Rational Development of a Potent 15-Lipoxygenase-1 Inhibitor with in Vitro and ex Vivo Anti-inflammatory Properties

Article abstract of DOI:10.1021/acs.jmedchem.5b01121

Human 15-lipoxygenase-1 (h-15-LOX-1) is a mammalian lipoxygenase and plays an important role in several inflammatory lung diseases such as asthma, COPD, and chronic bronchitis. Novel potent inhibitors of h-15-LOX-1 are required to explore the role of this enzyme further and to enable drug discovery efforts. In this study, we applied an approach in which we screened a fragment collection that is focused on a diverse substitution pattern of nitrogen-containing heterocycles such as indoles, quinolones, pyrazoles, and others. We denoted this approach substitution-oriented fragment screening (SOS) because it focuses on the identification of novel substitution patterns rather than on novel scaffolds. This approach enabled the identification of hits with good potency and clear structure-activity relationships (SAR) for h-1-5-LOX-1 inhibition. Molecular modeling enabled the rationalization of the observed SAR and supported structure-based design for further optimization to obtain inhibitor 14d that binds with a K_i of 36 nM to the enzyme. In vitro and ex vivo biological evaluations of our best inhibitor demonstrate a significant increase of interleukin-10 (IL-10) gene expression, which indicates its anti-inflammatory properties.

Full text of DOI:10.1021/acs.jmedchem.5b01121

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Brief Article
Rational development of a potent 15-lipoxygenase-1 inhibitor
with in vitro and ex vivo anti-inflammatory properties
Nikolaos Eleftheriadis, Constantinos Neochoritis, Niek Leus, Petra
van der Wouden, Alexander Dömling, and Frank Jacobus Dekker
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01121 • Publication Date (Web): 02 Sep 2015
Downloaded from http://pubs.acs.org on September 6, 2015
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Rational development of a potent 15ꢀlipoxygenaseꢀ1
inhibitor with in vitro and ex vivo antiꢀinflammatory
properties
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†
‡
†
Nikolaos Eleftheriadis, Constantinos G. Neochoritis, Niek G.J. Leus, Petra E. van der
†
‡
†,
Wouden, Alexander Dömling, Frank J. Dekker *
†
Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy,
University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
‡
Department of Drug Design, Groningen Research Institute of Pharmacy, University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
KEYWORDS
15ꢀlipoxygenase, indoleꢀbased inhibitor, fragment screening, antiꢀinflammatory, interleukinꢀ10,
ex vivo studies, precisionꢀcut lung slices
ABSTRACT
Human 15ꢀlipoxygenaseꢀ1 (hꢀ15ꢀLOXꢀ1) is an important mammalian lipoxygenase and plays an
important role in several inflammatory lung diseases such as asthma, COPD and chronic
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bronchitis. Novel potent inhibitors of hꢀ15ꢀLOXꢀ1 are required to explore the role of this enzyme
further and to enable drug discovery efforts. In this study, we applied an approach in which we
screened a fragment collection that is focused on a diverse substitution pattern of nitrogen
containing heterocycles such as indoles, quinolones, pyrazoles etc. We denoted this approach
Substitution Oriented fragment Screening (SOS), because it is focuses on identification of novel
substitution patterns rather than on novel scaffolds. This approach enabled the identification of
hits with good potency and clear structure activity relationships (SAR) for hꢀ1ꢀ5ꢀLOXꢀ1
inhibition. A molecular modeling enabled the rationalization of the observed SAR and supported
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structureꢀbased design for further optimization to obtain inhibitor 14d that binds with a K of 36
i
nM to the enzyme. In vitro and ex vivo biological evaluations of our best inhibitor demonstrate a
significant increase of interleukinꢀ10 (ILꢀ10) gene expression, which indicates antiꢀinflammatory
properties.
INTRODUCTION
Inflammatory lung diseases like asthma and chronic obstructive pulmonary disease (COPD) can
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have detrimental effects on patients’ health. Fortunately, nowadays these diseases can be
alleviated by various therapeutic agents. Nevertheless, expansion of the therapeutic possibilities
is needed, because for some patients the currently available medicines are ineffective or cause
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severe side effects. Therefore, the development of novel compounds targeting enzymes that are
involved in inflammatory lung diseases is highly important.
The regulatory function of macrophages is gaining increasing attention in drug discovery,
because they play key regulatory roles in the different disease stages of asthma and COPD as
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they polarize into different subclasses according to the cytokines they encounter in their
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environment. Based upon the signals they receive, their role, and cytokine profile, macrophages
are categorized in to three subpopulations: M1 (induced by LPS/IFNγ), M2 (induced by ILꢀ4/ILꢀ
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3) and M2ꢀlike subsets (combination of Tollꢀlike receptor stimulation). M1 macrophages play a
role in inflammatory responses to intracellular pathogens and M2 are involved in scavenging
debris, promoting angiogenesis, help in tissue remodeling/repair, and are therefore considered as
woundꢀhealing macrophages. The third class are the M2ꢀlike macrophages; as the name implies
these are macrophages which resemble M2. M2ꢀlike macrophages are able to produce TGFꢀβ
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and ILꢀ10 implying an antiꢀinflammatory role.
An enzyme class highly expressed in macrophages and other immune cells are the lipoxygenases
(
LOXs). These enzymes are nonꢀheme iron containing enzymes that metabolize polyunsaturated
fatty acids (PUFAs) such as arachidonic acid and linoleic acid into lipid signaling molecules
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such as leukotrienes and lipoxins.
2/15ꢀLOX) is an important mammalian lipoxygenase, responsible for the biosynthesis of antiꢀ
inflammatory and proꢀinflammatory mediators (signaling molecules) such as lipoxins and
Human 15ꢀlipoxygenaseꢀ1 (hꢀ15ꢀLOXꢀ1, also denoted
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eoxins.
This enzyme is highly expressed in monocytes, bronchoꢀalveolar epithelial cells, and
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in eosinophils and macrophages of asthmatic patients.
Growing evidence suggests that hꢀ15ꢀ
LOXꢀ1 may modulate inflammatory responses. It was demonstrated that hꢀ15ꢀLOXꢀ1 regulates
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the expression of ILꢀ12 in a cellꢀtype and stimuliꢀrestricted manner. In addition, in lungs, it has
been demonstrated that signaling products of hꢀ15ꢀLOXꢀ1 can stimulate inflammation and
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mucus secretion. The crucial regulatory role of hꢀ15ꢀLOXꢀ1 in several respiratory diseases
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such as asthma, COPD and chronic bronchitis
and their role in modulating the
inflammatory response calls for development of novel potent and selective inhibitors.
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Despite the fact that the key role of hꢀ15ꢀLOXꢀ1 was exemplified as target in drug discovery for
several inflammatory diseases, the discovery of very potent hꢀ15ꢀLOXꢀ1 inhibitors and their role
as a novel therapeutic strategy is still in an early phase (Figure 1). Indoleꢀbased inhibitors, such
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as PDꢀ146176 by Pfizer and tryptamine sulfonamides by BristolꢀMyers Squibb (BMS)
exhibited inhibitory potency against r12/15ꢀLOX with IC50 value of 3.81 ꢁM and 21 nM
respectively. The inhibitor PDꢀ146176 also showed downregulation of interleukinꢀ12 (ILꢀ12)
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after stimulation with LPS. However, the inhibitory potency of the PDꢀ146176 is relatively
low. Furthermore, fiveꢀmembered heterocycles like pyrazoleꢀbased sulfonamides and sulfamides
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IC = 1.4 nM, r12/15ꢀLOX) have been reported. Also oxadiazole or oxazole derivatives such
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as ML094 (IC = 10 nM, hꢀ15ꢀLOXꢀ1) and ML351 (IC = 200 nM, hꢀ15ꢀLOXꢀ1) are highly
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potent. Another class of potent inhibitors are imidazoleꢀbased (IC = 75 nM, r12/15ꢀLOX) .
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Less potent 15ꢀLOX inhibitors are indolizine (IC = 25 ꢁM, r12/15ꢀLOX), thiourea (IC = 1.8
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ꢁ
M, soybean 15ꢀLOX) and thiadiazine (IC50 = 9 ꢁM, soybean 15ꢀLOX) derivatives.
Recently, anacardic acid derived salicylates were described by our research group as LOX
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inhibitors.
Although the potency of these inhibitors is moderate or good they often suffer
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from unfavourable physicochemical properties and limited ligand efficiency values (LE),
which limits their utility as drug candidates. Taking into account all the above, new chemotypes
are needed in order to explore the structure activity relationships (SAR) and the drugability of
this enzyme further.
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Figure 1. Examples of previously reported 15ꢀLOX inhibitors
The currently available inhibitors are frequently built around nitrogen containing heterocycles
such as indoles, imidazoles or pyrazoles. We anticipate that the identification of the right
substitution pattern is more demanding than finding a scaffold that provides inhibition. We
therefore developed an approach for screening of fragments that is focused on specific nitrogen
containing heterocycles, such as indoles, quinolones, pyrazoles, etc. that have a very diverse
substitution pattern. We denoted this approach Substitution Oriented fragment Screening (SOS).
After having identified fragment hits with the right substitution pattern to give inhibition in the
micromolar range we assume that the active hits are sufficiently potent to provide initial SAR.
This will enable docking studies and structureꢀbased design to optimize the substitution pattern
further.
Here we set out to test the SOS approach for fragmentꢀbased screening to find potent substituted
scaffolds that inhibit the enzyme hꢀ15ꢀLOXꢀ1. The hits were used to establish an initial SAR and
docking studies were performed to enable the proposal of a plausible binding mode for the new
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hits in line with the SAR. The hits were further optimized using structureꢀbased design to yield
inhibitors with potency in the nanomolar range. The effects of the most potent inhibitor on proꢀ
and antiꢀinflammatory gene expression were investigated in RAW 264.7 macrophages and
precisionꢀcut lung slices (PCLS) of mouse lung tissue.
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RESULTS AND DISCUSSION
Initial screening and discovery of potent inhibitors. For our SOS approach we employed a
library of 200 fragments containing 26 indoles, 73 quinolones, 22 pyrroles and 79 other
heterocycles with a diverse substitution pattern (Figure 2). This library consists mostly of diꢀ or
triꢀsubstituted scaffolds with substituents varying from EWG, EDG, aliphatic and aromatic
moieties.
For the hꢀ15ꢀLOXꢀ1 activity studies an enzyme assay was used that employs the UV absorption
of the hꢀ15ꢀLOXꢀ1 product (λmax 234 nm) that is formed upon enzymatic conversion from
linoleic acid (Figure S1). This assay was done in a 96ꢀwell format, which is suited for mediumꢀ
throughput screening, measurement of inhibition concentration 50% (IC ), and enzyme kinetic
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studies. Using this assay the SOS library was screened and four hits were identified that provide
more than 80% inhibition of the enzyme activity at 50 ꢀM, which indicates an inhibitory potency
in the low micromolar range. The potency was confirmed by determination of the IC values for
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compounds 1 and 2 (Figure3). The identified hits contain an indole scaffold as previously found
in hꢀ15ꢀLOXꢀ1 inhibitors such as for example PDꢀ146176 or tryptamine sulfonamides (Figure
1). Interestingly, the hits 6ꢀchloroꢀ1Hꢀindoleꢀ2ꢀethyl acetate (1) and 6ꢀchloroꢀ3ꢀformylꢀ1Hꢀ
indoleꢀ2ꢀethyl acetate (2) have both a unique substitution pattern around the indole scaffold,
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which has not been identified previously. Hits 1 and 2 have beneficial properties for further
development, because halogen substitution in the 5 or 6 position could prevent hydroxylation of
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the indole thereby increasing its metabolic stability.
This stresses the utility of the SOS
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approach to identify novel substitution patterns around known scaffolds.
Figure 2. Substitution Oriented fragment Screening (SOS) enables the identification of unique substitution patterns around
known scaffolds, which can be further optimized by identification of Structure Activity Relationships (SAR) to support structureꢀ
based design.
An initial SAR was derived by comparison of 1 and 2 with a selection of compounds with a
closely related structure (Figure 3). We investigated the importance of the indole NH as potential
hydrogen bond donor by methylation using MeI/K CO to give compound 3, which inhibits the
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enzyme activity less than 50% at 50 ꢀM. This indicates that the free NH is important for binding,
possibly due to hydrogen bond formation. Importantly, also compounds 4, 5, 7 and 8 are
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inactive, which indicates that the carboxy ethyl substitution in the 2ꢀposition is important for
binding to the enzyme.
Comparison of 1 and 2 indicates that the 3ꢀformyl substitution does not greatly change the
inhibitory potency thus indicating that larger substituents could be linked to this position. This
suggestion is in line with literature on related indole based inhibitors where substitution of the
indole heterocycle at the 3ꢀposition markedly improved the hꢀ15ꢀLOXꢀ1 inhibitory potency as
observed for inhibitors like PDꢀ146176 or tryptamine sulfonamides (Figure 1). This triggered us
to synthesize compounds 14c and 14l that are elongated on 3ꢀposition. Compounds 14c and 14l
were synthesized after acylation of the 6ꢀchloroꢀ1Hꢀindoleꢀ2ꢀethyl acetate (1) with butyric
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anhydride and 4ꢀchlorophenylacetyl chloride respectively via a FriedelꢀCrafts type reaction.
The IC values were calculated to be 0.7 ± 0.3 ꢁM for the compound 14c and 1.2 ± 0.5 ꢁM for
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the compound 14l which means 2 to 2.5 folds better inhibitory potency, probably due to
lipophilic interactions. The fact that these inhibitors are more potent then compound 1 and 2
indicates that substitution in this position is possible and could improve the potency of this type
of inhibitors. To conclude, we confirmed that both the –NH and ꢀethyl acetate groups are playing
a vital role in the inhibition of hꢀ15ꢀLOXꢀ1 while attachment of extended substitutions in 3ꢀ
position is possible with retention or even improvement of affinity. Having established these
qualitative SAR we performed molecular modeling studies and structureꢀbased design in order to
provide a rationale for our findings and optimize the substitution in the 3ꢀposition.
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Figure 3. A study of structureꢀactivity relations of compound 2. The compounds with red background do not inhibit hꢀ15ꢀLOXꢀ1
in concentration below 50 μM, whereas the compounds with green background are active in the low micromolar range.
In order to establish the mechanism of hꢀ15ꢀLOXꢀ1 inhibition, MichaelisꢀMenten enzyme
kinetics analysis in presence of inhibitor 14l was performed (Figure 4A,B). The Lineweaverꢀ
Burk double reciprocal plot shows that inhibitor 14l causes an increase in the K values, whereas
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the Vmax values remain more constant (Table S3), indicating competitive inhibition. Using the
ChengꢀPrusoff equation the binding affinities (K ) of all the inhibitors were calculated and shown
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in Table 1.
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Figure 4. SteadyꢀState kinetic characterization of hꢀ15ꢀLOXꢀ1 in the presence of different concentrations of compound 14l: A)
MichaelisꢀMenten representation and B) LineweaverꢀBurk representation.
In order to link the observed SAR to structural information, compound 2 was docked in the
active site of the enzyme in line with the identified competitive inhibition. Due to the fact that
there is no crystal structure of human 15ꢀLOXꢀ1 available, the docking study was performed
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with the crystal structure of rabbit 15ꢀLOX (PDB: 1LOX) as determined by Gillmor et al. This
is justified by the high sequence similarity (87%) between rabbit 15ꢀLOX and human 15ꢀLOXꢀ1
with the main differences located in the Nꢀterminal domain and not in the catalytic domain. In
three of the top five highest scoring poses, inhibitor 2 binds in the same orientation in the active
site of the enzyme, forming a hydrogen bond with Glu357 (Figure S4). Subsequent energy
minimization in the active site provided the final binding model for inhibitor 2 (Figure 5). The
modeling shows the presence of two hydrogen bonds, the first between Gln548 and the ꢀethyl
acetate group and the second between the –NH group and the Glu357 (Figure 5B). The formyl
group in 3ꢀposition gives an interaction with the iron of the enzyme and there is space for
extension of the substitution in this position due to presence of free space in the substrate binding
pocket (Figure 5A). The 2ꢀcarboxy ethyl of inhibitor 2 is tightly embedded in the binding pocket,
which is in line with the lack of inhibition observed for the 2ꢀcarboxy tertbutyl substituted
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compound 8 (Figure 3). These features in this model are consistent with the observed SAR thus
justifying the application of this model as a basis for structureꢀbased design to optimize the
substituent in the 3ꢀposition.
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Figure 5. A) Binding orientation of inhibitor 2 in the active site of hꢀ15ꢀLOXꢀ1 proposed by molecular modeling studies. The
surface of the active site of the enzyme is presented with grey while the light green vectors in the compound shows the direction
for possible extension of the molecule. B) 2D representation of the interactions of compound 2 with the active site of the enzyme.
Structureꢀbased design. Our SAR based model indicates that optimization of the inhibitor by
acylations in the indole 3ꢀposition is feasible. The free space in the enzyme binding pocket that
should be addressed this way has mainly a hydrophobic character in line with the character
required for binding to its natural substrate linoleic acid. Taking this into account, we
synthesized a library of 14 derivatives to explore the SAR for substitution in this position.
Exclusive acylation on the 3ꢀposition of inhibitor 1 as starting material was achieved using a
FriedelꢀCraft type acylation with different acid chlorides or anhydrides. The reactions were
performed in the presence of SnCl and CH NO in DCM, yielding the corresponding
4
3
2
compounds 14aꢀn in good to excellent yields (Scheme 1). All the compounds were analytically
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pure after simple washing steps, underscoring the facility of their synthetic preparation. The acid
chlorides, which are not commercially available, were synthesized from the corresponding acids
using oxalyl chloride in DCM in quantitative yields and used without further purification. The
enantiomerically pure products 14d,e were prepared in a four step reaction sequence starting
from the commercially available (R)ꢀ(+)ꢀ or (S)ꢀ(ꢀ)ꢀCitronellal. Initially, the aldehydes 10d,e
were oxidized to carboxylic acids 11d,e using Tollens' reagent and the double bonds were
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reduced with Pd/C under H atmosphere to give 12d,e. Next, the acid chlorides 13d,e were
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synthesized and coupled with the 6ꢀchloroꢀ1Hꢀindoleꢀ2ꢀethyl acetate (1) resulting in the desired
products 14d,e (Scheme 1).
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o
Scheme 1. General synthesis. Reagents and conditions: a) POCl , DMF, 60 C, overnight; b) K CO , CH I, acetone, rt, 2 h; c)
3
2
3
3
t
LiOH, EtOH/H O, rt, 24 h; d) H SO , MgSO , DCM, BuOH, rt, overnight; e) SnCl , CH NO , DCM, rt, overnight; f) (COCl) ,
2
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4
4
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o
DCM, 0 °C for 30 min then rt for 3 h; g) Ag O, H O, rt, overnight; h) H , Pd/C, EtOH, 40 C, overnight.
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All the newly synthesized compounds were subjected for IC determination in a hꢀ15ꢀLOXꢀ1
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0
inhibition assay and the values are shown in Table 1. We first explored inhibitors with a phenyl
group in the 3ꢀsubstituent in which we varied both the position of the phenyl group as well as the
electronic and steric properties (14jꢀn). However, changing the phenyl substitution in 14j to
14m, 14l and 14k did not improve the inhibition. Also changing the position of the phenylꢀgroup
in 14j to 14n did not greatly affect the inhibition although the potency with 0.56 ꢀM is
reasonably good. We secondly explored another route of optimization with compounds 14aꢀi, in
which the length of the lipophilic tale is varied from one to eleven carbon atoms. This series of
inhibitors showed a clear structure activity relationship with optimal binding for 14g with 9
aliphatic carbons in the lipophilic tail. The clear activity dependence on the length of the
aliphatic tale, from 2.3 ꢁM to 0.4 ꢁM (Table 1), indicates the role for lipophilic interactions in
the binding, which is according to the molecular modeling study. The successful optimization of
the inhibitors with an aliphatic acylation in the 3ꢀposition triggered our interest to include an
32
aliphatic tail that was identified in a previous study as reflected in inhibitors 14d,e. Previously,
we found that an aliphatic tail that is branching at 3,7ꢀposition improves the inhibitory activity
3
2
for hꢀ15ꢀLOXꢀ1. We included these structural features in our inhibitors as a further
optimization of the aliphatic substituent in the 3ꢀposition. The resulting inhibitors 14e and 14d
contain a stereogenic center and the stereochemistry being either R or S affected the affinity by a
3ꢀfold difference in potency. The S enantiomer (14d) provided the highest potency thus reaching
an IC value of 0.09 ± 0.03 ꢁM, which reflects a K value of 0.03 ± 0.01 ꢁM.
5
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Molecular modeling of the inhibitor 14d in the active site of the enzyme using procedures
applied for inhibitor 2 provided a binding configuration in which the 6ꢀchloroꢀ1Hꢀindoleꢀ2ꢀethyl
acetate moiety binds similar to the docking of inhibitor 2. The extension of the substituent in the
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3ꢀposition fills the lipophilic binding pocket available in this position. The docking shows again
the presence of the two hydrogen bonds with Gln548 and Glu357 and the interaction with the
iron, while the branched aliphatic tail occupies the lipophilic binding pocket completely (Figure
6, S5). This SAR is in total agreement with the observed activity data in which 14g is the most
potent inhibitor with a linear aliphatic tail and 14d reaches the highest affinity with a branched
aliphatic tail.
Figure 6. A) Binding of compound 14d in the active site of 15ꢀLOXꢀ1 after molecular modeling studies. B) 2D illustration of the
interactions of compound 14d with the active site of the enzyme.
Table 1. Compound collection of the synthesized inhibitor with IC and K values.
50
i
IC value
K value
(ꢁM)
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i
Compound
4a
R
(ꢁM)
1
2.31 ± 0.71
1.78 ± 0.27
0.73 ± 0.29
0.93 ± 0.28
0.72 ± 0.10
0.29 ± 0.12
14b
14c
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14d
14e
0.09 ± 0.03
0.03 ± 0.01
0.30 ± 0.15
0.43 ± 0.27
0.20 ± 0.07
0.27 ± 0.10
0.54 ± 0.30
0.60 ± 0.31
2.08 ± 1.33
0.12 ± 0.06
0.17 ± 0.10
0.08 ± 0.03
0.10 ± 0.04
0.21 ± 0.12
0.25 ± 0.12
0.83 ± 0.53
1
4f
4g
14h
4i
4j
4k
4l
14m
4n
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1
1.19 ± 0.48
0.48 ± 0.19
0.77 ± 0.30
0.56 ± 0.28
0.31 ± 0.12
0.22 ± 0.11
1
Ligand efficiency metrics. The SOS based development strategy provided 14d as an inhibitor
with nanomolar potency that can be further investigated in biochemical studies. To support
development of inhibitors from this stage towards successful application in drug discovery
projects they need to show acceptable physicochemical properties to achieve an acceptable
ADMEꢀTox (absorption, distribution, metabolism, excretion and toxicity) profile in vivo. In this
perspective, ligand efficiency metrics rendered into a generally accepted tool to estimate the
3
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value of lead compounds in this perspective. Therefore, ligand efficiency metrics and indices
such as ligand efficiency (LE), binding efficiency index (BEI) and the surface efficiency index
(
SEI) were calculated for 14d in comparison to previously described inhibitors (table 2). Ligand
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efficiency is defined as the binding affinity of a ligand in relation to the number of nonꢀhydrogen
39
atoms (HA) according the Equation LE = (1.37/HA) x pK . Inhibitor 14d has 26 heavy atoms
i
(
nonꢀhydrogen) and the K value is 0.03 ꢁM, so the LE is equal to 0.40 kcal per mol per heavy
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atom. LE values > 0.3 kcal per mol are considered to be acceptable values for LE of drug
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candidates. Inhibitor 14d satisfies the requirements and exceeds the values calculated for
previously described inhibitors, except the inhibitor PDꢀ146176. Another parameter is BEI,
which is the binding efficiency index relating potency to molecular weight on a per kDa scale
and SEI is the surface efficiency index monitoring the potency gains as related to the increase in
2 41
polar surface area (PSA) referred to 100 Å . The BEI and the SEI for 14d and the other
inhibitors were calculated according to the Equations BEI = pKi/MW and SEI = pKi/(PSA/100
2
41
Å ). The MW of inhibitor 14d is 377 Da or 0.377 KDa, so the BEI is 19.90. The polar surface
area (PSA) was estimated from MarvinSketch software to 59.16 and according to that the SEI for
the compound 14d is equal to 12.71. Reference values for these parameters were obtained from
mapping of the surfaceꢀbinding and binding efficiency indices (SEI–BEI values) for 92 examples
of marketed drugs, which provided average values for SEI of 17.9 ± 15.7 and BEI of 28.0 ±
3
8
11.5. The parameters for inhibitor 14d lay well with this range (Table 2). In addition, we
calculated the lipophilic ligand efficiency (LLE) to estimate the balance between affinity and
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lipophilicity. The LLE for 14d has a value comparable to other compounds except for the
Pyrazoleꢀbased sulfamide and ML094, which show more favourable values. This indicates that
14d can be improved with respect to its lipophilicity. In conclusion, the ligand efficiency
metrics for 14d demonstrate a favourable binding efficiency compared to previously described
inhibitors, which justifies further investigation of this molecule in biochemical studies.
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Table 2. Binding affinity and ligand efficiency metrics and indices
shown in Figure 1 as well as the newly synthesized inhibitor 14d
of the previously published 15ꢀLOXꢀ1 inhibitors as
*
1
5ꢀLOX inhibitors
K (µM)
i
LE
BEI
SEI
LLE
14d
0.036
3.81
0.40
0.43
0.30
0.32
0.24
0.36
0.19
19.90
22.79
15.71
15.54
11.93
19.12
10.47
12.71
34.31
10.78
8.26
0.6
1.0
ꢀ0.5
4.9
0.9
3
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PDꢀ146176
Tryptamine sulfonamide
Pyrazoleꢀbased sulfamide
Imidazoleꢀbased sulfamide
ML094
0.021
0.001
0.073
0.010
25.0
7.88
12.26
9.63
Indolizine
ꢀ2
*
The units for LE are (kcal/mol·HA)
Gene expression profiling. Having identified 14d as a potent inhibitor for hꢀ15ꢀLOXꢀ1 we
moved on to study its effects in model systems for inflammatory lung diseases using in vitro
studies with RAW 264.7 macrophages and ex vivo studies with precisionꢀcut lung slices (PCLS).
As a first step the cytotoxicity of compound 14d was investigated in RAW 264.7 cells as well as
in PCLS in order to identify the concentration range in which this inhibitor does not affect cell
viability. The inhibitor 14d did not inhibit cell viability at concentration below 12.5 ꢁM in RAW
264.7 cells (Figure 7A) as determined using an MTS assay. The viability of PCLS was
determined by measuring the release of lactate dehydrogenase (LDH) into the medium upon
incubation with different concentrations of inhibitor 14d. The LDH release demonstrated that
inhibitor 14d is not affecting the PCLS viability at concentrations of 500 nM and lower (Figure
7B).
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Figure 7. A) LC50 graph after MTS assay. The compound 14d was incubated with RAW 264.7 cells at concentrations from 0.05
ꢁM to 100 ꢁM. Red bars are statistically different compared to nonꢀtreated control cells. B) Viability of precisionꢀcut lung slices
(PCLS) measured by release of lactate dehydrogenase (LDH) into the incubation medium. PCLS were treated with various
concentrations of 14d (1, 10, 100 and 500 nM) and 10 ꢁM linoleic acid for 20 h. Maximal LDH content of the PCLS was
determined by lysis with 1% Triton Xꢀ100. LDH release was plotted relative to maximal LDH. Data are presented as mean values
±
SD of 3 independent experiments.
Having identified the nonꢀtoxic concentration for inhibitor 14d its influence on gene expression
was investigated. In these models LPS/INFγ was applied as inflammatory stimulus and the
influence of 14d on the expression of proꢀinflammatory genes ILꢀ1β, ILꢀ6 or ILꢀ12 and the antiꢀ
inflammatory gene ILꢀ10 was investigated. Since the cellular concentration of free fatty acids
(preferred substrate of mammalian LOXs) is rather low, we supplemented the culture medium
with linoleic acid (C18:ꢂ2, n – 6) which is the most abundant polyenoic fatty acid in mammalian
cells and serves as a natural substrate for 15ꢀLOXꢀ1. Linoleic acid itself did not alter the
expression of ILꢀ1β, ILꢀ6, ILꢀ10 and ILꢀ12 in RAW 264.7 cells and PCLS (data not shown).
In LPS/INFγꢀstimulated RAW 264.7 macrophages incubation with the hꢀ15ꢀLOXꢀ1 inhibitor
14d did not change the expression of the genes ILꢀ1β, ILꢀ6, and ILꢀ12 but provided a more then
2
ꢀfold increase of the ILꢀ10 expression at concentrations as low as 1 nM (Figure 8A). Since ILꢀ
0 has antiꢀinflammatory properties, and is connected with the antiꢀinflammatory M2
1
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macrophage phenotype, the observed changes in gene expression are indicative for a potential
antiꢀinflammatory effect. Next, the effect of 14d on the expression of LPS/INFγꢀinduced
cytokines in PCLS was assessed. In line with the results obtained in macrophages the expression
of ILꢀ10 is upregulated in PCLS. In addition, there is a small but significant downregulation of
the proꢀinflammatory genes ILꢀ1β and ILꢀ6 (Figure 8B). Remarkably, the expression of ILꢀ12
remained unchanged while others have reported that hꢀ15ꢀLOXꢀ1 regulates the production of ILꢀ
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12 in macrophages. The induction of IL10 in PCLS seems to show a bellꢀshaped pattern. This
could mean that hꢀ15ꢀLOXꢀ1 inhibition in this system is effective in a limited range of
concentrations with 10 nM as the optimal dose. Nevertheless, further research is needed on this
observation. The effect on ILꢀ10 expression is remarkable because the important role of ILꢀ10 in
inflammatory lung diseases has been previously highlighted in a study that showed diminished
ILꢀ10 production in lung tissue of COPD patients after LPS stimulation as compared to lung
4
2
tissue of patients with normal lung function. In addition, it has been demonstrated that the level
of ILꢀ10 and ILꢀ10ꢀpositive macrophages in sputum of COPD patients and healthy smokers was
4
3
decreased as compared to healthy nonꢀsmokers. This suggests that macrophages in COPD have
lost their ability to produce antiꢀinflammatory cytokines like ILꢀ10 and are therefore unable to
effectively dampen inflammation. Our newly developed inhibitor 14b could, among others,
alleviate the lack of IL10 production in these diseases. Nevertheless, we note that overꢀ
4
4
expression of IL10 can be associated with certain types of cancer, which indicates that the final
effect of this inhibitor in disease models need to be evaluated carefully. Taken together, we
demonstrate that the effects observed for treatment with inhibitor 14d are in agreement with an
antiꢀinflammatory effect that could be beneficial in inflammatory lung diseases. This sets the
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stage for further investigation and optimization of inhibitor 14d for in vivo applications towards
the development of novel therapeutics for inflammatory lung diseases.
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Figure 8. Effect of hꢀ15ꢀLOXꢀ1 inhibition on interleukin expression in RAW 264.7 cells and precisionꢀcut lung slices (PCLS).
Stimulated RAW 264.7 cells (A) and PCLS (B) were subjected to 14d in combination with 10 ꢁM linoleic acid for 20 h and
stimulated with LPS/IFNγ for the last 4 h. Subsequently, RAW 264.7 cells and PCLS were lysed and gene expression was
assessed by RTꢀqPCR and expressed as fold change compared to control (LPS/IFNγ/linoleic acidꢀtreated) group. Data are
presented as mean values ± SEM of 4ꢀ6 independent experiments. * p < 0.05; *** p < 0.001 compared to control.
CONCLUSIONS
In this study, we employed an efficient strategy to identify structurally new inhibitors for the
enzyme hꢀ15ꢀLOXꢀ1, which is an emerging drug target in various inflammatory diseases. Our
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approach started with a substitution oriented fragment screening (SOS) of a focused fragment
library containing diversely substituted heterocycles. After the discovery of four hits, two
inhibitors were selected to derive structure activity relationships and subjected to enzyme kinetic
analysis, which indicated competitive inhibition. These observations were applied to support a
molecular modeling study proposing a binding configuration in the active site of the enzyme hꢀ
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15ꢀLOXꢀ1. Based on this model the substitution of the inhibitor was further optimized using
structureꢀbased design to provide inhibitor 14d with a K of 36 nM and good ligand efficiency
i
metrics. This inhibitor was evaluated in cellꢀbased studies using RAW 264.7 macrophages and
ex vivo studies in mouse precisionꢀcut lung slices. The inhibitor provided an increase in the
expression of ILꢀ10 both in macrophages and mouse lung tissue. In the future, these inhibitors
could be used as lead compounds for further optimization or as a starting point of drug discovery
efforts targeting hꢀ15ꢀLOXꢀ1.
EXPERIMENTAL SECTION
Chemistry
General. The solvent and reagents were purchased from SigmaꢀAldrich and Acros chemicals
and were used without further purification unless otherwise noted. Reactions were monitored by
thin layer chromatography (TLC). Merck silica gel 60 F254 plates were used and spots were
detected under UV light or after staining with potassium permanganate for the non UVꢀactive
1
compounds. MP Ecochrom silica 32ꢀ63, 60Å was used for flash column chromatography. H
13
NMR (500 MHz) and C NMR (126 MHz) spectra were recorded with a Bruker Avance 4ꢀ
channel NMR Spectrometer with TXI probe. Chemical shifts were referenced to the residual
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proton and carbon signal of the deuterated solvent CDCl : δ = 7.26 ppm ( H) and 77.05 ppm
3
1
3
(
C). The following abbreviations were used for spin multiplicity: s = singlet, br s = broad
singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double
of doublet of doublets, m = multiplet. Fourier Transform Mass Spectrometry (FTMS) and
electrospray ionization (ESI) were recorded on an Applied Biosystems/SCIEX API3000ꢀtriple
quadrupole mass spectrometer. All the compounds were analyzed by Waters Investigator Semiꢀ
prep 15 SFCꢀMS instrument confirming purity ≥ 95%. The corresponding chromatographs are
shown in the SI.
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8
9
0
Synthetic procedure 1: indole 3ꢀacylation. Indole (1.0 mmol) was dissolved in 4.0 mL DCM
o
under nitrogen atmosphere and the solution was cooled down to 0 C. To the stirring solution,
SnCl (1.2 mmol) was added in a single portion via syringe. After the ice bath was removed, the
4
mixture was stirred at room temperature for 30 min and then the acyl chloride or anhydride (1.0
mmol) was added in small portions, followed by the addition of nitromethane (3.0 mL). The
reaction mixture was stirred at room temperature overnight. After being quenched with cold
water (10 mL), the organic material extracted with EtOAc (2 x 15 mL), dried over MgSO4,
filtered and concentrated under reduced pressure.
Ethyl 3ꢀacetylꢀ6ꢀchloro-1H-indoleꢀ2ꢀcarboxylate (14a, DN397). The final product was
1
obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Pink solid, yield 75%. H
NMR (500 MHz, CDCl ) δ 9.28 (br s, 1H), 8.00 (d, J = 10.0 Hz, 1H), 7.41 (d, J = 0.5 Hz, 1H),
3
13
7
.22 (dd, J = 8.7, 1.7 Hz, 1H), 4.48 (q, J = 7.2 Hz, 2H), 2.75 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 198.3, 160.8, 135.4, 132.3, 127.3, 125.4, 124.0, 123.9, 121.7, 111.8,
3
+
.
62.5, 32.1, 14.3. MS (ESI): m/z 264.0 [MꢀH]
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Ethyl 6ꢀchloroꢀ3ꢀpropionylꢀ1H-indoleꢀ2ꢀcarboxylate (14b, N239). The final product was
obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Orange solid, yield 82%.
1
H NMR (500 MHz, CDCl ) δ 9.17 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 1.6 Hz, 1H),
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
.20 (dd, J = 8.7, 1.6 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 3.08 (q, J = 7.3 Hz, 2H), 1.43 (t, J = 7.1
1
3
Hz, 3H), 1.23 (t, J = 7.3 Hz, 3H). C NMR (126 MHz, CDCl ) δ 201.7, 160.6, 135.3, 132.3,
3
+
.
1
26.1, 125.2, 123.7, 123.6, 121.9, 111.5, 62.1, 37.3, 14.2, 8.6. MS (ESI): m/z 302.1 [M+Na]
Ethyl 3ꢀbutyrylꢀ6ꢀchloroꢀ1H-indoleꢀ2ꢀcarboxylate (14c, N238). The final product was
obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Orange solid, yield 83%.
1
H NMR (500 MHz, CDCl ) δ 9.16 (s, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H),
3
7.20 (dd, J = 8.7, 1.8 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.04 (t, J = 7.4 Hz, 2H), 1.76 (m, 2H),
13
1.43 (t, J = 7.1 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 201.3, 160.6,
3
135.2, 132.2, 126.1, 125.2, 123.6, 123.5, 122.2, 111.5, 62.1, 46.1, 18.2, 14.3, 13.9. MS (ESI):
+
.
m/z 316.1 [M+Na]
Synthetic procedure 2: oxidation with Tollens' reagent. Fresh silver oxide was prepared by
dropwise addition of a solution of AgNO (2.3 mmol) in water (4.0 mL) to a stirred solution of
3
o
NaOH (4.9 mmol) in water (4.0 mL) at 0 C and further stirring for 30 min in the dark while a
brown precipitate formed. Under continuous stirring, the aldehyde (1.0 mmol) was added
dropwise and the reaction was stirred overnight. The resulting suspension was filtered, washed
with hot water (2 x 10 mL) and the clear solution was acidified with aqueous HCl (4N) to pH<1.
The solution was extracted with Et O (3 x 20 mL), dried over MgSO , filtered and concentrated
2
4
under reduced pressure.
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Synthetic procedure 3: reduction of the alkene. The alkene (1.0 mmol) and Pd/C (10%) (0.10
mmol) were suspended in EtOH (10 mL). The suspension was stirred under H atmosphere at 40
2
o
C overnight. Subsequently, the mixture was filtered through Celite and after that, the solvent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was evaporated under reduced pressure.
Synthetic procedure 4: conversion of carboxylic acids to acid chlorides. The acid was
dissolved in dry DCM (5.0 mL) under nitrogen atmosphere and the solution was cooled down to
o
0
C. A solution of oxalyl chloride (1.2 mmol) in dry DCM (4.0 mL) was added dropwise and
the solution was stirred under nitrogen atmosphere at room temperature for an additional 3 h.
The solvent and the excess of oxalyl chloride were evaporated under reduced pressure yielding
the product as yellow oil.
(S)ꢀ3,7ꢀdimethyloctꢀ6ꢀenoic acid (11d, N240). The final product was obtained after oxidation of
(S)ꢀ3,7ꢀdimethyloctꢀ6ꢀenal with Tollens' reagent, using the synthetic procedure 2. Yellow oil,
1
yield 94%. H NMR (500 MHz, CDCl ) δ 10.40 (s, 1H), 5.08 (t, J = 7.1 Hz, 1H), 2.35 (dd, J =
3
15.0, 5.8 Hz, 1H), 2.14 (dd, J = 15.0, 8.3 Hz, 1H), 2.07 – 1.92 (m, 3H), 1.67 (s, 3H), 1.59 (s,
1
3
3
H), 1.37 (m, 1H), 1.24 (m, 1H), 0.97 (d, J = 6.7 Hz, 3H). C NMR (126 MHz, CDCl ) δ 179.9,
3
131.6, 124.2, 41.6, 36.7, 29.8, 25.7, 25.4, 19.6, 17.6.
(S)ꢀ3,7ꢀdimethyloctanoic acid (12d, N242). The final product was obtained after reduction of
the alkene of (S)ꢀ3,7ꢀdimethyloctꢀ6ꢀenoic acid using synthetic procedure 3. Yellow oil,
1
quantitative yield. H NMR (500 MHz, CDCl ) δ 10.68 (s, 1H), 2.33 (dd, J = 15.0, 5.9 Hz, 1H),
3
2.13 (dd, J = 15.0, 8.2 Hz, 1H), 1.95 (m, 1H), 1.51 (m, 1H), 1.34 – 1.12 (m, 6H), 0.95 (d, J = 6.8
1
3
Hz, 3H), 0.85 (d, J = 7.0 Hz, 6H). C NMR (126 MHz, CDCl ) δ 180.0, 41.7, 39.0, 36.9, 30.2,
3
27.9, 24.7, 22.7, 22.6, 19.7.
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(
R) or (S)ꢀethyl 6ꢀchloroꢀ3ꢀ(3,7ꢀdimethyloctanoyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14e (N246), 14d
(N247)). The final product was obtained after 3ꢀacylation of indole 1 using the synthetic
1
procedure 1. White solid, yield 89%. H NMR (500 MHz, CDCl ) δ 9.20 (s, 1H), 7.84 (d, J = 8.7
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hz, 1H), 7.41 (s, 1H), 7.20 (d, J = 8.7 Hz, 1H), 4.55 (q, J = 7.1 Hz, 2H), 3.09 (dd, J = 15.8, 5.7
Hz, 1H), 2.88 (dd, J = 15.8, 8.2 Hz, 1H), 2.11 (m, 1H), 1.43 (t, J = 7.1 Hz, 3H), 1.35 – 1.06 (m,
1
3
7
H), 0.94 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.7 Hz, 6H). C NMR (126 MHz, CDCl ) δ 201.3,
3
160.6, 135.3, 132.2, 126.0, 125.2, 123.6, 123.5, 122.5, 111.6, 62.1, 51.6, 39.1, 37.3, 30.4, 27.9,
+
.
24.8, 22.7, 22.6, 20.0, 14.3. MS (ESI): m/z 400.2 [M+Na]
Ethyl 6ꢀchloroꢀ3ꢀdodecanoylꢀ1H-indoleꢀ2ꢀcarboxylate (14f, DN433). The final product was
1
obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Red solid, yield 91%. H
NMR (500 MHz, CDCl ) δ 9.28 (br s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H),
3
7.20 (dd, J = 8.7, 1.8 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.06 (t, J = 7.6 Hz, 2H), 1.75ꢀ1.71 (m,
1
3
2
H), 1.36 (t, J = 7.1 Hz, 3H), 1.33ꢀ1.24 (m, 16H), 0.89ꢀ0.86 (m, 3H). C NMR (126 MHz,
CDCl ) δ 201.7, 160.9, 135.5, 132.4, 126.2, 125.4, (2x) 123.7, 122.4, 111.7, 62.3, 44.5, 32.1,
3
+.
29.8, 29.7, 29.5, 24.9, 22.9, 14.4, 14.3. MS (ESI): m/z 428.2 [M+Na]
Ethyl 6ꢀchloroꢀ3ꢀdecanoylꢀ1H-indoleꢀ2ꢀcarboxylate (14g, DN432). The final product was
1
obtained after 3ꢀacylation of indole 1 using synthetic procedure 1. Red solid, yield 91%. H
NMR (500 MHz, CDCl ) δ 9.25 (br s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H),
3
7.21 (dd, J = 8.7, 1.8 Hz, 1H), 4.45 (q, J = 7.2 Hz, 2H), 3.05 (t, J = 7.5 Hz, 2H), 1.74ꢀ1.71 (m,
1
3
2H), 1.43 (t, J = 7.2 Hz, 3H), 1.33ꢀ1.24 (m, 14H), 0.87 (t, J = 7.1 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 201.7, 160.8, 135.5, 132.4, 126.2, 125.4, 123.8, 123.7, 122.4, 111.7, 62.3, 44.5, 32.1,
3
+
.
29.6, 29.6, 29.5, 29.4, 24.9, 22.9, 14.4, 14.3. MS (ESI): m/z 400.2 [M+Na]
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Ethyl 6ꢀchloroꢀ3ꢀoctanoylꢀ1H-indoleꢀ2ꢀcarboxylate (14h, N214). The final product was
1
obtained after 3ꢀacylation of indole 1 using synthetic procedure 1. Orange solid, yield 93%. H
NMR (500 MHz, CDCl ) δ 9.25 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 1.8 Hz, 1H), 7.19
3
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
(dd, J = 8.7, 1.8 Hz, 1H), 4.45 (q, J = 7.2 Hz, 2H), 3.05 (t, J = 7.5 Hz, 2H), 1.74 – 1.68 (m, 2H),
1
3
1.43 (t, J = 7.2 Hz, 3H), 1.37 – 1.23 (m, 8H), 0.86 (t, J = 6.7 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 201.4, 160.6, 135.3, 132.2, 126.0, 125.2, 123.6, 123.5, 122.2, 111.5, 62.1, 44.2, 31.7,
3
+
.
29.3, 29.2, 24.7, 22.6, 14.2, 14.1. MS (ESI): m/z 372.2 [M+Na]
Ethyl 6ꢀchloroꢀ3ꢀhexanoylꢀ1H-indoleꢀ2ꢀcarboxylate (14i, DN441). The final product was
obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Yellow solid, yield 95%.
1
H NMR (500 MHz, CDCl ) δ 9.17 (br s, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H),
3
7.20 (dd, J = 8.7, 1.8 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 3.06 (t, J = 7.4 Hz, 2H), 1.76ꢀ1.70 (m,
1
3
2
H), 1.37 (t, J = 7.1 Hz, 3H), 1.33ꢀ1.26 (m, 4H), 0.90ꢀ0.88 (m, 3H). C NMR (126 MHz,
CDCl ) δ 201.4, 160.8, 135.4, 132.4, 126.2, 125.5, 123.9, 123.7, 122.4, 111.7, 62.3, 44.4, 31.7,
3
+
.
24.6, 24.6, 22.8, 14.4. MS (ESI): m/z 322.1 [M+H]
Ethyl 6ꢀchloroꢀ3ꢀ(2ꢀphenylacetyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14j, DN309). The final product
was obtained after 3ꢀacylation of indole 1 using synthetic procedure 1. Yellow solid, yield 99%.
1
H NMR (500 MHz, CDCl ) δ 9.49 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.36 (s, 1H), 7.28 (d, J =
3
7.2 Hz, 2H), 7.27ꢀ7.20 (m, 3H), 7.15 (d, J = 8.7, 1.7 Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 4.45 (s,
1
3
2
H), 1.43 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 198.3, 160.5, 135.2, 134.7, 133.2,
3
129.6, 128.5, 126.8, 126.4, 125.4, (2x)123.6, 123.5, 121.5, 111.6, 62.2, 50.5, 14.3. MS (ESI): m/z
+.
340.2 [MꢀH]
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Ethyl 6ꢀchloroꢀ3ꢀ(2ꢀ(pꢀtolyl)acetyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14k, N225). The final product
was obtained after 3ꢀacylation of indole 1 using synthetic procedure 1. White solid, yield 94%.
1
H NMR (500 MHz, CDCl ) δ 9.18 (s, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 7.21 – 7.11
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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52
53
54
55
56
57
58
59
60
13
(
m, 5H), 4.42 (q, J = 7.1 Hz, 2H), 3.62 (s, 2H), 2.33 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). C NMR
(126 MHz, CDCl ) δ 177.4, 162.2, 137.2, 137.0, 131.3, 130.3, 129.4, 129.2, 128.1, 126.0, 123.5,
3
+
.
1
21.8, 111.8, 108.7, 61.4, 40.6, 21.1, 14.4. MS (ESI): m/z 378.1 [M+Na]
Ethyl 6ꢀchloroꢀ3ꢀ(2ꢀ(4ꢀchlorophenyl)acetyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14l, DN522). The final
product was obtained after 3ꢀacylation of indole 1 using synthetic procedure 1. Yellow solid,
1
yield 95%. H NMR (500 MHz, DMSOꢀd6) δ 12.5 (br s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.53 (s,
1
H), 7.35 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 4.44 (q, J = 6.6
1
3
Hz, 2H), 4.37 (s, 2H), 1.36 (t, J = 6.6 Hz, 3H). C NMR (126 MHz, DMSOꢀd6) δ 197.1, 161.1,
1
36.2, 134.8, 132.0, 131.7, 130.4, 128.8, 128.6, 125.1, 123.7, 123.2, 120.1, 112.7, 62.3, 48.9,
+.
1
4.5. MS (ESI): m/z 374.0 [MꢀH]
Ethyl 6ꢀchloroꢀ3ꢀ(2ꢀ(4ꢀfluorophenyl)acetyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14m, DN312). The
final product was obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Yellow
1
solid, yield 95%. H NMR (500 MHz, CDCl ) δ 9.20 (br s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.40 (s,
3
1H), 7.23ꢀ7.18 (m, 3H), 6.98ꢀ7.01 (m, 2H), 4.50 (q, J = 7.2 Hz, 2H), 4.43 (s, 2H), 1.46 (t, J = 7.2
13
Hz, 3H). C NMR (126 MHz, CDCl ) δ 198.3, 160.6, 135.4, 132.7, (2x) 131.4, 126.6, 125.7,
3
+.
1
24.0, 123.9, 115.6, 115.5, 111.7, 62.5, 49.6, 40.1, 14.5. MS (ESI): m/z 358.0 [MꢀH]
Ethyl 6ꢀchloroꢀ3ꢀ(3ꢀphenylpropanoyl)ꢀ1H-indoleꢀ2ꢀcarboxylate (14n, DN427). The final
product was obtained after 3ꢀacylation of indole 1 using the synthetic procedure 1. Red solid,
1
yield 96%. H NMR (500 MHz, CDCl ) δ 9.20 (br s, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.39 (s, 1H),
3
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2
2
2
2
2
2
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3
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7
.28ꢀ7.17 (m, 6H), 4.41 (q, J = 7.1 Hz, 2H), 3.42 (t, J = 7.7 Hz, 2H), 3.09 (t, J = 7.7 Hz, 2H),
1
3
1.38 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 200.1, 160.7, 141.4, 135.4, 134.8, 132.5,
3
(2x) 128.60, 125.5, 124.0, (2x) 123.8, 111.7, 62.4, 45.7, 30.8, 14.4. MS (ESI): m/z 378.1
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
+.
[
M+Na]
4
5,46
1
Ethyl 6ꢀchloroꢀ1H-indoleꢀ2ꢀcarboxylate (1).
White solid, yield 75%. H NMR (500 MHz,
CDCl ) δ 8.89 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.20 (d, J = 8.7 Hz, 1H), 7.13 (dd, J
3
=
8.7, 0.5 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H).
4
7,48
1
Ethyl 6ꢀchloroꢀ3ꢀformylꢀ1H-indoleꢀ2ꢀcarboxylate (2).
Yellow solid, yield 85%. H NMR
(500 MHz, CDCl ) δ 10.7 (s, 1H), 9.34 (br s, 1H), 8.38 (d, J = 8.7 Hz, 1H), 7.45 (s, 1H), 7.30 (d,
3
J = 8.7 Hz, 1H), 4.52 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H).
4
7
1
Ethyl 6ꢀchloroꢀ3ꢀformylꢀ1ꢀmethylꢀ1H-indoleꢀ2ꢀcarboxylate (3). Yellow solid, yield 95%. H
NMR (500 MHz, CDCl ) δ 10.6 (s, 1H), 8.43 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.32 (d, J = 8.7
3
13
Hz, 1H), 4.53 (q, J = 7.1 Hz, 2H), 1.48 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 188.4,
3
161.0, 134.8, 132.6, (2x) 125.1, 123.2, 120.0, (2x) 110.6, 62.5, 32.8, 14.5.
4
7
1
6
ꢀchloroꢀ3ꢀformylꢀ1H-indoleꢀ2ꢀcarboxylic acid (7). Pink solid, yield 75%. H NMR (500
MHz, CDCl ) δ 10.7 (s, 1H), 8.27 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 0.5 Hz, 1H), 7.23 (dd, J =
3
8.77, 0.5 Hz, 1H).
4
9
1
tertꢀbutyl 6ꢀchloroꢀ1H-indoleꢀ2ꢀcarboxylate (8). Pink solid, yield 75%. H NMR (500 MHz,
CDCl ) δ 7.57 (d, J = 10 Hz, 1H), 7.41 (s, 1H), 7.11ꢀ7.09 (m, 2H), 1.62 (s, 9H).
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Enzyme inhibition studies
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Journal of Medicinal Chemistry
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Human 15ꢀLOXꢀ1 screening UV assay. The hꢀ15ꢀLOXꢀ1 enzyme was expressed and purified
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as described before. The hꢀ15ꢀLOXꢀ1 activity was determined by the conversion of linoleic
acid to 13Sꢀhydroperoxyꢀ9Z,11Eꢀoctadecadienoic acid (13(S)ꢀHpODE) (λmax of 234 nm). The
conversion rate was followed by UVꢀabsorbance at 234 nm over time. The linear increase in
absorbance was used to determine the enzyme activity. That conversion rate was evaluated at the
linear part of the plot before the substrate depletion. The linear part usually covers the first 10 to
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16 minutes depending on the enzyme concentration. The optimum concentration of hꢀ15ꢀLOXꢀ1
was determined by an enzyme activity assay (x 640 times dilution). The raw data of the
conversion of linoleic acid in present (Positive Control) or absent of the enzyme (Blank)
following with UVꢀabsorbance assay at 234 nm over time are presenting in the Figure S1.
The assay buffer consists of 25 mM HEPES titrated to pH 7.5 using a concentrated aqueous
solution of NaOH. The substrate, linoleic acid (LA) (Sigma Aldrich, L1376) was diluted in
ethanol to 500 µM. The inhibitor (100 mM in DMSO) was diluted with the assay buffer to 125
µM. The inhibitor solution of 80 µL was mixed with 60 µL assay buffer, 50 µL of 1:160 enzyme
solution and incubated for 10 min at rt. After incubation, 10 µL of 500 µM LA was added to the
mixture which provided a mixture with a final dilution of the enzyme of 1:640 and 25 µM LA.
The linear absorbance increase in absence of the inhibitor was set as 100%, whereas the
absorbance increase in absence of the enzyme was set to 0%. All experiments were performed in
triplicate and the average triplicate values and their standard deviations are plotted. The half
maximal inhibitor concentration (IC ) of the inhibitors for hꢀ15ꢀLOXꢀ1 was determined using
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the same assay. The inhibitor was diluted with assay buffer. Using a serial dilution, the desired
concentrations of the inhibitor were obtained ranging from 0.09ꢀ50 µM or 0.009ꢀ20 µM
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