Constantinou-Kokotou, Violetta

Creason, Michael

Dennis, Edward A.

Kokotos, George

Magrioti, Victoria

Nicolaou, Aikaterini

Shah, Ishita

Smyrniotou, Anneta

Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

POTENT AND SELECTIVE INHIBITION OF HUMAN Ca2+-INDEPENDENT PHOSPHOLIPASE A2 BY FLUOROKETONESIntroductionThe superfamily of phospholipases A2 (PLA2) consists of hydrolytic enzymes that actupon the sn-2 ester bond of phospholipids, releasing free fatty acids and lysophospholipids.The main representative of these fatty acids is arachidonic acid, which can be transformedinto eicosanoids (prostaglandins, leukotriens, etc) by the action of other enzymes.Lysophospholipids are precursors for other bioactive compounds, such as platelet-activatingfactor (PAF). PAF and eicosanoids constitute basic mediators of inflammation. The threepredominant groups of phospholipase A2 found in human tissues are the cytosolic PLA2(cPLA2), the calcium-independent PLA2 (iPLA2) and the secreted PLA2 (sPLA2). iPLA2 hasbeen proposed as a homeostatic enzyme involved in basal metabolism within the cell, buthas also been found to be involved in diseases of the brain, the heart and the central nervoussystem, which makes this enzyme an attractive drug target. We have recently demonstratedthat Ca2+-independent phospholipase A2 (GVIA iPLA2) plays a key-role in experimentalautoimmune encephalomyelitis and that GVIA iPLA2 is a novel target for the developmentof new therapies for multiple sclerosis.1A series of fluoroketones has been presented as iPLA2 inhibitors and the structure-activity relationship has been evaluated.2 To extend this research, we synthesized a varietyof polyfluoroketones containing an aromatic ring and a four carbon atom chain between thering and the polyfluoroketone group.SynthesisCommercially available aromatic aldehydes 1a-f were used as starting materials, toprepare the desired fluoroketones (4a-f, 5a-f, 6a). Each aldehyde underwent a Horner –Wadsworth – Emmons reaction with triethyl-4-phosphonocrotonate in the presence of LiOHto produce the corresponding unsaturated ester 2a-f. After catalytic hydrogenation andsaponification with 1 N NaOH in ethanol we acquired the corresponding carboxylic acids3a-f which were converted to acyl chlorides by the oxalyl chloride/DMF method. In situ,acyl chlorides were treated with pyridine and trifluoroacetic anhydride orpentafluoropropionic anhydride or heptafluorobutanoic anhydride to providetrifluoromethyl ketones 4a-f, pentafluoroethyl ketones 5a-f and the heptafluoropropylketone 6a, respectively (Scheme 1).Anneta Smyrniotou,a Aikaterini Nicolaou,b Victoria Magrioti,b Michael Creason,c Ishita Shah,c Violetta Constantinou-Kokotou,aEdward A. Dennis,c George KokotosbaChemical Laboratories, Agricultural University of Athens, Athens, Greece; bDepartment of Chemistry, University of Athens, Athens, Greece; cDepartment of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, USA.Reagents and conditions: a) C2H5OOCCH=CHCH2P(=O)(OC2H5)2, LiOH, THF, 77oC, b) i) H2,10% Pd/C, EtOH, ii) NaOH 1N, EtOH, c) (COCl)2, DMF, CH2Cl2, d) pyridine, (CF3CO)2O, CH2Cl2,0 oC to r.t., e) pyridine, (CF3CF2CO)2O, CH2Cl2, 0oC to r.t., f) pyridine, (CF3CF2CF2CO)2O, CH2Cl2,0 oC to r.t.In vitro resultsAll synthesized compounds were tested for inhibition on human GVIA iPLA2,GIVA cPLA2 and GV sPLA2. (Table 1) The percentage of inhibition for each enzymewas determined at 0.091 mol fraction, and XI(50) values were measured for inhibitorswhich showed more than 93% inhibition for an enzyme. When compared to the knowninhibitor FKGK18, changing the position of the carbon chain on the naphthalene ring,leads to less potency, but higher selectivity. Increasing the number of fluorine atoms,GK172 and GK173 become less potent inhibitors of iPLA2. When a methoxy group isadded to the naphthalene ring, inhibitors GK213 and GK214 lose selectivity towardsiPLA2. The biphenyl group instead of the naphthalene shows a similar inhibition forboth iPLA2 and cPLA2 (GK174, GK175). The insertion of a trifluoromethyl group onthe aromatic ring presented excellent potency (GK178 and GK189), but poorselectivity. In the case of GK176 and GK188 the fluorine on the aromatic ring showsboth great potency and selectivity towards iPLA2. When the fluorine atom is replacedby a methoxy group, compounds GK177 and GK187 are currently the most potentand selective inhibitors for iPLA2. Inhibitors GK176, GK177, GK187 and GK188can be used to study the role of GVIA iPLA2 in neurological disorders.Table 1: Inhibition of PLA2 by fluoroketonesReferences1.Kalyvas, A.; Baskakis, C.; Magrioti, V.; Constantinou-Kokotou, V.; Stephens, D.;Dennis, E. A.; Kokotos G.; David, S. Brain 2009, 132, 1221-12352. Kokotos, G.; Hsu, Y.-H.; Burke, J. E.; Baskakis, C.; Kokotos, C.G.; Magrioti, V.;Dennis, E. A. J. Med. Chem. 2010, 53, 3602-361.AcknowledgementThis research has been co-financed by the European Union (European Social Fund –ESF) and Greek national funds through the Operational Program "Education andLifelong Learning" of the National Strategic Reference Framework (NSRF) - ResearchFunding Program: Heracleitus II. Investing in knowledge society through the EuropeanSocial Fund.1-6 ArabcdefScheme 1: Synthesis of polyfluoroketonesNo Structure iPLA2 cPLA2 sPLA2% inhibitionXI(50) % inhibitionXI(50) % inhibitionFKGK11 99.4 0.0014 ±0.0001N.D. 28FKGK18 99.9 0.0002 ±0.000093.1 0.039 ±0.00136.8GK171,4a98.7 0.0018 ±0.000277.4 31.9GK172,5a96.4 0.0034 ±0.000264.0 29.4GK173,6a76.7 60.4 57.8GK174,4e97.9 0.0189 ±0.004596.5 0.0074 ±0.000340.5 GK175,5e94.6 0.0134 ±0.001772.6 38.3GK176,4b98.5 0.0002 ±0.000041.4 N.D.GK177,4d99.8 0.0001 ±0.000054.3 N.D.GK178,4c99.9 0.0002 ±0.000083.9 30.4GK187,5d99.8 0.0001 ±0.000012.9 32.8GK188,5b98.2 0.0003 ±0.000022.2 29.3GK189,5c98.9 0.0003 ±0.000162.5 36.9GK213,4f92.6 85.5 41.7GK214,5f92.5 54.7 63.1N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).

Potent and selective inhibition of human Ca2+-independent phospholipase A2 by fluoroketones

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Potent and selective inhibition of human Ca2+-independent phospholipase A2 by fluoroketones

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Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)

Potent and selective inhibition of human Ca2+-independent phospholipase A2 by fluoroketones

Abstract

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Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)