Barbayianni, Efrosini

Constantinou-Kokotou, Violetta

Dennis, Edward A.

Kokotos, George

Shah, Ishita

Smyrniotou, Anneta

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

INHIBITION OF Ca2+- INDEPENDENT PHOSPHOLIPASE A2 BY 2-OXOAMIDES BASED ON DIPEPTIDES AND ETHER PSEUDODIPEPTIDESAnneta Smyrniotou,a Efrosini Barbayianni,b Ishita Shah,c Violetta Constantinou-Kokotou,aEdward A. Dennis,c George KokotosbaChemical Laboratories, Agricultural University of Athens, Athens 11855, Greece; b Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece; cDepartment of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093-0601, USA.IntroductionPhospholipases A2 (PLA2) catalyze the hydrolysis of the sn-2 ester bond ofglycerophospholipids producing free fatty acids and lysophospholipids. The free arachidonicacid (AA) that is released may be converted to a variety of proinflammatory eicosanoids.Therefore, inhibiting AA release is of great therapeutic relevance for the development of newanti-inflammatory drugs. Moreover, selective inhibition of the less studied enzyme of thethree main categories, GVIA iPLA2, may offer valuable information about the enzyme’sphysiological role. A new class of PLA2 inhibitors has been developed by our group: longchain 2-oxoamides based on amino acids.1-3 Among others, we have shown that twomolecules based on a pseudodipeptide ethyl ester and a dipeptide tert-butyl ester are the first2-oxoamide derivatives, which preferentially inhibit GVIA iPLA2 with ΧΙ(50) values of 0.017and 0.011 μΜ, respectively.4 To extend our studies, we synthesized a variety of 2-oxoamidesbased on dipeptides and ether pseudodipeptides, bearing an aryl terminal group on their 2-oxoamide aliphatic chain and we studied their in vitro activity on three human PLA2 classes:GIVA cPLA2, GVIA iPLA2 and GV sPLA2.SynthesisAs starting materials for the synthesis of 2-hydroxy-acids we used 5-phenyl-1-pentanol (1)and 2-naphthaldehyde (4). Alcohol 1 was oxidized to aldehyde using the AcNH-TEMPOmethod, which was treated with NaHSO3/KCN to provide the corresponding cyanhydrin 2.The latter was converted to the 2-hydroxy-acid 3 after treatment with condensed hydrochloricacid and subsequently with potassium hydroxide in a solution of ethanol/water. 2-Naphthaldehyde (4) underwent a Horner – Wadsworth – Emmons olefination, in order toextend the carbon chain, followed by catalytic hydrogenation. The resulting ester 6 wasreduced to the corresponding alcohol 7 using DIBALH. Following the same chemical steps aspreviously, cyanhydrin 8 was obtained, which was converted to the 2-hydroxy-methylester 9after treatment with methanolic hydrochloride. The desired 2-hydroxy-acid 10 was producedafter saponification (Scheme 1). N-Protected L-norleucines 11a,b and glycine esters 12a,bwere coupled using WSCI.HCl as a condensing agent in the presence of HOBt to provide thedesired dipeptides (13a,b) (Scheme 2), while the pseudodipeptide 20 was obtained afterreaction of L-norleucinol with tert-butyl bromoacetate under phase transfer conditions(Scheme 3).Scheme 1. Reagents and conditions: a) i. NaOCl, NaBr, AcNH-TEMPO, NaHCO3, AcOEt/toluene/H2O 3:3:0.5, -5oC; ii. NaHSO3, KCN, CH2Cl2, H2O; b) i. conc. HCl, ii. KOH, EtOH/H2O; c) C2H5OOCCH=CHCH2P(=O)(OC2H5)2,LiOH, THF, reflux; d) H2, 10% Pd/C, EtOH; e) DIBALH, anhydrous Et2O; f) 3N HCl/ MeOH; g) NaOH 1N, MeOH.Scheme 2. Reagents and conditions: a) WSCI, HOBt, Et3N, CH2Cl2; b) H2, 10% Pd/C, THF; c) 4N HCl/Et2O; d)Dess-Martin periodinane, CH2Cl2; e) 50% TFA/CH2Cl2.13, 14 P R Yield (%) a b c a Z But 81 97  b Boc  Et 72  98     15-17 Ar R Yield (%) a d e a Phenyl But 66 79 88 b Phenyl Et 58 80  c Naphthyl But 47 99 93 d Naphthyl Et 42 80   Coupling reaction between the free amino group of the dipeptides or the etherpseudodipeptide and the 2-hydroxy acid took place in the presence of WSCI/HOBt and theresulting 2-hydroxy amides 15a-d and 22a,b were oxidized to 2-oxoamides 16a-d and 23a,busing the Dess-Martin method. Oxoamides 17a,c and 24a,b containing a free carboxyl group,were obtained by treatment of 16a,c and 23a,b with trifluoroacetic acid (Schemes 2 and 3).Scheme 3. Reagents and conditions: a) i. NMM, ClCOOEt, THF; ii. NaBH4, CH3OH; b) BrCH2COOBut, 50%NaOH, Bu4NHSO4, benzene; c) H2, 10% Pd/C; d) WSCI, HOBt, Et3N, CH2Cl2; e) Dess-Martin periodinane, CH2Cl2;f) 50% TFA/CH2Cl2.AcknowledgementsThis research has been co-financed by the European Union (European Social Fund – ESF) and Greek national fundsthrough the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework(NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European SocialFund.References1. Stephens, D.; Barbayianni, E.; Constantinou-Kokotou, V.; Peristeraki, A.; Six, D. A.; Cooper, J.;Harkewicz, R.; Deems, R. A.; Dennis, E. A.; Kokotos, G. J. Med. Chem. 2006, 49, 2821.2. Six, D. A.; Barbayianni, E.; Loukas, V.; Constantinou-Kokotou, V.; Hadjipavlou-Litina, D.;Stephens, D.; Wong, A. C.; Magrioti, V.; Moutevelis-Minakakis, P.; Baker, S.; Dennis, E. A.;Kokotos, G. J. Med. Chem. 2007, 50, 4222.3. Antonopoulou, G.; Barbayianni, E.; Magrioti, V.; Cotton, N.; Stephens, D.; Constantinou-Kokotou, V.; Dennis, E. A.; Kokotos, G. Bioorg. Med. Chem. 2008, 16, 10257.4. Barbayianni, E.; Stephens, D.; Grkovich, A.; Magrioti, V.; Hsu, Y.-H.; Cotton, N.; Dolatzas, P.;Kalogiannidis, D.; Dennis, E. A.; Kokotos, G. Bioorg. Med. Chem. 2009, 17, 4833.Results and discussionPreliminary PLA2 inhibition results are summarized in Table 1. Derivatives 24 and 25 havebeen also included for comparison reasons. Among the seven compounds tested, 16a and 16c,based on Nle-Gly tert-butyl ester, bearing phenyl and naphthyl side chain terminal moietiesrespectively, appear to inhibit preferentially GVIA iPLA2. This observation is in fullagreement with our previous report, that the 2-oxoamide derivative based on Nle-Gly tert-butyl ester (24) is potent and selective inhibitor of GVIA iPLA2.4 The other two tert-butylesters of this series, namely 23a and 23b, based on ether pseudodipeptides, exhibitedconsiderable but lower activity towards GVIA iPLA2, though they inhibit also GIVA cPLA2 toa similar level. 2-Oxoamide derivatives containing free carboxylic acids (17a and 17c) do notinhibit GVIA iPLA2.Compound Structure% InhibitioncPLA2 iPLA2 sPLA216a 46.9 91.1 43.416c 57.5 89.1 44.816d 70.5 77.1 54.417a 11.2 - -17c 43.1 - -23a 82.3 76.0 57.523b 85.5 88.4 63.924a 72XI(50)0.0115925a 52XI(50)0.01781Table 1. In vitro results of the human phospholipases A2 inhibition  by 2-oxoamides.22-24 Ar Yield (%) d e f a Phenyl 53 95 98 b Naphthyl 69 84 97  a Data taken from Ref. 4

Inhibition OF Ca2+- Independent phospholipase A2 by 2-oxoamides based on dipeptides and ether pseudodipeptides

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Inhibition OF Ca2+- Independent phospholipase A2 by 2-oxoamides based on dipeptides and ether pseudodipeptides

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Inhibition OF Ca2+- Independent phospholipase A2 by 2-oxoamides based on dipeptides and ether pseudodipeptides

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