Synthesis of selective inhibitors of sphingosine kinase 1

Article abstract of DOI:10.1039/c3cc00181d

Sphingosine kinase isoform 1 (SK1) inhibitors may serve as therapeutic agents for proliferative diseases, including hypertension. We synthesized a series of sphingosine-based SK1-selective inhibitors, the most potent of which is RB-005 (IC₅₀ = 3.6 μM), which also induced proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells.

Full text of DOI:10.1039/c3cc00181d

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Synthesis of selective inhibitors of sphingosine
kinase 1†
Dong Jae Baek,a Neil MacRitchie,b Nigel J. Pyne,b Susan Pyneb and
Robert Bittman*a
Cite this: Chem. Commun., 2013,
49, 2136
Received 9th January 2013,
Accepted 24th January 2013
DOI: 10.1039/c3cc00181d
www.rsc.org/chemcomm
Sphingosine kinase isoform 1 (SK1) inhibitors may serve as therapeutic
agents for proliferative diseases, including hypertension. We synthesized
a series of sphingosine-based SK1-selective inhibitors, the most potent of
which is RB-005 (IC50 = 3.6 lM), which also induced proteasomal
degradation of SK1 in human pulmonary arterial smooth muscle cells.
S1P is formed from sphingosine by two isoforms of sphingosine
kinase (SK1 and SK2) that are encoded by distinct genes and differ in
their biochemical properties, subcellular localization, and function.1
Binding of S1P to 5 cell-surface G-protein coupled S1P receptors
(termed S1P1–5) induces a multitude of cell responses. S1P also has
intracellular targets.2–4 Targeting SK1 is also of particular relevance to
cancer.1 Many human tumor types exhibit increased levels of SK1
mRNA and/or protein which correlate, in several studies, with
increased tumor grade, reduced patient survival, and development
of chemotherapeutic resistance to anti-cancer agents. Indeed,
knockdown of SK1 reduces proliferation of glioblastoma cells5
and androgen-independent PC-3 prostate cancer cells.6
We have demonstrated that sustained hypoxia induces an
increase in the transcription of the SK1 gene in proliferating
human pulmonary arterial smooth muscle cells (PASMC) that
might account for their increased survival7 and thereby contribute
to vascular remodeling in pulmonary arterial hypertension (PAH).
Moreover, others have reported lower levels of ceramide in
vascular smooth muscle cells isolated from spontaneously hyper-
tensive rats,8 suggesting that hypertension is associated with
increased conversion of ceramide to sphingosine and S1P. Here,
we report the synthesis and evaluation of new sphingosine-based
SK1-selective inhibitors, which may have potential as inhibitors of
proliferative diseases, including PAH.
Fig. 1 Structures of SK inhibitors.
Several SK inhibitors have been previously identified. For example,
the non-sphingoid molecule 2-(p-hydroxyanilino)-4-(p-chlorophenyl)-
thiazole (SKi, also referred to as SKI-II; see Fig. 1) reduced intracellular
S1P, inhibited proliferation, and induced apoptosis in various cancer
cell lines.9 SKi is orally bioavailable, inhibits tumor growth10 and
ulceritive colitis,11 and reduces airway hyper-responsiveness.12
The sphingosine analogue (2R,3S,4E)-N-methyl-5-(40-pentylphenyl)-
2-aminopent-4-ene-1,3-diol (BML-258, also referred to as SK1-I;
Fig. 1) is a SK1-selective inhibitor.13
Inhibition of SK activity in cells with the non-selective isoform
inhibitors SKi, N,N-dimethylsphingosine, and FTY720 (Fig. 1) induces
proteasomal degradation and removal of SK1 from PASMC and
cancer cell lines.14–16 Removal of SK1 reduces intracellular S1P and
increases C22:0-ceramide levels, thereby promoting apoptosis.14
Scheme 1 outlines our synthetic route from 4-octylphenethyl
alcohol (1, see Scheme S1, ESI†) to a series of FTY720-like
analogues. The tertiary amines shown in Table 1 were prepared
in good yields by displacement of mesylate ion from 2 with
a Department of Chemistry and Biochemistry, Queens College of the City University
of New York, Flushing, New York 11367-1597, USA.
E-mail: robert.bittman@qc.cuny.edu; Tel: +1 718-997-3279
b Cell Biology Group, Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow G4 0RE, UK
Scheme 1 Synthesis of tertiary amine derivatives (RB-001–010, 019 and 020)
and quaternary ammonium salts (RB-011–018) of FTY720. (a) MsCl, Et3N, CH2Cl2;
(b) MeI, K2CO3, MeCN.
† Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedures, NMR and mass spectra, enzyme assay procedures, and hPASMC results.
See DOI: 10.1039/c3cc00181d
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Table 1 Synthesized compounds for evaluation as SK inhibitors
Compound
RB-001
R
Compound
RB-011
R
Scheme 2 Synthesis of phenylsulfone analogues of RB-021 and RB-022. (a)
PhSO2Na, THF, DMF; (b) 2-(4-bromophenyl)-ethanol, Pd(PPh3)4, CuI, Et3N; (c) 10%
Pd/C, H2, EtOAc; (d) MsCl, Et3N, CH2Cl2; (e) piperidine or 4-OH-piperidine, MeCN.
RB-002
RB-003
RB-004
RB-012
RB-013
RB-014
RB-005
RB-006
RB-007
RB-015
RB-016
RB-017
RB-008
RB-009
RB-010
RB-018
RB-019
RB-020
amines in acetonitrile. Some of the quaternary ammonium
salts were prepared by N-alkylation of the tertiary amines
(and the secondary amine RB-006) with an excess of MeI and
K2CO3 in acetonitrile (Scheme S2, ESI†), whereas others (RB-
011, RB-012, RB-017, and RB-018) were prepared by N-alkylation
of 2 with tertiary N-methylamines (see ESI†).
We have established SK1 and SK2 assays using sphingosine/
[32P]-ATP with over-expressed recombinant SK1 in HEK 293 cell
lysates or purified SK2.15,16 By using sphingosine concentra-
tions of 3 mM and 10 mM, which correspond to the Km values
Fig. 2 Effect of inhibitors on SK1 or SK2 activity. SK1 activity was measured using
3 mM sphingosine and 250 mM ATP. SK2 activity was assayed using 10 mM sphingosine
and 250 mM ATP (n = 3 for each compound, results expressed as mean inhibition ꢀ SD).
RB series compounds were used at 50 mM. BML-258 (50 mM) inhibited SK1 activity by
74.5 ꢀ 3.3% (n = 3). The control is 100% and equals activity against Sph alone.
of SK116 and SK2,17 respectively, we demonstrate here that hypothesis that RB-005 is a highly selective inhibitor of SK1. The
RB-001–003, RB-005, RB-007–009, RB-019 (Table 1), and RB-021 hydroxyl group in the heterocyclic ring is important for inhibition of
(Scheme 2) are selective inhibitors of SK1 over SK2.
SK1 by RB-005. Replacement of the 4-OH group with a 4-methyl group
The data in Fig. 2 show that the lead molecules for further affords RB-004, which is a moderate inhibitor of both SK1 and SK2;
structural optimization are 1-(4-octylphenethyl)piperidin-4-ol (RB- the 3-OH regioisomer RB-019 is a selective but less potent SK1
005) and 1-(4-octylphenethyl)piperidin-3-ol (RB-019) because these inhibitor. The size of the heterocycle is important as RB-003 is less
compounds have the highest selectivity for SK1 over SK2 (15.0- and selective than RB-005. The hydrophobic alkyl chain is also a key
6.1-fold, respectively). These compounds feature an n-octylphenyl feature as sulfonation18 in this region reduces inhibition of SK1; the
group linked in a 2-carbon tether to the nitrogen of 4-hydroxy- or sulfones RB-021 and RB-022 were prepared by Sonogashira reaction of
3-hydroxypiperidine, respectively. Small changes in the structure of alkyne 3 with 2-(4-bromophenyl)-ethanol, followed by catalytic hydro-
this tertiary amine result in large changes in selectivity, arguing for the genation and mesylation of 4, and finally reaction with piperidine or
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Table 2 RB-020 (50 mM) is a substrate of SK1 and SK2 and an inhibitor of SK1
and also possess a –(CH2)2OH and –(CH2)3OH group, respectively.
However, RB-008 and RB-009 are not substrates for SK1 or SK2 (data
not shown) but are inhibitors of SK1 (Fig. 2).
activity against sphingosine (Sph)a
SK1 activity (% control)
SK2 activity (% control)
RB-005 inhibited SK1 activity with an IC50 value of 3.6 ꢀ 0.38 mM
(Fig. 3A). Finally, to examine whether RB-005 may function as more
than a reversible inhibitor of SK1 activity, we assessed its ability to
promote the proteasomal degradation of SK1 in cells.
Importantly, we found that treatment of PASMC with RB-005
(10 mM, 24 h) reduced the expression of SK1. In common with
(S)-FTY720 vinylphosphonate,16 the effect of RB-005 on SK1
expression was reversed by pre-treatment of the cells with the
proteasomal inhibitor MG132 (Fig. 3B).
We conclude that the FTY720 scaffold can produce selective SK1
inhibitors with low mM potency and which also induce the protea-
somal degradation of SK1 in cells. Therefore, RB-005 represents a
molecule for optimization to produce effective therapeutic agents.
This work was supported by a British Heart Foundation
grant (29476) to NJP/SP and by NIH Grant HL-083187 to RB.
Sph
Sph + RB-020
RB-020
100 ꢀ 4.4
34.4 ꢀ 4.1
27.5 ꢀ 3.0
100 ꢀ 12.8
315 ꢀ 5.1
228 ꢀ 17.9
a
Sph was used at 3 and 10 mM for SK1 and SK2, respectively. Results are
represented as % activities ꢀ SD (n = 3 assays) of control (where 100%
equals activity against Sph alone).
its 4-hydroxy derivative (Scheme 2 and Scheme S3, ESI†). Conversion
of the heterocyclic amines RB-003–RB-006 by N-methylation to the
corresponding quaternary ammonium compounds, RB-013–RB-016,
reduces inhibition of both SK1 and SK2 (Fig. 2). Relocation of
the quaternary nitrogen functionality to an exocyclic position as in
RB-016–RB-018 also afforded nonselective SK inhibitors (Fig. 2). This
contrasts with a study that suggested that quaternary ammonium
compounds are selective inhibitors of SK2 over SK1.19 The length of
the intervening C–C link between the benzene ring and heterocyclic
ring remains to be examined.
Notes and references
Next, the possibility that the six inhibitors bearing a hydroxyl
group may also serve as SK substrates was examined. At 50 mM,
none of these compounds are SK substrates, with the exception of
RB-020, which is a weak substrate of SK1 but a good substrate of
SK2 (Table 2), and RB-019, which is a very weak substrate of SK2
(o10% of Sph, data not shown).
RB-020 is less efficiently phosphorylated by SK1 than sphingo-
sine, and probably overlaps the catalytic site of SK1 to inhibit
phosphorylation of sphingosine (Table 2). However, this is not the
case for SK2 where phosphorylation of sphingosine and RB-020
appear mutually exclusive (Table 2). RB-019 also inhibits SK1 activity
against sphingosine (Fig. 2). Both RB-019 and RB-020 contain a
hydroxyl group that is likely to be phosphorylated by SK (Table 2). It
is noteworthy that RB-020 has a primary hydroxyl group attached to
the heterocyclic ring through a 4-CH2 group, while RB-019 has a
secondary hydroxyl group directly attached to C-3 of the heterocyclic
ring; therefore, the latter hydroxyl group may be too far removed
from the catalytic determinants of SK1 to be phosphorylated, but
probably overlaps the substrate binding site to inhibit SK1 activity.
RB-008 and RB-009 have a second N atom in the heterocyclic ring,
1 N. J. Pyne and S. Pyne, Nat. Rev. Cancer, 2010, 10, 489.
2 N. C. Hait, J. Allegood, M. Maceyka, G. M. Strub, K. B. Harikumar,
S. K. Singh, C. Luo, R. Marmorstein, T. Kordula, S. Milstien and
S. Spiegel, Science, 2009, 325, 1254.
3 S. E. Alvarez, K. B. Harikumar, N. C. Hait, J. Allegood, G. M. Strub,
E. Y. Kim, M. Maceyka, H. Jiang, C. Luo, T. Kordula, S. Milstien and
S. Spiegel, Nature, 2010, 465, 1084.
4 G. M. Strub, M. Paillard, J. Liang, L. Gomez, J. C. Allegood, N. C. Hait,
M. Maceyka, M. M. Price, Q. Chen, D. C. Simpson, T. Kordula,
S. Milstien, E. J. Lesnefsky and S. Spiegel, FASEB J., 2011, 25, 600.
5 J. R. Van Brocklyn, C. A. Jackson, D. K. Pearl, M. S. Kotur, P. J. Snyder
and T. W. Prior, J. Neuropathol. Exp. Neurol., 2005, 64, 695.
6 Y. Akao, Y. Banno, Y. Nakagawa, N. Hasegawa, T. J. Kim, T. Murate, Y.
Igarashi and Y. Nozawa, Biochem. Biophys. Res. Commun., 2006, 342, 1284.
7 M. Ahmad, J. S. Long, N. J. Pyne and S. Pyne, Prostaglandins Other
Lipid Mediators, 2006, 79, 278.
8 D. G. Johns, R. C. Webb and J. R. Charpie, J. Hypertens., 2001, 19, 63.
9 K. J. French, R. S. Schrecengost, B. D. Lee, Y. Zhuang, S. N. Smith,
J. L. Eberly, J. K. Yun and C. D. Smith, Cancer Res., 2003, 63, 5962.
10 K. J. French, J. J. Upson, S. N. Keller, Y. Zhuang, J. K. Yun and
C. D. Smith, J. Pharmacol. Exp. Ther., 2006, 318, 596.
11 L. W. Maines, L. R. Fitzpatrick, K. J. French, Y. Zhuang, Z. Xia,
S. N. Keller, J. J. Upson and C. D. Smith, Dig. Dis Sci., 2008, 53, 997.
12 Y. Chiba, H. Takeuchi, H. Sakai and M. Misawa, J. Pharmacol. Sci.,
2010, 114, 304.
13 S. W. Paugh, B. S. Paugh, M. Rahmani, D. Kapitonov, T. Kordula,
S. Milstien, J. K. Adams, R. E. Zipkin, S. Grant and S. Spiegel, Blood,
2008, 112, 1382.
14 C. Loveridge, F. Tonelli, T. Leclercq, K. G. Lim, S. Long,
E. Berdyshev, R. J. Tate, V. Natarajan, S. M. Pitson, N. J. Pyne and
S. Pyne, J. Biol. Chem., 2010, 285, 38841.
15 F. Tonelli, K. G. Lim, C. Loveridge, J. Long, S. M. Pitson, G. Tigyi,
R. Bittman, S. Pyne and N. J. Pyne, Cell. Signalling, 2010, 22, 1536.
16 K. G. Lim, F. Tonelli, Z. Li, X. Lu, R. Bittman, S. Pyne and N. J. Pyne,
J. Biol. Chem., 2011, 286, 18633.
17 K. G. Lim, S. Chaode, R. Bittman, N. J. Pyne and S. Pyne, Cell.
Signalling, 2011, 23, 1590.
18 A non-lipid based phenylsulfone (PF-543) was recently reported to be a
potent SK1 inhibitor: M. E. Schnute, M. D. McReynolds, T. Kasten,
M. Yates, G. Jerome, J. W. Rains, T. Hall, J. Chrencik, M. Kraus,
C. N. Cronin, M. Saabye, M. K. Highkin, R. Broadus, S. Ogawa,
K. Cukyne, L. E. Zawadzke, V. Peterkin, K. Iyanar, J. A. Scholten,
J. Wendling, H. Fujiwara, O. Nemirovskiy, A. J. Wittwer and
M. M. Nagiec, Biochem. J., 2012, 444, 79.
Fig. 3 Effect of RB-005 on SK1 activity and expression. (A) Concentration-dependent
inhibition of SK1 activity by RB-005 using 3 mM sphingosine and 250 mM ATP (results
expressed as mean inhibition ꢀ SD of control) (n = 3, the control is 100% and equals
activity against sphingosine alone). (B) Effects of RB-005 on SK1 down-regulation.
PASMC were treated with or without MG132 (10 mM, 30 min) before RB-005 (10 mM,
24 h). Cell lysates were western blotted with anti-SK1 and -actin antibodies. Results are
representative of three experiments.
19 M. R. Raje, K. Knott, Y. Kharel, P. Bissel, K. R. Lynch and
W. L. Santos, Bioorg. Med. Chem., 2012, 20, 183.
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2138 Chem. Commun., 2013, 49, 2136--2138
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