A New Generation of Arachidonic Acid Analogues as Potential Neurological Agent Targeting Cytosolic Phospholipase A2

Cytosolic phospholipase A2 (cPLA2) is an enzyme that releases arachidonic acid (AA) for the synthesis of eicosanoids and lysophospholipids which play critical roles in the initiation and modulation of oxidative stress and neuroinflammation. In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.


Synthesis of 1 and 2
Commercially available reagents were bought from Sigma Aldrich, Alfa Aesar, Acros and Tokyo Chemical Industry and used without purification. AACOCF 3 , CDIBA and pyrrophenone were bought from Sigma Aldrich, Axon Medchem and Santa Cruz respectively. Solvents such as hexane, ethyl acetate, dichloromethane, and methanol were pre-distilled while others were used without further purification. Flash column chromatography was carried out on Merck silica gel 60. Thin-layer chromatography (TLC) was performed on precoated F254 silica plates from Merck and visualized with UV light. TLC were heated with potassium permanganate stain whenever necessary. Preparative TLC was from Analtech Silica GEL GP (Cat 02015). 1 H and 13 C NMR spectra were recorded on Bruker ACF300 (300MHz) or AMX 500 (500MHz) spectrometer at 298K. All J values are reported in Hz and chemical shift (δ) reported in parts per million (ppm) relative to tetramethylsilane (TMS). Mass spectra were determined by high resolution mass spectrometry (HRMS) electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI). General procedure for the synthesis of 1a-l and 1n-o. To the respective ester (0.31 mmol) was added 4M NaOH (5 mL) and the reaction mixture was microwaved for 1 h at 120 o C. Upon cooling to ambient temperature, the reaction mixture was quenched with 3M HCl (7 mL) and extracted with CH 2 Cl 2 (3 x 20 mL).
The combined organic phase was dried with anhydrous MgSO 4 , concentrated and used for the next step of the reaction without further purification. Trifluoromethylation was performed by adapting the procedure reported by Ackermann et al. 2 The acid obtained in the aforementioned reaction (0.31 mmol) was dissolved in CH 2 Cl 2 (5 mL). Thereafter, pyridine (0.26 mL, 3.20 mmol) and trifluoroacetic/chlorodifluoroacetic anhydride (0.44 mL, 3.123 mmol) were added. For 1a-h, the reaction mixture was stirred for 2 h. For 1n and 1o, the mixture was stirred overnight. Upon completion of reaction (based on TLC), the reaction was quenched by shaking the mixture with saturated NaHCO 3 (50 mL) and subsequently extracting the aqueous layer with ethyl acetate (3 x 20 mL). The combined organic layer was then dried with anhydrous MgSO 4 , concentrated and purified using flash column chromatography with a 98:2 hexane-ethyl acetate eluent system to afford the respective product as a yellow oil.
General procedure for the alkylation of 12a-d to form 13a-d. The general procedure employed was adapted from the protocol reported by Yoshida et al. 3 To propargyl alcohol (116 μL, 2.00 mmol) in THF (3 mL) was added HMPA (1.1 mL, 6.3 mmol) and the reaction mixture was cooled to -78 o C. 2 M n-Butyllithium in cyclohexane (2.0 mL, 4 mmol) was then added to the mixture via a canella and stirred vigorously.
Thereafter, the respective 12 (1.00 mmol) dissolved in THF (3 mL) was transferred to the reaction mixture via a canella. The temperature of the reaction mixture was allowed to rise to room temperature while stirring overnight. Upon completion of the reaction (based on TLC), the reaction was quenched by adding an equivalent amount of ethyl acetate and washed with saturated NH 4 Cl (3 x 20 mL). The organic layer was dried with MgSO 4 , concentrated and purified by flash column chromatography using a 5:1 hexane-ethyl acetate eluent system to afford the respective product as a colourless oil or white solid.
Upon the completion of reaction, the reaction was quenched by adding an equivalent amount of ethyl acetate and washed with aqueous NaHCO 3 (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried with MgSO 4 and purified by flash column chromatography.
General procedure for the synthesis of 20n and 20o. The respective acid 19 (0.12mmol) was dissolved in CH 2 Cl 2 (3.5 mL). Thereafter, oxalyl chloride (21.2 μL, 0.243 mmol) and 1 drop of DMF were added and the mixture was stirred for 1 h at room temperature. After which, the solvent was removed. The formed acid chloride was re-dissolved in DMF (1.0 mL), the respective amine (0.243 mmol) and TEA (34 μL, 0.243 mmol) and stirred at room temperature overnight. When no more starting material was observed, ethyl acetate and brine were added to quench the reaction. The mixture was extracted with ethyl acetate (3x 20mL) and the organic layer was combined, dried with MgSO 4 and purified by flash column chromatography.

1-bromopentadeca
Ligand preparation. The 3D structures of the test compounds were built using Maestro and minimized using the Macromodel module employing the OPLS-2005 force field in Schrodinger 9.0 [7][8] . All the inhibitors were then prepared with Ligprep that generates low energy tautomers and enumerates realistic protonation states at physiological pH.
Ligand docking. The prepared inhibitors were docked into the binding pockets of the models of cPLA 2 using Glide 9 . A box of size 10 x 10 x 10 Ǻ for molecular docking centered on the selected active site residue (Ser228) was used to confine the search space of each docked ligand. For the grid generation, the default Glide settings were used. A rigid receptor docking (RRD) protocol was used which fixes the protein conformation while allowing the ligands to be flexible. All inhibitors were docked into the active sites of cPLA 2 using this protocol and the docked conformation of each ligand was evaluated using the Glide Extra Precision (XP) scoring function. Docking was carried out on several conformational substrates of cPLA 2 identified by clustering of MD trajectories. The partial charges and force field parameters for each inhibitor