Novel Highly Potent and Metabolically Resistant Oxoeicosanoid (OXE) Receptor Antagonists That Block the Actions of the Granulocyte Chemoattractant 5-Oxo-6,8,11,14-Eicosatetraenoic Acid (5-oxo-ETE)

Article abstract of DOI:10.1021/acs.jmedchem.8b00154

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent lipid mediator that induces tissue eosinophilia via the selective OXE receptor (OXE-R), which is an attractive therapeutic target in eosinophilic diseases. We previously identified indole OXE-R antagonists that block 5-oxo-ETE-induced primate eosinophil activation. Although these compounds possess good oral absorption, their plasma levels decline rapidly due to extensive oxidation of their hexyl side chain. We have now succeeded in dramatically increasing antagonist potency and resistance to metabolism by replacing the hexyl group with phenylpentyl or phenylhexyl side chains. Compared with our previous lead compound S-230, our most potent antagonist, S-C025, has an IC₅₀ (120 pM) over 80 times lower and a substantially longer plasma half-life. A single major metabolite, which retains antagonist activity (IC₅₀, 690 pM) and has a prolonged lifetime in plasma was observed. These new highly potent OXE-R antagonists may provide a novel strategy for the treatment of eosinophilic disorders like asthma.

Full text of DOI:10.1021/acs.jmedchem.8b00154

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Article
Novel highly potent and metabolically resistant oxoeicosanoid
(OXE) receptor antagonists that block the actions of the granulocyte
chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)
Shishir Chourey, Qiuji Ye, Chintam Nagendra Reddy, Rui Wang, Chantal Cossette, Sylvie
Gravel, Irina Slobodchikova, Dajana Vuckovic, Joshua Rokach, and William S. Powell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00154 • Publication Date (Web): 04 Jul 2018
Downloaded from http://pubs.acs.org on July 4, 2018
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Novel highly potent and metabolically resistant oxoeicosanoid (OXE) receptor antagonists
that block the actions of the granulocyte chemoattractant 5ꢀoxoꢀ6,8,11,14ꢀeicosatetraenoic
acid (5ꢀoxoꢀETE)
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a
a
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a
b
Shishir Chourey , Qiuji Ye , Chintam Nagendra Reddy , Rui Wang , Chantal Cossette ,
b
c
c
a
Sylvie Gravel , Irina Slobodchikova , Dajana Vuckovic , Joshua Rokach , and William S.
b*
Powell
a
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150
West University Boulevard, Melbourne, FL 32901ꢀ6982, USA
b
MeakinsꢀChristie Laboratories, Centre for Translational Biology, McGill University Health
Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada
c
Department of Chemistry and Biochemistry and PERFORM Centre, Concordia University,
7141 Sherbrooke St. W., Montréal, QC H4B 1R6, Canada
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ABSTRACT
ꢀOxoꢀ6,8,11,14ꢀeicosatetraenoic acid (5ꢀoxoꢀETE) is a potent lipid mediator that induces tissue
5
eosinophilia via the selective OXE receptor (OXEꢀR), which is an attractive therapeutic target in
eosinophilic diseases. We previously identified indole OXEꢀR antagonists that block 5ꢀoxoꢀETEꢀ
induced primate eosinophil activation. Although these compounds possess good oral absorption,
their plasma levels decline rapidly due to extensive oxidation of their hexyl side chain. We have
now succeeded in dramatically increasing antagonist potency and resistance to metabolism by
replacing the hexyl group with phenylpentyl or phenylhexyl side chains. Compared with our
previous lead compound S-230, our most potent antagonist, SꢀC025, has an IC (120 pM) over
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80 times lower and a substantially longer plasma halfꢀlife. A single major metabolite, which
retains antagonist activity (IC , 690 pM) and has a prolonged lifetime in plasma was observed.
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These new highly potent OXEꢀR antagonists may provide a novel strategy for the treatment of
eosinophilic disorders like asthma.
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INTRODUCTION
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ꢀOxoꢀETE (5ꢀoxoꢀ6,8,11,14ꢀeicosatetraenoic acid) is a metabolite of arachidonic acid formed
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along with leukotrienes by the 5ꢀlipoxygenase pathway. It is produced by the oxidation of the 5ꢀ
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lipoxygenase product 5S-HETE (5Sꢀhydroxyꢀ6,8,11,14ꢀeicosatetraenoic acid) by the selective
+
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enzyme 5ꢀhydroxyeicosanoid dehydrogenase in the presence of NADP (Fig. 1). 5ꢀOxoꢀETE is
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5, 6
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a potent chemoattractant for human eosinophils, neutrophils, basophils, and monocytes. Its
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ꢀ10
actions are mediated by the OXE receptor (OXEꢀR), which is most highly expressed on
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eosinophils. This receptor is highly selective for 5ꢀoxoꢀETE, with a variety of closely related
2
compounds, including various 5ꢀoxoꢀETE metabolites, having only weak effects. The response
to activation of OXEꢀR is mediated by the G protein subfamily, primarily through the βγ
i
11
subunits.
Figure 1. Inhibition of 5ꢀoxoꢀETEꢀinduced eosinophil
activation by the OXEꢀR antagonists 230 and 264.
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ꢀOxoꢀETE induces a variety of responses in eosinophils, including calcium mobilization, actin
2, 12
polymerization, increased surface expression of CD11b and CD69, and Lꢀselectin shedding.
It also elicits the respiratory burst and promotes degranulation, especially in the presence of
1
3, 14
cytokines such as GMꢀCSF.
Among lipid mediators, 5ꢀoxoꢀETE is the most powerful
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chemoattractant for human eosinophils. It is a potent inducer of the transendothelial migration
of these cells, due to both its chemoattractant properties and its ability to activate protease
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pathways, resulting in the degradation of matrix components, thereby facilitating the passage of
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eosinophils.
Intradermal injection of 5ꢀoxoꢀETE in humans results in infiltration of
eosinophils into the skin, a response that is much more intense in asthmatic subjects compared to
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healthy controls. Because of its potent effects on eosinophils, 5ꢀoxoꢀETE may be an important
proinflammatory mediator in eosinophilic diseases such as asthma and allergic rhinitis. The OXE
receptor is therefore an attractive target for novel therapies to treat these diseases.
The first OXE receptor antagonist to be reported was 5ꢀoxoꢀ12SꢀHETE, a metabolite formed by
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the action of platelet 12ꢀlipoxygenase on 5ꢀoxoꢀETE. This substance inhibited 5ꢀoxoꢀETEꢀ
induced calcium mobilization in human neutrophils with an IC of about 500 nM, but was not
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suitable for clinical development because of its instability and susceptibly to metabolism. More
recently, the benzobisthiazole derivative Gue1654 was identified from a library screen as an
1
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OXEꢀR antagonist. We identified stable indoleꢀbased antagonists bearing substituents
resembling the regions of 5ꢀoxoꢀETE that are required for agonist activity. The most potent of
these are 230 and 264, which are chloroindoles bearing adjacent 3ꢀmethylꢀ5ꢀoxovalerate and
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hexyl groups on the pyrrole ring of the indole (Fig. 1). They are about 10 times more potent
1
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than Gue1654 as OXEꢀR antagonists. Both contain a single chiral carbon, with the S-
19, 20
enantiomers (IC ~10 nM) being several hundred times more potent than the R-enantiomers.
5
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We recently investigated the pharmacokinetics and metabolism of 230 and 264 in cynomolgus
monkeys. 264 rapidly appears in the blood following oral administration of a dose of 30 mg/kg,
reaching concentrations of over 25 µM within 1 h and then rapidly declining to below 500 nM by
2
1
8
h. The major metabolites identified in plasma were ω2ꢀhydroxyꢀ264 and ω2ꢀoxoꢀ264 (Fig. 1).
A somewhat more favorable PK profile was obtained with the same dose of (racemic) 230, with
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plasma concentrations of over 100 µM being achieved within 30 min to 1 h, which declined
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fairly rapidly to about 2.7 µM by 8 h. ω2ꢀOxidation is also the major metabolic pathway
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detected for 230, but in this case, substantial amounts of αꢀhydroxy products were also formed.
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The S enantiomer of 230 (S-230) appears to be metabolized more rapidly than the Rꢀenantiomer,
with plasma levels of about 36 µM 1 h after oral administration of an identical dose, dropping to
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.6 µM by 8 h.
In view of the rapid decline in the plasma concentrations of 230 and 264 following oral
administration, due at least in part to ω2ꢀoxidation of the hexyl side chain common to both
antagonists, we sought to reduce the rate of metabolism and increase potency by modifying their
alkyl side chain. The resulting compounds were tested for antagonist activity in a calcium
mobilization assay and the plasma levels of the most potent of these were measured in monkeys,
as this is the species we have chosen for further preclinical studies, given the lack of an OXEꢀR
ortholog in rodents. We found that replacement of the hexyl group of 230 and 264 with a
phenylalkyl group dramatically enhances antagonist potency, reduces metabolism and improves
the pharmacokinetic profiles of the resulting antagonists.
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RESULTS
Effect of a terminal trifluoromethyl group on 5ꢀoxoꢀETEꢀinduced calcium mobilization
We initially attempted to block metabolism of our OXE antagonist by replacing the hexyl group
of 230 with a trifluoromethylpentyl group (7), which we hoped would be resistant to ωꢀ
oxidation. This compound was synthesized as shown in Scheme 1. A Wittig reaction between the
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Wittig salt 2 and the aldehyde 3 using LiHMDS as a base yielded a mixture of cis and trans
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olefins (cis/trans:70/30), which were hydrogenated using 10% Pd/C to afford 5. A Friedelꢀ
Craft’s acylation of 5 using Me AlCl as a Lewis acid and 3ꢀmethylglutaric anhydride (6) as an
2
acylating agent yielded compound 7.
Scheme 1. Synthesis of 7. Reagents and
conditions: (a) PPh , CH CN, reflux, 16 h, 95%;
3
3
o
(
b) LiHMDS, THF, ꢀ78 C – rt, 2 h, 44%; (c)
10% Pd/C, EtOH, H , rt, 3 h, 99%; (d) 6,
2
Me AlCl, CH Cl , rt, 45 min, 58%.
2
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2
Figure 2. Effect of the ωꢀtrifluoro analog of 230 (7) (ꢀ) on 5ꢀoxoꢀ
ETEꢀinduced calcium mobilization in neutrophils. The response to 230
(
○) is shown for comparison.
We examined the antagonist potency of the trifluoro compound (7) by determining its ability to
block 5ꢀoxoꢀETEꢀinduced calcium mobilization in human neutrophils, which is our standard
screening assay for OXE receptor antagonists. Unfortunately, this compound is less potent than
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30, with an IC of 38 ± 11 nM compared to 17 ± 3 nM for racemic 230 (Fig. 2). For this
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reason, and because we subsequently identified much more potent antagonists (see below), we
did not conduct any further studies with the
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trifluoro compound.
Scheme 2. Synthesis of Nꢀacyl series
antagonists (12 aꢀd). Reagents and conditions:
o
o
(
a) KHMDS/LiHMDS, THF, ꢀ78 C – 0 C, 3 h,
8
0ꢀ99%; (b) 10%Pd/C, EtOH, H , rt, 3 h, 90 –
2
t
95%; (c) 6, BuOK, THF, 0 °C – rt, 3 h, 65 –
7
5%.
Synthesis of phenylalkyl analogs of 230 and 264
Our next strategy to block ωꢀoxidation was to replace the hexyl group in 230 and 264 by a
phenyl group separated from the indole structure by a spacer containing different numbers of
methylene groups. To this end, we synthesized a series of racemic compounds (12aꢀd)
containing a 3ꢀmethylꢀ5ꢀoxovaleric acid substituent in the 1ꢀposition of 6ꢀchloroindole and in the
2ꢀposition, a phenyl group separated from the indole by 3, 4, 5, or 6 methylene groups as shown
in Scheme 2. The number of methylene units between the indole and the phenyl ring were varied
1
9
by altering the length of the Wittig salts (compounds 9aꢀd). The aldehyde 8 was reacted with
the Wittig salts 9aꢀd using KHMDS or LiHMDS as a base to yield the olefins 10aꢀd, which were
subsequently hydrogenated using 10% Pd/C to afford 11aꢀd. Finally, acylation of the indole ring
t
at the 1ꢀpostion in 11aꢀd with 3ꢀmethylglutaric anhydride (6) using BuOK as a base yielded a
series of Nꢀacyl antagonists (compounds 12aꢀd).
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Figure 3. Effects of phenylalkyl indole OXEꢀR antagonists on 5ꢀoxoꢀETEꢀinduced calcium mobilization
in human neutrophils. The effects on the response to 5ꢀoxoꢀETE (10 nM) of replacing the hexyl groups of
A: 264 (∆) and B: 230 (∆) with a phenyl group connected to the 2ꢀposition of the indole by a
polymethylene spacer containing 3 (■), 4 (∇), 5 (▲), or 6 (ꢀ) carbons were investigated. All compounds
shown in A and B are racemic mixtures. C: Separation of the S and R enantiomers of C025
(phenylhexamethylene analog of 230) by chiral HPLC on a celluloseꢀ1 column with
o
hexane/MeOH/HOAc (97.9:2: 0.1) as the mobile phase (flow rate 2 ml/min; temperature 30 C). D:
Effects of S-C025 (ꢀ) and R-C025 (○), purified from racemic C025, on 5ꢀoxoꢀETEꢀinduced calcium
mobilization in human neutrophils. The effects of S-C025 (∆), prepared by chiral synthesis (see below)
are show for comparison. E: Structures of the S and R enantiomers of C025.
Potencies of phenylalkyl OXE receptor antagonists
The antagonist potencies of each of the above Nꢀacylindoles (12aꢀd) were determined by
assessing their effects on 5ꢀoxoꢀETEꢀinduced calcium mobilization (Fig. 3A). Insertion of a 3ꢀ
carbon spacer (12b) between the phenyl group and the indole reduced potency by over 10ꢀfold
compared to racemic 264 (Table 1), whereas 4ꢀcarbon (12c) or 6ꢀcarbon (12d) spacers resulted
in antagonists that had potencies similar to that of 264. In contrast, insertion of a pentamethylene
spacer (C149) between the phenyl group and the indole moiety increased potency by about 8ꢀ
fold (IC , 2.1 nM).
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Table 1. Potencies of OXE receptor antagonists. The IC values (± SE with the number of independent
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experiments shown in brackets) of 1ꢀacylindoles related to 264 are shown on the left, whereas those for 3ꢀ
acyl indoles related to 230 are shown on the right. All compounds are racemic unless otherwise indicated.
ꢀ
ꢀꢀ 1ꢀacyl series ꢀꢀꢀ
ꢀꢀꢀ 3ꢀacyl series ꢀꢀꢀ
Cpd #
64
C149 (12a)
2b
2c
2d
n
ꢀ
IC (nM)
Cpd #
230
n
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IC (nM)
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17 ± 2 (11)
26 ± 4 (13)
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2.1 ± 0.3 (6)
210 ± 90 (2)
18 ± 1 (3)
C025 (17a)
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ꢀ
0.28 ± 0.07 (5)
223 ± 18 (2)
38 ± 8 (4)
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12 ± 3 (5)
17d
11 ± 1 (7)
a
S-230
S-C025
R-C025
6 ± 2 (4)
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0.12 ± 0.05 (6)
23 ± 2 (3)
a
From ref. 19
A similar series of 3ꢀacylꢀ2ꢀphenylalkylꢀ1ꢀmethylindoles was synthesized as shown in Scheme 3.
In this case, the olefins 13aꢀd, containing different numbers of methylene units in between the
indole and the phenyl ring, were synthesized by reacting the aldehyde 3 with the Wittig salts 9aꢀ
d. Subsequent hydrogenation of 13aꢀd using 10% Pd/C furnished compounds 14aꢀd, which were
acylated at the 3ꢀposition of indole using Me AlCl as a Lewis acid and methyl 5ꢀchloroꢀ3ꢀ
2
methylꢀ5ꢀoxopentanoate (15) as an acylating agent to afford 16aꢀd. Finally, basic hydrolysis of
the methyl esters 16aꢀd
yielded the desired 3ꢀacyl
series of antagonists
(compounds 17aꢀd).
Scheme 3. Synthesis of 3ꢀ
acyl series antagonists. (a)
KHMDS/LiHMDS, THF, ꢀ
o
o
7
8 C – 0 C, 3 h, 80 – 90%; (b) 10%Pd/C, H , EtOH, rt, 3 h, 95%ꢀquant; (c) 17, Me AlCl, CH2Cl , rt, 3
2 2 2
h, 90 – 95%; (d) LiOH.H O, THF/H O (4:1), MeOH, rt, 12 – 18 h, 85 – 95%.
2
2
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1
1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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3
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5
5
5
5
6
As in the 1ꢀacyl series, the presence of a phenyl group with a trimethylene spacer (17b) in the 3ꢀ
acyl series reduced potency by about 8ꢀfold compared to 230 (Fig. 3B and Table 1). The
corresponding compound with a tetramethylene spacer (17c) is slightly less potency than 230,
whereas that with a pentamethylene spacer (17d) is about twice as potent. However, in contrast
to the N-acyl series, further lengthening of the spacer by one additional methylene group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
compound C025) dramatically increased potency by nearly 100ꢀfold (IC , 0.28 ± 0.07 nM)
50
compared to 230.
In view of the selectivity of the OXE receptor for the Sꢀenantiomer of 230, we separated the two
enantiomers of C025 (17a) by chiral HPLC (Fig. 3C) and examined their antagonist potencies.
We initially assumed that the earlierꢀeluting peak was the Sꢀenantiomer by analogy with the S
and R enantiomers of 230, and this was subsequently confirmed by chiral synthesis of S-C025
(20, Scheme 4), as discussed below. As with 230, the Sꢀenantiomer of C025 was nearly 200
times more potent than the Rꢀenantiomer in blocking the response to 5ꢀoxoꢀETE (Fig. 3D and
Table 1). The response to authentic S-C025, prepared by chiral synthesis as discussed below, is
also included in Fig. 3D for comparison. The structures of the S and R enantiomers of C025 are
shown in Fig. 3E.
Metabolism and pharmacokinetics of C149
To investigate its pharmacokinetics and in vivo metabolism, C149 (12a) was administered by
oral gavage to a cynomolgus monkey at a dose of 30 mg/kg and its concentration measured by
RPꢀHPLC in plasma samples obtained at various times up to 24 h (Fig. 4A). The maximal level
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Metabolism and pharmacokinetics of C149 and C025 (both racemic mixtures) in monkeys. A:
Plasma levels of racemic 149 (▲; n=1), and racemic 264 (∆; shown for comparison from ref. 21), both at
doses of 30 mg/kg. B: RPꢀHPLC of an extract of a plasma sample taken 12 h after administration of C149
by oral gavage. The stationary phase was a C18 Novapak column (4 µm particle size; 3.9 x 150 mm),
whereas the mobile phase was a gradient over 15 min between 70 and 100% MeOH in H O, both
2
o
containing 0.02% HOAc. The flow rate was 1 ml/min and the temperature 35 C. The internal standard
was 12c (the tetramethylene analog of C149). C: UV spectra of C149 and metabolites X and Y from
panel B. D: Plasma levels of racemic C025 (●; n=2; error bars indicate the range of values) and 230 (○;
shown for comparison from ref. 22) at doses of 30 mg/kg. E: RPꢀHPLC of an extract of a plasma sample
taken 12 h after administration of C025 by oral gavage. The chromatography conditions were identical to
those used for C149. The internal standard was 17c (the tetramethylene analog of C025). F: For
comparison, an RPꢀHPLC (conditions as described above) is shown for a plasma extract from a monkey 8
h after oral administration of racemic 230 (data from ref. 22). G: UV spectra of C025 and C025M,
captured from the chromatogram shown in panel E.
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6
Table 2. PK data for OXEꢀR antagonists. Antagonists were administered by oral gavage and blood
samples were collected at 0.5, 1, 2, 4, 8, 12, and 24 hours. tmax: time to reach maximal concentration after
oral administration; Cmax: maximum plasma concentration; t : time difference between t and the time
1/2
max
at which the plasma concentration dropped to 50% of Cmax. AUC: area under the curve between 0 and 24
h.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Dose
AUC
(nmol x h x ml )
ꢀ1
Antagonist n (mg/ml) tmax (h) Cmax (µM)
t½ (h)
1.2 ± 0.5
a
Rac 264
4
30
0.9 ± 0.1 27 ± 11
55 ± 17
Rac C149
1
30
2.0 55
3.0
294
b
Rac 230
4
2
30
30
15
15
0.6 ± 0.1 121 ± 22
2.5 ± 1.5 108 ± 39
0.8 ± 0.3 32 ±15
2.5 ± 1.5 79 ± 20
0.7 ± 0.2
198 ± 40
Rac C025
7.4 ± 0.6 1,250 ± 321
3.4 ± 1.3 169 ± 54
9.3 ± 1.5 1,081 ± 266
C025→S 2
C025→R 2
c
S-230
3
3
3
3
3
3
30
1
1.0 ± 0.0 36 ± 7
1.6 ± 0.0
100 ± 20
13 ± 1
SꢀC025
0.7 ± 0.2 4.5 ± 1.1 0.9 ± 0.3
0.7 ± 0.2 7.8 ± 1.8 1.1 ± 0.3
1.2 ± 0.4 18 ± 3.8 1.9 ± 0.2
“
“
“
“
2
26 ± 3
5
78 ± 12
171 ± 13
684 ± 119
10
30
1.2 ± 0.4 35 ± 1
1.3 ± 0.3 106 ± 21
2.8 ± 1
d
d
e
3.5 ± 0.6
a
b
c
d
e
Calculated from data in ref 21
Calculated from data in ref 22
Calculated from data in ref 23. Note that data was available for only 1, 4, 6, 12, and 24 h
p < 0.05 compared to S-230 (30 mg/kg)
p < 0.01 compared to S-230 (30 mg/kg)
of C149 (55 µM) was double that which we previously observed for 264 and was reached by 2 h,
with a t1/2 of 3 h, compared to 1.2 h for 264 (Table 2). The chromatographic profile of
metabolites in plasma obtained 12 h after administration of C149, revealed two major peaks
labeled X and Y in Fig. 4B, along with several minor peaks. The UV spectra of these two
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metabolites are quite similar to that of C149 (Fig. 4C), suggesting that the indole structure
remained intact and that the modifications were most likely on the phenylpentamethylene side
chain. Because of the much more favorable pharmacokinetic profile of C025 as discussed below,
we did not conduct any further studies with C149.
1
1
1
1
1
1
1
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0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Metabolism and pharmacokinetics of racemic C025
Administration of racemic C025 (17a, 30 mg/kg) to monkeys resulted in much higher and more
sustained plasma levels compared to C149 (Fig. 4D). The maximal concentration of C025 (>100
1
9
µM) was comparable to that achieved with 230, shown from a previous study for comparison,
and occurred after ~2.5 h, subsequently declining with a t1/2 of over 7 h, about 10 times longer
than that previously observed for 230 (Table 2). The RPꢀHPLC profile of C025 and its
metabolites in a plasma sample obtained 12 h after administration is shown in Fig. 4E. Note that
there is a 20ꢀfold difference in the scale of the Yꢀaxis between Figures 4B and 4E. It is obvious
from the figure that the level of C025 after 12 h is much higher and its metabolic profile much
simpler than those of C149 and 230 (the latter shown for comparison in Fig. 4F), with only one
major metabolite (C025M) detected. The UV spectrum of C025M (Fig. 4G) is very similar to
that of 025 except for a bathochromic shift in the λmax at 304 nm in C025 to 309 nm in C025M.
This is similar to the shift that we previously observed for one of the plasma metabolites of 230,
which we identified as a hydroxy derivative with the hydroxyl group α to the indole (i.e. αꢀ
2
3
hydroxyꢀ230).
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Chiral analysis of plasma C025 and its major metabolite C025M
The chirality of plasma C025 following oral administration of racemic C025 was examined by
chiral HPLC (Fig. 5A). The plasma concentration of the Rꢀenantiomer was higher than that of
the Sꢀenantiomer at all time points (Fig. 5B), and increased with time from 62% of total C025 at
0
1
2
3
4
5
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9
0
1
2
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1
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4
5
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8
9
0
3
0 min to 95% at 24 h (Fig. 5C). The maximal concentration reached by R-C025 was about 2.5
times higher than that for S-C025 and the t almost 3 times longer (Table 2). The plasma
½
concentration of R-C025 was still above 20 µM after 24 h, in contrast to S-C025, which was
about 1 µM at this time.
Figure 5. Chiral analysis of C025 and C025M in plasma following administration of C025. A: Chiralꢀ
HPLC of C025, isolated after RPꢀHPLC of an extract of plasma taken 4 h after oral administration of
racemic C025 (30 mg/kg). The stationary phase was a Celluloseꢀ1 column, and the mobile phase
o
hexane/MeOH/HOAc (97.5:2.5:0.1) (2 ml/min; 30 C). B: Plasma levels of the S (▲) and R (▼)
enantiomers of C025 followed oral administration as described above (n=2). The levels of racemic C025
(○) from Fig. 4D are included for comparison. C: Plasma levels of the Sꢀ (ꢀ) and Rꢀ (▲) enantiomers of
C025 (n = 2), expressed as percentages of total C025. Also shown are the plasma levels of the two
stereoisomers of C025M: M1 (∆) and M2 (○) (n = 1), expressed as percentages of total C025M in
samples taken at different times after administration of racemic C025. D and E: Chiral HPLC of C025M,
isolated from plasma taken 4 h (D) and 24 h (E) after administration of racemic C025. A Celluloseꢀ1
o
column was used with a mobile phase of hexane/MeOH/HOAc (97:3:0.1) (2 ml/min; 30 C).
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We also examined the chirality of C025M, which was isolated by RPꢀHPLC as shown in Fig.
4
E, and then subjected to chiral HPLC. Two peaks (M and M ) with UV spectra identical to that
1 2
of C025M were observed, with M2 predominating after 4 h (Fig. 5D) and M predominating
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
after 24 h (Fig. 5E). As shown in Fig. 5C, the proportion of M gradually increased, following a
1
time course similar to, but lagging behind, R-C025, and accounting for 83% of total C025M
after 24 h. Similarly, the time course for M lagged behind that of S-C025, suggesting that M is
2
2
derived from S-C025 (i.e. S-methyl) and M from R-C025 (i.e. R-methyl). This was
1
subsequently confirmed by studies on the metabolism of S-C025 as well as by total chemical
synthesis of M (see below).
2
Scheme 4. Synthesis of 20 (S-C025). Reagents and conditions: (a) 18, Me AlCl, CH Cl , rt, 3 h, 93%; (b)
2
2
2
LiOH.H O, THF/H O (4:1), MeOH, rt, 16 h, 95%.
2
2
Pharmacokinetics of SꢀC025
To directly assess the PK of the Sꢀenantiomer of C025 (20), this compound was prepared by
chiral synthesis (Scheme 4), using a method similar to that used for C025, but employing the
2
5
chiral synthon 18, which was prepared as described previously. Analysis of plasma extracts by
RPꢀHPLC following administration of S-C025 revealed a pattern virtually identical to that
obtained with racemic C025, with a single major metabolite (S-C025M) with chromatographic
and UV properties identical to those of C025M (data not shown). The PK profile of S-C025 (30
mg/kg) is compared to those of racemic C025 and S-230 (the latter taken from a previous
2
3
study ) in Fig. 6A. The maximal plasma level (~100 µM) of S-C025 was similar to that for
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2
2
2
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racemic C025, but its concentration declined more rapidly (t 3.5 h compared to 7.4 h for C025;
½
Table 2). However, the PK profile of S-C025 was substantially better than that of S-230, with
increased values for Cmax (p < 0.05), t , (p < 0.05) and AUC (p < 0.01) (Table 2). A caveat to the
½
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
above conclusion is that the previously published data for S-230 did not include time points at
0
.5 and 2 h. However, removal of these time points from the data for S-C025 in the present study
had relatively little effect on the calculated values for Cmax (92 ± 15; p < 0.05), t (3.7 h; p <
½
0
.01), and AUC (652 ± 100; p < 0.01). Low but measurable levels of S-C025 were detected in
plasma for up to 1 wk after administration (160 and 40 nM after 3 and 7 days, respectively),
whereas higher levels of S-C025M (1.5 µM and 0.2 µM) were observed at these time points
(Fig. 6B).
Figure 6. Pharmacokinetics of S-C025. A: The plasma levels of S-C025 (ꢀ; n=3) following oral
administration of a dose of 30 mg/kg are compared to those obtained after identical doses of racemic
C025 (∆; Fig. 4F) and S-230.(∇; ref. 23). B: The levels of S-C025 (●) and S-C025M (ꢀ) were followed
in 2 monkeys over 7 days. Plasma levels of S-C025 (panel C) and S-C025M (panel D) following
administration of S-C025 at doses of 1 (▼), 2 (∆), 5 (▲), 10 (○), and 30 (ꢀ) mg/kg. Data for S-230 (□)
from ref. 23 are shown for comparison. E: Concentrationꢀresponse curves for the inhibition of 5ꢀoxoꢀ
ETEꢀinduced calcium mobilization in neutrophils by S-C025 (○) and S-C025M (ꢀ), isolated from plasma
after administration of S-C025.
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In view of the enhanced PK profile of S-C025, we investigated a series of lower doses, including
1
0, 5, 2, and 1 mg/kg (Fig. 6C; note that a log scale is used for the Yꢀaxis in this figure). S-C025
was detectable at all time points within a period of 24 h for all doses tested. Compared to the 30
mg/kg dose, maximal levels of S-C025 were observed at shorter time points (0.5 to 1 h) for all
the lower doses. Higher plasma concentrations of S-C025 were achieved with a dose of 5 mg/kg
between 4 and 8 h compared to a dose of 30 mg/kg of S-230, and at later time points, a dose of 2
mg/kg gave plasma concentrations similar to the much higher dose of S-230 (Fig. 6C). The
plasma concentrations of S-C025M rose more slowly than S-C025, reaching maximal levels
between 2 and 4 hours, which remained fairly constant up to 12 h and then declined somewhat
by 24 h (Fig. 6D). To determine whether it has antagonist activity, we isolated S-C025M from
monkey plasma and examined its effects on 5ꢀoxoꢀETEꢀinduced calcium mobilization in human
neutrophils. As shown in Fig. 6E, S-C025M is a potent OXE receptor antagonist with an IC of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
0.69 ± 0.36 nM, compared to 0.12 ± 0.05 nM for S-C025.
Figure 7. Mass spectra of S-C025M isolated
2
from plasma. A: MS fragmentation of the [Mꢀ
ꢀ
3
H] ion at m/z 468 for S-C025M. B: MS
fragmentation of the ion at m/z 292 shown in
panel A. The ion at m/z 206 is formed as a result
of a McLafferty rearrangement.
Identification S-C025M
ꢀ
LCꢀMS/MS analysis of S-C025M revealed an [MꢀH] ion at m/z 468.1958, compared to the
theoretical value of m/z 468.1942 expected for a hydroxy metabolite of S-C025 (mass accuracy,
2
3.4 ppm). MS fragmentation of this ion (Fig. 7A) resulted in a single major ion at m/z 292 (loss
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of phenylhydroxyalkyl side chain), consistent with the presence of a hydroxyl group α to the
indole, and a smaller ion at m/z 450 (loss of H O). In addition, ions of low intensity were
2
observed at m/z 406 (loss of H O and CO ), m/z 274 (loss of H O from m/z 292), 248 (loss of
2
2
2
0
1
2
3
4
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9
0
1
2
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9
0
1
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9
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1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
CO from m/z 292) and 206 (loss of CH =CHꢀCH ꢀCO H from m/z 292 due to a McLafferty
2
2
2
2
3
rearrangement). The MS profile obtained from the major ion at m/z 292 included intense ions at
m/z 274, 248, and 206, identical to the latter 3 ions listed above, along with an additional ion at
m/z 180 (Fig. 7B). A similar fragmentation pattern was previously observed for αꢀhydroxyꢀS-
23
2
30 , which also had an intense ion at m/z 292 due to loss of the hydroxyhexyl side chain.
Scheme 5. Synthesis of αꢀOHꢀC025. Reagents and conditions: (a) Mg, THF, reflux; (b) THF, 0 °C – rt, 3
h, 56%; (c) TBDMSCl, imidazole, CH Cl , rt, 3 h, 88%; (d) 15, Me AlCl, CH Cl , 0 °C – rt, 12 h, 44%;
2
2
2
2
2
(e) LiOH. H O, THF/H O (4:1), MeOH, rt, 48 h, 89%.
2
2
To provide further evidence for the structure of S-C025M and to investigate the chirality of the
αꢀhydroxyl group and the antagonist potency of the different stereoisomers of this compound, we
prepared a mixture of αꢀhydroxyꢀC025 stereoisomers (26) by total chemical synthesis as shown
in Scheme 5. The αꢀOH center was generated using a Grignard reaction between the aldehyde 3
and the Grignard reagent 22. The free alcohol group of 23 was protected with a silyl ether
(OTBDMS) group to obtain 24, which was then acylated at the 3ꢀposition of the indole ring. In
the acylation reaction of 24 using Me AlCl as a Lewis acid, the TBDMS group was removed by
2
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Me AlCl to obtain the alcohol 25. Subsequent hydrolysis of 25 under basic conditions furnished
2
2
6 (αꢀOHꢀC025).
Synthetic αꢀhydroxyꢀC025, which consisted of 4 diastereomers in equal proportions, was
separated into two peaks by NP (normalꢀphase)ꢀHPLC, labeled A and B in Fig. 8A. S-C025M
from plasma had a retention time identical to that of peak B (Fig. 8B) and, when mixed with the
synthetic material, cochromatographed with peak B (Fig. 8C). The material in peak A in Fig. 8A
was separated into 2 peaks (A and A ) by chiral HPLC (Fig. 8D). Similarly, the material in peak
1
1
1
1
1
1
1
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1
1
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2
2
2
2
2
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0
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0
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9
0
1
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9
0
1
2
B was separated into B and B by chiral HPLC (Fig. 8E). S-C025M gave a single peak when
1
2
analyzed by chiral HPLC (Fig. 8F), which cochromatographed with synthetic peak B (Fig. 8G).
1
By analogy with our previous study on the αꢀhydroxylation of S-230 in the monkey, it seemed
likely that S-C025M/peak B was identical to αS-hydroxyꢀS-C025. To confirm this, we prepared
1
the latter compound by chiral synthesis as shown in Scheme 6 using a method similar to that
23
which we described previously for the synthesis of αSꢀOHꢀS-230. In this synthesis, an (S)ꢀ
BINOLꢀmediated enantioselective addition of the Grignard reagent 22 to the prochiral aldehyde
3 yielded the chiral alcohol 27, 92.2% of which was the Sꢀenantiomer. The alcohol 27 was
protected using a silyl ether to obtain 28. Acylation of 28 using the acyl chloride 18 and
Me AlCl, followed by removal of TBDMS and ester hydrolysis yielded 29 (αSꢀOHꢀS-C025).
2
Authentic αSꢀOHꢀS-C025 (Fig. 8J) had the same retention time as stereoisomer B and S-
1
C025M, isolated from plasma, when chromatographed by NPꢀHPLC (data not shown) and chiral
HPLC (Fig. 8H).
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9
0
Figure 8. Identification of S-C015M by cochromatography and its effect on 5ꢀoxoꢀETEꢀinduced
calcium mobilization. A: NPꢀHPLC of a synthetic mixture of αꢀhydroxyꢀC025 stereoisomers on an
Econosphere silica column using hexane/iPrOH/HOAc (98.5:1.5:0.1) at flow rate of 1 ml/min as the
o
mobile phase and a column temperature of 30 C. B: NPꢀHPLC as described above of S-C025M, isolated
from plasma. C: Cochromatography of synthetic αꢀhydroxyꢀC025 and S-C025M by NPꢀHPLC as
described above. D: Chiral HPLC of peak A from panel A using a Celluloseꢀ2 column with
hexane/MeOH/HOAc (95:5:0.1) at a flow rate of 1 ml/min as the mobile phase and a column temperature
o
of 45 C. E: Chiral HPLC of peak B from panel A (conditions as for panel D). F: Chiral HPLC as
described above of S-C025M, isolated from plasma. G: Cochromatography of synthetic αꢀhydroxyꢀC025
(
peak B) and S-C025M by chiral HPLC as described above. H: Chiral HPLC of synthetic αSꢀhydroxyꢀSꢀ
C025. I: Concentrationꢀresponse curves for the inhibitory effects on 5ꢀoxoꢀETEꢀinduced calcium
mobilization of S-C025 (●), S-C025M isolated from plasma (○), and the 4 stereoisomers purified from
synthetic αꢀhydroxyꢀC025: A (▼), A (∇), B (▲), and B (∆) (n = 3). J: Structure of αSꢀhydroxyꢀS-
1
2
1
2
C025 (SꢀC025M).
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Scheme 6. Synthesis of 29 (αS-OHꢀS-C025).
Reagents and conditions: (a) 22, (S)ꢀBINOL,
t
Ti(OiPr) , BuOMe, BDMAEE, THF, ꢀ15 °C – rt,
4
36 h, 38%; (b) TBDMSCl, imidazole, CH Cl , rt,
2
2
1
6 h, 94%; (c) (1) 18, Me AlCl,CH Cl ; (2)
2
2
2
1
1
1
1
1
1
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1
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0
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8
9
0
HF·Pyridine, CH CN; (3) LiOH·H O, THF/H O
3
2
2
(
4:1), MeOH, rt, 14 h, 23% over three steps.
The effects of all 4 stereoisomers of αꢀhydroxyꢀC025 on 5ꢀoxoꢀETEꢀinduced calcium
mobilization were compared to those of synthetic S-C025 and plasma S-C025M (Fig. 8I).
Stereoisomer B (IC ~0.4 nM) and S-C025M (IC ~0.7 nM) had similar IC values and were
1
50
50
50
only a little less potent than S-C025 (IC ~0.1 nM) in blocking the response to 5ꢀoxoꢀETE
5
0
(
Table 3), whereas the other three αꢀhydroxyꢀC025 diastereomers were approximately 1000 to
6000 times less potent.
Table 3. Potencies of synthetic and
metabolicallyꢀformed αꢀOHꢀC025
stereoisomers in inhibiting 5ꢀoxoꢀETEꢀ
induced calcium mobilization in human
neutrophils.
Compound
Chirality
IC (nM)
50
S-C025
0.12 ± 0.05 (6)
0.69 ± 0.36 (5)
120 ± 60 (3)
S-C025M
A (M )
αS-OHꢀS-C025
αS-OHꢀR-C025*
αR-OHꢀS-C025*
αS-OHꢀS-C025
αR-OHꢀ R-C025
1
1
A2
260 ± 30 (3)
* Assignment of the chirality of these
isomers is based on comparison of their
relative retention times with those of the
B (M )
0.38 ± 0.09 (3)
750 ± 140 (3)
1
2
B2
23
corresponding derivatives of 230.
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6
DISCUSSION
We recently identified a series of indoleꢀbased OXE receptor antagonists, which have potencies
1
9, 22
in the nanomolar range and are absorbed following oral administration to primates.
Of these,
0
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9
0
the most potent were S-230 and Sꢀ264, with IC values of ~10 nM, suggesting that these
5
0
compounds might be credible drug candidates. However, although very high initial levels of both
2
2
21
2
30 and 264 appeared in the blood shortly after oral administration to monkeys, their
concentrations dropped sharply shortly thereafter, due in large part to the rapid ω2ꢀoxidation of
the hexyl side chain common to both antagonists.
In an attempt to increase resistance to ωꢀoxidation we introduced a phenyl group at the end of the
alkyl side chain, a strategy that was used in the case of prostaglandins. Replacement of the three
terminal carbons of PGF by a phenyl group, as in 17ꢀphenylꢀ18,19,20ꢀtrinorꢀPGF , resulted in
2
α
2α
2
6
27
a substantially longer in vivo halfꢀlife as well as increased agonist potency at the FP receptor.
A similar analog of PGE , 17ꢀphenylꢀ18,19,20ꢀtrinorꢀPGE , is a potent EP and EP receptor
2
2
1
3
28, 29
agonist.
In the present study we initially explored a similar approach with both the 1ꢀacyl
(264) and 3ꢀacyl (230) series of OXEꢀR antagonists and replaced the last 3 carbons of the hexyl
side chain with a phenyl group, resulting in the “phenylꢀtrinor” compounds 12b and 17b,
analogous to the above prostaglandin derivatives. However, in contrast to prostaglandins, this
reduced potency by about 10ꢀfold. We then examined the effects of lengthening the
polymethylene spacer and found the optimal length to be 5 carbons (C149) in the 1ꢀacyl series
and 6 carbons (C025) in the 3ꢀacyl series. Compound C025 is remarkably potent, with an IC of
5
0
1
20 pM for the active S-enantiomer (S-C025), 50 times lower than that of S-230.
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Although the large increase in potency of the phenyl analogs was very encouraging, the primary
goal of the current study was to reduce the rate of in vivo metabolism of our OXEꢀR antagonists.
To determine whether higher and more prolonged plasma levels of antagonist can be achieved
with these compounds, we conducted pharmacokinetic experiments in cynomolgus monkeys.
Although the plasma levels of C149 (30 mg/kg) were above 13 µM over the first 8 h and much
higher than those achieved with an identical dose of 264, they were not nearly as high as those
obtained with C025, which were above 20 µM for at least 24 h. For this reason, we focused on
C025 in our further studies.
1
1
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0
We previously found that following oral administration of racemic 230 there was a transitory
22
decrease in the ratio of the active S-enantiomer to the inactive R-enantiomer in plasma, ,
suggesting that there might be modest differences in the rates of absorption or metabolism of the
two enantiomers. However, chiral analysis of plasma C025 revealed much larger differences in
the case of the phenyl analog, with the S-enantiomer accounting for only 5% of the total C025
after 24 h. Although selective uptake of the Rꢀenantiomer from the gut might contribute to some
extent to its higher plasma levels, the selective disappearance of S-C025 from plasma suggests
that the main determinant is its increased metabolism or clearance compared to R-C025. The
much higher degree of enantiomeric selectivity in the clearance of C025 compared to 230 might
be due to the different metabolic pathways for these two antagonists. Whereas the principal
plasma metabolites of 230 are formed by ω2ꢀoxidation, along with a smaller degree of αꢀ
23
oxidation, the only major plasma metabolite of C025 that we observed was formed by αꢀ
oxidation, which may have a higher degree of chiral selectivity. In support of this, we found that
the ratio of M (derived from S-C025) to M (derived from R-C025) was much higher than the
2
1
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ratio of S-C025 to R-C025 (Fig. 5C). This suggests that a higher proportion of S-C025 is
metabolized by this pathway, compared to its R-enantiomer, which could, at least in part, account
for its more rapid disappearance in plasma. In spite of its shorter t compared to racemic C025,
½
0
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S-C025 is still far superior to our previous OXEꢀR antagonist, S-230, with respect to plasma
halfꢀlife, Cmax, and AUC, and is a highly credible candidate for further preclinical testing.
It is interesting that the αꢀhydroxylation of S-C025 appears to be highly stereospecific, resulting
in the selective formation of the single stereoisomer (i.e. B , M ) with appreciable antagonist
1
2
activity. αS-hydroxyꢀS-C025 is a potent OXEꢀR antagonist in its own right, with an IC below 1
5
0
nM, only several fold higher than that of its precursor. This contrasts with the αSꢀhydroxy
23
metabolite of S-230, which is over 100 times less potent than S-230. Thus, in the case of a
hexyl side chain, the presence of a hydroxyl group α to the indole has a strong negative effect on
interaction with OXEꢀR, whereas in the case of a phenylhexyl side chain, a hydroxyl group in
this position has little or no effect. This could possibly be because the phenyl group in
αSꢀhydroxyꢀS-C025 affects the conformation of the molecule and changes the orientation of the
αꢀhydroxyl group, resulting in less interference with binding to the receptor. The potency of S-
C025M and its predominance over S-C025 in plasma at time points longer than 12 h suggest that
this metabolite could contribute substantially to antagonist activity. The longer apparent halfꢀlife
of S-C025M compared to its precursor could possibly be due to stabilization of the molecule by
hydrogen bonding to the nearby oxo group, which might make it more resistant to further
metabolism and clearance.
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9
Because of the large differences in the potencies and rates of clearance between the S and R
enantiomers of C025, and the selective metabolism of the Sꢀenantiomer to a highly potent
metabolite, it would clearly be preferable to use the pure S-enantiomer in preclinical or clinical
studies, rather than the racemate. Fortunately, we previously developed a procedure for the
synthesis of a chiral synthon that can be used to introduce the critical 3Sꢀmethylꢀ5ꢀoxovalerate
side chain to this series of indoleꢀbased antagonists. We initially used this procedure for the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
4
4
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5
5
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5
5
5
5
5
5
5
6
0
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0
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6
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8
9
0
2
5
synthesis of S-230 and Sꢀ264, but in the present study, it was adapted for the synthesis of the
substantial amounts of S-C025 required for these studies.
The high potency of S-C025, along with its resistance to metabolism and its persistence in the
circulation, suggest that it could be a useful therapeutic agent in diseases in which 5ꢀoxoꢀETE
plays a role. Largely because of the lack of an OXEꢀR ortholog in the mouse and other rodents,
the pathophysiological role of 5ꢀoxoꢀETE has not yet been established. However, the high
10
expression of OXEꢀR in eosinophils and the ability of 5ꢀoxoꢀETE to induce the infiltration of
17
these cells into the skin, especially in asthmatics, suggests that S-C025 could be a novel
therapeutic agent in asthma and other eosinophilic diseases, a hypothesis that we are currently
exploring in monkeys. Because the synthesis of 5ꢀoxoꢀETE is dependent on elevated levels of
+
NADP , which occur during oxidative stress and cell death, it is likely to be formed at
inflammatory sites, and may contribute to chronic disease characterized by prolonged
30
inflammation. Another possible use of an OXEꢀR antagonist might be in the treatment of some
3
1
types of cancer. 5ꢀOxoꢀETE was reported to induce the proliferation of prostate cancer cells
and siRNA directed at OXEꢀR has been shown to reduce the viability of these cells, suggesting a
3
2
role for 5ꢀoxoꢀETE in maintaining prostate cancer cell survival.
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1
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1
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2
2
2
2
2
2
2
2
2
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3
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5
5
6
CONCLUSIONS
In conclusion, addition of a phenyl group at the end of the hexyl side chain of the OXEꢀR
antagonist S-230 results in dramatic increases in both in vitro potency and halfꢀlife in the
circulatory system when administered orally to monkeys. S-C025 (20) is a highly potent OXEꢀR
antagonist with an IC in the picomolar range. It appears rapidly in the blood following oral
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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0
5
0
administration to monkeys and its concentration in plasma is sustained at a much higher level
than S-230 over 24 h. Unlike Sꢀ230, which is converted to several plasma metabolites that
possess little antagonist activity, SꢀC025 is converted to a single major plasma metabolite (Sꢀ
C025M) with substantial antagonist activity and a prolonged lifetime in plasma. Future work
will be aimed at preparing sufficient amounts of this metabolite, which contains two chiral
centres, to evaluate its properties following oral administration and to determine whether it may
also be a potential drug candidate.
The potency and favorable PK profile of SꢀC025 will permit its use at substantially lower doses
than would be required for our earlier OXEꢀR antagonists. Because of the potent chemoattractant
effects of 5ꢀoxoꢀETE on eosinophils, S-C025 is a promising candidate as a novel therapeutic
agent in eosinophilic diseases such as asthma, allergic rhinitis, or atopic dermatitis, used either
on its own or to complement the effects of currently available drugs that act by different
mechanisms. S-C025 could also potentially be useful for the treatment of some forms of cancer
3
2, 33
in which 5ꢀoxoꢀETE has been shown to have a proliferative effect on cancer cells.
Preclinical studies in monkeys are currently underway to determine whether an OXEꢀR
antagonist can inhibit allergenꢀinduced dermal eosinophilia.
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9
EXPERIMENTAL SECTION
Evaluation of OXE receptor antagonist potency. The potency of potential OXEꢀR antagonists
was examined by evaluating their abilities to inhibit calcium mobilization in human neutrophils
1
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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5
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5
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0
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9
0
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2
3
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9
0
in response to 5ꢀoxoꢀETE as described previously. Neutrophils were prepared from the blood
of healthy subjects following dextran sedimentation of red blood cells and removal of
mononuclear cells by centrifugation over FicollꢀPaque. The cells were then labelled with the
o
calciumꢀsensitive dye indoꢀ1 and fluorescence was measured at 37 C with a Cary Eclipse
spectrofluorometer (Agilent Technologies, Santa Clara, CA) equipped with a temperatureꢀ
controlled cuvette holder and a magnetic stirrer. After the fluorescence had stabilized, various
concentrations of potential antagonists were added, followed 2 min later by 5ꢀoxoꢀETE (10 nM).
Digitonin (final concentration 0.1%) was added 1 min later to determine maximal fluorescence.
Monkey experiments
Female cynomolgus monkeys weighing between 2.5 and 3.5 kg, housed at INRSꢀInstitut
ArmandꢀFrappier, Laval, Quebec were used for studies on the metabolism and pharmacokinetics
of OXEꢀR antagonists. All experiments were performed in accordance with the guidelines of the
Canadian Council on Animal Care and were approved by the local institutional animal care
committee. Racemic C149 (12a), racemic C025 (17a), or the Sꢀenantiomer of C025 (20),
synthesized as described below, were dissolved in EtOH to give a concentration of 75 mg/mL.
To administer a dose of 30 mg/kg, 0.4 mL/kg body weight of this solution was diluted in 10
volumes of 20 mM NaHCO , pH 8.0. The resulting suspension (4.4 mL/kg; 9.1% EtOH) was
3
immediately vortexed and administered by oral gavage to a monkey. Lower doses of SꢀC025
were administered in the same way, except that the volume administered was 2.2 mL/kg (9.1%
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EtOH in 20 mM NaHCO , pH 8.0). Blood samples (1 to 2 ml) were collected in heparinized
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tubes just prior to gavage and 0.5, 1, 2, 4, 8, 12, and 24 h after gavage. In some cases, blood was
also collected after 3 and 7 days. After centrifugation, plasma samples were frozen and stored at
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80 C prior to extraction and analysis.
Quantitation of C149 and C025 in plasma
After they were thawed, plasma samples were diluted with 2 volumes of MeOH, followed by the
addition of the appropriate internal standard, which were the tetramethylene analogs of C149
(compound 12c; 1 µg) and C025 (compound 17c; 1.3 µg). The samples were then stored
o
overnight at ꢀ80 C and centrifuged to remove the precipitated material. The concentration of
MeOH in the supernatant was then adjusted to 30% by the addition of water and the sample was
loaded onto a C18 SepꢀPak cartridge (Waters Corp), which was washed with 30% MeOH,
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followed by the elution of the antagonist and its metabolites with 100% MeOH. After removal
of the solvent in vacuo using a rotary evaporator, the residue was dissolved in 30% MeOH and
analyzed by precolumn extractionꢀRPꢀHPLC as described below. In some cases, the peaks for
C025 and C025M were collected and analyzed by chiral HPLC and/or LCꢀMS/MS.
HPLC analysis of OXEꢀR antagonists
HPLC analyses were carried out using a modified Waters 2695 Alliance system (Waters
Corporation, Mississauga, Ontario, Canada) equipped with a Waters model 2996 photodiode
array detector. A C18 SecurityGuard cartridge (4 x 3 mm; Phenomenex, Torrance, CA) was used
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for automated precolumn extraction as described previously. The stationary phase for RPꢀ
HPLC was a Novapak C column (3.9 x 150 mm; 4 µm particle size; Waters Corp). An
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Econosphere column (250 x 4.6 mm; 5 µm particle size; Alltech Associates, Deerfield, IL) was
used for normalꢀphase HPLC. Chiral HPLC was carried out using either a Celluloseꢀ1 or a
Celluloseꢀ2 column (4.6 x 250 mm; 5 µm particle size; Phenomenex, Torrance, CA). Additional
details about the chromatographic conditions are described in the appropriate figure legends. All
solvents used for extraction and chromatographic analysis were purchased from Fisher Scientific,
Markham, ON, Canada.
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Analysis of the major plasma metabolite of C025 by LCꢀMS/MS
To identify C025M, an extract of plasma prepared from a blood sample taken 4 h after
administration of C025 (17a, 30 mg/kg) was analyzed by LCꢀMS/MS, using a model 1100
HPLC system (Agilent Technologies, Santa Clara, CA) connected to an LTQ Velos Orbitrap
high resolution mass spectrometer by a heated electrospray ionization source (Thermo Scientific,
San Jose, CA). The stationary phase was a Phenomenex Kinetex C18 column (2.6 µm particle
size; 50 x 2.1 mm). The mobile phase was a linear gradient between solvents A (0.02% HOAc in
water) and B (0.02% HOAc in MeCN) as follows: 0 min, 30% B; 1 min, 30% B; 25 min, 55% B;
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2 min, 55% B. The flow rate was 0.3 mL/min, the column temperature 25°C and the injection
volume 10 µL. Analyses were performed in negative electrospray ionization (ESI) mode with the
o
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following settings: capillary temperature: 350 C; source heater temperature: 300 C; sheath gas
flow: 20; auxiliary gas flow: 10; source voltage: ꢀ 3.0 kV. The MS settings were: S lens RF
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level: 60%; automatic gain control (AGC) target: 1 x 10 ions; mass range: m/z 250 to m/z 700;
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resolution: 100,000. Multiple levels of MS analysis in data dependent acquisition (DDA) mode
were used for the identification and elucidation of C025M. In DDA mode, the selection of the
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precursor ion for MS analysis was based on the chlorine isotope pattern and/or isolation of the
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