(OXE) receptor antagonists that block the actions of the granulocy

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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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Journal of Medicinal Chemistry

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 Choureya, Qiuji Yea, Chintam Nagendra Reddya, Rui Wanga, Chantal Cossetteb, Sylvie Gravelb, Irina Slobodchikovac, Dajana Vuckovicc, Joshua Rokacha, and William S. Powellb*

a

Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150

West University Boulevard, Melbourne, FL 32901-6982, USA b

Meakins-Christie 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

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT 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-ETEinduced 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.


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INTRODUCTION 5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a metabolite of arachidonic acid formed along with leukotrienes by the 5-lipoxygenase pathway.1 It is produced by the oxidation of the 5lipoxygenase product 5S-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by the selective enzyme 5-hydroxyeicosanoid dehydrogenase in the presence of NADP+ (Fig. 1).2 5-Oxo-ETE is a potent chemoattractant for human eosinophils,3 neutrophils,4 basophils,5, 6 and monocytes.7 Its actions are mediated by the OXE receptor (OXE-R),8-10 which is most highly expressed on eosinophils.10 This receptor is highly selective for 5-oxo-ETE, with a variety of closely related compounds, including various 5-oxo-ETE metabolites, having only weak effects.2 The response to activation of OXE-R is mediated by the Gi protein subfamily, primarily through the βγ subunits.11

Figure 1. Inhibition of 5-oxo-ETE-induced eosinophil activation by the OXE-R antagonists 230 and 264.

5-Oxo-ETE induces a variety of responses in eosinophils, including calcium mobilization, actin polymerization, increased surface expression of CD11b and CD69, and L-selectin shedding.2, 12 It also elicits the respiratory burst and promotes degranulation, especially in the presence of cytokines such as GM-CSF.13, 14 Among lipid mediators, 5-oxo-ETE is the most powerful chemoattractant for human eosinophils.3 It is a potent inducer of the transendothelial migration of these cells, due to both its chemoattractant properties and its ability to activate protease



pathways, resulting in the degradation of matrix components, thereby facilitating the passage of eosinophils.15, 16 Intradermal injection of 5-oxo-ETE in humans results in infiltration of eosinophils into the skin, a response that is much more intense in asthmatic subjects compared to healthy controls.17 Because of its potent effects on eosinophils, 5-oxo-ETE 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 5-oxo-12S-HETE, a metabolite formed by the action of platelet 12-lipoxygenase on 5-oxo-ETE.18 This substance inhibited 5-oxo-ETEinduced calcium mobilization in human neutrophils with an IC50 of about 500 nM, but was not 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 OXE-R antagonist.11 We identified stable indole-based antagonists bearing substituents resembling the regions of 5-oxo-ETE that are required for agonist activity. The most potent of these are 230 and 264, which are chloroindoles bearing adjacent 3-methyl-5-oxovalerate and hexyl groups on the pyrrole ring of the indole (Fig. 1).19 They are about 10 times more potent than Gue1654 as OXE-R antagonists.19 Both contain a single chiral carbon, with the Senantiomers (IC50 ~10 nM) being several hundred times more potent than the R-enantiomers.19, 20 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 8 h.21 The major metabolites identified in plasma were ω2-hydroxy-264 and ω2-oxo-264 (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 fairly rapidly to about 2.7 µM by 8 h.22 ω2-Oxidation is also the major metabolic pathway detected for 230, but in this case, substantial amounts of α-hydroxy products were also formed.23 The S enantiomer of 230 (S-230) appears to be metabolized more rapidly than the R-enantiomer, with plasma levels of about 36 µM 1 h after oral administration of an identical dose, dropping to 1.6 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 ω2-oxidation 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 OXE-R 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.



RESULTS Effect of a terminal trifluoromethyl group on 5-oxo-ETE-induced 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 ωoxidation. This compound was synthesized as shown in Scheme 1. A Wittig reaction between the Wittig salt 2 and the aldehyde 322 using LiHMDS as a base yielded a mixture of cis and trans olefins (cis/trans:70/30),24 which were hydrogenated using 10% Pd/C to afford 5. A FriedelCraft’s acylation of 5 using Me2AlCl as a Lewis acid and 3-methylglutaric anhydride (6) as an acylating agent yielded compound 7.

Scheme 1. Synthesis of 7. Reagents and conditions: (a) PPh3, CH3CN, reflux, 16 h, 95%; (b) LiHMDS, THF, -78 oC – rt, 2 h, 44%; (c) 10% Pd/C, EtOH, H2, rt, 3 h, 99%; (d) 6, Me2AlCl, CH2Cl2, rt, 45 min, 58%.

Figure 2. Effect of the ω-trifluoro analog of 230 (7) () on 5-oxoETE-induced 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 5-oxo-ETE-induced calcium mobilization in human neutrophils, which is our standard screening assay for OXE receptor antagonists. Unfortunately, this compound is less potent than 6 ACS Paragon Plus Environment

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230, with an IC50 of 38 ± 11 nM compared to 17 ± 3 nM for racemic 230 (Fig. 2). For this reason, and because we subsequently identified much more potent antagonists (see below), we did not conduct any further studies with the trifluoro compound. Scheme 2. Synthesis of N-acyl series antagonists (12 a-d). Reagents and conditions: (a) KHMDS/LiHMDS, THF, -78 oC – 0 oC, 3 h, 80-99%; (b) 10%Pd/C, EtOH, H2, rt, 3 h, 90 – 95%; (c) 6, tBuOK, THF, 0 °C – rt, 3 h, 65 – 75%.

Synthesis of phenylalkyl analogs of 230 and 264 Our next strategy to block ω-oxidation 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 (12a-d) containing a 3-methyl-5-oxovaleric acid substituent in the 1-position of 6-chloroindole and in the 2-position, 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 by altering the length of the Wittig salts (compounds 9a-d). The aldehyde 819 was reacted with the Wittig salts 9a-d using KHMDS or LiHMDS as a base to yield the olefins 10a-d, which were subsequently hydrogenated using 10% Pd/C to afford 11a-d. Finally, acylation of the indole ring at the 1-postion in 11a-d with 3-methylglutaric anhydride (6) using tBuOK as a base yielded a series of N-acyl antagonists (compounds 12a-d).



Figure 3. Effects of phenylalkyl indole OXE-R antagonists on 5-oxo-ETE-induced calcium mobilization in human neutrophils. The effects on the response to 5-oxo-ETE (10 nM) of replacing the hexyl groups of A: 264 (∆) and B: 230 (∆) with a phenyl group connected to the 2-position 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 cellulose-1 column with hexane/MeOH/HOAc (97.9:2: 0.1) as the mobile phase (flow rate 2 ml/min; temperature 30 oC). D: Effects of S-C025 () and R-C025 (○), purified from racemic C025, on 5-oxo-ETE-induced 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 N-acylindoles (12a-d) were determined by assessing their effects on 5-oxo-ETE-induced calcium mobilization (Fig. 3A). Insertion of a 3carbon spacer (12b) between the phenyl group and the indole reduced potency by over 10-fold compared to racemic 264 (Table 1), whereas 4-carbon (12c) or 6-carbon (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 8fold (IC50, 2.1 nM).


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Table 1. Potencies of OXE receptor antagonists. The IC50 values (± SE with the number of independent experiments shown in brackets) of 1-acylindoles related to 264 are shown on the left, whereas those for 3acyl indoles related to 230 are shown on the right. All compounds are racemic unless otherwise indicated. --- 1-acyl series ---

a

--- 3-acyl series ---

Cpd #

n

IC50 (nM)

Cpd #

n

IC50 (nM)

264

-

17 ± 2 (11)

230

-

26 ± 4 (13)

C149 (12a)

5

2.1 ± 0.3 (6)

C025 (17a)

6

0.28 ± 0.07 (5)

12b

3

210 ± 90 (2)

17b

3

223 ± 18 (2)

12c

4

18 ± 1 (3)

17c

4

38 ± 8 (4)

12d

6

12 ± 3 (5)

17d

5

11 ± 1 (7)

S-230

-

6 ± 2 (4)a

S-C025

6

0.12 ± 0.05 (6)

R-C025

6

23 ± 2 (3)

From ref. 19

A similar series of 3-acyl-2-phenylalkyl-1-methylindoles was synthesized as shown in Scheme 3. In this case, the olefins 13a-d, 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 9ad. Subsequent hydrogenation of 13a-d using 10% Pd/C furnished compounds 14a-d, which were acylated at the 3-position of indole using Me2AlCl as a Lewis acid and methyl 5-chloro-3methyl-5-oxopentanoate (15) as an acylating agent to afford 16a-d. Finally, basic hydrolysis of the methyl esters 16a-d yielded the desired 3-acyl series of antagonists (compounds 17a-d). Scheme 3. Synthesis of 3acyl series antagonists. (a) KHMDS/LiHMDS, THF, 78 oC – 0 oC, 3 h, 80 – 90%; (b) 10%Pd/C, H2, EtOH, rt, 3 h, 95%-quant; (c) 17, Me2AlCl, CH2Cl2, rt, 3 h, 90 – 95%; (d) LiOH.H2O, THF/H2O (4:1), MeOH, rt, 12 – 18 h, 85 – 95%.



As in the 1-acyl series, the presence of a phenyl group with a trimethylene spacer (17b) in the 3acyl series reduced potency by about 8-fold 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 (compound C025) dramatically increased potency by nearly 100-fold (IC50, 0.28 ± 0.07 nM) compared to 230.

In view of the selectivity of the OXE receptor for the S-enantiomer 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 earlier-eluting peak was the S-enantiomer 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 S-enantiomer of C025 was nearly 200 times more potent than the R-enantiomer in blocking the response to 5-oxo-ETE (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 RP-HPLC in plasma samples obtained at various times up to 24 h (Fig. 4A). The maximal level


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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: RP-HPLC 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 H2O, both containing 0.02% HOAc. The flow rate was 1 ml/min and the temperature 35 oC. 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: RP-HPLC 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 RP-HPLC (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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Table 2. PK data for OXE-R 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; t1/2: time difference between tmax and the time at which the plasma concentration dropped to 50% of Cmax. AUC: area under the curve between 0 and 24 h.

Dose Antagonist n (mg/ml) tmax (h) Cmax (µM) t½ (h) Rac 264a 4 30 0.9 ± 0.1 27 ± 11 1.2 ± 0.5 Rac C149

1

30

2.0

Rac 230b

4

30

Rac C025

2

55

AUC (nmol x h x ml-1) 55 ± 17

3.0

294

0.6 ± 0.1 121 ± 22

0.7 ± 0.2

198 ± 40

30

2.5 ± 1.5 108 ± 39

7.4 ± 0.6

1,250 ± 321

C025→S 2

15

0.8 ± 0.3

32 ±15

3.4 ± 1.3

169 ± 54

C025→R 2

15

2.5 ± 1.5

79 ± 20

9.3 ± 1.5

1,081 ± 266

S-230c

3

30

1.0 ± 0.0

36 ± 7

1.6 ± 0.0

100 ± 20

S-C025

3

1

0.7 ± 0.2

4.5 ± 1.1

0.9 ± 0.3

13 ± 1

“

3

2

0.7 ± 0.2

7.8 ± 1.8

1.1 ± 0.3

26 ± 3

“

3

5

1.2 ± 0.4

18 ± 3.8

1.9 ± 0.2

78 ± 12

“

3

10

1.2 ± 0.4

35 ± 1

2.8 ± 1

“

3

30

1.3 ± 0.3 106 ± 21

d

3.5 ± 0.6

171 ± 13 d

684 ± 119 e

a

Calculated from data in ref 21

b

Calculated from data in ref 22

c

Calculated from data in ref 23. Note that data was available for only 1, 4, 6, 12, and 24 h

d

p < 0.05 compared to S-230 (30 mg/kg)

e

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 12 ACS Paragon Plus Environment

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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.

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 µM) was comparable to that achieved with 230, shown from a previous study19 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 RP-HPLC 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 20-fold difference in the scale of the Y-axis 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. αhydroxy-230).23



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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 R-enantiomer was higher than that of the S-enantiomer at all time points (Fig. 5B), and increased with time from 62% of total C025 at 30 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 t½ almost 3 times longer (Table 2). The plasma 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: ChiralHPLC of C025, isolated after RP-HPLC of an extract of plasma taken 4 h after oral administration of racemic C025 (30 mg/kg). The stationary phase was a Cellulose-1 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 S- () and R- (▲) 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 Cellulose-1 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 RP-HPLC as shown in Fig. 4E, and then subjected to chiral HPLC. Two peaks (M1 and M2) with UV spectra identical to that of C025M were observed, with M2 predominating after 4 h (Fig. 5D) and M1 predominating after 24 h (Fig. 5E). As shown in Fig. 5C, the proportion of M1 gradually increased, following a time course similar to, but lagging behind, R-C025, and accounting for 83% of total C025M after 24 h. Similarly, the time course for M2 lagged behind that of S-C025, suggesting that M2 is derived from S-C025 (i.e. S-methyl) and M1 from R-C025 (i.e. R-methyl). This was subsequently confirmed by studies on the metabolism of S-C025 as well as by total chemical synthesis of M2 (see below).

Scheme 4. Synthesis of 20 (S-C025). Reagents and conditions: (a) 18, Me2AlCl, CH2Cl2, rt, 3 h, 93%; (b) LiOH.H2O, THF/H2O (4:1), MeOH, rt, 16 h, 95%.

Pharmacokinetics of S-C025 To directly assess the PK of the S-enantiomer of C025 (20), this compound was prepared by chiral synthesis (Scheme 4), using a method similar to that used for C025, but employing the chiral synthon 18, which was prepared as described previously. 25 Analysis of plasma extracts by RP-HPLC 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 study23) in Fig. 6A. The maximal plasma level (~100 µM) of S-C025 was similar to that for



racemic C025, but its concentration declined more rapidly (t½ 3.5 h compared to 7.4 h for C025; 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), t½, (p < 0.05) and AUC (p < 0.01) (Table 2). A caveat to the 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), t½ (3.7 h; p < 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: Concentration-response curves for the inhibition of 5-oxoETE-induced 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 10, 5, 2, and 1 mg/kg (Fig. 6C; note that a log scale is used for the Y-axis 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 5-oxo-ETE-induced calcium mobilization in human neutrophils. As shown in Fig. 6E, S-C025M is a potent OXE receptor antagonist with an IC50 of 0.69 ± 0.36 nM, compared to 0.12 ± 0.05 nM for S-C025.

Figure 7. Mass spectra of S-C025M isolated from plasma. A: MS2 fragmentation of the [MH]- ion at m/z 468 for S-C025M. B: MS3 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 LC-MS/MS analysis of S-C025M revealed an [M-H]- 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, 3.4 ppm). MS2 fragmentation of this ion (Fig. 7A) resulted in a single major ion at m/z 292 (loss



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 H2O). In addition, ions of low intensity were observed at m/z 406 (loss of H2O and CO2), m/z 274 (loss of H2O from m/z 292), 248 (loss of CO2 from m/z 292) and 206 (loss of CH2=CH-CH2-CO2H from m/z 292 due to a McLafferty rearrangement). The MS3 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 α-hydroxy-S23023, which also had an intense ion at m/z 292 due to loss of the hydroxyhexyl side chain.

Scheme 5. Synthesis of α-OH-C025. Reagents and conditions: (a) Mg, THF, reflux; (b) THF, 0 °C – rt, 3 h, 56%; (c) TBDMSCl, imidazole, CH2Cl2, rt, 3 h, 88%; (d) 15, Me2AlCl, CH2Cl2, 0 °C – rt, 12 h, 44%; (e) LiOH. H2O, THF/H2O (4:1), MeOH, rt, 48 h, 89%.

To provide further evidence for the structure of S-C025M and to investigate the chirality of the α-hydroxyl group and the antagonist potency of the different stereoisomers of this compound, we prepared a mixture of α-hydroxy-C025 stereoisomers (26) by total chemical synthesis as shown in Scheme 5. The α-OH 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 3-position of the indole ring. In the acylation reaction of 24 using Me2AlCl as a Lewis acid, the TBDMS group was removed by 18 ACS Paragon Plus Environment

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Me2AlCl to obtain the alcohol 25. Subsequent hydrolysis of 25 under basic conditions furnished 26 (α-OH-C025).

Synthetic α-hydroxy-C025, which consisted of 4 diastereomers in equal proportions, was separated into two peaks by NP (normal-phase)-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 (A1 and A2) by chiral HPLC (Fig. 8D). Similarly, the material in peak B was separated into B1 and B2 by chiral HPLC (Fig. 8E). S-C025M gave a single peak when analyzed by chiral HPLC (Fig. 8F), which cochromatographed with synthetic peak B1 (Fig. 8G).

By analogy with our previous study on the α-hydroxylation of S-230 in the monkey, it seemed likely that S-C025M/peak B1 was identical to αS-hydroxy-S-C025. To confirm this, we prepared the latter compound by chiral synthesis as shown in Scheme 6 using a method similar to that which we described previously for the synthesis of αS-OH-S-230.23 In this synthesis, an (S)BINOL-mediated enantioselective addition of the Grignard reagent 22 to the prochiral aldehyde 3 yielded the chiral alcohol 27, 92.2% of which was the S-enantiomer. The alcohol 27 was protected using a silyl ether to obtain 28. Acylation of 28 using the acyl chloride 18 and Me2AlCl, followed by removal of TBDMS and ester hydrolysis yielded 29 (αS-OH-S-C025). Authentic αS-OH-S-C025 (Fig. 8J) had the same retention time as stereoisomer B1 and SC025M, isolated from plasma, when chromatographed by NP-HPLC (data not shown) and chiral HPLC (Fig. 8H).



Figure 8. Identification of S-C015M by cochromatography and its effect on 5-oxo-ETE-induced calcium mobilization. A: NP-HPLC of a synthetic mixture of α-hydroxy-C025 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: NP-HPLC as described above of S-C025M, isolated from plasma. C: Cochromatography of synthetic α-hydroxy-C025 and S-C025M by NP-HPLC as described above. D: Chiral HPLC of peak A from panel A using a Cellulose-2 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 α-hydroxy-C025 (peak B) and S-C025M by chiral HPLC as described above. H: Chiral HPLC of synthetic αS-hydroxy-SC025. I: Concentration-response curves for the inhibitory effects on 5-oxo-ETE-induced calcium mobilization of S-C025 (●), S-C025M isolated from plasma (○), and the 4 stereoisomers purified from synthetic α-hydroxy-C025: A1 (▼), A2 (∇), B1 (▲), and B2 (∆) (n = 3). J: Structure of αS-hydroxy-SC025 (S-C025M).


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Scheme 6. Synthesis of 29 (αS-OH-S-C025). Reagents and conditions: (a) 22, (S)-BINOL, Ti(OiPr)4, tBuOMe, BDMAEE, THF, -15 °C – rt, 36 h, 38%; (b) TBDMSCl, imidazole, CH2Cl2, rt, 16 h, 94%; (c) (1) 18, Me2AlCl,CH2Cl2; (2) HF·Pyridine, CH3CN; (3) LiOH·H2O, THF/H2O (4:1), MeOH, rt, 14 h, 23% over three steps.

The effects of all 4 stereoisomers of α-hydroxy-C025 on 5-oxo-ETE-induced calcium mobilization were compared to those of synthetic S-C025 and plasma S-C025M (Fig. 8I). Stereoisomer B1 (IC50 ~0.4 nM) and S-C025M (IC50 ~0.7 nM) had similar IC50 values and were only a little less potent than S-C025 (IC50 ~0.1 nM) in blocking the response to 5-oxo-ETE (Table 3), whereas the other three α-hydroxy-C025 diastereomers were approximately 1000 to 6000 times less potent.

Compound

Chirality

IC50 (nM) 0.12 ± 0.05 (6)

S-C025

Table 3. Potencies of synthetic and metabolically-formed α-OH-C025 stereoisomers in inhibiting 5-oxo-ETE-

S-C025M

αS-OH-S-C025

0.69 ± 0.36 (5)

A1 (M1)

αS-OH-R-C025*

120 ± 60 (3)

neutrophils.

A2

αR-OH-S-C025*

260 ± 30 (3)

* Assignment of the chirality of these

B1 (M2)

αS-OH-S-C025

0.38 ± 0.09 (3)

isomers is based on comparison of their

B2

αR-OH- R-C025

750 ± 140 (3)

relative retention times with those of the

induced calcium mobilization in human

corresponding derivatives of 230.23



DISCUSSION We recently identified a series of indole-based OXE receptor antagonists, which have potencies in the nanomolar range and are absorbed following oral administration to primates.19, 22 Of these, the most potent were S-230 and S-264, with IC50 values of ~10 nM, suggesting that these compounds might be credible drug candidates. However, although very high initial levels of both 23022 and 26421 appeared in the blood shortly after oral administration to monkeys, their concentrations dropped sharply shortly thereafter, due in large part to the rapid ω2-oxidation of the hexyl side chain common to both antagonists.

In an attempt to increase resistance to ω-oxidation 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 PGF2α by a phenyl group, as in 17-phenyl-18,19,20-trinor-PGF2α, resulted in a substantially longer in vivo half-life26 as well as increased agonist potency at the FP receptor.27 A similar analog of PGE2, 17-phenyl-18,19,20-trinor-PGE2, is a potent EP1 and EP3 receptor agonist.28, 29 In the present study we initially explored a similar approach with both the 1-acyl (264) and 3-acyl (230) series of OXE-R antagonists and replaced the last 3 carbons of the hexyl side chain with a phenyl group, resulting in the “phenyl-trinor” compounds 12b and 17b, analogous to the above prostaglandin derivatives. However, in contrast to prostaglandins, this reduced potency by about 10-fold. We then examined the effects of lengthening the polymethylene spacer and found the optimal length to be 5 carbons (C149) in the 1-acyl series and 6 carbons (C025) in the 3-acyl series. Compound C025 is remarkably potent, with an IC50 of 120 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 OXE-R 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.

We previously found that following oral administration of racemic 230 there was a transitory decrease in the ratio of the active S-enantiomer to the inactive R-enantiomer in plasma,22, 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 R-enantiomer 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 ω2-oxidation, along with a smaller degree of αoxidation,23 the only major plasma metabolite of C025 that we observed was formed by αoxidation, which may have a higher degree of chiral selectivity. In support of this, we found that the ratio of M2 (derived from S-C025) to M1 (derived from R-C025) was much higher than the



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 t½ compared to racemic C025, S-C025 is still far superior to our previous OXE-R antagonist, S-230, with respect to plasma half-life, Cmax, and AUC, and is a highly credible candidate for further preclinical testing.

It is interesting that the α-hydroxylation of S-C025 appears to be highly stereospecific, resulting in the selective formation of the single stereoisomer (i.e. B1, M2) with appreciable antagonist activity. αS-hydroxy-S-C025 is a potent OXE-R antagonist in its own right, with an IC50 below 1 nM, only several fold higher than that of its precursor. This contrasts with the αS-hydroxy metabolite of S-230, which is over 100 times less potent than S-230.23 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 OXE-R, 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 αS-hydroxy-S-C025 affects the conformation of the molecule and changes the orientation of the α-hydroxyl group, resulting in less interference with binding to the receptor. The potency of SC025M 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 half-life 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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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 S-enantiomer 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 3S-methyl-5-oxovalerate side chain to this series of indole-based antagonists. We initially used this procedure for the synthesis of S-230 and S-264,25 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 5-oxo-ETE plays a role. Largely because of the lack of an OXE-R ortholog in the mouse and other rodents, the pathophysiological role of 5-oxo-ETE has not yet been established. However, the high expression of OXE-R in eosinophils10 and the ability of 5-oxo-ETE to induce the infiltration of these cells into the skin, especially in asthmatics,17 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 5-oxo-ETE 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 inflammation.30 Another possible use of an OXE-R antagonist might be in the treatment of some types of cancer. 5-Oxo-ETE was reported to induce the proliferation of prostate cancer cells31 and siRNA directed at OXE-R has been shown to reduce the viability of these cells, suggesting a role for 5-oxo-ETE in maintaining prostate cancer cell survival.32



CONCLUSIONS In conclusion, addition of a phenyl group at the end of the hexyl side chain of the OXE-R antagonist S-230 results in dramatic increases in both in vitro potency and half-life in the circulatory system when administered orally to monkeys. S-C025 (20) is a highly potent OXE-R antagonist with an IC50 in the picomolar range. It appears rapidly in the blood following oral administration to monkeys and its concentration in plasma is sustained at a much higher level than S-230 over 24 h. Unlike S-230, which is converted to several plasma metabolites that possess little antagonist activity, S-C025 is converted to a single major plasma metabolite (SC025M) 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 S-C025 will permit its use at substantially lower doses than would be required for our earlier OXE-R antagonists. Because of the potent chemoattractant effects of 5-oxo-ETE 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 in which 5-oxo-ETE has been shown to have a proliferative effect on cancer cells.32, 33 Preclinical studies in monkeys are currently underway to determine whether an OXE-R antagonist can inhibit allergen-induced dermal eosinophilia.


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EXPERIMENTAL SECTION Evaluation of OXE receptor antagonist potency. The potency of potential OXE-R antagonists was examined by evaluating their abilities to inhibit calcium mobilization in human neutrophils in response to 5-oxo-ETE as described previously.19 Neutrophils were prepared from the blood of healthy subjects following dextran sedimentation of red blood cells and removal of mononuclear cells by centrifugation over Ficoll-Paque. The cells were then labelled with the calcium-sensitive dye indo-1 and fluorescence was measured at 37 oC with a Cary Eclipse spectrofluorometer (Agilent Technologies, Santa Clara, CA) equipped with a temperaturecontrolled cuvette holder and a magnetic stirrer. After the fluorescence had stabilized, various concentrations of potential antagonists were added, followed 2 min later by 5-oxo-ETE (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 INRS-Institut Armand-Frappier, Laval, Quebec were used for studies on the metabolism and pharmacokinetics of OXE-R 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 S-enantiomer 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 NaHCO3, pH 8.0. The resulting suspension (4.4 mL/kg; 9.1% EtOH) was immediately vortexed and administered by oral gavage to a monkey. Lower doses of S-C025 were administered in the same way, except that the volume administered was 2.2 mL/kg (9.1%



EtOH in 20 mM NaHCO3, pH 8.0). Blood samples (1 to 2 ml) were collected in heparinized 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 -80 oC 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 overnight at -80 oC 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 Sep-Pak cartridge (Waters Corp), which was washed with 30% MeOH, followed by the elution of the antagonist and its metabolites with 100% MeOH.34 After removal of the solvent in vacuo using a rotary evaporator, the residue was dissolved in 30% MeOH and analyzed by precolumn extraction-RP-HPLC as described below. In some cases, the peaks for C025 and C025M were collected and analyzed by chiral HPLC and/or LC-MS/MS.

HPLC analysis of OXE-R 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 for automated precolumn extraction as described previously.35 The stationary phase for RPHPLC was a Novapak C18 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 normal-phase HPLC. Chiral HPLC was carried out using either a Cellulose-1 or a Cellulose-2 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.

Analysis of the major plasma metabolite of C025 by LC-MS/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 LC-MS/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; 32 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 following settings: 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 for the identification and elucidation of C025M. 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 for fragmentation. MS2 settings were: collision-induced dissociation (CID); signal threshold: 5,000; normalized collision energy: 35; isolation width: 2 Da; activation time: 50 ms. MS3 used parent and product mass lists to trigger MS3 for selected ions and was performed with the same settings as MS2 except normalized collision energy of 45 was used.

Chemical syntheses All reactions were carried out under an inert atmosphere (nitrogen or argon) using oven-dried glassware and dry solvents. Reaction progress was monitored using TLC Silica gel 60 F254 plates. Silica gel column chromatography was carried out on silica gel 40-60 µm, 60 Å pore size. 1

H NMR and 13C NMR spectra were recorded on a BRUKER AMX 400 MHz spectrometer in

CDCl3, using TMS as an internal standard. High resolution mass spectrometry (HRMS) was performed using an AccuTOF mass spectrometer with positive ion ESI mode and DART as an ion source. The purity of all tested compounds was determined to be >95% by a combination of HPLC, NMR and HRMS.

Synthesis of 5-(5-Chloro-1-methyl-2-(6,6,6-trifluorohexyl)-1H-indol-3-yl)-3-methyl-5oxopentanoic acid (7; ω-trifluoro-230; see Scheme 1) Triphenyl(5,5,5-trifluoropentyl)phosphonium bromide (2). To a stirred solution of 5-bromo1,1,1-trifluoropentane (1) (1.00 g, 4.90 mmol) in CH3CN (10 ml) was added PPh3 (2.00 g, 7.63 mmol). The reaction flask was attached to a condenser and refluxed for 16 h. The reaction mixture was distilled by a rotary evaporator, and the crude sample was purified by silica gel column chromatography using CH2Cl2/MeOH (10/1) as eluent. The product was obtained as a


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white solid (2.18 g, 95%). HRMS (ESI) m/z calcd for [C23H23F3P]+: 387.1484 found 387.1458. 1

H NMR (400 MHz, CDCl3): δ 7.93 – 7.84 (m, 6H), 7.85 – 7.76 (m, 3H), 7.75 – 7.68 (m, 6H),

3.97 (td, J = 13.1, 8.3 Hz, 2H), 2.30 – 2.14 (m, 2H), 1.98 (p, J = 7.3 Hz, 2H), 1.80 – 1.69 (m, 2H). 13C NMR: δ 135.08 (d, J = 3.0 Hz, 3C), 133.71 (d, J = 10.1 Hz, 6C), 130.52 (d, J = 12.7 Hz, 6C), 127.01 (q, J = 276.9 Hz), 118.18 (d, J = 86.0 Hz, 3C), 32.78 (q, J = 27.9 Hz), 22.78 – 2.71 (m), 22.62 (d, J = 50.8 Hz), 21.58 (d, J = 4.0 Hz).

(Z)-5-chloro-1-methyl-2-(6,6,6-trifluorohex-1-en-1-yl)-1H-indole (4). To a stirred suspension of triphenyl(5,5,5-trifluoropentyl)phosphonium bromide (2) (2.30 g, 4.92 mmol) in THF (10 ml) was added KHMDS (1.0 M in THF, 6.00 ml, 6.00 mmol) at -78 °C. The reaction mixture was allowed to warm up to rt, and stirred for 20 min. The indole aldehyde 3 (1.0 g, 5.16 mmol) was dissolved in 5 mL of THF, and was added to the reaction flask slowly at -78 °C. The reaction mixture was allowed to warm up to rt, stirred for 2 hours, and quenched with saturated aqueous NH4Cl solution (10 ml) at -78 °C. The aqueous phase was extracted with EtOAc, and the combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and purified using silica gel column chromatography using hexane/EtOAc (7/1) as eluent. The product (4) was obtained as a light-yellow viscous oil (0.65 g, 44%). HRMS (ESI) m/z calcd for [C15H15ClF3N + H]+: 302.0918 found 302.0876. 1H NMR (400 MHz, CDCl3): δ 7.54 (d, J = 1.9 Hz, 1H), 7.22 – 7.09 (m, 2H), 6.45 (d, J = 11.5 Hz, 1H), 6.39 (s, 1H), 5.84 (dt, J = 11.7, 7.0 Hz, 1H), 3.68 (s, 3H), 2.51 (qd, J = 7.4, 1.7 Hz, 2H), 2.21 – 2.05 (m, 2H), 1.77 (p, J = 7.8 Hz, 2H). 13

C NMR: δ 137.0, 135.5, 134.4, 128.6, 127.1 (q, J = 276.2), 125.3, 121.9, 119.7, 118.8, 110.1,

101.5, 33.4 (q, J = 28.7 Hz), 30.0, 28.1, 21.8 (q, J = 2.7 Hz).



5-Chloro-1-methyl-2-(6,6,6-trifluorohexyl)-1H-indole (5). To a stirred solution of (Z)-5chloro-1-methyl-2-(6,6,6-trifluorohex-1-en-1-yl)-1H-indole (4) (0.65 g, 2.15 mmol) in EtOH (6 mL) was added 10% Pd/C (65 mg) under an H2 atmosphere. The reaction mixture was stirred at rt for 3 h, and then filtered over Celite. The residue was washed with EtOAc, and the combined filtrate was concentrated under reduced pressure to afford the product 5 as a light-yellow solid (0.65 g, 99%).HRMS (ESI) m/z calcd for [C15H17ClF3N + H]+: 304.1080 found 304.0682. 1H NMR (400 MHz, CDCl3): δ 7.48 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 8.6, 2.0 Hz, 1H), 6.18 (s, 1H), 3.65 (s, 3H), 2.74 (t, J = 7.6 Hz, 2H), 2.19 – 2.00 (m, 2H), 1.75 (p, J = 7.6 Hz, 2H), 1.69 – 1.58 (m, 2H), 1.54 – 1.45 (m, 2H). 13C NMR: δ 142.1, 135.8, 128.8, 127.1 (q, J = 276.4), 125.0, 120.8, 119.1, 109.7, 98.5, 33.7 (q, J = 28.4 Hz), 29.6, 28.4, 28.0, 26.6, 21.8 (q, J = 2.9 Hz).

5-(5-Chloro-1-methyl-2-(6,6,6-trifluorohexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid (7). To a stirred solution of 5-chloro-1-methyl-2-(6,6,6-trifluorohexyl)-1H-indole (5) (41 mg, 0.135 mmol) in CH2Cl2 was added Me2AlCl (1.0 M in hexanes, 0.42 ml) at -20 °C. The reaction mixture was allowed to warm up to rt and stirred for 30 min. The stirred solution was cooled back to -20 °C, and was added with the CH2Cl2 solution of 3-methylglutaric anhydride (6) (43 mg, 0.336 mmol). After stirring at rt for 45 min, the reaction was quenched with H2O, and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and rotary evaporated. The crude material was purified by silica gel column chromatography with hex/EtOAc (3/1, with 0.1% AcOH) to afford the product (7) as an off-white solid (34 mg, 58%). HRMS (ESI) m/z calcd for [C21H25ClF3NO3 + H]+: 432.1553 found 432.0809. 1H NMR (400 MHz, CDCl3): δ 7.86 (d, J = 1.8 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.23 (dd, J = 8.7, 1.8 Hz, 1H),


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3.72 (s, 3H), 3.26 – 3.15 (t, J = 7.5 Hz, 2H), 3.08 – 2.94 (m, 2H), 2.73 (dq, J = 13.3, 6.7 Hz, 2H), 2.56 (dd, J = 15.1, 5.7 Hz, 1H), 2.38 (dd, J = 15.1, 7.2 Hz, 1H), 2.16 – 2.03 (m, 2H), 1.75 – 1.48 (m, 6H), 1.16 (d, J = 6.7 Hz, 3H). 13C NMR: δ 195.4, 175.9, 150.7, 135.2, 128.1, 127.3 (q, J = 157.6 Hz), 126.8, 122.4, 120.3, 113.12, 110.8, 49.1, 40.8, 33.6 (q, J = 28.7 Hz), 29.7, 28.7, 28.6, 26.5, 26.1, 21.7 (q, J = 2.7 Hz), 20.5.

Synthesis of compound C149 (12a, n = 5) and other N-acyl antagonists (see Scheme 2) (E)-6-chloro-2-(5-phenylpent-1-en-1-yl)-1H-indole (10a, n = 3). To a stirred suspension of triphenyl(4-phenylbutyl)phosphonium bromide (9a, n = 3) (1.56 g, 3.28 mmol) in THF (5 mL) was added LiHMDS (1.0 M in THF, 2.95 mL, 2.95 mmol) at -78 °C. The mixture was stirred for 30 min, cooled back to -78 °C, and 6-chloro-1H-indole-2-carbaldehyde (8) (281 mg, 1.56 mmol) in THF (10 ml) was added dropwise. The reaction mixture was allowed to warm to rt and stirred for 3 h. Saturated aqueous NH4Cl solution was added, and the aqueous phase was extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4 and the solvents were evaporated under reduced pressure. The resulting crude material was purified by silica gel chromatography (15% EtOAc/hex) to afford (E)-6-chloro-2-(5-phenylpent-1-en-1-yl)1H-indole (10a, n = 3) (455 mg, 98.6%). HRMS (ESI) m/z calcd for [C19H18ClN+H]+: 296.1206, found 296.1205. 1H NMR (400 MHz, CDCl3): δ 8.00 (s, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.35 – 7.12 (m, 6H), 7.02 (d, J = 6.5 Hz, 1H), 6.48 – 6.26 (m, 2H), 6.10 – 5.96 (m, 1H), 2.76 – 2.64 (m, 2H), 2.27 – 2.24 (m, 2H), 1.90 – 1.75 (m, 2H). 13C NMR: δ 142.1, 137.2, 136.8, 130.3, 128.5 (2C), 128.4 (2C), 127.9, 127.5, 125.8, 121.1, 120.9, 120.7, 110.4, 101.4, 35.3, 32.4, 30.8.



6-chloro-2-(5-phenylpentyl)-1H-indole (11a, n = 5). To a stirred solution of (E)-6-chloro-2-(5phenylpent-1-en-1-yl)-1H-indole (10a, n = 3) (429 mg, 1.45 mmol) in EtOH (10 mL) was added 10% Pd/C (27 mg) under an H2 atm. The reaction mixture was stirred at rt for 8 h and then filtered through Celite. The solid residue was washed with EtOAc, and the combined filtrate was concentrated under reduced pressure. The crude material was purified by silica gel chromatography (10% EtOAc/hexane) to afford 6-chloro-2-(5-phenylpentyl)-1H-indole (11a, n = 5) (385 mg, 89%). HRMS (ESI) m/z calcd for [C19H20ClN+H]+: 298.1363, found 298.1361. 1H NMR (400 MHz, CDCl3): δ 7.71 (s, 1H), 7.45 – 7.34 (m, 1H), 7.32 – 7.12 (m, 6H), 7.03 (dd, J = 4.7, 1.9 Hz, 1H), 6.17 (s, 1H), 2.75 – 2.71 (m, 2H), 2.61 – 2.5 (m, 2H), 1.79 – 1.60 (m, 4H), 1.52 – 1.30 (m, 2H). 13C NMR: δ 142.5, 140.6, 136.1, 128.4 (2C), 128.3 (2C), 127.4, 126.7, 125.7, 120.5, 120.2, 110.3, 99.6, 35.8, 31.1, 28.9, 28.8, 28.1.

5-(6-chloro-2-(5-phenylpentyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid 149 (12a, n = 5). To a stirred solution of 6-chloro-2-(5-phenylpentyl)-1H-indole (11a, n = 5) (365 mg, 1.23 mmol) in THF (8 mL) was added tBuOK (1.0 M solution in THF, 1.25 mL) at rt. The reaction mixture was stirred for 30 min and 3-methylglutaric anhydride (6) (628 mg, 4.9 mmol) was added. After 3 h of stirring, the reaction was quenched with saturated aqueous NH4Cl, extracted with EtOAc, and the organic layers were combined, washed with brine and dried over Na2SO4. The solvents were evaporated under reduced pressure and the crude material was purified by silica gel chromatography using 30% EtOAc/hex as eluent to afford 5-(6-chloro-2-(5phenylpentyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid (12a, n = 5) (344 mg, 65.8%). HRMS (ESI) m/z calcd for [C25H28ClNO3+H]+: 426.1836, found 426.1832. 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.20 – 7.11 (m, 4H), 6.35


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(s, 1H), 3.09 (dd, J = 16.2, 6.2 Hz, 1H), 2.93 – 2.82 (m, 3H), 2.78 – 2.69 (m, 1H), 2.68 – 2.53 (m, 2H), 2.49 – 2.37 (m, 2H), 1.78 – 1.62 (m, 4H), 1.53 – 1.41 (m, 2H), 1.14 (d, J = 1.9 Hz, 3H). 13

C NMR: δ 177.9, 172.1, 143.1, 142.5, 136.5, 129.3, 128.4 (2C), 128.3 (2C), 125.7, 123.5,

120.7, 115.3, 107.9, 44.9, 40.3, 37.7, 35.8, 31.3, 30.5, 29.0, 28.7, 27.2, 20.1.

5-(6-chloro-2-(3-phenylpropyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid (12b, n = 3). HRMS (ESI) m/z calcd for [C23H24ClNO3+H]+: 398.1517, found 398.1363. 1H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.32 – 7.27 (m, 2H), 7.25 – 7.15 (m, 4H), 6.38 (s, 1H), 3.11 – 2.93 (m, 3H), 2.87 (dd, J = 16.2, 7.0 Hz, 1H), 2.79 – 2.69 (m, 3H), 2.55 (dd, J = 15.6, 5.9 Hz, 1H), 2.45 – 2.35 (m, 1H), 2.10 – 1.98 (m, 2H), 1.13 (d, J = 6.6 Hz, 3H). 13C NMR: δ 177.1, 172.1, 142.6, 141.7, 136.6, 129.5, 128.47 (2C), 128.45 (2C), 128.2, 126.0, 123.5, 120.8, 115.3, 108.1, 44.9, 40.1, 35.5, 30.5, 30.1, 27.2, 20.1.

5-(6-chloro-2-(4-phenylbutyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid (12c, n = 4). HRMS (ESI) m/z calcd for [C24H26ClNO3+H]+: 412.1679, found 412.1676. 1H NMR (400 MHz, CDCl3): δ 7.87 (s, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.29 – 2.21 (m, 2H), 7.21 – 7.08 (m, 4H), 6.33 (s, 1H), 3.07 (d, J = 15.8 Hz, 1H), 3.02 – 2.91 (m, 2H), 2.88 (d, J = 15.1 Hz, 1H), 2.77 – 2.61 (m, 3H), 2.55 (d, J = 14.9 Hz, 1H), 2.40 (d, J = 11.5 Hz, 1H), 1.82 – 1.64 (m, 4H), 1.13 (d, J = 4.6 Hz, 3H). 13C NMR: δ 178.1, 172.1, 142.9, 142.2, 136.5, 129.4, 128.4 (2C), 128.3 (2C), 128.3, 125.8, 123.5, 120.7, 115.7, 108.0, 44.9, 40.3, 35.7, 31.1, 30.4, 28.4, 27.2, 20.1.

5-(6-chloro-2-(6-phenylhexyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid (12d, n = 6). HRMS (ESI) m/z calcd for [C26H30ClNO3+H]+: 440.1992, found 440.1832. 1H NMR: δ 7.89 (s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.28 – 7.24 (m, 2H), 7.19 – 7.16 (m, 4H), 6.35 (s, 1H), 3.09 (dd, J 35 ACS Paragon Plus Environment


= 16.2, 6.2 Hz, 1H), 2.96 – 2.88 (m, 3H), 2.79 – 2.71 (m, 1H), 2.63 – 2.55 (m, 3H), 2.41 (dd, J = 15.7, 7.0 Hz, 1H), 1.73 – 1.60 (m, 4H), 1.51 – 1.34 (m, 4H), 1.14 (d, J = 6.7 Hz, 3H). 13C NMR: δ 177.9, 172.1, 143.1, 142.7, 136.6, 129.4, 128.4 (2C), 128.29 (2C), 128.26, 125.6, 123.5, 120.7, 115.3, 107.9, 44.9, 40.3, 35.9, 31.4, 30.6, 29.3, 29.1, 28.8, 27.2, 20.1.

Synthesis of compound C025 (17a, n = 6) and other 3-acyl antagonists (see Scheme 3) (E)-5-chloro-1-methyl-2-(6-phenylhex-1-en-1-yl)-1H-indole (13a, n = 4). To a stirred solution of triphenyl(5-phenylpentyl)phosphonium bromide (9a, n = 4) (1.4 g, 2.860 mmol) in THF (3 ml) was added LiHMDS (4 mL, 1.0 M in THF, 4 mmol) at -78 °C. After 30 min, the aldehyde 3 (300 mg, 1.549 mmol) in THF (4 mL) was added dropwise at -78 °C. The reaction mixture was brought to rt and stirred for 3 h. Saturated aqueous NH4Cl solution (6 mL) was added and the aqueous layer extracted with EtOAc (3 x 6 mL). The combined organic extracts were washed with brine (20 mL), dried over Na2SO4, and the crude material was purified by silica gel column chromatography (5% EtOAc/hex) to obtain the trans olefin 13a (n = 6) as a major product in the form of a yellow viscous oil, 451 mg, 90%. HRMS (ESI) m/z calcd for [C21H22ClNO3+H]+: 324.1514, found 324.1840. 1H NMR (400 MHz, CDCl3): δ 7.48 (d, J = 1.5 Hz, 1H), 7.31 – 7.26 (m, 2H), 7.17 (dd, J = 15.3, 7.6 Hz, 4H), 7.09 (d, J = 1.9 Hz, 1H), 6.48 (s, 1H), 6.40 – 6.26 (m, 2H), 3.68 (s, 3H), 2.70 – 2.62 (m, 2H), 2.32 – 2.27 (m, 2H), 1.75 – 1.63 (m, 2H), 1.61 – 1.51 (m, 2H). 13C NMR: δ 142.5, 140.0, 135.3, 135.2, 128.9, 128.4 (2C), 128.3 (2C), 125.7, 125.2, 121.3, 119.3, 118.6, 109.9, 97.3, 35.8, 33.3, 31.0, 29.9, 28.8.


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5-Chloro-1-methyl-2-(6-phenylhexyl)-1H-indole (14a, n = 6). To a stirred solution of (E)-5chloro-1-methyl-2-(6-phenylhex-1-en-1-yl)-1H-indole (13a, n = 6) (280 mg, 0.865 mmol) in EtOH (5 mL) was added 10% Pd/C (28 mg) at rt. The mixture was degassed under vacuum and the reaction flask charged with an H2 balloon. The reaction mixture was stirred at rt for 4 h. The mixture was diluted with CH2Cl2, and filtered through Celite. The residue was washed with CH2Cl2.The combined filtrate was concentrated and purified using silica gel column chromatography (5% EtOAc/hex) to yield the desired product (14a, n = 6) as a pale-yellow solid, 271 mg, 96%. HRMS (ESI) m/z calcd for [C21H24ClN+H]+: 326.1670, found 326.1843. 1H NMR (400 MHz, CDCl3): δ 7.47 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 7.9 Hz, 2H), 7.21 – 7.13 (m, 4H), 7.08 (dd, J = 8.7, 2.0 Hz, 1H), 6.17 (s, 1H), 3.63 (s, 3H), 2.70 (t, J = 7.7 Hz, 2H), 2.62 (t, J = 7.7 Hz, 2H), 1.71 (dt, J = 11.6, 5.7 Hz, 2H), 1.68 – 1.60 (m, 2H), 1.45 (m, 4H). 13C NMR: δ 142.7, 138.6, 135.0, 129.4, 128.4 (2C), 128.3 (2C), 125.7, 124.3, 120.6, 117.5, 109.4, 106.1, 35.9, 31.4, 29.7, 29.7, 29.2, 29.1, 24.5.

Methyl 5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoate (16a, n = 6). To a stirred solution of 5-methoxy-3-methyl-5-oxopentanoic acid (370 mg, 2.31 mmol) in dichloromethane (3 mL) was added oxalyl chloride solution (2.0 M in dichloromethane, 2.1 mL) at 0 °C followed by DMF (2 µL). The reaction mixture was stirred at rt for 3 h. The solvents were evaporated under reduced pressure to obtain the crude methyl 5chloro-3-methyl-5-oxopentanoate (15). To a stirred solution of 5-chloro-1-methyl-2-(6phenylhexyl)-1H-indole (14a, n = 6) (185 mg, 0.568 mmol) in dichloromethane (2 mL) was added Me2AlCl (1.0 M in hexane, 12.3 mL, 12.3 mmol) at 0 °C dropwise and the mixture was stirred at rt. After 20 min, 5-chloro-3-methyl-5-oxopentanoate (200 mg, 1.12 mmol) in CH2Cl2 (2



mL) was added dropwise at rt and the reaction mixture stirred for 2 h. The reaction was quenched by adding aqueous saturated NH4Cl (10 mL), the two layers were separated, and the aqueous layer extracted with EtOAc (3 x 10 mL). The combined organic extracts were washed with brine (20 mL) and dried over Na2SO4. The solvents were evaporated under reduced pressure and the crude product was purified by silica gel chromatography using 25% EtOAc/hex as eluent to afford 5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoate (16a, n = 6) (244 mg, 92%). HRMS (ESI) m/z calcd for [C28H34ClNO3+H]+: 468.2305, found 468.2357. 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.28 – 7.15 (m, 7H), 3.68 (m, 6H), 3.16 (t, J = 7.7 Hz, 2H), 3.02 (dd, J = 16.2 Hz, 6.3 Hz, 1H), 2.89 (dd, J = 16.3 Hz, 6.8 Hz, 1H), 2.79 – 2.71 (m, 1H), 2.60 (t, J = 7.7 Hz, 2H), 2.52 (dd, J = 14.9 Hz, 5.7Hz, 1H), 2.31 (dd, J = 7.5 Hz, 14.8 Hz, 1H), 1.67 – 1.58 (m, 4H), 1.53 – 1.37 (m, 4H), 1.09 (d, J = 6.5 Hz, 3H). 13C NMR: δ 194.9, 173.2, 150.6, 142.7, 135.1, 128.4 (2C), 128.3 (2C), 127.8, 126.9, 125.6, 122.2, 120.4, 113.2, 110.6, 51.5, 49.4, 41.1, 35.9, 31.4, 29.7, 29.6, 29.09, 29.1, 26.5, 26.3, 20.4.

5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid C025 (17a, n = 6). To a stirred solution of 5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3methyl-5-oxopentanoate (16a, n = 6) (210 mg, 0.449 mmol) in THF/H2O:4/1 (4 mL) was added LiOH·H2O (54 mg, 1.287 mmol) followed by 2 drops of MeOH. The reaction mixture was stirred at rt for 16 h. 2N HCl (2 mL) was added dropwise at 0 °C, pH ~ 1. The aqueous layer was extracted with EtOAc (3 x 5 mL), the combined organic extracts were washed with brine (15 mL), dried over Na2SO4, and the solvents were evaporated to obtain the desired product C025 as a yellow viscous oil (193 mg, 95%). HRMS (ESI) m/z calcd for [C27H32ClNO3+H]+: 454.2143, found 454.2357. 1H NMR (400 MHz, CDCl3): δ 7.88 (d, J = 1.04 Hz, 1H), 7.27 – 7.14 (m, 7H),


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3.66 (s, 3H), 3.14 (t, J = 7.8 Hz, 2H), 3.02 (dd, J = 16.0 Hz, 7.0 Hz, 1H), 2.92 (dd, J = 16.0 Hz, 6.7 Hz, 1H), 2.79 – 2.69 (m, 1H), 2.61 – 2.54 (m, 3H), 2.34 (dd, J = 15.3 Hz, 7.5 Hz, 1H), 1.66 – 1.57 (m, 4H), 1.52 – 1.37 (m, 4H), 1.13 (d, J = 6.7 Hz, 3H). 13C NMR: δ 193.7, 176.8, 149.5, 141.2, 133.7, 126.9 (2C), 126.8 (2C), 126.5, 125.5, 124.2, 120.8, 119.0, 111.6, 109.2, 47.6, 39.5, 34.4, 29.9, 29.2, 28.2, 27.6, 27.6, 24.9, 24.9, 18.9.

5-(5-chloro-1-methyl-2-(3-phenylpropyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid (17b, n = 3). HRMS (ESI) m/z calcd for [C24H26ClNO3+H]+: 412.1674, found 412.1523. 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.29 (t, J = 7.4 Hz, 2H), 7.24 – 7.17 (m, 5H), 3.56 (s, 3H), 3.23 – 3.14 (m, 2H), 3.06 – 2.88 (m, 2H), 2.80 (t, J = 7.4 Hz, 2H), 2.78 – 2.65 (m, 1H), 2.55 (dd, J = 15.2, 5.6 Hz, 1H), 2.36 (dd, J = 15.2, 7.3 Hz, 1H), 2.02 – 1.86 (m, 2H), 1.14 (d, J = 6.7 Hz, 3H). 13

C NMR: δ 195.4, 171.7, 150.6, 141.5, 135.2, 128.48 (4C), 128.1, 126.9, 126.1, 122.4, 120.4,

113.3, 110.7, 49.0, 40.9, 35.7, 30.5, 29.5, 26.5, 25.8, 20.5.

5-(5-chloro-1-methyl-2-(4-phenylbutyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid (17c, n = 4). HRMS (ESI) m/z calcd for [C25H28ClNO3+H]+: 426.1836, found 426.1828. 1H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.27 – 7.14 (m, 7H), 3.63 (s, 3H), 3.18 (t, J = 7.8 Hz, 2H), 3.01 (dd, J = 16.1 Hz, 6.9 Hz, 1H), 2.92 (dd, J = 16.0 Hz, 6.6 Hz, 1H), 2.75 – 2.65 (m, 3H), 2.55 (dd, J = 15.3 Hz, 5.7 Hz, 1H), 2.34 (dd, J = 15.3 Hz, 7.5 Hz, 1H), 1.83 – 1.75 (m, 2H), 1.69 – 1.61 (m, 2H), 1.12 (d, J = 6.7 Hz, 3H). 13C NMR: δ 195.2, 178.1, 150.8, 142.1, 135.1, 128.4 (2C), 128.3 (2C), 128.0, 126.9, 125.8, 122.3, 120.4, 113.2, 110.7, 49.1, 41.0, 35.6, 31.3, 29.6, 28.6, 26.4, 26.2, 20.4.



5-(5-chloro-1-methyl-2-(5-phenylpentyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid (17d, n = 5). HRMS (ESI) m/z calcd for [C26H30ClNO3+H]+: 440.1992, found 440.1831. 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.28 – 7.15 (m, 7H), 3.68 (s, 3H), 3.16 (t, J = 7.8 Hz, 2H), 3.05 – 2.92 (m, 2H), 2.77 – 2.69 (m, 1H), 2.64 – 2.51 (m, 3H), 2.35 (dd, J = 15.2, 7.3 Hz, 1H), 1.73 – 1.60 (m, 4H), 1.51 – 1.45 (m, 2H), 1.14 (d, J = 6.6 Hz, 3H). 13C NMR: δ 195.1, 178.3, 150.9, 142.5, 135.1, 128.4 (2C), 128.3 (2C), 128.0, 126.9, 125.7, 122.3, 120.4, 113.1, 110.7, 49.1, 41.0, 35.8, 31.2, 29.6, 29.4, 28.9, 26.4, 26.3, 20.4.

Synthesis of S-C025 (compound 19, see Scheme 4) Methyl (S)-5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5oxopentanoate (19). To a stirred solution of 5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indole (14d, n = 6) (2.0 g, 6.14 mmol) in dichloromethane was added Me2AlCl (1.0 M in hexane, 12.3 mL, 12.3 mmol) at 0 °C. After 45 min, freshly prepared methyl (R)-5-chloro-3-methyl-5oxopentanoate (18) (1.31 g, 7.36 mmol) in CH2Cl2 (10 mL) was added dropwise at rt and the reaction mixture stirred for 1 h. The reaction was quenched by adding water and extracted with EtOAc. The organic layers were combined, washed with brine and dried over Na2SO4. The solvents were evaporated under reduced pressure and the crude product was purified by silica gel chromatography using hex/EtOAc (4/1) as eluent to afford 19 (2.6 g, 90%). Spectral properties of 19 were the same as compound 16d (n = 6).

(S)-5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid 20 (S-C025). To a stirred solution of 19 (1.14 g, 2.44 mmol) in THF/H2O (4/1, 10 mL) was added LiOH·H2O (1.02 g, 24.4 mmol). The reaction mixture was stirred for 48 h in rt and the THF was


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evaporated under reduced pressure. The aqueous layer was acidified with 4 N HCl and then extracted with EtOAc, the organic layers were combined, washed with brine, and dried over Na2SO4. The organic solvents were evaporated under reduced pressure, and the crude product was purified by silica gel chromatography with hexane/EtOAc (3/1, with 0.1% AcOH) to afford the final product S-C025 (1.05 g, 95%). Spectral properties of S-C025 was the same as C025.

Synthesis of mixture of α-hydroxy-C025 diastereomers (compound 26, see Scheme 5) 1-(5-chloro-1-methyl-1H-indol-2-yl)-6-phenylhexan-1-ol (23). To a freshly prepared Grignard reagent 22 (1.2 M solution in THF, 4 mL) was added the aldehyde 3 (393 mg, 2.03 mmol) in THF (4 mL) at 0 °C. The mixture was allowed to warm to rt and stirred for 2 h. Saturated aqueous NH4Cl (5 mL) was added, the aqueous layer was extracted with EtOAc (3 x 5 mL), the combined organic layers were washed with brine (15 mL), dried over Na2SO4 and concentrated. The product was then purified using silica gel column chromatography (15% EtOAc/hex) to isolate the desired product 23 as a yellow solid (518 mg, 75%). HRMS (ESI) m/z calcd for [C21H24ClNO + H]+: 342.1624, found 342.1673. 1H NMR (400 MHz, CDCl3): δ 7.52 (d, J = 1.4 Hz, 1H), 7.29 – 7.25 (m, 2H), 7.21 – 7.13 (m, 5H), 6.34 (s, 1H), 4.81 – 4.76 (m, 1H), 3.76 (s, 3H), 2.61 (t, J = 7.6 Hz, 2H), 2.00 – 1.88 (m, 2H), 1.69 – 1.37 (m, 6H). 13C NMR (CDCl3): δ 143.3, 142.6, 136.4, 128.4 (2C), 128.3 (2C), 128.1, 125.7, 125.2, 122.0, 120.0, 110.1, 98.6, 67.2, 36.1, 35.8, 31.3, 30.3, 29.0, 25.9.

2-(1-((tert-butyldimethylsilyl)oxy)-6-phenylhexyl)-5-chloro-1-methyl-1H-indole (24). To a stirred solution of 1-(5-chloro-1-methyl-1H-indol-2-yl)-6-phenylhexan-1-ol (23) (116 mg, 0.339 mmol) in CH2Cl2 (5.0 mL) was added TBDMSCl (72 mg, 0.478 mmol) in a single portion



followed by imidazole (63 mg, 0.925 mmol) at rt. The reaction mixture was stirred at rt for 8 h. Water (12 mL) was added, the aqueous phase extracted with CH2Cl2 (3 x 6 mL), and the combined organic extracts were dried over Na2SO4 and concentrated in vacuo. After purification by silica gel column chromatography (3% EtOAc/hex) the product 24 was isolated as a lightyellow viscous oil (115 mg, 74%). HRMS (ESI) m/z calcd for [C27H38ClNOSi + H]+: 456.2489, found 456.2371. 1H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 1.7 Hz, 1H), 7.28 – 7.25 (m, 2H), 7.20 – 7.11 (m, 5H), 6.22 (s, 1H), 4.83 (t, J = 7.1 Hz, 1H), 3.80 (s, 3H), 2.58 (t, J = 7.6 Hz, 2H), 1.94 – 1.85 (m, 1H), 1.80 – 1.71 (m, 1H), 1.64 – 1.56 (m, 2H), 1.50 – 1.21 (m, 4H), 0.87 (s, 9H), 0.00 (s, 3H), -0.18 (s, 3H). 13C NMR (CDCl3): δ 143.6, 142.7, 136.6, 128.40 (2C), 128.35, 128.3 (2C), 125.7, 125.0, 121.4, 119.8, 109.9, 99.6, 70.0, 38.0, 35.9, 31.4, 31.0, 29.0, 26.0, 25.8 (3C), 18.1, -4.8, -5.2.

Methyl 5-(5-chloro-2-(1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5oxopentanoate (25). To a stirred solution of 2-(1-((tert-butyldimethylsilyl)oxy)-6-phenylhexyl)5-chloro-1-methyl-1H-indole (24) (45 mg, 0.099 mmol) and the acyl chloride 15 (50 mg, 0.280 mmol) in CH2Cl2 (12 mL) was added Me2AlCl (0.2 mL, 1.0 M solution in hexanes) at 0 °C dropwise. After stirring the reaction mixture at 0 °C for 2 h, it was allowed to warm to rt and stirred for 10 h. Saturated aqueous NaHCO3 (3.5 mL) was added, the two layers separated, the aqueous phase extracted with EtOAc (3 × 4 mL), and the combined organic extracts dried over Na2SO4, concentrated. After purification using silica gel column chromatography (10% EtOAC/hex), the alcohol 25 was obtained as a pair of diastereomers in the form of a pale-yellow viscous oil, 21 mg, 44%. HRMS (ESI) m/z calcd for [C28H34ClNO4 + H]+: 484.2254, found 484.2251.


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5-(5-chloro-2-(1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid 26 (α-OH-C025). To a stirred solution of methyl 5-(5-chloro-2-(1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3yl)-3-methyl-5-oxopentanoate (25) (15 mg, 0.031 mmol) in THF/H2O (4:1, 4 mL) was added LiOH·H2O (31 mg, 0.739 mmol) and MeOH (2 drops). The reaction mixture was stirred at rt for 2 days. H2O (20 mL) was added, the aqueous phase extracted with EtOAc (3 x 5 mL), and the combined organic extracts were washed with brine (5 mL), dried over Na2SO4, and concentrated. Purification using silica gel column chromatography (5% MeOH/CH2Cl2) afforded the desired product 26 (α-OH-C025) as a mixture of four diastereomers, 13 mg, 89%. HRMS (ESI) m/z calcd for [C27H32ClNO4 + H]+: 470.2098, found 470.2080.

Synthesis of αS-hydroxy-S-C025 (compound 29, see Scheme 6) (S)-1-(5-chloro-1-methyl-1H-indol-2-yl)-6-phenylhexan-1-ol (27). Flask A: To a stirred solution of (S)-BINOL (196 mg, 0.685 mmol) in tBuOMe (18 mL) was added Ti(OiPr)4 (1.2 mL, 4.053 mmol) dropwise at rt, and the resulting red solution was stirred for 30 min. Flask B: to a stirred solution of BDMAEE (1.72 mL, 9.015 mmol) in tBuOMe (18 mL) was added (5phenylpentyl)magnesium bromide (22) (1.03 M solution in THF, 8.8 mL) at 0 °C. The resulting suspension was stirred at 0 °C for 35 min. The mixture from flask ‘A’ was then transferred into flask ‘B’ via a syringe at 0 °C. The reaction mixture was allowed to warm to rt and stirred for 1 h. The mixture was cooled to -15 °C and added 5-chloro-1-methyl-1H-indole-2-carbaldehyde (3) (641 mg, 3.310 mmol) in THF (8 mL) dropwise. The reaction mixture was stirred at rt for 36 h and the reaction quenched by adding aqueous NH4Cl (25 mL). The two layers were separated,



the aqueous layer extracted with EtOAc (3 x 20 mL), and the combined organic extracts were washed with brine (40 mL), dried over Na2SO4, filtered, and concentrated. Purification by silica gel column chromatography (25% EtOAc/hexane) afforded product 27 as a yellow viscous oil (430 mg, 38%), the spectral properties of which were identical to those of the racemic compound 23. The %S enantiomer was determined to be 92.2% by chiral HPLC.

(S)-2-(1-((tert-butyldimethylsilyl)oxy)-6-phenylhexyl)-5-chloro-1-methyl-1H-indole (28). To a stirred solution of (S)-1-(5-chloro-1-methyl-1H-indol-2-yl)-6-phenylhexan-1-ol (27) (241 mg, 0.705 mmol) in CH2Cl2 (8 mL) was added TBDMSCl (216 mg, 1.433 mmol), followed by imidazole (198 mg, 2.908 mmol) at rt. The reaction mixture was stirred at rt for 16 h. Water (20 mL) was added, the aqueous phase extracted with CH2Cl2 (3 x 10 mL), and the combined organic extracts were dried over Na2SO4, and concentrated in vacuo. After purification by silica gel column chromatography (3% EtOAc/hex) the product 28 was isolated as a light-yellow viscous oil (302 mg, 94%), the spectral properties of which were identical to those of the racemic compound 24.

(S)-5-(5-chloro-2-((S)-1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5oxopentanoic acid 29 (αS-OH-S-C025). To a stirred solution of (S)-2-(1-((tertbutyldimethylsilyl)oxy)-6-phenylhexyl)-5-chloro-1-methyl-1H-indole (28) (115 mg, 0.252 mmol) and the acyl chloride 18 (126 mg, 0.705 mmol) in CH2Cl2 (20 mL) was added Me2AlCl (0.45 mL, 1.0 M solution in hexanes) at 0 °C dropwise. After stirring the reaction mixture at 0 °C for 1 h, it was allowed to warm to rt and stirred for a further 16 h. The reaction was quenched by adding saturated aqueous NaHCO3 (8 mL), the two layers separated, the aqueous phase extracted with EtOAc (3 x 10 mL), and the combined organic extracts dried over Na2SO4, filtered, and 44 ACS Paragon Plus Environment

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concentrated. Silica gel column chromatography (5% EtOAc/hexane) afforded the acylated product along with some inseparable impurities, which was used as is without further purification. The mixture was dissolved in CH3CN (8 mL) and HF.Pyridine (70% HF, 30% pyridine, 0.3 mL) was added at 0 °C. The reaction mixture was warmed to rt and stirred for 5 h. Aqueous NaHCO3 (10 mL) was added, the aqueous layer extracted with EtOAc (3 x 8 mL) and the organic extracts dried over Na2SO4. The solvents were removed in vacuo, the crude product was filtered through Celite, and the combined filtrate was concentrated to obtain a yellow viscous oil that was subjected to ester hydrolysis under basic conditions. To a stirred suspension of the yellow viscous oil in THF/H2O:4/1 (4 mL) was added LiOH·H2O (80 mg, 2.108 mmol) and MeOH (2 drops). The reaction mixture was stirred at rt for 14 h. H2O (20 mL) was added and the aqueous layer was extracted with EtOAc (3 x 6 mL), and the organic extract dried over Na2SO4, and concentrated. Purification using silica gel column chromatography (10%MeOH/CH2Cl2) yielded the final product 29 (αS-OH-S-C025), as a yellow viscous oil, 27 mg, 23% over three steps). HRMS (ESI) m/z calcd for [C27H32ClNO4 + H]+: 470.2098, found 470.2080. 1H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H), 7.31 – 7.23 (m, 2H), 7.18 – 7.12 (m, 5H), 4.96 – 4.94 (m, 1H), 3.74 (s, 3H), 3.17 – 3.11 (m, 1H), 3.00 – 2.64 (m, 1H), 2.81 – 2.71 (m, 1H), 2.64 – 2.53 (m, 3H), 2.42 – 2.31 (m, 1H), 2.01 – 1.91 (m, 2H), 1.67 – 1.58 (m, 2H), 1.40 – 1.22 (m, 4H), 1.12 (d, J = 6.8 Hz, 3H). 13C NMR (CDCl3): δ 197.7, 172.1, 142.6, 135.0, 128.8, 128.8, 128.4 (2C), 128.2 (2C), 126.9, 126.9, 125.6, 123.1, 120.6, 120.6, 67.2, 56.0, 49.2, 35.8, 31.3, 31.0, 29.7, 28.9, 26.5, 25.9, 20.4.



ASSOCIATED CONTENT Supporting information Molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding author *Tel: 1-514-934-1934 ext. 76414 E-mail: [email protected]

ORCID William S Powell: 0000-0002-8507-4038 Joshua Rokach: 0000-0003-1814-7505 Dajana Vuckovic: 0000-0002-7764-8492 Shishir Chourey: 0000-0002-2629-9632 Qiuji Ye: 0000-0002-3812-3001

ACKNOWLEDGMENTS This work was supported by the Canadian Institutes of Health Research (WSP: Grants MOP6254 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 Santé du Québec 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 46 ACS Paragon Plus Environment

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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.

ABBREVIATIONS USED 5-oxo-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; 230, 5-(5-chloro-2-hexyl-1-methyl-1Hindol-3-yl)-3-methyl-5-oxopentanoate; S-230, (S)-5-(5-chloro-2-hexyl-1-methyl-1H-indol-3-yl)3-methyl-5-oxopentanoate; 264, 5-(6-chloro-2-hexyl-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid; S-264, (S)-5-(6-chloro-2-hexyl-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid; C149, 5-(6chloro-2-(5-phenylpentyl)-1H-indol-1-yl)-3-methyl-5-oxopentanoic acid; C025, 5-(5-chloro-1methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid; S-C025, (S)-5-(5chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid; R-C025, (R)5-(5-chloro-1-methyl-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid; C025M, 5-(5-chloro-2-(1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoic acid; S-C025M, (S)-5-(5-chloro-2-((S)-1-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3methyl-5-oxopentanoic acid; BDMAEE, bis[2-(N,N-dimethylamino)ethyl] ether; (S)-BINOL, (S)-(−)-1,1’-bi(2-naphthol); TBDMSCl, tert-butyldimethylchlorosilane; TMS, tetramethylsilane; NP-HPLC, normal-phase-high performance liquid chromatography; RP-HPLC, reversed-phasehigh performance liquid chromatography.



REFERENCES (1) Powell, W. S.; Gravelle, F.; Gravel, S. Metabolism of 5(S)-hydroxy-6,8,11,14eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes. J. Biol. Chem. 1992, 267, 19233-19241. (2) Powell, W. S.; Rokach, J. Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid. Biochim. Biophys. Acta 2015, 1851, 340-355. (3) Powell, W. S.; Chung, D.; Gravel, S. 5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent stimulator of human eosinophil migration. J. Immunol. 1995, 154, 4123-4132. (4) Powell, W. S.; Gravel, S.; MacLeod, R. J.; Mills, E.; Hashefi, M. Stimulation of human neutrophils by 5-oxo-6,8,11,14- eicosatetraenoic acid by a mechanism independent of the leukotriene B4 receptor. J. Biol. Chem. 1993, 268, 9280-9286. (5) Iikura, M.; Suzukawa, M.; Yamaguchi, M.; Sekiya, T.; Komiya, A.; Yoshimura-Uchiyama, C.; Nagase, H.; Matsushima, K.; Yamamoto, K.; Hirai, K. 5-Lipoxygenase products regulate basophil functions: 5-Oxo-ETE elicits migration, and leukotriene B4 induces degranulation. J. Allergy Clin. Immunol. 2005, 116, 578-585. (6) Sturm, G. J.; Schuligoi, R.; Sturm, E. M.; Royer, J. F.; Lang-Loidolt, D.; Stammberger, H.; Amann, R.; Peskar, B. A.; Heinemann, A. 5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent chemoattractant for human basophils. J. Allergy Clin. Immunol. 2005, 116, 1014-1019. (7) Sozzani, S.; Zhou, D.; Locati, M.; Bernasconi, S.; Luini, W.; Mantovani, A.; O'Flaherty, J. T. Stimulating properties of 5-oxo-eicosanoids for human monocytes: synergism with monocyte chemotactic protein-1 and -3. J. Immunol. 1996, 157, 4664-4671.


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(8) Hosoi, T.; Koguchi, Y.; Sugikawa, E.; Chikada, A.; Ogawa, K.; Tsuda, N.; Suto, N.; Tsunoda, S.; Taniguchi, T.; Ohnuki, T. Identification of a novel eicosanoid receptor coupled to Gi/o. J. Biol. Chem. 2002, 277, 31459-31465. (9) Takeda, S.; Yamamoto, A.; Haga, T. Identification of a G protein-coupled receptor for 5oxo-eicosatetraenoic acid. Biomedical Research-Tokyo 2002, 23, 101-108. (10) Jones, C. E.; Holden, S.; Tenaillon, L.; Bhatia, U.; Seuwen, K.; Tranter, P.; Turner, J.; Kettle, R.; Bouhelal, R.; Charlton, S.; Nirmala, N. R.; Jarai, G.; Finan, P. Expression and characterization of a 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor highly expressed on human eosinophils and neutrophils. Mol. Pharmacol. 2003, 63, 471-477. (11) Blättermann, S.; Peters, L.; Ottersbach, P. A.; Bock, A.; Konya, V.; Weaver, C. D.; Gonzalez, A.; Schroder, R.; Tyagi, R.; Luschnig, P.; Gab, J.; Hennen, S.; Ulven, T.; Pardo, L.; Mohr, K.; Gutschow, M.; Heinemann, A.; Kostenis, E. A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer. Nat. Chem. Biol. 2012, 8, 631-638. (12) Urasaki, T.; Takasaki, J.; Nagasawa, T.; Ninomiya, H. Pivotal role of 5-lipoxygenase in the activation of human eosinophils: platelet-activating factor and interleukin-5 induce CD69 on eosinophils through the 5-lipoxygenase pathway. J. Leukoc. Biol. 2001, 69, 105-112. (13) O'Flaherty, J. T.; Kuroki, M.; Nixon, A. B.; Wijkander, J.; Yee, E.; Lee, S. L.; Smitherman, P. K.; Wykle, R. L.; Daniel, L. W. 5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes. J. Immunol. 1996, 157, 336-342. (14) Czech, W.; Barbisch, M.; Tenscher, K.; Schopf, E.; Schröder, J. M.; Norgauer, J. Chemotactic 5-oxo-eicosatetraenoic acids induce oxygen radical production, Ca2+-mobilization, and actin reorganization in human eosinophils via a pertussis toxin-sensitive G-protein. J. Invest. Dermatol. 1997, 108, 108-112.



(15) Guilbert, M.; Ferland, C.; Bosse, M.; Flamand, N.; Lavigne, S.; Laviolette, M. 5-Oxo6,8,11,14-eicosatetraenoic acid induces important eosinophil transmigration through basement membrane components: comparison of normal and asthmatic eosinophils. Am. J. Respir. Cell Mol. Biol. 1999, 21, 97-104. (16) Dallaire, M. J.; Ferland, C.; Page, N.; Lavigne, S.; Davoine, F.; Laviolette, M. Endothelial cells modulate eosinophil surface markers and mediator release. Eur. Respir. J. 2003, 21, 918924. (17) Muro, S.; Hamid, Q.; Olivenstein, R.; Taha, R.; Rokach, J.; Powell, W. S. 5-Oxo6,8,11,14-eicosatetraenoic acid induces the infiltration of granulocytes into human skin. J. Allergy Clin. Immunol. 2003, 112, 768-774. (18) Powell, W. S.; Gravel, S.; Khanapure, S. P.; Rokach, J. Biological inactivation of 5-oxo6,8,11,14-eicosatetraenoic acid by human platelets. Blood 1999, 93, 1086-1096. (19) Gore, V.; Gravel, S.; Cossette, C.; Patel, P.; Chourey, S.; Ye, Q.; Rokach, J.; Powell, W. S. Inhibition of 5-oxo-6,8,11,14-eicosatetraenoic acid-induced activation of neutrophils and eosinophils by novel indole OXE receptor antagonists. J. Med. Chem. 2014, 57, 364-377. (20) Patel, P.; Reddy, C. N.; Gore, V.; Chourey, S.; Ye, Q.; Ouedraogo, Y. P.; Gravel, S.; Powell, W. S.; Rokach, J. Two potent OXE-R antagonists: assignment of stereochemistry. ACS Med. Chem. Lett. 2014, 5, 815-819. (21) Reddy, C. N.; Alhamza, H.; Chourey, S.; Ye, Q.; Gore, V.; Cossette, C.; Gravel, S.; Slobodchikova, I.; Vuckovic, D.; Rokach, J.; Powell, W. S. Metabolism and pharmacokinetics of a potent N-acylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in rats and monkeys. Eur. J. Pharm. Sci. 2018, 115, 88-99.


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(22) Cossette, C.; Chourey, S.; Ye, Q.; Nagendra Reddy, C.; Gore, V.; Gravel, S.; Slobodchikova, I.; Vuckovic, D.; Rokach, J.; Powell, W. S. Pharmacokinetics and metabolism of selective oxoeicosanoid (OXE) receptor antagonists and their effects on 5-oxo-6,8,11,14eicosatetraenoic acid (5-oxo-ETE)-induced granulocyte activation in monkeys. J. Med. Chem. 2016, 59, 10127-10146. (23) Chourey, S.; Ye, Q.; Reddy, C. N.; Cossette, C.; Gravel, S.; Zeller, M.; Slobodchikova, I.; Vuckovic, D.; Rokach, J.; Powell, W. S. In vivo α-hydroxylation of a 2-alkylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid in monkeys. Biochem. Pharmacol. 2017, 138, 107-118. (24) Gore, V.; Chourey, S.; Ye, Q. J.; Patel, P.; Ouedraogo, Y.; Gravel, S.; Powell, W. S.; Rokach, J. Base-dependent formation of cis and trans olefins and their application in the synthesis of 5-oxo-ETE receptor antagonists. Bioorg. Med. Chem. Lett. 2014, 24, 3385-3388. (25) Reddy, C. N.; Ye, Q. J.; Chourey, S.; Gravel, S.; Powell, W. S.; Rokach, J. Stereoselective synthesis of two highly potent 5-oxo-ETE receptor antagonists. Tetrahedron Lett. 2015, 56, 6896-6899. (26) Granström, E. Metabolism of 17-phenyl-18,19,20-trinor-prostaglandin-F2α in cynomolgus monkey and human female. Prostaglandins 1975, 9, 19-45. (27) Graves, P. E.; Pierce, K. L.; Bailey, T. J.; Rueda, B. R.; Gil, D. W.; Woodward, D. F.; Yool, A. J.; Hoyer, P. B.; Regan, J. W. Cloning of a receptor for prostaglandin F2α from the ovine corpus luteum. Endocrinology 1995, 136, 3430-3436. (28) Miller, W. L.; Weeks, J. R.; Lauderdale, J. W.; Kirton, K. T. Biological activities of 17phenyl-18,19,20-trinorprostaglandins. Prostaglandins 1975, 9, 9-18.



(29) Kiriyama, M.; Ushikubi, F.; Kobayashi, T.; Hirata, M.; Sugimoto, Y.; Narumiya, S. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br. J. Pharmacol. 1997, 122, 217-224. (30) Powell, W. S.; Rokach, J. The eosinophil chemoattractant 5-oxo-ETE and the OXE receptor. Prog. Lipid Res. 2013, 52, 651-665. (31) Ghosh, J.; Myers, C. E. Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. Proc. Natl. Acad. Sci. USA 1998, 95, 13182-13187. (32) Sundaram, S.; Ghosh, J. Expression of 5-oxoETE receptor in prostate cancer cells: critical role in survival. Biochem. Biophys. Res. Commun. 2006, 339, 93-98. (33) O'Flaherty, J. T.; Rogers, L. C.; Paumi, C. M.; Hantgan, R. R.; Thomas, L. R.; Clay, C. E.; High, K.; Chen, Y. Q.; Willingham, M. C.; Smitherman, P. K.; Kute, T. E.; Rao, A.; Cramer, S. D.; Morrow, C. S. 5-Oxo-ETE analogs and the proliferation of cancer cells. Biochim. Biophys. Acta 2005, 1736, 228-236. (34) Powell, W. S. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins 1980, 20, 947-957. (35) Powell, W. S. Precolumn extraction and reversed-phase high-pressure liquid chromatography of prostaglandins and leukotrienes. Anal. Biochem. 1987, 164, 117-131.


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Table of contents graphic 209x54mm (300 x 300 DPI)

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Figure 1 46x28mm (600 x 600 DPI)





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Scheme 1 37x16mm (600 x 600 DPI)





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48x15mm (600 x 600 DPI)





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(OXE) receptor antagonists that block the actions of the granulocy

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