Differential inhibition of group IVA and group VIA phospholipases A 2 by 2-oxoamides

Article abstract of DOI:10.1021/jm050993h

Inhibitors of the Group IVA phospholipase A₂ (GIVA CPLA ₂) 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

Full text of DOI:10.1021/jm050993h

J. Med. Chem. 2006, 49, 2821-2828
2821
Differential Inhibition of Group IVA and Group VIA Phospholipases A2 by 2-Oxoamides
Daren Stephens,†,| Efrosini Barbayianni,‡,| Violetta Constantinou-Kokotou,§ Anna Peristeraki,§ David A. Six,† Jennifer Cooper,†
Richard Harkewicz,† Raymond A. Deems,† Edward A. Dennis,*,† and George Kokotos*,‡
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093-0601, Laboratory of Organic
Chemistry, Department of Chemistry, UniVersity of Athens, Panepistimiopolis, Athens 15771, Greece, and Chemical Laboratories, Agricultural
UniVersity of Athens, Athens 11855, Greece
ReceiVed October 5, 2005
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 building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although 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
and GVIA PLA2,22,23 whereas methyl arachidonyl fluorophos-
phonate (MAFP) functions as an irreversible inhibitor of both
enzymes.24 Variants of the trifluoromethyl ketones show dif-
ferential 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 toward GVIA iPLA2.25 Interestingly, the trifluoro-
methyl ketone 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 it 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
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, based on exhaustive mutagenesis and a crystal structure,
was for GIVA cPLA2, which confirmed that the catalytic dyad
is present in a noncanonical R/â 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 homologue 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
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. On the basis of the similarity of substrates, the
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-
Arachidonyl trifluoromethyl ketone (ATFK) has been shown
to function as a tight binding, reversible inhibitor of both GIVA
* To whom correspondence should be addressed. Tel: 858-534-3055.
Fax: 858-534-7390. E-mail: edennis@ucsd.edu (E.A.D.). Tel: 30210
7274462. Fax: 30210 7274761. E-mail: gkokotos@chem.uoa.gr (G.K.).
† University of California, San Diego.
‡ University of Athens.
§ Agricultural University of Athens.
| These authors contributed equally to this work.
10.1021/jm050993h CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/13/2006

2822 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Stephens et al.
Scheme 1
Scheme 3a
Scheme 2a
a Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/
toluene/H2O, -5 °C; (b) Ph3PdCHCOOCH3, THF, reflux; (c) 4 N HCl in
THF; (d) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (e) 1 N
NaOH/MeOH; (f) Dess-Martin periodinane, CH2Cl2.
a Reagents and conditions: (a) H2N(CH2)3COOCH3, Et3N, WSCI, HOBt,
CH2Cl2; (b) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/toluene/H2O, 0 °C;
(c) Dess-Martin periodinane, CH2Cl2; (d) 1 N NaOH/MeOH; (e) NaOCl,
TEMPO, NaBr, NaHCO3, EtOAc/toluene/H2O, 0 °C, then HCl.
compounds listed in Table 1, five show at least partial inhibition
of GVIA iPLA2 at the highest concentrations tested.
Among 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 no detectable
inhibition of GIVA cPLA2. Four 2-oxoamides containing a
substituted phenyl chain at the R1 position (4a,b, 5a,b)
(AX035-AX038) did not inhibit GVIA iPLA2. This is some-
what 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 the increased
negative charge at the micelle surface because of the 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. Com-
pound 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 XI(50) value of 0.017 mole fraction.28
Compound 4c is an inhibitor of GVIA iPLA2 with an XI(50)
value of 0.067 mole fraction. Interestingly, it also inhibits GIVA
cPLA2 with an XI(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 XI(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 XI(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
triacylglycerol analogues33,34 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.
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-R,â-unsaturated
acid is shown. It should be noted that the oxidation of
unsaturated 2-hydroxyamides 2c, 8, and 9 was carried out using
Dess-Martin periodinane,37 instead of NaOCl/TEMPO, to avoid
the oxidation of the double bonds.
Selective Inhibition of GIVA and GVIA PLA2 by 2-Oxo-
amide Inhibitors. Fourteen 2-oxoamides were tested for
inhibition of GVIA iPLA2 in our in vitro assay system27,28 and
compared with GIVA cPLA2 inhibition. The data, summarized
in Table 1, is represented as XI(50) values. XI(50) is defined as
the inhibitor concentration in a 2D 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.
XI(50) is utilized as opposed to the more common IC50 because
GIVA and GVIA PLA2 are active at a 2D lipid interface
containing the substrate phospholipids rather than in a 3D
solution with soluble, monomeric substrates.22,25,38,42 Because
the 2-oxoamide inhibitors also partition to the micelle interface,
the relevant concentration of the inhibitor for membrane-bound
enzymes is the surface concentration (mole fraction) and not
the bulk concentration (molar units).22,25,28,38,39,42 Of the fourteen

Group IVA and Group VIA Phospholipases
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2823
Table 1. Structures of 2-Oxoamide Inhibitors and Their Effects on
GIVA and GVIA PLA2
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 (b), 4c (O), and 11 (1) was increased as
shown. A logarithmic or linear fit function was used to calculate the
XI(50) values shown in Table 1.
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 (noncovalent and readily reversible)
for GVIA iPLA2 is palmitoyl trifuoromethyl ketone (PATK).
Previous tests of this compound in our lab have confirmed that
the XI(50) value of PATK for GVIA iPLA2 is 0.0075 mole
fraction.22 A further study tested an expanded panel of hydro-
phobic trifluoromethyl ketones and found that most are slow,
tight-binding inhibitors of GIVA cPLA2 and fast, reversible
inhibitors of GVIA iPLA2; therefore, 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 min and then assayed in the
standard GVIA iPLA2 assay mix with 5 µM of inhibitor. The
final concentration of the inhibitors in the assay mix was 0.01
mole fraction, and the samples were incubated for 30 min at
40 °C. Both 18 and 11 showed no increased potency with
prolonged incubation, demonstrating a fast-binding and revers-
ible mode of inhibition (Figure 2A). We next preincubated 25
ng of GVIA iPLA2 with 10 µM 18 or 11 for 10 min before
diluting the enzyme, 1:50, into the standard GVIA iPLA2 assay
mix lacking the inhibitor and incubating for 30 min at 40 °C.
The final inhibitor concentration in these assays was 0.0004
mole fraction, well below surface concentrations at which 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).
a ND: negligible inhibition (0-25%) at the highest dose. Unless
otherwise indicated, the highest dose tested was 0.091 mole fraction. b LD:
limited inhibition (25-50%) at the highest dose. c Data taken from ref
28. d XI(50) is the surface concentration of the inhibitor at which there is
50% inhibition. e 0.01 mole fraction. f 0.02 mole fraction.
preferable to a saturated one. This is consistent with the presence
of unsaturated fatty acids at the sn-2 position of many phos-
pholipids. The inhibition dose-response curve for 18 appears
to plateau at the higher mole fractions tested. The in vitro assay
contains detergents and phospholipids 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 (noncovalent and readily revers-
ible) for GIVA cPLA2 is not commercially available, but a
patented inhibitor of GIVA cPLA2, pyrrophenone, is described
in the literature.40,41 A comprehensive analysis of pyrrophenone
demonstrated that it inhibits GIVA cPLA2 with an XI(50) of
0.002 mole fraction under a variety of assay conditions.42 This
level of potency is similar to that of the most potent GIVA
cPLA2 2-oxoamide inhibitors, (4S)-4-[(2-oxododecanoyl)amino]-
Inhibition of PGE2 Production by 2-Oxoamide Inhibitors.
We tested several 2-oxoamides in the long-term lipopolysac-
charide (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). On the basis of previous
work, this is the fraction of PGE2 release attributable to GIVA
cPLA2.44,45 At 1 and 5 µM concentrations, small activations were

2824 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Stephens et al.
2-oxoamide compounds that significantly inhibit GVIA iPLA2
are promising leads for selective inhibitors of GVIA iPLA2 that
would 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 in CDCl3 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 a 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-hydroxybenzotri-
azole (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, 1 N HCl, brine, 5% NaHCO3, and brine
and dried over Na2SO4 and evaporated under reduced pressure. The
residue was purified by column-chromatography using CHCl3 as
eluent.
Figure 2. Immediate and reversible inhibition of GVIA iPLA2 by 18
and 11. (A) Time-dependent binding of 18 and 11 was tested by
preincubating no inhibitor (black bar), 5 µM 18 (light gray bars), or 5
µM 11 (dark gray 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 of the 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 min prior to diluting 1:50 into mixed
micelles consisting of 100 µM DPPC and 400 µM Triton X-100 and
assaying for activity.
4-(2-Hydroxy-5-phenyl-pentanoylamino)-butyric Acid Methyl
1
Ester (2a). Yield 82%; white solid; mp 34-35 °C; H 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
1
Ester (2b). Yield 85%; white solid; mp 50-51 °C; H 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; mp 55-57 °C; H NMR: δ
Figure 3. Inhibition of PGE2 production in RAW 264.7 cells by
2-oxoamides containing a methyl ester. Increasing concentrations of
4c (O) or 11 (1) were added to cells for 30 min prior to stimulation
with 100 ng/mL of LPS for 24 h. The media were harvested and assayed
for PGE2 production as described in the Experimental Section.
1
6.80 (1H, m, NHCO), 5.33 (2H, m, CHdCH), 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×CH2CHdCH), 1.85 (2H, m, CH2-
CH2NH), 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.
often seen, suggesting minor stimulation of the cells from
membrane-perturbing compounds.
In conclusion, building upon 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
noncharged 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
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 R-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; mp 40-42 °C; 1H NMR: δ 6.85 (1H, dd,
J1 ) 5.2 Hz, J2 ) 15.4 Hz, CHCHdCH), 6.60 (1H, d, J ) 9.2 Hz,
NHCO), 5.87 (1H, d, J ) 15.4 Hz, CHdCHCOOCH3), 4.62 (1H,

Group IVA and Group VIA Phospholipases
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2825
m, CH), 4.14 (1H, m, CH), 3.73 (3H, s, COOCH3), 2.77 (1H, m,
OH), 1.98-1.01 (32H, m, 16×CH2), 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.
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, CHdCH), 3.67 (3H, s, CH3O), 3.33 (2H, m, CH2-
NH), 2.91 (2H, t, J ) 7.2 Hz, CH2COCO), 2.38 (2H, t, J ) 7.4
Hz, CH2COO), 1.98 (4H, m, 2×CH2CHdCH), 1.88 (2H, m, CH2-
CH2NH), 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) [Μ + H]+. Anal. (C24H43NO4) C, H, N.
4-(2-Oxohexadecanoylamino)oct-2-enoic Acid Methyl Ester
(11). Yield 81%, white solid; mp 48-50 °C; [R]D -19.7 (c 0.95,
Oxidation of 2-Hydroxyamides. Method A. To a solution of
2-hydroxyamide (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 Es-
ter (4a). Yield 67%; white solid; mp 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.
1
CHCl3); Η 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, CHdCHCOOCH3), 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 pro-
cedure is the same as that followed in method B except that the
organic layer was not washed with 10% aqueous NaHCO3. Yield
1
69%, white solid; mp 65-67 °C; [R]D -7.7 (c 0.84, CHCl3); Η
NMR: δ 7.0 (1H, m, NHCOCO), 6.82 (1H, dd, J1 ) 6 Hz, J2 )
16 Hz, CHCHdCH), 5.87 (1H, d, J ) 16 Hz, CHdCHCOOCH3),
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.
4-(2-Oxo-6-phenyl-hexanoylamino)butyric Acid Methyl Ester
1
(4b). Yield 75%; white solid; mp 52-54 °C; H NMR: δ 7.29-
Saponification of Methyl Esters. To a stirred solution of a
methyl ester (2.00 mmol) in a mixture of dioxane/H2O (9:1, 20
mL), 1 N 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 1 N
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 recrystal-
lization (EtOAc/petroleum ether (bp 40-60 °C)).
4-(2-Hydroxy-5-phenylpentanoylamino)butyric Acid (3a). Yield
79%; white solid; mp 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; mp 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; mp 46-48 °C; 1H NMR: δ 6.92 (1H, m, NHCO),
6.76 (1H, dd, J1 ) 6 Hz, J2 ) 16 Hz, CHCH dCH), 5.87 (1H, d,
J ) 16 Hz, CHdCHCOOH), 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, 2×CH3);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.
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, which is
described above, except that in this case the aqueous layer was
acidified before the workup 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; mp 65-67 °C; 1Η
NMR: δ 7.25-7.11 (6H, m, C6H5, NHCOCO), 3.33 (2H, m, CH2-
NH), 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) [Μ - H]-. Anal. (C15H19-
NO4) 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; mp 60-62 °C; 1Η NMR: δ 7.27-7.15 (6H, m, C6H5,
NHCOCO), 3.35 (2H, m, CH2NH), 2.94 (2H, t, J ) 7.4 Hz, CH2-
COCO), 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) [Μ + 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 and 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)).
Inhibitor 12 was prepared by following procedures similar to
those described above.
4-(2-Oxononadec-10-enoylamino)butyric Acid (12). Yield
69%; white solid; mp 57-59 °C; 1Η NMR: δ 10.05 (1H, br,
COOH), 7.23 (1H, m, NHCOCO), 5.33 (2H, m, CHdCH), 3.38

2826 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Stephens et al.
(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×CH2CHdCH), 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. The 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 iPLA2, kindly provided by
Dr. Brian Kennedy at Merck-Frost, modified with a six-residue
histidine tag and three amino acid linker to the amino terminus
using PCR with oligonucleotides 5′-ATGCAGTTCCACCATCAC-
CATCACCATTTTGGAGCGCTGGTCAATACC-3′ and 5′-CCT-
CAGGGTGAGAGCAGCAGCTG-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 via
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/mL was infected with the recombinant baculovirus
with an MOI of approximately 0.1. Infections were carried out for
72 h, and the cells were harvested by centrifugation at 3000g for
10 min and stored at -80 °C. The frozen cell pellets from 200 mL
of suspension culture Sf9 cells were resuspended in 25 mL of
resuspension buffer (25 mM Tris at pH 8.0, 150 mM NaCl, 10
mM DTT, 5 mM EDTA, 2 mM ATP, 0.2% methyl-â-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 min and then clarified by centrifugation at 15 000g for
30 min at 4 °C. The resulting pellet was resuspended in a
solubilization buffer (25 mM Tris at pH 8.0, 150 mM NaCl, 10
mM â-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 1
h followed by centrifugation at 15 000g for 30 min 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 min 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 at 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 at pH 7.4, 100 mM NaCl, 50
mM urea, 2 mM ATP, and 200 mM imidazole, 30% v/v glycerol).
The eluate was collected as 1.5 mL fractions into tubes containing
15 µL of 500 mM DTT (5 mM DTT final). The fractions containing
protein were pooled, measured for activity and protein concentration
and stored as 200 µL aliquots at -80 °C.
(190 ng) was added to start the reaction followed by incubation
for 30 min 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 of
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. The reactions were quenched,
extracted, and analyzed using the modified Dole assay.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. The cells were grown in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 10% fetal calf serum (HyClone Labs, Provo,
Utah), 100 units/mL of penicillin, and 100 µg/mL of streptomycin
(Invitrogen, Carlsbad, California). Prior to stimulation, the cells
were plated at a density of 5 × 105 cells/well in standard 12-well
tissue culture plates and were allowed to adhere for 24 h. They
were then washed with a serum-free medium and allowed to adjust
for 18 h. The cells were then exposed to 100 ng/mL of LPS (Sigma
L4130 from E. coli 0111:B4) for 24 h. Following stimulation, the
medium was removed, and the cells were scraped into 1 mL of
PBS and counted. Deuterated PGE2 internal standard (10 ng) was
added to the medium of each sample, and the media were cleared
of cellular debris by centrifugation (3000g, 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 of methanol followed by
2 mL of water. The sample was loaded and the column washed
with 2 mL of 0.5% methanol. The sample was eluted from the
column with 1 mL of 100% methanol.
The inhibitors, when included, were dissolved in DMSO and
diluted into serum-free media prior to the addition to cells. The
DMSO concentration was kept below 0.5% v/v in all studies. All
inhibitors were added 30 min prior to stimulation.
The 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 × 250 mm) run with a
gradient beginning with 100% Buffer A (63:37:0.02 water/aceto-
nitrile/formic acid) and ending with 100% Buffer B (50:50
acetonitrile/2-propanol). 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 ) -4.5 kV, source temperature ) 525
°C, source gas 1 ) 60, source gas 2 ) 60, and declustering potential
) -50 V. The PGE2 was detected via CID with a precursor ion of
351, a product ion of 189 amu, a collision energy of -27 V, and
high Q2 collision gas.
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 nmol of dipalmitoyl
phosphatidylcholine (DPPC) mixed with 1 × 105 cpm of 1-palmi-
toyl, 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 at pH 7.5, 50 mM EDTA, 20 mM
DTT, 10 mM ATP, and 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 at pH 7.5 to give a
final volume of 500 µL upon the addition of the enzyme and the
inhibitor. The inhibitors were dissolved in DMSO to a stock
concentration of 5 mM and diluted with DMSO prior to the 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. The purified enzyme
Acknowledgment. We thank Dr. Brian Kennedy at Merck-
Frost for a generous gift of the cDNA coding for human Group
VIA-2 iPLA2 and Dr. Ruth Kramer at Lilly Research Labora-
tories for a generous gift of pure, native human GIVA cPLA2.
We thank Dr. Karin Lucas for her input and valuable discussions
while preparing this manuscript. This work was supported by
NIH Grants GM 20501 and GM 064611 (E.A.D.) and by
AnalgesiX/UC Discovery Biotechnology Grant # B1002-10303.
The Regents of the University of California, G.K., and E.A.D.
hold equity in AnalgesiX.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.

Group IVA and Group VIA Phospholipases
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