Structure-Activity relationships and molecular modeling of sphingosine kinase inhibitors

Article abstract of DOI:10.1021/jm401399c

The design, synthesis, and evaluation of the potency of new isoform-selective inhibitors of sphingosine kinases 1 and 2 (SK1 and SK2), the enzyme that catalyzes the phosphorylation of d-erythro-sphingosine to produce the key signaling lipid, sphingosine 1-phosphate, are described. Recently, we reported that 1-(4-octylphenethyl)piperidin-4-ol (RB-005) is a selective inhibitor of SK1. Here we report the synthesis of 43 new analogues of RB-005, in which the lipophilic tail, polar headgroup, and linker region were modified to extend the structure-activity relationship profile for this lead compound, which we explain using modeling studies with the recently published crystal structure of SK1. We provide a basis for the key residues targeted by our profiled series and provide further evidence for the ability to discriminate between the two isoforms using pharmacological intervention.

Full text of DOI:10.1021/jm401399c

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Article
Structure Activity Relationships and Molecular
Modeling of Sphingosine Kinase Inhibitors
Dongjae Baek, Neil MacRitchie, Nahoum G. Anthony, Simon
Paul Mackay, Susan Pyne, Nigel Pyne, and Robert Bittman
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Oct 2013
Downloaded from http://pubs.acs.org on November 3, 2013
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Structure Activity Relationships and Molecular Modeling of Sphingosine Kinase
Inhibitors
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†,a
‡,a
‡
‡
‡
Dong Jae Baek, Neil MacRitchie, Nahoum G. Anthony, Simon P. Mackay, Susan Pyne, Nigel J.
‡
*,†
Pyne, and Robert Bittman
†
Department of Chemistry and Biochemistry, Queens College, The City University of New York,
Flushing, New York 11367ꢀ1597, United States
‡
Cell Biology and Drug Discovery & Design Groups, Strathclyde Institute of Pharmacy and Biomedical
Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom
a
These authors contributed equally.
Abstract
The design, synthesis, and evaluation of the potency of new isoformꢀselective inhibitors of sphingosine
kinase 1 and 2 (SK1 and SK2), the enzyme that catalyzes the phosphorylation of
to produce the key signaling lipid, sphingosine 1ꢀphosphate, are described. Recently, we reported that 1ꢀ
4ꢀoctylphenethyl)piperdinꢀ4ꢀol (RB-005) is a selective inhibitor of SK1. Here we report the synthesis of
3 new analogues of RB-005, in which the lipophilic tail, polar headgroup, and linker region were
Dꢀerythroꢀsphingosine
(
4
modified to extend the structureꢀactivity relationship profile for this lead compound, which we explain
using modeling studies with the recently published crystal structure of SK1. We provide a basis for the
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key residues targeted by our profiled series, and provide further evidence for the ability to discriminate
between the two isoforms using pharmacological intervention.
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Introduction
The lipid kinase, sphingosine kinase (SK), plays a myriad of roles in regulating cell survival, growth,
and migration of mammalian cells through its product, sphingosine 1ꢀphosphate (S1P). S1P is a ligand
for five cellꢀsurface Gꢀprotein coupled receptors and for several intracellular targets, such as histone
1
deacetylase 1 and 2 (HDAC1/2, which regulate gene expression). There are two isoforms of
sphingosine kinase, SK1 and SK2, that are encoded by different genes and which exhibit distinct
biochemical properties, substrate and inhibitor sensitivities, and subꢀcellular distribution. SK1 and SK2
have redundant roles to some extent as knockout of both genes is embyronically lethal in mice, whereas
2
animals survive when either gene is removed alone. Moreover, there is evidence that both SK1 and SK2
3
,4
play a similar role in certain cancers. Surprisingly, SK1 and SK2 may have opposing roles in
inflammation, being largely proꢀinflammatory and antiꢀinflammatory, respectively. On the other hand, a
3
proꢀinflammatory role for SK2 in other cell types has also been reported. The differing roles of SK1
and SK2 in inflammatory disease and supporting data from knockout mice make a compelling case for
the development of isoformꢀselective inhibitors in order to elucidate the functions and roles of each
isozyme and for designing drugs for therapeutic intervention in pathophysiological processes such as
inflammatory diseases and cancer.
Various SK inhibitors have been identified (see Fig. 1 for examples). Enzyme kinetic studies show
that many of these are competitive with sphingosine (Sph). The initial SK inhibitors were Sph analogues,
3
such as
D
,
L
-threoꢀdihydrosphingosine which has a K of ~5 ꢀM. N,NꢀDimethylsphingosine also inhibits
i
both SK isoforms. The first SK1ꢀselective inhibitor to be reported was the waterꢀsoluble Sph analogue
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SK1-I (also known as BML-258; K ~10 ꢀM). The most potent nanomolar SK1ꢀselective inhibitor is
i
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PF-543. However, this compound is also a substrate for SK1, thereby compromising data interpretation.
Analogues of the immunosuppressive agent FTY720 (Gilenya) have been prepared using a synthetic
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route starting with 4ꢀoctylphenethyl alcohol and were found to be SK1ꢀselective inhibitors (e.g., RB-
7
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005). FTY720 is an inhibitor of SK1, and also inhibits other sphingolipidꢀmetabolizing enzymes, such
9,10
11
as ceramide synthase and S1P lyase. We also recently showed that SK1 contains an allosteric site.
Replacement of the amino group in (S)ꢀFTY720ꢀvinylphosphonate with an azido group changes this
1
2
compound from an allosteric inhibitor to an activator of SK1. Therefore, allosteric inhibitors of SK1
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are also an exciting option for future study. With regard to SK2ꢀselective inhibitors, ABC294640, (R)ꢀ
1
4
FTY720 methyl ether (ROMe), K145 (3ꢀ(2ꢀaminoꢀethyl)ꢀ5ꢀ[3ꢀ(4ꢀbutoxylꢀphenyl)ꢀpropylidene]ꢀ
1
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thiazolidineꢀ2,4ꢀdione), and SLR080811 ((S)ꢀ2ꢀ[3ꢀ(4ꢀoctylphenyl)ꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl]pyrrolidineꢀ1ꢀ
1
6
carboximidamide) have K values in the range of ~1ꢀ10 ꢁM. Taken together, it is clear that there is still
i
a need to develop new SK1 and SK2 inhibitors, both to increase the number of selective tools that can be
used to interrogate the biology of these enzymes and to increase the possibility of having useful new
therapeutic agents to treat disease.
7
In a recent study, we showed that RB-005 is a highly selective SK1 inhibitor. Herein, we describe
8
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18,19
the synthesis of analogues of the known SK1 inhibitors RB-005, FTY720, SKi, compounds 36a
2
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and 82, BML-258, CB5468139, and SLR080811 These new analogues, which were designed to
possess some degree of structural similarity to the known inhibitors, were evaluated through enzyme
activity studies as inhibitors of SK1 and SK2. Structureꢀactivity relationship (SAR) studies are discussed,
which are supported by an analysis of molecular modeling poses of the inhibitors in the active site of
human SK1.
.
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Fig. 1. Structures of selected SK inhibitors.
Fig. 2. Modifications introduced into the known SK inhibitors RB-005, SKi, SLR080811, 36a
VPC96091), and 82.
(
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Results and Discussion
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The structures of the new compounds and their key structural modifications are shown in Tables 1ꢀ4.
Table 1 Piperidyl analogues of RB-005 synthesized for evaluation as SK inhibitors
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Table 2 Pyrrolidine analogues of RB-005 synthesized for evaluation as SK inhibitors
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Table 3 Benzamide analogues of RB-005 synthesized for evaluation as SK inhibitors
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Table 4 Pyridine, pyridinium, and triazole analogues of RB-005 synthesized for evaluation as SK
inhibitors
Chemical Synthesis.
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Linker Length. The synthetic routes we employed to prepare compounds with oneꢀ, threeꢀ, and fourꢀ
carbon tethers are displayed in Scheme 1. The sole carbon atom of the hydroxymethyl group of 4ꢀ
iodobenzyl alcohol provided the tether in RB-023, whereas the three carbons of propargyl alcohol were
the source of the tether in RB-024. In each of these compounds, a Sonogashira reaction followed by
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catalytic hydrogenation of the alkyne intermediate (1 and 3) and a S 2 reaction of a mesylate
N
intermediate derived from 2 and 4 with 4ꢀhydroxypiperidine gave the desired compounds. We used 4ꢀ(4ꢀ
2
2
octylphenyl)butanꢀ1ꢀol (5) as the starting material for the preparation of RB-025.
a
Scheme 1 Synthesis of RB-023 – RB-025
a
Reagents and conditions: (a) 1ꢀoctyne, Pd(PPh ) , CuI, Et N, 50 °C, 12 h; (b) Pd/C, H , EtOAc, rt, 12 h;
3
4
3
2
(
c) i) MsCl, Et N, CH Cl , 3 h, rt; ii) 4ꢀhydroxypiperidine, MeCN, 50 °C, 12 h; (d) propargyl alcohol,
3 2 2
Pd(PPh ) , CuI, Et N, 50 °C, 12 h.
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Modifications of the 4-Alkylphenyl and the Piperidyl Groups. Scheme 2 shows the synthetic pathways
employed to prepare RB-026 – RB-033. After a Sonogashira reaction was used to install an alkynyl
group with six to twelve carbons, catalytic hydrogenation of 6 and 7 afforded alkyl derivatives 8 and 9,
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and S 2 displacement as in Scheme 1 gave the desired compounds in good yield. To assess the role of
N
the hydroxyl group in RB-005, RB-026, and RB-028 in inhibition of SK, we replaced this group with an
azido group via mesylation of the alcohol and reaction with sodium azide in DMF to obtain 10, RB-029,
and RB-030. Reduction of the azide afforded the amino derivatives, RB-031, RB-032, and RB-033.
a
Scheme 2 Synthesis of RB-026 – RB-033
a
Reagents and conditions: (a) 1ꢀhexyne or 1ꢀdodecyne, Pd(PPh ) , CuI, Et N, 50 °C, 12 h; (b) Pd/C, H ,
3
4
3
2
EtOAc; (c) i) MsCl, Et N, CH Cl ; ii) 4ꢀhydroxypiperidine, MeCN, 50 °C, 12 h; (d) i) MsCl, Et N,
3
2
2
3
CH Cl , 3 h, rt; ii) NaN , DMF, 80 ꢀ100 °C, 12 h; (e) Pd/C, H , CH Cl /MeOH (1:3), rt, 12 h.
2
2
3
2
2
2
Fluorination of RB-005 with diethylaminosulfur trifluoride (DAST) gave RB-034 in 90% yield
(Scheme 3). The 4ꢀketo derivative RB-035 was synthesized by oxidation of the 4ꢀhydroxyl group of
7
RB-005 with pyridinium chlorochromate. Reaction of mesylate 11 with 4ꢀmethoxypiperidine provided
RB-036. Similarly, reaction of commercially available chiral pyrrolidines containing a 3ꢀhydroxyl or a
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ꢀhydroxymethyl substituent gave RB-037 – RB-043.
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a
Scheme 3 Synthesis of RB-034 − RB-043
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Reagents and conditions: (a) DAST, CH Cl , 0 °C ꢀ rt, 5 h, rt; (b) PCC, CH Cl , 4 h, rt; (c) cyclic amine,
2
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2
2
MeCN, 50 °C, 12 h.
Benzamide derivatives. Benzamideꢀcontaining analogues RB-044 – RB-050 were prepared from 4ꢀ
iodobenzoic acid as outlined in Scheme 4. Alkyne intermediate 12 was reduced to carboxylate 13, which
was converted to the acyl chloride with thionyl chloride and then treated with the desired cyclic amine in
the presence of potassium carbonate.
a
Scheme 4 Synthesis of RB-044 – RB-050 from 4ꢀiodobenzoic acid
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a
Reagents and conditions: (a) 1ꢀoctyne, Pd(PPh ) , CuI, Et N, 60 °C, 12 h; (b) Pd/C, H , EtOAc, 12 h, rt;
3
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(c) i) SOCl , CH Cl , reflux, 12 h; ii) cyclic amine, K CO , MeCN, 50 °C, 12 h.
2 2 2 2 3
Quaternary Ammonium Derivatives. To synthesize RB-052, which contains a pyridinium ion in the polar
headgroup, we used the halogens in 1ꢀbromoꢀ4ꢀiodobenzene to carry out two separate Sonogashira
reactions, as shown in Scheme 5. First, we used 1ꢀoctyne to prepare alkyne 14, which reacted with 3ꢀ
ethynylpyridine to give dialkyne 15. Reduction of 15 yielded RB-051, and Nꢀalkylation with methyl
iodide (5 equiv) in acetonitrile afforded the Nꢀmethylpyridinium salt RB-052.
a
Scheme 5 Synthesis of RB-051 and RB-052 from 1ꢀbromoꢀ4ꢀiodobenzene
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a
Reagents and conditions: (a) 1ꢀoctyne, Pd(PP ) , CuI, Et N, 50°C, 12 h; (b) 3ꢀethynylpyridine,
3
4
3
Pd(PPh ) , CuI, Et N, 80 °C, 3 d; (c) Pd/C, H , EtOAc, 12 h, rt; (d) K CO , MeI, MeCN, overnight, rt.
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Pyridinium salt RB-053 was prepared by a similar route, as shown in Scheme 6. A Sonogashira
reaction of 4ꢀiodobenzyl bromide with 1ꢀoctyne provided alkyne 16; displacement of bromide ion with
4
ꢀ(4ꢀmethylpiperidinꢀ1ꢀyl)pyridine (17) afforded alkyne 18, which provided RB-053 on catalytic
hydrogenation in MeOH/CH Cl (1:3).
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a
Scheme 6 Synthesis of pyridinium salt RB-053 from 4ꢀiodobenzyl bromide and of 4ꢀ(4ꢀ
methylpiperidinꢀ1ꢀyl)pyridine (17) from 4ꢀchloropyridine hydrochloride
a
Reagents and conditions: (a) 1ꢀoctyne, Pd(PPh ) , CuI, Et N; (b) 4ꢀmethylpiperidine, DIPEA, MeCN,
3
4
3
microwave heating, 160 °C, 1 h; (c) 17, 2ꢀbutanone, 100 °C, 3 d; (d) Pd/C, H , MeOH/CH Cl (1:3).
2
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Triazole Derivatives. A Pd/Cuꢀcatalyzed Sonogashira reaction followed by a Cu(I)ꢀcatalyzed azideꢀ
alkyne 1,3ꢀdipolar addition (click reaction) were employed to prepare RB-054, in which a triazole ring
bearing a pyridine substituent is the headgroup (Scheme 7). Thus, Sonogashira coupling of 1ꢀoctyne
with 4ꢀiodoaniline afforded alkynylꢀaniline derivative 19. After conversion of amine 19 to aryl azide 20,
the triazole ring was installed in a click reaction with 3ꢀethynylpyridine; finally, catalytic hydrogenation
of alkyne 21 gave RB-054.
0
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Scheme 7 Synthesis of RB-054 from 4ꢀiodoaniline
a
Reagents and conditions: (a) 1ꢀoctyne, Pd(PPh ) , CuI, Et N, 60 °C, 12 h; (b) i) 10% aq. HCl, NaNO ;
3 4
3
2
ii) NaN ; (c) 3ꢀethynylpyridine, sodium ascorbate, CuSO , tꢀBuOH/H O (1:1); (d) Pd/C, H , EtOAc, rt,
3
4
2
2
2
d.
We reversed the location of the triazole in analogues RB057 – RB-065. As shown in Scheme 8, the
headgroup in these analogues is a piperidyl derivative and the lipophilic tail contains an alkylꢀsubstituted
triazole. Azides 22 and 23 were prepared from commercially available 4ꢀaminophenol and 2ꢀ(4ꢀ
aminophenyl)ethanol, respectively. The click reaction with terminal alkynes afforded triazoles RB-055,
2
4, 25, and RB-056. To prepare RB-059 – RB-065, alcohols 24, 25, and RB-056 were converted to their
corresponding mesylates 26, 27, and 28, which were treated with piperidine, 1ꢀmethylpiperidine, or 4ꢀ
hydroxypiperidine.
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a
Scheme 8 Synthesis of RB-055 – RB-065
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a
Reagents and conditions: (a) i) 10% aq. HCl, NaNO ; ii) NaN ; (b) 1ꢀdecyne, sodium ascorbate, CuSO ,
2
3
4
tꢀBuOH/H O (1:1), 12 h, rt; (c) acetylenic substrate (24: 1ꢀhexyne; 25: 1ꢀheptyne, RB-056: 1ꢀdecyne),
2
sodium ascorbate, CuSO , tꢀBuOH, H O; (d) MsCl, Et N, CH Cl , 0 °C ꢀ rt, 5 h; (e) piperidine, MeCN
4
2
3
2
2
(RB-059 – RB-061), 1ꢀmethylpiperidine, MeCN (RB-060 – RB-062) or 4ꢀhydroxypiperidine, MeCN,
5
0 °C, 12 h (RB-063 – RB-065).
Effects on SK1 and SK2 Activity and SAR.
We previously demonstrated the importance of the 4ꢀhydroxypiperidinyl group in the selective
7
inhibition of SK1, which was subsequently confirmed by Gustin et al., who generated chiral piperidyl
2
0
analogues bearing hydroxyl and hydroxymethyl groups. To examine SAR among a panel of related
compounds, we prepared a series of analogues bearing a 4ꢀhydroxypiperidinyl group but varied the
linker length between the aryl group and the piperidine. We also assessed the role of the alkyl substituent
in the aryl group. To evaluate compound selectivity against SK1 or SK2, the assays were performed (see
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Supporting Information) using Sph at concentrations of 3 and 10 ꢀM (the K values of SK1 and SK2,
m
respectively), which corresponds to 50% substrate saturation and enables a qualitative estimation of
selectivity by comparing the % inhibition of each kinase using a fixed concentration of inhibitor. We
consider this approach to be an appropriate comparison of selectivity since both enzymes exhibit 50%
0
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occupancy with the substrate. Compounds that were found to be effective inhibitors were then analyzed
in more detail by performing doseꢀresponse curves.
We have previously shown that RB-005 (the ‘parent compound’) is a selective inhibitor of SK1 and
exhibits an IC = 3.6 ± 0.38 ꢀM at 3 ꢀM Sph (which corresponds to the K of SK1) and reduces SK1
5
0
m
7
activity by ~90% at 50 ꢀM RB-005. The effect of linker length on potency was assessed by comparing
the % inhibition of SK1 and SK2 obtained with RB-023 (which has a oneꢀcarbon tether), RB-024
(threeꢀcarbon tether), and RB-025 (fourꢀcarbon tether). The linker length did not significantly alter the
ability of RB-023, RB-024, and RB-025 to inhibit SK1 activity (Fig. 3). RB-023 – RB-025 also retained
selectivity for SK1 over SK2.
The aliphatic chain at the para position of the benzene ring of FTY720 is C H , which is known to
8
17
2
3
24
be optimal for the action of FTY720 on its targets such as S1P receptors. Knott et al. reported that the
ability of quaternary ammonium salts with a phenylꢀsubstituted cyclohexylamine scaffold to inhibit SK2
was affected by the alkyl chain length. To examine the role of the alkyl substituent on the benzene ring
of RBꢀ005, and thus the lipophilicity of the molecule, we compared the inhibitory activity of RB-026
(
which has a methyl group as the alkyl substituent), RB-027 (which has a nꢀhexyl group), RB-005
which has a nꢀoctyl group), and RB-028 (which has a nꢀdodecyl group). SK1 inhibition was decreased
(
by more than 6ꢀfold in RB-026 compared with RB-023. The almost complete lack of inhibition
displayed by RB-026 against SK1 indicates that a larger alkyl group than a methyl group is required for
inhibitory activity. We also evaluated the effect of alkyl chain length in the lipophilic tail of compounds
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in which the 4ꢀhydroxypiperidinyl group was replaced by a 4ꢀaminopiperidinyl group (see below).
Changing the nꢀoctyl group of RB-032 to a methyl or nꢀdodecyl group gave RB-031 and RB-033,
respectively, and eliminated the inhibitory activity toward SK1. These results confirm the critical
requirement for the nꢀoctyl group.
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Next, we probed the role of the 4ꢀhydroxyl group of RB-005 by replacing it with an azido, amino,
fluoro, keto, or methoxy group (RB-029 – RB-036). Azido replacement (RB-029, RB-030) reduced
SK1 inhibition markedly, while replacement of the 4ꢀhydroxyl group with an amino group (RB-032)
diminished the potency of SK1 inhibition (Fig. 4). The isoform selectivity of SK1 over SK2 was
retained for RB-032, suggesting that the amino group replacement maintains efficient binding to SK1.
Replacement of the 4ꢀhydroxyl group of RB-005 with a fluoro (RB-034) or methoxy group (RB-036)
eliminated inhibitory activity against SK1, while replacement with a keto group to produce RB-035
increased inhibition of SK2 and maintained inhibition of SK1 but eliminated the isoform selectivity.
To examine the role of the piperidyl group in inhibition of SK, we replaced it with a pyrrolidine ring;
the hydroxylꢀcontaining substituent was retained (as either a chiral hydroxyl or a chiral hydroxymethyl
group) but its orientation was varied, as shown in compounds RB-037 – RB-043. RB-037 and RB-038
retained inhibitory activity against SK1 despite having opposite configurations at Cꢀ3 of the pyrrolidinꢀ
3
ꢀol group. Stereoisomers RB-040 and RB-042, which differ in the length of the aliphatic chain (C H17
8
vs. C H ) but possess the R configuration at Cꢀ2 of the 2ꢀhydroxymethyl pyrrolidinyl group, were
12
5
equipotent inhibitors of SK1 and SK2 (Fig. 3 and Fig. 5A,B). The corresponding S enantiomers RB-041
and RB-043 were much less active (Fig. 3). To establish whether RB-041 and RB-043 were capable of
inhibiting SK1 and SK2 activity in a concentrationꢀdependent manner, we used a higher concentration of
each (100 ꢀM, compared to the 50 ꢀM concentration data shown in Fig. 3) and found that the inhibition
of SK1 and SK2 with RB-041 was 72.2 ± 5.9% and 45.7 ± 2.6%, respectively, whereas with RB-043 the
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inhibition of SK1 and SK2 was 49.9 ± 6.2% and 49.7 ± 7%, respectively. These findings indicate that
RB-041 and RB-043 can inhibit SK1 and SK2, but that the sensitivity of inhibition compared with RB-
40 and RB-042 is considerably reduced. Interestingly, the S enantiomers RB-041 and RB-043 are
0
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substrates for SK2 (see Supporting Information, Fig. S1).
To further examine the influence of the length of the alkyl substituent on the benzene ring on SK
activity, we assessed the extent of SK inhibition afforded by pyrrolidine derivatives RB-039, RB-042,
and RB-043. The ability of the compound to inhibit SK1 is abolished in RB-039 and RB-043, which
have a methyl and a nꢀdodecyl group in the lipophilic tail, respectively.
An amidine or proline headgroup incorporated into a benzamideꢀcontaining scaffold was shown to
18,19
provide potent SK1 inhibitors,
as in compound 36a (Fig. 1). When we replaced the methylene linker
between the aryl group and the heterocycle with a keto group to produce the benzamide analogues RB-
0
44 – RB-050, inhibition of SK1 was effectively abolished (Fig. 3), as were the pyridine derivatives
25
(RB-048 and RB-051). The report that quaternary ammonium salts are selective SK2 inhibitors
prompted us to prepare equivalent derivatives RB-052, RB-053, RB-060, RB-061, and RB-062, which
were ineffective as SK inhibitors, although RB-053 demonstrated a moderate selectivity for SK2 (Fig. 3).
We also prepared aliphatic quaternary ammonium salts (not shown) that were inactive.
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Fig. 3. Effect of inhibitors on SK1 or SK2 activity. SK1 activity was measured using 3 ꢀM Sph and 250
ꢀM ATP. SK2 activity was assayed using 10 ꢀM Sph and 250 ꢀM ATP (n = 3 for each compound,
results are expressed as % of control ± S.D.). RB series compounds were used at 50 ꢀM. BMLꢀ258 (50
ꢀM) inhibited SK1 activity by 74.5 ± 3.3 % (n = 3). The control is 100% and equals activity against Sph
alone.
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Fig. 4. Effect of RB-032 on SK1 activity. Concentrationꢀdependent inhibition of SK1 activity by RB-
0
32 using 3 ꢀM Sph and 250 ꢀM ATP. The results are expressed as % of control ± S.D.; n = 3; the
control is 100% and equals activity against Sph alone. RB-032 inhibits SK1 activity with an IC = 16.9
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7
±
1.6 ꢀM; RB-005 inhibits SK1 activity with an IC = 3.6 ± 0.4 ꢀM.
50
SK inhibitors containing a central thiazole group have been reported (e.g., SKi and compound 82,
Fig. 1). 1,2,3ꢀTriazoles are mimics of thiazoles, and are easily prepared by Cu(I)ꢀcatalyzed azide/alkyne
click chemistry. In our series of triazole analogues of RB-005 (RB-054 – RB-065), we found that RB-
0
65 was a highly selective SK1 inhibitor, whereas the other ten triazole analogues, all of which lack the
4
ꢀhydroxypiperidinyl group, were inactive (Fig. 3).
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Fig. 5. Effect of RB-040 and RB-042 on (A) SK1 activity and (B) SK2 activity. Concentrationꢀ
dependent inhibition of SK activity by RB-040 and RB-042 using 3 ꢀM Sph (SK1) or 10 ꢀM Sph (SK2)
and 250 ꢀM ATP. The results are expressed as % of control ± S.D. (n = 3). The control is 100% and
equals activity against Sph alone. RB-040 inhibits SK1 activity with an IC = 2.2 ± 0.22 ꢀM, and SK2
5
0
with an IC = 5.2 ± 0.82 ꢀM (Fig. 5). RB-042 inhibits SK1 activity with an IC = 5.3 ± 0.5 ꢀM and
5
0
50
7
SK2 with an IC = 5.0 ± 1.3 ꢀM.
50
Modeling the Inhibitors in the Atomic Structure of SK1.
2
6
The crystal structures of human SK1 in a complex with ADP and SKi were determined recently.
We demonstrate here that the chemical modifications of the highly selective SK1 inhibitor RB-005
produced SAR that can be explained using this crystal structure. Fig. 6A displays the result of a
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modeling analysis of RB-005 in the active site of human SK1; the piperidyl hydroxyl group is hydrogen
bonded to D81 and the protonated amine of the head group forms a salt bridge with the carboxylate of
D178. Fluoroꢀ (RB-034) or methoxyꢀ(RB-036) containing compounds do not exhibit inhibitory activity
against SK1 (Fig. 3), as these groups no longer have hydrogen bond donating capacity, suggesting that
the interaction of the 4ꢀhydroxypiperidinyl group of RB-005 is with a hydrogen bond acceptor in the
protein. The lack of SK1 inhibitor activity of the azideꢀcontaining compounds (RB-029, RB-030)
supports this possibility. One of the two oxygens of the carboxylate ion of D81 is hydrogen bonded to
the backbone NH of L116, and the other is hydrogen bonded to the backbone NH of A115; these
interactions prevent the carboxylate of D81 from being catalytic and shift the catalytic role to D178 (Fig.
0
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). The latter carboxylate oxygen also forms a hydrogen bond to the hydroxyl group of the inhibitor. If
these compounds formed hydrogen bonds with the side chain of S168, then RB-029, RB-034, and RB-
36 could also bind to the donor/acceptor hydroxyl group of S168 or water and therefore act as
0
inhibitors. Since RB-029, RB-034, and RB-036 cannot form hydrogen bonds with D81 and are not
inhibitors, we propose that the key interaction of the hydroxyl group of RB-005 (Fig. 6A), RB-025 (Fig.
6
B), and RB-028 (Fig. 6C) is with D81 and not with S168. In contrast, modeling of RBꢀ035 (which
contains a 4ꢀketo group instead of a 4ꢀhydroxyl group, yet maintains inhibition of SK1) suggests that the
carbonyl group can form a hydrogen bond with the hydroxyl group of S168 and water (see Supporting
Information, Fig. S2).
The inhibitory effect of RB-032 (Fig. 6D) and its absence for RB-033 (Fig. 6E) can be explained by
protonation of the primary amine. When the protonated primary amine rather than the piperidyl group
forms a salt bridge with D178, the inhibitors are pushed deeper into the J channel, which was identified
2
6
by Wang et al. as the region that accommodates the alkyl chain of Sph. Since RB-032 has a shorter
alkyl chain than RB-033, it can be accommodated in the substrate pocket, whereas the dodecyl group of
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RB-033 cannot fit into the channel formed when the protonated primary amine forms a salt bridge with
D178. Therefore, RB-033, which is inactive, does not bind to D81, D178, S168 or L268, indicating that
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these are key aminoꢀacid residues required for binding SK inhibitors.
0
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+
The salt bridge between the NH group of RB-032 and D178 (Fig. 6D) is the only polar interaction
3
with the protein, which might explain the lower SK1 inhibitor activity when compared with RB-005; the
latter forms a salt bridge and hydrogen bonds with D81. Interestingly, the Rꢀenantiomers RB-040 and
RB-042 are equipotent inhibitors of SK1 and SK2, while the S-enantiomers RB-041 and RB-043 are
weak substrates for SK2, implying that the spatial orientation of the hydroxyl group in RB-041 and RB-
0
43 required for catalysis is different in SK2 compared with SK1. The protonated amine in RB-040 (Fig.
6
F) and RB-042 (Fig. 6G) can form a salt bridge with D178 and can also form a hydrogen bond with the
carbonyl oxygen of L268. Both inhibitors can orientate the hydroxymethyl group of the pyrrolidine (R
enantiomer) to also form a hydrogen bond with the side chain of D81. The protonated amino group of
RB-041 and RB-043 can form a salt bridge with D178 but, because of the orientation of the
hydroxymethyl group of the pyrrolidine (S enantiomer), cannot form a hydrogen bond between their
hydroxyl group and D81, as found in our modeling study. Instead, the hydroxymethyl group could form
a hydrogen bond to D178. As the experimental evidence shows that RB-041 and RB-043 do not inhibit
SK1, this suggests that dynamic factors (accessing the binding site), which are not taken into account by
docking studies, prevent the binding of these compounds.
RB-044ꢀRB-050 are ineffective inhibitors of SK1. There are three possible explanations: first, the
nitrogen in an amide cannot be protonated, thus preventing salt bridge formation. Second, the link
between nitrogen and phenyl is constrained and planar compared with a methylene group, which
prevents optimization of the hydrogen bonding network with the hydroxyl group. Third, the carbonyl
group of the amide would be proximal to the side chain of D178, which would result in electrostatic
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repulsion.
The pyridinium salts RB-052 and RB-053 and the quaternary ammonium salts RB-060, RB-061,
and RB-062 were also ineffective SK1 inhibitors. The absence of a hydroxyl group in these compounds
rules out hydrogen bonding with D81 or D178. The triazole moiety in RB-065 forms a hydrogen bond
with T196 (Fig. 6H) and, furthermore, adds a kink in the chain that helps orientate the alkyl group into
the J channel, which may account for its SK1 inhibitory activity.
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Our modeling studies suggest that RB-005 (Fig. 6A), RB-025 (Fig. 6B), and RB-028 (Fig. 6C)
interact with D81 and not with S168. These findings are consistent with D81 not acting as a base,
because RB-005, RB-025, or RB-028 are not substrates for SK1. As depicted in Fig. 7, modeling of Sph
+
into the catalytic site of SK1 suggests that S168 can form hydrogen bonds with the NH group of Sph
3
and, via a water molecule, with the secondary hydroxyl group of Sph. The water molecule also hydrogen
bonds with A339, thereby linking this amino acid residue to the secondary hydroxyl group of Sph (Fig.
7
). Thus D178 functions as the deprotonating base in this model to enable nucleophilic attack by Sph on
the γꢀphosphate group of ATP, with subsequent transfer of this phosphate to Sph.
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Fig. 6. Various modeled poses of SK1 inhibitors in the catalytic site of SK1. (A) RB-005, (B) RB-025,
C) RB-028, (D) RB-032, (E) RB-033, (F) RB-040, (G) RB-042, and (H) RB-065.
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Fig. 7. Schematic model of the proposed mechanism of the phosphorylation of Sph catalyzed by SK1.
Conclusion
In this study we have identified a series of SK1ꢀselective inhibitors and have used molecular
modeling to define their interactions with the catalytic site of the enzyme. These studies reveal a
substantial flexibility in the catalytic site in terms of binding SK1 inhibitors. For instance, RB-005 is
proposed to interact with D81 and D178, while RB-025 appears to interact with D81 and L268. The
findings obtained from the modeling study fully account for the SAR of the inhibitors and explain why
some of these compounds are inactive. These findings also reveal the architecture of the SK1 catalytic
site and suggest a major role for D178 as the deprotonating base that facilitates phosphorylation of Sph
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by ATP. In summary, the novel information presented here should enable development of new SK1
inhibitors with improved potency and selectivity. Similarly, resolution of the atomic structure of SK2
(
yet to be achieved) along with information provided herein will enable better insights into the
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molecular basis of the selectivity of these inhibitors for SK1 over SK2.
Experimental Section
Docking studies. The crystal structure of SK1 in complex with Sph (PDB entry 3VZB) was used for
docking studies. Chain A of the complex was kept along with a single water molecule found to be tightly
bound to the complex which hydrogen bonds to the side chain hydroxyl group of S168, the backbone –
NH of G342, and the secondary hydroxyl group of Sph (water number 680). Hydrogen atoms were
added to the protein and water using Accelrys Discovery studio 3.1 (Accelrys Software, San Diego, CA),
and all of the inhibitors presented in this study were docked using GOLD 5.1 for Windows (Cambridge
Crystallographic Data Centre, Cambridge, UK). Default software settings were used, keeping ChemPLP
as a scoring function after redocking Sph in place in the 3VZB crystal structure as well as the SKi
inhibitor in the 3VZD pdb entry (RMSD of 1.8 and 0.2 Å, respectively) as validation.
Synthesis. General Methods. All chemicals were reagent grade and used as purchased. Reactions
were run under nitrogen and were monitored by TLC using silica gel 60 F254 aluminumꢀbacked plates.
Flash column chromatography was performed on silica gel grade 60 (230–400 mesh). THF was distilled
over sodium/benzophenone immediately prior to use; dichloromethane was distilled over CaH ; Et N
2
3
was distilled over KOH pellets. All other solvents were of anhydrous quality and were used as received.
1
13
H NMR and C NMR spectra were recorded on a Bruker Avance I spectrometer, and chemical shifts
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are reported in δ units relative to deuterated solvents, which served as internal references, at 400 and 100
MHz, respectively. Highꢀresolution mass spectra (HRMS) were recorded at the CUNY Mass
Spectrometry Facility on an Agilent Technologies G6520A QꢀTOF mass spectrometer using electrospray
ionization (ESI). Microwave reactions were performed in a Biotage Emrys Creator Synthesizer. HPLC
was carried out using a reverseꢀphase column with a gradient of acetonitrile/water from 50/50 to 90/10,
with detection at 214 and 254 nm. Elemental analysis was performed at Columbia Analytical Services,
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1
Tucson, AZ. All compounds were ≥95% pure as determined by examining their HRMS and H NMR
spectra.
(4-(Oct-1-ynyl)phenyl)methanol (1)
To a solution of 4ꢀiodobenzyl alcohol (200 mg, 0.85 mmol), bis(triphenylphosphine)palladium
dichloride (49 mg, 0.040 mmol), and copper(I) iodide (7.6 mg, 0.04 mmol) in anhydrous triethylamine
(10 mL) was added 1ꢀoctyne (283 mg, 2.56 mmol) at rt. After the reaction mixture was heated at 50 °C
for 12 h, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with
EtOAc. The combined solution was washed with water, brine, and dried. Flash column chromatography
1
with hexanes/EtOAc (5:1) as the eluent gave alkyne 1 (180 mg, 98%) as a yellow oil; H NMR (400
MHz, CDCl ) δ 0.90 (t, J = 6.9 Hz, 3H), 1.28–1.36 (m, 4H), 1.41–1.49 (m, 2H), 1.60 (quin, J = 7.3 Hz,
3
2
2
1
H), 1.89 (br s, 1H), 2.40 (t, J = 7.1 Hz, 2H), 4.65 (s, 2H), 7.25 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz,
1
3
H); C NMR (100 MHz, CDCl ) δ 14.1, 19.4, 22.6, 28.6, 28.7, 31.4, 65.0, 80.3, 90.6, 123.4, 126.8,
3
+
31.7, 140.1; ESIꢀHRMS (M+H) m/z calcd for C H O 217.1592, found 217.1588.
1
5
21
(4-Octylphenyl)methanol (2)
Compound 1 (180 mg, 0.83 mmol) was dissolved in EtOAc (10 mL), and 10% Pd/C (90 mg, 50 wt %)
was added. The reaction mixture was hydrogenated at rt for 12 h. The catalyst was removed by filtration
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through a pad of Celite, which was rinsed with EtOAc. Product 2 was obtained, without purification, as
1
a colorless oil; H NMR (400 MHz, CDCl ) δ 0.87 (t, J = 6.6 Hz, 3H), 1.26–1.30 (m, 10H), 1.56–1.63
3
(m, 2H), 1.78 (br s, 1H), 2.59 (t, J = 7.7 Hz, 2H), 4.64 (s, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0
0
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Hz, 2H); C NMR (100 MHz, CDCl ) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.9, 35.7, 65.3, 127.1, 128.6,
3
+
138.1, 142.6; ESIꢀHRMS (M+H) m/z calcd for C H O 221.1905, found 221.1887.
1
5
25
1
-(4-Octylbenzyl)piperidin-4-ol (RB-023)
To a solution of 2 (37 mg, 0.17 mmol) and triethylamine (0.23 mL, 1.7 mmol) in CH Cl (5 mL) at
2
2
0
°C was added methanesulfonyl chloride (40 ꢀL, 0.50 mmol). After being stirred at rt for 3 h, the
reaction mixture was evaporated, diluted with water, and the product was extracted with EtOAc. The
extract was washed with brine, dried, and evaporated. To a solution of the reaction mixture (0.17 mmol)
in MeCN (3 mL) was added 4ꢀhydroxypiperidine (86 mg, 0.85 mmol). The reaction mixture was stirred
at 50 °C for 12 h and concentrated. Purification by silica gel chromatography, eluting with
1
CH Cl /MeOH (5:1), gave 35 mg (69%, 2 steps) of RB-023 as a colorless oil; H NMR (400 MHz,
2
2
CDCl ) δ 0.88 (t, J = 6.8 Hz, 3H), 1.23–1.30 (m, 10H), 1.56–1.73 (m, 4H), 1.97–2.06 (m, 2H), 2.35–2.47
3
(m, 2H), 2.59 (t, J = 7.7 Hz, 2H), 2.85–2.90 (m, 2H), 3.64 (s, 2H), 3.78–3.81 (m, 1H), 7.15 (d, J = 7.9
13
Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H); C NMR (100 MHz, CDCl ) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.5, 31.9,
3
+
3
3.2, 35.7, 50.1, 62.2, 128.5, 129.7, 137.2, 142.8; ESIꢀHRMS (M+H) m/z calcd for C H NO 304.2640,
20 34
found 304.2637.
3-(4-Octylphenyl)prop-2-yn-1-ol (3)
Compound 3 was prepared from 1ꢀbromoꢀ4ꢀnꢀoctylbenzene and propargyl alcohol according to a
1
Sonogashira reaction procedure similar to that described for 1; yield = 82%; H NMR (400 MHz, CDCl )
3
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δ 0.86 (t, J = 6.8 Hz, 3H), 1.22–1.30 (m, 10H), 1.57–1.60 (m, 2H), 2.59 (t, J = 7.7 Hz, 2H), 4.49 (s, 2H),
4
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3
7
3
2
.11 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.7 Hz, 2H); C NMR (100 MHz, CDCl ) δ 14.1, 22.7, 29.2, 29.4,
3
+
1.2, 31.9, 35.9, 51.7, 85.9, 86.5, 119.6, 128.4, 131.6, 143.7; ESIꢀHRMS (M+H) m/z calcd for C H O
1
7
25
0
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45.1905, found 245.1903.
3
-(4-Octylphenyl)propan-1-ol (4)
Compound 4 was prepared from 3 by a catalytic hydrogenation procedure similar to that described
1
for 2; H NMR (400 MHz, CDCl ) δ 0.88 (t, J = 6.8 Hz, 3H), 1.24–1.30 (m, 10H), 1.59 (quin, J = 7.4 Hz,
3
2
H), 1.88 (quin, J = 6.5 Hz, 2H), 2.56 (t, J = 7.7 Hz, 2H), 2.67 (t, J = 7.7 Hz, 2H), 3.66 (t, J = 6.4 Hz,
1
3
2
H), 7.10 (s, 4H); C NMR (100 MHz, CDCl ) δ 14.1, 22.7, 29.3, 29.4, 29.5, 31.6, 31.7, 31.9, 34.3,
3
+
35.6, 62.4, 128.3, 128.4, 138.9, 140.5; ESIꢀHRMS (M+H) m/z calcd for C H O 249.2218, found
1
7
29
2
49.2210.
1
-(3-(4-Octylphenyl)propyl)piperidin-4-ol (RB-024)
Compound RB-024 was prepared from 4 according to a procedure similar to that described for RB-
1
023; yield = 73%; H NMR (400 MHz, CDCl ) δ 0.87 (t, J = 6.8 Hz, 3H), 1.25–1.30 (m, 10H), 1.55–
3
1
.65 (m, 4H), 1.79–1.92 (m, 4H), 2.16–2.20 (m, 2H), 2.40–2.43 (m, 2H), 2.53–2.63 (m, 4H), 2.80–2.83
1
3
(
m, 2H), 3.67–3.69 (m, 1H), 7.08 (s, 4H); C NMR (100 MHz, CDCl ) δ 14.1, 22.7, 28.5, 29.3, 29.4,
3
+
29.5, 31.6, 31.9, 33.3, 34.0, 35.6, 50.9, 57.9, 128.2, 128.4, 138.9, 140.4; ESIꢀHRMS (M+H) m/z calcd
for C H NO 332.2953, found 332.2951.
2
2
38
1
-(4-(4-Octylphenyl)butyl)piperidin-4-ol (RB-025)
22
Compound RB-025 was prepared from 4ꢀ(4ꢀoctylphenyl)butanꢀ1ꢀol (5) according to a procedure
2
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