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

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.

The diverse bioactive lipids produced by the cPLA 2 activity regulate normal physiological processes and disease pathogenesis, and as a consequence, great attention has been given to the development of selective GIVA cPLA 2 inhibitors. The structural diversity of the synthetic inhibitors is summarized in a number of review articles 1, [14][15][16] . The first synthetic inhibitor of GIVA cPLA 2 was an arachidonic acid derivative, arachidonoyl trifluoromethyl ketone, containing an activated carbonyl functionality 17 . Shionogi developed a series of pyrrolidine-based inhibitors, including pyrrophenone (1, Fig. 1), following a high throughput screening approach 18,19 . Wyeth has expended major efforts to develop novel indole-based inhibitors, for example, ecopladib (2a, Fig. 1), efipladib (2b, Fig. 1) and giripladib (2c, Fig. 1) as novel therapeutics for inflammatory diseases [20][21][22][23] . Giripladib was the most promising among them as it was advanced into a Phase II clinical trial for osteoarthritis, however in 2007 the trial was terminated due to gastrointestinal side effects 24 . A structurally related GIVA cPLA 2 inhibitor is currently on phase I/II clinical study in healthy volunteers and patients with moderate to severe dermatitis and the estimated date of completion is June 2017 25 . Our groups have designed and developed long chain 2-oxoamides based on unnatural amino acids, for example compound 3, as GIVA cPLA 2 inhibitors [26][27][28][29][30][31] . Lehr and coworkers studied a variety of activated carbonyl-based indol-1-yl-propan-2-ones, for example compound 4 (Fig. 1) containing a variety of substituents on the heterocyclic ring to optimize the enzyme-inhibitor binding [32][33][34][35][36] . Recently, we have reported the new thiazolyl ketone GK470 37 (5, Fig. 1) as a GIVA cPLA 2 inhibitor, while Tomoo and colleagues demonstrated a new series of indole-based inhibitors, such as inhibitor 6 38 .
To fully understand the role that each particular PLA 2 type plays in physiological and pathological conditions, and to develop new candidates for the treatment of various inflammatory diseases, potent and selective GIVA cPLA 2 inhibitors are needed. In this work, we present a novel class of potent and selective GIVA cPLA 2 inhibitors and our studies on their synthesis and study of their in vitro inhibitory potency and selectivity.

Results
Design and synthesis of inhibitors. Upon activation by intracellular calcium binding to the C2 domain of GIVA cPLA 2 , the enzyme is translocated to the surface of the phospholipid membrane where it extracts a single phospholipid substrate into the active site 39,40 . Then, the catalytic active site serine attacks the ester bond of the phospholipid substrate initiating the hydrolysis step. A number of the existing potent GIVA cPLA 2 inhibitors, for example arachidonoyl trifluoromethyl ketone 17 , 2-oxoamides [26][27][28][29][30][31] , indolyl-propanones [32][33][34][35][36] , thiazolyl ketones 37 contain an activated carbonyl group able to interact with the active site serine. In our quest for novel potent and selective GIVA cPLA 2 inhibitors, we envisaged that the 2-oxoester (or α-keto ester) functionality could serve as such an activated carbonyl group. In 1990, it was demonstrated that α-keto ester derivatives of N-protected amino acids and peptides inhibit serine and cysteine proteinases 41 , while peptidyl α-keto esters inhibit the serine proteases porcine pancreatic elastase and human neutrophil elastase 42 . Later on, various peptide α-keto-esters and α-keto acids were reported as inhibitors of calpains and other cysteine proteases 43 and of hepatitis C virus NS3 protease 44 . It is quite clear that a potential GIVA cPLA 2 inhibitor, in addition to a functionality targeting the active site serine, should contain a lipophilic chain able to mimic the interactions of the substrate arachidonoyl chain with the lipophilic binding site of the enzyme. In addition, a free carboxyl group may contribute significantly to the overall binding of the inhibitor to the enzyme. As we have proposed in the past 26 , and according to the results of our mechanistic studies using a combination of hydrogen-deuterium exchange mass spectrometry with molecular dynamics simulations 31 , such a carboxyl group may interact with the side chain of the enzyme residue Arg-200. Taken together, we designed compounds containing a 2-oxoester functionality, a lipophilic chain and a free carboxyl group (Fig. 2).
A variety of 2-hydroxy acids, required for the synthesis of 2-oxoesters, were synthesized as described in Fig. 3. Aldehydes 7a-d were converted into cyanohydrins 8a-d and consequently to 2-hydroxy methyl esters 9a-d by treatment with HCl in methanol. 2-Hydroxy acids 11a-d were obtained by alkaline hydrolysis of 9a-d.
The general route for the synthesis of the designed 2-oxoesters carrying a free carboxyl group is quite straightforward and is depicted in Fig. 4. The key-step was the reaction between the cesium salt of the appropriate 2-hydroxy acids 11a, 11c, 11d and 13a,b with omega-bromo esters 14a,b. The resulting 2-hydroxy esters 15a-h ware then oxidized to the corresponding 2-oxoesters 16a-h using preferably the Dess-Martin periodinane reagent 45 . Removal of the tert-butyl ester protecting group under acidic conditions led to the target compounds 17a-h.
2-Oxoester 19 carrying an ethyl ester group and 2-hydroxyester 20 carrying a free carboxyl group were synthesized as depicted in Fig. 5.
In vitro inhibition of GIVA cPLA 2 , GVIA iPLA 2 and GV sPLA 2 . All synthesized 2-oxoesters were tested for their in vitro activity on recombinant human GIVA cPLA 2 using mixed micelle assays. In addition, their selectivity over human GVIA iPLA 2 and GV sPLA 2 was also studied using group specific mixed micelle assays . The activity of these PLA 2 s was tested on mixed-micelles containing 100 µM PAPC and 400 µM Triton-X.
The in vitro inhibition of human GIVA cPLA 2 , GVIA iPLA 2 and GV sPLA 2 was carried out using previously described mixed micelle-based assays 27,28,30 . The inhibition results are presented in Table 1, either as percent inhibition or as X I (50) values. At first, the percent of inhibition for each PLA 2 enzyme at 0.091 mole fraction of each inhibitor was determined. Then, the X I (50) values were measured for compounds that displayed greater than 95% inhibition of GIVA cPLA 2 . The X I (50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%.
Representative curves for the concentration dependence of the inhibition of GIVA cPLA 2 by 2-oxoesters 17a, 17b and 17d were fit to sigmoidal curves and are presented in Fig. 6.

Discussion
Methyl 2-oxopalmitate 10e (entry 1, Table 1) weakly inhibited, at a high concentration, both the intracellular enzymes GIVA cPLA 2 and GVIA iPLA 2 . However, 2-oxopalmitic acid 12e (entry 2, Table 1) inhibited weakly, but selectively, GIVA cPLA 2 . Interestingly, when the 2-oxoester functionality was combined with a long aliphatic chain together with a free carboxyl group at a distance of three carbon atoms, potent inhibition of GIVA cPLA 2 was observed and the inhibitor 17a (GK161) showed a X I (50) value of 0.00008 (entry 3, Table 1). In addition, this inhibitor was selective and did not inhibit the activities of GVIA iPLA 2 and the secreted GV sPLA 2 . This selectivity is in agreement with our previous observations that 2-oxoamides containing a free carboxyl group selectively inhibit GIVA cPLA 2 28, 30 . Given that for the most potent 2-oxoamides present X I (50) values are not lower than 0.003 30 , the present 2-oxoester was proven to be a much more potent inhibitor of GIVA cPLA 2 . The corresponding 2-hydroxy ester derivative 20 did not present any inhibition of either GIVA cPLA 2 or GVIA iPLA 2 (entry 4, Table 1), demonstrating the importance of the oxoester functionality for the inhibition.
When the long aliphatic chain was replaced by a chain of a similar size containing an aromatic ring, the inhibitory activity over GIVA cPLA 2 was considerably reduced (entry 5, Table 1). Compounds 16c and 19 containing a medium chain carrying an aromatic ring and a protected carboxyl group (either ethyl ester or tert-butyl ester) totally abolished any inhibitory activity (entries 6 and 7, Table 1). In accord with our expectation, the replacement of the long aliphatic chain by a more drug-like chain of four carbon atoms carrying a biphenyl system led again to a potent and selective inhibition of GIVA cPLA 2 (entry 8, Table 1). Inhibitor 17d (GK200) was found to be eight times less potent than 17a showing a X I (50) value of 0.00068. To extend the structure-activity relationship studies, we either increased the distance between the free carboxyl group and the oxoester functionality or decreased the distance between the aromatic rings and the oxoester functionality. Inhibitor 17e (GK433) proved to be highly potent, slightly better than 17a, presenting a X I (50) value of 0.00007 (entry 9, Table 1). The importance of the four-carbon atoms distance between the free carboxyl group and the oxoester functionality was clearly demonstrated by the inhibitor 17f (GK452), which presented highly potent inhibition of GIVA cPLA 2 with a X I (50) value of 0.000078 (entry 10, Table 1). Decrease of the distance between the biphenyl aromatic system and the oxoester functionality (compounds 17g and 17h) resulted in considerable reduction of the potency (entries 11 and 12, Table 1). All the highly potent GIVA cPLA 2 inhibitors 17a, 17d, 17e and 17f presented selectivity, because none of them exhibited any appreciable inhibition of GVIA iPLA 2 . In addition, none of the synthesized and tested 2-oxoesters inhibited GV sPLA 2 .
Since both the intracellular enzymes GIVA cPLA 2 and GVIA iPLA 2 are serine hydrolases and both utilize a catalytic dyad in their catalytic mechanism, it is likely that cross reactivity may be observed for inhibitors designed to carry a functionality targeting the active site serine. Indeed, such cross reactivity has been observed for several inhibitors containing an activated carbonyl group initially developed to target GIVA cPLA 2 . For example, arachidonoyl trifluoromethyl ketone was found to inhibit not only GIVA cPLA 2 , but also GVIA iPLA 2 . It is apparent that the presence of other groups able to develop appropriate hydrophobic and/or hydrophilic interactions contributes to the overall binding of the inhibitor to the enzyme, determining the inhibitory selectivity over GIVA cPLA 2 or GVIA iPLA 2 . We have previously shown that pentafluoroethyl or trifluoromethyl ketones of a four-carbon atom chain carrying an aromatic ring are selective inhibitors of GVIA iPLA 2 46-48 . Inspired by the structures of FKGK11 46 and FKGK18 47 , we designed simple methyl 2-oxoesters with a linker of four methylene groups between the activated carbonyl group and the aromatic ring. Unfortunately, compound 10a (entry 13, Table 1) carrying a phenyl ring only weakly inhibited GVIA iPLA 2 at a high concentration. On the contrary, compound 10b (GK451)    (entry 14, Table 1) carrying a naphthalene ring presented interesting inhibition of GVIA iPLA 2 with a X I (50) value of 0.0052. At the same time, it presents selectivity, because it only weakly inhibits GIVA cPLA 2 at a high concentration (55% at 0.091 mole fraction), while it does not inhibit at all GV sPLA 2 .
To better understand the interaction of 2-oxoesters with GIVA cPLA 2 and GVIA iPLA 2 , the most potent GIVA cPLA 2 inhibitor 17f was docked in the active site of either GIVA cPLA 2 or GVIA iPLA 2 . For the docking calculations, the structures of GIVA cPLA 2 and GVIA iPLA 2 with two different fluoroketone compounds in the active site were used (GK174: orange color in Fig. 7a and FKGK18: magenta color in Fig. 7b). The binding mode of these two fluoroketones was validated using H/D exchange and MD simulations in a previously published study 49 . A theoretical score of 10.2 kcal/mol indicated that 17f is a tight binder for GIVA cPLA 2 . The oxoester moiety forms hydrogen-bonding with the oxyanion hole (Gly197/Gly198), while the carboxylic moiety interacts with Arg200, which was found to stabilize the phosphate group of a phospholipid substrate molecule 39 . Compared to GK174 (orange color in Fig. 7a) the addition of the carboxylic moiety is responsible for increasing the potency of 17f by 10-fold. This compound exhibits no activity towards GVIA iPLA 2 and it received a low theoretical binding score of 6.3 kcal/mol indicating that is a weak binder. Compared to fluoroketone FKGK18 (magenta color in Fig. 7b) the addition of the carboxylic moiety increases the size of the compound and it cannot be accommodated in the active site of GVIA iPLA 2 .
All the above data, clearly demonstrate that 2-oxoesters consisting of a quite long chain (aliphatic or incorporating aromatic systems like the biphenyl system) in combination with a free carboxyl group at a distance of four or three carbon-atoms from the oxoester functionality are highly potent and selective inhibitors of GIVA cPLA 2 . Decreasing the size of the synthetic compound and eliminating the free carboxyl group may change the selectivity. Indeed, a methyl 2-oxoester based on a short chain carrying a naphthalene ring was found to inhibit preferentially GVIA iPLA 2 . In other words, it seems that the selectivity of compounds based on the 2-oxoester functionality may be tuned choosing the structural features that ensure the appropriate interactions with each enzyme (either GIVA cPLA 2 or GVIA iPLA 2 ).
To compare our novel highly potent 2-oxoester inhibitors of GIVA cPLA 2 with the existing inhibitors, we studied the benchmark GIVA cPLA 2 inhibitor 4 in our mixed-micelle assay. This inhibitor, developed by Lehr 32 , is the most potent inhibitor in the literature presenting an IC 50 value of 4.3 nM in a vesicle assay 32 . In the mixed micelle assay, it was proved equipotent with oxoester 17a with a X I (50) value of 0.00008 (entry 15, Table 1). In addition, several 2-oxoesters were found to be more potent than the other benchmark GIVA cPLA 2 inhibitor 1 (pyrrophenone), which presents an X I (50) value of 0.002 26,27 . Another important property of a GIVA cPLA 2 inhibitor, is the ClogP value, which is a measure of the hydrophobicity. ClogP represents the calculated partition coefficient in octanol/water on a logarithmic scale. Usually, GIVA cPLA 2 inhibitors suffer from high lipophilicity. For example, the ClogP value of inhibitor 4 is 8.50, while pyrrophenone 1 and giripladib 2c present high lipophilicities too (ClogP 8.29 and 10.75, respectively). Inhibitors with such high values are not expected to present favorable ADME properties according to Lipinski's rule of five 50 . Although 2-oxoesters 17a and 17e contain a long aliphatic chain, they present lower lipophilicity (ClogP 6.76 and 6.68, respectively), while the 2-oxoesters 17d and 17f carrying the biphenyl system have considerably lower ClogP values (4.78 and 4.70, respectively). The logP value of 17f, measured by HPLC, was found 3.5. Thus, the lipophilicity of 17f is encouraging and this inhibitor is the first example of a highly potent GIVA cPLA 2 inhibitor, which presents a ClogP value lower than 5.
The cellular effect of the most potent GIVA cPLA 2 inhibitor 17f on eicosanoid biosynthesis was studied in macrophages. RAW264.7 macrophages were used as a model system to determine if 17f displays inhibitory activity toward GIVA cPLA 2 in vivo. It is well established that the toll-like receptor 4 (TLR4)-specific agonist Kdo2-lipid A (KLA) leads to GIVA cPLA 2 activation 51, 52 and release of arachidonic acid in macrophages that is then converted into eicosanoids by cyclooxygenase-2 [53][54][55] . Previous work has demonstrated that the major eicosanoid produced by KLA stimulated RAW264.7 macrophages is prostaglandin D 2 (PGD 2 ) 56 . The high levels of PGD 2 compared to background in culture supernatants following KLA stimulation makes it an ideal marker for GIVA cPLA 2 activity in macrophages. Inhibitor 17f did not show cellular toxicity at any concentrations tested as measured by trypan blue exclusion (data not shown). RAW264.7 macrophages were pre-treated with vehicle control, DMSO or 17f (5 μM) for one hour prior to stimulation with KLA (100 ng/mL). Culture supernatants were collected after 24 hours for eicosanoid quantification by LC-MS/MS. Treatment with inhibitor 17f resulted in over 50% decrease in KLA-elicited PGD 2 production by macrophages (Fig. 8). A similar reduction in other minor products including PGE 2 , PGF 2α , 11-HETE and 15-HETE was observed (data not shown), suggesting that the inhibition was not specific to PGD 2 . This data is consistent with 17f inhibition of GIVA cPLA 2 in living cells.  32 .
In conclusion, we describe a novel class of GIVA cPLA 2 inhibitors based on the 2-oxoester functionality. This reactive 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 cPLA 2 inhibitors. Inhibitors 17a, 17e and 17f present X I (50) values of 0.00007-0.00008 and are equipotent to the most potent known GIVA cPLA 2 inhibitor. In particular, inhibitors incorporating the biphenyl system, like 17f, present interesting favorable lipophilicity (ClogP values lower than 5). The novel highly potent and selective GIVA cPLA 2 inhibitors may be excellent tools for the study of the role of the enzyme in cells and in animals and may contribute to the development of novel medicinal agents for the treatment of inflammatory diseases.

Methods
General. Chromatographic purification of products was accomplished using Merck Silica Gel 60 (70-230 or 230-400 mesh). Thin-layer chromatography (TLC) was performed on Silica Gel 60 F254 aluminum plates. TLC pots were visualized with UV light and/or phosphomolybdic acid in EtOH. Melting points were determined using a Büchi 530 apparatus and were uncorrected. 1 H and 13 C NMR spectra were recorded on a Varian Mercury (200 MHz and 50 MHz respectively) in CDCl 3 . Chemical shifts are given in ppm, and coupling constants (J) in Hz. Peak multiplicities are described as follows: s, singlet, d, doublet, t, triplet and m, multiplet. Electron spray ionization (ESI) mass spectra were recorded on a Finnigan, Surveyor MSQ Plus spectrometer. Dichloromethane was dried by standard procedures and stored over molecular sieves. All other solvents and chemicals were reagent grade and used without further purification. The purity of all compounds subjected to biological tests was determined by analytical HPLC, and was found to be ≥95%. HPLC analyses were carried out on a Shimadzu LC-2010AHT system and a Merck Chromolith Performance (100 × 4.6 mm) analytical column, using H 2 O/ MeOH 10/90 v/v, at a flow rate of 1.0 mL/min. HRMS spectra were recorded on a Bruker Maxis Impact QTOF Spectrometer.

Synthesis of 2-hydroxy acids 11α-e.
To a stirred solution of 2-hydroxy ester 9a-e (1 mmol) in methanol (10 mL), aqueous NaOH (1.1 mL, 1 N) was added and the reaction mixture was stirred overnight at room temperature. The organic solvent was evaporated in vacuo to dryness and then aqueous HCl 1 N was added until acidic pH. The aqueous phase was washed with EtOAc (3 × 10 mL). Finally, the organic phase was dried (Na 2 SO 4 ) and evaporated under reduced pressure.

2-Oxohexadecanoic acid (12e).
To a stirred solution of 9e (0.35 mmol, 100 mg) in MeOH (3.5 mL), aqueous Cs 2 CO 3 20% (w/v) (1.7 mL, 1.0 mmol) was added, and the reaction mixture was stirred at room temperature. The reaction progress was monitored by TLC, until completion. The organic solvent was evaporated in vacuo to dryness, water was added (10 mL) and then aqueous HCl 1 N was added until acidic pH. The aqueous phase was washed with EtOAc (3 × 10 mL). Finally, the organic phase was dried over Na 2