Design, synthesis and analysis of novel sphingosine kinase-1 inhibitors to improve oral bioavailability

Article abstract of DOI:10.1016/j.bmcl.2021.128329

The sphingomyelin pathway is important in cell regulation and determining cellular fate. Inhibition of sphingosine kinase isoform 1 (SK1) within this pathway, leads to a buildup of sphingosine and ceramide, two molecules directly linked to cell apoptosis, while decreasing the intracellular concentration of sphingosine-1-phosphate (S1P), a molecule linked to cellular proliferation. Recently, an inhibitor capable of inhibiting SK1 in vitro was identified, but also shown to be ineffective in vivo. A set of compounds designed to assess the impact of synthetic modifications to the hydroxynaphthalene ring region of the template inhibitor with SK1 to obtain a compound with increased efficacy in vivo. Of these fifteen compounds, 4A was shown to have an IC₅₀ = 6.55 μM with improved solubility and in vivo potential.

Full text of DOI:10.1016/j.bmcl.2021.128329

Bioorg. Med. Chem. Lett. 50 (2021) 128329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and analysis of novel sphingosine kinase-1 inhibitors to
improve oral bioavailability
Kendarius J. Butlera, Angel A. Castroa, Tiffany S. Dwyera, Louise M. Hardwicka,
Melody C. Iacinoa, Sara G. Manorea, Kevin M. Maysa, Caylie A. McGladea, Lisa N. Haira,
Erin W. Parkera, Mikala R. Smitha, Morgan T. Turnowa, Matthew R. Wilsona,
Stephanie R. Woodsona, William E. Cothamb, Michael D. Wallab, Jason C. Hurlberta,
,
*
T. Christian Grattana
a Winthrop University, Department of Chemistry, Physics and Geology, SIMS Building Rm 101, Rock Hill, SC 29733, United States
b University of South Carolina, Department of Chemistry and Biochemistry, GSRC Rm 108M, Columbia, SC 29208, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The sphingomyelin pathway is important in cell regulation and determining cellular fate. Inhibition of sphin-
gosine kinase isoform 1 (SK1) within this pathway, leads to a buildup of sphingosine and ceramide, two mole-
cules directly linked to cell apoptosis, while decreasing the intracellular concentration of sphingosine-1-
phosphate (S1P), a molecule linked to cellular proliferation. Recently, an inhibitor capable of inhibiting SK1
in vitro was identified, but also shown to be ineffective in vivo. A set of compounds designed to assess the impact
of synthetic modifications to the hydroxynaphthalene ring region of the template inhibitor with SK1 to obtain a
Sphingosine kinase
Kinase inhibition
Bioorganic synthesis
Microwave synthesis
compound with increased efficacy in vivo. Of these fifteen compounds, 4A was shown to have an IC50 = 6.55
with improved solubility and in vivo potential.
μM
Targeted therapy is a developing form of cancer treatment capable of
interfering with specific molecular entities involved in cell growth and
survival.1 Traditionally, chemotherapeutic agents attack all actively
dividing cells, whether they are cancerous or not. Targeted therapies
have the ability to attack cancer cells and specific pathways within
them, doing little to no harm to the surrounding healthy cells.2 The
research described herein focuses on the sphingomyelin pathway
(Fig. 1), which is important in cell regulation, signaling, and cell fate.3
One of the therapeutic targets of interest in this pathway is the
phosphorylation of sphingosine into sphingosine-1-phosphate (S1P),
catalyzed by sphingosine kinase isoform 1 (SK1). Inhibition of SK1 is
important because S1P has been shown to promote cell proliferation,
angiogenesis, and inflammation. SK1 has also been shown to be over-
expressed in cancer cells.4 Inhibition of SK1 should result in a decreased
cellular concentration of S1P and a buildup of ceramide and sphingo-
sine, two molecules linked to apoptosis.5 Because of this, SK1 provides a
potential target for new anticancer targeted therapeutics.6,7
I), which showed success against SK1 in vitro, but, in a follow-up study,
not in vivo3, presumably due to its low solubility. The objective of this
work focuses on the design and synthesis of potential inhibitors that
assess modifications to the hydroxynaphthalene ring (Fig. 2) that may
improve the in vivo effectiveness as SK1 inhibitors.
Due to the complexity of the SKI-I template structure, several po-
tential areas of chemical modification are available, and we have
focused our initial efforts on making synthetic modifications to the
hydroxynaphthalene ring (Fig. 2), with a specific goal of decreasing the
theoretical log P value of the compound thereby increasing its aqueous
solubility and in vivo activity. Log P values are good predictors of a
compound’s ability to penetrate through the lipid bilayer, a component
of the cellular membrane which acts as a barrier for a cell.10–12 SKI-I has
a calculated log P value of 5.613, which is too high to be effectively
solubilized and circulated through the serum. Candidate modifications
made to the SKI-I hydroxynaphthalene ring template were designed to
improve the hydrophilic nature of the structure by as much as four times
relative to SKI-1 which would give a calculated log P value of at least
5.0.
In this study, modifications to a known SK1 inhibitor are being
investigated (Fig. 2). Initial research conducted by Smith et al.4 led to the
discovery of a template compound, sphingosine kinase inhibitor-I (SKI-
We designed a library of candidate compounds and obtained initial
* Corresponding author.
https://doi.org/10.1016/j.bmcl.2021.128329
Received 7 May 2021; Received in revised form 16 July 2021; Accepted 11 August 2021
Available online 19 August 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

K.J. Butler et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128329
(LifeTechnologies Corporation, Madison, WI) to determine IC50
values.17 The compounds were assayed at a starting concentration of 10
μ
M in 1% DMSO with threefold serial dilutions at [Km,app] ATP, which
was conducted at 100 μM ATP. These inhibitors were evaluated against a
known inhibitor of sphingosine kinase 1, PF-54319, which has a pub-
lished Ki of 4.3 nM.
The bioavailability of SKI-1 is a major concern, likely preventing an
effective dosage from accumulating in the blood stream and reaching the
target sites, and may be responsible for its lack of success in vivo.10 With
increased hydrophilicity and improved enzyme binding, we hypothesize
that these inhibitors will be more readily bioavailable and potent in vivo.
The binding of each new inhibitor was assessed comparatively to the
binding of SKI-I. These results are provided in Table 1 along with the
synthetic yield, in silico docking energies and calculated log P values.
The synthesis of SK1 inhibitors was accomplished in three steps
(Schemes 1 – 3), adapted from Sharma et. al.8 The synthesis of 2 was
done via a Claisen-like reaction in the microwave reactor with 1 (1
equiv), sodium ethoxide in ethanol (21%) (1.5 equiv) and diethyl oxa-
late (1.2 equiv) (Scheme 1), reacting to a very high yield of 96%.20
Previous efforts involved running this reaction overnight while the
method described has reduced that time down to one hour, dramatically
improving the reaction efficiency while consistently maintaining very
high yields, >90%, which is a significant improvement over previous
methodology.8
Fig. 1. Stress induced degradation of sphingomyelin, a component of the cell
membrane, to form proapoptotic ceramide and sphingosine as well as prolif-
erative sphingosine-1-phosphate.
Synthesis of 3 was adapted from Sharma et al8 using a 50% hydrazine
hydrate solution (Scheme 2). This reaction is traditionally performed
under reflux for 6 h. Substituting the microwave in this step resulted in a
99% yield21 after just one hour, which is an improvement over previous
work based on yield alone with a substantial reduction in reaction time.
The final step was prepared by reacting 3 with an appropriate
aldehyde derivative via an imine formation (Scheme 3)22. The aldehyde
derivatives are responsible for the modifications and were chosen in an
attempt to assess improvements in the hydrophilicity of the final in-
hibitors and potentially the in vivo potency. Log P values for the in-
hibitors are mostly lower than SKI-1 (Table 1) due to these modifications
with improved bioavailability.
Fig. 2. The template inhibitor (SKI-I) compound by Smith et al.4. The area of
modification in the current work is shown in the brackets.
estimates of their efficacy via in silico docking experiments using the
proposed derivatives and the known crystal structure of an inhibitor
bound form of the enzyme (PDB ID: 3VZB).9 The inhibitor compounds
were prepared following a modified method8 using microwave synthesis
to improve time and overall yield. These purified derivatives were then
submitted for bioassay testing to assess whether improvements were
made in lipid bilayer absorption and penetration and ultimately
increased inhibition success rates in vivo.
A total of fifteen potential SK1 inhibitors have been synthesized
using this process. These derivatives were all formed in good to high
yields with high purity. While the yields are an improvement, the
reduction in overall reaction time from two overnight reactions plus a 6
h reflux to approximately 4 reaction hours overall is perhaps one of the
most significant aspects of this research. Percent yields for the final re-
actions, aldehyde derivatives, and calculated log P values are included
(Table 1). Table 1 also includes the in silico binding energy values
calculated with AutoDock Vina and Thermo Fisher Scientific’s Select-
ScreenServicesTM assay results compared to PF-543.
Previously, the synthetic scheme for the SKI-I template and its de-
rivatives followed a traditional three step process, however this work
performed all three synthetic steps in a microwave reactor. These
modified inhibitors are synthesized using microwave-assisted organic
synthesis (MAOS) to improve yields and promote cleaner reactions in a
timely manner which have become more popular as safe alternatives to
traditional synthetic strategies with little to no environmental impact
and greater efficiency of the reactions.14–17 Utilizing MAOS to synthe-
size inhibitors with improved hydrophilicity decreasing the overall re-
action time while simultaneously increasing purity and product yields.
Using MAOS, a total of fifteen SK1 inhibitors with modifications to the
SKI-I hydroxynaphthalene ring moiety have been synthesized success-
fully and are described herein.
Observations of the crystal structure of human sphingosine kinase in
complex with sphingosine (PDB ID: 3VZB), Fig. 3A, Fig. 3B, reveal a
distinctly cylindrical binding pocket bounded by Phe288 and His311 at
one end, the hydrophobic main body of the pocket defined by amino
acids Ile174, Val177, Thr196, Leu 268 and Met272 and the end closest to
the ATP binding pocket by Ser168. The C-2 amino group of the bound
sphingosine substrate forms a hydrogen bond with the hydroxyl oxygen
of Ser168, serving to orient the C-1 hydroxyl towards the ATP binding
site of the enzyme. Using the dimensions of this sphingosine binding site
as the boundaries for the in silico studies, docking of the SKI-2 molecule
In silico docking: The x-ray crystallographic structure of sphingosine
kinase isoform 1 in complex with sphingosine (PDB ID: 3VZB) was used
as a base structure for all docking runs. SKI-I and design analogues were
built in MarvinSketch.18 Proteins and ligands were converted to PDBQT
files using the DockPrep module of Chimera and were submitted for
molecular docking with Autodock Vina using the dimensions of the
sphingosine binding pocket of SK1 as the receptor. The results of the
docking calculations were subsequently viewed in the ViewDock mod-
ule of Chimera.
˜
into the crystal structure of hSK1 resulted in a less than 0.5 A… RMSD
between the location of the bound, crystallized SKI-2 molecule and the
placement of the same molecule in the binding site by Autodock Vina,
thereby validating the method.9
Of the eight interactions observed between sphingosine and SK1 in
the crystal structure, seven are hydrophobic in nature, Fig. 3A, an
observation that complicates the design of targeted inhibitors with
increased hydrophilicity. Inhibitors 4F and 4 K have log P values
matching that of the template inhibitor and were synthesized to deter-
mine the role of the hydroxyl group on the naphthyl ring interacting
In vitro studies: The inhibitors were tested in biochemical kinase ac-
tivity assays using the Invitrogen SelectScreen Kinase Profiling Service
2

K.J. Butler et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128329
Table 1
Final reaction aldehyde derivatives, percent yieldsa and Log P values.
R1
N
NH
N
NH2
NH
N
NH
NH
O
R
H+
H
+
O
O
EtOH
80 oC, 2 h
microwave
3
4
Inhibitor
Aldehyde Derivative (R)
Yield (%)
Binding Energy with SK1
(kcal/mol)b
Mean SinglePoint(% inhibition)c
IC50
(μ
M)c
Log P valued
[Std. Dev (n ¼ 4)]
4A
4B
3,4-dimethoxybenzaldehyde
99
68
80
72
58
64
92
85
69
47
62
47
98
91
92
88
ꢀ 11.2
ꢀ 11.9
ꢀ 11.2
ꢀ 11.1
ꢀ 10.8
ꢀ 12.5
ꢀ 11.2
ꢀ 11.2
ꢀ 10.5
ꢀ 11.0
ꢀ 12.3
ꢀ 11.0
ꢀ 10.9
ꢀ 11.4
ꢀ 11.4
ꢀ 12.4
57 [9.9]
1 [7.9]
6.55
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.222
4.466
4.632
4.584
4.608
5.740
4.072
4.072
4.516
4.736
5.735
4.580
3.340
5.253
4.510
5.675
1,4-benzodioxan-6-carboxaldehyde
p-methoxybenzaldehyde
o-methoxybenzaldehyde
m-methoxybenzaldehyde
2-methoxynaphthaldehyde
p-hydroxybenzaldehyde
m-hydroxybenzaldehyde
o-hydroxybenzaldehyde
cyclohexane carboxaldehyde
1-naphthaldehyde
4C
ꢀ 2 [5.4]
4 [4.9]
4D
4E
ꢀ 3 [7.5]
6 [4.2]
4F
4G
4H
4I
17 [7.9]
10 [3.0]
ꢀ 6 [6.2]
ꢀ 9 [12.7]
ꢀ 7 [8.6]
10 [3.5]
18 [5.0]
ꢀ 15 [8.3]
ꢀ 2 [3.8]
3 [2.2]
4 J
4 K
4L
benzaldehyde
4 M
4 N
4O
SKI-1
3-pyridinecarboxaldehyde
p-chlorobenzaldehyde
m-nitrobenzaldehyde
2-hydroxynapthaldehyde
a
b
c
Reagents and conditions: Pyrazole/hydrazide (1 mmol), aldehyde (1 mmol), ethanol (3 ml), 12 M HCl (1 drop).
Binding energy values recorded from Auto Dock Vina with 3vzb model.
Data obtained from Thermo Fisher Scientific SelectScreenTM Profiling Service, SSBK-Adapta, Madison, WI.
Log P values calculated using www.molinspiration.com.13
d
Scheme 1. Claisen-like reaction to produce compound 2.
to Ser168, an amino acid integral to sphingosine binding in the active
site. This flip may explain the lower binding energy values for the in-
hibitors without the retained naphthalene ring.
Within the enzyme binding pocket, there are eight amino acids that
can interact with these new inhibitors. Compared to SKI-I, five new in-
hibitors scored better in the single point inhibition assay and in each of
these structures the calculated log P values are 4.58 or below. This in-
dicates that the drugs may have reoriented in the binding site, as pre-
dicted by the docking study leading to a higher efficacy of inhibition
during in vivo testing as they have in these in vitro studies.
Scheme 2. Pyrazole and hydrazide formation of compound 3.
with the enzyme. Despite the increased hydrophobic character of the
two compounds, both showed improved interactions with SK1 based
upon the docking studies, but did not inhibit sphingosine kinase 1
significantly better than SKI-1 in the in vitro kinase assay (Table 1).
Closer examination of these compounds, in silico, shows a preference for
the naphthalene in the modified area remaining in the hydrophobic
pocket, while in the other examples tested removal of this naphthalene
ring flips the inhibitor within the binding site placing the “zone 1”
naphthalene ring in this region (Fig. 3B and 3D). This results in a new
orientation of the structure, where the modified ring is in close contact
The five best inhibitors based on the single point titration assay data
(4A, 4G, 4 M, 4L and 4H) show strong interactions within the binding
pocket with the primary improvement being the interaction with the
polar side chain, Ser168, as well as the backbone interactions with
Thr196. The best inhibitor using both the single point and IC50value, 4A,
has the methoxy group in the meta position which places it in close
proximity (2.540 Å) to Ser168 for a more stabilizing binding interaction
R1
N
NH
N
NH2
NH
N
NH
NH
O
R
H+
H
+
O
O
EtOH
3
4
Scheme 3. Acid catalyzed imine formation to yield the final derivatives.
3

K.J. Butler et al.
Bioorganic & Medicinal Chemistry Letters 50 (2021) 128329
Fig. 3. Ligands occupying the binding pocket of SK1. A) Crystallographic structure of sphingosine bound to the enzyme surrounded by hydrophobic side chains; B)
Crystallographic structure of SKI-1 bound to the enzyme. C) Crystallographic structure of SKI-2 bound to the enzyme; D) Best conformer of compound 4A in the active
site of SK1 as determined by Autodock Vina. E) Best conformer of compound 4G in the active site of SK1 as determined by Autodock Vina.
with SK1 (Fig. 3D). There are also two potential interactions between
the Thr196 and the vinyl pyrazole nitrogen/carbonyl oxygen. For the 4G
derivative, the meta-hydroxy group interacts with Asp81 (2.646 Å) along
with interactions between the pyrazole nitrogens and the Thr198 (Fig.
3E). 4 M, 4L and 4H also show comparable interactions with Ser168 and
Thr198 as an explanation to their improved inhibition assay results.
To validate whether or not the correct products were produced,
proton and carbon NMR were performed along with IR and high-
resonance mass spectrometry. Carbon NMR spectra were extremely
complex due to the number of peaks in the aromatic region of the spectra
so attached proton test (APT) carbon NMR was performed to provide
greater spectral clarity. One interesting result that was noted for a
number of derivatives in the NMR spectra was the potential for geo-
metric isomers to be formed during the final step. This was especially
true for the methoxy derivatives (4C, 4D, 4E and 4F) where two peaks
appeared in the APT spectra in the methyl region (55 ppm) identifying
geometric isomerization.
Acknowledgments
We would like to acknowledge the funding and support provided by
an NIH-INBRE grant from the National Center for Research Resources
and the National Institute for General Medical Sciences (8 P20
GM103499), Winthrop University Research Council grants (529230,
529231, SC 10011, 381118-2555-200) and the Winthrop University
Department of Chemistry, Physics and Geology.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128329.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4

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5