Synthesis and biological evaluation of sphingosine kinase 2 inhibitors with anti-inflammatory activity

Article abstract of DOI:10.1002/ardp.201800298

The synthesis of inhibitors of SphK2 with novel structural scaffolds is reported. These compounds were designed from a molecular modeling study, in which the molecular interactions stabilizing the different complexes were taken into account. Particularly

Full text of DOI:10.1002/ardp.201800298

Received: 8 October 2018
Revised: 28 November 2018
Accepted: 5 December 2018
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DOI: 10.1002/ardp.201800298
FULL PAPER
Synthesis and biological evaluation of sphingosine kinase 2
inhibitors with anti-inflammatory activity
1
2,3
4
5
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Marcela Vettorazzi
Laura Vila
Santiago Lima
Lina Acosta
5
5
6
7
8
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Felipe Yépes
Alirio Palma
Justo Cobo
Jan Tengler
Ivan Malik
1
8
2,3
2,3
2,3
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Sergio Alvarez
Patrice Marqués
Nuria Cabedo
María J. Sanz
4
1
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Josef Jampilek
Sarah Spiegel
Ricardo D. Enriz
1
Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Instituto Multidisciplinario de Investigaciones Biológicas (IMIBIO-SL), San Luis,
Argentina
2
Department of Pharmacology, University of Valencia, Valencia, Spain
3
Institute of Health Research INCLIVA University Clinic Hospital of Valencia, Valencia, Spain
4
Department of Biology and Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia
5
Laboratorio de Síntesis Orgánica, Escuela de Química, Universidad Industrial de Santander, Bucaramanga, Colombia
6
Inorganic and Organic Department, University of Jaén, Jaén, Spain
7
Medis International a.s., Bolatice, Czech Republic
8
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Comenius University, Bratislava, Slovakia
Correspondence
Abstract
Dr. Ricardo D. Enriz, Facultad de Química,
Bioquímica y Farmacia, Universidad Nacional de
San Luis, Instituto Multidisciplinario de
Investigaciones Biológicas (IMIBIO-SL),
Chacabuco 915, 5700 San Luis, Argentina.
Email: denriz@unsl.edu.ar
The synthesis of inhibitors of SphK2 with novel structural scaffolds is reported. These
compounds were designed from a molecular modeling study, in which the molecular
interactions stabilizing the different complexes were taken into account.
Particularly interesting is that 7-bromo-2-(2-phenylethyl)-2,3,4,5-tetrahydro-1,4-
epoxynaphtho[1,2-b]azepine, which is a selective inhibitor of SphK2, does not exert
any cytotoxic effects and has a potent anti-inflammatory effect. It was found to
inhibit mononuclear cell adhesion to the dysfunctional endothelium with minimal
impact on neutrophil–endothelial cell interactions. The information obtained from
our theoretical and experimental study can be useful in the search for inhibitors of
SphK2 that play a prominent role in different diseases, especially in inflammatory and
cardiovascular disorders.
Funding information
Universidad Nacional de San Luis; Colombian
Institute for Science and Research,
Grant number: 110265842651; CONICET-
Argentina; Slovak Research and Development
Agency, Grant number: APVV-0516-12;
SANOFI-AVENTIS Pharma Slovakia; Spanish
Ministry of Economy and Competiveness,
Grant numbers: SAF2014-57845R, SAF2017-
89714-R, CP15/00150; Carlos III Institute of
Health (ISCIII); European Regional Development
Fund; Spanish Ministry of Innovation and
Competitiveness
K E Y W O R D S
anti-inflammatory activity, bioassays, molecular modeling, sphingosine kinase 2 inhibitors,
synthesis
[1,2]
1
|
INTRODUCTION
SphK2).
S1P regulates many important physiological functions;
however, it also has a pathological role in autoimmune dysfunction,
[
3–7]
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator
that is synthesized by two isoforms of sphingosine kinase (SphK1 and
inflammation, cancer, and many other diseases.
Most of its actions
are mediated by binding and signaling through a family of five G
|
1
Arch Pharm Chem Life Sci. 2019;1–14.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft

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VETTORAZZI ET AL.
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protein-coupled receptors (S1PR1-5) leading to downstream signaling
2
|
RESULTS AND DISCUSSION
Evaluating SphK2 inhibitory activity
[8,9]
important in inflammation, immunity, and cancer.
Although SphK1 and SphK2 share a high degree of homology, they
differ in molecular weight, tissue distribution, and subcellular
2.1
|
We first evaluated the SphK2-inhibitory activity of the 16 compounds
[10]
localization.
shares no homology with SphK1.
been focused on SphK1 due to its strong link to cancer and
Specifically, SphK2 has an additional domain that
[
25]
selected from our previous screen of SphK1 inhibitors.
Synthesis
[11]
Most of the research to date has
and structural characterization of compounds 1–16 has been
[25,26]
described
and their structures are shown in Table 1. SphK2
[
12–14]
inflammatory diseases.
The structural data reported by Wang
inhibitors were evaluated in a 384-well high-throughput format assay
and coworkers in 2013 on the active site of SphK1 has contributed to
the understanding of its mechanism of action and development of
[27]
as described.
Of these compounds, only three showed significant
SphK2 inhibitory activity: compounds 2, 4, and 13 (IC50 values lower
than 200 μM) (Figure 1a).
[15]
SphK1 inhibitors.
Numerous inhibitors of SphK1 have been
[16]
reported, including very potent ones, such as PF-543.
much less is known regarding SphK2.
In contrast,
Compound 2, which had the highest inhibitory effect on SphK1
(
IC50, 12 μM), also showed the strongest inhibitory activity against
Attention on SphK2 increased greatly after the discovery of the
immunosuppressant drug FTY720/Fingolimod. It is a prodrug that is
phosphorylated in vivo by SphK2 to its active form FTY720-
SphK2 (IC50, 27.8 μM). In turn compounds 4 and 13 displayed 56.8 and
1
40 μM inhibitory activity, respectively.
It is important to note that we have previously reported that K145
[
17]
phosphate, a mimetic of S1P that modulates S1PR functions.
In
[
27]
suppressed SphK2 activity with an IC50 of 33.7 µM,
which is
addition, intracellular S1P generated by SphK2 is an endogenous
comparable with the IC50 value determined with the conventional
[9]
[18]
inhibitor of histone deacetylases, stabilizes telomerase
and in
[23]
radioactive assay.
Thus, it is possible to consider that the inhibitory
[
19]
mitochondria, binds to prohibitin 2.
These studies indicate that
effects found for compound 2 are comparable to that reported for
K145 and therefore very significant. However, to search for a new
SphK2 inhibitor with a novel structural scaffold highly selective for
SphK2, we next carried out a molecular modeling study using starting
structures based on compounds 2, 4, and 13.
SphK2 is involved in epigenetic regulation, aging, and mitochondrial
respiratory complex function. Furthermore, SphK2 also regulates IL-
[
20]
2
pathways in T cells.
Therefore, it has been suggested that
inhibitors of SphK2 may have therapeutic utility in inflammatory
[20]
and/or autoimmune diseases.
However, despite these advances,
The assay used for screening SphK compounds has been
much remains still unknown, regarding the physiological and
pathological roles of this SphK isoenzyme. In order to better
understand functions of SphK2 in autoimmune/inflammatory
disease, there is a need to develop SphK2 inhibitors with selectivity
over SphK1.
extensively characterized and demonstrated to reliably reproduce
[27]
IC50s for well-known SphK inhibitors.
However, in screening assays
we used 50 μM K145 positive control for SphK2 inhibition. As shown
in Supporting Information Figure S1, no SphK2 activity was observed
under these conditions.
In contrast to SphK1, for which a wide number of inhibitors
have been developed, very few potent inhibitors of SphK2 have
been described and most of them only displayed moderate activity
against SphK2. Moreover, the vast majority of them are not SphK2
specific, thus further complicating the interpretation of their in
vitro and in vivo effects. The most well-known isotype specific
2
.2
|
Molecular modeling
We conducted a molecular simulation study to identify the critical
molecular interactions between compounds 2, 4, and 13 with active
site residues of SphK2. Since no crystal structure is currently available
for SphK2, we generated a structural model of SphK2 using
[
21]
[22]
inhibitors of SphK2 are: ABC294640,
(R)-FTY720-OMe,
[
23]
[24]
K145,
up to now.
We have recently described two types of structural scaffolds
and SLM6031434
one of the most potent reported
[28]
MODELLER,
a comparative protein structure modeling program,
using the structure of SphK1 (Homo sapiens) (PDB ID: 3VZB, 3VZC,
[15,29,30]
[
25]
for developing new SphK1 inhibitors.
These new compounds
3VZD, 4L02, 4V24) as a template.
were obtained through virtual screening, being the most active
compounds of these series (molecules 2–4 (Table 1)). Considering
there are relatively few inhibitors of SphK2 reported, we were
interested in determining whether some of the compounds in our
screen with moderate or no activity against SphK1 would inhibit
SphK2. Thus, in the current study we report the inhibitory activity
on SphK2 and a molecular modeling approach that allowed us to
design, synthesize, and evaluate the in vitro and in vivo effects of a
new SphK2 inhibitor. Furthermore, we have also examined the
potential anti-inflammatory effect of the most active compounds in
this series.
The main objective of this study was to assess the interactions that
stabilize or destabilize receptor-ligand (R-L) complexes. As a reference,
[23]
we also included the SphK2-isotype specific compounds K145
and
[24]
SLM6031434
that are strong and selective SphK2 inhibitors. It was
expected that this approach would produce a comparative analysis of
different SphK2 inhibitory activities of these molecules in relation to
their structural differences (Figure 2).
The molecular modeling study was conducted in three different
stages. First, a docking analysis was carried out using the AutoDock
[31]
program.
In the second stage, molecular dynamics (MD) simulations
[32]
were made using the AMBER software package.
From the

VETTORAZZI ET AL.
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[25]
TABLE 1 Structural features of compounds selected to evaluate SphK inhibitory effect
IC50 (μM)
Compounds
1
Structure
SphK1
SphK2
>650
>650
2
12
28
3
= −m-NHCOO(CH
2
)
3
CH
3
60
55
>650
4
5
= −p-NHCOO(CH
2
)
3
CH
3
57
>
650
>650
=
>
650
>650
>650
6
7
=
= −CH
3
>650
8
9
1
= −CH
2
CH
3
>650
>650
>650
>650
>650
>650
= −(CH
)
2 3
CH
3
0 = R
1
=−CH
3
R
2
=−(CH
2
)
3
CH
3
1
1
1
1 = R
1
=−(CH
=−(CH
2
)
)
3
CH
CH
3
>650
>650
>650
>650
>650
140
R
2
2
3
3
2 = R
1
=−CH
=−(CH
3
R
2
2
)
2
CH
3
3 = R
1 2 3
=−(CH) CH
R
2
=−p-OCH
3
14 = R
1
=−(CH
2
)
2
OCH
3
>650
>650
R
2
=−(2,6-dimethoxy)
(
Continues)

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VETTORAZZI ET AL.
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TABLE 1 (Continued)
IC50 (μM)
Compounds
Structure
SphK1
SphK2
1
5 = −o-O(CH
2
)
2
OCH
3
>650
>650
1
6 = −p-O(CH
2
)
3
CH
3
>650
>650
trajectories obtained with the MD simulations, a per-residue analysis
was performed for each compound.
be concluded that these compounds bind in a similar manner to K145
as they essentially interact with the same SphK2 residues. Based on
these results, we sought to obtain compounds that were structurally
related to compounds 2, 4, and 13, and had closely matching simulated
SphK2 binding histograms.
The docking analysis indicated that all compounds fit in the binding
pocket of SphK2 (Figure 3). Furthermore, the docking analysis and MD
simulations suggested that these compounds bind in a very compara-
ble manner and interact with similar amino acids. Per-residue analysis
helped to define the main interactions that stabilize the different
complexes (Figure 4). In general, the new SphK2 inhibitors described
here displayed the pharmacophoric portion at the active site in a similar
Furthermore, it is interesting to note that compound 3 is selective
for SphK1 with an IC50 of 60 μM, while compound 4 has an IC50 of 55
and 56.8 μM for SphK1 and SphK2, respectively (see Table 1).
Although these compounds are isomers, our docking study shows that
they are located very differently in the active site of the protein
(Supporting Information Figure S4). This could give us an indication
about why one is selective while the other is not.
[23]
manner to K145.
It should be noted that SLM6031434 is more active and selective
on SphK2, however, our docking studies and MD simulations indicate
that this compound does not bind in a similar way to K145 nor to our
compounds (Supporting Information Figure S2). Thus, for the
molecular modeling studies, K145 was taken as the reference
compound.
The docking was done using the AutoDock program, using a box
centered on the active site, with a size of 30 × 64 × 24 Å. Two hundred
conformations were obtained, of which the lowest energy was
graphed for each case.
MD simulations indicate that residues participating in the
stabilization of inhibitors–enzyme interactions are: Phe339, Val340,
Phe358, Leu540, Leu542, Leu549, Cys569, Phe584, Met587, His592,
and Leu600. Similar histograms were obtained for compounds 2, 4
2
.3
|
Design of novel SphK2 inhibitors
Several synthesizable compounds with modifications on the basic
structures of compounds 2 and 13 structures were simulated in the
docking analysis and MD computations. Two aspects were considered
for the selection of the candidate compounds. On one hand, a
(
Figure 4c and d), and 13 (Supporting Information Figure S3). It should
be noted that MD simulations predicted conservation between amino
acids stabilizing compounds 2, 4, and 13 with SphK2. Therefore, it can
FIGURE 1 SphK2 percent inhibition versus concentration plot for the compounds 2, 4, 13 (a), 17 and 18 (b)

VETTORAZZI ET AL.
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Of 12 potential SphK2 inhibitors (structures shown in Supporting
Information Table S1), only 7-bromo-2-(2-phenylethyl)-2,3,4,5-tetra-
hydro-1,4-epoxynaphtho[1,2-b]azepine (17) and N-[2-(4-fluorophe-
noxy)ethyl]-2-hydroxy-3-({4-[(propoxycarbonyl)amino]benzoyl)-oxy)-
propan-1-aminium chloride (18) (Figure 2) behaved similarly in our
simulations as known SphK 2 inhibitors. As shown in Figures 5 and 3
(
for compound 17), SphK2-active binding site was very similar to those
obtained for compounds 2, 4 (Figure 4c and d), and 13 (Supporting
Information Figure S3), as they were to the simulations with the
reference compound, K145 (Figure 4a).
2.4
|
Synthesis and inhibition studies for compounds
17 and 18
Compounds 17 and 18 were synthesized and evaluated for their
inhibitory activity against SphK2. Compound 17 was synthesized as
[33]
previously reported,
and involved the selective oxidation of the
2-allyl-4-bromo-N-(3-phenylpropyl)naphthalen-1-
corresponding
amine with an excess of hydrogen peroxide solution in the presence
of catalytic amount of sodium tungstate. The subsequent internal 1,3-
dipolar cycloaddition of the resulting nitrone toward the terminal CC
bond of the pendant allylic fragment connected to the ortho position
gave the tricyclic 1,4-epoxycycloadduct 17 (Scheme 1). In these
conditions, the thermal induced intramolecular 1,3-dipolar cycloaddi-
tion of the intermediate nitrone was entirely stereoselective leading to
the exclusive formation of the 2-exo-cycloadduct, as demonstrated by
FIGURE 2 Structural features of K145, SLM6031434 and
compounds 17 and 18. mSphK1 and mSphK2 correspond to mouse
sphingosine kinase 1 and 2, respectively. All other determinations
were performed on human cells
1
H NMR spectroscopy. The stereochemistry was deduced from
chemical shift and coupling constant values of protons at the 1,4-
epoxytetrahydroazepine ring protons (see Section 4), and especially
because of the absence of correlation between tertiary 2-H and 4-H
protons in the NOESY spectrum. Compound 17 was obtained as a
racemic mixture of (2R,4S) and (2S,4R) forms.
limitation was that the proposed compound was available in our
laboratory or that it could be synthesized without major problems for
us. The other condition was that it had a certain structural similarity (at
least considering its central scaffolding) with compounds 2, 4, and 13.
N-[2-(4-Fluorophenoxy)ethyl]-2-hydroxy-3-({4-[(propoxycar-
bonyl)amino]benzoyl}oxy)propan-1-aminium chloride (18) was syn-
thesized by the multiple-step reactions shown in Scheme 2 and
[34]
described by Tengler et al.
Briefly, oxiran-2-ylmethyl-4-[(propox-
ycarbonyl)amino]benzoate was prepared from 4-aminobenzoic acid
by reaction with propyl chloroformiate that yielded 4-[(propoxy-
carbonyl)amino]benzoic acid that was converted to propyl [4-
(
chlorocarbonyl)phenyl]carbamate by reaction with thionyl chloride.
The desired epoxide was formed after reaction of propyl [4-
chlorocarbonyl)phenyl]carbamate with 2,3-epoxypropan-1-ol. The
(
oxirane ring was opened by addition of 2-(4-fluorophenoxy)ethan-
amine prepared by Gabriel synthesis from 4-fluorophenol via 1-(2-
bromoethoxy)-4-fluorobenzene and 1-(2-bromoethoxy)-4-fluoro-
benzene. The product, 3-{[2-(4-fluorophenoxy)ethyl]amino}-2-hy-
droxypropyl 4-[(propoxycarbonyl)amino]benzoate, was transformed
to the hydrochloride salts with higher water solubility using ethereal
HCl.
FIGURE 3 Spatial view of the overlap of the following
compounds: K145 (blue), 2 (magenta), 4 (green), and 17 (yellow).
These compounds are interacting in the active site of SphK2. The
main amino acids involved in the formation of the complexes are
also shown in this figure. The structures were taken from a
clustering process in which the last 40 ns of each of the three
simulations were considered
SphK2 inhibition assays were performed as previously de-
[27]
scribed
with 17 and 18. Compound 17 showed a higher SphK2
inhibitory capacity (71.0 μM), while compound 18 expressed only
moderate activity (131.7 μM), see Figure 1b. Interestingly, compound

6
VETTORAZZI ET AL.
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FIGURE 4 Histograms show the interaction energies obtained for the specific inhibitors K145 (a), SLM6031434 (b) and compounds 2 (c)
and 4 (d) with the main amino acids involved in the complex formation
1
7 inhibits SphK2 but had no inhibitory effect on SphK1 (data shown in
Table 1).
As it has been suggested that development of SphK1/SphK2
selective inhibitors might be useful for treatment of inflammatory and/
inhibited both enzymes but weaker than 2 (IC50 SphK1 = 55 μM and
IC50 SphK2 = 56 μM); compound 3 was selective for SphK1 (IC50
SphK1 = 60 μM) and compound 17 selectively inhibited SphK2 (IC50
SphK2 = 71 μM). Interestingly compound 17 does not show any
inhibitory effect against SphK1 even at high concentration (650 μM).
[12,35]
or autoimmune diseases,
it was of interest to evaluate the
potential anti-inflammatory effect of 17. To establish proper
comparisons, compounds 2–4 and 17 were tested. In regard to this,
compound 2 was the most potent in the series, but non-isotype-
selective (IC50 SphK1 = 12 μM and IC50 SphK2 = 28 μM); compound 4
2
.5
|
Cell viability assay
The cytotoxicity effects of these compounds on cell viability were
determined by MTT assay. Compound 2, with a benzo[b]pyrimido[5,4-
f]azepine system, showed high cytotoxicity in human umbilical vein
endothelial cells (HUVEC) and human neutrophils, decreasing cell
viability by more than 40% at 10 μM (Figure 6a and b).
Compound 3 with a pyridinyl-piperazine phenyl 3-carbamate
structure, displayed cytotoxicity in HUVEC at 30 μM and was
cytotoxic for neutrophils at all concentrations assayed (10–100 μM)
(
Figure 6c and d). In contrast, compound 4, an isomer of 3, showed no
toxicity at 100 μM in HUVEC when it was compared with vehicle
0.04% DMSO in medium). Compound 17 with an epoxynaphtho[1,2-
(
b]azepine system, only showed significant toxicity at the maximum
dose of 100 μM in human neutrophils but cell viability remained higher
than 70% (Figure 6e–h).
2.6
|
Compound 17 reduces the adhesion of
leukocytes to dysfunctional endothelium
Inflammation and especially the adhesion of mononuclear cell to the
dysfunctional endothelium play an important role in atherosclerosis
[36]
development.
One of the key events in the inflammatory process
involves endothelial-leukocyte adhesion and their subsequent emigra-
[37]
tion to the extravascular space.
To examine the effect of SphK2
inhibition with the less toxic compounds 4 and 17 on leukocyte
adhesion, parallel-plate flow chamber assays were carried out. This
experimental setting allows studies of leukocyte–endothelial cell
FIGURE 5 Histograms show the interaction energies obtained for
1
7 (a) and 18 (b) with the main amino acids involved in the complex
formation

VETTORAZZI ET AL.
| 7
SCHEME 1 Synthesis of (2RS,4SR)-7-bromo-2-(2-phenylethyl)-2,3,4,5-tetrahydro-1,4-epoxynaphtho[1,2-b]azepine (17)
interactions under flow conditions mimicking the in vivo physiological
fluid dynamics within the blood vessels of the microcirculation. When
neutrophils were perfused across TNFα-stimulated endothelial cells,
significant leukocyte adhesion was observed compared to the
adhesion to unstimulated endothelial cells (0.04% DMSO,
Figure 7a). Preincubation with compound 4, the pyridinyl-piperazine
phenyl 4-carbamate, prior to TNFα stimulation, did not affect TNFα-
induced neutrophil adhesion to endothelial cells at the concentration
assayed (100 µM, Figure 7a). When endothelial cells were incubated
with compound 17, the epoxynaphtho[1,2-b]azepine, although some
inhibition of neutrophil arrest to the dysfunctional endothelium (TNFα
stimulated) was detected, the reduction in this parameter was below
In order to explore the mechanisms involved in the decreased
mononuclear cell adhesion to endothelial cells provoked by
compound 17, we next investigated its effect on endothelial cell
3
adhesion molecule (CAM) expression and fractalkine (CX CL1)
up-regulation. TNFα caused increased endothelial expression of
intercellular adhesion molecule-1 (ICAM-1, Figure 8a), vascular
cell adhesion molecule-1 (VCAM-1, Figure 8b), and the mem-
brane-bound chemokine fractalkine (CX
interesting to point out that both VCAM-1 and fractalkine
(CX CL1) are mainly expressed in the vasculature at sites prone
3
CL1, Figure 8c). It is
3
to atherosclerosis lesion formation and here we show that
preincubation of endothelial cells with compound 17 at 10 μM,
significantly reduced TNFα-induced ICAM-1 (a), VCAM-1 (b), and
5
0% (43.4%) at 100 µM (Figure 7a).
Compounds 4 and 17 were firstly assayed at 100 µM to evaluate
3
fractalkine (CX CL1) (c) expression by 60.0, 60.2, and 59.1%,
their impact on mononuclear cell adhesion. At this concentration, while
compound 4 did not significantly affect TNFα-induced mononuclear
cell-endothelial adhesion, compound 17 did (Figure 7b). Since this
effect was inhibited by 70%, at 100 µM, the effect of compound 17
was analyzed within a concentration range of 0.1–100 µM. Interest-
ingly, compound 17 inhibited in a concentration-dependent manner
mononuclear leukocyte-endothelial cell adhesion elicited by TNFα
respectively (Figure 8).
In conclusion, our results indicate that compound 17 which is a
SphK2 selective inhibitor, shows no (endothelial cells) or low toxicity
(neutrophils) toward human cells and is able to inhibit mononuclear
cell adhesion to the dysfunctional endothelium with a minimal
impact on neutrophil responses. These responses are in part
mediated through down-regulation of endothelial cell adhesion
(
Figure 7c) with an estimated IC50 of 3 μM. These results are relevant
3
molecule and fractalkine expression (CX CL1). Therefore, compound
given that the effect on mononuclear cell recruitment exerted by
compound 17 differs from those displayed on neutrophils suggesting
that it likely does not compromise neutrophilic responses which are
necessary for innate host defence.
17 is a potential candidate to be used in the treatment of
inflammatory disorders in which mononuclear cell recruitment plays
a prominent role such as cardiovascular diseases associated to
cardiometabolic disorders.
SCHEME 2 Synthesis of N-[2-(4-fluorophenoxy)ethyl]-2-hydroxy-3-({4-[(propoxycarbonyl)amino]benzoyl}oxy)propan-1-aminium chloride
[34]
(
18).
Reagents and conditions: a) acetone, pyridine; b) SOCl
2
, toluene; c) 2,3-epoxypropan-1-ol, THF, TEA; d) 1,2-dibromoethane, NaOH; e)
potassium phthalimide, KI, DMF; f) NH
2
NH .H O, ethanol; g) propan-2-ol; h) HCl, Et O
2
2
2

8
VETTORAZZI ET AL.
|
FIGURE 6 HUVEC and human neutrophil viability after 24 h incubation with four sphingosine kinase inhibitors. Cells were incubated with
compounds 2–4 and 17 at 10, 30, and 100 µM, just with medium (control) or medium plus vehicle (vehicle, 0.04% DMSO in medium) for 24 h
and MTT assay was performed. Data are presented as mean ± SEM of the percentage of viable cells in n = 5 independent experiments.
+
++
*
p < 0.05 or **p < 0.01 versus control, p < 0.05 or p < 0.01 versus vehicle
3
|
CONCLUSIONS
Particularly interesting is that compound 17 which is a selective
inhibitor of SphK2 and does not have exert any cytotoxic effects, has a
potent anti-inflammatory effect since it inhibits mononuclear cell
adhesion to the dysfunctional endothelium.
In this work, we used a molecular modeling approach that allowed us to
obtain new inhibitors of SphK2 with novel structural scaffolds.

VETTORAZZI ET AL.
| 9
It should be noted that compound 17 shows an anti-inflammatory
mechanisms responsible to produce the anti-inflammatory effect of
these compounds. However, it is reasonable to think that its biological
activity is due, at least in part, to the aforementioned inhibitory effect.
On the other hand, its profile makes itself an interesting starting
structure for the search for new selective inhibitors of SphK2, as well
as new anti-inflammatory agents.
activity in a concentration-dependent manner with an IC50 in the low
micromolar range. Therefore, based on our results we cannot say that
the anti-inflammatory activity of this compound is due only to the
action on this enzyme. In fact, it is logical to have doubts about the
It is important to note that 7-bromo-2-(2-phenylethyl)-2,3,4,5-
tetrahydro-1,4-epoxynaphtho[1,2-b]azepine (17) was obtained from a
molecular modeling study carried out based on the structure of 4-[(2E)-
2
-(4-chlorobenzylidene)hydrazinyl]-6,11-dimethyl-6,11-dihydro-5H-
pyrimido[4,5-b][1]benzazepine (2) and using K145 as control structure.
Therefore, the information obtained from this theoretical model based
on the molecular interactions involved in the stabilization of the
different complexes can be useful in the search for new inhibitors of
SphK2 recruitment, which plays a prominent role, such in inflammatory
and cardiovascular diseases.
4
4
4
|
EXPERIMENTAL
Chemistry
.1
|
.1.1 General
|
Commercially available compounds were used as received, unless
stated otherwise. Melting points were measured by a Barstead
electrothermal 9100 apparatus or a Kofler hot plate apparatus HMK
(
Franz Kustner Nacht GK, Dresden, Germany) and are uncorrected.
TLC was performed on silica gel 60 F254 on aluminum sheets (Merck,
Darmstadt, Germany) and visualized with UV light (254 nm). Com-
pounds were purified by silica gel 60 (40–63 μm, Merck 9385) column
3
FIGURE 7 (a) Compound 17 but not 4 modestly inhibits
neutrophil adhesion to the dysfunctional endothelium (TNFα-
stimulated). Cells were pretreated for 24 h with compound 4, 17
(
100 μM), just with medium (control) or medium plus vehicle
(
vehicle, 0.04% DMSO) prior to TNFα stimulation (20 ng/mL, 24 h).
Freshly isolated human neutrophils were perfused across the
2
endothelial monolayers for 5 min at 0.5 dynes/cm and neutrophil
adhesion was quantified. (b and c) Compound 17 inhibits
mononuclear cell adhesion to the dysfunctional endothelium (TNFα-
stimulated) in a concentration-dependent manner. (b) Cells were
pretreated for 24 h with compound 4, 17 (100 μM), just with
medium (control) or medium plus vehicle (vehicle, 0.04% DMSO)
prior to TNFα stimulation (20 ng/mL, 24 h). Freshly isolated human
mononuclear cells were perfused across the endothelial monolayers
2
for 5 min at 0.5 dynes/cm and mononuclear cells adhesion was
quantified. (c) Cells were pretreated for 24 h with compound 17
(
0.1–100 μM), just with medium (control) or medium plus vehicle
vehicle, 0.04% DMSO) prior to TNFα stimulation (20 ng/mL, 24 h).
(
Freshly isolated human mononuclear cells were perfused across the
2
endothelial monolayers for 5 min at 0.5 dynes/cm and
mononuclear cell adhesion was quantified. Results are the
mean ± SEM of 4–6 independent experiments. **p < 0.01 versus
+
+
FIGURE 7 Continued.
vehicle, p < 0.01 versus TNFα-stimulated cells

10
VETTORAZZI ET AL.
|
(
Bruker, Karlsruhe, Germany). The carbon typology (C, CH, CH
2
or
1
3
CH
3
) was deduced from C NMR DEPT experiments, which along
with the 2D experiments, COSY, HSQC, and HMBC correlations,
permitted the full assignation of all carbons and hydrogens. Chemical
shifts are relative to the solvent peaks used as reference and reported
in δ parts per million (ppm), and J values in Hz. High-resolution mass
spectra (HRMS) were measured using a high-performance liquid
®
chromatograph Dionex UltiMate 3000 (Thermo Scientific, West Palm
Beach, FL, USA) coupled with a LTQ Orbitrap XL™ Hybrid Ion Trap-
Orbitrap Fourier transform mass spectrometer (Thermo Scientific)
with injection into HESI II in the positive or negative mode, or on a
Waters Micromass AutoSpect NT (equipped with a direct inlet probe)
by electronic impact operating at 70 eV. IR spectra were recorded
on a Bruker Tensor 27 spectrometer (equipped with a platinum ATR
cell).
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
4.1.2
|
Synthesis of (2RS,4SR)-7-bromo-2-phenethyl-
2,3,4,5-tetrahydro-1,4-epoxy-naphtho[1,2-b]azepine
(17)
Sodium tungstate dihydrate Na
followed by 30% aqueous hydrogen peroxide solution (0.74 mL,
.72 mmol), were added to a stirred and cooled solution of the 2-
allyl-4-bromo-N-(3-phenylpropyl)naphthalen-1-amine (0.85 g,
.24 mmol) in acetone–water (30 mL, 10:3 v/v). The resulting
2 4 2
WO .2H O (0.072 g, 0.22 mol),
6
2
mixture was stirred at ambient temperature for 26 h, after that
was filtered and the solvent removed under reduced pressure.
Toluene (30 mL) was added to the organic residue and the resulting
solution was heated at 60°C for 6 h. After cooling the solution to
ambient temperature, the solvent was removed under reduced
pressure and the crude product was purified by chromatography on
silica using heptane–ethyl acetate (compositions ranged from 30:1 to
1
0:1 v/v) as eluent to afford 17 as a pale-yellow syrup in yield 43%.
1
3
H NMR (CDCl ) δ 8.39 (dd, J = 7.0, 2.2 Hz, 1H, CH-11), 8.17 (dd,
J = 7.0, 2.1 Hz, 1H, CH-8), 7.59 (td, J = 7.0, 2.2 Hz, 1H, CH-10), 7.54
td, J = 7.0, 2.1 Hz, 1H, CH-9), 7.50 (s, 1H, CH-6), 7.29 (dd, J = 8.2,
.7 Hz, 2H, CH-2″ and CH-6″), 7.28 (t, J = 8.3 Hz, 2H, CH-3″ and
CH-5″), 7.18 (td, J = 8.4, 1.7 Hz, 1H, CH-4″), 4.94 (ddd, J = 8.0, 5.5,
2.8 Hz, 1H, CH-4), 3.48 (dd, J = 16.9, 5.5 Hz, 1H, CH -5), 3.17 (dddd,
J = 11.0, 8.5, 5.5, 3.0 Hz, 1H, CH-2), 3.13 (ddd, J = 13.9, 11.0, 5.5 Hz,
H, CH -2′), 2.84 (ddd, J = 13.9, 11.0, 5.5 Hz, 1H, CH -2′), 2.52 (d,
J = 16.9 Hz, 1H, CH -5), 2.28 (dddd, J = 13.6, 11.0, 6.0, 5.5 Hz, 1H,
CH -1′), 2.16–2.22 (m, 2H, CH -3), 1.81 (ddt, J = 13.6, 11.0,
5.5 Hz, 1H, CH ) δ 145.4 (C-11b), 141.8 (C-1″),
31.2 (C-6), 130.9 (C-7a), 128.8 (C-11a), 128.6 (C-2″ and C-6″),
28.5 (C-3″ and C-5″), 127.3 (C-8), 127.1 (C-10), 126.9 (C-9), 126.0
(
1
FIGURE 8 Compound 17 inhibits TNFα-induced ICAM-1, VCAM-
, and CX CL1 expression on HUVEC. Some cells were pretreated
B
1
3
with compound 17 (10 μM) 24 h before TNFα stimulation (20 ng/
mL, 24 h) or with vehicle (0.04% DMSO in medium). ICAM-1 (a),
1
A
B
A
3
VCAM-1 (b), and CX CL1 (c) expression was determined by flow
A
A B
H
cytometry. Results (mean ± SEM) are expressed as the mean
1
3
fluorescence intensity (MFI) of n = 6 independent experiments.
B 3
-1′). C NMR (CDCl
+
*
+
p < 0.05 or **p < 0.01 relative to the vehicle group, p < 0.05 or
1
1
+
p < 0.01 relative to TNFα-stimulated cells
(
C-4″), 122.4 (C-11), 122.3 (C-5a), 119.6 (C-7), 74.6 (C-4), 72.3 (C-2),
1
13
chromatography. H NMR and C NMR spectra were recorded at
5°C with CDCl , CD OD or DMSO-d as solvents on Bruker AC-300,
AC-400, AC-500 or Avance III 400 MHz FT-NMR spectrometers
39.9 (C-3), 38.7 (C-1′), 35.2 (C-5), 33.7 (C-2′). HRMS (EI, 70 eV):
2
3
3
6
C22H20NOBr calcd. 393.0728 m/z. Found: 393.0722 m/z. IR (ATR,
−
1
cm ): νmax = 1497 (CC), 1258 (C-N), 1043 (C-O), 990 (N-O).

VETTORAZZI ET AL.
| 11
4.1.3
|
Synthesis of compound 18
4.3.4 MTT assay
|
The detailed synthetic pathway and full analytical characterization of
Cytotoxicity studies were performed with both HUVECs and freshly
the discussed compound 18 as well as intermediates are provided in
isolated human neutrophils using 3-(4,5-dimethylthiazol-2-yl)-2,5-
[39]
[34]
Tengler et al.
diphenyltetrazolium bromide (MTT) colorimetric assay.
Neutrophils
were obtained from buffy coats of healthy donors by Ficoll-Hypaque
[40]
density gradient centrifugation as described.
A total of 100 μL of
4.2
|
SphK2 inhibition assays
neutrophils and HUVEC suspension in supplemented RPMI medium
5
Potential SphK2 inhibitors were evaluated with fluorescence SphK2
(2 × 10 cells/mL (Biowest, Nuaillé, France) were added to each well of
[
27]
assays in 384-well format as described.
Briefly, compounds were
a 96-well microtiter plate. Cells were incubated in the absence or
presence of compounds at 37°C for 24 h. Compounds 2–4 and 17
were tested at three different concentrations: 10, 30, and 100 µM.
Some assays were carried out just with medium and others with vehicle
(0.04% DMSO in medium). MTT was freshly prepared at 2 mg/mL in
PBS. A total of 100 μL of MTT solution was added to each well and
incubated at 37°C for another 3 h. The supernatants were discarded
and 200 μL of DMSO were added to each well to dissolve the
formazan. The optical densities at dual wavelengths (560 and 630 nm)
were determined in a spectrophotometer (Infinite M200, Tecan,
Mannedorf, Switzerland). Results were presented as mean ± standard
errors of mean (SEM).
dissolved in DMSO and initially screened at 650 μM. Candidates
showing inhibition at this concentration were further characterized
to obtain IC50s. SphK2 activity was measured in 384-well plates
(
Greiner Bio-One, Frickenhausen, Germany) in buffer containing
0 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 200 mM KCl, and 10%
3
glycerol, in the presence of NBD-sphingosine (60 μM; Avanti Polar
Lipids), and recombinant SphK2 (15 nM). Reactions were initiated
with the addition of an ATP–Mg mixture (1 mM ATP, 2 mM MgCl
2
,
4
0 mM Tris-HCl pH 7.4), and followed in a TECAN Infinite M1000
fluorescence plate reader (Männedorf, Switzerland) at 37°C.
Excitation and emission wavelengths were 550 and 584 nm,
respectively. All data were analyzed using GraphPad Prism (La Jolla,
CA, USA)
4
.3.5 Leukocyte–endothelial cell interactions under
|
flow conditions
4
.3
|
Toxicological studies and study of potential
HUVECs up to passage 1 were grown to confluence and stimulated
with recombinant human TNFα (20 ng/mL) for 24 h. Cells were pre-
incubated for 24 h with compounds 4 or 17 at 100 µM. Some assays
were carried out just with medium or with vehicle (0.04% DMSO in
medium). Then, cells were stimulated with TNFα (20 ng/mL) for
another 24 h. In another set of experiments, cells were pretreated or
not with compound 17 (0.1–100 μM) for 24 h prior to TNFα
stimulation (20 ng/mL, 24 h). Human neutrophils and mononuclear
cells were obtained from buffy coats of healthy donors by Ficoll-
anti-inflammatory activity
4
.3.1 Human studies
|
All research with human samples in the current study complied with
the principles outlined in the Declaration of Helsinki and was approved
by the Institutional Ethics Committee of the University Clinic Hospital
of Valencia (Valencia, Spain). Written, informed consent was obtained
from all volunteers.
[40]
Hypaque density gradient centrifugation.
The Glycotech flow
chamber (GlycoTech, Gaithersburg, MD) was assembled and placed on
an inverted microscope stage. Freshly isolated neutrophils or
4
.3.2 Reagents and compounds
|
6
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and dimethyl sulfoxide (DMSO) and tumor necrosis factor-alpha
TNFα) were purchased from Sigma–Aldrich (Madrid, Spain). All tested
mononuclear cells (1 × 10 cells/mL) were then perfused across the
endothelial monolayers (HUVEC) unstimulated or stimulated with
(
20 ng/mL of TNFα for 24 h. Leukocyte interactions were determined
2
compounds 2–4 and 17 were dissolved in culture medium containing
after 5 min at 0.5 dyn/cm . Cells interacting on the surface of the
DMSO (0.04%).
endothelium were visualized and recorded (×20 objective, ×10
eyepiece) using phase-contrast microscope (Axio Observer A1 Carl
Zeiss microscope, Thornwood, NY).
4
.3.3 Cell culture
|
Human umbilical vein endothelial cells (HUVEC) were isolated by
4
.3.6 Determination of cell adhesion molecule and
|
[38]
collagenase treatment
and maintained in human specific endothe-
fractalkine (CX CL1) expression by flow cytometry
3
lial basal medium (EBM-2, Lonza, Verviers, Belgium), supplemented
with endothelial growth media (EGM-2, Lonza, Verviers, Belgium) and
HUVEC expression of intercellular adhesion molecule-1 (ICAM-1),
1
0% of fetal bovine serum (FBS, Lonza, Verviers, Belgium). Cells up to
3
vascular cell adhesion molecule-1 (VCAM-1), and fractalkine (CX CL1)
passage 1 were grown to confluence to preserve endothelial features.
Prior to every experiment, cells were incubated 16 h in medium
containing 1% FBS.
were determined by flow cytometry. Cells were pretreated for 24 h
with compound 17 (10 μM) or vehicle (0.04% DMSO in medium) and
stimulated for additional 24 h with TNFα (20 ng/mL). Then, endothelial

12
VETTORAZZI ET AL.
|
cells were detached from culture flasks by scraping in ice-cold PBS
4.4.2 Molecular docking
|
containing 2 mM EDTA. Next, cells were washed and incubated at
[
31]
AutoDock4
was used to dock each compound to the SphK2 active
6
2
× 10 cells/mL for 1 h at 4°C in the dark with FITC-conjugated mAb
site using a Lamarckian genetic algorithm with pseudo-Solis and Wets
against human ICAM-1 (diluted 1:25; 400 µg/mL; clone HA58,
BioLegend, San Diego, CA), an APC-conjugated mAb against human
VCAM-1 (diluted 1:30; 100 µg/mL; clone STA, BioLegend, San Diego,
[43]
local search.
The following parameters were used: the initial
population of trial ligands was constituted by 200 individuals; the
4
maximum number of generations was set to 2.7 × 10 . The maximum
CA) or with a PE–conjugated mAb against human CX
:25; 1.25 µg/mL; clone 51637, R&D Systems, Minneapolis, MN), all
diluted in PBS with 3% BSA. Samples were run in a flow cytometer
3
CL1 (diluted
6
number of energy evaluations was 25.0 × 10 . For each docking job,
1
2
00 conformations were generated. All other run parameters were
maintained at their default setting. The resulting docked conforma-
tions were clustered into families by considering the backbone rmsd.
The lowest docking-energy conformation was considered the most
(
(
FACSVerse, BD Biosciences, San Jose, CA). The expression of ICAM-1
FITC-fluorescence), VCAM-1 (APC-fluorescence) and CX CL1 (PE-
3
fluorescence) was expressed as the mean of fluorescence intensity
MFI).
[44]
favorable orientation.
(
4
.4.3 MD simulations
|
4
.4
|
Molecular modeling
Homology modeling
The complex geometries from docking were soaked in boxes of
[45]
4
.4.1
|
explicit water using the TIP3P model
and subjected to MD
simulation. All MD simulations were performed with the Amber
Since no crystal structure is currently available for SphK2, we
[
32]
software package
using periodic boundary conditions and
[28]
generated a structural model of SphK2 using MODELLER,
using
octahedral simulation cells. The particle mesh Ewald method
the structure of sphingosine kinase 1 (Homo sapiens) (PDB ID: 3VZB,
[
46]
(
PME)
interpolation order of 4 and a real space direct sum cutoff of
0 Å. The SHAKE algorithm was applied allowing for an integration
was applied using a grid spacing of 1.2 Å, a spline
[15,29,30]
3
VZC, 3VZD, 4L02, 4V24) as a template.
A high degree overall
homology to the template (52% in aligned regions) was found and
there is a considerable sequence and structural similarity at the
sphingosine binding (C4) domain.
1
time step of 2 fs. MD simulations were carried out at 310 K
temperature. Three MD simulations of 50 ns were conducted for
each system under different starting velocity distribution functions;
thus, in total 150 ns were simulated for each complex. The NPT
ensemble was employed using Berendsen coupling to a baro/
thermostat (target pressure 1 atm, relaxation time 0.1 ps). Post-MD
analysis was carried out with program PTRAJ.
Human SphK2 (code: Q9NRA0) sequence was obtained from the
UNIPROT database (www.uniprot.org/). A position specific iterated
[41,42]
BLAST
identified a kinase – sphingosine kinase 1 from Homo sapiens (PDB ID:
search against the database of Protein Data Bank proteins
4
4
V24), as the closest match to both proteins with 52.12% identity and
4% homology in the aligned regions of SphK. Unaligned regions in the
protein were deleted. The model for the protein with the lowest DOPE
discrete optimized protein energy) scores was chosen for further
refinement.
We refined the geometry by performing molecular dynamics
(
4.4.4 Binding energy calculations
|
The MM-GBSA protocol was applied to each MD trajectory in order to
calculate the relative binding energies of the SphK2-ligand complexes.
The MM-GBSA method was used in a hierarchical strategy, and the
simulations. The modeled protein was soaked in truncated octahe-
dral periodic boxes of explicit water using the TIP3P model and
subjected to MD simulation. All MD simulations were performed
with the Amber software package. Sodium ions were added to
neutralize the charge of the system. The entire system was subjected
to energy minimization.
[47]
details of this method have been presented elsewhere.
This
protocol was applied to 4000 equidistant snapshots extracted from the
last 40.0 ns and was used within the one-trajectory approximation.
Briefly, the binding free energy (ΔGbind) resulting from the formation of
a RL complex between a ligand (L) and a receptor (R) is calculated as
follows:
In the next place each system was then heated in the NVT
ensemble from 0 to 300 K in 500 ps and equilibrated at an isothermal
isobaric (NPT) ensemble for another 2 ns. A Langevin thermostat was
ΔGbind ¼ ΔEMM þ ΔGsol ꢀ TΔS
ΔEMM ¼ ΔEinternal þ ΔEelectrostatic þ ΔEvdW
ΔGsol ¼ ΔGPB þ ΔGSA
ð1Þ
ð2Þ
ð3Þ
−
1
used for temperature coupling with a collision frequency of 1.0 ps
.
The particle mesh Ewald (PME) method was employed to treat the
long-range electrostatic interactions in a periodic boundary condition.
The SHAKE method was used to constrain hydrogen atoms. The time
step for all MD is 2 fs, with a direct-space, non-bonded cutoff of 8 Å.
Finally, three MD simulations of 50 ns were conducted under different
starting velocity distribution functions; thus, in total 150 ns were
simulated.
where ΔEMM, ΔGsol, and −TΔS are the changes in the gas-phase MM
energy, the solvation free energy, and the conformational entropy

VETTORAZZI ET AL.
| 13
upon binding, respectively. ΔEMM includes ΔEinternal (bond, angle, and
dihedral energies), ΔEelectrostatic (electrostatic), and ΔEvdw (van der
Waals) energies. ΔGsolv is the sum of electrostatic solvation energy
[15] Z. Wang, X. Min, S.-H. Xiao, S. Johnstone, W. Romanow, D. Meininger,
H. Xu, J. Liu, J. Dai, S. An, S. Thibault, N. Walker, Structure 2013, 21(5),
7
98.
[
16] 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.
Highkin, R. Broadus, S. Ogawa, K. Cukyne, L. Zawadzke, V. Peterkin, K.
Iyanar, J. Scholten, J. Wendling, H. Fujiwara, O. Nemirovskiy, A.
Wittwer, M. Nagiec, Biochem. J. 2012, 444(1), 79.
(
polar contribution), ΔGPB, and the non-electrostatic solvation
component (nonpolar contribution), ΔGSA. Polar contribution is
calculated using the PB model, while the nonpolar energy is estimated
by solvent accessible surface area.
[
17] V. Brinkmann, A. Billich, T. Baumruker, P. Heining, R. Schmouder, G.
Francis, S. Aradhye, P. Burtin, Nat. Rev. Drug Discov. 2010, 9(11),
8
83.
ACKNOWLEDGMENTS
[
18] S. Panneer Selvam, R. M. De Palma, J. J. Oaks, N. Oleinik,
Y. K. Peterson, R. V Stahelin, E. Skordalakes, S. Ponnusamy, E.
Garrett-Mayer, C. D. Smith, B. Ogretmen, Sci. Signal. 2015, 8(381),
ra58.
Grants from Universidad Nacional de San Luis (UNSL-Argentina) and
Colombian Institute for Science and Research (Grant No.
1
10265842651) partially supported this work. Marcela Vettorazzi
[19] 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. Lesnefsky, S. Spiegel, FASEB J. 2011, 25(2), 600.
thanksa doctoral fellowshipofCONICET-Argentina. Thisstudywasalso
partially supported by the Slovak Research and Development Agency
[
[
[
[
[
[
20] E. T. Samy, C. A. Meyer, P. Caplazi, C. L. Langrish, J. M. Lora, H.
(
Grant No. APVV-0516-12) and by SANOFI-AVENTIS Pharma Slovakia.
Bluethmann, S. L. Peng, J. Immunol. 2007, 179(9), 5644.
This work was also supported by grants SAF2014-57845R and
SAF2017-89714-R, and the CP15/00150 from the Spanish Ministry
ofEconomy and Competiveness, the CarlosIII InstituteofHealth(ISCIII),
andtheEuropean Regional Development Fund. Pre-doctoral grants: PM
21] J. Yang, C. Yang, S. Zhang, Z. Mei, M. Shi, S. Sun, L. Shi, Z. Wang, Y.
Wang, Z. Li, C. Xie, Cancer Biol. Ther. 2015, 16(8), 1194.
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Kellogg, S. Grant, S. Spiegel, S. Zhang, PLoS ONE 2013, 8(2), e56471.
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Santos, K. R. Lynch, J. Pharmacol. Exp. Ther. 2015, 355(1), 23.
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T. Padrtova, P. Mokry, P. Bobal, L. M. Acosta, L. Acosta, A. Palma, J.
Cobo, J. Bobalova, J. Csollei, I. Malik, S. Alvarez, S. Spiegel, J. Jampilek,
R. Enriz, Eur. J. Med. Chem. 2017, 139, 461.
(
FPI) from the Spanish Ministry of Innovation and Competitiveness.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
[
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ORCID
Ricardo D. Enriz
http://orcid.org/0000-0002-4846-9452
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How to cite this article: Vettorazzi M, Vila L, Lima S, et al.
Synthesis and biological evaluation of sphingosine kinase 2
inhibitors with anti-inflammatory activity. Arch Pharm Chem
Life Sci. 2019;1–14.
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https://doi.org/10.1002/ardp.201800298