Two potent OXE-R antagonists: Assignment of stereochemistry

Article abstract of DOI:10.1021/ml500161v

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed by the oxidation of 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), which is a major metabolite of enzymatic oxidation of arachidonic acid (AA). 5-Oxo-ETE is the most potent lipid chemoattractant for human eosinophils. Its actions are mediated by the selective OXE receptor, which is therefore an attractive target in eosinophilic diseases such as allergic rhinitis and asthma. Recently, we have reported two excellent OXE receptor antagonists that have IC₅₀ values at low nanomolar concentrations. Each of these antagonists has a chiral center, and the isolation of the individual enantiomers by chiral high-performance liquid chromatography (HPLC) revealed that in each case one enantiomer is over 300 times more potent than the other. To unambiguously assign the stereochemistry of these enantiomers and to provide access to larger amounts of the active compounds for biological testing, we report here their total synthesis.

Full text of DOI:10.1021/ml500161v

Letter
pubs.acs.org/acsmedchemlett
Two Potent OXE‑R Antagonists: Assignment of Stereochemistry
Pranav Patel,†,§ Chintam Nagendra Reddy,† Vivek Gore,†,§ Shishir Chourey,† Qiuji Ye,†
Yannick P. Ouedraogo,†,∥ Sylvie Gravel,‡ William S. Powell,‡ and Joshua Rokach*,†
†Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 West University Boulevard, Melbourne,
Florida 32901, United States
‡Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec H2X 2P2,
Canada
S
* Supporting Information
ABSTRACT: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)
is formed by the oxidation of 5-hydroxy-6E,8Z,11Z,14Z-
eicosatetraenoic acid (5-HETE), which is a major metabolite
of enzymatic oxidation of arachidonic acid (AA). 5-Oxo-ETE is
the most potent lipid chemoattractant for human eosinophils.
Its actions are mediated by the selective OXE receptor, which is
therefore an attractive target in eosinophilic diseases such as
allergic rhinitis and asthma. Recently, we have reported two
excellent OXE receptor antagonists that have IC50 values at low
nanomolar concentrations. Each of these antagonists has a
chiral center, and the isolation of the individual enantiomers by
chiral high-performance liquid chromatography (HPLC)
revealed that in each case one enantiomer is over 300 times more potent than the other. To unambiguously assign the
stereochemistry of these enantiomers and to provide access to larger amounts of the active compounds for biological testing, we
report here their total synthesis.
KEYWORDS: 5-Oxo-ETE, eosinophil, OXE receptor, antagonist, enantiomeric, optical purity, chiral auxiliary
sthma is a complex disease, which is essentially characterized
by a bronchoconstriction. In most cases it is composed of an
After the first total synthesis of 5-oxo-ETE,6 we have identified
and investigated the activities of its metabolites on the 5-oxo-
ETE receptor (OXE-R).7 We found that a metabolite of 5-oxo-
ETE formed by platelets, 5-oxo-12(S)-HETE, inhibits 5-oxo-
ETE-induced Ca2+ mobilization, though not increasing intra-
cellular Ca2+ concentration by itself.8 However, this compound
is not suitable for the development of an OXE-R antagonist as it
is not stable under physiological conditions. Studies on a variety
of 5-oxo-ETE analogues clearly showed that the carboxyl,
carbonyl (including the 5-oxo group), and the alkyl regions of the
molecule are essential for biological activity.9 We therefore
sought to develop synthetic OXE-R antagonists by incorporating
both 5-oxo-valeryl and hexyl groups onto an indole backbone.
The selection of the indole system looked more promising.
We synthesized other aromatic scaffolds such as naphthalene,
benzofuran, and quinoline, which turned out to be inactive.
These studies culminated in the identification of two potent OXE-R
antagonists (Figure 3) with IC50 values of less than 30 nM.10,11
Bothof these compounds (5 and 6) have an asymmetric center on
their acyl side chain, and it seemed likely that their activities would
be principally due to a single highly potent enantiomer. To test
this hypothesis we separated the two enantiomers of each of the
A
early phase (or acute) and a late-phase (or inflammatory) asthma.
One of the hallmarks of the disease is the accumulation of
eosinophils in the early phase, which is then responsible for
the late-phase. 5-Oxo-ETE is a potent eosinophil chemotactic
factor1,2 and responsible for the accumulation of eosinophils in
the lungs3 (Figure 1).
Figure 1. Implication of 5-oxo-ETE in asthma and other allergic
diseases.
It is formed from arachidonic acid (AA) by the 5-lipoxygenase
(5-LO) pathway, following oxidation of 5-hydroxy-
6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) by 5-hydrox-
yeicosanoid dehydrogenase in the presence of NADP+ (Figure 2).4,5
Received: April 24, 2014
Accepted: May 29, 2014
Published: May 29, 2014
© 2014 American Chemical Society
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Figure 2. Enzymatic oxygenation of eicosanoids.
We have also prepared 9 by the pig liver esterase (PLE)-catalyzed
hydrolysis of 8 using a modified literature method (Scheme 1).12
Precursor 8 was prepared by the hydrolysis of 3-methyl glutaric
anhydride (7) with HCl and MeOH under reflux conditions
in high yield (98%). For the PLE-catalyzed hydrolysis of 8,
we obtained optical purity of the product 9 comparable to the
commercial compound. The optical purity of 9 was determined
by chiral HPLC of its diastereomer 11. The phenyl derivative was
made in order to use UV-HPLC detection.
Figure 3. Structures of two racemic OXE-R antagonists.11
Scheme 2 shows the synthesis of the R isomers of 5 and 6 from
9. First, 9 was reacted with (COCl)2 to get the acyl chloride 12.
Friedel−Crafts acylation of 13 with 12 using Me2AlCl followed
by the basic hydrolysis of the ester 15 afforded the R isomer of 6,
6a. The R isomer of 5, 5a, was prepared by N-acylation of 14
using t-BuOK as a base to give the ester 16, which was hydrolyzed
with HCl under reflux conditions to give 5a. The intermediates
13 and 14 were synthesized from indole carboxylic acid as we
reported previously.10
When we compared the retention time of 5a to that of the
active enantiomer of 5 that we previously resolved by chiral
HPLC11 (cf. Figure 4B; data not shown), it was apparent that the
R enantiomer was the one without appreciable antagonist
activity. This was confirmed by measuring its ability to block
5-oxo-ETE-induced calcium mobilization in neutrophils, which
revealed that it was substantially less potent than the racemic
racemic antagonists by chiral high-performance liquid chroma-
tography (HPLC) and compared their potencies in a calcium
mobilization assay.11 In both cases, nearly all of the antagonist
activity resided in a single enantiomer (IC50, 6 to 7 nM), with the
inactive enantiomer being over 300 times less potent. Because
these indole-based OXE-R antagonists could be useful thera-
peutic agents in the treatment of asthma and other eosinophilic
disorders, it is important to identify the active enantiomers and to
develop procedures that will permit the synthesis of sufficient
quantities for in vivo biological testing. In this letter, we report the
stereospecific synthesis of the four enantiomers (Schemes 2 and 5)
and the assignment of stereochemistry to the R and S compounds
5a, 5b, 6a, and 6b.
Results. Synthesis of R Isomers 5a and 6a. We decided
to synthesize the R component first as the required precursor 9
was commercially available (enantiomeric ratio ≥90:10 (R/S)).
a
Scheme 1. PLE Catalyzed Enzymatic Hydrolysis of 8
a
Reagents and conditions: (a) conc. HCl, conc. H2SO4, MeOH, reflux, 12 h, 98%; (b) pig liver esterase, KH2PO4 buffer, −78−0 °C, 20 h, 89.6%;
(c) DCC, DMAP, CH2Cl2, rt, 12 h, 65%.
a
Scheme 2. Synthesis of the R-Isomers 5a and 6a
a
Reagents and conditions: (a) (COCl)2, cat. DMF, CH2Cl2, rt, 3 h, 90%; (b) Me2AlCl, CH2Cl2, rt, 1 h, 89.5%; (c) LiOH, i-PrOH/H2O (4:1), rt,
24 h, 89%; (d) t-BuOK, CH2Cl2, 0 °C to rt, 7 h, 60%; (e) 6 N HCl, THF, reflux, 7 h, 60%.
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that intermediate 18 could be prepared and purified by column
chromatography and used to react with the chiral auxiliaries 10,
21, 24, and 27 saving considerable time and effort (Scheme 3).
Although the resulting diastereomeric mixtures could be
separated by analytical chiral HPLC, none of them could be
separated by either TLC or silica gel column chromatography.
Diastereomeric separation of the menthol derivatives of the
other lead compound 5 was also attempted, but the correspond-
ing diastereomers could not be separated. We also attempted to
separate the diastereomers of the intermediate 30 by the same
approach as used for 5 and 6 (Scheme 4), but unfortunately,
these pairs of diastereomers could not be resolved by TLC or
silica gel column chromatography. However, the diastereomeric
pairs derived from 30 could be resolved by chiral HPLC, which
proved useful as a measure of optical purity in subsequent studies
utilizing this intermediate.
Synthesis of S Compounds 5b and 6b. We decided to
perform the total synthesis of the S enantiomers by inverting
the stereocenter using the (R) synthon 9 as starting material
(Scheme 5). This approach was possible because the synthon is
pro-symmetrical. It also relies on high quantitative and qualitative
steps to ensure chiral purity. For example, step b in Scheme 5
is very selective considering that it is a basic hydrolysis, which
could also have partially removed the silyl moiety (Scheme 6).
However, if that were the case the potential byproducts (9 and 42)
could be easily removed by column chromatography. The result
would be a lower yield but no dilution of the enantiomeric purity.
In our case, the basic hydrolysis of 37 with 5 equiv of LiOH in
i-PrOH/H2O (4:1) at rt afforded the desired product 38 in 60%
yield. A smaller amount of the byproduct 42 (∼10%) was also
generated, but the other potential byproduct 9 was not observed.
Chiral HPLC Analysis of the Enantiomers of 5 and 6 (5a, 5b,
6a, and 6b). All four enantiomers 5a, 5b, 6a, and 6b as well as
the two racemic mixtures 5 and 6 were analyzed by chiral HPLC.
The S and R enantiomers of racemic 5 were nearly completely
separated (Figure 4A) with the two peaks having identical areas.
Analysis of 5b (Figure 4B) revealed that the S enantiomer
Figure 4. Chiral HPLC of OXE-R antagonists. Racemic 5 (A), the
synthetic S-enantiomer 5b (B), racemic 6 (C), and the synthetic
S-enantiomer 6b (D) were analyzed by chiral HPLC as described in the
experimental section (Supporting Information).
compound. We therefore decided to develop methods for the
total synthesis of the S isomers in both series. Unfortunately,
in this case the required enantiomer of 9 was not commercially
available. There are previously reported syntheses of the
enantiomer of 9, which rely on the catalytic desymmetrization
of 3-methyl glutaric anhydride.13−19 We elected to proceed with
the methods described herein.
Chiral Resolution of 5, 6, and 30. Although we could readily
separate the R and S enantiomers of 5 and 6 by analytical scale
chiral HPLC, it was not practical to do this on a preparative scale
because of the limited capacity and high cost of these columns.
We therefore attempted to resolve the racemic compounds
following derivatization with chiral auxiliaries, which we hoped
would permit large scale separation of the resulting diastereo-
meric pair on silica columns. We originally prepared the
diastereomeric derivatives of 6 individually but then realized
a
Scheme 3. Attempted Chiral Resolution of Racemic 6
a
Reagents and conditions: (a) 17, DMAP, CH2Cl2, rt, 12 h, 80%; (b) DMAP, CH2Cl2, rt, 12 h, 65−70%.
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a
Scheme 4. Attempted Chiral Resolution of Racemic 30
a
Reagents and conditions: (a) MeOH, reflux, 12 h, 90%; (b) 17, DMAP, CH2Cl2, rt, 12 h, 80%; (c) DMAP, CH2Cl2, rt, 12 h, 70%.
a
Scheme 5. Synthesis of S Antagonists 5b and 6b
a
Reagents and conditions: (a) 17, DMAP, CH2Cl2, rt, 10 h, 90%; (b) LiOH, i-PrOH/H2O (4:1), rt, 7 h, 60%; (c) (COCl)2, cat. DMF, CH2Cl2, rt,
3 h, 89%; (d) 14, t-BuOK, CH2Cl2, 0 °C to rt, 6 h, 60%; (e) TiCl4, CH2Cl2, 0 °C to rt, 0.5 h, 80%; (f) 13, Me2AlCl, CH2Cl2, 0 °C to rt,1 h, 25%;
(g) LiOH, i-PrOH/H2O (4:1), rt, 24 h, 85%.
a
Scheme 6. Possible Products of Basic Hydrolysis of 37
AUTHOR INFORMATION
■
Corresponding Author
*(J.R.) Tel: 321-674-8578. Fax: 321-674-7743. E-mail: jrokach@
fit.edu.
a
Reagents and conditions: (a) LiOH, i-PrOH/H2O (4:1), rt, 7 h, 60%.
Present Addresses
§(P.P. and V.G.) Navinta LLC, 1499 Lower Ferry Road, Ewing,
New Jersey 08618, United States. E-mail: pranav.patel@navinta.
com(P.P.) and vivek.gore@navinta.com (V.G.). Phone: 609-
883-1135.
predominated by a ratio of 93:7 (S/R). The main peak (tR,
14.4 min) had a retention time similar to that of the active
component (IC50, 7 nM) of the racemate that we previously
reported,11 whereas the minor peak (tR, 16.1 min) corresponded
to the enantiomer with low activity (IC50, 2.2 μM). Separation
of the S and R enantiomers of 6 by chiral HPLC is shown in
Figure 4C. The earlier eluting S enantiomer (tR, 25 min) cor-
responded to the active component (IC50, 6 nM) of the racemate that
we had previously reported,11 whereas the later eluting R enantiomer
(tR, 26.2 min) corresponded to the weakly active component
(IC50, 2.7 μM). Chiral HPLC of the synthetic S enantiomer 6b
indicated that the ratio of S/R enantiomers in this preparation is 91:9
(Figure 4D). Similar results were obtained with the synthetic R
enantiomers 5a and 6a. The peak assigments of all of the synthetic
enantiomers were confirmed by coinjections with the racemates 5
and 6. The slight variation observed in the enantioselectivity is the
result of variation in the preparation of the R enantiomers.
∥(Y.P.O.) Intel Corp., 2501 NW 229th Avenue, Hillsboro,
Oregon 97124, United States. E-mail: yannick.p.ouedraogo@
intel.com. Phone: 503−840−7685.
Funding
This work was supported by the American Asthma Foundation
(to J.R.; Award Number 12-0049) and the Canadian Institutes of
Health Research (to W.S.P.; MOP-6254 and PPP-99490). The
Meakins-Christie LaboratoriesMUHC-RI are supported in
́
part by a Center grant from Le Fond de la Recherche en Sante du
́
Quebec as well as by the J. T. Costello Memorial Research Fund.
J.R. wishes to acknowledge the National Science Foundation for
the Bruker 400 MHz (Grant Number CHE-03-42251) NMR
instrument.
Conclusions. We have performed the synthesis of the
R and S enantiomers of two low nanomolar OXE-R antagonists.
The availability of these compounds has made it possible to
unambiguously assign the S configuration to the active
enantiomers of each of these antagonists.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
5-HETE, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5-
oxo-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; AA,
arachidonic acid; DMF, dimethylformamide; DCC, N,N′-
dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine;
TMS, tetramethylsilane; HPLC, high-performance liquid
chromatography
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of in vitro assay, HPLC conditions, and synthetic procedures
and analytical data for compounds reported. This material is
available free of charge via the Internet at http://pubs.acs.org.
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