Novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from Lipopolysaccharide-Induced Cytotoxicity

Article abstract of DOI:10.1021/acs.jmedchem.9b00212

Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.

Full text of DOI:10.1021/acs.jmedchem.9b00212

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Article
A novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages
from Lipopolysaccharides-Induced Cytotoxicity
Hao Guo, Iris Verhoek, Gerian Prins, Ramon van der Vlag, Petra E. van der Wouden,
Ronald van Merkerk, Wim J. Quax, Peter Olinga, Anna K. H. Hirsch, and Frank J. Dekker
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00212 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 10, 2019
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A novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from
Lipopolysaccharides-Induced Cytotoxicity
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a
a
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Hao Guo, Iris C. Verhoek, Gerian G. H. Prins, Ramon van der Vlag, Petra E. van der
a
a
a
b
c,d,e
Wouden, Ronald van Merkerk, Wim J. Quax, Peter Olinga, Anna K. H. Hirsch,
Frank J.
Dekker*,
a
Affiliations:
a
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands
b
Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands
c
Department of Chemical Biology 2, Stratingh Institute for Chemistry, University of Groningen,
Nijenborgh 7, 9747 AG Groningen, The Netherlands
d
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz Centre for
Infection Research (HZI), Department of Drug Design and Optimization, Campus Building E8.1,
6
6123 Saarbrücken, Germany
e
Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany
*
Corresponding author.
E-mail address: f.j.dekker@rug.nl (Frank J. Dekker).
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Abstract
Various mechanisms for regulated cell death include the formation of oxidative mediators
such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a
key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are
interconnected with nuclear factor-κB (NF-κB) signaling and NO production. Inhibition of 15-
LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this
study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure–activity
relationships were explored. In vitro, this inhibitors protected cells from LPS-induced cell death,
inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that
inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration
of these mechanisms.
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1
. Introduction
Over recent years, an increasing number of mechanisms for regulated cell death have been
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identified and versatile roles in numerous diseases were proposed. Cell death via a mechanism
other than apoptosis leads to plasma membrane rupture and release of the cellular content, thus
providing damage-associated molecular patterns (DAMPs) that can induce an auto-amplification
loop of regulated cell death and inflammation. Such amplification loops are expected to play key
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roles in diseases such as acute lung injury and acute respiratory distress syndrome. Understanding
the underlying mechanisms to develop of small molecule inhibitors to interfere with cell death
holds promise for therapeutic control of these disorders.
The discovery of multiple types of cell death provides new challenges to identify the
molecular mechanisms involved. One mechanism of non-apoptotic cell death is pyroptosis in
which macrophages die by excessive stimulation of Toll-like receptors (TLRs) and activation of
the NF-κB pathway by, for example, lipopolysaccharides (LPS).2–6 Normally, pyroptosis is a
mechanism to protect multicellular organisms from invading pathogens, such as microbial
infections. However, under pathogenic conditions pyroptosis can be involved in the onset of
chronic inflammation. Another mechanism for non-apoptotic cell death is ferroptosis, which is a
process in which excessive levels of lipid peroxides cause cell death. It is anticipated that
lipoxygenases (LOXs) play key roles in ferroptosis by catalyzing lipid peroxidation.2,7 The
identification of pyroptosis, ferroptosis and other mechanisms for regulated cell death raises the
question how these mechanisms can be exploited for drug discovery.
Although distinct mechanisms for regulated cell death were described, the mechanisms
involved are often closely related and cross talk exists. In this study, we aim to address the cross-
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talk between macrophages cell death upon LPS stimulation and the enzymatic activity of 15-
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lipoxygenase-1 (15-LOX-1) as a regulator of cellular lipid peroxidation (Figure 1). Activation of
the NF-κB pathway results in transcription of downstream genes, such as inducible nitric oxide
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synthase (iNOS), that plays a critical role in inflammatory responses. iNOS catalyzes the
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formation of NO radicals that play key roles in many physiological processes. On the other hand,
excessive NO production can lead to the formation of reactive nitrogen species (RNOS), which
induces cell death and tissue damage.11
Reactive oxygen species (ROS) such as lipid peroxides have been shown to augment LPS-
mediated NF-κB activation and thus increase expression of NF-κB target genes,8,12 which
represents a mechanism of crosstalk between lipid peroxidation and NF-κB activation. 15-LOX-1
is a non-heme iron-containing enzyme producing lipid peroxides from poly-unsaturated fatty acids
(
PUFAs), such as arachidonic acid (AA) and linoleic acid (LA).13–15 15-LOX-1 oxidizes either AA,
to form the corresponding 15-hydroxyeicosatetraenoic acid (15-HpETE), or LA, to form the
corresponding 13-hydroperoxyoctadecadienoic acid (13-HpODE).16,17 Apart from these
hydroperoxy fatty acids, lipoxins are also derived from the 15-LOXs pathway and play a role as
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anti-inflammatory mediators. On the other hand, the 15-LOXs metabolites, eoxins are proposed
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to be a family of pro-inflammatory eicosanoids. Altogether, lipid peroxides and can be converted
further in distinct lipid signaling molecules that have key regulatory roles in immune responses,20–22
2
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and numerous diseases. Importantly, if the production of lipid peroxides is not balanced by the
cellular anti-oxidant system, this can result in ferroptotic cell death and in enhanced activation of
the NF-κB pathway, thus providing synergistic crosstalk between two mechanisms of regulated
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cell death. Thus 15-LOX-1 is a key enzyme in oxidative stress and regulated cell death in
numerous diseases.13,25,26
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Figure 1. Several mechanisms of lipopolysaccharide (LPS) signaling in macrophages are connected to cell death. LPS-
mediated activation of NF-κB pathway results in the overexpression of inducible nitric oxide synthase (iNOS). This
leads to the production of nitric oxide (NO) and reactive nitrogen species (RNOS), which is involved in cell death. In
the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (13-HpODE), the metabolite of 15-LOX-1 activity, can
also induce cell death. Both mechanisms act in concert and cross talk exist.
For 15-LOX-1, roles have been described in diseases such as asthma,14 stroke,15
2
16,17
cancer,20,21 Alzheimer’s disease,22,23 and Parkinson’s disease. This
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atherogenesis, diabetes,
triggered the interest in the development of 15-LOX-1 inhibitors for drug discovery. In an early
phase, indole-based inhibitors, PD-146176 was identified as r-12/15-LOX inhibitor with an IC50
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value of 3.81 μM (Figure 2). This stimulated efforts to develop inhibitors with an indolyl core
(Figure 2). More researchers reported the discovery of indole-based or indole-like 15-LOX-1
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8,29
inhibitors, 371 and Haydi-4b (with IC of 0.006 and 3.84 μM, respectively).
In our group, we
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previously discovered 15-LOX-1 inhibitor Eleftheriadis-14d, which also contains an indole core
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and demonstrates good potency (IC = 90 nM). Furthermore, 1,3-oxazole based compound
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(ML351), purine-based (Anders-6b) compound, and pyrrole-based compound (21B10) were
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identified as 15-LOX-1 inhibitors as well (Figure 1). These inhibitors proved to be effective in
various disease models, thus indicating the potential of 15-LOX-1 inhibitors for drug discovery.
Complementary to development of inhibitors, efforts were made to engineer 15-LOX-1
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substrates for detection of enzyme activity. We developed activity-based probe N144 as a chemical
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reporter for lipoxygenase activity in cell lysates and tissue samples. Another study employed the
omega-alkynyl fatty acid (aAA) to identify the intracellular targets of 12/15-LOX generated lipid-
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5
derived electrophiles. This sets the stage for the development of potent 15-LOX-1 inhibitors and
to study their cellular activity.
Figure 2. Examples of previously reported 15-LOX-1 inhibitors and chemical tools to study lipoxygenase activity. (A)
Indole-based 15-LOX-1 inhibitor and inhibitors based on other nitrogen containing heterocycles. (B) Substrate-based
chemical tools to study lipoxygenase activity in cell-based systems.
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In this study, we investigated novel substitutions of the indole core and investigated the
structure–activity relationships (SAR) for 15-LOX-1 inhibition. For the most potent inhibitor the
effects on cellular 15-LOX-1 inhibition, the effects on formation of reactive oxygen species (ROS)
and regulated cell death were investigated on RAW 264.7 macrophages to provide insight in the
cellular potency of this type of inhibitors.
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2
. RESULTS AND DISCUSSION
2
.1. Chemistry. Scheme 1 presents the general methodology for the synthesis of
compounds 5a and 5b. The synthesis started with the assembly of ethyl 6-chloro-1H-indole-2-
carboxylate (1) and corresponding aldehyde (2) using known literature procedures.30,36
Subsequently, the 2-formyl functionality of the aldehyde 2 was oxidized into its corresponding
carboxylic acid (3) via Pinnick oxidation using sodium chlorite (NaClO ) giving a yield of 78%.
2
Attempts to use KMnO or Tollens' reagent did not provide the desired product. Finally, the amide
4
bonds in products 5a and 5b were generated by using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI) and N-hydroxybenzotriazole (HOBt) as coupling reagents, giving yields of
about 85%.
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Scheme 1. Synthetic route to compounds 5a–b. Reagents and conditions: (a) POCl
t-BuOH, 50 C, 4 h; (c) amine (4a–b), EDCI, HOBT, TEA, DCM, r.t., 4 h.
3
2
, DMF, 60 C, 48 h; (b) NaClO ,
Compounds 9a–j were synthesized using procedures as shown in Scheme 2. As a first step
the 2-formyl functionality of aldehyde 2 was employed for Wittig reaction with (tert-
butoxycarbonylmethylene) triphenylphosphorane to provide the α,β-unsaturated ester 6. Initially,
attempts were made to obtain compound 7 by refluxing aldehyde 2 with the Wittig reagent in
toluene overnight. However, this provided compound 6 as a mixture of E- and Z-isomers (E/Z =
3
7
approximately 9/1). Changing the solvent from toluene to ethanol at 80 C enabled the reaction
to finish in 2 hours with the E-alkene as major product that could be isolated in a yield of 70% after
purification. The E- and Z-isomer could be distinguished by their J values of 16.0 Hz and 7.0 Hz,
respectively. Finally, intermediate 7 was converted into the amides 9a–j by removal of the tert-
butyl protective group using trifluoroacetic acid (TFA) treatment and subsequent coupling of the
corresponding amines using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and N-
hydroxybenzotriazole (HOBt) as coupling reagents in yields between 80 and 90% over two steps.
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Scheme 2. Synthetic route to compounds 9a–j. Reagents and conditions: (d) (tert-Butoxycarbonylmethylene)
triphenylphosphorane, EtOH, reflux, 2 h; (e) TFA, DCM, r.t., overnight; (f) amine (8a–j), EDCI, HOBT, TEA, DCM,
r.t., 4 h.
Products 12a–g were produced starting from compound 10 as shown in Scheme 3. The
carboxylic acids 10 was coupled to amines 11a–g using EDCI and HOBT as coupling reagents,
that gives the desired products in 80–90% yield. Afterwards, compound 13 was obtained from
compound 12c by using a Mannich reaction with dimethyl amine, formaldehyde and acetic acid to
provide the desired product in a yield of 93%. As shown in Scheme 4, compounds 14–17 were
obtained from the corresponding 2-carboxy ethyl indoles (9c, Eleftheriadis-14d and Eleftheriadis-
1
4e) over two steps in the similar way as for 12a–g in a high yield (80–90%).
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Scheme 3. Synthetic route to compounds 12a–g and 13. Reagents and conditions: (g) amine (11a–g), EDCI, HOBT,
TEA, DCM, r.t., 4 h; (h) dimethylamine, formaldehyde, acetic acid, MeOH, reflux, 4 h.
Scheme 4. Synthetic route to compounds 14–17. Reagents and conditions: (i) LiOH, THF, H O, 50 C, 2 h; (j) EDCI,
2
HOBT, TEA, DCM, r.t., 4 h.
2
.2. Structure–activity relationships. Inhibition of 15-LOX-1 enzyme activity was
performed using an activity assay as described previously by us.30,33,38 The activity of 15-LOX-1
was monitored by measuring the conversion of LA into the UV-active 13-HpODE (λmax 234 nm).
This assay was used to determine the half maximal inhibitory concentration (IC ) of each
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compound. SARs for binding to 15-LOX-1 were investigated starting from ethyl 6-chloro-1H-
indole-2-carboxylate (1). We aimed to introduce structural modifications to replace the lipid chain
in the 3-position and the ethylcarboxylate in the 2-position. Key structural modifications are shown
in Figure 3 starting from previously identified inhibitors 1 and Eleftheriadis-14d.30
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Systematic modifications of 15-LOX-1 inhibitor 1 as core scaffold and the previously described inhibitor
3
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Eleftheriadis-14d to provide the new inhibitors 5b, 9c, 15–17 and their IC50 values for 15-LOX-1 inhibition.
The SAR of the previously identified inhibitor Eleftheriadis-14d, was explored with respect
to aliphatic acyl substitutions in the indole 3-position. In order to expand the SAR in novel
directions, the carboxyl ethyl ester in the indole 3-position was replaced by an amide in order to
gain metabolic stability. Both compounds 5a and 5b (Scheme 1, Table 1) provided IC values
5
0
above 1 µM, which is much higher compared to the previously reported series of inhibitors with
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the carboxyl ethyl ester. Apparently, replacement of the carboxyl ethyl ester in the indole 2-
position for an amide is unfavorable for 15-LOX-1 inhibition.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. IC50 values for 15-LOX-1 inhibition by amide substituted indoles in the 3-position (analogues 5a–b).
R
NH
IC50 (μM)
O
Compound
R
[
± SD (μM)]
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5a
5b
> 20
N
O
Cl
H
2.87 ± 1.81
The SAR in the indole 3-position was further explored by replacement of the carbonyl in
-position for a double bond using Wittig chemistry. Using this chemistry, we aimed to replace the
3
aliphatic lipid chain in Eleftheriadis-14d for less flexible substituents. Thus, we investigated a
series of E-alkenes as shown in Table 2. Clear SARs were observed for this series of compounds.
Compound 9c (i472) proved to be the most potent 15-LOX-1 inhibitor with an IC value of 0.19
5
0
µM. Comparison of inhibitor 9c (i472) to inhibitor 9f indicates that the ortho-methoxy substitution
on the benzyl provides a ten-fold gain in potency compared to a non-substituted benzyl.
Importantly, the IC values decrease if the methoxy is moved from the ortho to the meta or para
5
0
position on the benzyl 9d and 9e. Extending the benzyl to an ethylphenyl in inhibitors 9g–j did not
improve their potencies either. Taken together, the 2-methoxybenzyl group in 9c (i472) provides
the most potent inhibitor in this series.
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Table 2. IC50 values against 15-LOX-1 with different variations in R (analogues 9a–j).
R
NH
IC50 (μM)
O
Compound
R
[
± SD (μM)]
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Cl
Cl
9a
2.95 ± 1.28
0.74 ± 0.22
N
O
Cl
H
Cl
9
b (i44)
O
9c (i472)
0.19 ± 0.03
0.40 ± 0.01
0.75 ± 0.31
9
d
O
O
9
e
9
f
2.43 ± 0.96
>20
9
g
Cl
9
h
1.29 ± 0.42
0.19 ± 0.05
0.27 ± 0.01
Cl
Cl
9
i
O
9
j
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5
5
5
5
5
6
In order to further explore the SAR of previously identified inhibitor Eleftheriadis-14d,
variations were made in the indolyl 2-position. Towards this aim, the ester group was replaced with
various amides to provide inhibitors 12a–g as shown in Table 3. The results indicate that amide
substitution provides inhibitors with potencies in the micromolar range. However, the SAR for
modifications with methyl, ethyl, n-propyl, n-butyl, n-pentyl and branched alkyl groups proved to
be relatively flat with potency differences of no more than two- to three-fold. Remarkably,
cyclopropyl substitution in 12g turns out to be inactive (IC > 20 µM). Taken together, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
investigated series of amide substituted indoles did not provide improved potency and the most
potent inhibitor in this series is compound 12c with a propyl substitution.
Table 3. IC50 values against 15-LOX-1 with different variations in the amide tail (analogues 12a–g).
H
IC50 (μM)
N
R
Compound
R
Cl
N
[± SD (μM)]
O
H
1
2a
12b
2c
2d
9.38 ± 2 .63
6.88 ± 2.32
4.09 ± 1.11
13.02 ± 2.73
10.94 ± 2.21
1
1
12e
12f
9.82 ± 1.41
>20
1
2g
As a next step, we combined ethyl- or propyl-substituted amides in the indole 2-position
with substitutions in the 3-position to provide inhibitors 14–17 as shown in Table 4. Unfortunately,
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combination of both modifications provided inhibitors with low potency. Apparently, the more
polar amide bond is not well tolerated for enzyme inhibition and combination of modifications in
the 2- and 3-position caused a greatly reduced potency. Inhibitor 13 also showed a complete loss
in potency against 15-LOX-1. Taken together, we concluded that inhibitor 9c (i472) has the highest
potency of this series and that the IC value is in the same range as for the previously identified
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
inhibitor Eleftheriadis-14d.
1
2
Table 4. IC50 values against 15-LOX-1 with different variations in R and R (analogues 14–18).
R1
IC50 (μM)
R1
R2
H
Compound
2
N
R
[
± SD (μM)]
Cl
N
O
H
1
1
3
4
N
>20
O
O
>20
>20
1
1
5
6
O
O
O
O
5.81 ± 1.18
N
H
1
7
>20
N
H
2
.3. Docking 15-LOX-1. In order to understand the observed SAR, a molecular modeling
study was performed in which key inhibitors were docked in the 15-LOX-1 active site. In this
study, docking was performed using Discovery studio (Dassault systèmes) version 2018. And the
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rabbit reticulocyte 15-LOX-1 crystal structure (PDB ID: 1LOX) was used for docking because of
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its high sequence similarity in the active site. In this crystal structure, the ligand in the crystal
structure was removed and the center of the binding sphere was set at the same position. Based on
this position, CDocker, a CHARMm based method, was used and resulted in ten highest ranked
poses for all selected inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on the observed SAR, the most potent inhibitor 9c (i472) was docked and compared
to 9f (lacking the methoxy group) and 16 (in which the ester is replaced for an amide). In both
cases the potency decreased by at least ten-fold. The docking model suggested several interactions
between the active site of 15-LOX-1 and 9c (i472) as shown in Figure 4(A). Upon comparison of
the docking of 9c (i472) and 9f, the 2-methoxy on the benzyl functionality provides two hydrogen
bonds with GLN 548 and ILE 593, respectively (Figure 4(A)). This provides an explanation for the
ten-fold potency difference between 9c (i472) and 9f. In addition, because of the hydrophobic
character of 15-LOX-1, except from the edge of the active site, of the majority of the pocket is
hydrophobic, as shown in brown in Figure 4 (B) and (C). The hydrophilic sites are shown in blue.
Docking of compound 16, in which an amide replaces the carboxy ethyl ester at the indole 2-
position, shows a positionally inverted orientation compared to 9c (i472) and 9f (Figure 4 (C) and
S1 (C)). Apparently, the amide with an additional hydrogen bond donor does not fit in the same
position as the ester in 9c (i472). This change in orientation upon docking is also in line with the
observed drop in potency for 16 compared to 9c (i472) and 9f.
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1
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2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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3
4
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5
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5
5
5
5
5
6
0
1
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5
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Molecular modeling of selected compounds in the active site of 15-LOX-1 (PDB ID: 1LOX). The surface
in the pocket is colored based on the relative hydrophobicity: brown for hydrophobic and blue for hydrophilic areas
(
A) Interactions of compound 9c (i472) with the active site of the enzyme. (B) The preferred orientation of compound
c (i472) in the active site of the enzyme. (C) The preferred orientation of compound 16 in the active site of the enzyme
that is opposite to 9c (i472).
9
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.4. Physicochemical properties of inhibitor 9c (i472). The α,β-unsaturated amide
functionality in 9c (i472) is, as a Michael-acceptor, reactive towards conjugate addition by
nucleophiles, such as thiols. In order to monitor this reactivity the UV spectrum of 9c (i472) was
recorded upon incubation with 2-mercaptoethanol at pH 7.4. No changes in the UV spectrum were
observed, which indicates that the chromophore, including the α,β-unsaturated system, did not
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
change (Figure S2), thus indicating a reasonable stability of compound 9c (i472) towards
nucleophilic substitution. This stability might be attributed to the conjugation of the α,β-
unsaturated double bond with the aromatic indole core.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
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9
0
Inhibitor 9c (i472) has a calculated cLogP (ChemDraw Professional version 12.0) of 4.7,
which is more than two order of magnitude lower compared to the previously identified inhibitor
Eleftheriadis-14d (cLogP = 6.9), whereas both inhibitors have molecular weights around 400 g/mol.
Considering the physicochemical properties, the newly identified inhibitor 9c (i472) has fewer
rotatable bonds and a cLogP that is more favorable for cellular permeability compared to the
previously identified inhibitor Eleftheridis-14d.
2
.5. LOX inhibitory potency of 9c (i472) in cells by activity-based labeling. As a next
step in the characterization of inhibitor 9c (i472), the inhibition of cellular LOX activity was
investigated. Towards this aim we employed a method for activity-based labeling of LOX activity
in cell-based systems that we developed recently.34 In this method, a covalent inhibitor of
lipoxygenase activity is equipped with a terminal alkene for bioorthogonal labeling with biotin
4
0
using the oxidative Heck reaction. Here we employed this method to estimate the inhibition of
cellular lipoxygenase activity by inhibitor 9c (i472) in RAW264.7 macrophages. Inhibitor treated
and non-treated cell-lysates were exposed to covalent inhibitor N144 (Figure 2) for 2 minutes, and
subsequently the samples were subjected to oxidative Heck reaction to link a biotinylated
phenylboronic acid for detection. In parallel to the labeling the same samples were subjected to
staining for β-actin as a loading control and antibody based detection of the amount of 15-LOX-1.
Representative blots are shown in Figure 5. We observed a decreased intensity for the band for
activity-based lipoxygenase labeling as detection by streptavidin-HRP. For comparison, the bands
normalized with the β-actin antibody and the 15-LOX antibody were included as well, which show
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comparable intensities. Quantifications of the bands from three independent experiments are shown
in Figure 6. From these results, we conclude that 15-LOX-1 inhibitor 9c(i472) is able to inhibit the
activity of cellular lipoxygenases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Detection of the effect of inhibitor 9c (i472) on the activity of 15-LOX-1 by activity-based probe. Labeling
was performed on cell lysis of RAW 264.7 cells. Positive control (with probe and without inhibitor), negative control
(without probe or inhibitor) and incubation of 9c (i472) were performed with the 15-LOX antibody, β-actin antibody
and streptavidin-HRP (n=3).
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0
1
2
3
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9
0
1
2
3
4
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Figure 6. Quantification of Western blots for detection and analysis of activity-based labeling: The values are
measured by integrating the gray values by ImageJ 1.44. The integrated gray values of streptavidin-HRP and 15-LOX
are normalized to β-actin, respectively. All the values were expressed as means ± SEM. The results were normalized
by three independent experiments (n=3). *p < 0.05; **p < 0.005 and ***p < 0.001 compared to control by two-tailed
test.
2
.6. Protection of RAW 264.7 macrophages from LPS-induced cytotoxicity. After
identifying compound 9c (i472) as a potent inhibitor for recombinant expressed 15-LOX-1 and
cellular LOX activity, we moved on to investigate the potency of this compound in cell-based
studies. As the insight into the mechanism, we presume that 15-LOX-1 inhibitors inhibit the
formation of lipid peroxides in cells, thereby preventing ferroptotic cell death. Additionally, we
expect this mechanism to have cross-talk with the NF-κB pathway. Activation of this pathway can
also lead to cell death. To test this hypothesis we employed a model in which we stimulated
3
RAW264.7 macrophages with LPS to cause cell death as reported previously. This study reported
an LD of 89.5 μg/mL for LPS induced cell death in macrophages. In our experiments, 40%
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0
inhibition of cell viability was obtained at 100 µg/mL (Figure S3). Subsequently, as shown in
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Figure 7, we employed an LPS concentration of 100 µg/mL and investigated the protection from
cell death by treatment with lipoxygenase inhibitors. The 5-LOX inhibitor Zileuton and the
previously identified 15-LOX-1 inhibitor Eleftheriadis-14d comparably improved the viability of
LPS-treated RAW 264.7 macrophages. In addition, inhibitor 9c (i472) showed stronger, dose-
dependent effects with a 20% viability increase at 5 µM. Thus, these data demonstrated that
inhibiting 15-LOX-1 by compound 9c (i472) can protect RAW 264.7 macrophages from LPS-
induced cell death.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
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0
1
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0
1
2
3
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0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Inhibitor 9c (i472) protects RAW 264.7 macrophages from LPS-induced cytotoxicity. RAW 264.7
macrophages were treated with lipopolysaccharides (LPS) (100 μg/mL) and 9c (i472) together for 24 hours. Then, the
cell viability was determined by MTS assay (n=3–13). All the values were expressed as means ± SEM. *p < 0.05; **p
<
0.005 and ***p < 0.001 compared to positive control by two-tailed test that is only with the treatment of LPS (100
μg/mL) for 48 hours.
2.7. NF-κB activity determination. To gain further insight in the mechanism of protection
for LPS-induced cell death, we investigated the effect of inhibitor 9c (i472) on NF-κB activity
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2
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2
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using an NF-κB reporter assay in RAW-Blue macrophages (modified RAW 264.7 macrophages).
These RAW-Blue macrophages can stably express a secreted embryonic alkaline phosphatase
(SEAP) gene that is inducible by NF-κB and AP-1 transcription factors. Previous evidence
demonstrated that the product of 15-LOX-1, 13-HpODE, can increase NF-κB activation but has no
0
1
2
3
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0
1
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effect on AP-1. The cells were stimulated with LPS, interferon gamma (IFNγ) and inhibitor 9c
4
1
(
i472). This provided significant but not complete inhibition of NF-κB transcriptional activation
upon LPS/INF stimulation (Figure 8). These results are in line with the anticipated cross-talk
between 15-LOX-1 inhibition and NF-B signaling.
Figure 8. Inhibitor 9c (i472) reduces NF-κB activity. RAW-Blue macrophages were pre-treated with inhibitors 9c
(i472) at 0.2, 1 and 5 μM for 20 hours, after which an inflammatory lipopolysaccharides (LPS) and interferon gamma
(IFNγ) stimulus (10 ng/mL of each) was given for another 4 hours in continued presence of inhibitor 9c. All the values
were expressed as means ± SEM (n=8–16). *p < 0.05; **p < 0.005 and ***p < 0.001 compared to positive control that
is treated with LPS and IFNγ by two-tailed test.
2
.8. Gene Expression Profiling. Subsequently, we turned our attention to the influence of
15-LOX-1 inhibition by 9c (i472) on the gene expression of NF-κB related gene iNOS (Figure 9).
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As a model we used RAW 264.7 macrophages that were activated by LPS and IFNγ (10 ng/mL of
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each). The gene expression of iNOS was downregulated by approximately 50% at 5 μM. This
finding is in line with but more pronounced than the observed decrease in NF-κB transcriptional
activity (Figure 8).
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Figure 9. Effect of inhibition of 15-LOX-1 by 9c (i472) on iNOS in RAW 264.7 cells: Lipopolysaccharides (LPS)/
interferon gamma (IFNγ) (10 ng/mL of each) stimulated cells were normalized to the positive control. All experimental
groups were treated with compound 9c (i472) at 0.2, 1 and 5 μM for 20 h and stimulated with LPS/IFNγ for another 4
hours (n=3–4). All the values were expressed as means ± SEM. *p < 0.05; **p < 0.005 and ***p < 0.001 compared to
LPS/IFNγ treated positive control group by two-tailed test.
2
.9. Quantification of Nitric oxide (NO) production. Gene transcription of iNOS is
connected to NO production, which plays an important role in the regulation of immune response
and apoptosis. In our study, we compared the inhibitory effects of 9c (i472) on the ratio of total
nitrate/nitrite in RAW 264.7 macrophages (Figure 10). We demonstrated that 9c (i472) as 15-LOX-
1 inhibitor provided dose-dependent inhibition of NO production, which is consistent with the
results of reduced activity of NF-κB and the gene expression of iNOS. The observations that 15-
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LOX inhibition inhibits NF-κB reporter gene activity, iNOS expression and NO levels are in line
with the idea that there is cross-talk between 15-LOX-1 activity and cell-death via activity of the
NF-κB pathway and NO production.
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Figure 10. Dose-dependent effect of 9c (i472) on the expression of total nitrate/nitrite in RAW 264.7 cells:
Lipopolysaccharides (LPS)/ interferon gamma (IFNγ) (10 ng/mL of each) stimulated cells were corrected to 100% as
positive control. All experimental group were treated with compound 9c (i472) at 0.2, 1 and 5 μM for 20 h and
stimulated with LPS/IFNγ for another 4 hours (n=3). All the values were expressed as means ± SEM. *p < 0.05; **p
< 0.005 and ***p < 0.001 compared to LPS/IFNγ treated control group by two-tailed test.
2.10. Lipid peroxidation. Oxidative stress can cause a series of toxic effects through the
43
production of lipid peroxides that play a role in cell death. The effect of lipoxygenase inhibitor
9
c (i472) on lipid peroxidation RAW 264.7 cells was investigated using the fluorescent dye
4
4
BODIPY 581/591 C and fluorescence-activated cell sorting (FACS). As shown in Figure 11,
1
1
1
5-LOX-inhibitor PD-146176 and 9c (i472) revealed comparable effects that both of them
significantly attenuated the boost of lipid peroxides at 5 µM after the treatment of LPS/IFNγ (10
ng/mL of each). This result could be attributed to loss of 15-LOX products, such as 13-HpODE.
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Furthermore, compared to 5-LOX inhibitor, Zileuton, both 15-LOX-1 inhibitors showed a more
pronounced effect in lipid peroxidation fitting the result of LPS-induced cell death assay that 15-
LOX-1 inhibitor has a better rescue effect. Although 15-LOX-1 is not the only pathway that can
trigger lipid peroxide formation, these results indicate that inhibition of 15-LOX-1 has a strong
influence on lipid peroxidation in this model system.
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Figure 11. Analysis lipid peroxidation using BODIPY 581/591 C11 staining and FACS analysis. Cells were treated
with lipopolysaccharides (LPS)/ interferon gamma (IFNγ) (10 ng/mL of each) and PD-146176 (5 μM), Zileuton (5
μM) or 9c (i472) (5 μM). Results are represented as means ± SEM (n = 3). *p < 0.05; **p < 0.005 and ***p < 0.001
compared to LPS/IFNγ treated control group by two-tailed test.
3
. Conclusions
In this study, compound 9c (i472) was developed as a potent 15-LOX-1 inhibitor with a
novel substituent pattern (IC = 0.19 μM) and its SARs were explored. Using activity-based
5
0
labeling we demonstrated that inhibitor 9c (i472) was able to inhibit cellular lipoxygenases. Further
characterization of this compound demonstrated that it was able to protect RAW264.7
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macrophages from LPS-induced cell death. We explored the influence of inhibitor 9c (i472) on
different pathways of cell death. We investigated NF-κB activation, iNOS expression and NO
formation as a line of events that can trigger cell death. Treatment with inhibitor 9c (i472) enabled
downregulation of NF-κB transcriptional activity in a reporter gene assay. Furthermore, we
demonstrated that iNOS gene expression and the levels NO in RAW 264.7 macrophages decreased
significantly upon 9c (i472) treatment. As a direct effect of inhibiting lipoxygenase activity, we
investigated inhibition of cellular lipid peroxidation upon 9c (i472) treatment for which we
observed a clear reduction back to baseline levels. Having explored both mechanisms, we can
conclude that inhibitor 9c (i472) influences both NO production and lipid peroxidation, potentially
via a cross-talk mechanism. Thus, we conclude that we provide a novel 15-LOX-1 inhibitor 9c
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(i472) with cellular activity that inhibits the formation of oxidative mediators, such as NO and lipid
peroxides, that are connected to different mechanisms for cell death.
4
. Experimental section
4
.1. General. All reagents, solvents and catalysts were purchased from commercial sources
(Acros Organics, Sigma-Aldrich and abcr GmbH, Netherlands) and used without purification. All
reactions were performed in oven-dried flasks in open or under nitrogen atmosphere, and monitored
by thin layer chromatography on TLC precoated (250 µm) silica gel 60 F254 aluminum foil (EMD
Chemicals Inc.). Visualization was achieved using UV light. Alternatively, non UV-active
compounds were detected after staining with potassium permanganate. Flash column
1
chromatography was performed on silica gel (32-63 µm, 60 Å pore size). H NMR (500 MHz) and
13C NMR (126 MHz) spectra were recorded with a Bruker Avance 4-channel NMR Spectrometer
with TXI probe. Chemical shifts (δ) are reported in ppm. Abbreviations are as follows: s (singlet),
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d (doublet), t (triplet), q (quartet), m (multiplet). Fourier Transform Mass Spectrometry (FTMS)
and electrospray ionization (ESI) were done on an Applied Biosystems/SCIEX API3000-triple
quadrupole mass spectrometer. High performance liquid chromatography (HPLC) analysis was
performed for confirming purity with a Shimadzu LC-10AT HPLC with a Shimadzu SP-M10A
ELSD detector and a Shimadzu SPD-M10A photodiode array detector. Analytical HPLC was
performed using a Kinetex C18 column (150 mm × 4.6 mm, 5 μm) with 5−95% MeCN gradient in
1
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H O as mobile phase, confirming purity ≥ 95%. Retention time (RT) of HPLC was also reported.
2
4
4
.2. Synthesis and Characterization.
.2.1. Ethyl 6-chloro-3-formyl-1H-indole-2-carboxylate (2): To a solution of POCl3
(0.30 mL, 3.2 mmol, 1.2 eq.) in DMF (6.0 mL) stirred at 0 °C for 0.5 h, 1 (0.60 g, 2.7 mmol), was
added to the reaction mixture and heated to 50 C for 46 h. After completion, the reaction mixture
was slowly poured into a mixture of crushed ice and H O (300 mL). Product was obtained by
2
filtration of the resulting suspension. The residue was washed with acetonitrile and dried at r.t.
giving the title intermediate 2 as yellow solid in a yield of 83% (0.55 g, 2.2 mmol). The NMR
spectra were the same as reported previously.36
4
.2.2. 6-chloro-2-(ethoxycarbonyl)-1H-indole-3-carboxylic acid (3): To a solution of 2
(
0.10 g, 0.39 mmol) and NaClO (71 mg, 0.78 mmol) mixed in 5.0 mL t-BuOH at 50 °C for 4h.
2
After completion, the mixture was concentrated and H O (50 mL) was added, followed by
2
extraction with EtOAc (3 × 15 mL). The organic phases were collected and evaporated giving a
white crude product without further purification in 82% yield.
4
.2.3. General synthetic procedure 1: Amide bond formation. The respective carboxylic
acid (1.0 equiv.) was added to a mixture of HOBt (0.40 equiv.), EDCI (2.0 equiv.) and Et N (1.0
3
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eq) in CH Cl (20 mL) at room temperature for 30 minutes. After the stirring, the respective amine
2
2
(1.5 equiv.) was added to this reaction mixture that was subsequently stirred at room temperature
for 4 h. Then the reaction mixture was washed with 1.0 M aqueous HCl (5.0 mL), sat. aqueous
NaHCO (5.0 mL), brine (5.0 mL), dried over MgSO , filtered, and concentrated under reduced
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pressure. The crude product was purified by column chromatography eluted with 20% ethyl acetate
in DCM as solvent to reach a white solid product with a general yield from 70% to 80%.
4.2.4. Ethyl 6-chloro-3-(propylcarbamoyl)-1H-indole-2-carboxylate (5a): The product
was obtained using general procedure 1 starting from carboxylate 3 and propylamine. The product
1
was obtained as yellow solid in a 86% yield. H NMR (500 MHz, DMSO-d ) δ 12.96 (s, 1H), 8.92
6
(t, J = 5.5 Hz, 1H), 8.57 (d, J = 2.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.35 (dd, J = 8.5, 2.5 Hz,1H),
4.31 (q, J = 7.0 Hz, 2H), 3.26-3.23 (m, 2H), 1.59-1.52 (m, 2H), 1.32 (t, J = 7.0 Hz, 3H), 0.91 (t, J
13
=
7.0 Hz, 3H). C NMR (126 MHz, DMSO-d ) δ 167.37, 160.16, 154.97, 139.38, 136.90, 130.43,
6
1
30.37, 124.08, 120.02, 119.90, 63.31, 41.49, 22.49, 14.26, 11.88. HRMS, calculated for
+
C H ClO N [M + H] : 309.1000, found 309.1002. HPLC: purity 96%, retention time 17.7 min.
1
5
18
2
3
4
.2.5. Ethyl (R)-6-chloro-3-((2,6-dimethylheptyl)carbamoyl)-1H-indole-2-carboxylate
(5b): The product was obtained using general procedure 1 starting from carboxylate 3. The product
1
was obtained as yellow solid in a 82% yield. H NMR (500 MHz, CDCl ) δ 10.75 (s, 1H), 9.40 (s,
3
1H), 8.43 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 8.5, 2.0 Hz, 1H), 5.75 (s, 1H),
4
.56 (q, J = 7.0 Hz, 2H), 3.25-3.06 (m, 1H), 1.54 (m, 1H), 1.51 (t, J = 5.0 Hz, 3H), 1.36-1.15 (m,
1
3
8
H), 0.96 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H). C NMR (126 MHz, DMSO-d ) δ 166.71,
6
160.13, 154.91, 139.28, 136.81, 131.58, 130.40, 124.03, 120.33, 119.92, 63.29, 45.55, 38.70, 36.64,
+
3
3
6.34, 27.81, 24.04, 22.87, 20.86, 14.26, 14.18. HRMS, calculated for C H ClN O [M + H] :
2
1
30
2
3
93.1939, found 393.1938. HPLC: purity 99%, retention time 14.3 min.
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.2.6.
Ethyl
(E)-3-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-6-chloro-1H-indole-2-
carboxylate (6): 2 (0.10 g, 0.39 mmol), (tert-butoxycarbonylmethylene) triphenylphosphorane
(0.17 g, 0.50 mmol) and EtOH (10 mL) were mixed under an atmosphere of nitrogen in an oven-
dried flask. The mixture was heated at reflux for 2 h. Then the solvent was evaporated under
reduced pressure. The product was purified by column chromatography eluted with 20% ethyl
acetate in petroleum as solvent and obtained as a yellow solid in 84% yield. The NMR spectra were
the same as reported previously.45
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.2.7. (E)-3-(6-chloro-2-(ethoxycarbonyl)-1H-indol-3-yl)acrylic acid (7): 6 (0.50g,
1
.7mmol) was dissolved in DCM (1mL). Then Trifluoroacetic acid (1.0 mL, 2.0 mmol) was added
and the mixture was stirred at 0 C for 2 h. After evaporation of the solvent, the crude product did
not need further purification. The NMR spectra were the same as reported previously.45
4
.2.8. Ethyl (E)-6-chloro-3-(3-((2,4-dichlorobenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-
indole-2-carboxylate (9a): The product was obtained using general procedure 1 starting from 7.
1
The product was obtained as white solid in a 89% yield. H NMR (500 MHz, DMSO-d ) δ 12.31
6
(
s, 1H), 8.63 (t, J = 6.0 Hz, 1H), 8.34 (d, J = 16.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.65 (d, J =
2
.0 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.48-7.43 (m, 2H), 7.28 (dd, J = 8.5, 2.0 Hz, 1H), 7.24 (d, J
1
3
= 16.0 Hz, 1H), 4.48 (d, J = 6.0 Hz, 2H), 4.41 (q, J = 7.0 Hz, 2H). 1.39 (t, J = 7.0 Hz, 3H). C
NMR (126 MHz, DMSO-d ) δ 166.16, 161.20, 137.57, 136.10, 133.64, 132.81, 132.02, 131.12,
6
1
30.47, 129.09, 127.88, 127.66, 123.97, 123.71, 122.58, 122.37, 117.17, 113.10, 105.90, 61.59,
+
1
4.69. HRMS, calculated for C H Cl N O [M + H] : 451.0378, found 451.0377. HPLC: purity
21 18
3
2
3
95%, retention time 19.2 min.
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