Differential Inhibition of Group IVA and Group VIA Phospholipases A₂ by 2-Oxoamides

Abstract

Inhibitors of the Group IVA phospholipase A₂ (GIVA cPLA₂) and GVIA iPLA₂ are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA₂ based on the 2-oxoamide backbone that are uncharged, containing ester groups. While the most potent inhibitors of GVIA iPLA₂ also inhibited GIVA cPLA₂, there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA₂. We further show that several potent 2-oxoamide inhibitors of GIVA cPLA₂, containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891–2893), do not inhibit GVIA iPLA₂, and are therefore selective GIVA cPLA₂ inhibitors.

Introduction

Phospholipase A₂ (PLA₂) constitutes a superfamily of enzymes that catalyze the hydrolysis of the fatty acid ester from the sn-2 position of a membrane phospholipid, yielding a free fatty acid and a lysophospholipid. Among the intracellular PLA₂s are the cytosolic Group IVA PLA₂ (GIVA cPLA₂), which is generally considered a pro-inflammatory enzyme, and the calcium-independent Group VIA iPLA₂ (GVIA iPLA₂), which is typically referred to in the literature as iPLA₂. GVIA iPLA₂ is actually a group of cytosolic enzymes ranging from 85 to 88 kDa and expressed as several distinct splice variants of the same gene, only two of which have been shown to be catalytically active (Group VIA-1 and VIA-2 iPLA₂).1 The role of GVIA iPLA₂ in the inflammatory process is unclear, but this enzyme appears to be the primary PLA₂ for basal metabolic functions within the cell, reportedly including membrane homeostasis,^2–7 insulin receptor signaling^5,8 and calcium channel regulation.^9–11

The GVIA iPLA₂ enzymes all contain a consensus lipase motif, Gly-Thr-Ser*-Thr-Gly, with the catalytic serine confirmed by site-directed mutagenesis.^1,12 More recently the homologous Group VIB iPLA₂ was confirmed to have an active site catalytic dyad consisting of the conserved Ser and an equally conserved Asp.¹³ The first identification of the novel catalytic Ser/Asp dyad was for GIVA cPLA₂ based on exhaustive mutagenesis and a crystal structure, which confirmed that the catalytic dyad is present in a non-canonical α/β hydrolase and that the mechanism involves an acyl-enzyme intermediate on the serine.^14–19 A similar structure, topology, and conserved catalytic dyad were also found in patatin, a distant plant homolog of both GIV and GVI PLA₂.20 The growing family of lipid hydrolases ulilizing a catalytic Ser-Asp dyad now includes bacterial ExoU, fungal phospholipase B/Spo1, plant patatins, and the many mammalian enzymes in the GIV PLA₂, GVI PLA₂, and neuropathy target esterase groupings.²¹

Arachidonyl trifluoromethyl ketone (ATFK) has been shown to function as a tight binding, reversible inhibitor of both GIVA and GVIA PLA₂,22,23 while methyl arachidonyl fluorophosphonate (MAFP) functions as an irreversible inhibitor of both enzymes.²⁴ Variants of the trifluoromethyl ketones show differential potencies for GIVA and GVIA PLA₂; oleic acid- and phenyl-containing compounds are more potent than ATFK with GVIA iPLA₂ and less potent than ATFK with GIVA cPLA₂.25 Similar trends in potency are seen with the fluorophosphonate inhibitors; oleic acid and phenyl derivatives are more potent than MAFP towards GVIA iPLA₂.25 Interestingly, the trifluoromethylketone and fluorophosphonate inhibitors all show fast binding to GVIA iPLA₂ and slow binding to GIVA cPLA₂,22,25,26 suggesting subtle differences in the active sites of GIVA and GVIA PLA₂. Bromoenol lactone (BEL) is an irreversible, covalent inhibitor of GVIA iPLA₂ but does not inhibit GIVA cPLA₂. Because of this, BEL is commonly used to selectively inhibit GVIA iPLA₂ in cellular systems.^3,5,7,9,22 However, it has been shown that in addition to inhibiting GVIA iPLA₂, BEL inhibits numerous cellular enzymes including the magnesium-dependent phosphatidate phosphohydrolase 1.²⁷

We have recently reported that 2-oxoamides containing a free carboxyl group are potent inhibitors of human GIVA cPLA₂.28,29 The aim of the present work was to develop inhibitors based on the 2-oxoamide backbone that are selective for GIVA or GVIA PLA₂. Based upon the similarity of substrates, classes of common inhibitors, and the homologous Ser-Asp catalytic dyad, it is very likely that the active sites of GIVA and GVIA PLA₂ are similar such that inhibitors of GIVA cPLA₂ may show cross-reactivity with GVIA iPLA₂. There are, however, significant differences in substrate preference, known inhibitor profiles, and the primary sequence between GIVA and GVIA PLA₂ that could be exploited in designing selective inhibitors.

Design and Synthesis of 2-Oxoamide Inhibitors

We have developed a strategy for the design of inhibitors of serine-containing lipolytic enzymes, which is based on the principle that the inhibitor should consist of two components: (a) an electrophilic group that is able to react with the active-site serine residue, and (b) a lipophilic segment that contains chemical motifs necessary for both specific interactions and a proper orientation in the substrate binding cleft of the enzyme.³⁰ This strategy has been successfully applied in the development of lipophilic 2-oxoamides,^31,32 2-oxoamide and bis-2-oxoamide triacylglycerol analogues,^33,34 as well as lipophilic aldehydes³⁵ and trifluoromethyl ketones³⁶ as effective inhibitors of pancreatic and gastric lipases. Accordingly, we have recently developed a novel class of 2-oxoamides that inhibit GIVA cPLA₂.28,29 The noted homology of GVIA iPLA₂ to GVIB PLA₂, patatin and GIVA cPLA₂ (lipases known to possess a catalytic Ser-Asp dyad) and the confirmation of its catalytic serine strongly suggest that GVIA iPLA₂ would be susceptible to inhibition by 2-oxoamides.¹² Thus, we studied a number of 2-oxoamides of the generic structure shown in Scheme 1 in an effort to understand the effect of R¹ and R² groups on GVIA iPLA₂ inhibition.

Scheme 1.

2-Oxoamide inhibitors containing either a free carboxyl group or a carboxymethyl ester group and 2-oxoacyl residues based on oleic acid or phenyl groups were synthesized using methods previously developed,²⁹ as depicted in Scheme 2. In Scheme 3, the synthesis of inhibitors based on a γ-amino-α,β-unsaturated acid is shown. It should be noted that the oxidation of the unsaturated 2-hydroxyamides 2c, 6 and 7 was carried out using Dess-Martin periodinane,³⁷ instead of NaOCl/TEMPO, to avoid oxidation of the double bonds.

Scheme 2^a.

^aReagents and conditions: (a) H₂N(CH₂)₃COOCH₃, Et₃N, WSCI, HOBt, CH₂Cl₂; (b) NaOCl, TEMPO, NaBr, NaHCO₃, EtOAc/toluene/H₂O, 0 ºC; (c) Dess-Martin periodinane, CH₂Cl₂; (d) 1N NaOH/MeOH; (e) NaOCl, TEMPO, NaBr, NaHCO₃, EtOAc/toluene/H₂O, 0 ºC, then HCl.

Scheme 3^a.

^aReagents and conditions (a) NaOCl, TEMPO, NaBr, NaHCO₃, EtOAc/toluene/H₂O, −5 ºC; (b) Ph₃P=CHCOOCH₃, THF, reflux; (c) 4 N HCl in THF; (d) CH₃(CH₂)₁₃CHOHCOOH, Et₃N, WSCI, HOBt, CH₂Cl₂; (e) 1N NaOH/MeOH; (f) Dess-Martin periodinane, CH₂Cl₂.

Selective Inhibition of GIVA and GVIA PLA₂ by 2-Oxoamide Inhibitors

Fourteen 2-oxoamides were tested for inhibition of GVIA iPLA₂ in our in vitro assay system^27,28 and compared with GIVA cPLA₂ inhibition. The data, summarized in Table 1, are represented as X_I(50) values. X_I(50) is defined as the inhibitor concentration in a two-dimensional micellar surface that produces 50% inhibition. The surface concentration (mole fraction units) is calculated as the moles of inhibitor divided by the total moles of inhibitor, detergent, and phospholipid in the micelle surface. X_I(50) is utilized as opposed to the more common IC₅₀ because GIVA and GVIA PLA₂ are active at a two-imensional lipid interface containing the substrate phospholipids rather than in three-dimensional solution with soluble, monomeric substrates.^22,25,38,42 Because the 2-oxoamide inhibitors also partition to the micelle interface, the relevant concentration of inhibitor for membrane-bound enzymes is the surface concentration (mole fraction) not the bulk concentration (molar units).^{22,25,28,38,39,42} Of the fourteen compounds listed in Table 1, five show at least partial inhibition of GVIA iPLA₂ at the highest concentrations tested.

Table 1.

Structures of 2-Oxoamide Inhibitors and their Effects on GIVA and GVIA PLA₂.

Number	Structure	Inhibition of GVIA iPLA2	Inhibition of GIVA cPLA2
13		NDa,f	NDe
14		ND	ND
15		LDb,f	NDe
16		LDf	NDe
17		ND	XI(50) = 0.017 ± 0.009c,d
18		LD	ND
5a		ND	ND
4a		ND	ND
5b		ND	ND
4b		ND	ND
12		ND	XI(50) = 0.011 ± 0.003
4c		XI(50) = 0.067 ± 0.003	XI(50) = 0.012 ± 0.014
11		XI(50) = 0.032 ± 0.010	XI(50) = 0.018 ± 0.010
10		ND	XI(50) = 0.003 ± 0.001

^aND: negligible inhibition (0–25%) at highest dose. Unless otherwise indicated the highest dose tested was 0.091 mole fraction.

^bLD: limited inhibition (25–50%) at highest dose.

^cData taken from Ref. 28.

^dX_I(50) is the surface concentration of inhibitor at which there is 50% inhibition.

^e0.01 mole fraction.

^f0.02 mole fraction.

Among the primary 2-oxoamides 13 (AX001)²⁹ and 14 (AX015),²⁹ neither exhibits significant inhibition of GIVA or GVIA PLA₂. The secondary 2-oxoamides, 15 (AX002)²⁹ and 16 (AX009),²⁹ with long carbon chains either at the R¹ or at the R² position present limited inhibition of GVIA iPLA₂, but Four 2-oxoamides containing a substituted phenyl chain at the no detectable inhibition of GIVA cPLA₂. R¹ position (4a,b, 5a,b) (AX035-AX038) did not inhibit GVIA iPLA₂. This is somewhat unexpected given previous reports of the selectivity of phenyl-containing fluoroketones or fluorophosphonates. None of the phenyl-containing 2-oxoamides inhibits GIVA cPLA₂.

The 2-oxoamides containing a free carboxyl group 17 (AX006),²⁹ 12 (AX040), and 10 (AX074) inhibit GIVA cPLA₂ but do not inhibit GVIA iPLA₂. In fact, in all cases these compounds enhance GVIA iPLA₂ enzymatic activity. The increased GVIA iPLA₂ activity may be due to increased negative charge at the micelle surface due to addition of inhibitors with a free carboxyl group. Unlike the inhibitors of GIVA cPLA₂, the inhibitors of GVIA iPLA₂ 18 (AX010),²⁹ 4c (AX041), and 11 (AX073) are uncharged. The effect of charge is highlighted when comparing 17 to 18, where 18 possesses a carboxymethyl ester in place of the free carboxyl found in 17. Compound 18 exhibits limited inhibition of GVIA iPLA₂ but does not significantly inhibit GIVA cPLA₂. Compound 17 does not significantly inhibit GVIA iPLA₂ at concentrations up to 0.091 mole fraction but is a potent inhibitor of GIVA cPLA₂ with an X_I(50) value of 0.017 mole fraction.28 Compound 4c is an inhibitor of GVIA iPLA₂ with an X_I(50) value of 0.067 mole fraction. Interestingly, it also inhibits GIVA cPLA₂ with an X_I(50) value of 0.012 mole fraction. Compound 12, the charged variant of 4c, does not inhibit GVIA iPLA₂ but is an inhibitor of GIVA cPLA₂ with an X_I(50) value of 0.011 mole fraction. Consistent results were seen with compounds 11 and 10. These compounds are also variants that contain either a carboxymethyl ester (11) or a free carboxyl (10). Compound 10 is the most potent 2-oxoamide inhibitor of GIVA cPLA₂ reported to date with an X_I(50) of 0.003 mole fraction. By observing the trend of inhibition of GVIA iPLA₂ by 18, 4c, and 11, it appears that an unsaturated chain at R¹ or R² is preferable to a saturated one. This is consistent with the presence of unsaturated fatty acids at the sn-2 position of many phospholipids. The inhibition dose-response curve for 18 appears to plateau at the higher mole fractions tested. The in vitro assay contains detergent and phospholipid that should readily form mixed micelles with 18, which has a similar hydrophobicity (ClogP) to many other compounds that behave normally. Most other lower potency 2-oxoamide inhibitors possess a linear dose-response. Compound 18 is unique as a lower potency inhibitor with a logarithmic dose-response.

A known reference inhibitor (non-covalent and readily reversible) for GIVA cPLA₂ is not commercially available, but a patented inhibitor of GIVA cPLA₂, pyrrophenone, is described in the literature^40,41. Comprehensive analysis of pyrrophenone demonstrated that it inhibits GIVA cPLA₂ with an X_I(50) of 0.002 mole fraction under a variety of assay conditions.42 This level of potency is similar to the most potent GIVA cPLA₂ 2-oxoamide inhibitors, (4S)-4-[(2-oxododecanoyl)amino]octanoic acid (AX007)²⁹ and 10 (this work). Pyrrophenone was reported to have no effect on the activity of GVIA iPLA₂.42 A known reference inhibitor (non-covalent and readily reversible) for GVIA iPLA₂ is palmitoyl trifuoromethyl ketone (PATK). Previous tests of this compound in our lab have confirmed the X_I(50) of PATK for GVIA iPLA₂ is 0.0075 mole fraction.²² A further study tested an expanded panel of hydrophobic trifluoromethyl ketones and found that most are slow, tight-binding inhibitors of GIVA cPLA₂ and fast, reversible inhibitors of GVIA iPLA₂, so the inhibition of the two enzymes by these compounds are not readily comparable.²⁵

Mechanism of GVIA PLA₂ Inhibition by 2-Oxoamide Inhibitors

We tested 18 and 11 to determine if these inhibitors showed either time-dependent or irreversible inhibition of GVIA iPLA₂. GVIA iPLA₂ (25 ng) was preincubated with either 18 or 11 (5 μM) for 0, 5, 15 or 30 minutes and then assayed in the standard GVIA iPLA₂ assay mix containing 5 μM inhibitor. The final concentration of the inhibitors in the assay mix was 0.01 mole fraction, and the samples were incubated for 30 minutes at 40°C. Both 18 and 11 showed no increased potency with prolonged incubation, demonstrating a fast-binding and reversible mode of inhibition (Figure 2A). We next preincubated 25 ng of GVIA iPLA₂ with 10 μM 18 or 11 for 10 minutes before diluting the enzyme 1:50 into the standard GVIA iPLA₂ assay mix lacking inhibitor, and incubating for 30 minutes at 40 °C. The final inhibitor concentration in these assays was 0.0004 mole fraction, well below surface concentrations that either 18 or 11 inhibit the enzyme. GVIA iPLA₂ showed full activity in this system, demonstrating that both 18 and 11 are freely reversible inhibitors (Figure 2B).

Figure 2.

Immediate and reversible inhibition of GVIA iPLA₂ by 18 and 11. A. Time-dependent binding of 18 and 11 was tested by pre-incubating no inhibitor (black bar), 5 μM 18 (light bars), or 5 μM 11 (dark bars) with GVIA iPLA₂ prior to adding to mixed micelles consisting of 100 μM DPPC and 400 μM Triton X-100 containing 0.01 mole fraction inhibitor. B. Reversibility of 18 and 11 was tested by preincubating no inhibitor, 10 μM 18, or 10 μM 11 with GVIA iPLA₂ for 10 minutes prior to diluting 1:50 into mixed micelles consisting of 100 μM DPPC and 400 μM Triton X-100 and assaying for activity.

Inhibition of PGE₂ Production by 2-Oxoamide Inhibitors

We tested several 2-oxoamides in the long-term lipopolysaccharide (LPS) stimulation pathway in the murine RAW 264.7 macrophage-like cell line.^43,44 This pathway requires GIVA cPLA₂ activity for maximal extracellular release of many eicosanoid compounds, including the prostaglandin PGE₂.45 Compound 18, which does not significantly inhibit GIVA cPLA₂ in vitro, also did not inhibit PGE₂ release from the RAW cells (data not shown). In the low μM range, 4c and 11 reduced PGE₂ release by roughly 40% (Figure 3). Based on previous work, this is the fraction of PGE₂ release attributable to GIVA cPLA₂.44,45 At 1 μM and 5 μM concentrations, small activations were often seen, suggesting minor stimulation of the cells from membrane perturbing compounds.

Figure 3.

Inhibition of PGE₂ production in RAW 264.7 cells by 2-oxoamides containing a methyl ester. Increasing concentrations of 4c (○) or 11 (▼) were added to cells for 30 minutes prior to stimulation with 100 ng/ml LPS for 24 hours. Media was harvested and assayed for PGE₂ production as described in the experimental section.

In conclusion, based on the 2-oxoamide backbone structure we have developed inhibitors that selectively inhibit GIVA cPLA₂ or inhibit both GIVA and GVIA PLA₂. The selective 2-oxoamide inhibitors of GIVA cPLA₂ were found to be charged, containing a free carboxyl group. Interestingly some non-charged 2-oxoamides showed dual specificity in inhibiting both GIVA cPLA₂ and GVIA iPLA₂. Inhibitors selective for GIVA cPLA₂ or dual specificity inhibitors reduced PGE₂ levels in cellular assays that test for inhibition of GIVA cPLA₂. Several 2-oxoamide compounds that significantly inhibit GVIA iPLA₂ are promising leads for selective inhibitors of GVIA iPLA₂ that would significantly improve investigations into the role of GVIA iPLA₂ in cellular systems. As we have previously demonstrated for 2-oxoamide inhibitors of GIVA cPLA₂, the inhibitors of GVIA iPLA₂ are also fast-binding and freely reversible. Such selective inhibitors of GIVA and GVIA enzymes will be a significant asset in examining the role of these enzymes in cellular signaling and inflammation.

Experimental Section

Synthesis of 2-Oxoamide Inhibitors

Melting points were determined on a Buchi 530 apparatus and are uncorrected. Specific rotations were measured at 25 °C on a Perkin-Elmer 343 polarimeter using a 10 cm cell. NMR spectra were recorded on a Varian Mercury (200 MHz) spectrometer. Fast atom bombardment (FAB) mass spectra were recorded using a VG analytical ZAB-SE instrument. Electron spray ionization (ESI) mass spectra were recorded on a Finnigan, Surveyor MSQ Plus spectrometer. TLC plates (silica gel 60 F₂₅₄) and silica gel 60 (70–230 or 230–400 mesh) for column chromatography were purchased from Merck.

Coupling of 2-hydroxy acids with amino components

To a stirred solution of 2-hydroxy acid (2.0 mmol) and hydrochloride amino component (2.0 mmol) in CH₂Cl₂ (20 mL), Et₃N (0.61 mL, 4.4 mmol) and subsequently 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (WSCI) (0.42 g, 2.2 mmol) and 1-hydroxybenzotriazole (HOBt) (0.27 g, 2.0 mmol) were added at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and overnight at room temperature. The solvent was evaporated under reduced pressure and EtOAc (20 mL) was added. The organic layer was washed consecutively with brine, 1N HCl, brine, 5% NaHCO₃, and brine, dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by column-chromatography using CHCl₃ as eluent.

4-(2-Hydroxy-5-phenyl-pentanoylamino)-butyric acid methyl ester (2a)

yield 82%; white solid; m.p. 34–35 °C; ¹H NMR: δ 7.24-7.11 (5H, m, C₆H₅), 6.82 (1H, m, NHCO), 4.06 (1H, m, CH), 3.62 (3H, s, CH₃O), 3.53 (1H, d, J = 5.2 Hz, OH), 3.26 (2H, m, CH₂NH), 2.59 (2H, t, J = 7.8 Hz, CH₂C₆H₅), 2.30 (2H, t, J = 6.8 Hz, CH₂COO), 1.82-1.70 (6H, m, 3×CH₂); 13C NMR: δ 174.2, 173.8 142.0, 128.3, 128.2, 125.7, 71.7, 51.7, 38.3, 35.5, 34.3, 31.3, 26.8, 24.6; MS (ESI): m/z (%): 316 (100) [M + Na]⁺. Anal. (C₁₆H₂₃NO₄) C, H, N.

4-(2-Hydroxy-6-phenyl-hexanoylamino)-butyric acid methyl ester (2b)

yield 85%; white solid; m.p. 50–51 °C; ¹H NMR: δ 7.31-7.15 (5H, m, C₆H₅), 6.76 (1H, m, NHCO), 4.08 (1H, m, CH), 3.68 (3H, s, CH₃O), 3.32 (2H, m, CH₂NH), 3.10 (1H, d, J = 4.8 Hz, OH), 2.62 (2H, t, J = 7.8 Hz, CH₂C₆H₅), 2.36 (2H, t, J = 7.4 Hz, CH₂COO), 1.91-1.49 (8H, m, 4×CH₂); 13C NMR: δ 174.0, 142.3, 128.3, 128.2, 125.7, 72.0, 51.7, 38.4, 35.7, 34.7, 31.4, 31.1, 24.6; MS (ESI): m/z (%): 330 (88) [M + Na]⁺, 308 (100) [M + H]⁺. Anal. (C₁₇H₂₅NO₄) C, H, N.

4-(2-Hydroxy-nonadec-10-enoylamino)-butyric acid methyl ester (2c)

yield 82%; white solid; m.p. 55–57 °C; ¹H NMR: δ 6.80 (1H, m, NHCO), 5.33 (2H, m, CH=CH), 4.07 (1H, m, CH), 3.67 (3H, s, CH₃O), 3.30 (2H, m, CH₂NH), 2.37 (2H, t, J = 7.2 Hz, CH₂COO), 1.98 (4H, m, 2×CH₂CH=CH), 1.85 (2H, m, CH₂CH₂NH), 1.26 (24H, br s, 12×CH₂), 0.87 (3H, t, J = 6.6 Hz, CH₃); ¹³C NMR: δ 174.2, 173.8, 129.9, 129.7, 72.1, 51.7, 38.4, 34.8, 31.8, 31.3, 29.7, 29.5, 29.4, 29.3, 29.2, 27.2, 25.0, 24.6, 22.6, 14.1. Anal. (C₂₄H₄₅NO₄) C, H, N.

4-(2-Hydroxy-hexadecanoylamino)-oct-2-enoic acid methyl ester (9)

The oxidation of compound 4 follows method A. The Wittig reaction of the resulting N-protected α-aminoaldehyde with a stabilized ylide and the general method for the removal of the Boc group was carried out as described previously.²⁹ The coupling reaction to yield compound 9 is as described above. The overall yield 52%; white solid; m.p 40–42 °C; ¹H NMR: δ 6.85 (1H, dd, J₁ = 5.2 Hz, J₂ = 15.4 Hz, CHCH =CH), 6.60 (1H, d, J = 9.2 Hz, NHCO), 5.87 (1H, d, J = 15.4 Hz, CH=CHCOOCH₃), 4.62 (1H, m, CH), 4.14 (1H, m, CH), 3.73 (3H, s, COOCH₃), 2.77 (1H, m, OH), 1.98-1.01 (32H, m, 16xCH₂), 0.86 (6H, t, J = 7 Hz, 2×CH₃); ¹³C NMR: δ 173.3, 166.7, 148.0, 120.5, 72.3, 51.6, 49.6, 37.0, 34.9, 34.0, 31.9, 29.7, 29.5, 29.3, 27.7, 25.0, 24.9, 22.7, 22.3, 14.1, 13.8; MS (ESI): m/z (%): 448 (100) [M + Na]⁺. Anal. (C₂₅H₄₇NO₄) C, H, N.

Oxidation of 2-hydroxy-amides

Method A

To a solution of 2-hydroxy-amide (5.00 mmol) in a mixture of toluene-EtOAc 1:1 (30 mL), a solution of NaBr (0.54 g, 5.25 mmol) in water (2.5 mL) was added followed by TEMPO (11 mg, 0.050 mmol). To the resulting biphasic system, which was cooled at −5 °C, an aqueous solution of 0.35 M NaOCl (15.7 mL, 5.50 mmol) containing NaHCO₃ (1.26 g, 15 mmol) was added dropwise under vigorous stirring, at −5 °C over a period of 1 h. After the mixture had been stirred for a further 15 min at 0 °C, EtOAc (30 mL) and H₂O (10 mL) were added. The aqueous layer was separated and washed with EtOAc (20 mL). The combined organic layers were washed consecutively with 5% aqueous citric acid (30 mL) containing KI (0.18 g), 10% aqueous Na₂S₂O₃ (30 mL), and brine and dried over Na₂SO₄. The solvents were evaporated under reduced pressure and the residue was purified by column chromatography [EtOAc-petroleum ether (bp 40–60 °C), 1:9].

4-(2-Oxo-5-phenyl-pentanoylamino)butyric acid methyl ester (4a)

yield 67%; white solid; m.p. 30–31 °C; ¹H NMR: δ 7.19-7.15 (6H, m, C₆H₅, NHCO), 3.67 (3H, s, CH₃O), 3.35 (2H, m, CH₂NH), 2.94 (2H, t, J = 7.4 Hz, CH₂COCO), 2.65 (2H, t, J = 7.8 Hz, CH₂C₆H₅), 2.36 (2H, t, J = 7.0 Hz, CH₂COO), 1.91 (4H, m, 2×CH₂); ¹³C NMR: δ 198.7, 173.2, 160.0, 141.1, 128.3, 128.2, 125.8, 51.6, 38.5, 35.9, 34.8, 31.1, 24.6, 24.1; MS (ESI): m/z (%): 314 (63) [M + Na]⁺. Anal. (C₁₆H₂₁NO₄) C, H, N.

4-(2-Oxo-6-phenyl-hexanoylamino)butyric acid methyl ester (4b)

yield 75%; white solid; m.p. 52–54 °C; ¹H NMR: δ 7.29-7.16 (6H, m, C₆H₅, NHCO), 3.69 (3H, s, CH₃O), 3.37 (2H, m, CH₂NH), 2.95 (2H, t, J = 7.0 Hz, CH₂COCO), 2.64 (2H, t, J = 7.0 Hz, CH₂C₆H₅), 2.38 (2H, t, J = 7.0 Hz, CH₂COO), 1.89-1.66 (6H, m, 3×CH₂); 13C NMR: δ 198.8, 173.2, 160.1, 141.9, 128.21, 128.15, 125.6, 51.6, 38.5, 36.4, 35.4, 31.1, 30.6, 24.2, 22.6; MS (ESI): m/z (%): 328 (75) [M + Na]⁺. Anal. (C₁₇H₂₃NO₄) C, H, N.

4-(2-Oxo-5-phenyl-pentanoylamino)butyric acid (5a)

The procedure is the same as that followed in method A described above, with the difference that in this case the aqueous layer was acidified before the work-up, and then extracted with EtOAc, and the combined organic layers were washed with 5% aqueous citric acid containing KI, and 10% aqueous Na₂S₂O₃ (30 mL). The residue was purified by column chromatography [EtOAc-petroleum ether (bp 40–60 °C)]. Yield 48%; white solid; m.p. 65–67 °C; ¹H NMR: δ 7.25-7.11 (6H, m, C₆H₅, NHCOCO), 3.33 (2H, m, CH₂NH), 2.86 (2H, t, J = 7.4 Hz, CH₂COCO), 2.60 (2H, m, CH₂), 2.36 (2H, m, CH₂), 1.86 (4H, m, 2×CH₂); ¹³C NMR: δ 198.8, 178.5, 160.3, 141.2, 128.41, 128.37, 126.0, 38.5, 36.1, 34.9, 31.2, 24.7, 24.0; MS (ESI) : m/z (%): 276 (100) [M – H]⁻. Anal. (C₁₅H₁₉NO₄) C, H, N.

4-(2-Oxo-6-phenyl-hexanoylamino)-butyric acid (5b)

The procedure is the same as that followed for 5a. Yield 47%; white solid; m.p. 60–62 °C; ¹H NMR: δ 7.27-7.15 (6H, m, C₆H₅, NHCOCO), 3.35 (2H, m, CH₂NH), 2.94 (2H, t, J = 7.4 Hz, CH₂COCO), 2.60 (2H, m, CH₂), 2.38 (2H, m, CH₂), 1.86 (2H, m, CH₂), 1.64 (4H, m, 2×CH₂); 13C NMR: δ 198.8, 178.8, 160.3, 142.0, 128.33, 128.27, 125.7, 38.6, 36.5, 35.5, 31.4, 30.7, 24.2, 22.6; MS (FAB) : m/z (%): 292 (100) [M + H]⁺. Anal. (C₁₆H₂₁NO₄) C, H, N.

Oxidation of 2-hydroxyamides

Method B

To a solution of 2-hydroxyamide (1 mmol) in dry CH₂Cl₂ (20 mL) Dess-Martin periodinane was added (0.64 g, 1.5 mmol) and the mixture was stirred for 2 h at room temperature. The organic solution was washed with 10% aqueous NaHCO₃, dried over Na₂SO₄ and the organic solvent was evaporated under reduced pressure. The residue was purified by recrystallization [EtOAc/petroleum ether (bp 40–60 °C)].

4-(2-Oxononadec-10-enoylamino)butyric acid methyl ester (4c)

yield 82%; oily solid; ¹H NMR: δ 7.13 (1H, m, NHCOCO), 5.33 (2H, m, CH=CH), 3.67 (3H, s, CH₃O), 3.33 (2H, m, CH₂NH), 2.91 (2H, t, J = 7.2 Hz, CH₂COCO), 2.38 (2H, t, J = 7.4 Hz, CH₂COO), 1.98 (4H, m, 2×CH₂CH=CH), 1.88 (2H, m, CH₂CH₂NH), 1.59 (2H, m, CH₂CH₂COCO), 1.26 (20H, br s, 10×CH₂), 0.87 (3H, t, J = 6.6 Hz, CH₃); 13C NMR: δ 199.2, 173.3, 160.3, 129.9, 129.7, 51.7, 38.0, 36.7, 31.8, 31.3, 29.7, 29.6, 29.5, 29.3, 29.2, 29.0, 28.98, 27.2, 27.1, 24.3, 23.1, 22.6, 14.1; MS (FAB): m/z (%): 410 (100) [M + H]⁺. Anal. (C₂₄H₄₃NO₄) C, H, N.

4-(2-Oxohexadecanoylamino)oct-2-enoic acid methyl ester (11)

yield 81%, white solid; m.p. 48–50 °C; [α]_D −19.7 (c 0.95 CHCl₃); ¹H NMR: δ 6.93 (1H, d, J = 8 Hz, NHCOCO), 6.85 (1H, dd, J₁ = 6 Hz, J₂ = 16 Hz, CHCH=CH), 5.87 (1H, d, J = 16 Hz, CH=CHCOOCH₃), 4.58 (1H, m, CH), 3.73 (3H, s, COOCH₃), 2.91 (2H, t, J = 7 Hz, CH₂COCO), 1.61 (4H, m, 2×CH₂), 1.30 (26H, m, 13×CH₂), 0.88 (6H, t, J = 7 Hz, 2×CH₃); 13C NMR: δ 199.3, 166.7, 159.8, 146.9, 121.4, 51.9, 50.4, 37.0, 34.1, 32.1, 29.9, 29.8, 29.6, 29.5, 29.3, 27.9, 23.4, 22.9, 22.5, 14.3, 14.0; MS (ESI): m/z (%): 446 (85) [M + Na]⁺. Anal. (C₂₅H₄₅NO₄) C, H, N.

4-(2-Oxohexadecanoylamino)oct-2-enoic acid (10)

The procedure is the same as that followed in method B with the difference that the organic layer was not washed with 10% aqueous NaHCO₃. Yield 69%, white solid; m.p. 65–67 °C; [α]_D −7.7 (c 0.84 CHCl₃); ¹H NMR: δ 7.0 (1H, m, NHCOCO), 6.82 (1H, dd, J₁ = 6 Hz, J₂ = 16 Hz, CHCH=CH), 5.87 (1H, d, J = 16 Hz, CH=CHCOOCH₃), 4.62 (1H, m, CH), 2.91 (2H, t, J = 7 Hz, CH₂COCO), 1.61 (4H, m, 2×CH₂), 1.44-1.25 (26H, m, 13×CH₂), 0.88 (6H, t, J = 7 Hz, 2×CH₃); 13C NMR: δ 199.0, 170.8, 159.6, 149.0, 120.8, 50.2, 36.7, 33.7, 31.9, 29.6, 29.4, 29.3, 29.0, 27.7, 23.1, 22.7, 22.3, 14.1, 13.8; MS (ESI): m/z (%): 408 (100) [M – H]⁻. Anal. (C₂₄H₄₃NO₄) C, H, N.

Saponification of methyl esters

To a stirred solution of methyl ester (2.00 mmol) in a mixture of dioxane-H₂O (9:1, 20 mL), 1N NaOH (2.2 mL, 2.2 mmol) was added and the mixture was stirred for 12 h at room temperature. The organic solvent was evaporated under reduced pressure and H₂O (10 mL) was added. The aqueous layer was washed with EtOAc, acidified with 1N HCl, and extracted with EtOAc (3 × 12 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, and evaporated under reduced pressure. The residue was purified after recrystallization [EtOAc-petroleum ether (bp 40–60 °C)].

4-(2-Hydroxy-5-phenylpentanoylamino)butyric acid (3a)

yield 79%; white solid; m.p. 63–65 °C; ¹H NMR: δ 7.26-7.12 (6H, m, C₆H₅, NHCO), 4.09 (1H, m, CH), 3.27 (2H, m, CH₂NH), 2.59 (2H, t, J = 6.6 Hz, CH₂C₆H₅), 2.31 (2H, t, J = 6.6 Hz, CH₂COOH), 1.78 (6H, m, 3×CH₂); 13C NMR: δ 177.3, 175.5, 142.0, 128.3, 125.8, 71.8, 38.4, 35.5, 34.1, 31.3, 26.8, 24.3. Anal. (C₁₅H₂₁NO₄) C, H, N.

4-(2-Hydroxy-6-phenylhexanoylamino)butyric acid (3b)

yield 86%; white solid; m.p. 78–80 °C; ¹H NMR: δ 7.30-7.13 (6H, m, C₆H₅, NHCO), 4.11 (1H, m, CH), 3.30 (2H, m, CH₂NH), 2.60 (2H, t, J = 7.8 Hz, CH₂C₆H₅), 2.35 (2H, t, J = 6.6 Hz, CH₂COOH), 1.81-1.47 (8H, m, 4×CH₂); ¹³C NMR: δ 177.4, 175.5, 142.4, 128.3, 128.2, 125.7, 71.9, 38.4, 35.7, 34.3, 31.4, 31.1, 24.7, 24.4; MS (ESI): m/z (%): 316 (54) [M + Na]⁺, 294 (100) [M + H]⁺. Anal. (C₁₆H₂₃NO₄) C, H, N.

4-(2-Hydroxyhexadecanoylamino)oct-2-enoic acid (8)

yield 62%; white solid; m.p 46–48 °C; ¹H NMR: δ 6.92 (1H, m, NHCO), 6.76 (1H, dd, J₁ = 6 Hz, J₂ = 16 Hz, CHCH =CH), 5.87 (1H, d, J = 16 Hz, CH=CHCOOH), 4.64 (1H, m, CH), 4.20 (1H, m, CH), 3.42 (1H, br, OH), 1.95-1.25 (32H, m, 16xCH₂), 0.88 (6H, t, J = 7 Hz, 2xCH₃); ¹³C NMR: δ 172.3, 170.5, 150.0, 120.5, 72.6, 49.9, 35.1, 34.2, 32.1, 29.9, 29.6, 28.0, 25.3, 22.9, 22.7, 22.5, 14.3, 14.1; MS (ESI): m/z (%): 434 (100) [M + Na]⁺. Anal. (C₂₄H₄₅NO₄) C, H, N.

Inhibitor 12 was prepared by similar procedures.

4-(2-Oxononadec-10-enoylamino)butyric acid (12)

yield 69%; white solid; m.p. 57–59 °C; ¹H NMR: δ 10.05 (1H, br, COOH), 7.23 (1H, m, NHCOCO), 5.33 (2H, m, CH=CH), 3.38 (2H, m, CH₂NH), 2.90 (2H, t, J = 7.2 Hz, CH₂COCO), 2.41 (2H, t, J = 6.8 Hz, CH₂COOH), 1.98 (4H, m, 2×CH₂CH=CH), 1.89 (2H, m, CH₂CH₂NH), 1.58 (2H, m, CH₂CH₂COCO), 1.26 (20H, br s, 10×CH₂), 0.87 (3H, t, J = 6.6 Hz, CH₃); 13C NMR: δ 199.1, 178.4, 160.4, 129.9, 129.7, 38.5, 36.7, 32.7, 31.8, 31.2, 29.7, 29.6, 29.5, 29.3, 29.2, 29.02, 28.96, 27.1, 24.1, 23.1, 22.6, 14.1; MS (ESI): m/z (%): 418 (95) [M + Na]⁺. Anal. (C₂₃H₄₁NO₄) C, H, N.

Inhibitors 13–18 were prepared as described previously.^28,29

Expression and Purification of Recombinant Group VIA PLA₂

Protein was produced in Sf9 insect cells using a recombinant baculovirus. The virus had been constructed using the cDNA coding for human Group VIA-2 iPLA_2, kindly provided by Dr. Brian Kennedy at Merck-Frost, modified with a six residue histidine tag added three amino acids from the amino terminus using PCR with oligonucleotides 5′-ATGCAGTTCCACCATCACCATCACCATTTTGGAGCGCTGGTCAATACC-3′ and 5′-CCTCAGGGTGAGAGCAGCAGCTG-3′. Gateway cloning ends were added to the histidine-tagged Group VIA-2 cDNA followed by insertion into pDONOR201 (Invitrogen) to produce a Gateway entry clone. The gene construct was then transferred to pDEST8 using Gateway cloning technology and used to make recombinant baculovirus using the Bac-to-Bac system (Invitrogen).

A suspension culture of Sf9 insect cells at a density of 1.1 to 1.5 million cells per mL was infected with the recombinant baculovirus with an MOI of approximately 0.1. Infections were carried out for 72 hours and the cells were harvested by centrifugation at 3,000 × g for 10 minutes and stored at −80 °C. The frozen cell pellets from 200 mL of suspension culture Sf9 cells were resuspended in 25 mL resuspension buffer (25 mM Tris pH 8.0, 150 mM NaCl, 10 mM DTT, 5 mM EDTA, 2 mM ATP, 0.2% methyl-β-cyclodextrin (Sigma-Aldrich) and 1X protease inhibitor cocktail). The cells were lysed by repeated sonication, and the lysate was allowed to sit on ice for 10 minutes and then clarified by centrifugation at 15,000 × g for 30 minutes at 4 °C. The resulting pellet was resuspended in solubilization buffer (25 mM Tris pH 8.0, 150 mM NaCl, 10 mM β-mercaptoethanol, 2 mM ATP, 1 M urea and 1X protease inhibitor cocktail) by 20 passes of a Dounce homogenizer with the tight pestle. The resuspended pellet was then stirred at 4 °C for one hour followed by centrifugation at 15,000 × g for 30 minutes at 4 °C to remove insoluble material. At this point, 2.5 mL of Fast-flow Ni-NTA resin per 200 mL cell pellet was mixed with the soluble protein fraction and allowed to incubate at 4 °C for 30 minutes for batch binding. The protein/resin slurry was poured into a column and allowed to settle. The column was washed with 15 column volumes of Ni-wash buffer (25 mM NaHPO₄ pH 7.4, 250 mM NaCl, 2 mM ATP, 0.2% dodecyl maltoside (Anatrace) and 1X protease inhibitor cocktail) and eluted with 10 column volumes of Ni-elution buffer (25 mM NaHPO₄ pH 7.4, 100 mM NaCl, 50 mM urea, 2 mM ATP and 200 mM imidazole, 30% v/v glycerol). Eluate was collected as 1.5 mL fractions into tubes containing 15 μL 500 mM DTT (5 mM DTT final). Fractions containing protein were pooled, measured for activity and protein concentration, and stored as 200 μL aliquots at −80 °C.

Group VIA iPLA₂ Activity Assays

The standard Group VIA iPLA₂ activity assay utilizes DPPC/Trition X-100 mixed micelles at a ratio of 1:4 as previously described.^46,47 A stock solution of lipid was generated by drying down 50 nmoles of dipalmitoyl phosphatidylcholine (DPPC) mixed with 1 × 10⁵ cpm of 1-palmitoyl, 2-[1-¹⁴C]-palmitoyl PC per assay tube under a stream of nitrogen gas. The dried lipids were solubilized in 50 μL of 10X assay buffer (100 mM HEPES pH 7.5, 50 mM EDTA, 20 mM DTT, 10 mM ATP, 4 mM Triton X-100) per assay tube by repeated vortexing and heating to 40 °C. The resulting 10X substrate mixture was combined with 100 mM HEPES pH 7.5 to give a final volume of 500 μL upon addition of enzyme and inhibitor. Inhibitors were dissolved in DMSO to a stock concentration of 5 mM and diluted with DMSO prior to addition of 5 μL to the reaction tube, yielding a final DMSO concentration of 1%. The final substrate concentration in this mixed-micelle assay is 100 μM DPPC and 400 μM Triton X-100. Purified enzyme (190 ng) was added to start the reaction followed by incubation for 30 minutes at 40 °C. The reaction was quenched, extracted and analyzed using the modified Dole assay.⁴⁸

Group IVA cPLA₂ Activity Assays

The GIVA cPLA₂ assays have been described previously.^28,29,46 Pure, native, human GIVA cPLA₂ was a generous gift from Dr. Ruth Kramer of Lilly Research Laboratories. Briefly, the final assay conditions were 10 ng GIVA cPLA₂ in 100 mM HEPES (pH 7.5), 80 μM CaCl₂, 0.1 mg/mL fatty acid free bovine serum albumin, 2 mM DTT, 97 μM 1-palmitoyl-2-[¹⁴C]-arachidonoyl phosphatidylcholine (100,000 cpm), 3 μM phosphatidylinositol 4,5-bisphosphate, and 400 μM Triton X-100 in 500 μL. The reaction contained 1% DMSO with varying amounts of inhibitors added as described above. The assays were incubated at 40 °C for 30 min. Reactions were quenched, extracted and analyzed using the modified Dole assay as above.⁴⁸

Cell Culture and PGE₂ Assay

The RAW 264.7 macrophage-like cell line was maintained at 37 °C in a humidified 5% CO₂ atmosphere. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal calf serum (HyClone Labs, Provo Utah), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad California). Prior to stimulation, cells were plated at a density of 5 × 10⁵ cells per well in standard 12 well tissue culture plates and were allowed to adhere for 24 hours. They were then washed with serum-free medium and allowed to adjust for 18 hours. Cells were then exposed to 100 ng/mL LPS (Sigma L4130 from E. coli 0111:B4) for 24 hours. Following stimulation, the media was removed and the cells were scraped into 1 mL PBS and counted. Deuterated PGE₂ internal standard (10 ng) was added to the media of each sample, and the media was cleared of cellular debris by centrifugation (3000 × g, 10 min). Methanol and acetic acid were added to the cleared supernate to a final concentration of 10% and 2% respectively. Prostaglandins were extracted using 60 mg/3 mL Strata-X columns (Phenomenex). The columns were preconditioned with 2 mL methanol followed by 2 mL water. The sample was loaded and the column washed with 2 mL 0.5% methanol. The sample was eluted from the column with 1 mL 100% methanol.

Inhibitors, when included, were dissolved in DMSO and diluted into serum-free medium prior to addition to cells. The DMSO concentration was kept below 0.5% v/v in all studies. All inhibitors were added 30 minutes prior to stimulation.

PGE₂ released by the cells was quantitated by the following LCMS procedure. The chromatography was performed on a Grace-Vydac reverse phase C18 column (2.1 mm X 250 mm) run with a gradient beginning with 100% Buffer A (63:37:0.02 water:acetonitrile:formic acid) and ending with 100% Buffer B (50:50 acetonitrile:isopropanol). PGE₂ was detected on an ABI 4000 Qtrap mass spectrometer in MRM mode with the electrospray ion source operating in negative ion mode using the following settings: curtain gas = 10, spray voltage = −4.5kV, source temperature = 525 °C, source gas 1 = 60, source gas 2 = 60, declustering potential = −50V. The PGE₂ was detected via CID with a precursor ion of 351 and a product ion of 189 amu and a collision energy = −27V and Q2 collision gas = high.

Supplementary Material

1si20060222_11. Supporting Information Available.

Elemental analysis data. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1.

Dose-response curves for 2-oxoamide inhibitors of GVIA iPLA₂. The activity of human GVIA iPLA₂ was tested on mixed-micelles containing 100 μM DPPC and 400 μM Triton X-100. The surface concentration of 18 (●), 4c (○), and 11 (▼) was increased as shown. A logarithmic or linear fit function was used to calculate the X_I(50) values shown in Table 1.