Structure-activity relationship studies of the lipophilic tail region of sphingosine kinase 2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.03.041

Sphingosine-1-phosphate (S1P) is a ubiquitous, endogenous small molecule that is synthesized by two isoforms of sphingosine kinase (SphK1 and 2). Intervention of the S1P signaling pathway has attracted significant attention because alteration of S1P levels is linked to several disease states including cancer, fibrosis, and sickle cell disease. While intense investigations have focused on developing SphK1 inhibitors, only a limited number of SphK2-selective agents have been reported. Herein, we report our investigations on the structure-activity relationship studies of the lipophilic tail region of SLR080811, a SphK2-selective inhibitor. Our studies demonstrate that the internal phenyl ring is a key structural feature that is essential in the SLR080811 scaffold. Further, we show the dependence of SphK2 activity and selectivity on alkyl tail length, suggesting a larger lipid binding pocket in SphK2 compared to SphK1.

Full text of DOI:10.1016/j.bmcl.2015.03.041

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4956–4960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship studies of the lipophilic tail region
of sphingosine kinase 2 inhibitors
Molly D. Congdon a, Elizabeth S. Childress a, Neeraj N. Patwardhan a, James Gumkowski a, Emily A. Morris a,
Yugesh Kharel b, Kevin R. Lynch b, Webster L. Santos a,
⇑
a Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA
b Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine-1-phosphate (S1P) is a ubiquitous, endogenous small molecule that is synthesized by two
isoforms of sphingosine kinase (SphK1 and 2). Intervention of the S1P signaling pathway has attracted
significant attention because alteration of S1P levels is linked to several disease states including cancer,
fibrosis, and sickle cell disease. While intense investigations have focused on developing SphK1 inhibi-
tors, only a limited number of SphK2-selective agents have been reported. Herein, we report our
investigations on the structure–activity relationship studies of the lipophilic tail region of SLR080811,
a SphK2-selective inhibitor. Our studies demonstrate that the internal phenyl ring is a key structural fea-
ture that is essential in the SLR080811 scaffold. Further, we show the dependence of SphK2 activity and
selectivity on alkyl tail length, suggesting a larger lipid binding pocket in SphK2 compared to SphK1.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 5 February 2015
Revised 12 March 2015
Accepted 16 March 2015
Available online 23 March 2015
Keywords:
Sphingosine kinase
SphK1
SphK2
Sphingosine-1-phosphate
S1P
O
Sphingosine 1-phosphate (S1P) is both an intermediate in the
catabolism of sphingolipids and an extracellular signaling mole-
cule. The synthesis of S1P in vivo is controlled by two isoforms of
sphingosine kinase (SphK1 and SphK2), which phosphorylate
sphingosine (Sph) to S1P. S1P is involved in a variety of important
intracellular and extracellular functions through a complex net-
work of signaling pathways including G-protein coupled receptors
S1P1–5. S1P signaling has been associated with a variety of dis-
eases including cancer, fibrosis, multiple sclerosis, and sickle cell
disease.1–4 As a result of its key role in Sph and S1P metabolism,
regulation of SphKs has attracted an increasing amount of atten-
tion as a therapeutic target. The ability to control SphK function
would also aid in the understanding of their in vivo function as well
as their effects in the sphingolipid signaling pathway.
Cl
NH2
N
O
Me
N
O
S
O
HN
ABC294640
K145
NH
⋅HCl
H2N
Me
Me
O
Me
N
N
N
Me
N
Me
N
I
VT-ME6
SLR080811
NH
⋅HCl
H2N
O
N
SLP120701
Me
N
Many differences exist between SphK1 and SphK2 including
size, cellular localization, and intracellular roles.5,6 While double
knockout studies in mice suggests that SphKs are the sole source
of S1P, some functional redundancy exists as SphK1 or SphK2 null
mice are viable and fertile. Although inhibitor development
towards SphK1 has been a focus of intense studies,7 inhibitors of
SphK2 are emerging (Fig. 1). For example, ABC294640
Figure 1. Structure of sphingosine kinase 2 inhibitors.
been deployed in a variety of disease models including lupus
nephritis, diabetic nephropathy, Crohn’s disease, ulcerative colitis,
and osteoarthritis.8,9 However, it was recently reported to inhibit
estrogen receptors in breast cancer cells by acting as a partial ago-
nist similar to tamoxifen.10 Another inhibitor, thiazolidine-2,4-
(Ki = 10 lM) was the first inhibitor with SphK2 activity that has
dione K145 (Ki = 6.4
lM), which is an analog of sphingosine was
⇑
Corresponding author. Tel.: +1 540 231 5742; fax: +1 540 231 3255.
E-mail address: santosw@vt.edu (W.L. Santos).
recently reported as a selective SphK2 inhibitor.11 K145 was shown
http://dx.doi.org/10.1016/j.bmcl.2015.03.041
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

M. D. Congdon et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4956–4960
4957
Linker
elevated S1P levels in mice. Further structure–activity relationship
studies on the guanidine core revealed that an azetidine-contain-
ing derivative SLP1201701 improved the half-life to 8 h in mice.16
In this report, we detail our investigations on the tail region of the
scaffold (Fig. 2). Our studies demonstrate that the internal phenyl
ring is essential to maintain inhibitory activity for SphK2 and that
the alkyl tail length has a significant effect on the potency and
selectivity towards SphK2.
NH
NH2
O
N
HN
Me
N
Head
Tail
Figure 2. Pharmacophore of guanidine-based inhibitors.
The synthesis of SLR080811 derivatives with varying alkyl
length as well as heterocycles attached to the phenyl ring is shown
in Schemes 1 and 2. In Scheme 1, 4-iodobenzonitrile was cross-
coupled to a series of alkynes or hydroborated intermediates under
standard Sonogashira or Suzuki–Miyaura conditions. Subsequent
reaction with hydroxylamine afforded amidoximes 2a–e, which
were cyclized to 1,2,4-oxadiazoles 3a–f in the presence of HCTU
to inhibit leukemia cell growth in vitro as well as in a xenograft
mouse model.
Due to our interest in understanding the in vivo function of
SphK2 and the lack of highly potent and selective inhibitors,12
we focused our studies in developing unique scaffolds to achieve
our goals. Our first generation inhibitor, VT-ME6, contained a qua-
ternary ammonium group as a warhead and established that a
positively charged moiety is necessary for engaging key amino
acid residues in the enzyme binding pocket.13,14 This compound
is moderately potent (Ki = 8 lM) and displays three-fold selectivity
for SphK2 over SphK1. Subsequent improvement resulted in a
scaffold that featured a 1,2,4-oxadiazole linker and guanidine as
and Boc-L-Proline. Deprotection with HCl and reduction of alkynyl
groups with tosylhydrazine at refluxing conditions yielded amines
4a–h. To install the guanidine moiety, the amines were treated
with DIEA and N,N0-Di-Boc-1H-pyrazole-1-carboxamidine for sev-
eral days at room temperature and deprotected with HCl to pro-
duce the desired derivatives 5a,d,f–h. A similar synthetic strategy
was employed to access the remaining phenyl/alkyl derivatives
(7c and 7f–g); however, heterocycles 7d–e were obtained via
Buchwald–Hartwig coupling conditions as shown in Scheme 2.
warhead: SLR080811 possesses a Ki of 13.3 lM and 1.3 lM for
SphK1 and SphK2, respectively.15 A significant finding from these
studies was that pharmacological inhibition of SphK2 resulted in
Similarly, Scheme
3
illustrates the synthesis of various
Boc
O
H
O
OH
N
N
N
N
N
f
d
CN
c
N
N
NH2
R
R
= I
R
= I
R
1a
1b
1c
1d
1e
3a
3b
3c
3d
3e
3f
= I
2a
4a = I
4b = n-CH3(CH2)7C
4c = n-CH3(CH2)9C
4d = n-CH3(CH2)13
4e = PhCH2CH2
4f = n-CH3(CH2)5
4g = n-CH3(CH2)9
4h = n-CH3(CH2)11
= n-CH3(CH2)7C
≡
≡
C
C
2b
2c
2d
2e
= n-CH3(CH2)7C
≡
C
C
≡
≡
C
C
= n-CH3(CH2)7C
≡
C
C
a
b
a, e
= n-CH3(CH2)9C
= n-CH3(CH2)13
= PhCH2CH2
= n-CH3(CH2)9C
= n-CH3(CH2)13
= PhCH2CH2
≡
= n-CH3(CH2)9C
= n-CH3(CH2)13
= PhCH2CH2
≡
e
= n-CH3(CH2)5
NH2
·HCl
HN
5a = I
O
5d
= n-CH3(CH2)13
N
N
g, f
5e = PhCH2CH2
5f = n-CH3(CH2)5
N
5g
= n-CH3(CH2)9
R
5h = n-CH3(CH2)11
Scheme 1. (a) Alkyne (2 equiv), TEA (5 equiv), DMF, PdCl2(PPh3)2 (0.05 equiv), CuI (0.03 equiv), 80 °C, 18 h, (72–93%); (b) (i) alkene, 0.5 M 9-BBN, in THF, rt, 12 h; (ii)
-Proline (1.4 equiv), DIEA (1.4 equiv), HCTU
Pd(dppf)Cl2, Cs2CO3, DMF, 70 °C, 18 h, (75–93%); (c) NH2OHꢀHCl (3 equiv), TEA (3 equiv), EtOH, 80 °C, 6 h, (43–95%); (d) Boc-
L
(1.8 equiv), DMF, 110 °C, 18 h, (25–65%); (e) DME (20 vol/wt), 4-toluenesulfonyl hydrazide (10 equiv), TEA (5 equiv), reflux, (67–71%); (f) HCl/MeOH, (35–100%); (g) DIEA
(3 equiv), N,N0-Di-Boc-1H-pyrazole-1-carboxamidine (1.05 equiv), CH3CN, rt, 3 days, (27–76%).
HN
NH2
·HCl
Boc
N
Boc
N
O
O
O
N
N
N
b, c
or d
N
a
f, g, f
2a
N
N
N
I
R
R
5
6a = n-CH3(CH2)3C
6b
6c = Ph
≡
C
C
7c = Ph
7d
= morpholine
= n-CH3(CH2)7C
≡
7e = 1-methylpiperazine
7f = n-CH3(CH2)5
6d = morpholine
e
7g = n-CH3(CH2)9
6e
= 1-methylpiperazine
6f = n-CH3(CH2)5
6g = n-CH3(CH2)9
Scheme 2. (a) Boc-L-azetidine (1.4 equiv), DIEA (1.4 equiv), HCTU (1.8 equiv), DMF, 110 °C, 18 h, (63%); (b) alkyne (2 equiv), TEA (5 equiv), DMF, PdCl2(PPh3)2 (0.05 equiv), CuI
(0.03 equiv), 80 °C, 18 h, (33–57%); (c) phenylboronic acid (1.3 equiv), Cs2CO3 (equiv.), DMF, PdCl2(dppf) (0.04 equiv), 80 °C, 18 h, (91%); (d) amine, Pd(dba)3, Cs2CO3, PtBu3,
toluene, 120 °C, 6 d, (81–83%); (e) DME (20 vol/wt), 4-toluenesulfonyl hydrazide (10 equiv), TEA (5 equiv), reflux, (60–71%); (f) HCl/MeOH, (78–96%); (g) DIEA (3 equiv), N,N0-
Di-Boc-1H-pyrazole-1-carboxamidine (1.05 equiv), CH3CN, rt, 3 days, (43–66%).

4958
M. D. Congdon et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4956–4960
amidopiperazine tail surrogates 10a–d using Buchwald–Hartwig
and amide coupling reactions.
liquid scintillation counting. The kinase inhibition was determined
as the amount of 32P-S1P produced as a function of inhibitor con-
Compounds 14 and 17 were synthesized as shown in Scheme 4.
4-(3-Ethoxymethyl)-5-methylbenzyl)benzonitrile 13 was formed
in two steps via mono-substitution of 1,3-palladium-catalyzed
cross coupling reaction with 4-cyanophenylboronic acid to afford
13. Alternatively, benzonitrile 16 was achieved using sodium ben-
zenesulfonate and 15. Standard oxadiazole formation, guanidyla-
tion, and deprotection afforded 14 and 17. Finally, a series of
alkyl tails directly linked to the oxadiazole ring were synthesized
(Scheme 5). Treatment of alkylbromides with potassium cyanide
gave alkylnitriles 19a–c, which were converted to amidoximes
20a–c. Transformation to oxadiazoles 21a–c was effected either
by HCTU-mediated cyclization at 110 °C or by two-step coupling/
TBAF-catalyzed cyclization, which eventually led to 22a–c.
With the library of putative inhibitors synthesized, the inhibi-
tory effects of the compounds were determined for hSphK1 and
centration. Compounds were screened at 10
concentrations.
lM inhibitor
As shown in Table 1, replacement of the octyl chain of
SLR080811 with iodide, phenyl or phenethyl groups did not
improve inhibitory activity (entries 1–3). Decreasing or increasing
the lipophilic alkyl tail length from hexyl to tetradecyl in two-car-
bon increments resulted in compounds with similar inhibitory
activity as SLR080811 (entries 4–10), although the hexyl chain
was slightly less active. In cases where the kinase activity was
similar to SLR080811 at 10
inhibitor concentration (1
l
M, rescreening at a more stringent
l
M) was performed: the results indi-
cated that none of these analogs had improved activity compared
to SLR080811. We also note that the pyrrolidine and azetidine
rings have been shown to have similar potency, but with the
advantage of improved in vivo half-life for the azetidine deriva-
tives.16 Interestingly, as the alkyl tail increased to a decyl group,
SphK2 selectivity decreased as SphK1 inhibition increased.
However, as the chain length increased further to a dodecyl and
tetradecyl, inhibition of SphK1 decreased while maintaining
SphK2 activity. These results suggest that the lipid binding pocket
mSphK2 using
a
previously published protocol (Table 1).16
Briefly, Sph and cell lysate containing recombinant SphK1 or
SphK2 were incubated with or without inhibitor in the presence
of c
-[32P]ATP. After 20 min, the reaction mixtures were extracted,
separated using thin layer chromatography, and quantified using
Boc
N
NH2
·HCl
HN
O
O
Boc
O
N
N
N
N
N
N
N
a
b
N
2a
c,d,c
N
N
N
N
8
R
N
HN
R
9a = isovaleryl
10a = isovaleryl
9b = 1-adamantanecarbonyl
9c = phenylacetyl
9d = benzyl
10b = 1-adamantanecarbonyl
10c = phenylacetyl
10d = benzyl
Scheme 3. (a) Piperazine, Pd2(dba)3, PtBu3, Cs2CO3, toluene, 120 °C, 3 days, (52%); (b) acid chloride, CH2Cl2, 0 °C–rt, 2 h, (66–88%); (c) HCl/MeOH, (76–95%); (d) DIEA
(3 equiv), N,N0-Di-Boc-1H-pyrazole-1-carboxamidine (1.05 equiv), CH3CN, rt, 3 days, (23–74%).
NH2
HN
·HCl
CH3
CH3
CH3
O
CH3
N
N
CN
a
c-f, e
b
N
EtO
Br
EtO
Br
Br
EtO
11
12
13
14
HN
CN
CN
N
N
O
NH
·HCl
O
O
c-f, e
g
Br
S
S
N
Ph
O
O
15
16
17
Scheme 4. (a) NaH, EtOH, 0 °C–rt, (46%); (b) 4-cyanophenylboronic acid, Pd(PPh3)4 (10 mol %), Na2CO3, THF/H2O, (94%); (c) NH2OHꢀHCl (3 equiv), TEA (3 equiv), EtOH, 80 °C,
6 h, (71–93%); (d) Boc-
-Proline (1.4 equiv), DIEA (1.4 equiv), HCTU (1.8 equiv), DMF, 110 °C, 18 h, (46–82%); (e) HCl/MeOH, (33–91%); (f) DIEA (3 equiv), N,N0-Di-Boc-1H-
L
pyrazole-1-carboxamidine (1.05 equiv), CH3CN, rt, 3 days, (53–83%); (g) sodium benzenesulfonate (1.5 equiv), DMF, 60 °C, 2 h, (89%).
NH
H2N
·HCl
Boc
OH
O
N
O
N
N
N
b
N
N
a
c or d, e
f,g,f
Br
N
R
N
R
R
NH2
R
R
18
19
21
20
22a
= octyl
22b = dodecyl
22c
= hexadecyl
Scheme 5. (a) KCN (2 equiv), 9:1 EtOH:H2O, 80 °C, 18 h, (20–93%); (b) NH2OHꢀHCl (3 equiv), TEA (3 equiv), EtOH, 80 °C, 12 h, (53–69%); (c) Boc-
(1.4 equiv), HCTU (1.8 equiv), DMF, 110 °C, 18 h, (50%); (d) Boc- -Proline (1.4 equiv), DIEA (1.4 equiv), HCTU (1.8 equiv), CH2Cl2, rt, 4 h, (57–80%); (e) TBAF (1.0 M, 1 equiv),
THF, rt, 1 h, (93–95%); (f) HCl/MeOH, (66–100%); (g) DIEA (3 equiv), N,N0-Di-Boc-1H-pyrazole-1-carboxamidine (1.05 equiv), CH3CN, rt, 3 days, (51–71%).
L-Proline (1.4 equiv), DIEA
L

M. D. Congdon et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4956–4960
4959
Table 1
Inhibitory effects of SLR080811 derivatives on SphK1 and SphK2a
NH2
HN
·HCl
O
N
N
N
R
n
Entry
R
n
% kinase activityb
SphK1 SphK2
Entry
R
n
% kinase activityb
SphK1 SphK2
⁄
⁄
I
*
1
2
5a
7c
1
0
100
101
2
1
63
91
1
12
13
22b
22c
C12H25
–
–
1
1
60
37
1
5
57
52
1
2
*
1
C16H33
3
4
5e
5f
1
1
78
1
2
70
35
7
3
14
15
7d
7e
0
0
90
97
4
2
94
90
2
3
O
N
*
*
C6H13
C6H13
*
103
N
N
*
(76 6)
45
(86 6)
2
5
6
7f
0
1
94
60
18
2
1
9
*
89
2
89
3
16
10d
1
N
N
N
N
*
*
SLR
080811
9
4
C8H17
*
(44 4)
17
18
10a
10c
1
1
79
90
2
2
90
86
2
2
O
9
7
7
5g
1
C10H21
*
(64 4)
(46 5)
N
N
*
O
C10H21
C12H25
*
*
8
9
7g
5h
0
1
11
37
4
2
12
15
2
1
19
10b
1
81 28
87
46
3
9
N
N
*
O
CH
3
(82 3)
(56 1)
*
20
21
14
17
1
1
96
99
1
1
(99 3)
EtO
75
(88 3)
1
16 10
(52 4)
10
11
5d
C14H29
*
1
1
*
88
2
S
O
O
⁄
22a
C8H17
–
96
1
88
1
a
Values represent percent activity of human SphK1 or mouse SphK2 with 10 and 5
experiments. Lower SphK activity level indicates better inhibition.
lM Sph, respectively, in the presence of 10 lM inhibitor. Each value is an average of two
b
Values in parenthesis indicate compounds assayed at 1 lM.
in SphK2 is much larger than that of SphK1 and is consistent with
the prediction based on a crystal structure of SphK1 bound to
SphK1 inhibitor PF-543.17 We next investigated the effect of the
phenyl substituent next to the 1,2,4-oxadiazole ring. Removal of
this ring while maintaining the overall length of the molecule
resulted not only in diminished SphK2 selectivity but also inhibi-
tory activity (entries 11–13). Our data indicate that the phenyl
ring is necessary for selectivity and potency using this scaffold.
To further determine features of the lipid binding pocket, mor-
pholine and a series of heterocyclic rings were synthesized (entries
14–19). In particular, a piperazine ring is attractive because of
increased conformational rigidity as well as an anchor point in
which various groups can be appended. Morpholine, N-methyl or
N-benzyl piperazine derivatives were inactive. As these sub-
stituents are positively charged and the likelihood that the lipid
binding pocket is lined with hydrophobic groups, neutral amide
versions with increasing steric bulk were tested. Isovaleryl, phena-
cetyl, and adamantylcarbonyl groups were also inactive. Finally,
trisubstituted aryl 14 as well as sulfonate 17 bearing groups, fea-
tured in SphK1 inhibitor PF-543, were tested and also found to
be poor inhibitors (entries 20–21).18
of the pharmacophore were synthesized. Our studies demonstrate
the dependence of SphK2 inhibitory activity on alkyl chain length;
the most optimal length includes octyl and decyl substituents,
which suggests an ideal ‘head-to-tail’ (positive charge to terminal
methyl group) length of approximately 18–21 atoms.
Furthermore, our studies provide evidence for the much larger
lipophilic binding cavity in SphK2 over SphK1. In the SLR080811
scaffold, the internal phenyl ring appears to be essential for activity
and is likely interacting with residues in the kinase binding pocket.
These predictions can be aided by a SphK2 crystal structure, which
is currently unavailable.
Acknowledgments
We acknowledge financial support by NIH (Grants R01
GM104366 and R01 GM067958).
Supplementary data
Supplementary data (characterization data for final com-
pounds) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmcl.2015.03.041.
In summary, a focused library of SphK2-selective inhibitor
SLR080811 derivatives that interrogated the lipophilic tail region

4960
M. D. Congdon et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4956–4960
10. Antoon, J. W.; White, M. D.; Meacham, W. D.; Slaughter, E. M.; Muir, S. E.;
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