2-Oxoesters: A Novel Class of Potent and Selective Inhibitors of Cytosolic Group IVA Phospholipase A2

Abstract

Cytosolic phospholipase A₂ (GIVA cPLA₂) is the only PLA₂ that exhibits a marked preference for hydrolysis of arachidonic acid containing phospholipid substrates releasing free arachidonic acid and lysophospholipids and giving rise to the generation of diverse lipid mediators involved in inflammatory conditions. Thus, the development of potent and selective GIVA cPLA₂ inhibitors is of great importance. We have developed a novel class of such inhibitors based on the 2-oxoester functionality. This functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA₂ inhibitors (X _I(50) values 0.00007-0.00008) and docking studies aid in understanding this selectivity. A methyl 2-oxoester, with a short chain carrying a naphthalene ring, was found to preferentially inhibit the other major intracellular PLA₂, the calcium-independent PLA₂. In RAW264.7 macrophages, treatment with the most potent 2-oxoester GIVA cPLA₂ inhibitor resulted in over 50% decrease in KLA-elicited prostaglandin D₂ production. The novel, highly potent and selective GIVA cPLA₂ inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of inflammatory diseases.

Figure 1

Common inhibitors of phospholipases A₂.

Figure 2

Design of 2-oxoesters.

Figure 3

Synthesis of 2-hydroxy acids and 2-oxoacids. (a) (i) aq. sol. NaHSO₃, CH₂Cl₂, (ii) KCN, H₂O; (b) 4 N HCl/CH₃OH; (c) Dess-Martin periodinane reagent, dry CH₂Cl₂; (d) NaOH 1 N, CH₃OH; (e) 20% aq. sol. Cs₂CO₃, CH₃OH.

Figure 4

Synthesis of 2-oxoesters. (a) i. 20% aq. sol. Cs₂CO₃, THF, H₂O, ii. Br(CH₂)_nCH₂CH₂COOBu^t, DMF, reflux overnight; (b) Dess-Martin periodinane reagent, dry CH₂Cl₂; (c) 50% CF₃COOH in CH₂Cl₂.

Figure 5

Synthesis of compounds 19 and 20. (a) i. 20% aq. sol. Cs₂CO₃, THF, H₂O, ii. BrCH₂CH₂CH₂COOEt, DMF, reflux overnight; (b) Dess-Martin periodinane reagent, dry CH₂Cl₂; (c) 50% CF₃COOH in CH₂Cl₂.

Figure 6

Inhibition curves for 17a, 17b and 17d. The curves were generated using GraphPad Prism with a nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition versus log(inhibitor concentration). The reported X _I(50) values were calculated from the resultant plots.

Figure 7

Binding mode of inhibitor 17f in the active site of (a) GIVA cPLA₂ and (b) GVIA iPLA₂.

Figure 8

Inhibitor 17f inhibits KLA-elicited prostaglandin D₂ biosynthesis by macrophages. Macrophages were pre-treated with media (control), vehicle control (DMSO, 0.1%) or inhibitor 17f (5 μM) 1 hr before KLA (100 ng/mL, ■) or mock (□) treatment. Supernatants were collected 24 hr following stimulation for eicosanoid quantification. Graph displays the mean ± SEM of a single experiment containing technical duplicates that is representative of 2 independent experiments. * indicates statistical difference compared to KLA treatment (P ≤ 0.05).