Synthesis, Biological Evaluation and Structure-Activity Relationships of Diflapolin Analogues as Dual sEH/FLAP Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.8b00415

A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesized, and characterized by ¹H NMR, ¹³C NMR, and elemental analysis. These novel compounds were biologically evaluated for their inhibitory activity against sEH and FLAP. Molecular modeling tools were applied to analyze structure-activity relationships (SAR) on both targets. Results show that even small modifications on the lead compound diflapolin markedly influence the inhibitory potential, especially on FLAP, suggesting very narrow SAR.

Full text of DOI:10.1021/acsmedchemlett.8b00415

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Letter
Synthesis, biological evaluation and structure-activity
relationships of diflapolin analogs as dual sEH/FLAP inhibitors
Lisa Vieider, Erik Romp, Veronika Temml, Jana Fischer, Christian Kretzer,
Martin Schoenthaler, Abdulla Taha, Victor Hernández-Olmos, Sonja Sturm,
Daniela Schuster, Oliver Werz, Ulrike Garscha, and Barbara Matuszczak
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.8b00415 • Publication Date (Web): 29 Nov 2018
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Synthesis, biological evaluation and structure-activity
relationships of diflapolin analogs as dual sEH/FLAP
inhibitors
Lisa Vieider1, Erik Romp2, Veronika Temml3, Jana Fischer2, Christian Kretzer2, Martin
Schoenthaler1, Abdulla Taha2, Victor Hernández-Olmos5, Sonja Sturm3, Daniela Schuster1,4,
Oliver Werz2, Ulrike Garscha2*, Barbara Matuszczak1*
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1
Institute of Pharmacy, Department of Pharmaceutical Chemistry, Center for Chemistry and Biomedicine,
University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
2 Chair of Pharmaceutical / Medicinal Chemistry, Friedrich-Schiller University Jena, Philosophenweg 14, D-07743
Jena, Germany.
3 Institute of Pharmacy, Department of Pharmacognosy, Center for Chemistry and Biomedicine, University of
Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria
4 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Paracelsus Medical University
Salzburg, Strubergasse 21, A-5020 Salzburg, Austria
5 Institute of Pharmaceutical Chemistry, Department of Biochemistry, Chemistry and Pharmacy, Goethe-
University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
KEYWORDS: soluble epoxide hydrolase (sEH), 5-lipoxygenase activating protein (FLAP), arachidonic acid cascade, diflapolin,
inflammation
ABSTRACT: A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein
1
(FLAP) inhibitor diflapolin was designed, synthesized and characterized by H NMR, 13C NMR and elemental analysis.
These novel compounds were biologically evaluated for their inhibitory activity against sEH and FLAP. Molecular
modelling tools were applied to analyze structure-activity relationships (SAR) on both targets. Results show that even small
modifications on the lead compound diflapolin markedly influence the inhibitory potential, especially on FLAP, suggesting
very narrow SAR.
A wide range of chronic pathologies (e.g. rheumatoid
arthritis, hashimoto thyroiditis, and asthma bronchiale)
is related to unresolved or misdirected inflammatory
processes. Bioactive lipid mediators deriving from
polyunsaturated fatty acids play central roles in
inflammation on the one hand, but also in resolution of
inflammatory events.1 Upon cell stimulation, cytosolic
phospholipase A2 (cPLA2) releases arachidonic acid (AA)
from phospholipids of cellular membranes, where
specific oxygenases such as cyclooxygenases (COX)-1/2,
5-, 12-, and 15-lipoxygenases (LO) as well as cytochrome
(CYP) P450 enzymes convert AA towards pro-
inflammatory, pro-resolving as well as anti-inflammatory
lipid mediators.2, 3 COX inhibitors are still the standard
treatment for acute inflammatory conditions and have
been intensively investigated over the last decades.
However, in long-term use they cause severe
gastrointestinal and cardiovascular side effects.4, 5 CYP
and LO metabolic routes attracted attention to develop
new anti-inflammatory drugs with less deleterious side
effects. Moreover, it is well known that crosstalk between
the branches of AA cascade occurs. Thus, selective
inhibition of one of the three pathways may cause a
shunt towards the other two.2, 6 The current strategy to
combat chronic inflammation is often the
administration of a drug cocktail, sometimes co-
formulated in one tablet to improve patient compliance.
Such a multicomponent therapy can suffer from drug-
drug interactions, unexpected pharmacokinetic
properties or changes in pharmacodynamics. Drugs that
interfere with multiple targets might provide a solution
for these problems. Therefore, current research focuses
on the development of designed multiple ligands
(DMLs).7, 8 Compared to multicomponent drugs, there is
only one active agent interfering with more than one
target, which lowers the risk of toxicity, drug-drug
interactions and offers the possibility for a low dose
treatment.2, 8, 9
Recently, diflapolin was identified as new dual inhibitor
interfering with both, the CYP and 5-LO pathway within
the AA cascade.10, 11 On the one hand, diflapolin (1)
(Figure 1) targets the soluble epoxide hydrolase (sEH),
which is responsible for the rapid degradation of CYP-
produced epoxyeicosatrienoic acids (EETs) towards the
corresponding
dihydroxyeicosatrienoic
In contrast to the anti-inflammatory
acids
12
(DHETrEs).3,
EETs, DHETrEs possess less beneficial properties and
are discussed to be rather related to inflammatory
processes. Thus, sEH inhibition leads to increased EETs
levels accompanied with advantageous effects.6 Despite
intense investigations, so far no sEH inhibitor has
reached the market but currently, some promising
candidates are undergoing clinical trials.13, 14 Common
to all sEH inhibitors is a urea or carboxamide
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substructure that forms hydrogen bonds with key
4
5
residues within the binding site (Tyr383, Tyr466 and
Asp355).15
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Diflapolin also suppresses leukotriene (LT) formation by
targeting the 5-LO-activating protein (FLAP).10, 11 AA is
converted by 5-LO in a two-step reaction to LTA4 the
precursor of chemotactic LTB4 and vasoactive cysteinyl
LTs.5 Early FLAP inhibitors such as MK-886 and MK-
591 showed promising results but were never brought to
the market due to detrimental pharmakokinetics.
However, novel inhibitors are currently undergoing
clinical trials up to phase III.16
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The increased interest is based on the fact that FLAP
inhibitors are effective against chronic inflammatory
diseases and show a better risk-benefit profile compared
to direct 5-LO inhibitors.17 The combination of sEH
inhibition and the inhibition of LT biosynthesis has
been shown to be beneficial as Meirer et al. reported a
dual 5-LO/sEH inhibitor with a imidazo-[1,2-a]pyridine
core, linked with an aryl urea moiety by a carbon chain.2,
Figure 1. Structural overview of the lead compound
diflapolin (1)
To determine the effects of the substitution pattern on
subunit V and the influence of the methyl group and its
position on subunit III, compounds 11-27 were prepared
using a synthetic strategy developed in our group.
Treatment of 2-chloromethylbenzothiazole (2) with 3-
methyl-4-nitrophenol, 2-methyl-4-nitrophenol, or 4-
nitrophenol in the presence of caesium carbonate,
sodium carbonate, and potassium iodide in refluxing
acetone led to compounds 3-5. Subsequent reduction of
the nitro function with hydrogen and Raney-Nickel as
catalyst afforded the corresponding substituted aniline
derivatives 7-9. Finally, the desired target compounds
with ether subunit (i.e. compounds 11-27) became
accessible by reaction of 7-9 with the appropriate phenyl
isocyanates. In addition to the 3,4-dichlorophenyl
substituted compounds, the corresponding 3,5-
dichlorophenyl, the three different monochlorophenyl
and derivatives with unsubstituted phenyl were
prepared.
18
The target combination of FLAP and sEH is
promising, since inhibition of FLAP might specifically
block LT formation without targeting other LOs that are
related to the biosynthesis of anti-inflammatory
specialized-pro-resolving mediators (SPM).19
Encouraged by the promising pharmacological profile
and the in vivo results of the dual sEH/FLAP inhibitor
diflapolin11, we elucidated structural essential moieties
in this SAR study and aimed to improve the activity
profile of diflapolin by modifying the lead compound.
Molecular modelling was used to rationalize the activity
data and to guide the optimization process. sEH
inhibitors in general possess a urea or a carboxamide
moiety and FLAP inhibitors frequently consist of a
bicycle, so we decided to keep substructure
I
(benzothiazole) and substructure IV (urea) intact, and
focused on subunits II, III and V. (Figure 1)
Scheme 1. General procedure for the synthesis of compounds 3-28
a Reagents and conditions: (a) methyl substituted or unsubstituted 4-nitrophenol or thiophenol, Na2CO3, Cs2CO3, KI, acetone,
reflux; (b) H2, Raney-Nickel in methanol; (c) corresponding phenylisocyanates in THF, room temperature.
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Moreover, three compounds with modified spacer unit
2-(4-aminostryryl) and the 2-(4-aminophenylethyl)
substituted benzothiazole (29, 30) (Scheme 2). All
diflapolin derivatives were tested for their inhibitory
potency against sEH and FLAP in biological assays.
(Table 1). IC50 values were determined for both assays
using GraphPad Prism 8 software (Graphpad Software
Inc., San Diego, CA).
II were prepared. Derivative 28, which is characterized
by a formal exchange of the oxygen by sulphur was
synthesized in an analogous manner as diflapolin using
4-nitrothiophenol instead of 4-nitrophenol (Scheme 1.).
The derivatives with a two-carbon spacer unit (i.e.
compounds 31 and 32) became accessible by reaction of
3,4-dichlorophenylisocyanate with the already known20
Scheme 2. Procedure for the synthesis of compounds 31 and 32
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a Reagents and conditions: (a) 3,4-dichlorophenylisocyanate in THF, room temperature.
Table 1. Activity data for compounds 11-28 and 31-32.
IC50 (µM) 5-LOX product
IC50 (nM) sEH activity
(mean ± SEM)
formation
(mean ± SEM )
compound
X-Y
R
R'
diflapolin (1)
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
2-methyl
2-methyl
2-methyl
2-methyl
2-methyl
2-methyl
3,4-dichloro
H
0.23
0.73
0.83
0.16
0.57
0.08
±
±
±
±
±
±
0.0
0.2
0.3
0.0
0.2
0.0
23.00
61.9
±
±
±
±
±
±
6.8
22.9
519.1
2.0
11
12
13
14
15
2-chloro
3-chloro
4-chloro
3,5-chloro
1499.5
15.8
5.2
1.5
20.0
5.0
16
17
18
19
20
21
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
3-methyl
3-methyl
3-methyl
3-methyl
3-methyl
3-methyl
H
2.07
2.17
0.68
2.09
±
±
±
±
1.2
0.6
0.2
1.0
6.5
0.6
35.1
2.0
4.1
0.8
±
±
±
±
±
±
1.5
0.2
7.1
0.5
1.7
0.1
3,4-dichloro
2-chloro
3-chloro
4-chloro
> 10 µM
3,5-dichloro
0.72
±
0.2
22
23
24
25
26
27
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
CH2-O
H
H
H
H
H
H
H
0.42
0.57
0.36
0.17
1.01
0.09
±
±
±
±
±
±
0.2
0.1
0.2
0.0
0.1
0.0
111.5
10.3
278.9
25.9
13.9
15.2
±
±
±
±
±
±
19.1
0.8
3,4-dichloro
2-chloro
84.0
3.1
3-chloro
4-chloro
3.7
3,5-dichloro
4.4
28
31
32
CH2-S
CH=CH
CH2-CH2
H
H
H
3,4-dichloro
3,4-dichloro
3,4-dichloro
1.13
±
±
0.0
3.1
25.2
9.1
±
±
±
10.7
1.8
10.76
> 10 µM
11.7
3.2
a 0 to 1 µM range for 5-LO product formation and 0 to 50 nM range for sEH activity were chosen for bar chart presentation (red)
of IC50 values (n ≥ 3) in order to visualize the inhibitory activity of the test compounds.
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The following SAR regarding subunit III and V was
generally beneficial for FLAP inhibition (-0.404), while
chloro substitution in the meta positions result in
positive activity contributions.
Most substitutions enhanced or did not affect sEH
activity, only a 2-chloro substitution on subunit V clearly
lowers it and received a negative contribution in the
Free-Wilson analysis (-0.910). 3-Methyl at subunit III is
preferred but not required for sEH inhibition.
observed for FLAP-mediated 5-LO product formation in
human neutrophils: Evaluation of the subunit III shows
that the 3-methyl (16-21) group impairs FLAP activity,
compared to the derivatives with a 2-methyl group (1 and
11-15). Removing the methyl group altogether (22-27)
leads to a slight increase in FLAP activity, with the
exception of 26.
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Within the 2-methyl substituted series, the 3,5-chloro
(15) and the 3-chloro (13) modifications lead to superior
inhibition than diflapolin.
In the generally less potent series of 3-methyl derivatives,
the 3-chloro substituted compound 18 was found to be
the most active. Within the generally more active series
without the methyl group, also the 3,5- and the 3-chloro
substitution (25, 27) displayed the highest FLAP
inhibitory activity.
Thus, we conclude that for FLAP inhibition, methyl
substitution of the phenylene ring is clearly preferred at
2-position but is not essential. The chlorine substitution
pattern (subunit V) can be varied but the most promising
derivatives are 3-chloro and 3,5-chloro.
Regarding inhibition of sEH, compounds with a 3-
methyl moiety at the phenylene ring (subunit III) are
most effective, but also 2-methyl and unsubstituted
phenylene lead to active compounds, many of them are
superior to diflapolin (13-17, 19-21, 23, 26, 27) Notably,
a chloro substituent in the ortho-position of the terminal
phenyl reduces the inhibitory activity against sEH (12,
18, 24).
To further investigate the SAR, the ligands were docked
into the sEH binding site. Docking on FLAP is not
shown, because the crystal structure was not suitable for
docking.
In the sEH docking simulation, modifications in
compound 13 (modified substitution on subunit V) and
21 (3-methyl on subunit III) did not affect the typical
binding pattern between the urea moiety and the
catalytic triad of sEH, that is, Asp 355, Tyr 466 and
Tyr383 (Figure 3). The least active compound 24, with
the chlorine in ortho-position leads to a turn of the urea
moiety, which prohibits the hydrogen bonding with the
catalytic site (Figure 4), explaining the loss of activity.
In conclusion, our SAR study of novel diflapolin
derivatives revealed important information regarding
relevant substructures to target FLAP and sEH,
respectively. While both activities can be improved on
one of the targets, it is challenging to modify the
molecule and optimize both activities at the same time.
For compounds 15 and 27 both activities were increased.
Modifications at the spacer unit II, had little effect on
sEH, but strongly affected FLAP activity. While a
thioether (28) only reduced FLAP activity, compounds
31 and 32 lose their inhibitory effects against FLAP,
which might be explained by the loss of the hydrogen
bond acceptor.
The SARs are summarized in Figure 2.
Figure 3. Diflapolin (blue) shown together with 21 (pink)
and 13 (white) in sEH. The simulation indicates that the
binding mode is not affected by the modifications and that
all form the key interactions with the catalytic triad of sEH,
that is, Asp355, Tyr466 and Tyr383. Yellow spheres
represent hydrophobic contacts, green arrows represent
hydrogen bond donors and red arrows mark hydrogen bond
acceptors.
Figure 2. Summary of the SAR for FLAP and sEH
inhibition (green: favorable moieties, red unfavorable
moieties)
We also conducted
a Free-Wilson analysis on
compounds 1 and 11-27 to quantify the influence of the
specific modifications on the overall activity on the two
targets (see supporting information).
The model showed that the 3-methyl modification on
subunit III is lowering FLAP inhibitory activity,
assigning a negative score of -0.923 to this modification
Also, the 4-chloro modification at subunit V is not
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The manuscript was written through contributions of all
authors and all authors have given approval to the final
version of the manuscript.
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Funding Sources
German Research Council (SFB1278 Polytarget, to O.W.,
and GA 2101/2-1, to U.G.)
“Doktoratsstipendium der Nachwuchsförderung” by
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University of Innsbruck (L.V.; M.S.)
ACKNOWLEDGMENT
We thank the German Research Council (SFB1278
Polytarget, to O.W., and GA 2101/2-1, to U.G.) for
financial support. L.V. and M.S. acknowledge the support
by the University of Innsbruck (“Doktoratsstipendium der
Nachwuchsförderung”). V.T. is funded by the FWF
Firnberg project T 942.
Figure 4. Diflapolin is shown in blue and inactive
compound 24 in green. The indicated hydrogen bond
interactions with sEH are only given for diflapolin. Because
of the turn caused by the steric hindrance of the chlorine in
the ortho-position in compound 24, the urea moiety is no
longer forming the key interactions.
ABBREVIATIONS
AA, arachidonic acid; BSA, bovine serum albumin; COX,
cyclooxygenase; cPLA2, cytosolic phospholipase A2; CYP,
cytochrome; DHETrEs, dihydroxyeicosatrienoic acids;
ASSOCIATED CONTENT
Supporting Information
DMLs,
designed
multiple
ligands;
DMSO,
The Supporting Information is available free of charge on
the ACS Publications website.
dimethylsulfoxide; EETs, epoxyeicosatrienoic acids; FLAP,
5-LO-activating protein; LO, lipoxygenase; LT, leukotriene;
Synthetic procedures, spectral data and elemental
analysis for key compounds, computational methods,
Free Wilson analysis, biological assay protocols.
(PDF)
PHOME,
3-phenyl-cyano(6-methoxy-2-
acid; SAR,
naphthalenyl)methylester-2-oxiraneacetic
structure-activity relationships; sEH, soluble epoxide
hydrolase; SPM, specialized-pro-resolving mediators; THF,
tetrahydrofuran.
AUTHOR INFORMATION
Corresponding Author
REFERENCES
*E-mail: Barbara.matuszczak@uibk.ac.at. Phone: +43 512
507-58262. Fax: +43 512 507-58299
*E-mail: Ulrike.garscha@uni-jena.de. Phone: +49 3641
949-811. Fax: +49 3641 949-802
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Synthesis, biological evaluation and structure-activity relationships of diflapolin analogs as dual
sEH/FLAP inhibitors
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Lisa Vieider1, Erik Romp2, Veronika Temml3, Jana Fischer2, Christian Kretzer2, Martin Schoenthaler1, Abdulla
Taha2, Victor Hernández-Olmos5, Sonja Sturm3, Daniela Schuster1,4, Oliver Werz2, Ulrike Garscha2*, Barbara
Matuszczak1*
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