Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Bioorg Med Chem Lett. 2011 Feb 12;21(7):1987–1990. doi: 10.1016/j.bmcl.2011.02.021

C20-trifluoro-5-oxo-ETE: A metabolically stable 5-oxo-ETE derivative

Pranav Patel a, Vivek Gore a, William S Powell b, Joshua Rokach a,✉
PMCID: PMC3062511  NIHMSID: NIHMS274051  PMID: 21377873

Abstract

The total synthesis of C20-trifluoro-6(E),8(Z),11(Z),14(Z) 5-oxo-ETE is reported. This compound was designed as an ω-oxidation-resistant analog of 5-oxo-ETE that would be resistant to metabolism. The trifluoro derivative of 5-oxo-ETE stimulated calcium mobilization in neutrophils and desensitized these cells to subsequent exposure to 5-oxo-ETE.

Arachidonic acid (AA) 1 is converted to a large number of biologically active products (eicosanoids) that are important in a variety of pathological conditions, including inflammatory and allergic diseases.1–5 Because of the complex array of products with different chiral centers and double bond configurations, chemical synthesis of eicosanoids has been critical for their identification and determination of their physiological and pharmacological properties. Over the past several years, our work has focused on the synthesis and biological effects of a variety of AA-derived products formed both enzymatically by lipoxygenase (LO) and cyclooxygenase (COX) pathways6–9 and nonenzymatically by reactive oxygen species (ROS)-induced oxidation.10–12 5-LO, which catalyzes the 5-peroxidation of AA leads to 5-HPETE 413–15 and leukotriene (LTA4).16 LTA4 is transformed by LTA4 hydrolase to LTB4, a potent neutrophil chemoattractant that acts principally via the BLT1 receptor.17 LTA4 is also converted to the cysteinyl leukotriene LTC4 by the addition of glutathione (GSH). LTC4 is further metabolized to LTD4. These substances, previously known as “slow reacting substance of anaphylaxis”, are potent stimulators of bronchoconstriction and vascular permeability and are critical mediators in asthma.

5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) 6 is the most potent chemotactic agent for eosinophils among lipid mediators.18 It is formed from 5-HETE 5 by the action of a very specific and selective dehydrogenase and exerts its biological actions through a dedicated receptor. Because of its effects on eosinophils, 5-oxo-ETE may be an important mediator in diseases in which these cells are prominent, including asthma and other allergic conditions, and diseases of the gastrointestinal system.

5-oxo-ETE 6 is synthesized by 5-hydroxyeicosanoid dehydrogenase (5-HEDH), which is highly specific for eicosanoids containing a 5(S)-hydroxyl group followed by a 6,7-trans-double bond (Scheme 1). This enzyme is present in neutrophils,19 monocytes,20 lymphocytes,20 eosinophils,21 platelets,22 and some types of structural cells. 5-oxo-ETE 6 acts through a specific Gi-coupled receptor,23–25 which has been cloned26,27 and is highly expressed by eosinophils > neutrophils > monocytes as well as by certain tumor cell lines. This receptor has been designated as the OXE receptor (OXE-R). 5-oxo-ETE is active in vivo, eliciting infiltration of eosinophils into rat lung28 and both eosinophils and neutrophils into human skin.29 OXE-R is highly selective for 5-oxo-ETE 6 over a variety of its metabolites, LTs and other eicosanoids. Although 5-oxo-ETE 6 is a chemoattractant for both neutrophils23 and monocytes,30 it is less potent than LTB4. In contrast, among lipid mediators, 5-oxo-ETE 6 is the strongest chemoattractant known for human eosinophils21 and also induces a variety of other responses in these cells,31–33 some of which are markedly enhanced by the proinflammatory cytokines GM-CSF and TNFα.34

Scheme 1.

Scheme 1

Open in a new tab

Eicosanoids

An obstacle in the biological evaluation of 5-oxo-ETE is its susceptibility to ω-oxidation24 and incorporation into cellular lipids,25 both of which result in dramatic losses in biological activity. One way to block metabolism by ω-oxidation would be to modify the ω-methyl group of 5-oxo-ETE. This could also potentially interfere with incorporation into lipids. However, alteration of the C20 methyl group could also have a marked effect on biological activity, as 20-hydroxy-5-oxo-ETE is about 100 times less potent than 5-oxo-ETE.24

Substitution of a trifluoromethyl group for the C20 methyl group of 5-oxo-ETE is one choice for a metabolically stable 5-oxo-ETE agonist. This would prevent ω-oxidation, the major pathway for the metabolism of 5-oxo-ETE, and may also possibly reduce the rate of incorporation in lipids. Scheme 2 shows the synthesis of the CF3 trifluoro derivative 23.35

Scheme 2. Total synthesis of long lasting agonist C20-Trifluoro-5-oxo-ETE.

Scheme 2

Open in a new tab

Reagents and Conditions: a) 10-camphor sulphonic acid, CH3CN, rt, 80%; b ) PCC, Al2O3, dry CH2Cl2, 0 °C - rt, 75%; c) 13, dry THF, LiHMDS, −78 °C - rt, 73%; d) THF, TBAF, rt, 87%; e) PPh3, imidazole, I2, dry CH2Cl2, rt, 90%; f) PPh3, CH3CN, 50 °C, 2 days, 92%; g) 15, dry THF, 1.6 M BuLi, −78 °C-rt, 75%; h) THF/H20, TFA, 78%; i) dry CH2Cl2, Na2CO3, LTA, 74%; j) 19, dry THF, −78 °C-rt, n-BuLi, 84%; k) LiOH.H2O, isopropanol, rt, 93%; l) TBAF, AcOH, THF, 50 °C, 12 h, 89%; m) Dess-Martin periodinane, dry CH2Cl2, rt, 88%.

The synthesis of trifluoro derivative 23 was achieved as described in detail in Scheme 2. The aldehyde 12 was obtained by oxidation of 11 which in turn was obtained by protecting triol 9 with dimethoxy propane. The α,β-unsaturated aldehyde 16 was prepared as previously described starting from D-arabinose.36 The trifluoro phosphonium salt 19 was prepared readily from commercial 1,1,1-trifluoro-6-bromohexane37 by refluxing with triphenyl phosphine in acetonitrile.

The use of D-arabinose (Scheme 3) for the preparation of 25 and ultimately 23, although not chirally economical, is nevertheless a very convenient procedure. The starting material 24 is very cheap and 25 can be prepared in batches of 200g at a time. Also, 16 can be used for the preparation of 5-HETE and other lipoxygenase products.

Scheme 3. Synthesis of α,β-unsaturated aldehyde 16.

Scheme 3

Open in a new tab

Reagents and Conditions: a) TBDPS-Cl, imidazole, CH2Cl2, rt, 98%; b) periodic acid, THF, ether, 0 °C, 83.3 %; c) 27, THF,91.5%; d) Pd/C H2, EtOH, rt, 96.5%; e) periodic acid, THF, ether, rt, 77.5%; f) 30, benzene, reflux, 76.3%.

To determine the biological potency of 23, we conducted preliminary experiments to examine its effects on intracellular calcium levels in human neutrophils. Neutrophils were prepared from blood from healthy subjects as previously described by removing erythrocytes with dextran 500 and mononuclear cells by centrifugation over Ficoll-Paque.38 The neutrophils were suspended in phosphate-buffered saline (PBS) and loaded with indo-1 acetoxymethyl ester.24 Five minutes prior to data acquisition, CaCl2 (1.8 mM) and MgCl2 (1 mM) were added. Fluorescence was measured using a spectrofluorometer with a temperature-controlled cuvette holder equipped with a magnetic stirrer. After stabilization of the baseline, various concentrations of 6 or 23 were added, followed 1.5 min later by addition of 6 (10 nM) to evaluate agonist-induced desensitization. After another 0.5 min, digitonin (0.1% final concentration) was added.

The response of neutrophils to 5-oxo-ETE (10 nM) following initial addition of vehicle is shown in Fig. 1, top curve. There was a strong increase in fluorescence, which peaked a few seconds after addition of 5-oxo-ETE. Digitonin was added before fluorescence returned to baseline to permit measurement of the maximal fluorescence. Addition of C20-trifluoro-5-oxo-ETE (F3-5o) (100 nM) also resulted in a sharp increase in fluorescence, although somewhat less than that for 5-oxo-ETE and completely abolished the response to 5-oxo-ETE (Fig. 1, bottom curve).

Figure 1.

Figure 1

Open in a new tab

Effects of 5-oxo-ETE and trifluoro-5-oxo-ETE on intracellular calcium levels in indo-1-labeled neutrophils. Either vehicle or trifluoro-5-oxo-ETE (100 nM) was added after 1 min, followed by 5-oxo-ETE (10 nM) at 2.5 min and digitonin (0.1%) at 3.5 min. Fluorescence due to the binding of Ca++ to indo-1 was monitored using excitation and emission wavelengths of 331 and 410 nm.

The concentration-response curves for compounds 6 and 23 on calcium mobilization are shown in Fig. 2A. 5-oxo-ETE was a potent inducer of calcium mobilization with an EC50 of 4 nM. C20-trifluoro-5-oxo-ETE was somewhat less potent, with a maximal response about 50% of that for 5-oxo-ETE. Both of the above agonists desensitized neutrophils to subsequent exposure to 5-oxo-ETE, as illustrated by Fig. 1. The IC50 for 5-oxo-ETE-induced desensitization was 3 nM, whereas that for C20-trifluoro-5-oxo-ETE was 7.5 nM (Fig. 2B).

Figure 2.

Figure 2

Open in a new tab

Concentration-response relationships for the effects of 5-oxo-ETE and trifluoro-5-oxo-ETE on intracellular calcium levels in neutrophils. A: Agonist effects of 5-oxo-ETE and trifluoro-5-oxo-ETE on intracellular Ca++ levels in neutrophils. B: Inhibition of 5-oxo-ETE (10 nM)-induced Ca++ mobilization in neutrophils due to prior treatment with different concentrations of 5-oxo-ETE or trifluoro-5-oxo-ETE as shown in Fig. 1.

Although C20-trifluoro-5-oxo-ETE is some what less potent an agonist than 5-oxo-ETE, it is considerably more potent than the 5-oxo-20-HETE 7, a C20-hydroxy metabolite of 5-oxo-ETE, indicating that addition of a substituent at this position does not necessarily dramatically reduce biological activity. The potent inhibitory effect of C20-trifluoro-5-oxo-ETE on 5-oxo-ETE-induced calcium mobilization (Fig. 2B) suggests that the trifluoro derivative binds strongly to the 5-oxo-ETE receptor, whereas the limited maximal response suggests that it is a partial agonist, having some antagonist properties. These results suggest that further modification of the C20 methyl group may lead to the identification of 5-oxo-ETE analogs with potent agonist or antagonist effects. Also, Figure 2B suggests that an appropriately radio-labeled C20-trifluoro-5-oxo-ETE could be useful in generating an OXE-R binding assay and for long term in vivo experimentation.

Supplementary Material

01

Acknowledgments

This work was supported by grants from the National Heart, Lung, And Blood Institute (J.R.; Award Number R01HL081873), the Canadian Institutes of Health Research (W.S.P.; MOP-6254), and the Quebec Heart and Stroke Foundation (W.S.P.). The Meakins-Christie Laboratories - MUHC-RI are supported in part by a Center grant from Le Fonds de la Recherche en Santé du Québec as well as by the J. T. Costello Memorial Research Fund. J.R. also wishes to acknowledge the National Science Foundation for the AMX-360 (Grant Number CHE-90-13145) and Bruker 400 MHz (Grant Number CHE-03-42251) NMR instruments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, And Blood Institute or the National Institutes of Health.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Rokach J. Leukotrienes and Lipoxygenases, Chemical, Biological and Clinical Aspects. 1. Vol. 11 Elsevier Science; Amsterdam, The Netherlands: 1989. [Google Scholar]
  • 2.Atkinson JG, Rokach J. Synthesis of the leukotrienes and other lipoxygenase-derived products. In: Willis AL, editor. CRC Handbook of Eicosanoids: Prostaglandins and Related Lipids. Chemical and Biochemical Aspects, Part B. I. CRC Press, Inc; Boca Raton, FL: 1987. p. 175. [Google Scholar]
  • 3.Vane J, O’Grady J. Therapeutic Applications of Prostaglandins. Edward Arnold; London: 1993. p. 277. [Google Scholar]
  • 4.Nicolaou KC, Petasis NA. Synthesis of eicosanoids. In: Willis AL, editor. CRC Handbook of Eicosanoids: Prostaglandins and Related Lipids. Chemical and Biochemical Aspects, Part B. I. CRC Press, Inc; Boca Raton, FL: 1987. p. 1. [Google Scholar]
  • 5.Spokas EG, Rokach J, Wong PY-K. Leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids. In: Lianos EA, editor. Methods in Molecular Biology. Humana Press; Totowa, NJ: 1999. p. 213. [DOI] [PubMed] [Google Scholar]
  • 6.Lau CK, Adams J, Guindon Y, Rokach J. Chemistry of the leukotrienes and other lipoxygenase products. In: Rokach J, editor. Leukotrienes and Lipoxygenase: Chemical, Biological, and Clinical Aspects. Chapter 1. Elsevier Press; Amsterdam: 1989. p. 1. [Google Scholar]
  • 7.Rokach J, Guindon Y, Young RN, Adams J, Atkinson JG. Synthesis of the leukotrienes. In: ApSimon JW, editor. The Total Synthesis of Natural Products. Vol. 7. Wiley-Interscience; New York: 1988. p. 141. [Google Scholar]
  • 8.Rokach J, Adams J. Acc Chem Res. 1985;19:87. [Google Scholar]
  • 9.Adams J, Fitzsimmons BJ, Girard Y, Leblanc Y, Evans JF, Rokach J. J Am Chem Soc. 1985;107:464. [Google Scholar]
  • 10.Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ., II Proc Natl Acad Sci USA. 1990;87:9383. doi: 10.1073/pnas.87.23.9383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Hwang SW, Adiyaman M, Khanapure S, Schio L, Rokach J. J Am Chem Soc. 1994;116:10829. [Google Scholar]
  • 12.Praticò D, Barry OP, Lawson JA, Adiyaman M, Hwang SW, Khanapure SP, Iuliano L, Rokach J, FitzGerald GA. Proc Natl Acad Sci USA. 1998;95:3449. doi: 10.1073/pnas.95.7.3449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zamboni R, Rokach J. Tetrahedron Lett. 1983;24:999. [Google Scholar]
  • 14.Corey EJ, Albright JO, Barton AE, Hashimoto S. J Am Chem Soc. 1980;102:1435. [Google Scholar]
  • 15.Dussault P, Lee I. J Org Chem. 1995;60:218. [Google Scholar]
  • 16.Murphy RC, Hammarstrom S, Samuelsson B. Proc Natl Acad Sci USA. 1979;76:4275. [PubMed] [Google Scholar]
  • 17.Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T. Nature. 1997;387:620. doi: 10.1038/42506. [DOI] [PubMed] [Google Scholar]
  • 18.Powell WS, Rokach J. Prog Lipid Res. 2005;44:154. doi: 10.1016/j.plipres.2005.04.002. [DOI] [PubMed] [Google Scholar]
  • 19.O’Flaherty JT, Cordes J, Redman J, Thomas MJ. Biochem Biophys Res Commun. 1993;192:129. doi: 10.1006/bbrc.1993.1391. [DOI] [PubMed] [Google Scholar]
  • 20.Zhang Y, Styler A, Powell WS. J Leukoc Biol. 1996;59:847. doi: 10.1002/jlb.59.6.847. [DOI] [PubMed] [Google Scholar]
  • 21.Powell WS, Chung D, Gravel S. J Immunol. 1995;154:4123. [PubMed] [Google Scholar]
  • 22.Powell WS, Gravel S, Khanapure SP, Rokach J. Blood. 1999;93:1086. [PubMed] [Google Scholar]
  • 23.Powell WS, Gravel S, MacLeod RJ, Mills E, Hashefi M. J Biol Chem. 1993;268:9280. [PubMed] [Google Scholar]
  • 24.Powell WS, MacLeod RJ, Gravel S, Gravelle F, Bhakar A. J Immunol. 1996;156:336. [PubMed] [Google Scholar]
  • 25.O’Flaherty JT, Taylor JS, Thomas MJ. J Biol Chem. 1998;273:32535. doi: 10.1074/jbc.273.49.32535. [DOI] [PubMed] [Google Scholar]
  • 26.Hosoi T, Koguchi Y, Sugikawa E, Chikada A, Ogawa K, Tsuda N, Suto N, Tsunoda S, Taniguchi T, Ohnuki T. J Biol Chem. 2002;277:31459. doi: 10.1074/jbc.M203194200. [DOI] [PubMed] [Google Scholar]
  • 27.Jones CE, Holden S, Tenaillon L, Bhatia U, Seuwen K, Tranter P, Turner J, Kettle R, Bouhelal R, Charlton S, Nirmala NR, Jarai G, Finan P. Mol Pharmacol. 2003;63:471. doi: 10.1124/mol.63.3.471. [DOI] [PubMed] [Google Scholar]
  • 28.Stamatiou P, Hamid Q, Taha R, Yu W, Issekutz TB, Rokach J, Khanapure SP, Powell WS. J Clin Invest. 1998;102:2165. doi: 10.1172/JCI1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Muro S, Hamid Q, Olivenstein R, Taha R, Rokach J, Powell WS. J Allergy Clin Immunol. 2003;112:768. doi: 10.1016/s0091-6749(03)01888-8. [DOI] [PubMed] [Google Scholar]
  • 30.Sozzani S, Zhou D, Locati M, Bernasconi S, Luini W, Mantovani A, O’Flaherty JT. J Immunol. 1996;157:4664. [PubMed] [Google Scholar]
  • 31.Guilbert M, Ferland C, Bosse M, Flamand N, Lavigne S, Laviolette M. Am J Respir Cell Mol Biol. 1999;21:97. doi: 10.1165/ajrcmb.21.1.3517. [DOI] [PubMed] [Google Scholar]
  • 32.Powell WS, Gravel S, Halwani F. Am J Respir Cell Mol Biol. 1999;20:163. doi: 10.1165/ajrcmb.20.1.3141. [DOI] [PubMed] [Google Scholar]
  • 33.Schwenk U, Schroder JM. J Biol Chem. 1995;270:15029. doi: 10.1074/jbc.270.25.15029. [DOI] [PubMed] [Google Scholar]
  • 34.O’Flaherty JT, Kuroki M, Nixon AB, Wijkander J, Yee E, Lee SL, Smitherman PK, Wykle RL, Daniel LW. J Immunol. 1996;157:336. [PubMed] [Google Scholar]
  • 35.13C NMR CDCl3: 199.1, 174.1, 140.09, 136.92, 129.66, 129.61, 129.51, 128.05, 126.99, 126.31, 39.78, 33.78, 33.50, 28.59, 26.80, 26.69, 25.69, 21.47, 19.04. 1NMR CDCl3: δ 7.52–7.47 (1H, dd, J = 15.2, 11.5), 6.04–6.14 (2H, m), 5.83–5.76 (1H, q), 5.41–5.23 (4H, m), 3.00–3.04 (2H, t, J = 7.2), 2.74–2.77 (2H, t), 2.57–2.61 (2H, t, J = 7.12), 2.35–2.38 (2H, m), 1.87–2.06 (6H, m) 1.46–1.54 (2H, m), 1.34–1.41(2H, m).
  • 36.Adams J, Fitzsimmons BJ, Rokach J. Tetrahedron Lett. 1984;25:4713. [Google Scholar]
  • 37.Oakwood Products, Inc. West Columbia, SC: [Google Scholar]
  • 38.Powell WS, Gravelle F, Gravel S. J Biol Chem. 1992;267:19233. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS274051-supplement-01.doc (356.5KB, doc)

RESOURCES