Novel highly potent OXE receptor antagonists with prolonged plasma lifetimes that are converted to active metabolites in vivo in monkeys

Article abstract of DOI:10.1111/bph.14874

Background and Purpose: The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), acting through the OXE receptor, is a potent eosinophil chemoattractant that may be an important proinflammatory mediator in eosinophilic diseases su

Full text of DOI:10.1111/bph.14874

Powell William (Orcid ID: 0000-0002-8507-4038)
Novel highly potent OXE receptor antagonists with prolonged plasma lifetimes that are
converted to active metabolites in vivo in monkeys
Running title: Potent OXE receptor antagonists with prolonged lifetimes
Qiuji Ye1,†, Shishir Chourey1,‡, Chintam Nagendra Reddy1,§, Rui Wang1, Chantal
Cossette2, Sylvie Gravel2, Irina Slobodchikova3, Dajana Vuckovic3, Joshua Rokach1,
and William S. Powell2
1Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150
West University Boulevard, Melbourne, FL 32901-6982, USA
2Meakins-Christie Laboratories, Centre for Translational Biology, McGill University Health
Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada
3 Department of Chemistry and Biochemistry and PERFORM Centre, Concordia University,
7141 Sherbrooke St. W., Montréal, QC H4B 1R6, Canada
†Present address: Dept. Chemistry, Rice University, 6100 Main Street, Houston, TX 77005,
USA
‡Present address: Chemical Development Dept., Albany Molecular Research Inc., 21
Corporate Circle, Albany, NY 12203, USA
§Present address: Olon Ricerca Bioscience, 7528 Auburn Road, Concord, OH 44077, USA
Word count: 4547 (Introduction: 941; Results: 2175; Discussion: 1431)
Abstract: 244 words
References: 37
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Acknowledgements
This work was supported by the Canadian Institutes of Health Research (WSP: Grants MOP-
6254 and PP2-133388), the American Asthma Foundation (JR: Grant 12-0049), and the
National Heart, Lung, and Blood Institute (JR: Grant R01HL081873) and by AmorChem
(Montreal, QC). The Meakins-Christie Laboratories-MUHC-RI are supported in part by a
Centre grant from Le Fond de la Recherche en Santꢀ du Quꢀbec as well as by the J. T.
Costello Memorial Research Fund. JR also wishes to acknowledge the National Science
Foundation for the AMX-360 (Grant CHE-90-13145) and Bruker 400 MHz (Grant CHE-03-
42251) NMR instruments. DV and IS were supported by the Natural Sciences and
Engineering Research Council of Canada (Grant RGPIN/435814-2103). IS also wishes to
acknowledge the Centre for Biological Applications of Mass Spectrometry at Concordia
University for PhD scholarship funding. 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.
Author contributions
Q.Y., S.C., C.N.R. and R.W. performed the chemical syntheses. S.G. evaluated antagonist
potencies. C.C. conducted HPLC analyses. I.S. and D.V. performed mass spectrometric
analyses. W.S.P. and J.R. designed the study. W.S.P, J.R., Q.Y., S.C. and D.V. wrote or
contributed to the writing of the manuscript.
Conflict of interest
W.S.P. and J.R. have been granted a patent covering 230 and have applied for a patent
covering S-Y048.
Declaration of transparency and scientific rigour
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research as stated in the BJP guidelines for
Design & Analysis, and Animal Experimentation, and as recommended by funding agencies,
publishers and other organisations engaged with supporting research.
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Abstract
Background and Purpose: The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic
acid (5-oxo-ETE), acting through the OXE receptor, is a potent eosinophil chemoattractant
that may be an important proinflammatory mediator in eosinophilic diseases such as asthma.
We previously identified a series of indole-based OXE receptor antagonists that rapidly
appear in the blood following oral administration but have limited lifetimes. The objective of
this study was to increase the potency and plasma half-lives of these compounds and thereby
identify the optimal candidate for future preclinical studies in monkeys, since rodents do not
have an OXE receptor ortholog.
Experimental Approach: We synthesized a series of substituted phenylalkyl indoles and
compared their antagonist potencies, pharmacokinetics, and metabolism to those of our
earlier compounds. The potencies of some of their metabolites were also investigated.
Key Results: Among the compounds tested, the S-enantiomer of the m-chlorophenyl
compound (S-Y048) was the most potent, with an pIC50 of about 10.8 for inhibition of 5-oxo-
ETE-induced calcium mobilization in human neutrophils. When administered orally to
cynomolgus monkeys, S-Y048 rapidly appeared in the blood and had a half-life in plasma of
over 7 h, considerably longer than any of the other OXE analogs tested. A major
hydroxylated metabolite, with a potency close to that of its precursor, was identified in
plasma.
Conclusion and Implications: Because of its highly potent antagonist activity and its long
lifetime in vivo, S-Y048 may be a useful anti-inflammatory agent for the treatment of
eosinophilic diseases such as asthma, allergic rhinitis, and atopic dermatitis.
What is already known
 The eicosanoid 5-oxo-ETE is a potent eosinophil chemoattractant that acts via the
OXE receptor.
 OXE receptor activation can be blocked by indole-based antagonists mimicking the
structure of 5-oxo-ETE.
What this study adds
 We have structurally modified our OXE antagonists to substantially increase both
potency and plasma half-life.
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 S-Y048 and its active hydroxylated metabolite have potencies in the picomolar range.
What is the clinical significance
 S-Y048 is a good candidate for in vivo efficacy studies in monkeys.
 This OXE receptor antagonist may be a novel therapeutic agent in eosinophilic
diseases in humans.
Abbreviations
230, 5-(5-chloro-2-hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoate
5-LO, 5-lipoxygenase
5-oxo-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid
C025, 5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid
S-C025, (S)-5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-
oxopentanoic acid
LT, leukotriene
MMP-9, matrix metalloproteinase-9
NP-HPLC, normal-phase-high performance liquid chromatography
RP-HPLC, reversed-phase-high performance liquid chromatography
S-230, (S)-5-(5-chloro-2-hexyl-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoate
S-Y048, (S)-5-(5-chloro-2-(6-(3-chlorophenyl)hexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-
oxopentanoate
S-Y048M, (S)-5-(5-chloro-2-((S)-6-(3-chlorophenyl)-1-hydroxyhexyl)-1-methyl-1H-indol-3-
yl)-3-methyl-5-oxopentanoate
uPAR, urokinase-type plasminogen activator receptor
Y048, 5-(5-chloro-2-(6-(3-chlorophenyl)hexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-
oxopentanoate.
Keywords: Lipid mediators; eicosanoids; 5-lipoxygenase products; 5-oxo-ETE;
inflammation; asthma; GPCR antagonists
Introduction
Arachidonic acid metabolites formed by the 5-lipoxygenase (5-LO) pathway are important
mediators in asthma and drugs targeting their biosynthesis reduce disease symptoms in
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humans (Haeggstrom, 2018; Peters-Golden & Henderson, 2007). While the focus has
principally been on leukotriene (LT) D4, acting through the selective cysLT1 receptor (Peters-
Golden & Henderson, 2007; Yokomizo et al., 2018), other 5-LO products may also play a
role. LTB4, LTD4, and other cysLTs are only very weak chemoattractants for human
eosinophils (Morita et al., 1989; Powell et al., 1995; Sun et al., 1991), which play a major
role in asthma and other allergic diseases (McBrien & Menzies-Gow, 2017). In contrast,
another 5-LO product, 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), is a potent
eosinophil chemoattractant, both in vitro (Powell et al., 1995) and in vivo (Muro et al., 2003).
Its actions are mediated by the G protein-coupled oxoeicosanoid (OXE) receptor (Bäck et al.,
2014; Hosoi et al., 2002; Jones et al., 2003; Takeda et al., 2002), which is encoded by the
OXER1 gene. The OXE receptor is highly expressed on eosinophils and basophils and to a
lesser extent on neutrophils, monocytes and macrophages (Iikura et al., 2005; Jones et al.,
2003; Sturm et al., 2005).
The pathophysiological role of 5-oxo-ETE has been difficult to establish, in part due to the
lack of an ortholog of the OXE receptor in rodents. Nevertheless, its potent effects on human
eosinophils suggest that it might be involved in eosinophilic diseases. In addition to its direct
chemoattractant effects on these cells, 5-oxo-ETE has been shown to induce the
transendothelial migration of eosinophils (Dallaire et al., 2003). This is due not only to its
effect on cell migration, but also to its ability to increase the expression and release of matrix
metalloproteinase-9 (MMP-9) as well as the expression of the urokinase-type plasminogen
activator receptor (uPAR), resulting in degradation of components of the extracellular matrix,
thereby facilitating the passage of eosinophils into the tissues (Guilbert et al., 1999; Langlois
et al., 2006). Our prior experiments showing that 5-oxo-ETE can elicit the infiltration of
eosinophils into the skin in humans, especially in asthmatic subjects (Muro et al., 2003),
would be consistent with a role for this mediator in human inflammatory diseases. We have
detected 5-oxo-ETE in feline bronchoalveolar lavage fluid (Cossette et al., 2015) and
appreciable levels of this substance have also recently been detected in exhaled breath
condensate from humans (Kowal et al., 2017). Furthermore, allergen challenge of house dust
mite-sensitive human subjects resulted in significant increases in 5-oxo-ETE, suggesting that
it may play a role in asthma (Kowal et al., 2017). Consistent with this, anti-IgE was shown to
elicit the release of 5-oxo-ETE from human bronchial segments (Kolmert et al., 2018). 5-
Oxo-ETE may also play a role in the development of nasal polyps, as it is formed by
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epithelial cells from nasal polyps and was shown to increase the levels of eosinophil cationic
protein in organ cultures of nasal polyps (Lin et al., 2018).
Since the potent effects of 5-oxo-ETE are due to its interaction with a selective receptor,
rather than to its electrophilic properties (cf. (Schopfer et al., 2011)), a selective OXE
receptor antagonist that could block 5-oxo-ETE signaling would be an important asset in
determining the pathophysiological role of this lipid mediator and could offer a novel
therapeutic approach to alleviate the symptoms of eosinophilic diseases such as asthma,
allergic rhinitis and atopic dermatitis. We therefore sought to develop synthetic OXE
antagonists with high potency and resistance to metabolism. Using 5-oxo-ETE-induced
calcium mobilization in human neutrophils to screen potential antagonists, we identified
indoles containing adjacent hexyl and 3S-methyl-5-oxovalerate side chains (e.g. S-230, Fig.
1) as selective OXE antagonists (pIC50 ~ 8) (Gore et al., 2014). Because of the absence of the
OXE receptor in rodents we looked for an alternative animal model to test the effects of our
OXE antagonists in vivo. We first considered cats, which are prone to develop asthma, but,
although feline leukocytes respond well to 5-oxo-ETE, our antagonists were not very potent
in this species (Cossette et al., 2015), presumably due to differences between the feline and
human receptors, which are ~75% identical. We finally settled on cynomolgus monkeys,
which have an OXE receptor that is 95% identical to the human receptor. In contrast to its
modest effects on feline granulocytes, 230 is a potent inhibitor of 5-oxo-ETE-induced
activation of monkey granulocytes (Cossette et al., 2016). High levels of 230 could be
detected in the blood shortly after oral administration to monkeys at a dose of 30 mg/kg, but
its concentration declined rapidly due to extensive ω-oxidation of the hexyl side chain. In an
attempt to reduce ω-oxidation, we replaced the hexyl side chain of S-230 with a phenyl
group, resulting in an antagonist (S-C025) that is considerably more potent than S-230 and,
despite being cleared fairly rapidly from the blood, displays improved pharmacokinetic
properties (Chourey et al., 2018).
Although the monkey is an excellent animal model for investigation of human diseases
because of its similarity to humans, the cost of these experiments is very high, and the
experimental design is consequently limited due to the relatively small numbers of animals
that can be used. Before initiating studies using monkey models of allergic disease, we
wished to optimize the properties of the OXE antagonist to be tested in these costly
experiments. We therefore sought to optimize our lead compound, S-C025, with respect to
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both potency and pharmacokinetic properties by investigating the effects of a variety of
substituents on the phenyl ring. This resulted in the identification of S-Y048, which has
enhanced potency and an increased lifetime in blood, and is converted to an abundant long-
lasting plasma metabolite with nearly equivalent potency.
Methods
Ligands and antagonists
5-Oxo-ETE (Khanapure et al., 1998) and LTB4 (Zamboni & Rokach, 1982) were synthesized
as previously described. 5-Oxo-ETE was purified by reversed-phase (RP)-HPLC prior to use.
Eotaxin-1 (CCL11) was obtained from Cedarlane, Burlington, Ont, Canada, whereas
prostaglandin (PG) D2 was purchased from Cayman Chemical, Ann Arbor, MI, USA. The
procedures for the synthesis of all of the OXE receptor antagonists evaluated in this study
have been provided as Supplementary Material.
Measurement of intracellular calcium levels in human neutrophils.
The potencies of OXE receptor antagonists were evaluated by determining their effects on 5-
oxo-ETE-induced calcium mobilization in human neutrophils (Gore et al., 2014). Neutrophils
were prepared from healthy human subjects after removal of red cells by dextran
sedimentation and mononuclear cells by centrifugation over Ficoll-Paque. After labelling the
cells with indo-1, fluorescence was measured at 37 °C using a Cary Eclipse
spectrofluorometer (Agilent Technologies, Santa Clara, CA) equipped with a temperature-
controlled cuvette holder and a magnetic stirrer. After stabilization of the fluorescence,
antagonists were added, followed by addition of 5-oxo-ETE (10 nM) 2 min later and
digitonin (final concentration 0.1%) after a further 1 min to determine maximal fluorescence.
Measurement of polymerized actin.
F-Actin was measured in unfractionated leukocytes from whole blood obtained from healthy
human subjects, following removal of red blood cells using dextran and hypotonic lysis as
described previously (Gore et al., 2014), with some minor modifications. The leukocytes
were initially incubated with anti-CD16-Pe/Cy5 (1.5 µL/106 cells; mouse IgG1, clone 3G8;
BioLegend, San Diego, CA; catalogue number 302010; Lot no: B15039; RRID: AB_314210)
for 30 min on ice. After centrifugation and washing once with PBS, the cells were suspended
in PBS containing Ca++ (1.8 mM) and Mg++ (1 mM) at a concentration of 5.5 × 106 cells/mL.
Aliquots (90 μL) of the labeled cells were preincubated for 5 min at 37 °C with vehicle (1 μL
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DMSO) or S-Y048 (10 or 1000 nM), followed by addition of either vehicle (10 μL PBS
containing Ca++, Mg++, and 0.1% BSA), 5- oxo-ETE (final concentration, 10 nM), PGD2 (10
nM), LTB4 (10 nM) or eotaxin-1 (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
MeOH; final concentration, 0.3 μM) was added to each sample, followed by incubation
overnight in the dark at 4 °C. Immediately prior to data acquisition by flow cytometry (BD
LSRFortessa X-20, BD Biosciences, San Jose, CA), 300 μL of PBS was added to each
sample. Eosinophils were identified by high side scatter and low expression of CD16,
whereas neutrophils also displayed high side scatter but high CD16 expression.
Pharmacokinetic experiments in cynomolgus monkeys
Pharmacokinetic experiments were performed using six female cynomolgus monkeys (2.5 to
5 kg), housed at INRS-Institut Armand-Frappier, Laval, Quebec. All experiments were
performed in accordance with the guidelines of the Canadian Council on Animal Care as well
as the ARRIVE and BJP guidelines and were approved by the local institutional animal care
committee. Animals were housed in pairs in cages with a 12 h light/12 h dark schedule. They
received a diet consisting of Teklad Global chow for nonhuman primates supplemented with
fresh fruit and vegetables and breakfast cereal and had unlimited access to water. Monkeys
were first habituated to the oral gavage procedure on three successive days and then weighed
to determine the correct dose. Three days later all food was withdrawn at the end of the
afternoon and a blood sample was taken. The following morning, various doses of S-Y048,
S-70, or S-71 were delivered by oral gavage and blood samples (1 ml) were taken from the
femoral vein 0.5, 1, 2, 4, 8, 12, and 24 h later. Feeding was resumed after the first blood
sample was obtained at 30 min.
To provide a dose of 5 mg kg-1, OXE receptor antagonists (S-Y048, S-70, and S-71) were
dissolved in ethanol (25 mg mL-1) and stored at -80 °C before use. On the morning of the
experiment, the ethanolic solution was thawed and vortexed and the required amount added
to 10 volumes of 20 mM NaHCO3 (pH 8.0). The resulting suspension (2.2 mL kg-1; 9.1%
EtOH) was immediately vortexed and administered by oral gavage to a monkey. An identical
procedure was followed for doses of 2 mg kg-1, except that the antagonist (i.e. S-Y048) was
dissolved in ethanol at a concentration of 10 mg mL-1. In some cases, a second identical dose
of antagonist was administered 8 h after the first dose, immediately after obtaining an 8-h
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blood sample. Blood samples (1 to 2 mL) were collected in heparinized tubes 1 h prior to
gavage and 0.5, 1, 2, 4, 8, 12, and 24 h after gavage. If a second dose was administered, an
additional sample was collected 1 h later. The blood samples were centrifuged, and the
resulting plasma was frozen and stored at -80 °C prior to extraction and analysis.
Measurement of OXE antagonists and their metabolites in plasma
Plasma samples were thawed and diluted with 2 volumes of MeOH, prior to the addition of
internal standards. For analysis of S-Y048, the tetramethylene analog of C-025 (i.e. 5-(5-
chloro-2-(4-phenylbutyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid; 1.3 µg) was used as
the internal standard, whereas for S-70 and S-71, the corresponding pentamethylene analog
(i.e. 5-(5-chloro-2-(5-phenylpentyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic; 1 µg) was
used. The samples were stored overnight at -80 °C and the precipitated material was removed
by centrifugation. After adjusting the concentration of MeOH in the supernatant to 30% by
the addition of water, the sample was loaded onto a C18 Sep-Pak cartridge (Waters Corp),
which was washed with 30% MeOH, followed by the elution of the antagonist and its
metabolites with 100% MeOH. The solvent was then removed in vacuo using a rotary
evaporator, and the residue dissolved in 30% MeOH containing 2.5 mM H3PO4 and analyzed
by precolumn extraction-RP-HPLC using a modified Waters 2695 Alliance system (Waters
Corporation, Mississauga, Ontario, Canada) equipped with a Waters model 2996 photodiode
array detector. Automated precolumn extraction was performed as described previously
(Powell, 1987) using a C18 SecurityGuard cartridge (4 x 3 mm; Phenomenex, Torrance, CA)
coupled with a Kinetex C18 column (Phenomenex; see figure legends for precise
chromatographic conditions). All solvents used for extraction and chromatographic analysis
were purchased from Fisher Scientific, Markham, ON, Canada.
Normal-phase (NP) HPLC
NP-HPLC of α-hydroxy-Y048 stereoisomers was performed using an Econosphere column
(250 x 4.6 mm; 5 µm particle size; Alltech Associates, Deerfield, IL). The mobile phase was
hexane/isopropanol/acetic acid (98.4:1.5:0.1) at a flow rate of 1.5 mL min-1 and a temperature
of 30 °C.
Chiral HPLC
The S- and R- enantiomers of compounds 69-72 and Y-048 were separated by chiral HPLC
with a Cellulose-1 column (5 µm particle size; 250 x 4.6 mm; Phenomenex) as the stationary
phase. Except for compound 70, isocratic elution was used with a mobile phase consisting of
hexane/methanol/acetic acid (98.7:1.2:0.1) with a flow rate of 1.2 mL min-1 and a column
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temperature of 30 oC. For compound 70, the mobile phase was hexane/methanol/acetic acid
(98.4:1.5:0.1) and the flow rate 1.5 mL min-1.
Stereoisomers of synthetic α-hydroxy-Y048 as well as S-Y048M from plasma were analyzed
by chiral HPLC using a Cellulose-2 column (4.6 x 250 mm; 5 µm particle size; Phenomenex)
with hexane/ethanol/acetic acid (93:7:0.1) as the mobile phase. The flow rate was 1 mL min-1
and the column temperature 45 °C.
Identification of the major plasma metabolite of S-Y048 by liquid chromatography-tandem
mass spectrometry (LC-MS/MS)
To identify S-Y048M, column fractions corresponding to the peak at 23.5 min in Fig. 4F
were collected during the analysis of a plasma extract from blood obtained 12 h after
administration of S-Y048 (5 mg kg-1; see legend to Fig. 5F for further details). The combined
fractions were concentrated to dryness in vacuo and dissolved in methanol (100 µL) prior to
analysis by LC-MS/MS, which was performed 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), whereas the mobile phase was a linear gradient between solvents A (0.02% HOAc in
water) and B (0.02% HOAc in MeCN) as follows: 0.0 min, 30% B; 1.0 min, 30% B; 25.0
min, 55% B; 32.0 min, 55% B; 32.1 min, 90% B; 37.0 min, 90% B. The flow rate was 0.3
mL min-1, the column temperature 25°C and the injection volume 10 µL. Analyses were
performed in negative electrospray ionization (ESI) mode as follows: capillary temperature:
350 oC; source heater temperature: 300 oC; sheath gas flow: 20; auxiliary gas flow: 10;
source voltage: - 3.0 kV. The MS settings were: S lens RF level: 60%; automatic gain control
(AGC) target: 1 x 106 ions; mass range: m/z 250 to m/z 700; resolution: 100,000. Multiple
levels of MSn analysis in data-dependent acquisition (DDA) mode were used to identify S-
Y048M. In DDA mode, the selection of the precursor ion for MS2 analysis was based on the
chlorine isotope pattern and/or isolation of the top three most intense ions from the full MS
scan. MS2 settings were: collision-induced dissociation; signal threshold: 5,000; normalized
collision energy: 35; isolation width: 2 Da; activation time: 50 ms. MS3 was performed using
parent and product mass lists to trigger MS3 for selected ions and was performed with the
same settings as MS2 except that a normalized collision energy of 45 was used.
Data analysis
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Data are represented either as individual values or as means ± standard error. The statistical
significance of differences among multiple groups was evaluated using one-way ANOVA
with the Bonferroni test as a multiple comparison method. Differences between two groups
were evaluated using t-tests. Differences with a p value of less than 0.05 were considered to
be statistically significant.
Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).
Results
Effects of phenyl ring substituents on OXE antagonist potency
We first examined the abilities of the hydroxyphenyl compounds 74-76 to inhibit 5-oxo-ETE-
induced calcium mobilization in human neutrophils, but none was as potent as racemic C025
(pIC50, 9.61 ± 0.07; Fig. 2A). The most potent of these was the p-hydroxy compound 76,
which had a pIC50 about 0.3 units lower than that of C025, whereas those of the meta and
ortho OH-substituted phenyl analogs were about 0.9 and 1.4 units lower (Fig. 2A, 2F). A
different pattern emerged when we examined the methoxy-substituted analogs 71-73 (Fig.
2B). In this case, the p-methoxy compound (73) was the least potent with a pIC50 about 0.4
units lower than that of C025, whereas the ortho-methoxy compound (71) was equipotent and
the meta-methoxy compound (72) was more potent. A similar pattern was observed for the
fluoro-substituted analogs, with the m-fluoro compound (69) having a pIC50 about 0.5 units
higher than C025 and the ortho and para compounds being less potent (Fig. 2C). However,
the most potent among this series of compounds proved to be the m-chloro analog Y048,
which had a pIC50 of 10.47, 0.86 units higher than that of C025 (Fig. 2D). In contrast, the
ortho- and para- chloro compounds 66 and 67 were slightly less potent than C025.
Effect of the length of the methylene chain of Y048 on antagonist potency
To examine the effect of the length of the polymethylene chain connecting the m-
chlorophenyl group to the indole moiety we prepared the pentamethylene and
heptamethylene analogs of Y048. Reducing the polymethylene spacer by a single methylene
group (compound 83) had a large impact on antagonist potency, reducing it by over 50-fold
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(Fig. 2E). In contrast, addition of a methylene group, as in the heptamethylene analog 84, had
relatively little impact on potency.
Potencies of S and R enantiomers of OXE receptor antagonists
All of the data shown in Fig. 2 are for racemic compounds, whereas we have previously
shown that most of the antagonist activity of 5-oxo-3-methylvalerate-substituted indoles
resides in the S-enantiomers (Gore et al., 2014; Reddy et al., 2015). We therefore separated
the S and R enantiomers of some of the above antagonists by chiral HPLC and examined the
potencies of the purified enantiomers. We initially investigated the p-fluoro and o-methoxy
compounds, since they were among the first members of this series to be synthesized, and
then investigated the more potent meta-substituted compounds. Although the S/R potency
ratios differed from one substituent to another, in all cases the S-enantiomer was considerably
more potent than the corresponding R-enantiomer (Table 1). The greatest difference was seen
with the methoxy derivatives (ortho and meta), in which case the S-enantiomers had pIC50
values approximately 2.5 to 3 units higher than the R-enantiomers (Table 1 and Fig. 3B, 3C).
The difference between enantiomers was less with the fluoro compounds (Fig. 3A, 3D), for
which the S-enantiomers had pIC50 values about 1.1 to 1.7 units higher than the R-
enantiomers. Of all the compounds tested, the S-enantiomer of the m-chloro compound S-
Y048 (pIC50,10.81) was the most potent, with the corresponding R-enantiomer having a pIC50
1.6 units lower (Fig. 3E).
We also investigated the effects of increasing concentrations of S-Y048 on the concentration-
response curve to 5-oxo-ETE (Fig. 3F). Indo-1-loaded neutrophils were preincubated with S-
Y048 (20, 200, and 2000 pM) for 2 min prior to the addition of increasing concentrations (2,
20, 200, and 2000 nM) of 5-oxo-ETE and changes in intracellular calcium levels were
measured. The lowest concentration of S-Y048 tested (20 pM) resulted in a rightward shift in
the concentration-response curve for 5-oxo-ETE and appeared to reduce the maximal
response by about 20%. Higher concentrations of S-Y048 further reduced the maximal
response to 5-oxo-ETE, which was nearly undetectable at a concentration of 2 nM.
S-Y048 is selective for the OXE receptor
To ensure that S-Y048 is selective for the OXE receptor we compared its effects to other
mediators that activate eosinophils and/or neutrophils. S-Y048 (10 nM) completely blocked
actin polymerization induced in human eosinophils by 5-oxo-ETE (10 nM) (Fig. 4A). In
contrast, a 100-fold higher concentration of S-Y048 (i.e. 1 µM) had no effect on actin
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polymerization in eosinophils in response to identical concentrations of PGD2, LTB4 or
human eotaxin-1. Similarly, S-Y048 blocked 5-oxo-ETE-induced actin polymerization in
neutrophils but had no effect on the response to LTB4 (Fig. 4B). As expected, neutrophils did
not respond to either PGD2 or eotaxin-1.
In vivo metabolism of OXE receptor antagonists
Because of their much higher potency, we decided to synthesize the S-enantiomers of some
of the above compounds for further in vivo pharmacokinetic and metabolic studies. We
initially prepared the S-enantiomers of some of the first compounds that we synthesized for
the above in vitro studies, including the p-fluoro compound 70 and the o-methoxy compound
71. Once we realized the high potency of S-Y048, purified from racemic Y048, we also
prepared this compound by total synthesis. Our initial PK experiments were done with the S-
enantiomers of compounds 70 and 71. The antagonists were dissolved in ethanol, which was
added to bicarbonate, and the resulting suspension was administered by oral gavage at a dose
of 5 mg kg-1. Analysis of plasma samples taken 12 h later revealed that, as with the
unsubstituted phenyl compound S-C025 (Fig. 5A), substantial levels of S-70 (Fig. 5B) and S-
71 (Fig. 5C) were still present. At this time point, for each antagonist we observed a major
metabolite with a tR lower than that of the parent compound. Small amounts of other
unidentified metabolites were also present, but we did not observe any other major
metabolites with similar or different UV spectra. It is likely that glucuronide conjugates were
also formed, but did not reach high concentrations in plasma due to their rapid clearance. We
previously detected glucuronides in monkey plasma after administration of the related
compound S-230 (Chourey et al., 2017).
The UV spectrum for each of the above major metabolites and their parent compounds are
shown in Fig. 5, panels D and E. For each of these metabolites the peak at 303 nm for the
parent compound underwent a bathochromic shift to 311 nm, similar to what we previously
observed for the major plasma metabolite of S-C025, which contains a hydroxyl group on the
hexamethylene chain on the methylene group adjacent (i.e. alpha) to the indole (Chourey et
al., 2018).
We subsequently investigated the in vivo metabolism of the more potent m-chloro antagonist
S-Y048, which was also converted to a major plasma metabolite (S-Y048M; Fig. 5F) with
similar UV properties (Fig. 5G). To conclusively identify this metabolite, we isolated it from
plasma by RP-HPLC following oral administration of S-Y048 (5 mg/kg) and analyzed it by
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LC-MS/MS. S-Y048M had an [M-H]- ion at m/z 502.1598, compared to the theoretical value
of m/z 502.1557 expected for a hydroxy metabolite of S-Y048 (mass accuracy, 8 ppm). MS2
fragmentation of this ion (Fig. 5H) resulted in intense ions at m/z 484 (loss of H2O), 440 (loss
of H2O + CO2), and 292 (base peak, loss of the chlorophenylhydroxyalkyl side chain),
consistent with the presence of a hydroxyl group α to the indole. Fragmentation of the ion at
m/z 292 gave an MS3 spectrum (Fig. 5I) with intense ions at m/z 274 (loss of H2O), 248 (loss
of CO2), 206 (loss of CH2=CH-CH2-CO2H due to a McLafferty rearrangement), and 180. The
latter four ions were also observed in the MS2 spectrum shown in Fig. 5H. We previously
observed an intense ion at m/z 292 in the MS2 spectrum of the α-hydroxy metabolites of S-
230 (hexyl group in the 2-position of the indole) (Chourey et al., 2017) and S-C025
(phenylhexyl group in the 2-position of the indole) (Chourey et al., 2018), suggesting that S-
C048M also has a hydroxyl group in this position, and is therefore identical to α-hydroxy-S-
Y048.
Pharmacokinetics of OXE receptor antagonists
Fig. 6 shows the plasma levels of the above three compounds as well as S-C025 (Fig. 6A)
and their major plasma metabolites over 24 h following oral administration. Preliminary
experiments with S-70 (Fig. 6B; n=1) and S-71 (Fig. 6C; n=2) suggest that these compounds
have similar PK profiles to that of S-C025 (Table 2). In contrast, S-Y048 appears to have a
slightly lower but more prolonged maximal plasma concentration (Fig. 6D). Furthermore, the
t for S-Y048 is about 4 times longer than that for S-C025 (p < 0.05) and the area under the
½
curve is about 70% greater (p < 0.05) (Table 2). Thus, although the Cmax for S-Y048 tended
to be a little lower than that for S-C025 (not statistically significant), its concentration
dropped more slowly over the full 24 h period, at which time the plasma concentration of S-
Y048 was about 2.7 times higher than that of S-C025. However, the latter difference did not
quite reach statistical significance, probably due to the limited number of animals (n = 3) that
we could study due to the high cost of experiments with monkeys. Another difference
between these two antagonists is that the concentrations of the major plasma metabolite of S-
C025 (i.e. S-025M) exceeded those of S-C025 at times longer than 8 h (Fig. 6A), whereas the
plasma concentrations of S-Y048 exceeded those of S-Y048M at 24 h in all three animals in
this group (Fig. 6D).
With a single dose of 5 mg kg-1, the plasma levels of S-Y048 were maintained between about
2 and 12 µM over a period of 24 h (Fig. 6D). When a second dose was administered 8 h after
the first, the plasma levels of S-Y048 remained between about 5 and 12 µM over 24 h (Fig.
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6E). We also examined a lower dose of S-Y048 (2 x 2 mg kg-1, given 8 h apart), which
resulted in plasma levels between about 3 and 7 µM over a period of 24 h (Fig. 6F). In a
limited number of animals (n = 2) we measured the plasma concentrations of S-Y048 and S-
Y048M over 72 h following administration of S-Y048 at doses of 5 (Fig. 6G) and 2 x 2 (Fig.
6H) mg kg-1. The concentrations of S-Y048M declined much more slowly than those of its
precursor, and between 48 and 72 h were about 2 to 3 times higher with both dosing
regimens.
Potencies of major plasma metabolites of OXE receptor antagonists
To determine whether they possess antagonist activity, the metabolites of the o-methoxy, p-
fluoro, and m-chloro compounds were purified from plasma by RP-HPLC and their abilities
to block 5-oxo-ETE-induced calcium mobilization in neutrophils were compared to those of
their parent compounds (Fig. 7, panels A to C). In all cases, the plasma metabolites were
potent OXE receptor antagonists, their pIC50 values being only about 0.7 units lower than
their respective precursors (Table 1). S-Y048M was the most potent of the metabolites
examined, with a pIC50 of 10.15.
Chirality of the α-hydroxyl group in S-Y048M
To determine the chirality of the hydroxyl group alpha to the indole in S-Y048M and to
further investigate the antagonist properties of α-hydroxy-Y048 stereoisomers, we
synthesized a mixture of stereoisomers of this compound, using a procedure similar to that
used to prepare the corresponding α-hydroxy derivatives of S-230 (Chourey et al., 2017), and
separated them by a combination of normal phase and chiral HPLC. Although we had hoped
to synthesize principally the αS-OH and αR-OH stereoisomers of S-Y048, in our initial
synthesis appreciable amounts of the R-methyl stereoisomers were also formed (see
Supporting Information for further details), which enabled us to purify all four stereoisomers
and examine their antagonist potencies.
Synthetic α-OH-Y048 was separated into 2 peaks by normal phase-HPLC (Fig. 7D). The
material in peak A was then separated into 2 peaks, A1 (αR-OH-S-Y048) and A2 (αS-OH-R-
Y048) by chiral HPLC (Fig. 7E). Similarly, the material in peak B was separated into B1 (αS-
OH-S-Y048) and B2 (αR-OH-R-Y048) using identical chiral chromatography conditions (Fig.
7F). The above assignments of the chirality of the methyl and hydroxyl groups are based on a
combination of chiral synthesis and by analogy between the elution order of α-OH-Y048
stereoisomers and that of the corresponding stereoisomers of α-OH-230 (containing a hexyl
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side chain instead of a chlorophenylhexyl side chain), which we have characterized using
single crystal X-ray diffractometry (Chourey et al., 2017).
The effects of the 4 stereoisomers of α-OH-Y048 on 5-oxo-ETE-induced calcium
mobilization are shown in Fig. 7G. One of the 4 isomers (B1, αS-OH-S-Y048) was extremely
potent, with a pIC50 approximately equivalent to that of S-Y048, whereas the other 3
stereoisomers had pIC50 values approximately 2 to 3 units lower (Table 3).
A cochromatography experiment was performed to determine the chirality of the α-hydroxyl
group in S-Y048M. S-Y048M alone gave a single peak when examined by chiral HPLC (Fig.
7H). A chromatogram showing the elution positions of a mixture of synthetic αS-OH-S-Y048
and αR-OH-R-Y048 is shown in Fig. 7I, whereas Fig. 7J shows a mixture of these two
standards with S-Y048M. A chromatogram of all 4 of the synthetic stereoisomers, run
separately, is also shown in Fig. 7J (dashed lines). S-Y048 is clearly identical to αS-OH-S-
Y048 (Fig. 7K), based both on its antagonist potency and its chromatographic behaviour.
Discussion
The primary objective of the current study was to improve the pharmacokinetic properties
and the potency of S-C025 by modification of the phenyl ring. We initially tested the effects
of the ortho, meta, and para hydroxy derivatives of C025, which we had previously
synthesized as potential plasma metabolites of C025. However, we were unable to detect
significant amounts of any of these compounds in plasma, even with the highest dose (30 mg
kg-1) of C025 tested (data not shown). None of these hydroxy derivatives was as potent as
C025 in blocking the effects of 5-oxo-ETE, with pIC50 values between 0.3 and 1.4 units
lower than that of the racemic parent compound. Because of their reduced potency and the
fact that addition of a hydroxyl group might promote electrophilic substitution of the phenyl
ring, which could potentially lead to increased metabolism, we did not investigate the
hydroxyphenyl compounds further.
We next examined the antagonist potencies of a series of substituted phenyl compounds
containing methoxy, fluoro, and chloro substituents in different position of the phenyl ring.
Interestingly, in contrast to the hydroxyphenyl derivatives, among which the p-hydroxy
isomer was the most potent, among the halogen and methoxy substituted compounds that we
investigated, those with a meta substituent were consistently the most potent, and, in all
cases, were more potent than C025. This difference could possibly be related to the more
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polar nature of the hydroxyl group and its ability to form hydrogen bonds, either with
substituents on the receptor or with water molecules.
Of all the compounds we investigated, the m-chloro derivative Y048 was the most potent. In
our previous study, which focused on S-C025, we examined a series of phenylalkyl
compounds containing between 3 and 6 methylene groups and found the hexamethylene
analog (i.e. S-C025) to be the most potent. This also appears to be true for the m-
chlorophenyl series, in which case the hexamethylene compound (i.e. S-Y048) has a pIC50 is
over 1.7 units higher than the pentamethylene analog 83. In contrast, addition of another
methylene group (heptamethylene compound 84) did not have an appreciable effect on
potency.
To examine the relative potencies of the R- and S- enantiomers of some of the above
compounds, we initially used chiral chromatography to separate the enantiomers from the
racemic mixtures. As expected, in all cases the S-enantiomers were far more potent than the
R-enantiomers. We therefore decided to focus on the S-enantiomers for further in vivo
pharmacokinetic studies, and synthesized several of these compounds by chiral synthesis
using a procedure similar to the one we developed for the synthesis of S-230 (Reddy et al.,
2015). Because of the far greater quantities of antagonists required for studies in larger
animals, as well as the substantial cost of performing in vivo experiments with monkeys, we
were able to perform only a limited number of experiments with select compounds. We
initially prepared the S-enantiomer of the p-fluoro derivative 70, as the racemic form of this
compound was the first in this series to be synthesized. However, because its PK properties
did not appear to be substantially different from those of S-C025, and since it had somewhat
reduced potency, we did not pursue this compound further. We next prepared the S-
enantiomer of another of our early compounds, the o-methoxy derivative 71, which was
approximately equipotent with S-C025 in blocking the OXE receptor, but neither did this
compound offer any advantage over S-C025 with respect to its pharmacokinetic properties.
The last series of compounds that we synthesized and tested were those containing meta
substituents, of which the m-chloro derivative was the most potent, with the racemic
compound and the S-enantiomer having pIC50 values approximately 0.8 units higher than that
those of C025 and its S-enantiomer. We therefore prepared S-Y048 by total chemical
synthesis and found it to have a much longer half-life in blood as well as a higher area under
the curve compared to an equivalent dose of S-C025. Furthermore, the blood levels of S-
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Y048 could be maintained at a relatively constant level over 24 h by administration of 2
doses 8 h apart.
We previously showed that one of our earlier related antagonists (racemic 230) selectively
blocks 5-oxo-ETE-induced calcium mobilization in neutrophils, without affecting the
responses to the neutrophil agonists platelet-activating factor, fMLP and interleukin-8 (Gore
et al., 2014). Because our ongoing in vivo studies are focused on eosinophils, we wanted to
ensure that S-Y048 is selective for the OXE receptor compared to other receptors involved in
the activation of these cells. Taking advantage of flow cytometry to distinguish between
eosinophils and neutrophils we found that S-Y048 has no effect on responses of eosinophils
mediated by the DP2, BLT1, or CCR3 receptors or responses of neutrophils mediated by the
BLT1 receptor.
S-Y048 is a lipophilic molecule with a calculated Log P (cLogP) value of 5.50, and it is
possible that this may contribute to its long lifetime in the blood, as it will be bound to
plasma proteins. Although this could theoretically compromise its in vivo efficacy, we have
recently shown that this antagonist can block 5-oxo-ETE-induced eosinophil infiltration into
the skin of rhesus monkeys and significantly inhibit dermal eosinophilia in response to
intradermal injection of house dust mite antigen (Miller et al., 2019). These results clearly
demonstrate that the lipophilic nature of S-Y048 does not prevent its in vivo activity in
primates, possibly due to its strong binding to the OXE receptor, as it appears to be an
insurmountable antagonist, as shown in Fig. 3F. Prolonged receptor residence time due to
slow dissociation from a receptor has been shown to be an important determinant of in vivo
drug efficacy (Seow et al., 2016). Furthermore, its main plasma metabolite S-hydroxy-S-
Y048M) has nearly equivalent potency, a long lifetime in the circulation, and is less
lipophilic due to its additional hydroxyl group (cLogP 3.75), and is therefore likely to
contribute to the effects of S-Y048, especially at later time points.
The high potencies of αS-hydroxy-S-Y048 and the corresponding metabolite of S-C025, αS-
hydroxy-S-C025, are very interesting. We identified a similar αS-hydroxy metabolite of S-
230 (Chourey et al., 2017), but there was a much greater loss of potency (over 2 log units)
compared to the corresponding metabolites of the phenyl antagonists. It is clear from our
earlier studies that the terminal hydrophobic portion of 5-oxo-ETE is required for its
biological activity (Patel et al., 2008), and the addition of a hydrophilic group to this part of
the molecule might be expected to interfere with its interaction with the OXE receptor.
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However, the presence of a hydroxyl group in the equivalent position of 5-oxo-ETE in the
15-lipoxygenase-generated metabolite 5-oxo-15S-HETE, has a relatively modest inhibitory
effect on agonist activity (O'Flaherty et al., 1996; Powell et al., 1995; Schwenk & Schröder,
1995), suggesting that a hydroxyl group may be tolerated in this position. The αR-hydroxy
derivatives of S-Y048, S-C025, and S-230 were all far less potent than the metabolically-
formed αS-hydroxy derivatives. This might possibly be due to differences in the degree of
hydrogen bonding between the α-hydroxyl group and the oxo group of the 5-oxovalerate side
chain, which could possibly result in a less favorable conformation for the αR-hydroxy
compounds, thereby impeding their interaction with the OXE receptor.
Because of its potency and prolonged lifetime in the blood, αS-hydroxy-S-C048 (S-C048M)
might in itself be an interesting drug candidate, especially if it is well-absorbed from the GI
tract. The slow rate of decline in its plasma concentration over 3 days following
administration of S-Y048 suggests that once-daily administration of a relatively low dose
might be feasible. However, the two chiral centers of αS-hydroxy-S-Y048 make its large-
scale synthesis more challenging, and we have not yet been able to test this hypothesis.
In conclusion, we have identified a potent OXE receptor antagonist with a pIC50 of about
10.8 (~20 pM) in inhibiting calcium mobilization induced by a 500-fold higher concentration
of 5-oxo-ETE. S-Y048 appears rapidly in the blood after oral administration, and remains at
relatively high levels over 24 h, especially when administered twice daily. In the present
study, S-Y048 was administered as a suspension in bicarbonate buffer, but with improved
formulation, it should be possible to extend its lifetime in the blood even further. Our initial
data from in vivo studies in rhesus monkeys demonstrate that S-Y048 has in vivo efficacy in
inhibiting allergen-induced infiltration of eosinophils into the skin (Miller et al., 2019) and
lungs (unpublished data). The high potency of S-Y048 together with its favorable PK
properties suggest that this OXE receptor antagonist may be a credible drug candidate for the
treatment of eosinophilic diseases in humans, such as atopic dermatitis, asthma, and allergic
rhinitis.
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Figure 1. Structures of OXE receptor antagonists.
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Figure 2. Effects of ortho- meta- and para- substituents on antagonist potency. The effects
of analogs of racemic 025 containing hydroxyl (A), methoxyl (B), fluoro (C), and chloro (D)
substituents in the ortho (o; ∆), meta (m; ●), and para (p; ▼) positions on 5-oxo-ETE-induced
calcium mobilization were determined. E: Effect of the length of the polymethylene spacer (C5,
∆; C6 (i.e. Y048), ●; C7, ▼) on the potency of m-Cl-substituted antagonists. E: Table showing
the pIC50 values for all of the compounds depicted in panels A to E. The values are means ±
SE with the numbers of independent experiments indicated in brackets. All compounds are
racemic mixtures. Data for C025 (○; pIC50, 9.61 ± 0.07 (n = 10)), which was included in this
experiment for comparison, are also shown.
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Figure 3. Effects of the S and R enantiomers of OXE-R antagonists on 5-oxo-ETE-
induced calcium mobilization. The effects of the S (●) and R (○) enantiomers of A: p-fluoro
(70), B: o-methoxy (71), C: m-methoxy (72), D: m-fluoro (69), and E: m-chloro (Y048)
substituted phenyl antagonists on calcium mobilization induced in human neutrophils by 10
nM 5-oxo-ETE are shown. The response to S-C025 (Δ; pIC50, 10.06 ± 0.01) is included for
comparison. The values are means ± SE (See Table 1 for numbers of experiments). F: Effects
of increasing concentrations of S-Y048 (S48) on the concentration-response curve for 5-oxo-
ETE-induced calcium mobilization in human neutrophils. Either vehicle (●) or different
concentrations (20 pM, ; 200 pM, ▲, or 2000 pM, ) of S-Y048 were added to indo-1-loaded
neutrophils, followed 2 min later by the addition of different concentrations of 5-oxo-ETE, (see
Methods section for further details).
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Figure 4. Selectivity of S-Y048 for the OXE receptor. Unfractionated human leukocytes
labeled with anti-CD16-PE/Cy5 were incubated for 5 min with either vehicle or concentrations
of S-Y048 (S48) of either 10 nM (5-oxo-ETE) or 1 µM (all other agonists). 5-Oxo-ETE, PGD2,
LTB4, or eotaxin-1 (all 10 nM) were then added and incubations were terminated after a further
20 s by the addition of formaldehyde. Polymerized F-actin was measured in eosinophils and
neutrophils by flow cytometry as described in the Methods section.S-C025 (F, G), all at doses
of 5 mg/kg, were administered to monkeys by oral gavage and blood samples (1 ml) were taken
after 12 h. Internal standards (tetramethylene analog of C025 for S-C025 and S-Y048 and
pentamethylene analog of C025 for compounds S-70 and S-71) were added to the plasma
samples, which were extracted and analyzed by RP-HPLC. For S-C025 (panel A), the data was
taken from a previous study (Chourey et al., 2018) employing a Novapak C18 column and a
gradient of 70 to 100% MeCN over 15 min. For panels B and C a Kinetex C18 column (2.6
µm particle size; 100 x 4.6 mm) was used with a gradient between 38 and 65% MeCN over 35
min, followed by 5 min at 65% MeCN. For panel F, a Kinetex C18 column (5 µm particle size;
250 x 4.6 mm) was used with a gradient between 55 and 75% MeCN over 30 min, followed by
5 min at 75% MeCN. All solvents contained 0.02% HOAc. The flow rates were 1 ml/min and
the column temperature was maintained at 30 oC. The UV spectra of S-70, S-71, and S-Y048
(broken lines) along with their major plasma metabolites (red solid lines) are shown in panels
D, E, and G, respectively. H: MS2 fragmentation of the [M − H]− ion at m/z 502 for S-Y048M,
isolated from plasma as shown in panel F. I: MS3 fragmentation of the ion at m/z 292 shown
in panel H. The ion at m/z 206 is formed as the result of a McLafferty rearrangement.
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Figure 5. Identification of major plasma metabolites of OXE receptor antagonists. S-
C025 (A), S-70 (p-fluoro-S-C025) (B, D), S-71 ( (o-methoxy-S-C025) (C, E), and S-Y048 (m-
chloro-S-C025 (F, G), all at doses of 5 mg/kg, were administered to monkeys by oral gavage
and blood samples (1 ml) were taken after 12 h. Internal standards (tetramethylene analog of
C025 for S-C025 and S-Y048 and pentamethylene analog of C025 for compounds S-70 and
S-71) were added to the plasma samples, which were extracted and analyzed by RP-HPLC. For
S-C025 (panel A), the data was taken from a previous study (Chourey et al., 2018) employing
a Novapak C18 column and a gradient of 70 to 100% MeCN over 15 min. For panels B and C
a Kinetex C18 column (2.6 µm particle size; 100 x 4.6 mm) was used with a gradient between
38 and 65% MeCN over 35 min, followed by 5 min at 65% MeCN. For panel F, a Kinetex C18
column (5 µm particle size; 250 x 4.6 mm) was used with a gradient between 55 and 75%
MeCN over 30 min, followed by 5 min at 75% MeCN. All solvents contained 0.02% HOAc.
The flow rates were 1 ml/min and the column temperature was maintained at 30 oC. The UV
spectra of S-70, S-71, and S-Y048 (broken lines) along with their major plasma metabolites
(red solid lines) are shown in panels D, E, and G, respectively. H: MS2 fragmentation of the
[M − H]− ion at m/z 502 for S-Y048M, isolated from plasma as shown in panel F. I: MS3
fragmentation of the ion at m/z 292 shown in panel H. The ion at m/z 206 is formed as the
result of a McLafferty rearrangement.
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Figure 6. Levels of OXE receptor antagonists and their major metabolites in plasma
following oral administration. OXE receptor antagonists were administered to cynomolgus
monkeys by oral gavage as described in Methods. Blood samples were collected after various
times and the plasma was subjected to solid-phase extraction and the amounts of unmetabolized
antagonist (●) along with their major plasma metabolite (○) were measured by RP-HPLC, using
appropriate internal standards as shown in Fig. 5. Data for S-C025 (5 mg/kg; n = 3), taken from
a previous study (Chourey et al., 2018), are shown in panel A. B: S-70 (p-fluoro; 5 mg/kg; n =
1); C: S-71 (o-methoxy; 5 mg/kg; n = 2); D: S-Y048 (m-chloro; 5 mg/kg; n = 3); E: S-Y048
(5 mg/kg followed 8 h later by a second dose of 5 mg/kg); F: S-Y048 (2 mg/kg followed 8 h
later by a second dose of 2 mg/kg). In two animals, measurements of S-Y048 and S-Y048M
(M) were made up to 72 h for doses of S-Y048 of 5 mg/kg (G) and 2 x 2 mg/kg (H).
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Figure 7. Antagonist potencies of the major plasma metabolites and their synthetic
stereoisomers of some OXE receptor antagonists. The major plasma metabolites of S-71 (o-
methoxy-S-C025) (A), S-70 (p-fluoro-S-C025) (B), and S-Y048 (m-chloro-S-C025) (C)
weremobilization in neutrophils. The values for antagonists (●) and their metabolites (○) are
means ± SE (n = 3), except for the data shown in panel C (n = 6). Panels D-F: αRS-Hydroxy-
RS-Y048 was prepared using the chiral synthon methyl (R)-5-chloro-3-methyl-5-
oxopentanoate (R:S = 9:1), which in this preparation resulted in mixture of stereoisomers of α-
OH-Y048 in which the S:R ratio for the methyl group in the acyl side chain of the final product
was about 4:1. D: Separation of RR/SS enantiomer pair from RS/SR enantiomer pair by NP-
HPLC. E: Separation of the SR and RS enantiomers from Peak A in panel D by chiral HPLC.
F: Separation of the SS and RR enantiomers from Peak B in panel D by chiral HPLC. G: Effects
of synthetic S-Y048 (□), A1 (○; αR-hydroxy-S-Y048), A2 (▲; αS-hydroxy-R-Y048), B1 (●,
αS-hydroxy-S-Y048), and B2 (; αR-hydroxy-R-Y048) on 5-oxo-ETE-induced calcium
mobilization in human neutrophils. H: Chiral HPLC of S-Y048M after isolation from plasma
by RP-HPLC. I: Chiral HPLC of a mixture of synthetic αS-OH-S-Y048 and αR-OH-R-Y048;
J: Cochromatography of S-Y048M with the standards shown in panel I. The dashed blue curve
shows a mixture of all 4 synthetic stereoisomers, run separately. K: Structures of αS-hydroxy-
S-Y048 (SS) and αR-hydroxy-S-Y048 (SR). See the Methods section for the NP and chiral
HPLC conditions.
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