Inhibition of 5-oxo-6,8,11,14-eicosatetraenoic acid-induced activation of neutrophils and eosinophils by novel indole oxe receptor antagonists

Article abstract of DOI:10.1021/jm401292m

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that is a potent granulocyte chemoattractant, which induces the infiltration of eosinophils into human skin when injected intradermally. It could therefore be an important proinflammatory mediator in eosinophilic diseases such as asthma and allergic rhinitis, and the OXE receptor, which mediates its actions, is therefore an attractive drug target. Using a structure-based approach in which substituents mimicking the essential polar (C₁-C₅) and hydrophobic (C₁₅-C₂₀) regions of 5-oxo-ETE were incorporated on an indole scaffold, we identified two potent selective OXE antagonists with IC₅₀ values of about 30 nM. Neither compound displayed agonist activity and both inhibited 5-oxo-ETE-induced chemotaxis and actin polymerization and were relatively resistant to metabolism by rat liver homogenates. The active enantiomers of these racemic antagonists were even more potent, with IC₅₀ values of <10 nM. These selective OXE antagonists could potentially be useful therapeutic agents in allergic diseases such as asthma.

Full text of DOI:10.1021/jm401292m

Article
pubs.acs.org/jmc
Inhibition of 5‑Oxo-6,8,11,14-eicosatetraenoic Acid-Induced
Activation of Neutrophils and Eosinophils by Novel Indole OXE
Receptor Antagonists
Vivek Gore,‡,§ Sylvie Gravel,† Chantal Cossette,† Pranav Patel,‡,∥ Shishir Chourey,‡ Qiuji Ye,‡
Joshua Rokach,‡ and William S. Powell*,†
†Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec H2X 2P2,
Canada
‡Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 West University Boulevard, Melbourne,
Florida 32901, United States
ABSTRACT: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-
lipoxygenase product that is a potent granulocyte chemoattractant, which
induces the infiltration of eosinophils into human skin when injected
intradermally. It could therefore be an important proinflammatory mediator
in eosinophilic diseases such as asthma and allergic rhinitis, and the OXE
receptor, which mediates its actions, is therefore an attractive drug target.
Using a structure-based approach in which substituents mimicking the
essential polar (C1−C5) and hydrophobic (C15−C20) regions of 5-oxo-ETE
were incorporated on an indole scaffold, we identified two potent selective OXE antagonists with IC50 values of about 30 nM.
Neither compound displayed agonist activity and both inhibited 5-oxo-ETE-induced chemotaxis and actin polymerization and
were relatively resistant to metabolism by rat liver homogenates. The active enantiomers of these racemic antagonists were even
more potent, with IC50 values of <10 nM. These selective OXE antagonists could potentially be useful therapeutic agents in
allergic diseases such as asthma.
and, to a lesser extent, on macrophages.15 5-Oxo-ETE has also
INTRODUCTION
■
been reported to be an endogenous promoter of the proliferation
The 5-lipoxygenase (5-LO) pathway is responsible for the
conversion of arachidonic acid (AA) to a group of potent
proinflammatory lipid mediators. 5-LO initially converts AA to
5S-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE)
of tumor cells, which express both the OXE receptor19 and 5-
HEDH.20
Metabolism of 5-oxo-ETE by various pathways results in
dramatically reduced biological activity.21 The major metabolic
(Figure 1). A portion of the latter intermediate remains bound
pathway in neutrophils is ω-oxidation by CYP4F3A to 5-oxo-20-
hydroxy-ETE, which is 100 times less potent than 5-oxo-ETE.
to 5-LO and is cyclized to the unstable epoxide leukotriene (LT)
A4, the precursor for LTB4, LTC4, and LTD4.1 However,
Reduction of 5-oxo-ETE back to 5-HETE by 5-HEDH, which is a
substantial amounts of 5-HpETE are released from 5-LO and are
reversible enzyme, also reduces potency by 100 times. An even
rapidly reduced by peroxidase to 5S-hydroxy-6,8,11,14-eicosate-
traenoic acid (5-HETE). 5-HETE is then oxidized to 5-oxo-
6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) by 5-hydroxyeico-
greater 1000-fold reduction in potency occurs after reduction of
the 6-trans double bond by eicosanoid Δ6-reductase, a calcium-
sanoid dehydrogenase (5-HEDH), a highly selective NADP+-
dependent enzyme also found in neutrophils. A smaller 5-fold
reduction in potency is observed after isomerization of the 8-cis
dependent enzyme that is widely expressed in both inflammatory
and structural cells.2,3 5-Oxo-ETE synthesis is strongly enhanced
double bond to the trans configuration, whereas methylation of
the carboxyl group reduces potency by about 20 times.10,22 Other
oxo-ETEs such as 12-oxo-ETE and 15-oxo-ETE do not activate
the OXE receptor. Further extensive structure−activity studies
revealed that a fatty acid chain length of at least 18 carbons and a
5-oxo-Δ6,8 diene system are the minimum requirements for
activation of this receptor.23
by activation of the respiratory burst4 and in the presence of
oxidative stress,5,6 both of which elevate the levels of the essential
cofactor NADP+.
Both LTs7 and 5-oxo-ETE have potent biological actions that
are mediated by G protein-coupled receptors.8−10 LTB4, acting
through the BLT1 receptor, is a potent neutrophil chemo-
attractant, whereas LTD4 stimulates smooth muscle contraction,
vascular leak, mucus secretion, and cytokine release, which are
mediated by the cysLT1 and cysLT2 receptors. 5-Oxo-ETE is a
potent eosinophil chemoattractant, both in vitro11 and in vivo.12
Its actions are mediated by the OXE receptor,13−16 which is
highly expressed on eosinophils, basophils,17,18 and neutrophils
The potent chemoattractant effects of 5-oxo-ETE on
eosinophils suggest that it may play an important role in
eosinophilic diseases such as asthma and allergic rhinitis.
Received: August 20, 2013
Published: December 18, 2013
© 2013 American Chemical Society
364
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 1. Biosynthesis of 5-oxo-ETE and its interaction with the OXE receptor. Abbreviation: Perox, peroxidase.
However, progress in understanding its pathophysiological role
has been impeded by the lack of an orthologue of the OXE
receptor in rodents. An alternative approach to investigate its
biological role would be the use of selective antagonists. To date,
little information is available about such compounds. We
previously showed that 5-oxo-12-HETE, a metabolite of 5-oxo-
ETE formed by platelets, while not itself affecting intracellular
calcium levels in neutrophils, blocks 5-oxo-ETE-induced calcium
mobilization with an IC50 of 0.5 μM.24 However, this substance is
not very stable and is not suitable for development as an
antagonist. Another very recent report documented antagonist
properties for the benzobisthiazole derivative, Gue1654.25 The
goal of the present study was to develop an OXE receptor
antagonist using an indole scaffold containing substituents
mimicking both the polar 5-oxovalerate portion of 5-oxo-ETE as
well as the hydrophobic ω-end of the molecule (Figure 2A). In an
containing two substituents: a 5-oxo-valeryl group and a hexyl
group to mimic the carboxyl and alkyl portions, respectively, of 5-
oxo-ETE. Addition of 5-oxo-ETE (10 nM) to indo-1-loaded
neutrophils resulted in a strong calcium response (Figure 2B).
Parts C−G of Figure 2 show the effects of a series of N-(5-
oxovaleryl) indoles (10 μM) containing hexyl substituents in
different positions.
As we previously reported,26 5-oxo-ETE-induced calcium
mobilization was completely abolished by the indole containing a
hexyl group in the 2-position (10, Figure 2C) but was hardly
affected when the hexyl group was present in the 3-, 5-, or 6-
positions (Figure 2D−G) and was only modestly effected when
the hexyl group was in the 7-position (Figure 2G). In addition to
the N-acyl indoles described above, we now also prepared two
indoles containing a 5-oxovaleryl substituent in the 3-position.
The presence of a 2-hexyl group in this series inhibited calcium
mobilization by about 50% (Figure 2I), whereas a 1-hexyl
substituent had a smaller effect (Figure 2H). The synthesis of
derivatives in parts B−G of Figure 2 has been reported by us
previously.23 The compounds in parts H and I of Figure 2 and
their bioactivity have not been reported previously.
Effects of Modification of the Alkyl Group on the
Antagonist Potency of 1-Acylindoles. We initially focused
on the most potent antagonist, 10, and examined the effects of
various modifications of the alkyl side chain (Figure 3).
Reduction of its length to 5 carbons (9) resulted in a small
decrease in potency, whereas further shortening to a butyl group
(8) had a much more dramatic effect, reducing the potency by
over 10-fold. In contrast, increasing the length of the side chain to
seven (11), eight (12), or 11 (13) carbons appeared to result in
slightly increased potency. These compounds were synthesized
from the common precursor 1H-indole-2-carboxylic acid as
shown in Scheme 1, and the detailed synthesis was reported
previously.23 Introduction of a single double bond in conjugation
with the aromatic system reduced potency in the 6-carbon series
(Δ1Hx (14) vs Hx (10)) but had little effect in the 11-carbon
series (Δ1Un (15) vs Un (13)). The compounds shown in
Figure 2 were synthesized as shown in Schemes 2 and 3.
Effects of Modification of the 5-Oxovalerate Group of
1-Acyl-2-hexylindoles. We next investigated the effects of
various structural modifications of the carboxyl side chain in a
series of N-acyl-2-hexyl indoles (Figure 4). Shortening the side
chain to 4 carbons (29; 4-oxobutyrate) nearly eliminated
antagonist activity. Addition of two methyl groups to the 3-
position of 5-oxovalerate (17) reduced potency by about 10-fold,
whereas placing a cyclopropyl group (25) in this position had
relatively little effect. In contrast, addition of a single methyl
group to the 3-position (27) increased potency by over 10 times,
resulting in an IC50 of 90 nM. The structural modifications
shown in Figure 4 were performed as described in Schemes 3−6.
Effects of Halides and Other Substituents on the
Potency of 1-Acyl-2-hexylindoles. Focusing on compound
27, we investigated the effects of additional substituents on the
indole moiety. The presence of a methyl group in the 3-position
(47) or a chloro substituent in the 5-position (41) had relatively
little effect on antagonist potency (Figure 5). On the other hand,
Figure 2. Design and screening of indoles containing hexyl (Hx) and 5-
oxovalerate (oV) substituents in different positions. (A) Hexyl and 5-
oxovalerate groups were placed on an indole scaffold to mimic the
corresponding regions of 5-oxo-ETE. Either vehicle (B) or indole
derivatives containing 5-oxovalerate and hexyl substituents in the 1 and
2 (C), 1 and 3 (D), 1 and 5 (E), 1 and 6 (F), 1 and 7 (G), 3 and 1 (H), or
3 and 2 (I) positions, respectively, were added to indo-1 labeled
neutrophils as described in the Experimental Section. 5-Oxo-ETE (10
nM) was added 2 min later. One minute later, digitonin was added to
lyse the cells and released the indo-1 to give the maximal fluorescence
response (not shown).
initial study, we recently reported antagonist activity in the
micromolar range of N-acyl-2-hexyl indoles.26 We have now
developed a much more potent compound in this series and have
identified a second series of indoles with comparable OXE
receptor antagonist potency.
RESULTS
■
OXE Receptor Antagonist Effects of Indoles Contain-
ing Hexyl and 5-Oxovalerate Substituents. Our initial
strategy was to examine the effects of a series of indoles
365
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 3. Effects of modification of the alkyl group on antagonist potency. The effects of 1-(5-oxovalerate)-indoles containing different alkyl substituents
in the 2-position on 5-oxo-ETE-induced calcium mobilization in neutrophils were investigated. Either vehicle or different concentrations of indoles were
added to indo-1-loaded neutrophils, followed 2 min later by addition of 5-oxo-ETE (10 nM) as shown in Figure 2. The symbols represent means SEM.
The IC50 values shown in the table to the right are the means SE of IC50 values from the numbers of independent experiments shown in brackets.
a
Scheme 1. Synthesis of Alkyl-Substituted 1-(5-Oxovalerate)indoles
a
Reagents and conditions: (a) LiAlH4, THF, 0 °C−rt; (b) MnO2, CH3CN, rt; (c) LiHMDS, THF, −78 °C−rt; (d) 10% Pd/C, H2, EtOH, rt; (e)
KOH, DMSO, rt.
a
shown in Schemes 10−13. We focused primarily on the effects of
modifications analogous to those employed in the 1-acylindole
series. Addition of a chloro substituent in the 5-position of the
indole ring (51; IC50, 566 nM) increased potency by about 10
times, whereas addition of a methyl group at the 3-position of the
5-oxovalerate side chain (52; IC50, 314 nM) had a slightly greater
effect. Combination of these two modifications (compound 53,
IC50, 28 nM) resulted in a further increase in potency of over 10-
fold. In contrast to the 1-acyl series, the 5-chloro compound 53
was considerably more potent than the 6-chloro derivative 56.
The corresponding 5-bromo derivative 54 was equipotent with
53. The acyl side chain was substituted at C3 on the indole ring as
shown in Scheme 10. Further modifications were performed as
shown in Schemes 11−13.
Scheme 2. Synthesis of 14 and 15
a
Reagents and conditions: (a) KOH, DMSO, rt.
Scheme 3. Synthesis of 4-(2-Hexyl-indol-1-yl)-4-oxo-butyric
a
Acid
Compounds 53 and 34 are Selective OXE Receptor
Antagonists. The selectivities of the two most potent
compounds (53 and 34) were further investigated (Figure 7).
Following addition of vehicle to neutrophils, 5-oxo-ETE elicited
a robust calcium response that did not affect the responses to
subsequent addition of LTB4, PAF, fMLP, or IL-8 (bottom
tracings in Figure 7A−D, respectively). Prior addition of high
concentrations (10 μM) of 53 (middle tracings) and 34 (upper
tracings) completely blocked 5-oxo-ETE-induced calcium
mobilization but had no effect on the responses to any of the
other four agonists shown in Figure 7, indicating that these
antagonists are highly selective for the OXE receptor.
a
Reagents and conditions: (a) EtMgBr, THF, 0 °C−rt.
a 5-bromo substituent (42) or a 6-methoxy group (36) reduced
potency by several fold. In contrast, addition of either a chloro
(34) or a bromo (35) substituent at the 6-position resulted in
increases in potency of approximately 3-fold. The 6-chloroindole
34 is a potent OXE receptor antagonist, with an IC50 value of 28
nM. These aromatic substituted compounds were synthesized as
shown in Schemes 7−9.
Compounds 53 and 34 Block 5-Oxo-ETE-Induced Actin
Polymerization and Chemotaxis. The effects of 53 and 34 on
5-oxo-ETE-induced actin polymerization in both eosinophils and
neutrophils were investigated using flow cytometry. Leukocytes
were preincubated with either vehicle, 53 or 34, for 5 min
followed by incubation with 5-oxo-ETE (10 nM) for a further 20
s, and the amounts of polymerized F-actin were determined in
Antagonist Effects of a Second Series of Indoles.
Because of the modest antagonist effect of 2-hexyl-3-(5-oxo-
valerate)indole (48) as illustrated by Figure 2I, we decided to test
the effects of various modifications of this compound. Because
methylation of the indole nitrogen (50; Figure 6) slightly
increased potency, an N-methyl group was incorporated in all
further compounds in this series, the syntheses of which are
366
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 4. Effects of modification of the carboxyl side chain on the antagonist potency of 2-hexylindoles. Either vehicle or different indoles were added to
indo-1-loaded neutrophils, followed by 5-oxo-ETE (10 nM) as shown in Figure 2. The symbols represent means SEM. The IC50 values were
determined as described in Figure 3
Scheme 4. Synthesis of 5-(2-Hexyl-indol-1-yl)-3,3-dimethyl-
5-oxo-pentanoic Acid
Scheme 6. Synthesis of 5-(2-Hexyl-indol-1-yl)-3-methyl-5-
oxo-pentanoic Acid
a
a
a
a
Reagents and conditions: (a) EtMgBr, THF, 0 °C−rt.
Reagents and conditions: (a) KOH, DMSO, rt.
both eosinophils and neutrophils. Both 53 (IC50, 33 10 nM)
and 34 (IC50, 30 7 nM) were potent inhibitors of 5-oxo-ETE-
induced actin polymerization in eosinophils (Figure 8A) and had
similar effects on neutrophils (Figure 8B). 53 (IC50, 454 192
nM) and 34 (IC50, 402 120 nM) also inhibited 5-oxo-ETE
induced neutrophil chemotaxis (Figure 8C). The IC50 values
were higher in this assay because we used a higher concentration
of 5-oxo-ETE (100 nM) to induce neutrophil migration.
The benzobisthiazole derivative Gue1654 was recently
reported to block Gβγ- but not Gαi-mediated responses to 5-
oxo-ETE.25 We found that this compound is about 10 times less
potent than 53 and 34 in blocking 5-oxo-ETE-induced actin
polymerization in eosinophils (Figure 8A) and neutrophils
(Figure 8B).
Metabolism of 34 and 53 by Rat Liver Homogenates.
To determine whether 53 and 34 are susceptible to metabolism,
they were incubated with rat liver homogenates. Negligible
metabolism of either compound was observed in medium that
favored β-oxidation (containing ATP, CoA, L-carnitine and
NAD+;27 data not shown). Compound 34 was metabolized to a
small extent when incubated for 60 min with a liver homogenate
containing NADPH (1 mM), the cofactor required by
cytochrome P450 (Figure 9A). Only one significant product
was observed, which had a tR of 18.2 min, compared to 30.2 min
for unmetabolized 34. The UV spectrum of this product was
virtually identical to that of 34 (inset to Figure 9A). 53 was
metabolized to an even lesser extent by liver homogenates under
identical conditions (Figure 9B). A very small amount of a more
polar product (tR, 14.1 min) was detected, with a UV spectrum
nearly identical to that of 53 (inset to Figure 9B).
Antagonist Potencies of Purified Enantiomers of 34
and 53. Because of the presence of a methyl group in the 3-
position of the 5-oxo-valerate substituents of 34 and 53, both
compounds are racemic mixtures. To determine whether the
individual enantiomers of each pair differed from one another in
their antagonist potencies, they were separated by chiral HPLC.
The enantiomers of 34 were completely resolved in a single
chromatography to give two peaks, designated as 34a and 34b
(Figure 10A). It was not possible to completely separate the two
enantiomers of 53 (designated as 53a and 53b) in a single
chromatography (Figure 10D), and a second step of HPLC using
the same conditions was required to purify 53b. Chiral HPLC of
the purified enantiomers is shown in Figure 10B,C,E,F).
a
Scheme 5. Synthesis of 2-(1-(2-(2-Hexyl-1H-indol-1-yl)-2-oxoethyl)cyclopropyl)acetic Acid
a
Reagents and conditions: (a) I2, PPh3, imidazole, rt; (b) KCN, DMF, 45−50 °C; (c) MeOH, H2SO4, 60−65 °C; (d) LiOH·H2O, iPrOH, H2O, rt;
(e) 130 °C; (f) KOH, DMSO, rt.
367
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 5. Effects of additional substituents on antagonist potency. The effects of 1-(3-methyl-5-oxovalerate)-2-hexylindoles containing various
additional substituents on 5-oxo-ETE-induced calcium mobilization in neutrophils were investigated. Either vehicle or different indoles were added to
indo-1-loaded neutrophils, followed by 5-oxo-ETE (10 nM) as shown in Figure 2. The symbols represent means SEM. The IC50 values were
determined as described in Figure 3
a
Scheme 7. Synthesis of 34, 35, and 36
a
Reagents and conditions: (a) LiAlH4, THF, 0 °C−rt; (b) MnO2, CH3CN, rt; (c) LiHMDS, THF, −78 °C−rt; (d) 10% Pd/C, H2, EtOH, rt; (e)
KOH, DMSO, rt.
a
Scheme 8. Synthesis of 41and 42
a
Reagents and conditions: (a) LiAlH4, THF, 0 °C−rt; (b) MnO2, CH3CN, rt; (c) LiHMDS, THF, −78 °C−rt; (d) 10% Pd/C, H2, EtOH, rt; (e)
KOH, DMSO, rt.
a
Scheme 9. Synthesis of 5-(2-Hexyl-3-methyl-indol-1-yl)-3-methyl-5-oxo-pentanoic Acid (47)
a
Reagents and conditions: (a) LiAlH4, THF, 0 °C−rt; (b) MnO2, CH3CN, rt; (c) LiHMDS, THF, −78 °C−rt; (d) 10% Pd/C, H2, EtOH, rt; (e)
KOH, DMSO, rt.
The effects of the above enantiomers on 5-oxo-ETE-induced
calcium mobilization in neutrophils was investigated. The
purities of the four enantiomers after purification were: 34a
(99.2%), 34b (99.9%), 53a (98.8%), and 53b (98.7%). In both
cases, the earlier-eluting enantiomers were by far the more
potent. 34a and 53a had nearly identical IC50 values of 7 2 and
OXE receptor that mediates its biological activity. We used a
structure-based approach in which we incorporated segments of
the 5-oxo-ETE molecule that are essential for its biological
activity onto an indole backbone. We previously identified
several regions of 5-oxo-ETE that are critical for leukocyte
activation: the C1 carboxyl group, the 5-oxo-Δ6,8-diene system,
and the terminal hydrophobic ω-end of the molecule.23 On the
basis of this information, we designed a series of indole
derivatives containing both the C1−C5 portion of 5-oxo-ETE
(i.e., a 5-oxovalerate group) and the C15−C20 portion of the
molecule in the form of a hexyl group. Because the oxo group in
these compounds is conjugated with the aromatic indole system,
the indole moiety should mimic the conjugated diene portion of
5-oxo-ETE as well as fix the positions of the alkyl and acyl side
6
1 nM, respectively, whereas the later-eluting enantiomers
34b and 53b had IC50 values of 2175 357 and 2730 956 nM,
respectively.
DISCUSSION
■
Because of the potent chemoattractant effect of 5-oxo-ETE on
eosinophils and its possible involvement in asthma and other
allergic diseases, we set out to design a selective antagonist of the
368
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 6. Inhibition of 5-oxo-ETE-induced calcium mobilization by 2-hexyl-3-(5-oxovalerate)-indoles. Either vehicle or different concentrations of
indoles, followed by 5-oxo-ETE, were added as shown in Figure 2. The symbols represent means SEM. The IC50 values were determined as described
in Figure 3
antagonist activity, with an IC50 above 30 μM (data not shown),
suggesting that the angle between the two side chains may be
important (5- vs 6-membered ring).
Scheme 10. Synthesis of 5-(2-Hexyl-1H-indol-3-yl)-5-oxo-
pentanoic Acid (48)
a
We tested several modifications of the alkyl side chain.
Whereas reducing its length to 4 or 5 carbons reduced potency,
increasing the length to seven, eight, or 11 carbons appeared to
slightly increase potency. However, lengthening this side chain
also reduced solubility and, in the cases of high concentrations
(30 μM) of the C8 and C11 compounds, resulted in some calcium
mobilization, which was not observed in compounds containing
a hexyl substituent. Although carbon-15 of 5-oxo-ETE is part of
an olefinic group, it seemed unlikely that an unsaturated carbon
in the hexyl group would be required, as saturation of the Δ14-
double bond of 5-oxo-ETE (as in 5-oxo-6,8,11-eicosatrienoic
acid) did not reduce biological potency.23 Nevertheless, we also
investigated the effects of replacing the hexyl group with a Δ1-
hexenyl group but found that this reduced antagonist potency.
However, this was not true with a C11 side chain, in which case
there was little difference in the potencies of undecyl and Δ1-
undecenyl derivatives. For the above reasons, we focused on 2-
hexylindoles in subsequent studies.
We also investigated the effects of modification of the 5-
oxovalerate substituent, in part as an attempt to block any
potential metabolism by β-oxidation. Addition of bulky groups
(dimethyl or cyclopropyl) to the 3-position of the 5-oxovalerate
moiety either reduced potency or had little effect. In contrast,
addition of a single methyl group to this position dramatically
increased potency in both the 1-acyl and 3-acyl series. The 3-
methyl group presumably favors a conformation of the acyl side
chain that interacts strongly with the OXE receptor, resulting in
increased potency. This modification would also have the
additional advantage of blocking β-oxidation, and we found no
a
Reagents and conditions: (a) Me2AlCl, CH2Cl2, rt.
chains relative to one another. Different positional isomers can
thereby mimic various conformational forms of 5-oxo-ETE that
may bind to and subsequently activate the OXE receptor. An
ideal OXE receptor antagonist would possess a structure that
mimicked the conformation of 5-oxo-ETE that has high affinity
for the receptor but could not alter its conformation to mimic the
one that serves to initiate OXE receptor signaling via G-proteins.
We identified two series of indoles with potent antagonist
activities containing a 5-oxovalerate moiety in either the 1-
position or the 3-position. Our preliminary studies revealed that a
hydrophobic side chain is required for activity, as 1(5-
oxovalerate)indole had no detectable activity at a concentration
of 30 μM.26 The present results clearly indicate that the alkyl
group must be adjacent to the acyl side chain in both of the above
series because further separation of the two side chains resulted
in dramatic losses in antagonist potency. This suggests that both
series can mimic a hairpin conformation of 5-oxo-ETE that can
bind to the OXE receptor without activating it. The indole
structure itself also appears to play a role, as we synthesized an
analogous quinoline derivative with a hexyl group in the 2-
position and 5-oxovalerate in the 3-position. Although this
compound did not act as an agonist, it had only very weak
a
Scheme 11. Synthesis of 50 and 51
a
Reagents and conditions: (a) KOH, DMSO, 0 °C−rt; (b) Me2AlCl, CH2Cl2, rt.
369
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
a
Scheme 12. Synthesis of 52, 53, and 54
a
Reagents and conditions: (a) KOH, DMSO, 0 °C−rt; (b) Me2AlCl, CH2Cl2, rt.
a
Scheme 13. Synthesis of 5-(6-Chloro-2-hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxo-pentanoic Acid (56)
a
Reagents and conditions: (a) KOH, DMSO, 0 °C−rt; (b) Me2AlCl, CH2Cl2, rt.
Figure 7. Selectivity of 53 and 34 for the OXE receptor. The effects of
53 and 34 on calcium mobilization in response to subsequent addition
of 5-oxo-ETE followed by either (A) LTB4 (10 nM), (B) PAF (10 nM),
(C) fMLP (100 nM), or (D) IL-8 (10 nM). Either vehicle, 53 (10 μM)
or 34 (10 μM), were added to indo-1-loaded neutrophils. 5-Oxo-ETE
(10 nM) was added 2 min later, followed after a further 2 min by one of
the above additional agonists. The results are representative of two
independent experiments, which had virtually identical results.
Figure 8. Effects of 53 and 34 on 5-oxo-ETE-induced actin
polymerization and chemotaxis. Anti-CD49d-labeled leukocytes were
incubated with vehicle, 53 (upward pointing open triangles; n = 7), 34
(downward pointing solid triangles; n = 7), or Gue1654 (open circles; n
= 3) for 5 min. 5-Oxo-ETE (10 nM) was then added, and 20 s later the
incubations were terminated as described in the Experimental Section.
Polymerized F-actin was measured by flow cytometry in eosinophils (A)
and neutrophils (B). Neutrophil migration (C) was assessed using
modified Boyden chambers as described in the Experimental Section.
Neutrophils and 5-oxo-ETE (100 nM) were added to the top or bottom
chambers, respectively, whereas either vehicle, 53 (upward pointing
open triangles; n = 6) or 34 (downward pointing solid triangles; n = 8)
were added to both the top and bottom chambers. Cells that had
migrated through the filter were counted under a microscope (5 fields/
well). All data are means SE.
evidence for the metabolism of either 53 or 34 when they were
incubated with rat liver homogenates under conditions27 known
to favor this pathway (data not shown).
We previously showed that addition of a chloro group to the 6-
position of 10 in the 1-acyl series increases potency by 3−4-fold
(IC50 of 6-Cl derivative ∼0.4 μM), whereas addition of a 5-chloro
substituent has no effect.26 A similar increase in potency was
observed in the present study following the addition of a 6-Cl
substituent (34) to the 1-(3-methyl-5-oxo-valerate) compound
27, in contrast to the corresponding 5-Cl derivative (41) which
was, if anything, less potent than 27. Addition of a 6-Cl
substituent also increased potency by about 4-fold in the 3-acyl
series (56 compared to 52 in Figure 6). However, in this case, the
corresponding 5-Cl compound (53) was even more potent. This
may be because the relative positions of the three substituents (3-
methyl-5-oxovalerate, hexyl, and Cl) to one another is more
important than their absolute positions on the indole ring. If the
indole in 53 is inverted as shown in Figure 11, it can be seen that a
chloro substituent in the 5-position should mimic the 6-chloro
group of 34. For this reason we wondered whether the presence
of a 3-methyl group in the 1-acyl series might increase potency,
but this was not the case (compare 27 and 47, Figure 5). In both
the 1-acyl and 3-acyl series, Cl could be replaced by Br without
affecting potency.
The progression in the SAR from 1(5-oxovalerate)indole and
1026 to the novel compounds 27 and 34, accompanied by their
IC50 values, is shown in Figure 11. A similar progression from the
novel 2,3-substituted indole 48 to the highly potent 53 is also
shown. Because of their similarity, it would seem likely that both
53 and 34 interact similarly with the OXE receptor. However,
these two compounds could differ in their rates of absorption or
metabolism due to structural differences such as the presence or
absence of an amide bond, which could potentially affect their in
vivo potencies.
370
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
compound was identical to that of 34, it is unlikely that it was
formed by alteration or addition to the indole structure. It would
seem probable that it is an ω, ω-1, or ω-2 oxidation product
formed by hydroxylation of the hexyl group by cytochrome P450.
Work on the characterization of this metabolite is currently
underway. 53 appeared to be metabolized to a very small extent
(∼2%) by this pathway. Preliminary pharmacokinetic experi-
ments in rats suggest that both antagonists appear rapidly in the
blood following oral administration, along with smaller amounts
of polar metabolites similar to those formed by liver
homogenates in the presence of NADPH (unpublished data).
Because both 34 and 53 contain a methyl group in the 3-
position of their 5-oxovalerate substituent, they are racemates.
Separation of the enantiomers by chiral chromatography
revealed that in each case a single enantiomer was responsible
for the majority of antagonist activity, with the more active
enantiomer being several hundred times more potent than the
less active one. The preparation of 53b that we used contained
about 1.3% of the more active enantiomer 53a, which would have
contributed to the antagonist effect we observed. Therefore, the
true IC50 value of 53b is likely to be even higher than the value of
∼2.7 μM that we observed. This was not true for the inactive
enantiomer of 34 (i.e., 34b), which did not contain any
detectable amount of 34a.
Figure 9. Metabolism of 34 (A) and 53 (B) by rat liver homogenates.
Either 34 or 53 (20 μM) were incubated with rat liver homogenates in
the presence of NADPH (1 mM) for 60 min at 37 °C and the products
analyzed by reversed-phase HPLC as described in the Experimental
Section. The wavelengths monitored were 271 nm (34) and 305 nm
(53). The solid lines in the insets show the UV spectra of the polar
metabolites, whereas the broken lines show the spectra of 34 and 53
standards.
Other than our preliminary study,26 very little information is
available on compounds with OXE receptor antagonist activity.
We previously found that platelets convert 5-oxo-ETE to
metabolites that can block 5-oxo-ETE-induced calcium mobi-
lization in neutrophils.24 We identified these compounds as the
8-cis and 8-trans isomers of 5-oxo-12S-hydroxy-6,8,10,14-
eicosatetraenoic acid (i.e., 5-oxo-12-HETE and 8-trans-5-oxo-
12-HETE). They were also formed by transcellular biosynthesis
in coincubations of platelets with neutrophils. Neither
compound mobilized intracellular calcium in neutrophils, but
both inhibited 5-oxo-ETE-induced calcium mobilization with
IC50 values of 0.5 μM (5-oxo-12-HETE) and 2.5 μM (8-trans-5-
oxo-12-HETE). Although these metabolites would not be good
drug candidates because of their instability and susceptibility to
metabolism, these findings encouraged us to search for more
suitable OXE receptor antagonists.
Long-chain polyunsaturated fatty acids, including
4,7,10,13,16,19-docosahexanenoic acid, 5,8,11,14,17-eicosapen-
taenoic acid, 8,11,14-eicosatrienoic acid, and 11,14,17-eicosa-
trienoic acid, have been reported to antagonize the stimulatory
effects of 5-oxo-ETE on GTPγS binding to an OXE receptor-Gαi
fusion protein.14 The IC50 values for their inhibitory effects
ranged between 2 and 6 μM. Although these results are
interesting, these substances would not be of practical use as
OXE receptor antagonists because they have various other
effects, including, in some cases, activation of inflammatory
cells.28 Furthermore, ω3-polyunsaturated fatty acids can be
converted to anti-inflammatory resolvins and protectins that
have a variety of independent effects of their own.29
It was recently reported that the synthetic compound
Gue1654 selectively blocks 5-oxo-ETE-induced calcium mobi-
lization, shape change, and chemotaxis in neutrophils and
eosinophils.30 This compound was identified as a result of
screening a selective library using a calcium mobilization assay,
followed by virtual screening using a model of the OXE receptor.
Although Gue1654 blocked all of the above responses, it failed to
prevent the inhibitory effect of 5-oxo-ETE on adenylyl cyclase in
neutrophils, indicating that it is a biased ligand for this receptor.
In the present study, we found that Gue1654 is about one-tenth
Figure 10. Effects of purified enantiomers of 34 and 53 on 5-oxo-ETE-
induced calcium mobilization in neutrophils. Chiral HPLC of 34 on a
Lux Amylose-2 column resulted in two peaks (34a and 34b; A), which
were collected as separate fractions. Analysis of aliquots of the two
fractions under identical conditions is shown in panels (B) (34a) and
(C) (34b). Two fractions (53a and 53b) were also obtained after
chromatography of 53 on a Lux Cellulose-1 column (D). An aliquot
from the first peak was analyzed under identical conditions (E). The
second peak (53b) was further purified by a second chromatography to
remove a small amount of contaminating 53a, and an aliquot was then
analyzed using the same conditions (F). G: The materials in the peaks
shown in (B) (upward pointing solid blue triangles), (C) (upward
pointing open blue triangles), (E) (solid red circles), and (F) (open red
circles) were tested for their abilities to block 5-oxo-ETE-induced
calcium mobilization in neutrophils (n = 4) as described in the legend to
Figure 2.
Both 34 and 53 were resistant to metabolism by β-oxidation by
liver homogenates, whereas 34 was metabolized to a small extent
when incubated under conditions favoring metabolism by
cytochrome P450, in which case about 12% was converted to a
single more polar product. Because the UV spectrum of this
371
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
Figure 11. Summary of the structure−activity relationships leading to the OXE receptor antagonists 34 and 53.
Evaluation of Actin Polymerization in Neutrophils and
Eosinophils. F-Actin was measured in unfractionated leukocytes
prepared as described above. The leukocytes were first incubated for 30
min on ice with allophycocyanin-labeled anti-CD49d (BioLegend, San
Diego, CA). After washing with PBS, the cells were suspended in PBS
containing Ca2+ (1.8 mM) and Mg2+ (1 mM) at a concentration of 5.5 ×
106 cells/mL. Aliquots (100 μL) of the fluorescently labeled cells were
then preincubated for 5 min at 37 °C with vehicle (1 μL DMSO) or
different concentrations of 53, 34, or Gue1654, followed by addition of
either vehicle (10 μL of PBS containing Ca2+, Mg2+, and 0.1% BSA) or 5-
oxo-ETE (final concentration, 10 nM). After 20 s, the incubations were
terminated by addition of formaldehyde (37%) to give a final
concentration of 8.5% and kept on ice for 30 min. A mixture of
lysophosphatidylcholine (30 μg in 23.8 μL PBS) and N-(7-nitrobenz-2-
oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin; Molecular Probes; 49
pmol in 6.2 μL methanol; final concentration, 0.3 μM) was added to
each sample,33 followed by incubation overnight in the dark at 4 °C.
Immediately prior to data acquisition by flow cytometry (FACSCalibur,
BD Biosciences, San Jose, CA), 200 μL of PBS containing 1%
formaldehyde was added to each sample. Eosinophils were identified by
high side scatter and high expression of CD49d, whereas neutrophils
displayed high side scatter but low CD49d expression.
as potent as 53 and 34 in inhibiting 5-oxo-ETE-induced actin
polymerization in neutrophils and eosinophils.
CONCLUSIONS
■
5-Oxo-ETE is a potent chemoattractant for eosinophils, both in
vitro11 and in vivo,12 and can also enhance the survival of these
cells by stimulating the release of GM-CSF from monocytes.31
Because of these effects, 5-oxo-ETE may be an important
mediator in eosinophilic disorders such as asthma and allergic
rhinitis, and selective OXE receptor antagonists such as 53 and
34 may be useful therapeutic agents in these diseases. Both
compounds are highly potent, with IC50 values of about 30 nM in
inhibiting 5-oxo-ETE-induced calcium mobilization and actin
polymerization, and are relatively resistant to metabolism by rat
liver homogenates. They are both racemic mixtures, and their
potencies can be further enhanced by the use of the pure active
enantiomers, which have IC50 values below 10 nM. It will be very
interesting to determine whether these antagonists are effective
in disease models in animals possessing the OXE receptor.
Evaluation of Neutrophil Migration. Neutrophil migration was
measured using a modified Boyden chamber technique with 48-well
microchemotaxis chambers (Neuro Probe Inc., Cabin John, MD) and
Sartorius cellulose nitrate filters (8 μm pore size; 140 μm thickness;
Neuro Probe Inc.).10 Vehicle (PBS containing Ca2+, Mg2+, and 0.3%
BSA) or 5-oxo-ETE (100 ng) were added to the bottom wells, whereas
neutrophils (150000 cells in PBS containing Ca2+, Mg2+, and 0.4%
OVA) were added to each of the top wells. Both top and bottom wells
also contained either vehicle or different concentrations of 53 or 34. The
chambers were incubated for 2 h at 37 °C in 5% CO2 and humidified air.
The cells were then stained using hematoxylin (Canada Wide Scientific)
followed by chromotrope 2R (Sigma Chemical Co), and the numbers of
cells on the bottom surfaces were counted in five different fields at a
magnification of 400× for each well. All incubations were performed in
triplicate.
Metabolism of 34 and 53 by Rat Liver Homogenates. Liver
from male Sprague−Dawley rats was minced in ice-cold 0.25 M sucrose
(4 mL/g tissue) and homogenized using 8 strokes in a Potter−Elvehjem
homogenizer in an ice bath, followed by centrifugation at 600g for 10
min. Then 0.2 mL of phosphate buffer (pH 7.4, final concentration 50
mM) containing NADPH (final concentration 1 mM) was added to 0.8
mL of the supernatant (referred to as the homogenate), and the mixture
was incubated with either 34 or 53 (20 μM) for 60 min at 37 °C. The
incubations were terminated by the addition of ice-cold MeOH (0.67
mL) followed by cooling on ice. The products were analyzed by
precolumn extraction combined with reversed-phase-HPLC34 using a
modified Waters 2695 Alliance system (Waters Corp., Mississauga, ON)
with a photodiode array detector (Waters model 2996). The stationary
phase was a Nova-Pak C18 column (150 mm × 3.9 mm; Waters Corp).
The mobile phase was a gradient between 22% and 42% acetonitrile in
water with acetic acid (0.02%) and a flow rate of 1 mL/min.
EXPERIMENTAL SECTION
■
Preparation of Leukocytes. Whole blood from healthy subjects
was mixed with a solution of dextran 500 (from Leuconostoc; Sigma-
Aldrich), and the red cells were allowed to sediment under unit gravity at
4 °C for 45 min. The supernatant was centrifuged to give a pellet
containing unfractionated leukocytes. The cells in the pellet were
subjected to hypotonic lysis and then used to investigate the effects of
OXE receptor antagonists on 5-oxo-ETE-indued actin polymerization.
Alternatively, for experiments designed to measure intracellular calcium
mobilization and chemotaxis in neutrophils, the pellet was resuspended
in PBS and centrifuged over Ficoll,32 followed by hypotonic lysis of any
remaining red cells. In both cases, the cells were resuspended in PBS
(without calcium or magnesium).
Measurement of Intracellular Calcium Levels in Neutrophils.
Neutrophils were loaded with indo-1 acetoxymethyl ester (Invitrogen,
Carlsbad, CA) as described previously.10 The cells were then washed
and resuspended in PBS. Five minutes before commencing data
acquisition, the cells were placed in a cuvette and Ca2+ and Mg2+ were
added to give final concentrations of 1.8 and 1 mM, respectively. The
cuvette was then placed in a Deltascan 4000 spectrofluorometer
(Photon Technology International, Birmingham, NJ) in a cuvette
holder maintained at 37 °C and equipped with a magnetic stirrer.
Fluorescence was monitored at excitation and emission wavelengths of
331 and 410 nm, respectively, and the baseline allowed stabilization
before addition of either vehicle (10 μL of DMSO) or potential OXE
receptor antagonist. Two minutes later, 5-oxo-ETE (final concentration
10 nM in PBS containing 0.1% BSA) was added, followed 1 min later by
addition of digitonin (final concentration, 0.1%) to permit measurement
of maximal fluorescence. In some cases, a second agonist (LTB4, PAF,
fMLP, or IL-8) was added 2 min after addition of 5-oxo-ETE, followed 1
min later by digitonin. Calcium levels were calculated as previously
described.10
Purification of Enantiomers of 34 and 53. 34 was separated into
its two enantiomers 34a and 34b by chiral HPLC using the Waters 2695
Alliance system described above, except that the sample was injected
372
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
directly into a loop in a 6-port valve. The stationary phase was a Lux
Amylose-2 column (250 mm × 4.6 mm; 3 μm particle size;
Phenomenex, Torrance, CA). The mobile phase was hexane/
methanol/acetic acid (99:1:0.1) at a flow rate of 1.5 mL/min. The
two enantiomers of 53 were purified as described above except that the
stationary phase was a Lux Cellulose-1 column (250 mm × 4.6 mm; 5
μm particle size; Phenomenex, Torrance, CA), and the mobile phase
was hexane/methanol/acetic acid (98.5:1.5:0.1). Because the later
eluting peak (53b) was initially contaminated with about 4% of 53a, it
was further purified by a second step of chiral HPLC using identical
conditions.
mmol) in DMSO (0.2 mL) was added KOH (13 mg, 0.24 mmol) at rt.
The reaction mixture was then stirred for 30 min, followed by the
addition of 16 (34 mg, 0.24 mmol). The stirring was continued for 3 h.
The reaction mixture was quenched with saturated NH4Cl solution and
extracted with EtOAc and the organic layers combined and dried over
Na2SO4. The solvent was evaporated under reduced pressure, and the
crude residue was purified by silica gel chromatography using 80%
EtOAc/Hex as eluent to afford 17 as a white solid (13.7 mg, 67%). 1H
NMR (400 MHz, CDCl3): δ 7.71 (d, J = 8.1 Hz, 1H), 7.39 (dd, J = 9.3,
7.8 Hz, 1H), 7.18−7.09 (m, 2H), 6.32 (s, 1H), 2.94−2.87 (m, 2H), 2.57
(s, 2H), 2.52 (s, 2H), 1.63 (dt, J = 15.2, 7.4 Hz, 2H), 1.39−1.22 (m, 6H),
1.14 (d, J = 5.6 Hz, 6H), 0.87−0.79 (m, 3H). 13C NMR (CDCl3): δ
136.76, 135.85, 135.22, 129.38, 124.76, 123.51, 121.09, 120.23, 116.17,
111.43, 37.69, 29.55, 29.47, 29.30, 22.66, 20.06, 14.08.
Reagents and Methods. Platelet-activating factor (PAF; β-acetyl-
γ-O-hexadecyl-L-α-phosphatidylcholine), formyl-met-leu-phe (fMLP),
and Gue1654 (7-(methylthio)-2-[(2,2-diphenylacetyl)amino]benzo-
[1,2-d:4,3-d′]bisthiazole) were obtained from Sigma-Aldrich, whereas
interleukin-8 (IL-8) was purchased from R&D Systems. 5-Oxo-ETE35
Synthesis of 1,1-Bis(iodomethyl)cyclopropane (19). To a
stirred suspension of Ph3P (1.53 g, 5.87 mmol) in CH2Cl2 (30 mL)
was added imidazole (792 mg, 11.64 mmol) at 0 °C followed by I2 (1.49
g, 5.87 mmol) and the reaction mixture stirred at 0 °C for 30 min. A
solution of cyclopropane-1,1-diyldimethanol (18) (100 mg, 0.97 mmol)
in CH2Cl2 (5 mL) was added. The reaction mixture was allowed to
warm to rt and stirred for 2 h. The reaction was quenched with brine and
extracted with CH2Cl2. The combined organic layers were washed with
water and then 5% Na2S2O3 solution and then was dried over Na2SO4.
The solvent was evaporated under reduced pressure, and the crude was
purified using silica gel chromatography using 1% EtOAc/Hex as eluent
36
and LTB4 were prepared by chemical synthesis as previously
described. All reactions were carried out using dry solvents under
argon atmosphere. High-resolution mass spectra were recorded on an
AccuTOF mass spectrometer by positive ion ESI mode with DART as
an ion source. 1H NMR and 13C NMR spectra were recorded in CDCl3
using TMS as an internal standard on a BRUKER AMX 400 MHz
spectrometer at rt. All compounds were analyzed by TLC, HRMS, and
NMR. Prior to biological assay, the purity of all final compounds was
determined to be >95% by NMR and HPLC. HPLC conditions: Waters
2695 Alliance system with Waters Novapak C18 (150 mm × 3.9 mm)
column and photodiode array detector (Waters model 2996), gradient
mobile phase between H2O/MeCN/MeOH (56:22:22) to H2O/
MeCN/MeOH (16:42:42) over 30 min; both solvents contained 0.02%
acetic acid, and the flow rate was 1 mL/min.
1
to afford 19 (300 mg, 96%). H NMR (400 MHz, CDCl3): δ 3.35 (s,
4H), 1.03 (s, 4H).
Synthesis of 2,2′-(Cyclopropane-1,1-diyl)diacetonitrile (20).
To a round-bottom flask containing KCN (161.8 mg, 2.48 mmol) was
added a solution of 1,1-bis(iodomethyl)cyclopropane (19) (50 mg,
0.248 mmol) in DMF (2 mL). The reaction mixture was refluxed at 45
°C and stirred for 5 h. Saturated NH4Cl solution (5 mL) was added and
the organic layer was extracted with EtOAc, and the combined organic
layers were washed with brine and dried over Na2SO4. The solvents were
evaporated under reduced pressure, and the residue was purified by silica
gel chromatography using 30% EtOAc/Hex as eluent to afford 20 (26
mg, 87%). 1H NMR (400 MHz, CDCl3): δ 2.55 (s, 4H), 0.78 (s, 4H).
Synthesis of Dimethyl 2,2′-(Cyclopropane-1,1-diyl)diacetate
(21). To a round-bottom flask containing 20 (45 mg, 0.37 mmol) in
methanol (3 mL) was added concentrated sulfuric acid (1.3 mL)
dropwise at rt, and the reaction mixture was then refluxed at 60−65 °C
overnight. The reaction was quenched with cold water and extracted
with EtOAc; the combined organic layers were dried over Na2SO4 and
evaporated under reduced pressure to afford 21 as a colorless liquid (40
mg, 57%). 1H NMR (400 MHz, CDCl3): δ 3.67 (s, 6H), 2.40 (s, 4H),
0.54 (s, 4H).
Synthesis of 8, 9, 10, 11, 12, 13. These compounds were
synthesized from 1H-Indole-2-carboxylic acid (1) as described
previously.23
Synthesis of 5-(2-Hex-1-enyl-indol-1-yl)-5-oxo-pentanoic
Acid (14). To a stirred solution of “5, n = 3” (15 mg, 0.0753 mmol)
in DMSO (0.7 mL) was added KOH (17 mg, 0.301 mmol) at rt and
stirred for 1 h. Dihydro-2H-pyran-2,6(3H)-dione (7) (34 mg, 0.301
mmol) in DMSO (0.5 mL) was added to the reaction mixture and
stirred for 2 h. The mixture was quenched with saturated NH4Cl,
extracted in EtOAc, dried over Na2SO4, and purified using column
chromatography using 50% EtOAc/Hex as eluent to afford 14 (13 mg,
55%). HRMS (ESI) m/z calcd for [C19H23NO3 + H]+, 314.1751; found,
314.1763. 1H NMR (400 MHz, CDCl3): δ 8.29 (d, J = 8.6 Hz, 1H), 7.41
(dd, J = 8.3, 2.5 Hz, 2H), 7.29 (dd, J = 8.6, 1.6 Hz, 1H), 7.19 (s, 1H), 6.52
(d, J = 3.7 Hz, 1H), 6.38 (d, J = 15.8 Hz, 1H), 6.16 (dt, J = 15.7, 6.9 Hz,
1H), 3.08−2.59 (m, 3H), 2.43 (ddd, J = 47.5, 15.9, 6.5 Hz, 2H), 2.15 (d,
J = 6.6 Hz, 2H), 1.42−1.27 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3): δ 177.32, 169.95, 133.94, 130.75, 130.51, 129.76, 124.98,
123.31, 118.06, 116.57, 109.39, 41.84, 40.33, 32.74, 31.62, 27.07, 22.28,
20.00, 13.94.
Synthesis of (Z)-5-Oxo-5-(2-(undec-1-en-1-yl)-1H-indol-1-yl)-
pentanoic Acid (15). To a solution of “5, n = 8” (50 mg, 0.19 mmol) in
DMSO (1 mL) was added KOH (42 mg, 0.74 mmol) at rt. The reaction
mixture was stirred for 30 min, followed by the addition of 7 (85 mg,
0.74 mmol). After 3 h, the reaction was quenched by adding saturated
NH4Cl solution. The organic layer was extracted with EtOAc, and the
combined organic layers were washed with brine and dried over Na2SO4.
The solvents were evaporated under reduced pressure, and the crude
was purified by silica gel chromatography using 50% EtOAc/Hex as
eluent to afford 15 (37.4 mg, 52%). HRMS (ESI) m/z calcd for
[C24H33NO3 + H]+, 384.2533; found, 384.2485. 1H NMR (400 MHz,
CDCl3): δ 8.27 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.34−7.17
(m, 2H), 6.46 (d, J = 13.7 Hz, 1H), 5.93−5.82 (m, 1H), 3.03 (t, J = 7.0
Hz, 2H), 2.51 (t, J = 7.1 Hz, 2H), 2.34 (q, J = 7.0 Hz, 2H), 2.12 (p, J = 7.0
Hz, 2H), 1.25 (s, 14H), 0.86 (d, J = 6.8 Hz, 3H). 13C NMR (CDCl3): δ
177.04, 172.99, 136.76, 135.84, 135.25, 129.40, 124.77, 123.52, 121.10,
120.25, 116.19, 111.44, 37.73, 32.71, 31.89, 29.57, 29.49, 29.43, 29.32,
29.06, 22.68, 20.08, 14.11.
Synthesis of 2,2′-(Cyclopropane-1,1-diyl)diacetic acid (22).
To a stirred solution of 21 (40 mg, 0.21 mmol) in 2-propanol (2 mL)
was added LiOH·H2O (89 mg) in H2O (0.2 mL) dropwise and stirred at
rt for 6 h. The reaction was quenched with saturated NH4Cl solution,
the organic layer was extracted with EtOAc, the combined organic layers
were washed with brine and dried over Na2SO4, and the solvents were
1
evaporated under reduced pressure to afford 22 (26.5 mg, 78%). H
NMR (400 MHz, CDCl3): δ 2.39 (s, 4H), 0.64 (s, 4H). 13C NMR
(CDCl3): δ 178.81, 41.76, 14.25, 12.50.
Synthesis of 6-Oxaspiro[2.5]octane-5,7-dione (24). To a
round-bottom flask containing 22 (150 mg, 0.95 mmol) was added
acetic anhydride (23) (5 mL) and refluxed at 130 °C for 6 h. The
reaction mixture was gradually cooled to rt, and then excess acetic
anhydride was evaporated under vacuum to obtain a crude product
which was purified by silica gel chromatography using 50% EtOAc/Hex
as eluent to afford 24 (93 mg, 70%). 1H NMR (400 MHz, CDCl3): δ
2.63 (s, 4H), 0.64 (s, 4H). 13C NMR (CDCl3): δ 166.05, 39.07, 11.25,
10.63.
Synthesis of 2-(1-(2-(2-Hexyl-1H-indol-1-yl)-2-oxoethyl)-
cyclopropyl)acetic Acid (25). To a solution of “6 (n = 3)” (15 mg,
0.08 mmol) in DMSO was added KOH (22.3 mg, 0.40 mmol) at rt and
stirred for 30 min, followed by the addition of 24 (20 mg, 0.14 mmol).
The reaction mixture was stirred for 3 h. The reaction was quenched by
adding saturated NH4Cl solution, the organic layer was extracted with
Synthesis of 5-(2-Hexyl-1H-indol-1-yl)-3,3-dimethyl-5-oxo-
pentanoic Acid (17). To a solution of “6, n = 3” (12 mg, 0.06
373
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
EtOAc, and the combined organic layers were washed with brine and
dried over Na2SO4. The solvents were evaporated under reduced
pressure, and the crude was purified by silica gel chromatography (50%
EtOAc/Hex) to afford 25 (14.5 mg, 57%). HRMS (ESI) m/z calcd for
[C21H27NO3 + H]+, 342.2064; found, 342.2008. 1H NMR (400 MHz,
CDCl3): δ 7.70 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 7.0 Hz, 1H), 7.13 (ddd, J
= 11.7, 6.7, 4.7 Hz, 2H), 6.33 (s, 1H), 3.13 (s, 2H), 2.93 (t, J = 7.5 Hz,
2H), 2.56 (s, 2H), 1.69−1.55 (m, 2H), 1.29 (dd, J = 34.0, 4.6 Hz, 6H),
0.82 (dd, J = 6, 5.4 Hz, 3H), 0.67−0.50 (m, 4H). 13C NMR (CDCl3): δ
177.28, 171.48, 142.10, 134.94, 129.00, 122.32, 121.90, 119.19, 113.86,
107.22, 44.43, 40.05, 30.70, 29.72, 28.09, 27.84, 21.59, 13.31, 13.06,
11.53, 11.43.
Synthesis of 5-(2-Hexyl-1H-indol-1-yl)-3-methyl-5-oxopenta-
noic Acid (27). To a solution of “6, n = 3” (20 mg, 0.10 mmol) in THF
(1 mL) stirring at 0 °C was added EtMgBr (1 M in THF, 0.20 mL, 0.20
mmol) and 26 (26 mg, 0.20 mmol, dissolved in 1 mL THF). After
stirring at 0 °C for 15 min, the reaction mixture was warmed to rt and
stirred for 1.5 h. The reaction mixture was quenched with saturated
NH4Cl solution and extracted with EtOAc and the organic layers
combined and dried over Na2SO4. The solvent was evaporated under
reduced pressure, and the crude residue was purified by silica gel
chromatography to afford 27 (23 mg, 75%). HRMS (ESI) m/z calcd for
[C20H27NO3 + H]+, 330.2064; found, 330.1963. 1H NMR (400 MHz,
CDCl3): δ 7.70 (d, J = 7.9 Hz, 1H), 7.42−7.33 (m, 1H), 7.19−7.06 (m,
2H), 6.32 (s, 1H), 2.98 (m, J = 15.1, 6.6 Hz, 4H), 2.70 (dd, J = 13.3, 6.6
Hz, 1H), 2.42 (ddd, J = 22.8, 15.6, 6.7 Hz, 2H), 1.69−1.54 (m, 2H),
1.42−1.13 (m, 6H), 1.07 (d, J = 6.7 Hz, 3H), 0.81 (dd, J = 9.5, 4.4 Hz,
3H). 13C NMR (CDCl3): δ 178.16, 172.31, 143.02, 136.01, 130.03,
123.35, 122.89, 120.22, 114.72, 108.16, 45.18, 40.53, 31.71, 30.59, 29.12,
28.93, 27.26, 22.60, 20.04, 14.05.
Synthesis of 4-(2-Hexyl-indol-1-yl)-4-oxo-butyric Acid (29).
To a stirred solution of “6, n = 3” (39 mg, 0.194 mmol) in DMSO (0.4
mL) was added KOH (54 mg, 0.964 mmol) at rt and stirred for 1 h.
Dihydro-furan-2,5-dione (28) (58.0 mg, 0.582 mmol) in DMSO (0.3
mL) was added to the reaction mixture and stirred for 2 h. The mixture
was quenched with saturated NH4Cl solution, extracted in EtOAc, and
dried over Na2SO4. The solvents were evaporated, and the crude was
purified using column chromatography (50% EtOAC in hexane) to
afford 29 (5.5 mg, 10%). HRMS (ESI) m/z calcd for [C18H23NO3 +
H]+, 302.1751; found, 302.1691. 1H NMR (400 MHz, CDCl3): δ 7.78
(d, J = 7.7 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.22−7.10 (m, 2H), 6.35 (s,
1H), 3.28 (t, J = 6.4 Hz, 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.85 (t, J = 6.3 Hz,
2H), 1.70−1.55 (m, 2H), 1.30−1.11 (m, 6H), 0.83 (d, J = 6.2 Hz, 3H).
13C NMR (CDCl3): δ 176.99, 171.75, 143.19, 136.10, 130.16, 123.51,
Synthesis of 6-Chloro-2-hex-1-enyl-1H-indole (32, R = Cl). To a
suspension of (1-pentyl)triphenyl-phosphonium bromide (“4, n = 3”)
(30 g, 72.6 mmol) in THF (40 mL) was added LiHMDS (1.0 M in THF,
65 mL, 65 mmol) at −78 °C atmosphere. The mixture was stirred for 30
min and cooled back to −78 °C, and 6-chloro-1H-indole-2-
carbaldehyde (31) (5.8 g, 32.3 mmol) in THF (40 mL) was added
dropwise. The reaction mixture was allowed to warm to rt and stirred for
3 h. Saturated NH4Cl solution was added, and the organic layer was
extracted with EtOAc. The combined organic layers were washed with
brine and dried over Na2SO4, and the solvents were evaporated under
reduced pressure. The crude was purified by silica gel chromatography
(15% EtOAc/hexane) to afford 32 as a pale-yellow solid (6.4 g, 85.0%).
HRMS (ESI) m/z calcd for [C14H16ClN + H]+, 234.1050; found,
234.1050. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.42 (d, J = 8.4
Hz, 1H), 7.37−7.26 (m, 2H), 7.02 (d, J = 8.4 Hz, 1H), 6.42−6.31 (m,
2H), 6.11−6.00 (m, 1H), 2.24 (q, J = 7.1 Hz, 2H), 1.42 (ddd, J = 31.6,
14.9, 7.4 Hz, 4H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 137.31,
136.76, 130.92, 127.78, 127.56, 121.10, 120.63, 120.41, 110.34, 101.26,
32.67, 31.37, 22.24, 13.94.
Synthesis of 6-Chloro-2-hexyl-1H-indole (33, R = Cl). To a stirred
solution of “32, R = Cl” (1.9 g, 8.13 mmol) in EtOH (20 mL) was added
10% Pd/C (114 mg) under H2 atm. The reaction mixture was stirred at
rt for 6 h and then filtered through Celite/florisil. The residue was
washed with EtOAc, and the combined filtrate was concentrated under
reduced pressure. The crude was purified by flash column chromatog-
raphy using 10% EtOAc/Hex as an eluent to afford 33 as a brown solid
(1.7 g, 89%). HRMS (ESI) m/z calcd for [C14H18ClN + H]+, 236.1206;
found, 236.1224. 1H NMR (400 MHz, CDCl3): δ 7.84 (s, 1H), 7.41 (d, J
= 8.4 Hz, 1H), 7.26 (s, 2H), 7.03 (d, J = 7.9 Hz, 1H), 6.20 (s, 1H), 2.73
(t, J = 7.4 Hz, 2H), 2.63 (s, 1H), 1.70 (dd, J = 13.9, 6.8 Hz, 2H), 1.35 (d, J
= 26.4 Hz, 6H), 0.89 (s, 3H).
Synthesis of 5-(6-Chloro-2-hexyl-indol-1-yl)-3-methyl-5-oxo-pen-
tanoic Acid (34). To a stirred solution of “33, R = Cl” (1.2 g, 5.09 mmol)
in DMSO (10 mL) was added KOH (1.21 g, 21.4 mmol) at rt. The
reaction mixture was stirred for 30 min, followed by addition of 3-
methylglutaric anhydride (26) (2.6 g, 21.4 mmol). After 3 h, the reaction
was quenched with saturated NH4Cl solution and extracted with EtOAc,
and the organic layers were combined, washed with brine, and dried over
Na2SO4. The solvents were evaporated under reduced pressure, and the
crude was purified by silica gel chromatography using 20% EtOAc/Hex
as eluent to afford 34 as a light-brown solid (1.285 g, 69.5%). HRMS
(ESI) m/z calcd for [C20H26ClNO3 + H]+, 364.1679; found, 316.1674.
1H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H),
7.18 (d, J = 7.2 Hz, 1H), 6.36 (s, 1H), 3.10 (dd, J = 15.6, 5.1 Hz, 1H),
3.00−2.86 (m, 3H), 2.77 (dd, J = 13.1, 6.7 Hz, 1H), 2.57 (dd, J = 14.6,
4.7 Hz, 1H), 2.41 (dd, J = 15.6, 7.2 Hz, 1H), 1.68 (d, J = 6.1 Hz, 2H),
1.47−1.37 (m, 2H), 1.37−1.28 (m, 4H), 1.15 (d, J = 6.4 Hz, 3H), 0.89
(s, 3H). 13C NMR (CDCl3): δ 177.60, 172.23, 143.14, 136.58, 129.32,
128.28, 123.40, 120.60, 115.31, 107.84, 44.97, 40.47, 31.69, 30.60, 29.18,
28.96, 28.75, 27.27, 22.60, 20.09, 14.07.
123.09, 120.26, 114.90, 108.49, 34.01, 31.69, 30.71, 29.12, 28.96, 28.75,
22.59, 14.04.
Synthesis of 34, 35, 36, 41, 42, 47. The title compounds were
synthesized from the commercial corresponding 1H-indole-2-carboxylic
acid as described below for 34.
Synthesis of 6-Chloro-1H-indole-2-carbaldehyde (31, R = Cl). To a
stirred solution of “30, R = Cl” (20.2 g, 103 mmol) in THF (20 mL) was
added LiAlH4 (7.8 g, 205.5 mmol) slowly at 0 °C. Once the addition was
complete, the reaction mixture was allowed to warm to rt and stirred for
14 h. Saturated NH4Cl solution was added, and the organic layer was
extracted with EtOAc. The combined organic layers were washed with
brine and dried over Na2SO4. The solvents were evaporated under
reduced pressure to get the crude alcohol (17.5 g), which was used
without any further purification. To a stirred solution of the crude
alcohol (17.5 g) in acetonitrile (20 mL) was added activated MnO2
(33.47 g, 391 mmol) and stirred at rt for 18 h. The reaction mixture was
diluted with EtOAc and filtered through Celite and florisil layers. The
solvents were evaporated under reduced pressure, and the crude was
purified using flash column chromatography (10% EtOAc/Hex) to
afford “31” as a brown solid (12 g, 69%). HRMS (ESI) m/z calcd for
5-(6-Bromo-2-hexyl-1H-indol-1-yl)-3-methyl-5-oxo-pentanoic
Acid (35). Yield: 41%. HRMS (ESI) m/z calcd for [C20H26BrNO3 + H]+,
1
408.1169; found, 408.1078. H NMR (400 MHz, CDCl3): δ 8.01 (s,
1H), 7.26 (s, 1H), 7.20 (s, 1H), 6.31 (s, 1H), 3.13−2.80 (m, 4H), 2.70
(dq, J = 13.0, 6.5 Hz, 1H), 2.46 (ddd, J = 63.5, 15.6, 6.4 Hz, 2H), 1.68−
1.57 (m, 2H), 1.42−1.22 (m, 6H), 1.10 (d, J = 6.7 Hz, 3H), 0.84 (t, J =
7.0 Hz, 3H). 13C NMR (CDCl3): δ 172.15, 143.02, 136.99, 128.62,
126.17, 121.06, 118.09, 117.05, 107.93, 44.93, 31.66, 30.57, 29.10, 28.87,
27.26, 22.58, 20.06, 14.05.
5-(2-Hexyl-6-methoxy-1H-indol-1-yl)-3-methyl-5-oxo-pentanoic
Acid (36). Yield: 72%. HRMS (ESI) m/z calcd for [C21H29NO4 + H]+,
360.2169; found, 360.2152. 1H NMR (400 MHz, CDCl3): δ 7.49 (d, J =
1.6 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 6.85 (dd, J = 8.5, 2.1 Hz, 1H), 6.32
(s, 1H), 3.87 (d, J = 7.4 Hz, 3H), 3.17−2.90 (m, 4H), 2.75 (tt, J = 13.7,
6.9 Hz, 1H), 2.65−2.33 (m, 2H), 1.68 (dt, J = 15.1, 7.5 Hz, 2H), 1.36
(ddd, J = 10.9, 10.5, 5.5 Hz, 6H), 1.05 (t, J = 6.0 Hz, 3H), 0.89 (t, J = 7.0
Hz, 3H). 13C NMR (CDCl3): δ 157.11, 141.19, 137.19, 123.77, 120.30,
110.80, 108.09, 101.06, 77.34, 77.02, 76.70, 55.88, 44.96, 31.72, 30.71,
29.12, 29.03, 27.28, 22.61, 20.08, 14.08.
1
[C9H6ClNO + H]+, 180.0216; found, 180.0215. H NMR (400 MHz,
CDCl3): δ 9.77 (1H, s), 9.11 (1H, s), 7.58−7.60 (1H, d), 7.39 (1H, s),
7.18−7.19 (1H, m), 7.07−7.10 (1H, dd). 13C NMR (CDCl3): δ 181.96
(s), 124.41 (s), 122.48 (s), 121.43 (s), 120.66 (s), 114.68 (s), 112.23 (s),
110.88 (s), 100.13 (s).
374
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
5-(5-Chloro-2-hexyl-1H-indol-1-yl)-3-methyl-5-oxo-pentanoic
Acid (41). Yield: 26%. HRMS (ESI) m/z calcd for [C20H26ClNO3 + H]+,
364.1674; found, 364.1672. 1H NMR (400 MHz, CDCl3): δ 7.69 (d, J =
8.9 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.11 (dd, J = 8.9, 2.1 Hz, 1H), 6.28
(s, 1H), 3.16−2.80 (m, 4H), 2.69 (dq, J = 13.4, 6.7 Hz, 1H), 2.55−2.27
(m, 2H), 1.62 (dt, J = 15.2, 7.5 Hz, 2H), 1.43−1.13 (m, 6H), 1.08 (d, J =
6.7 Hz, 3H), 0.83 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3): δ 176.43,
171.06, 143.14, 133.50, 130.18, 127.53, 122.38, 118.67, 114.81, 106.50,
43.96, 39.21, 30.64, 29.60, 28.08, 27.82, 26.19, 21.57, 19.04, 13.03.
5-(5-Bromo-2-hexyl-1H-indol-1-yl)-3-methyl-5-oxo-pentanoic
Acid (42). Yield: 66%. HRMS (ESI) m/z calcd for [C20H26BrNO3 + H]+,
+ H]+, 364.1674; found, 364.1628. 1H NMR (400 MHz, CDCl3): δ 7.93
(s, 1H), 7.29 (d, J = 3.9 Hz, 1H), 7.23 (dd, J = 8.7, 1.0 Hz, 1H), 3.74 (s,
3H), 3.25−3.15 (m, 2H), 3.09 (t, J = 6.9 Hz, 2H), 2.65−2.52 (m, 2H),
2.44 (dt, J = 18.1, 7.3 Hz, 1H), 2.16 (p, J = 7.1 Hz, 2H), 1.73−1.57 (m,
2H), 1.53−1.31 (m, 6H), 0.92 (t, J = 6.7 Hz, 3H). 13C NMR (CDCl3): δ
195.26, 178.70, 151.00, 135.13, 127.95, 126.98, 122.23, 120.46, 112.83,
110.60, 41.63, 33.31, 31.59, 29.63, 29.52, 29.08, 26.39, 22.61, 19.05,
14.06.
Synthesis of 52, 53, 54, and 56. These compounds were
synthesized as described for 53.
Synthesis of 5-(5-Chloro-2-hexyl-1-methyl-1H-indol-3-yl)-3-meth-
yl-5-oxo-pentanoic Acid (53, R = Cl). To a stirred solution of “49, R =
Cl” (85 mg, 0.34 mmol) in CH2Cl2 (5 mL) was added Me2AlCl (1 M in
hexane, 1.37 mL, 1.37 mmol) at rt. After 30 min, 3-methylglutaric
anhydride (26) (175 mg, 1.37 mmol) was added and the reaction
mixture stirred at rt for 3 h. The reaction was quenched by adding
saturated NH4Cl solution. The organic layer was extracted with CH2Cl2,
and the combined organic layers were washed with brine and dried over
Na2SO4. The solvents were evaporated under reduced pressure, and the
crude was purified using silica gel chromatography (30% Et2O/hexane)
to afford 53 (106.7 mg, 82.6%). HRMS (ESI) m/z calcd for
[C21H28ClNO3 + H]+, 378.1836; found, 378.1835. 1H NMR (400
MHz, CDCl3) δ 7.90 (s, 1H), 7.26−7.19 (m, 2H), 3.72 (s, 3H), 3.22−
3.13 (m, 2H), 3.07−2.92 (m, 2H), 2.79−2.67 (m, 1H), 2.56 (dd, J =
15.1, 5.3 Hz, 1H), 2.37 (dd, J = 15.0, 7.2 Hz, 1H), 1.68−1.56 (m, 2H),
1.47 (d, J = 4.9 Hz, 2H), 1.38−1.28 (m, 4H), 1.15 (d, J = 6.6 Hz, 3H),
0.89 (s, 3H). 13C NMR (CDCl3): δ 195.40, 176.62, 151.24, 135.17,
128.05, 126.97, 122.33, 120.44, 113.06, 110.66, 48.99, 40.89, 31.59,
29.68, 29.50, 29.09, 26.57, 26.40, 22.60, 20.51, 14.06.
5-(2-Hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxo-pentanoic
Acid (52). Yield: 95%. HRMS (ESI) m/z calcd for [C21H29NO3 + H]+,
344.2220; found, 378.1702. 1H NMR (400 MHz, CDCl3): δ 7.91−7.85
(m, 1H), 7.36−7.30 (m, 1H), 7.26 (s, 2H), 3.71 (s, 3H), 3.24−3.13 (m,
2H), 3.04 (d, J = 6.8 Hz, 2H), 2.83 (dd, J = 16.9, 3.2 Hz, 1H), 2.72 (dq, J
= 13.0, 6.4 Hz, 1H), 2.55 (dd, J = 14.9, 5.2 Hz, 1H), 2.34 (ddd, J = 31.0,
17.2, 8.0 Hz, 2H), 1.68−1.54 (m, 2H), 1.51−1.27 (m, 6H), 1.12 (t, J =
5.1 Hz, 3H), 0.87 (t, J = 6.0 Hz, 3H). 13C NMR (CDCl3): δ 195.87,
177.31, 166.22, 150.44, 136.78, 125.91, 122.10, 120.70, 113.30, 109.80,
49.14, 41.12, 37.73, 31.63, 29.50, 29.47, 29.11, 26.68, 26.27, 24.00, 22.61,
20.49, 20.00, 14.06.
5-(5-Bromo-2-hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxo-
pentanoic Acid (54). Yield: 54%. HRMS (ESI) m/z calcd for
[C21H28BrNO3 + H]+, 422.1325; found, 422.1157. 1H NMR (400
MHz, CDCl3): δ 7.64 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.25
(dd, J = 8.9, 2.0 Hz, 1H), 6.27 (s, 1H), 3.10−2.81 (m, 4H), 2.69 (dq, J =
13.3, 6.6 Hz, 1H), 2.43 (ddd, J = 60.4, 15.7, 6.6 Hz, 2H), 1.62 (dt, J =
15.2, 7.6 Hz, 2H), 1.42−1.22 (m, 6H), 1.20 (d, J = 9.7 Hz, 3H), 1.08 (d, J
= 6.7 Hz, 3H), 0.82 (dd, J = 8.6, 5.2 Hz, 3H). 13C NMR (CDCl3): δ
177.15, 172.12, 144.02, 134.89, 131.70, 126.10, 122.76, 116.23, 107.39,
45.00, 40.18, 31.67, 30.59, 29.71, 29.10, 28.84, 27.21, 22.60, 20.07, 14.07.
5-(6-Chloro-2-hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxo-
pentanoic Acid (56). Yield: 68%. HRMS (ESI) m/z calcd for
[C21H28ClNO3 + H]+, 378.1830; found, 378.1702. 1H NMR (400
MHz, CDCl3): δ 7.82 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 1.8 Hz, 1H), 7.20
(dd, J = 8.6, 1.8 Hz, 1H), 3.68 (s, 3H), 3.17 (dd, J = 19.2, 11.4 Hz, 2H),
2.99 (ddd, J = 40.5, 16.0, 6.9 Hz, 2H), 2.73 (dq, J = 13.5, 6.7 Hz, 1H),
2.59−2.28 (m, 2H), 1.67−1.54 (m, 2H), 1.53−1.28 (m, 6H), 1.13 (t, J =
6.3 Hz, 3H), 0.89 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3): δ 195.32,
178.31, 150.60, 137.24, 127.99, 124.44, 122.43, 121.65, 113.43, 109.84,
49.16, 41.00, 31.59, 29.59, 29.48, 29.13, 26.46, 26.26, 22.60, 20.35, 14.06.
1
408.1169; found, 408.1150. H NMR (400 MHz, CDCl3): δ 8.01 (s,
1H), 7.26 (m, 2H), 6.31 (s, 1H), 3.13−2.81 (m, 4H), 2.71 (dt, J = 13.1,
6.6 Hz, 1H), 2.46 (ddd, J = 63.5, 15.6, 6.4 Hz, 2H), 1.70−1.55 (m, 2H),
1.46−1.22 (m, 6H), 1.10 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.0 Hz, 3H). 13C
NMR (CDCl3): δ 172.15, 143.02, 136.99, 128.62, 126.17, 121.06,
118.09, 117.05, 107.93, 44.93, 31.66, 30.57, 29.10, 28.87, 27.26, 22.58,
20.06, 14.05.
5-(2-Hexyl-3-methyl-1H-indol-1-yl)-3-methyl-5-oxo-pentanoic
Acid (47). Yield: 67%. HRMS (ESI) m/z calcd for [C21H29NO3 + H]+,
344.2220; found, 344.2191. 1H NMR (400 MHz, CDCl3): δ 7.77−7.71
(m, 1H), 7.48−7.42 (m, 1H), 7.28−7.20 (m, 2H), 3.20−2.94 (m, 4H),
2.79 (dq, J = 13.4, 6.7 Hz, 1H), 2.50 (ddd, J = 22.8, 15.6, 6.7 Hz, 2H),
2.20 (s, 3H), 1.63−1.52 (m, 2H), 1.43−1.23 (m, 6H), 1.15 (d, J = 6.7
Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (CDCl3): δ 177.66, 171.89,
138.24, 135.13, 131.46, 123.48, 122.63, 118.51, 115.31, 114.51, 45.24,
40.46, 31.69, 29.94, 29.20, 27.28, 27.16, 22.65, 20.07, 14.08, 8.69.
Synthesis of 5-(2-Hexyl-1-methyl-1H-indol-3-yl)-5-oxo-pen-
tanoic Acid (48). To a stirred solution of “6, n = 3” (19 mg, 0.09 mmol)
in CH2Cl2 (1 mL) was added Me2AlCl (1 M in hexane, 0.189 mL, 0.189
mmol) at rt. After 30 min, the anhydride (7) (21.5 mg, 0.189 mmol) was
added and the reaction mixture stirred at rt for 3 h. The reaction was
quenched by adding saturated NH4Cl solution and extracted with
CH2Cl2. The combined organic layers were washed with brine and dried
over Na2SO4. The solvents were evaporated under reduced pressure,
and the crude was purified using silica gel chromatography (80% Et2O/
hexane) to afford 48 (25.3 mg, 92%). HRMS (ESI) m/z calcd for
[C19H25NO3 + H]+, 316.1907; found, 316.1898. 1H NMR (400 MHz,
CDCl3): δ 7.97−7.86 (m, 1H), 7.41−7.31 (m, 1H), 7.31−7.20 (m, 2H),
3.74 (s, 3H), 3.17 (dt, J = 13.9, 7.4 Hz, 4H), 2.62−2.48 (m, 2H), 2.15 (p,
J = 7.0 Hz, 2H), 1.70−1.54 (m, 2H), 1.36 (dd, J = 31.8, 28.6 Hz, 6H),
0.89 (t, J = 6.5 Hz, 3H). 13C NMR (CDCl3): δ 159.79, 178.11, 150.26,
136.74, 125.91, 122.09, 122.00, 120.76, 113.01, 109.76, 41.79, 33.39,
31.64, 29.70, 29.54, 29.43, 29.10, 26.28, 22.63, 19.16, 14.08.
Synthesis of 5-Chloro-2-hexyl-1-methyl-1H-indole (49, R =
Cl). To a stirred solution of 5-chloro-2-hexyl-1H-indole (33, R = Cl) (87
mg, 0.368 mmol) in DMSO (3 mL) was added KOH (103 mg, 1.84
mmol) at rt. After 30 min, CH3I (0.12 mL, 1.84 mmol) was added
dropwise and the reaction mixture stirred at rt for 3 h. Saturated NH4Cl
solution was added and the reaction mixture extracted with EtOAc. The
organic layers were combined, washed with brine, and dried over
Na2SO4. The solvents were evaporated under reduced pressure, and the
crude was purified using silica gel chromatography (10% EtOAc/
hexane) to afford 49 (85.4 mg, 92.9%). HRMS (ESI) m/z calcd for
1
[C15H20ClN + H]+, 250.1363; found, 250.1362. H NMR (400 MHz,
CDCl3) δ 7.47 (s, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H),
6.18 (s, 1H), 3.63 (s, 3H), 2.7 (t, J = 7.52 Hz, 2H), 1.74−1.67 (m, 2H),
1.34−1.33 (m, 6H), 0.90 (t, J = 6.76 Hz, 3H). 13C NMR (CDCl3): δ
142.98, 135.74, 128.88, 124.83, 120.57, 119.06, 109.6, 98.36, 31.65,
29.57, 29.11, 28.46, 26.88, 22.60, 14.87.
Synthesis of 5-(2-Hexyl-1-methyl-1H-indol-3-yl)-5-oxo-pen-
AUTHOR INFORMATION
Corresponding Author
*Phone: 514-398-3864 ext 094071. Fax: 514-398-7483. E-mail:
William.Powell@McGill.ca.
■
tanoic Acid (50). Yield: 92%. HRMS (ESI) m/z calcd for [C20H27NO3
1
+ H]+, 330.2064; found, 330.1929. H NMR (400 MHz, CDCl3): δ
7.85−7.83 (t, 1H), 7.29 −7.27 (t, 1H), 7.22−7.18 (m, 2 H), 3.68 (s,
3H), 3.14 (t, 2H), 3.05 (t, 2H), 2.49 (t, 2H), 2.16−2.02 (m, 2H), 1.62−
1.50 (m, 2H), 1.44−1.35 (m, 2H), 1.32−1.16 (m, 4H), 0.79 (t, 3H). 13C
NMR (CDCl3): δ 195.8, 178.1, 150.3, 136.7, 125.9, 122.1, 121.9, 120.8,
113.0, 41.8, 33.4, 31.6, 29.5, 29.4, 29.1, 26.3, 22.6, 19.2, 14.1.
Present Addresses
§For V.G.: Navinta LLC, 1499 Lower Ferry Road, Ewing, New
Jersey 08618, United States; phone, 609-883-1135; E-mail, vivek.
gore@navinta.com.
5-(5-Chloro-2-hexyl-1-methyl-1H-indol-3-yl)-5-oxo-penta-
noic Acid (51). Yield: 81%. HRMS (ESI) m/z calcd for [C20H26ClNO3
375
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
∥For P.P.: Navinta LLC, 1499 Lower Ferry Road, Ewing, New
Jersey 08618, United States; phone, 609-883-1135; E-mail,
pranav.patel@navinta.com.
(6) Graham, F. D.; Erlemann, K. R.; Gravel, S.; Rokach, J.; Powell, W. S.
Oxidative stress-induced changes in pyridine nucleotides and chemo-
attractant 5-lipoxygenase products in aging neutrophils. Free Radical
Biol. Med. 2009, 47, 62−71.
Notes
(7) Nakamura, M.; Shimizu, T. Leukotriene receptors. Chem. Rev.
2011, 111, 6231−6298.
The authors declare the following competing financial
interest(s): WSP and JR have applied for US and European
patents covering the OXE receptor antagonists described in this
manuscript.
(8) Norgauer, J.; Barbisch, M.; Czech, W.; Pareigis, J.; Schwenk, U.;
Schroder, J. M. Chemotactic 5-oxo-icosatetraenoic acids activate a
̈
unique pattern of neutrophil responsesanalysis of phospholipid
metabolism, intracellular Ca2+ transients, actin reorganization, super-
oxide-anion production and receptor up-regulation. Eur. J. Biochem.
1996, 236, 1003−1009.
ACKNOWLEDGMENTS
■
This work was supported by the Canadian Institutes of Health
Research (grants MOP-6254 and PPP-99490), the American
Asthma Foundation (grant 12-0049), the National Heart, Lung,
and Blood Institute (grant R01HL081873), and the Quebec
Heart and Stroke Foundation. The Meakins-Christie Laborato-
ries-MUHC-RI are supported in part by a Center grant from Le
(9) O’Flaherty, J. T.; Taylor, J. S.; Kuroki, M. The coupling of 5-oxo-
eicosanoid receptors to heterotrimeric G proteins. J. Immunol. 2000,
164, 3345−3352.
(10) Powell, W. S.; MacLeod, R. J.; Gravel, S.; Gravelle, F.; Bhakar, A.
Metabolism and biologic effects of 5-oxoeicosanoids on human
neutrophils. J. Immunol. 1996, 156, 336−342.
(11) Powell, W. S.; Chung, D.; Gravel, S. 5-Oxo-6,8,11,14-
eicosatetraenoic acid is a potent stimulator of human eosinophil
migration. J. Immunol. 1995, 154, 4123−4132.
́ ́
Fond de la Recherche en Sante du Quebec as well as by the J. T.
Costello Memorial Research Fund. J.R. also acknowledges the
National Science Foundation for the AMX-360 (grant CHE-90-
13145) and Bruker 400 MHz (grant CHE-03-42251) NMR
instruments. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Heart, Lung, and Blood Institute or the National
Institutes of Health.
(12) Muro, S.; Hamid, Q.; Olivenstein, R.; Taha, R.; Rokach, J.; Powell,
W. S. 5-Oxo-6,8,11,14-eicosatetraenoic acid induces the infiltration of
granulocytes into human skin. J. Allergy Clin. Immunol. 2003, 112, 768−
774.
(13) Brink, C.; Dahlen, S. E.; Drazen, J.; Evans, J. F.; Hay, D. W.;
Rovati, G. E.; Serhan, C. N.; Shimizu, T.; Yokomizo, T. International
Union of Pharmacology XLIV. Nomenclature for the oxoeicosanoid
receptor. Pharmacol. Rev. 2004, 56, 149−157.
(14) Hosoi, T.; Koguchi, Y.; Sugikawa, E.; Chikada, A.; Ogawa, K.;
Tsuda, N.; Suto, N.; Tsunoda, S.; Taniguchi, T.; Ohnuki, T.
Identification of a novel eicosanoid receptor coupled to Gi/o. J. Biol.
Chem. 2002, 277, 31459−31465.
(15) Jones, C. E.; Holden, S.; Tenaillon, L.; Bhatia, U.; Seuwen, K.;
Tranter, P.; Turner, J.; Kettle, R.; Bouhelal, R.; Charlton, S.; Nirmala, N.
R.; Jarai, G.; Finan, P. Expression and characterization of a 5-oxo-
6E,8Z,11Z,14Z-eicosatetraenoic acid receptor highly expressed on
human eosinophils and neutrophils. Mol. Pharmacol. 2003, 63, 471−
477.
(16) Takeda, S.; Yamamoto, A.; Haga, T. Identification of a G protein-
coupled receptor for 5-oxo-eicosatetraenoic acid. Biomed. Res. Tokyo
2002, 23, 101−108.
(17) Iikura, M.; Suzukawa, M.; Yamaguchi, M.; Sekiya, T.; Komiya, A.;
Yoshimura-Uchiyama, C.; Nagase, H.; Matsushima, K.; Yamamoto, K.;
Hirai, K. 5-Lipoxygenase products regulate basophil functions: 5-oxo-
ETE elicits migration, and leukotriene B(4) induces degranulation. J.
Allergy Clin. Immunol. 2005, 116, 578−585.
(18) Sturm, G. J.; Schuligoi, R.; Sturm, E. M.; Royer, J. F.; Lang-
Loidolt, D.; Stammberger, H.; Amann, R.; Peskar, B. A.; Heinemann, A.
5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent chemoattractant for
human basophils. J. Allergy Clin. Immunol. 2005, 116, 1014−1019.
(19) Sarveswaran, S.; Ghosh, J. OXER1, a G protein-coupled
oxoeicosatetraenoid receptor, mediates the survival-promoting effects
of arachidonate 5-lipoxygenase in prostate cancer cells. Cancer Lett.
2013, 336, 185−195.
(20) Grant, G. E.; Rubino, S.; Gravel, S.; Wang, X.; Patel, P.; Rokach, J.;
Powell, W. S. Enhanced formation of 5-oxo-6,8,11,14-eicosatetraenoic
acid by cancer cells in response to oxidative stress, docosahexaenoic acid
and neutrophil-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid.
Carcinogenesis 2011, 32, 822−828.
(21) Powell, W. S.; Rokach, J. Biochemistry, biology and chemistry of
the 5-lipoxygenase product 5-oxo-ETE. Prog. Lipid Res. 2005, 44, 154−
183.
(22) O’Flaherty, J. T.; Cordes, J. F.; Lee, S. L.; Samuel, M.; Thomas, M.
J. Chemical and biological characterization of oxo-eicosatetraenoic acids.
Biochim. Biophys. Acta 1994, 1201, 505−515.
(23) Patel, P.; Cossette, C.; Anumolu, J. R.; Gravel, S.; Lesimple, A.;
Mamer, O. A.; Rokach, J.; Powell, W. S. Structural requirements for
activation of the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-
ABBREVIATIONS USED
■
5-HEDH, 5-hydroxyeicosanoid dehydrogenase; 5-HETE, 5S-
hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5-HpETE, 5S-
hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5-LO, 5-lip-
oxygenase; 5-oxo-12-HETE, 5-oxo-12S-hydroxy-
6E,8Z,10E,14Z-eicosatetraenoic acid; 5-oxo-ETE, 5-oxo-
6E,8Z,11Z,14Z-eicosatetraenoic acid; CDCl3, deuterated chloro-
form; DMF, dimethylformamide; DMSO, dimethylsulfoxide;
EtOAc, ethyl acetate; Et2O, diethyl ether; EtOH, ethanol; fMLP,
formyl-met-leu-phe; GM-CSF, granulocyte/macrophage colony
stimulating factor; Hex, hexanes; I2, iodine; IL-8, interleukin 8;
iPrOH, 2-isopropanol; KCN, potassium cyanide; KOH,
potassium hydroxide; LiAlH4, lithium aluminum hydride;
LiHMDS, lithium hexamethyldisilazide; LiOH, lithium hydrox-
ide; LT, leukotriene; MeCN, acetonitrile; MeOH, methanol;
MnO2, manganese dioxide; Na2SO4, sodium sulfate; NaI, sodium
iodide; NH4Cl, ammonium chloride; PAF, platelet-activating
factor; PPh3, triphenylphosphine; THF, tetrahydrofuran; TMS,
tetramethylsilane
REFERENCES
■
(1) Haeggstrom, J. Z.; Funk, C. D. Lipoxygenase and leukotriene
pathways: biochemistry, biology, and roles in disease. Chem. Rev. 2011,
111, 5866−5898.
(2) Grant, G. E.; Rokach, J.; Powell, W. S. 5-Oxo-ETE and the OXE
receptor. Prostaglandins Other Lipid Mediators 2009, 89, 98−104.
(3) Powell, W. S.; Gravelle, F.; Gravel, S. Metabolism of 5(S)-hydroxy-
6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a
specific dehydrogenase in human polymorphonuclear leukocytes. J. Biol.
Chem. 1992, 267, 19233−19241.
(4) Powell, W. S.; Gravelle, F.; Gravel, S. Phorbol myristate acetate
stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by
human neutrophils by activating NADPH oxidase. J. Biol. Chem. 1994,
269, 25373−25380.
(5) Erlemann, K. R.; Rokach, J.; Powell, W. S. Oxidative stress
stimulates the synthesis of the eosinophil chemoattractant 5-oxo-
6,8,11,14-eicosatetraenoic acid by inflammatory cells. J. Biol. Chem.
2004, 279, 40376−40384.
376
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377

Journal of Medicinal Chemistry
Article
ETE) receptor: identification of a mead acid metabolite with potent
agonist activity. J. Pharmacol. Exp. Ther. 2008, 325, 698−707.
(24) Powell, W. S.; Gravel, S.; Khanapure, S. P.; Rokach, J. Biological
inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets.
Blood 1999, 93, 1086−1096.
(25) Blattermann, S.; Peters, L.; Ottersbach, P. A.; Bock, A.; Konya, V.;
̈
Weaver, C. D.; Gonzalez, A.; Schroder, R.; Tyagi, R.; Luschnig, P.; Gab,
J.; Hennen, S.; Ulven, T.; Pardo, L.; Mohr, K.; Gutschow, M.;
Heinemann, A.; Kostenis, E. A biased ligand for OXE-R uncouples Ga
and Gbg signaling within a heterotrimer. Nature Chem. Biol. 2012, 8,
631−638.
(26) Gore, V.; Patel, P.; Chang, C. T.; Sivendran, S.; Kang, N.;
Ouedraogo, Y. P.; Gravel, S.; Powell, W. S.; Rokach, J. 5-Oxo-ETE
Receptor Antagonists. J. Med. Chem. 2013, 56, 3725−3732.
(27) Lawson, J. A.; Kim, S.; Powell, W. S.; FitzGerald, G. A.; Rokach, J.
Oxidized derivatives of w-3 fatty acids: identification of iPF3a-VI in
human urine. J. Lipid Res. 2006, 47, 2515−2524.
(28) Huang, Z. H.; Hii, C. S.; Rathjen, D. A.; Poulos, A.; Murray, A. W.;
Ferrante, A. N-6 and N-3 polyunsaturated fatty acids stimulate
translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and
enhance agonist-induced NADPH oxidase in macrophages. Biochem. J.
1997, 325 (Pt2), 553−557.
(29) Serhan, C. N.; Petasis, N. A. Resolvins and protectins in
inflammation resolution. Chem. Rev. 2011, 111, 5922−5943.
(30) Blattermann, S.; Peters, L.; Ottersbach, P. A.; Bock, A.; Konya, V.;
Weaver, C. D.; Gonzalez, A.; Schroder, R.; Tyagi, R.; Luschnig, P.; Gab,
J.; Hennen, S.; Ulven, T.; Pardo, L.; Mohr, K.; Gutschow, M.;
Heinemann, A.; Kostenis, E. A biased ligand for OXE-R uncouples Ga
and Gbg signaling within a heterotrimer. Nature Chem. Biol. 2012, 8,
631−638.
(31) Stamatiou, P. B.; Chan, C. C.; Monneret, G.; Ethier, D.; Rokach,
J.; Powell, W. S. 5-Oxo-6,8,11,14-eicosatetraenoic acid stimulates the
release of the eosinophil survival factor granulocyte-macrophage colony
stimulating factor from monocytes. J. Biol. Chem. 2004, 279, 28159−
28164.
(32) Boyum, A. Isolation of mononuclear cells and granulocytes from
̈
human blood. Isolation of mononuclear cells by one centrifugation, and
of granulocytes by combining centrifugation and sedimentation at 1 g.
Scand. J. Clin. Lab. Invest. 1968, 21 (Suppl. 97), 77−89.
(33) Howard, T. H.; Oresajo, C. O. The kinetics of chemotactic
peptide-induced change in F-actin content, F-actin distribution, and the
shape of neutrophils. J. Cell Biol. 1985, 101, 1078−1085.
(34) Powell, W. S. Precolumn extraction and reversed-phase high-
pressure liquid chromatography of prostaglandins and leukotrienes.
Anal. Biochem. 1987, 164, 117−131.
(35) Khanapure, S. P.; Shi, X. X.; Powell, W. S.; Rokach, J. Total
synthesis of a potent proinflammatory 5-oxo-ETE and its 6,7-dihydro
biotransformation product. J. Org. Chem. 1998, 63, 337−342.
(36) Zamboni, R.; Rokach, J. Simple Efficient Synthesis of LTB4 and
12-epi-LTB4. Tetrahedron Lett. 1982, 23, 2631−2634.
377
dx.doi.org/10.1021/jm401292m | J. Med. Chem. 2014, 57, 364−377