,3S,4R)-isomer (7) (2S,3S,4R)-isomer (8)
ribo-phytosphingosine derivative 14 prompted spontaneous cycli-
zation to give the tetrahydrofuran, and removal of the Boc group
with TFA provided the desired product, 6.20
Figure 2. Structures of pachastrissamine stereoisomers.
The primary hydroxy group of 14 was protected with a TIPS
group. Subsequent conversion of the resulting silyl ether to oxazo-
lidinone using MeC(OMe)3 in the presence of a catalytic amount of
BF3ꢀOEt2 afforded 17. Protection of the carbamate nitrogen of 17
with Boc2O and alcoholysis of the oxazolidinone successfully pro-
vided 18. Similar to the synthesis of 5, bis-tosylation, desilylation
and TBAF-promoted tetrahydrofuran formation afforded the de-
sired core scaffold that was successively deprotected with Mg
and TFA to furnish 7.
The silyl ether of 18 was cleaved with TBAF in THF to give the
triol 21. Selective monotosylation of the primary hydroxy group
followed by base treatment afforded tetrahydrofuran 22. The Boc
group was removed with TFA to give 8.
Pachastrissa sp. (Fig. 2).14 Pachastrissamine exhibits marked sub-
micromolar cytotoxicity against several cancer cell lines. Delgado
and co-workers reported the cytotoxicity of pachastrissamine dia-
stereomers with a series of C-2 and C-3 stereochemistries on the
tetrahydrofuran core, which were prepared via Sharpless asym-
metric dihydroxylation.15 Rao and co-workers also revealed that
pachastrissamine enantiomer is less cytotoxic than pachastriss-
amine.16 There has also been significant interest in the molecular
mechanisms of cell death induced by pachastrissamine. Salma et
al. revealed that pachastrissamine inhibits sphingomyelin synthase
to increase the intracellular level of Cer, inducing apoptotic cell
death by a caspase-dependent pathway.17 However, its activity
OR
H2N
OTs
C14H29
CHO
Boc
OR
C
15H31PPh3Br
OsO4
NMO
O
LHMDS
TsOH
Mg
O
O
N
N
C14H29
5
N
C14H29
Boc
O
Boc
TsCl, Et3N
Me3N·HCl
11 R = H
12 R =Ts
9
10
13
OH
BocHN
OH
TsCl
Et3N, DMAP
C14H29
TsOH
TFA
HO
BocHN
11
14
6
C14H29
O
15
OH
14
TIPSO
MeC(OMe)3
1) Boc2O
C14H29
OAc
OR
BocHN
TBAF
OTs
C14H29
OH
Et3N
TIPSCl
imidazole
1) Mg
2) TFA
C14H29
C14H29
DMAP
BF3·OEt2
TIPSO
TIPSO
HN
O
7
O
O
BocHN
OR
BocHN
OH
OH
2) NaOMe
TsCl, Et3N
Me3N·HCl
18 R = H
19 R = Ts
16
17
20
RHN
OH
1) TsCl, Et3N
Me3N·HCl
C14H29
TBAF
HO
BocHN
C14H29
O
18
OH
2) K2CO3
22 R = Boc
8 R = H
TFA
21
Scheme 1. Stereoselective synthesis of pachastrissamine stereoisomers 5–8.

5404
Y. Yoshimitsu et al. / Bioorg. Med. Chem. 19 (2011) 5402–5408
2.2. Sphingosine kinase inhibition by pachastrissamine
stereoisomers
SphK inhibitors 7 and 8 were profiled against each PKC isoform
(Fig. 3). Although numerous studies on Sph itself and DMS were re-
vealed,28,29 little is known about its isoform selectivity. The indi-
vidual PKC isoform is involved in different cellular process,30 so
the importance of the selectivity profiles of inhibitors is empha-
sized. For example, PKCf has been implicated in epidermal growth
To evaluate the SphK inhibitory activity of pachastrissamines,
in vitro SphK inhibition assays were undertaken for SphK1 and
SphK2 based on the partitioning of Sph and S1P using the Lab-
Chip3000 system.21 N,N-dimethylsphingosine (DMS) was em-
ployed as a reference SphK inhibitor (Table 1).22 Interestingly, all
of the pachastrissamine stereoisomers exhibited an inhibitory ef-
fect on both SphKs. Among the compounds tested, (2R,3S,4R)-
isomer 7 and (2S,3S,4R)-isomer 8 exhibited most potent inhibitory
activity upon SphK1 and SphK2, respectively, which are more po-
tent than DMS. These results indicate that a primary hydroxy
group in the substrate Sph, which is phosphorylated by SphKs,
may not be essential for enzyme recognition. The stereochemistry
of the accessory amino and hydroxyl groups on the tetrahydrofu-
ran core has significant influence on SphK inhibitory potency. This
apparently ambiguous recognition of stereochemistry resembles
factor (EGF)-stimulated chemotaxis of cancer cells, whereas PKCi is
an oncogenic protein required for the transformed growth and
tumorigenesis of human cancer cells.31 Pachastrissamines 1, 7,
and 8 exerted complete inhibition of PKCf and PKCi at 10 lM,
whereas modest inhibition against the other PKCs was observed
(see Supplementary data). The inhibitory effects of pachastriss-
amine isomers at 3
l
M against PKCf and PKC
i
were ꢁ50%, which
were more potent than the reference DMS (Fig. 3). To our best
knowledge, this is the first report on the Sph-based PKC inhibitors
with selectivity for atypical PKC isozymes in vitro.
The catalytic domain (motifs required for ATP-substrate binding
and catalysis) of PKC is highly conserved among three subfamilies:
the potent SphK inhibitory effect by
D- and
L-threo-sphingo-
conventional PKCs (PKC
a, b1, b2, c), novel PKCs (PKCd, e, h, g), and
sines.23–25 Inhibitory activity of 7 and 8 may be due to sphingo-
sine-like conformation of hydroxy and amino groups. The
hydrophobic tetradecyl moiety probably significantly contributes
to binding to SphK by hydrophobic interactions, corresponding to
known inhibitors with sphingosine-based structures.23–26 Whereas
the cis-olefin-containing hydrocarbon is indispensable for SphK
inhibition by B-5354c,27 the saturated aliphatic chain of pachas-
trissamines subserves effective SphK suppression.
atypical PKCs (PKCf, ), whereas the structural features of the reg-
i
ulatory domain vary.32 Conventional PKCs and novel PKCs have a
C1 domain that binds diacylglycerol (DAG) for kinase activation.
In the in vitro assay system for the present study, conventional
PKCs/novel PKCs were activated by the addition of DAG. The addi-
tion of DAG for conventional PKCs/novel PKCs may diminish the
potential inhibitory effects by pachastrissamines.26,33 In contrast,
atypical PKCs, including distinct nucleotide binding loop and ATP
binding pocket, bind phosphatidylinositol 3,4,5-trisphosphate
(PIP3) and Cer (not DAG) in the atypical C1 domain, whereas they
lack a DAG binding site in the regulatory domain. Considering that
sphingolipid Cer binds to the atypical C1 domain,34 PKC-isoform
selectivity of pachastrissamines could be derived from the binding
to the site in the domain.35
2.3. Protein kinase C (PKC) inhibition by pachastrissamine
stereoisomers
DMS was also reported to work as a protein kinase C (PKC)
inhibitor. Hence, the bioactivity of pachastrissamine 1, and potent
Table 1
3. Conclusions
Inhibitory activity of pachastrissamine and its stereoisomers against sphingosine
kinases
In conclusion, we identified pachastrissamine (jaspine B) as a
novel SphK1 and SphK2 inhibitor. Several non-natural stereo-
isomers are more potent SphK inhibitors than the naturally
occurring pachastrissamine. The distinct chiral tetrahydrofuran
scaffold with accessory groups could facilitate favorable inter-
actions with both enzymes. In addition, the inhibitory activities
a
Compound
IC50
SphK1
SphK2
1 (Pachastrissamine)
2
3
4
4.6
3.9
2.1
3.0
2.7
2.1
0.59
0.94
2.8
6.6
15.8
6.1
2.2
10.5
6.2
1.8
0.48
13.7
of these SphK inhibitors against atypical PKCs (PKCf, PKCi) were
5
6
7
8
revealed, suggesting that SphKs and PKCs represent alternative
molecular targets of pachastrissamines to induce apoptotic pro-
cess. As such, these pachastrissamine stereoisomers obtained by
our unique divergent synthesis could be promising leads to
design sphingolipid-based kinase inhibitors. Further optimization
of these inhibitors (especially for the alkyl side-chains) and inves-
tigations to elucidate the biological impact of these inhibitors are
underway.
DMSb
a
IC50 values are the concentrations for 50% inhibition of the sphingosine phos-
phorylation by SphK1 or SphK2. The data were derived from the dose–response
curves generated from triplicate data points.
b
N,N-Dimethylsphingosine.
Figure 3. Inhibitory activity of pachastrissamine stereoisomers against PKCf (A) and PKC
i
(B). Values are reported as percentages of maximum activity of PKCs from duplicate
data points.

Y. Yoshimitsu et al. / Bioorg. Med. Chem. 19 (2011) 5402–5408
5405
for 1 d at this temperature, the mixture was quenched by addition
of saturated NH4Cl at 0 °C and the whole was extracted with CH2Cl2.
The extract was washed with saturated NH4Cl and brine, and dried
over MgSO4. The filtrate was concentrated under reduced pressure
to give an oily residue, which was purified by flash chromatography
over silica gel with n-hexane–EtOAc (10:1 to 7:1) to give 12 as a col-
4. Experimental
4.1. Synthesis
4.1.1. General methods
1H NMR spectra were recorded using a JEOL AL-400 or a JEOL
ECA-500 spectrometer at 400 or 500 MHz frequency, and chemical
shifts are reported in d (ppm) relative to TMS (in CDCl3) as internal
standard. 1H NMR spectra are tabulated as follows: chemical shift,
multiplicity (b = broad, s = singlet, d = doublet, t = triplet, q = quar-
tet, br s = broad singlet, m = multiplet), number of protons, and
coupling constant(s). All 1H NMR spectra were in agreement with
those of the enantiomers of our previous report.18 Optical rotations
were measured with a JASCO P-1020 polarimeter. Exact mass
(HRMS) spectra were recorded on a JMS-HX/HX 110A mass spec-
trometer. The purity of the compounds was determined by 1H
NMR and elemental analysis (>95%).
orless oil (285 mg, 88% yield); ½a D25
ꢃ
+23.9 (c 0.68, CHCl3); 1H NMR
(500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.08–1.33 (m, 26H),
1.43–1.54 (m, 15H), 2.41 (s, 3H), 2.43 (s, 3H), 3.84 (dd, J = 9.2,
6.9 Hz, 1H), 3.92 (dd, J = 9.2, 2.9 Hz, 1H), 4.05 (m, 1H), 4.63 (m,
1H), 5.21 (m, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H),
7.73 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H); HRMS (FAB) calcd
for C40H64NO9S2 (MH+) 766.4017, found 766.4015.
4.1.5. (2R,3R,4R)-4-Amino-2-tetradecyltetrahydrofuran-3-yl 4-
methylbenzenesulfonate (13)
To a stirred solution of 12 (262 mg, 0.342 mmol) in MeOH
(11 mL) was added TsOHꢀH2O (65 mg, 0.342 mmol) at 70 °C. After
stirring for 8 h at this temperature, the mixture was concentrated
under reduced pressure to give an oily residue, which was purified
by flash chromatography over silica gel with CHCl3–MeOH–28%
NH4OH (95:4:1) to give 13 as a white solid (140 mg, 90% yield);
4.1.2. tert-Butyl (S,Z)-4-(hexadec-1-en-1-yl)-2,2-
dimethyloxazolidine-3-carboxylate (10)
To a stirred solution of C15H31PPh3Br (53.3 g, 96.3 mmol) in THF
(350 mL) was added LHMDS (1.0 M in THF; 88.0 mL, 88.0 mmol) at
0 °C, and the mixture was stirred for 0.5 h at room temperature. To
the resulting dark red solution was added dropwise Garner’s alde-
hyde 9 (9.60 g, 41.9 mmol) in THF (70.0 mL) at ꢂ78 °C. The mixture
was stirred for 30 min at this temperature and then 8 h at room
temperature. The mixture was quenched by addition of saturated
NH4Cl at 0 °C, and concentrated under reduced pressure. The resi-
due was extracted with Et2O. The extract was washed with brine,
and dried over MgSO4. The filtrate was concentrated under reduced
pressure followed by rapid filtration through a short pad of silica
gel with n-hexane–EtOAc (10:1) to give a crude mixture. Further
purification by flash chromatography over silica gel with n-hex-
ane–EtOAc (100:1 to 80:1) gave 10 (15.4 g, 87%) as a colorless
mp 65–66 °C; ½a 2D5
ꢃ
ꢂ18.6 (c 0.28, CHCl3); 1H NMR (500 MHz, CDCl3)
d 0.88 (t, J = 6.9 Hz, 3H), 1.02–1.32 (m, 24H), 1.39–1.63 (m, 4H), 2.46
(s, 3H), 3.43 (dd, J = 8.6, 8.6 Hz, 1H), 3.72 (m, 1H), 3.88 (ddd, J = 4.6,
4.6, 4.0 Hz, 1H), 3.99 (dd, J = 8.6, 8.6 Hz, 1H), 4.85 (dd, J = 4.6,
4.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H); HRMS
(FAB) calcd for C25H44NO4S (MH+) 454.2986, found 454.2993.
4.1.6. (2R,3R,4R)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (5)
To a stirred mixture of 13 (23 mg, 0.0551 mmol) in MeOH
(1.1 mL) was added Mg (13 mg, 0.551 mmol) at room temperature.
After stirring for 1.5 h at this temperature, silica gel was added to
the solution and the mixture was concentrated under reduced pres-
sure. The residue was purified by flash chromatography over silica
gel withCHCl3–MeOH–28%NH4OH (95:4:1)to give 5 as a white solid
oil; ½a 2D5
ꢃ
ꢂ59.4 (c 0.43, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88
(t, J = 6.9 Hz, 3H), 1.22–1.33 (m, 24H), 1.36–1.62 (m, 15H), 1.98–
2.18 (m, 2H), 3.64 (dd, J = 8.6, 4.0 Hz, 1H), 4.05 (dd, J = 8.6, 6.3 Hz,
1H), 4.66 (m, 1H), 5.35–5.54 (m, 2H); HRMS (FAB) calcd for
(13 mg, 79% yield); mp 95–96 °C; ½a D25
ꢃ
ꢂ9.61 (c 0.61, CHCl3); 1H NMR
(500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.20–1.49 (m, 24H), 1.49–
1.59 (br s, 2H), 1.60–1.70 (m, 2H), 3.52 (dd, J = 8.5, 7.1 Hz, 1H), 3.66
(m, 1H), 3.73 (ddd, J = 7.1, 7.1, 3.4 Hz, 1H), 3.87 (dd, J = 4.6, 3.4 Hz,
1H), 3.92 (dd, J = 8.5, 7.3 Hz, 1H). Anal. Calcd for C18H37NO2: C,
72.19; H, 12.45; N, 4.68. Found: C, 72.10; H, 12.63; N, 4.65.
C
26H50NO3 (MH+) 424.3785, found 424.3784.
4.1.3. tert-Butyl (R)-4-[(1R,2S)-1,2-dihydroxyhexadecyl]-2,2-
dimethyloxazolidine-3-carboxylate (11)
To the stirred solution of 10 (6.7 g, 15.8 mmol) and N-methyl-
morpholine N-oxide (2.8 g, 23.7 mmol) in t-BuOH (40 mL) and water
(40 mL) was added OsO4 (2.5 w/v% in t-BuOH, 8.0 mL, 0.791 mmol)
at 0 °C, and the mixture was stirred for 9.0 h at room temperature.
The mixture was quenched by addition of saturated Na2S2O3 at
0 °C, and concentrated under reduced pressure. The residue was ex-
tracted with Et2O and washed with brine, and dried over MgSO4. The
filtrate was concentrated under reduced pressure to give an oily res-
idue, which was purified by flash chromatography over silica gel
with n-hexane–EtOAc (5:1) to give 11 (4.5 g, 62% yield) and its C3/
C4-stereoisomer (1.4 g, 19% yield) as white solids. Recrystallization
from n-hexane–EtOAc gave pure 11 as colorless crystals; mp 55–
4.1.7. tert-Butyl [(2R,3R,4S)-1,3,4-trihydroxyoctadecan-2-
yl)]carbamate (14)
To a stirred solution of 11 (2.42 g, 5.29 mmol) in MeOH
(176 mL) was added TsOHꢀH2O (101 mg, 0.529 mmol) at 0 °C, and
the mixture was stirred for 10 h at room temperature. The mixture
was quenched by addition of Et3N at 0 °C and concentrated under
reduced pressure, followed by flash chromatography over silica gel
with n-hexane–EtOAc (1:1) to give 14 as a white solid (1.93 g, 87%
yield); mp 85–86 °C; ½a D25
ꢃ
ꢂ6.61 (c 1.61, CHCl3); 1H NMR (500 MHz,
CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.21–1.39 (m, 24H), 1.45 (s, 9H),
1.47–1.55 (m, 2H), 2.42 (d, J = 4.0 Hz, 1H), 2.98 (m, 1H), 3.29 (d,
J = 6.9 Hz, 1H), 3.62 (m, 1H), 3.68 (m, 1H), 3.77 (m, 1H), 3.85 (m,
1H), 3.92 (m, 1H), 5.30 (m, 1H); HRMS (FAB) calcd for C23H48NO5
(MH+) 418.3527, found 418.3533.
56 °C; ½a 2D5
ꢃ
+8.87 (c 1.02, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88
(t, J = 6.9 Hz, 3H), 1.19–1.33 (m, 24H), 1.47–1.52 (m, 15H), 1.58–
1.63 (m, 2H), 3.14–3.32 (m, 2H), 3.53–3.67 (m, 2H), 4.00 (m, 1H),
4.11–4.25 (m, 2H); Anal. Calcd for C26H51NO5: C, 68.23; H, 11.23;
N, 3.06. Found: C, 67.95; H, 11.38; N, 3.08.
4.1.8. tert-Butyl [(3R,4R,5S)-4-hydroxy-5-
tetradecyltetrahydrofuran-3-yl]carbamate (15)
To a stirred mixture of 14 (90 mg, 0.216 mmol) and Et3N
4.1.4. tert-Butyl (R)-4-[(1R,2S)-1,2-bis(tosyloxy)hexadecyl]-2,2-
dimethyloxazolidine-3-carboxylate (12)
(108 lL, 0.778 mmol) in CH2Cl2 (7.2 mL) were added TsCl (74 mg,
0.389 mmol) and DMAP (2.4 mg, 0.0214 mmol) at 0 °C, and the
mixture was stirred for 18 h at room temperature. The mixture
was quenched by addition of saturated NH4Cl at 0 °C, and the
whole was extracted with CH2Cl2. The extract was washed with
To a stirred solution of 11 (194 mg, 0.424 mmol) in CH2Cl2 (850
were added Et3N (588 L, 4.24 mmol), TsCl (404 mg, 2.12 mmol) and
Me3NꢀHCl (41 mg, 0.424 mmol) at room temperature. After stirring
lL)
l

5406
Y. Yoshimitsu et al. / Bioorg. Med. Chem. 19 (2011) 5402–5408
brine, and dried over MgSO4. The filtrate was concentrated under
reduced pressure to give an oily residue, which was purified by
flash chromatography over silica gel with n-hexane–EtOAc (5:1
to 3:1) followed by recrystallization from n-hexane–EtOAc to give
addition of saturated NH4Cl at 0 °C. The mixture was concentrated
under reduced pressure, and the residue was extracted with Et2O.
The extract was washed with brine, and dried over MgSO4. The fil-
trate was concentrated under reduced pressure to give an oily res-
idue, which was dissolved in MeOH (7.0 mL). NaOMe (1.13 g,
21.0 mmol) was added to this solution under stirring at 0 °C, and
the mixture was stirred for 10 min at room temperature. The mix-
ture was quenched by addition of saturated NH4Cl, and concen-
trated under reduced pressure. The residue was extracted with
EtOAc, washed with brine, and dried over MgSO4. The filtrate
was concentrated under reduced pressure to give an oily residue,
which was purified by flash chromatography over silica gel with
n-hexane–EtOAc (4:1) to give 18 as a colorless oil (562 mg, 70%
15 (76 mg, 88% yield) as colorless crystals; mp 80–81 °C; ½a D25
ꢃ
ꢂ7.76 (c 0.29, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t,
J = 6.9 Hz, 3H), 1.22–1.34 (m, 24H), 1.46 (s, 9H), 1.52–1.57 (m,
2H), 2.10 (m, 1H), 3.51 (m, 1H), 3.71 (m, 1H), 3.94 (m, 1H), 4.11–
4.17 (m, 2H), 4.95 (m, 1H); HRMS (FAB) calcd for C23H46NO4
(MH+) 400.3421, found 400.3413.
4.1.9. (2S,3R,4R)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (6)
To a stirred solution of 15 (45 mg, 0.113 mmol) in CH2Cl2
(800
l
L) was added TFA (800
l
L) at 0 °C, and the mixture was stir-
yield); ½a 2D5
ꢃ
ꢂ20.6 (c 3.21, CHCl3); 1H NMR (500 MHz, CDCl3) d
red for 30 min at room temperature. The mixture was concentrated
under reduced pressure to give an oily residue, which was purified
by flash chromatography over silica gel with CHCl3–MeOH–28%
NH4OH (95:4:1) followed by recrystallization from n-hexane–
EtOAc to give 6 as colorless crystals (31 mg, 92% yield); mp 104–
0.88 (t, J = 6.9 Hz, 3H), 1.07 (d, J = 5.7 Hz, 18H), 1.08–1.14 (m,
3H), 1.14–1.42 (m, 24H), 1.44 (s, 9H), 1.45–1.61 (m, 2H), 2.55 (m,
1H), 3.46 (m, 1H), 3.60 (m, 1H), 3.66–3.79 (m, 2H), 3.79–3.99 (m,
2H), 5.15 (d, J = 8.0 Hz, 1H); HRMS (FAB) calcd for C32H68NO5Si
(MH+) 574.4861, found 574.4858.
105 °C; ½a 2D5
ꢃ
ꢂ8.78 (c 0.75, CHCl3); 1H NMR (500 MHz, CDCl3) d
0.88 (t, J = 6.9 Hz, 3H), 1.19–1.34 (m, 24H), 1.34–2.37 (m, 5H),
3.40 (dd, J = 8.6, 6.9 Hz, 1H), 3.46 (m, 1H), 3.57–3.66 (m, 2H),
4.13 (dd, J = 8.6, 6.3 Hz, 1H). Anal. Calcd for C18H37NO2 ꢀ 0.25H2O:
C, 71.12; H, 12.43; N, 4.61. Found: C, 71.39; H, 12.39; N, 4.59.
4.1.13. (2R,3S,4S)-2-[(tert-Butoxycarbonyl)amino]-1-
(triisopropylsilyloxy)octadecane-3,4-diyl bis(4-
methylbenzenesulfonate) (19)
To a stirred solution of 18 (255 mg, 0.444 mmol) in CH2Cl2
(1.0 mL) were added Et3N (613 lL, 4.44 mmol), TsCl (423 mg,
4.1.10. tert-Butyl [(2R,3R,4S)-3,4-dihydroxy-1-
(triisopropylsilyloxy)octadecan-2-yl]carbamate (16)
2.22 mmol), and Me3NꢀHCl (42 mg, 0.444 mmol) at room temper-
ature, and the mixture was stirred 4 h at room temperature. The
mixture was quenched by addition of saturated NH4Cl at 0 °C,
and the whole was extracted with CH2Cl2, dried over MgSO4.
The filtrate was concentrated under reduced pressure to give
an oily residue, which was purified by flash chromatography
over silica gel with n-hexane–EtOAc (10:1) to give 19 as a color-
To a stirred solution of 14 (1.74 g, 4.17 mmol) in DMF (42 mL)
were added imidazole (1.14 g, 16.7 mmol) and TIPSCl (3.53 mL,
16.7 mmol) at 0 °C, and the mixture was stirred for 1 h at room
temperature. The mixture was quenched by addition of MeOH at
0 °C, and concentrated under reduced pressure. The residue was di-
luted with CH2Cl2, washed with saturated NH4Cl and brine, and
dried over MgSO4. The filtrate was concentrated under reduced
pressure to give an oily residue, which was purified by flash chro-
matography over silica gel with n-hexane–EtOAc (8:1) to give 16 as
less oil (373 mg, 95% yield); ½a D25
ꢃ
ꢂ18.8 (c 0.95, CHCl3); 1H NMR
(400 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 0.91–1.11 (m, 26H),
1.11–1.36 (m, 18H), 1.41 (s, 9H), 1.54 (m, 3H), 2.44 (s, 3H),
2.45 (s, 3H), 3.46 (dd, J = 10.3, 6.3 Hz, 1H), 3.56 (dd, J = 10.3,
4.6 Hz, 1H), 4.01 (m, 1H), 4.65 (m, 1H), 4.79 (d, J = 9.7 Hz, 1H),
5.03 (dd, J = 4.6, 3.4 Hz, 1H), 7.31 (d, J = 8.3 Hz, 2H), 7.34 (d,
J = 8.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3 Hz, 2H);
HRMS (FAB) calcd for C46H79NNaO9S2Si (MNa+) 904.4863, found
904.4863.
a colorless oil (2.15 g, 90% yield); ½a D25
ꢃ
ꢂ23.5 (c 0.25, CHCl3); 1H
NMR (500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.04–1.09 (m,
18H), 1.10–1.18 (m, 2H), 1.18–1.38 (m, 24H), 1.44 (s, 9H), 1.47–
1.75 (m, 3H), 2.55 (d, J = 5.7 Hz, 1H), 3.24 (m, 1H), 3.58 (m, 1H),
3.63 (m, 1H), 3.86 (m, 1H), 3.89 (m, 1H), 4.05 (m, 1H), 5.24 (d,
J = 8.0 Hz, 1H); HRMS (FAB) calcd for
C
32H68NO5Si (MH+)
574.4861, found 574.4855.
4.1.14. (2R,3S,4R)-4-[(tert-Butoxycarbonyl)amino]-2-
tetradecyltetrahydrofuran-3-yl 4-methylbenzenesulfonate (20)
To a stirred solution of 19 (172 mg, 0.195 mmol) in THF (3.9 mL)
was added TBAF (1.0 M in THF; 390 lL, 0.390 mmol) at 0 °C, and
4.1.11. (S)-1-{(4R,5S)-2-Oxo-4-[(triisopropylsilyloxy)
methyl]oxazolidin-5-yl}pentadecyl acetate (17)
To a stirred solution of 16 (1.56 g, 2.72 mmol) in CH2Cl2 (270 mL)
the mixture was stirred for 2 h at room temperature. The mixture
was quenched by addition of saturated NH4Cl at 0 °C, and concen-
trated under reduced pressure. The residue was extracted with
Et2O, and dried over MgSO4. The filtrate was concentrated under
reduced pressure to give an oily residue, which was purified by
flash chromatography over silica gel with n-hexane–EtOAc (7:1)
were added MeC(OMe)3 (2.0 mL, 16.3 mmol) and BF3ꢀOEt2 (67
lL,
0.544 mmol) at 0 °C, and the mixture was stirred for 1.5 h at room
temperature. The mixture was quenched by addition of MeOH at
0 °C, and concentrated under reduced pressure to give an oily resi-
due, which was purified by flash chromatography over silica gel with
n-hexane–EtOAc (4:1) to give 17 as a colorless oil (1.28 g, 87% yield);
to give 20 as a colorless oil (71 mg, 65% yield); ½a D25
ꢃ
+1.48 (c
½
a 2D5
ꢃ
+25.5 (c 0.66, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t,
0.056, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H),
1.10–1.36 (m, 26 H), 1.43 (s, 9H), 2.45 (s, 3H), 3.73 (dd, J = 9.7,
2.9 Hz, 1H), 3.78 (dd, J = 9.7, 5.2 Hz, 1H), 3.93 (m, 1H), 4.07 (m,
1H), 4.43 (m, 1H), 4.74 (m, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.83 (d,
J = 6.9 Hz, 3H), 1.03–1.16 (d, J = 5.7 Hz, 18H), 1.08–1.14 (m, 3H),
1.22–1.37 (m, 24H), 1.63–1.75 (m, 2H), 2.10 (s, 3H), 3.61–3.67 (m,
1H), 3.67–3.73 (m, 2H), 4.41 (dd, J = 4.6, 3.4 Hz, 1H), 5.00 (ddd,
J = 6.9, 6.9, 3.4 Hz, 1H), 5.28 (m, 1H); HRMS (FAB) calcd for
J = 8.0 Hz, 2H); HRMS (FAB) calcd for
552.3364, found 552.3370.
C
30H50NO6S ([MꢂH]ꢂ)
C
30H60NO5Si (MH+) 542.4235, found 542.4240.
4.1.12. tert-Butyl [(2R,3S,4S)-3,4-dihydroxy-1-
(triisopropylsilyloxy)octadecan-2-yl]carbamate (18)
To a stirred solution of 17 (760 mg, 1.40 mmol) in THF (14 mL)
were added Et3N (194 lL, 1.40 mmol), Boc2O (428 mg, 1.96 mmol),
and DMAP (343 mg, 2.81 mmol) at 0 °C, and the mixture was
4.1.15. (2R,3S,4R)-4-Amino-2-tetradecyltetrahydrofuran-3-ol
(7)
To a stirred solution of 20 (48 mg, 0.087 mmol) in MeOH
(2.9 mL) was added Mg (21 mg, 0.87 mmol) at room temperature,
and the mixture was stirred for 45 min at this temperature. The
mixture was concentrated under reduced pressure, and the residue
stirred 2 h at room temperature. The mixture was quenched by

Y. Yoshimitsu et al. / Bioorg. Med. Chem. 19 (2011) 5402–5408
5407
was diluted with EtOAc, washed with H2O, and dried over MgSO4.
4.2. Sphingosine kinase assay
The filtrate was concentrated under reduced pressure to give a
white solid, which was dissolved in CH2Cl2 (1.5 mL). TFA (1.5 mL)
was added to the mixture at 0 °C. After stirring for 15 min at room
temperature, the mixture was concentrated under reduced pres-
sure to give an oily residue, which was purified by flash chroma-
tography over silica gel with CHCl3–MeOH–28% NH4OH (95:4:1)
SphK inhibitory activities were evaluated by the off-chip mobil-
ity shift assay by the QuickScoutÒ 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 chroma-
tography. 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
to give 7 as a white solid (23 mg, 88% yield); ½a D25
ꢃ
+1.48 (c 0.056,
CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.20–
1.42 (m, 24H), 1.42–1.67 (m, 2H), 2.14–2.42 (m, 2H), 3.33 (m,
1H), 3.53–3.57 (m, 1H), 3.57–3.63 (m, 2H), 4.00 (dd, J = 9.2,
6.3 Hz, 1H). Anal. Calcd for C18H37NO2: C, 72.19; H, 12.45; N,
4.68. Found: C, 72.29; H, 12.16; N, 4.61.
of the compound, 1
600 M for SphK2) in reaction buffer (20
each SphK in 384-well plates at room temperature for 1 h (n = 4).
The reaction was terminated by addition of 60 L of termination
lM Sph, 5 mM MgCl2, ATP (25
lM for SphK1;
l
lL) were incubated with
l
4.1.16. tert-Butyl [(2R,3S,4S)-1,3,4-trihydroxyoctadecan-2-
yl]carbamate (21)
To a stirred solution of 18 (87 mg, 0.152 mmol) in THF (3.0 mL)
was added TBAF (1.0 M in THF; 304 lL, 0.304 mmol) at 0 °C, and
buffer (Carna Biosciences). Substrate and product were separated
by electrophoretic means using the LabChip3000 system. The ki-
nase reaction was evaluated by the product ratio, which was calcu-
lated 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).
the mixture was stirred for 20 min at room temperature. The mix-
ture was quenched by addition of saturated NH4Cl at 0 °C, and con-
centrated under reduced pressure. The residue was extracted with
EtOAc, dried over MgSO4. The filtrate was concentrated under re-
duced pressure to give an oily residue, which was purified by flash
chromatography over silica gel with n-hexane–EtOAc (1:1 to 1:2)
4.3. Protein kinase C (PKC) assay
to give 21 as a white solid (59 mg, 93% yield); mp 68–70 °C; ½a D25
ꢃ
PKC inhibitory activities were evaluated using the off-chip
mobility shift assay by the QuickScoutÒ service from Carna Biosci-
ence (Kobe, Japan). N-terminal GST-fusion protein were employed
ꢂ4.33 (c 1.14, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t,
J = 6.9 Hz, 3H), 1.20–1.39 (m, 26H), 1.45 (s, 9H), 2.29 (m, 1H),
2.65 (m, 1H), 2.99 (m, 1H), 3.61–3.68 (m, 2H), 3.69–3.77 (m, 2H),
3.89 (m, 1H), 5.27 (m, 1H); HRMS (FAB) calcd for C23H46NO5
([MꢂH]ꢂ) 416.3381, found 416.3391.
for the assays: PKC
697), PKCd(1-676), PKC
PKCh(1-706), and PKC (1-587). These were expressed using the
a
(1-672), PKCb1(1-671), PKCb2(1-673), PKC
c(1-
e
(1-737), PKCf(1-592), PKC (1-683),
g
i
baculovirus expression system and purified by glutathione sephar-
ose chromatography. Each chemical in DMSO at different concen-
trations was diluted fourfold with reaction buffer [20 mM HEPES
(pH 7.5), 0.01% Triton X-100, 2 mM DTT]. For the kinase reactions
4.1.17. tert-Butyl [(3R,4S,5S)-4-hydroxy-5-
tetradecyltetrahydrofuran-3-yl]carbamate (22)
To a stirred mixture of 21 (42 mg, 0.100 mmol) and Et3N
(111 lL, 0.800 mmol) in CH2Cl2 (3.3 mL) were added TsCl
except for PKCf and PKC
cyl glycerol (5 g/mL) were added. A combination of the com-
pound, 1 M PKCh substrate (N-FL), ATP (25 M for PKC /b1/
/h/ ; 10 M for PKCb2/d/ ; 5 M for PKCf), 5 mM MgCl2, and
50 M CaCl2 (for PKC /b1/b2/ ) in reaction buffer (20 L) were
incubated with each PKC in 384-well plates at room temperature
for 1 h (n = 2). The reaction was terminated by addition of 60
i, phosphatidylserine (50 lg/mL) and dia-
(76 mg, 0.400 mmol) and Me3NꢀHCl (10 mg, 0.100 mmol) at
ꢂ78 °C, and the mixture was stirred for 4 h at this temperature.
The mixture was quenched by addition of EtOH at 0 °C, and con-
centrated under reduced pressure. The residue was extracted
with Et2O, washed with saturated NH4Cl and brine, and dried
over MgSO4. The filtrate was concentrated under reduced pres-
sure to give an oily residue, which was dissolved in MeOH
(3.3 mL). K2CO3 (41 mg, 0.300 mmol) was added to the stirred
mixture at 0 °C, and the mixture was stirred for 30 min at room
temperature. The mixture was quenched by addition of saturated
NH4Cl, and concentrated under reduced pressure. The residue
was extracted with EtOAc, washed with brine, and dried over
MgSO4. The filtrate was concentrated under reduced pressure
to give an oily residue, which was purified by flash chromatog-
raphy over silica gel with n-hexane–EtOAc (5:1 to 3:1) to give
l
l
l
a
c/
g
i
l
e
l
l
a
c
l
lL
of termination buffer (Carna Biosciences). Substrate and product
were separated by electrophoretic means using the LabChip3000
system. The kinase reaction was evaluated by the product ratio,
which was calculated from the peak heights of the substrate (S)
and product (P) peptides: [P/(P + S)]. Inhibition data were calcu-
lated by comparing with no-enzyme controls for 100% inhibition
and no-inhibitor reactions for 0% inhibition.
Acknowledgments
22 as a white solid (21 mg, 53% yield); mp 105–107 °C; ½a D25
ꢃ
+26.3 (c 0.13, CHCl3); 1H NMR (500 MHz, CDCl3) d 0.88 (t,
J = 6.9 Hz, 3H), 1.24–1.35 (m, 24H), 1.45 (s, 9H), 1.59–1.66 (m,
2H), 2.27 (m, 1H), 3.44 (dd, J = 9.7, 4.0, 1H), 3.81 (m, 1H), 4.00
(m, 1H), 4.07 (m, 1H), 4.24 (m, 1H), 4.62 (m, 1H); HRMS (FAB)
calcd for C23H44NO5 ([MꢂH]ꢂ): 398.3276, found 398.3275.
This work was supported by Grants-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology of Japan, and Targeted Proteins Research Program.
Y.Y. and S.I. are grateful for Research Fellowships from the Japan
Society for the Promotion of Science (JSPS) for Young Scientists.
4.1.18. (2S,3S,4R)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (8)
By a procedure identical with that described for synthesis of 6
from 15, 22 (30 mg, 0.075 mmol) was converted into 8 as a white
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.061.
solid (22 mg, 98%); mp 80–82 °C; ½a D25
ꢃ
+2.33 (c 0.20, CHCl3); 1H
NMR (500 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H), 1.21–1.40 (m,
24H), 1.40–1.69 (m, 5H), 3.39 (dd, J = 9.7, 3.4 Hz, 1H), 3.47 (m,
1H), 3.81 (m, 1H), 3.90 (m, 1H), 4.21 (dd, J = 9.7, 6.3 Hz, 1H). Anal.
Calcd for C18H37NO2ꢀ0.25H2O: C, 71.12; H, 12.43; N, 4.61. Found: C,
71.19; H, 12.45; N, 4.40.
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