Inhibition of group IVA cytosolic phospholipase A2 by novel 2-oxoamides in vitro, in cells, and in vivo

Article abstract of DOI:10.1021/jm030485c

The Group IVA cytosolic phospholipase A₂ (GIVA PLA₂) is a particularly attractive target for drug development because it is the rate-limiting provider of proinflammatory mediators. We previously reported the discovery of novel 2-oxoa

Full text of DOI:10.1021/jm030485c

J . Med. Chem. 2004, 47, 3615-3628
3615
In h ibition of Gr ou p IVA Cytosolic P h osp h olip a se A2 by Novel 2-Oxoa m id es in
Vitr o, in Cells, a n d in Vivo
George Kokotos,*,† David A. Six,‡ Vassilios Loukas,† Timothy Smith,‡ Violetta Constantinou-Kokotou,§
Dimitra Hadjipavlou-Litina,| Stavroula Kotsovolou,† Antonia Chiou,† Christopher C. Beltzner,‡ and
Edward A. Dennis*,‡
Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece,
Department of Chemistry and Biochemistry, Department 0601, University of CaliforniasSan Diego,
La J olla, California 92093-0601, Chemical Laboratories, Agricultural University of Athens, Athens 11855, Greece, and
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki,
Thessaloniki 54124, Greece
Received September 25, 2003
The Group IVA cytosolic phospholipase A2 (GIVA PLA2) is a particularly attractive target for
drug development because it is the rate-limiting provider of proinflammatory mediators. We
previously reported the discovery of novel 2-oxoamides that inhibit GIVA PLA2 [Kokotos, G.;
et al. J . Med. Chem. 2002, 45, 2891-2893]. In the present work, we have further explored this
class of inhibitors and found that the 2-oxoamide functionality is more potent when it contains
a long 2-oxoacyl residue and a free carboxy group. Long-chain 2-oxoamides based on
γ-aminobutyric acid and γ-norleucine are potent inhibitors of GIVA PLA2. Such inhibitors act
through a fast and reversible mode of inhibition in vitro, are able to block the production of
arachidonic acid and prostaglandin E2 in cells, and demonstrate potent in vivo anti-
inflammatory and analgesic activity.
In tr od u ction
gathered through the use of many nonspecific and
imperfect inhibitors in cellular and animal studies over
the years. But perhaps the most compelling evidence
for the central role of GIVA PLA2 comes from the gene-
targeted mice that lack GIVA PLA2.9-14 Various studies
have found that these mice are much less prone to
inflammatory pathological responses to disease, stresses,
and physical injuries, which in essence protects them
from cellular and systemic damage.9-14 Furthermore,
the mice lacking GIVA PLA2 are fairly normal in all
other respects.9-11,13,14 The female null mice have some
fertility problems mostly associated with their lack of
labor-inducing prostaglandins and thus must be surgi-
cally delivered, but their pups are otherwise nor-
mal.9,11,15 Therefore, these data suggest that synthetic
inhibitors of GIVA PLA2 would be exceedingly valuable
for prevention and treatment of various inflammatory
conditions and would avoid many side effects and
drawbacks of current therapies and treatments.16 Re-
cently we discovered novel 2-oxoamides that inhibit
GIVA PLA2.17 In the present work, we demonstrate that
synthetic 2-oxoamides constitute a novel class of human
GIVA PLA2 inhibitors with promising anti-inflamma-
tory and analgesic activity.
Phospholipase A2 (PLA2) catalyzes the hydrolysis of
the sn-2 or middle fatty acyl chain from a phospholipid.
The PLA2 superfamily of enzymes consists of two large
branches: the histidine and serine PLA2 branches.1 The
histidine PLA2’s are similar small, secreted, Ca2+
-
dependent enzymes that utilize a nucleophilic water
molecule activated by the active site Ca2+, aspartates,
and especially a catalytic histidine (Groups I-III, V,
IX-XIV). The other branch of the superfamily is com-
posed of larger, typically cytosolic enzymes that utilize
a nucleophilic serine (Groups IV and VI-VIII). Groups
VII and VIII PLA2 are also known as platelet activating
factor (PAF) acetylhydrolases and utilize their nucleo-
philic serine as part of a typical catalytic triad of serine-
histidine-aspartate. In the case of Group IVA PLA2
(also referred to as the 85 kDa, cytosolic PLA2), the
nucleophilic serine is in a novel catalytic dyad involving
only a serine and an aspartate.2,3 The Group VI PLA2
appears to utilize a similar catalytic mechanism4 and
is indeed inhibited by many compounds that inhibit the
Group IVA PLA2.5-8
The Group IVA PLA2 (GIVA PLA2) is a particularly
attractive target for drug development because it is the
rate-limiting provider of free polyunsaturated fatty acids
and lysophospholipids that go on to form the eicosanoids
and PAF, respectively. This information has been
Ra tion a l Design of GIVA P LA2 In h ibitor s
A decade ago, a trifluoromethyl ketone analogue of
arachidonic acid (AA) in which the COOH group was
replaced by COCF3 (AATFK, 1, Scheme 1) was prepared
and found to be a tight- and slow-binding inhibitor of
the 85 kDa cytosolic human GIVA PLA2.18 As demon-
strated with NMR studies, the inhibitor was bound to
the enzyme’s active site in a reversible manner and was
in a slow exchange with the free ligand.19 Another
AA derivative, methylarachidonoyl fluorophosphonate
* To whom correspondence should be addressed. For G.K.: (phone)
30210 7274462; (fax) 30210 7274761; (e-mail) gkokotos@cc.uoa.gr. For
E.A.D.: (phone) 858-534-3055; (fax) 858-534-7390; (e-mail) edennis@
ucsd.edu.
† University of Athens.
‡ University of CaliforniasSan Diego.
§ Agricultural University of Athens.
| Aristotelian University of Thessaloniki.
10.1021/jm030485c CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/02/2004

3616 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
Sch em e 1. Structures of Known Reversible GIVA PLA2 Inhibitors
(MAFP), has been shown to irreversibly inactivate GIVA
PLA2.20 The inhibition of GIVA PLA2, in comparison
with GVI PLA2, by unsaturated and saturated fatty acid
tricarbonyls21 (2) and trifluoromethyl ketones8 has been
studied. Lehr has reported that (4-acylpyrrol-2-yl)-
alkanoic acids23 such as 3, and 3-acyl 3-dodecanoylindol-
2-carboxylic acids24 such as 4 inhibit the cytosolic PLA2
of platelets as measured by the blocking of AA release
induced by the calcium ionophore A23187. Seno et al.
have shown that pyrrolidine derivatives,25 having the
thiazolidinedione group at the end of the substitutent
in the 2-position of the pyrrolidine ring and the 2-ben-
zoyl benzoyl group at the 1-position (for example,
compound 5) are potent inhibitors of human GIVA
PLA2. An extension of this research led to the potent
inhibitor pyrrophenone (6).26 Despite these advances,
potent and specific reversible competitive inhibitors of
GIVA PLA2 are not commercially available.
Novel inhibitors of GIVA PLA2 may be rationally
designed, taking into consideration the mechanism of
the enzyme’s catalytic action and the structure of the
substrate. GIVA PLA2 is an enzyme that acts at the
lipid-water interface. Its catalytic mechanism proceeds
through a serine-acyl intermediate using Ser-228 as
the nucleophilic residue. The X-ray crystal structure of
GIVA PLA2 indicates that the enzyme consists of an
N-terminal calcium-dependent lipid-binding C2 domain
and a catalytic R/â hydrolase domain whose topology is
distinct from that of other lipases.3 GIVA PLA2 pos-
sesses an unusual catalytic Ser-Asp dyad located in a
deep cleft at the center of a hydrophobic funnel of the
catalytic domain.3
Our strategy for the rational design of novel GIVA
PLA2 inhibitors is based on the principle that the
inhibitor should consist of several parts: an electrophilic
functionality (activated carbonyl group), which may
react with the active-site serine; a lipophilic segment;
and the appropriate chemical motifs necessary for
specific interactions and proper orientation in the
enzyme’s active site. It has been demonstrated that
lipophilic 2-oxoamides27 and 2-oxoamide and bis-2-
oxoamide triacylglycerol analogues28 are efficient inhibi-
tors of pancreatic and gastric lipases, enzymes contain-
ing a classical catalytic triad (Ser-His-Asp). Thus,
among the various existing functionalities capable of
reacting with the active-site serine, the 2-oxoamide
functionality was chosen as the reactive group. The
GIVA PLA2 residue Arg-200 has been proposed to
interact with the negative charge of its substrate
phospholipids,3 and therefore, an analogous carboxy
group was incorporated into the 2-oxoamide backbone
(Scheme 2). Such a design led to the discovery of the
lead 2-oxoamide structures AX006 and AX007,17 ex-
plored in the present work in detail. In the same year,
another type of GIVA PLA2 inhibitor based on a 1,3-
disubstituted propan-2-one skeleton (7) was reported by
Connolly et al.29

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3617
Sch em e 2. Design of 2-Oxoamide Inhibitors
In Vitr o Da ta for AX006 a n d AX007
All the inhibitors synthesized were tested against
GIVA PLA2, and the results of inhibition are presented
in Table 1 using either percent inhibition or XI(50). The
XI(50) is the mole fraction of inhibitor in the total
substrate interface required to inhibit the enzyme by
50%. The reason that XI(50) is used instead of the more
common IC50 or KI is that GIVA PLA2 is active only at
phospholipid surfaces such as cell membranes, phos-
pholipid vesicles, and phospholipid micelles, where its
substrate phospholipids reside. Almost all inhibitors of
PLA2’s partition at least to some degree into the
phospholipid surface because they usually have a hy-
drophobic portion that complements the hydrophobic
active site of the PLA2. When these inhibitors partition
into the surface, an important physical effect called
surface dilution comes into play. In this case, the affinity
of the PLA2 for the inhibitor depends not on the three-
dimensional (bulk) concentration of the inhibitor in
molar units but rather on the two-dimensional (surface)
concentration of the inhibitor in mole fraction units. For
these reasons, IC50 values obtained in different assay
systems (even as minor changes as varied substrate
concentrations) cannot be compared.
Syn th esis of 2-Oxoa m id e In h ibitor s
For the synthesis of inhibitors containing γ-aminobu-
tyric acid (GABA), methyl 4-aminobutyrate (9) was
coupled with 2-hydroxycarboxylic acids 8a ,b using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (WSCI)30 as
a condensing agent in the presence of 1-hydroxybenzo-
triazole (HOBt) (Scheme 3). Alkaline hydrolysis of
10a ,b, followed by oxidation with NaOCl in the presence
of a catalytic amount of 4-acetamido-2,2,6,6-tetrameth-
ylpiperidine-1-yloxy free radical (AcNH-TEMPO)31 pro-
duced compounds AX005 and AX006. GABA derivatives
AX010 and AX011 were also synthesized (Scheme 3).
N-protected amino alcohols 11a -d (Scheme 4) were
prepared from the corresponding natural R-amino acids
32
by the mixed-anhydride/NaBH4 or the acyl fluoride/
NaBH4 method.33 Alcohols 11a -d were oxidized to the
corresponding aldehydes 12a -d by the NaOCl/AcNH-
TEMPO method (Scheme 4), and the aldehydes were
directly used in the next step without any purification.
Primary 2-oxoamides (AX001 and AX015) containing
either a long, linear chain or a phenyl-substituted chain
do not inhibit GIVA PLA2. Secondary 2-oxoamides
lacking a carboxylic acid functionality (AX002, AX003,
AX004, and AX009) also failed to inhibit the enzyme.
However, 2-oxoamides based on GABA and γ-norleucine
(AX006, AX007, and AX008) do inhibit GIVA PLA2.
There are three routes for the synthesis of γ-amino
acid-based 2-oxoamide inhibitors depending on the ylide
and the N protecting group used (Scheme 4). Wittig
reactions of Boc-protected amino aldehydes 12a -c with
benzyl or methyl (triphenylphosphoranylidene)acetate
led to R,â-unsaturated esters 13 and 14a -c. The Boc
group of 13 was removed, and the amino component was
coupled with 2-hydroxydodecanoic acid by the WSCI/
HOBt method. In a similar manner, 14a -c, after
hydrogenation, were converted into 17a -e. Catalytic
hydrogenation of 16 and saponification of 17a -e, fol-
lowed by oxidation with either pyridinium dichromate
(PDC) or NaOCl/AcNH-TEMPO, led to the target com-
pounds AX007, AX012, AX013, AX016, AX017, and
AX024. γ-Proline-based inhibitors, AX018 and AX019,
and AX008, the enantiomer of AX007, were also pre-
pared. The Z-protected amino aldehyde (12d ) was
treated with tert-butyl (triphenylphosphoranylidene)-
acetate to give compound 15. After catalytic hydrogena-
tion, the amino component was coupled with 2-hydrox-
ypentadecanoicacid bythe WSCI/HOBt method.Oxidation
of 18, followed by the removal of the tert-butyl group
with CF3COOH, led to AX014.
Figure 1 shows the data used to determine the XI(50)
values for entries 6, 7, and 8,17 now named AX006,
AX007, and AX008, respectively. The data for AX006
and AX007 were easily fit to logarithmic functions using
nonlinear least-squares fits to determine the empirical
XI(50) values. This fit is useful for obtaining XI(50)
values with error values and does not represent a fit to
any kinetic equations. The data for AX008 clearly do
not fit a logarithmic function but rather are linear.
Therefore, the data for AX008 were fit using a linear
least-squares fit. AX007 and AX008 are enantiomeric,
so the 8-fold difference in their XI(50) values and the
shape of their inhibition curve provide important in-
sights into the GIVA PLA2 active site. In particular, all
potent inhibitors fit logarithmic functions while poor
inhibitors fit linear functions. It seems that the chirality
introduced by a hydrophobic side chain adjacent to the
2-oxoamide nitrogen in a γ-acid backbone provides
stereospecific enhancements of the binding interaction.
The in vitro activity assays used to determine the
potency of the various 2-oxoamide compounds were done
in the presence of 0.006 mole fraction phosphatidyli-
nositol 4,5-bisphosphate (PIP2). This is a tight binding
activator of GIVA PLA2.34,35 Without PIP2 in these
particular substrate micelles, GIVA PLA2 activity drops
to almost undetectable levels. Therefore, it was impor-
tant to rule out any interference with the GIVA PLA2-
PIP2 interaction by the 2-oxoamide inhibitors. We have
tested this using an alternative substrate micelle that
allows the measurement of GIVA PLA2 activity without
any PIP2 present, as described in the Supporting
Information of our initial study,17 consisting of 1-palmi-
The synthesis of γ-serine-based inhibitors is depicted
in Scheme 5. Boc-L-Ser-OH (19) was reacted with a
medium-chain alkyl iodide and was converted into
serinol derivative 21. The R,â-unsaturated ester 22 was
prepared by oxidation and reaction with benzyl (triphe-
nylphosphoranylidene)acetate. After removal of the Boc
group, coupling with 2-hydroxydodecanoic acid, hydro-
genation, and oxidation, 2-oxoamide derivative AX027
was obtained. Its enantiomer, AX026, was prepared in
a similar manner.
AX001-AX004, AX009, and AX015 (see Table 1 for
structures) were prepared as described.27b

3618 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Sch em e 3. Synthesis of GABA-Based Inhibitorsa
Kokotos et al.
a
Reagents and conditions: (a) WSCI, Et3N, HOBt, CH2Cl2; (b) 1 N NaOH, dioxane/H2O 9:1; (c) NaOCl, AcNH-TEMPO, NaBr, NaHCO3,
EtOAc/PhCH3/H2O 3:3:0.5, -7 °C.
Sch em e 4. Synthesis of γ-Amino Acid Based Inhibitorsa
a
Reagents and conditions: (a) NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, -5 °C; (b) Ph3PdCHCOOBn, THF,
reflux, 1 h; (c) Ph3PdCHCOOMe, THF, reflux, 1 h; (d) Ph3PdCHCOOtBu, THF, reflux, 1 h; (e) 4 N HCl/THF; (f) CH3(CH2)nCHOHCOOH,
WSCI, HOBt, Et3N, CH2Cl2; (g) H2, 10% Pd/C; (h) NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, 0 °C, then HCl; (i)
1 N NaOH, dioxane/H2O 9:1, then HCl; (j) 50% CF3COOH, CH2Cl2.
toyl-2-arachidonoylphosphatidylcholine (PAPC) and Tri-
ton X-100 (TX-100) at a 1 mM to 3 mM ratio. As shown
in Figure 2, the presence of 0.01 mole fraction of AX006
in these micelles that lack PIP2 does indeed inhibit
GIVA PLA2 by about 50%. This is consistent with a true
inhibition of GIVA PLA2 by AX006 in the PIP2-contain-
ing micelles. We also tested AX007 in conditions that
lacked PIP2. Figure 3 shows the dose-dependent inhibi-
tion of GIVA PLA2 by AX007. As in Figure 1, the
inhibition data have been fit to a logarithmic function.
The XI(50) derived from the equation of the fit was
0.0027 ( 0.0012 mole fraction. This is the same mag-
nitude as the XI(50) determined in the presence of PIP2,
indicating a true inhibition of GIVA PLA2 independent
of PIP2. This also compares favorably with the XI(50)
reported for pyrrophenone (0.002 mole fraction).26

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3619
Sch em e 5. Synthesis of γ-serine Based Inhibitorsa
a
Reagents and conditions: (a) NaH, C10H21I, DMF, 0 °C; (b) ClCOOEt, NMM, THF, -10 °C, then NaBH4, MeOH; (c) NaOCl, AcNH-
h; (e) 4 HCl/THF; (f)
TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, -5 °C; (d) Ph3PdCHCOOBn, THF, reflux,
CH3(CH2)9CHOHCOOH, WSCI, HOBt, Et3N, CH2Cl2; (g) H2, 10% Pd/C; (h) NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O
3:3:0.5, 0 °C, then HCl.
1
N
Ta ble 1. In Vitro Inhibition of GIVA PLA2 by 2-Oxoamides
a
b
AX001-AX008 are entries 1-8 from Kokotos et al. J . Med. Chem. 2002, 45, 2891-2893. Not determinable.
Various inhibitors of GIVA PLA2 have been shown to
be slow-binding and only slowly reversible. AATFK and
MAFP inhibit GIVA PLA2 in both concentration- and
time-dependent manners in the presence and absence
of a substrate interface.5-8 To test the type of inhibition
by the 2-oxoamide inhibitors, AX006 was preincubated

3620 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
F igu r e 1. Dose-response curves for 2-oxoamide inhibitors.
The activity of GIVA PLA2 was tested on mixed micelles
composed of TX-100 (400 µM), 1-palmitoyl-2-(1-[14C]-arachi-
donoyl) PC (97 µM, 100 000 cpm), and PIP2 (3 µM, 0.6 mol %).
The surface concentration of various inhibitors in the mixed
micelles was increased as shown for AX006 (2), AX007 (9),
and AX008 (0). The data for AX006 and AX007 were fit to a
simple logarithmic function (AX006 (solid curve) and AX007
(dashed curve)). The XI(50) determined by this fit was 0.017
( 0.009 for AX006 and 0.009 ( 0.004 for AX007. The data for
AX008 were fit to a linear function. The XI(50) for AX008
determined by this fit was 0.068 ( 0.005.
F igu r e 3. Inhibition of GIVA PLA2 by AX007 is independent
of PIP2. The activity of GIVA PLA2 was tested on mixed
micelles composed of TX-100 (3 mM) and 1-palmitoyl-2-(1-[14C]-
arachidonoyl) PC (1 mM, 200 000 cpm) with no PIP2 present.
The surface concentration of AX007 was increased as shown.
The curve is a fit of the data to a simple log function. The curve
values give an XI(50) of 0.0027 ( 0.0012 mole fraction.
mode of inhibition of AX006. The concentration of
AX006 for preincubation was 200 µM but dropped to
20 µM (0.005 mole fraction) after dilution of the enzyme-
inhibitor mix into the assay solution. The assay results
show that preincubation at a higher concentration
followed by dilution did not yield any increased inhibi-
tion, suggesting that AX006 is a completely reversible
inhibitor.
We also tested the more potent, chiral AX007 to see
if it were a fast, reversible inhibitor of GIVA PLA2. We
preincubated GIVA PLA2 with 20 µM AX007 and then
diluted the preincubated GIVA PLA2 into the assay
solution that also contained 20 µM AX007. As shown
in Figure 4B, a 4 h preincubation did not change the
level of inhibition of AX007. This indicates that there
is no time-dependent increase in inhibition of GIVA
PLA2 by AX007. In contrast, Figure 4C shows the
results when AX007 was present at 20 µM during the
preincubation and was then diluted 10-fold into the
assay mix. This dilution brought the final AX007
concentration down to 2 µM (0.0005 mole fraction) and
resulted in a significant reduction in the inhibition of
GIVA PLA2 by AX007 (Figure 4C). The significantly
reduced inhibition by the diluted AX007 (at 0.0005 mole
fraction) is consistent with the results shown in Figure
1. Together, the results for AX006 and AX007 strongly
indicate that 2-oxoamide inhibitors of GIVA PLA2
function through a fast and fully reversible mode of
inhibition.
F igu r e 2. Inhibition of GIVA PLA2 by AX006 is independent
of PIP2. The relative activity of GIVA PLA2 was tested on
mixed micelles composed of TX-100 (3 mM) and 1-palmitoyl-
2-(1-[14C]-arachidonoyl) PC (1 mM, 200,000 cpm) with no PIP2
present. The surface concentration of AX006 was 0 mole
fraction (left bar) and 0.01 mole fraction (40 µM, right bar).
The data for both bars have been normalized to the control
(left bar).
Str u ctu r e-Activity Rela tion sh ip s
The discovery that the (S)-γ-norleucine-containing
2-oxoamide (AX007) enhances inhibitory potency in an
enantioselective fashion (8-fold more potent than (R)-
AX008 and 2-fold more potent than nonchiral AX006)
led to the further exploration of the nature of the (S)
side chain. Substitutions of side chains at the (S)-γ-
position revealed that short hydrophobic side chains
were preferred. Two leucine variants, AX012 and AX013,
and an alanine variant, AX014, were as potent as AX006
but only about half as potent as the norleucine-contain-
with GIVA PLA2 without substrate for varying lengths
of time, after which the mix was added to the assay
solution (Figure 4A). The resulting activity assay showed
that there was no increase in inhibition with preincu-
bation times up to 30 min. This indicated a fast-binding

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3621
F igu r e 4. Immediate and reversible inhibition of GIVA PLA2
by AX006 and AX007. The activity of GIVA PLA2 was tested
on mixed micelles composed of TX-100 (400 µM), 1-palmitoyl-
2-(1-[14C]-arachidonoyl) PC (97 µM, 100 000 cpm), and PIP2
(3 µM, 0.6 mol %). (A) The micelles also contained a final
concentration of 0.005 mole fraction of AX006 (20 µM). GIVA
PLA2 was first preincubated with AX006 for the indicated
times (at 200 µM) before 10-fold dilution of the enzyme and
inhibitor into the substrate-containing solution. (B) The mi-
celles also contained a final concentration of 0.005 mole
fraction of AX007 (20 µM). GIVA PLA2 was first preincubated
with AX007 (20 µM) for 0 or 4 h before it was added to the
substrate-containing solution that also contained AX007 (20
µM). (C) The micelles also contained a final concentration of
0.005 mole fraction of AX007 (20 µM). GIVA PLA2 was first
preincubated with AX007 (20 µM) for 0 or 4 h before it was
added to the substrate-containing solution that contained no
additional AX007. This resulted in a final concentration for
AX007 of 0.0005 mole fraction (2 µM).
F igu r e 5. Inhibition of cellular PGE2 production by AX006
and AX007. Varying concentrations of AX006 (2) or AX007
(9) were added to P388D1 cells 30 min. prior to stimulation.
(A) The cells were stimulated with LPS for 18 h as described.
After stimulation, the media was harvested and the PGE2
levels in the media were quantitated as described. The data
were normalized to control DMSO-treated LPS-stimulated
cells to appear as percent inhibition. The lines correspond to
a nonlinear least-squares fit of the data to logarithmic func-
tions (AX006 (solid curve) and AX007 (dashed curve)). These
functions were then used to calculate the IC50 for AX006 (7.8
µM) and AX007 (5.8 µM). (B) The cells were stimulated with
LPS for 1 h and PAF for 15 min as described. After stimula-
tion, the media was harvested and the PGE2 levels in the
media were quantitated as described. The data were normal-
ized to control DMSO-treated LPS/PAF stimulated cells to
appear as percent inhibition. The data points are connected
by a dashed line (AX006) and a solid line (AX007) for ease of
visualization.
ing AX007. Two phenylalanine variants (AX016 and
AX017) and two proline variants (AX018 and AX019)
were significantly less potent. AX016 was nearly 4-fold
less potent than AX007, while AX017 was more than
10-fold less potent. AX018 was also more than 10-fold
less potent than AX007, while AX019 had no detectible
inhibition at the highest concentration tested. Further-
more, the substitution of serine-based, long aliphatic
ether side chains (AX026 and AX027) led to poor GIVA
PLA2 inhibitors. These enantiomeric compounds exhib-
ited essentially identical linear modes of inhibition
typical of poor inhibitors (data not shown) and showed
no stereoselectivity. These results together indicate that
the inhibitory potency and shape of the inhibitory profile
relate to the ability of the 2-oxoamide’s short, hydro-
phobic side chain to bind properly in a stereoselective
manner to the GIVA PLA2 active site.
The role of the free carboxy group was shown to be
important in our preliminary work by comparing AX006
and AX002.17 We have further confirmed the need for
the negative charge by testing a variant of AX006 that
contained a carboxymethyl ester (AX010) in place of the
free carboxy group (AX006). AX010 had no measurable
inhibition up to 0.08 mole fraction, whereas AX006
almost completely inhibited GIVA PLA2 at this concen-
tration. The carboxylic acid group in the 2-oxoamide
compounds is a shared feature among some previously
identified inhibitors of the small, secreted histidine
36
PLA2 as well as other GIVA PLA2 inhibitors, com-
pounds 3, 4, and 7 (Scheme 1).23,24,29 The role of the

3622 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
2-oxoamide functionality becomes obvious in a compari-
son of the effective GIVA PLA2 inhibitor AX006 (2-
oxoamide) with AX011 (corresponding simple amide),
which barely inhibits GIVA PLA2.
Lipophilicity is a very important parameter in the
case of GIVA PLA2 inhibitors because this enzyme
exhibits its catalytic action at the lipid-water interface.
From the values of ClogP (Table 1) calculated for the
inhibitors by CQSAR, version 4.30 (Biobyte Corp.), it
seems that the ClogP values of the active compounds
fall into the range 5-8. The effect of the lipophilicity
on activity is obvious upon comparison of the active
AX006 (ClogP ) 6.6) with the inactive compound AX005
(ClogP ) 2.4). However, the lipophilicity by itself is not
sufficient for the expression of activity (see, for example,
AX002 and AX004). Major effects related to hydropho-
bicity are not seen for most of the inhibitors, since the
ClogP values for similarly potent inhibitors vary from
5.8 (AX013) to 8.0 (AX024), suggesting complete parti-
tioning over this range. It should be noted that a high
ClogP (>8) renders an inhibitor an unlikely candidate
for pharmaceutical purposes. For comparison, the ClogP
values of other recent lead GIVA PLA2 inhibitors are
9.6 for pyrrophenone26 and 7.85 for the lead propane-
2-one-based inhibitor.29
F igu r e 6. Model for the binding mode of 2-oxoamide inhibi-
tors in the active-site crevice of GIVA PLA2.
Ta ble 2. Ex Vivo and in Vivo Activity of Selected 2-Oxoamide
Inhibitors
P388D1 long-term
LPS IC50 (µM)
rat in vivo
AA-derived
PGE2
anti-inflammatory
analgesic
entry products production activity ED50 (mmol/kg)a activityb (%)
AX006 8.6 ( 3.5
AX007 7.6 ( 2.4
7.8 ( 1.8
5.8 ( 2.1
0.01
0.1
0.008
93
63
69
AX012 4.6 ( 1.5 10 ( 5
a
Statistical significance of results was established using the
b
Student’s t-test, P < 0.001. Analgesic activity (% analgesis) for
the corresponding ED50 doses.
By varying the 2-oxoacyl chain length, we have
explored the differences in inhibition based on hydro-
phobicity. Using phenylalanine variants, we looked
specifically at the saturated chain length of the 2-oxo-
acyl chain (13 carbons for AX016). When the length was
shortened to six carbons (AX017), the potency went
down at least 4-fold. This same effect was seen in the
preliminary study comparing AX005 and AX006 that
differed by eight carbons.17 Smaller changes in the chain
length for already hydrophobic compounds can be seen
to have smaller or negligible effects. This is seen by
comparing the γ-norleucine-containing AX007 (10-
carbon chain) and AX024 (13-carbon chain), which have
equivalent inhibitory potency. Likewise, the γ-leucine-
containing AX013 (10 carbons) and AX012 (13 carbons)
have a nearly equivalent inhibitory potency. It is
possible that partitioning to the micelles is affected by
the less hydrophobic shortened acyl chains (such as
AX005), but it is also possible that the shorter-chained
compounds could have a weaker interaction with the
deep hydrophobic active site of GIVA PLA2. By analogy
to phospholipid substrate, it is likely that this hydro-
phobic chain occupies part of the substrate (AA) binding
pocket.
Lehr23 and Connolly et al.29 have proposed models for
the interaction of cytosolic PLA2 with indole and pyrrole
inhibitors and 1,3-disubstituted propan-2-one based
inhibitors. According to the findings of this research,
we propose a four-point model for the binding of inhibi-
tors to GIVA PLA2, as shown in Figure 6. The enzyme-
inhibitor complex is likely to be stabilized by the
“oxyanion hole” corresponding to Gly 197, 198, and 229
within the enzyme active site. The lipophilic long carbon
chain of the inhibitor may be accommodated in the
hydrophobic binding site, where the sn-2 arachidonoyl
residue of the natural substrate binds. The carboxy
group may bind to the proposed polar binding site (Arg
200) in a manner similar to that of the phosphate group
of phospholipids. In addition, the results of this work
indicate the presence of an important fourth binding site
that interacts with the side chain adjacent to the amide.
This side chain binding site exhibits enantioselectivity
and seems to accommodate a short, preferably linear,
carbon chain. This part of the inhibitor may correspond
to the sn-1 acyl chain of the natural substrate phospho-
lipid. Alternatively, because inhibition is higher with
shorter side chains than with a typical sn-1 acyl chain,
it is possible that the side chain is fitting into a small
pocket of GIVA PLA2 not exploited in substrate binding.
Ex Vivo Da ta : Stim u la tion of th e P 388D1
Mu r in e Ma cr op h a ge Cell Lin e
AX006, AX007, and AX012 were tested on the murine
P388D1 macrophage-like cell line for inhibition of the
GIVA PLA2 in the long-term lipopolysaccharide (LPS)
stimulation pathway (Table 2). GIVA PLA2 is the
central enzyme for the long-term LPS pathway through
its ability to produce AA, which subsequently can be
made directly into PGE2.37 The AA can also be used to
up-regulate the message and protein levels of Group V
PLA2, which in turn produces its own AA that goes on
to form PGE2.38,39 Blocking GIVA PLA2 with 2-oxoamide
inhibitors potently inhibited the release of AA with IC50
values in the low micromolar range (Table 2). This
inhibition is even more potent than that seen previously
with 25 µM MAFP, which reached a maximal inhibition
at about 60% inhibition.38
Previous results showed that the substantial, but not
complete, inhibition of AA release by MAFP correlated
with complete abrogation of PGE2 production.38 As
shown in Figure 5A, both AX006 and AX007 were able
to completely block prostaglandin E2 (PGE2) release
with the addition of 50 µM inhibitor, and neither showed
cytotoxicity at this dose. Moreover, the dose-response
data nicely fit to logarithmic functions. The IC50 values
for the inhibition of PGE2 release derived from these
fits were 7.8 µM for AX006 and 5.8 µM for AX007
(Figure 5 and Table 2). Since MAFP interferes with the

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3623
measurement of PGE2 release,38 the ability of GIVA
PLA2 inhibitors to completely block PGE2 is shown here
for the first time. Previous reports did show that PGE2
production was totally blocked by the cyclooxygenase-
2-specific inhibitor, NS-398, at 5 µM and the secreted
PLA2 inhibitor LY311727 at 25 µM.38
are promising novel agents for the prevention and
treatment of various inflammatory conditions.
Exp er im en ta l Section
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 Mz) spec-
trometer. All amino acid derivatives were purchased from
Fluka Chemical Co. TLC plates (silica gel 60 F254) and silica
gel 60 (70-230 or 230-400 mesh) for column chromatography
were purchased from Merk. Visualization of spots was effected
with UV light and/or phosphomolybdic acid and/or ninhydrin,
both in EtOH stain. THF, toluene, and Et2O were dried by
standard procedures and stored over molecular sieves or Na.
All other solvents and chemicals were reagent grade and used
without further purification. All the products gave satisfactory
elemental analysis results.
A. Gen er a l Meth od for th e Cou p lin g of 2-Hyd r oxy
Acid s w it h Am in o Com p on en t s. To a stirred solution of
2-hydroxy acid (2.0 mmol) and the hydrochloride amino
component (2.0 mmol) in CH2Cl2 (20 mL), Et3N (6.2 mL, 4.4
mmol) and subsequently WSCI (0.42 g, 2.2 mmol) and HOBt
(0.32 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, dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromatography
using CHCl3 as the eluent.
Additionally, both AX006 and AX007 were tested in
the LPS/PAF stimulation conditions of the P388D1 cells
(Figure 5B). These activation conditions result in the
release of AA from cellular membranes by GIVA PLA2.37
Both of these inhibitors showed essentially identical
inhibition of the PGE2 release from the cells. The
inhibition reached close to 60% at the maximal dosage
tested (50 µM). This is also consistent with previous
results indicating that LPS/PAF stimulation produced
PGE2 by two separate pathways, through GIVA PLA2
and the Group V PLA2. MAFP and LY311727 have each
been shown to partially block AA and PGE2 produc-
tion.40 Thus, the data shown in Figure 5B indicate that
AX006 and AX007 inhibit the P388D1 GIVA PLA2 under
the LPS/PAF stimulation conditions, resulting in the
expected partial abrogation of PGE2 release. Together,
the data in the P388D1 cells confirm that the 2-oxoamide
inhibitors AX006, AX007, and AX012 can get into the
cells and target GIVA PLA2 without any detectible
cytotoxicity.
In Vivo Da ta
B. Gen er a l Meth od for Sa p on ifica tion of 2-Hyd r oxy-
a m id es Con ta in in g Ester Gr ou p s. To a stirred solution of
a 2-hydroxyamide containing an ester group (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 recrystallization [EtOAc-petroleum ether (bp 40-60 °C)].
C. Gen er a l Meth od for th e Oxid a tion of 2-Hyd r oxy-
a m id es Con ta in in g Ca r boxylic Gr ou p s. To a solution of a
2-hydroxyamide (2.00 mmol) in a mixture of toluene-EtOAc
(1:1, 12 mL), a solution of NaBr (0.22 g, 2.1 mmol) in H2O (1
mL) was added, followed by AcNH-TEMPO (4 mg, 0.02 mmol).
To the resulting biphasic system, which was cooled to 0 °C,
an aqueous solution of 0.35 M NaOCl (6.2 mL, 2.2 mmol),
containing NaHCO3 (0.50 g, 6 mmol), was added dropwise with
vigorous stirring at 0 °C over 1 h. The mixture was stirred for
15 min at 0 °C, and H2O (4 mL) was added. The aqueous layer
was separated, acidified with 1 N HCl, and extracted with
EtOAc (2 × 12 mL). The combined organic layers were washed
consecutively with 5% aqueous KI (12 mL), 10% aqueous
Na2S2O3 (12 mL), and brine, dried over Na2SO4, and evapo-
rated under reduced pressure. The residue was purified by
column chromatography using EtOAc as the eluent.
To evaluate the anti-inflammatory activity of 2-oxo-
amide inhibitors, the rat paw carrageenan-induced
edema assay41 was employed as a model for acute
inflammation. Indomethacin was used in these experi-
ments as a reference drug and gave 47% inhibition of
inflammation at 0.01 mmol/kg. The acetic acid writhing
test42 was used to assess the analgesic activity in rats.
Acetylsalicylate was used in these experiments as a
reference drug and gave 93.4% analgesis at 1 mmol/kg.
The results of in vivo anti-inflammatory and analgesic
activity of selected 2-oxoamide inhibitors are presented
in Table 2. AX006 and AX012 exhibit in vivo anti-
inflammatory activities comparable to that of indometha-
cin, whereas AX007 presented a weaker effect. Addi-
tionally all three compounds also showed analgesic
activity at the ED50 dosage determined by anti-inflam-
matory activity and were significantly more potent than
acetylsalicylate. There is no obvious reason for the
higher efficacy of AX006 and AX012 compared with
AX007, but the bioavailability and physiological half-
life will have to be considered.
Con clu sion
D. Gen er a l Meth od for th e Oxid a tion of 2-Hyd r oxy-
a m id es Con ta in in g Ester Gr ou p s. To a solution of a
2-hydroxyamide (5.00 mmol) in a mixture of toluene-EtOAc
(30 mL), a solution of NaBr (0.54 g, 5.25 mmol) in water (2.5
mL) was added, followed by AcNH-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 while stirring vigorously 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.
In conclusion, we have developed a novel class of
human GIVA PLA2 inhibitors. Structure-activity rela-
tionship studies have revealed that potent GIVA PLA2
inhibitors contain (a) the 2-oxoamide funtionality, (b) a
free carboxy group at a distance corresponding to
γ-amino acid, (c) a long 2-oxoacyl residue, and (d) a
short, preferably linear, aliphatic side chain. As dem-
onstrated by selected examples, 2-oxoamide compounds
inhibit GIVA PLA2 through a fast and reversible mode
of inhibition and block the production of AA and PGE2.
Furthermore, they exhibit potent in vivo anti-inflam-
matory and analgesic activity. Therefore, 2-oxoamides

3624 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
The solvents were evaporated under reduced pressure, and the
residue was purified by column chromatography [EtOAc-
petroleum ether (bp 40-60 °C) 1:9].
E. Gen er a l Meth od for th e Oxid a tion of N-P r otected
Am in o Alcoh ols to Ald eh yd es. Method D was followed, but
the residue was used immediately in the next step without
any purification.
F . Wittig Rea ction of N-P r otected r-Am in oa ld eh yd es
w ith Sta bilized Ylid es. To a solution of the N-protected
R-aminoaldehyde 12a -d (5.00 mmol) in dry THF (25 mL),
Ph3PdCHCOOX (X ) Me, tBu, Bn) (5.5 mmol) was added, and
the reaction mixture was refluxed for 1 h. Saturated aqueous
NH4Cl (20 mL) was added and extracted with Et2O (3 × 10
mL). The combined organic phases were washed with brine
and dried (Na2SO4). The solvent was evaporated under reduced
pressure, and the residue was purified by column chromatog-
raphy [EtOAc-petroleum ether (bp 40-60 °C) 2:8].
G. Gen er a l Meth od for th e Rem ova l of Boc Gr ou p s.
To a solution of 4 N HCl in MeOH (5.0 mL, 20 mmol), an
N-Boc-protected component (2 mmol) was added. After being
stirred at room temperature for 30 min, the mixture was
evaporated, dry Et2O was added, and the product was filtered
and used for coupling with a 2-hydroxy acid.
H. Gen er a l Meth od for Ca ta lytic Hyd r ogen a tion . To a
solution of the substrate (2.00 mmol) in MeOH (10 mL)
(through which N2 had been passed for 5 min), 10% Pd/C
catalyst (22 mg, 0.02 mmol) was added. The reaction mixture
was stirred under H2 atmosphere overnight at room temper-
ature. The catalyst was removed by filtration through a pad
of Celite, and the organic solvent was evaporated under
reduced pressure. The residue was used for the next step.
I. Rem ova l of ter t-Bu tyl Gr ou p s. To a stirred solution of
a tert-butyl ester (1 mmol) in CH2Cl2 (10 mL), CF3COOH (10
mL) was added. After the mixture was stirred for 1 h at room
temperature, the solvent was evaporated under reduced pres-
sure, and the residue was treated consecutively with petroleum
ether (bp 40-60 °C), Et2O, and dry Et2O. The residue was
purified by column chromatography using EtOAc as eluent
Com p ou n d s 10a ,b were synthesized from compounds 8a ,b
by procedure A.
Meth yl 4-[(2-h yd r oxyocta n oyl)a m in o]bu ta n oa te (10a ):
yield 92%; white solid; mp 68-69 °C; 1H NMR δ 6.75 (1H, t, J
) 7 Hz, NH), 4.04 (1H, m, CH), 3.67 (3H, s, COOCH3), 3.28
(2H, m, CH2NH), 3.19 (1H, br, OH), 2.37 (2H, t, J ) 7 Hz,
CH2COOCH3), 1.83 (4H, m, 2 × CH2), 1.65-1.20 (8H, m, 4 ×
CH2), 0.83 (3H, t, J ) 7 Hz, CH3); 13C NMR δ 174.1, 173.7,
72.2, 51.7, 38.4, 31.7, 31.0, 29.1, 24.9, 22.6, 14.1.
7 Hz, CH2CO), 2.38 (2H, t, J ) 7 Hz, CH2COOCH3), 1.88 (2H,
m, CH2CH2COOCH3), 1.58 (2H, m, CH2CH2CO), 1.25 (22H,
m, 11 × CH2), 0.83 (3H, t, J ) 7 Hz, CH3); 13C NMR δ 199.2,
173.4, 160.3, 51.7, 38.6, 36.5, 31.9, 31.5, 29.6, 29.5, 29.3, 29.0,
24.0, 22.6, 14.1.
4-(Hexa d eca n oyla m in o)bu tyr ic Acid (AX011). To a stir-
ring solution of 4-aminobutanoic acid (103 mg, 1 mmol) in THF
(5 mL) a solution of 1 N NaOH (1.1 mL, 1.1 mmol) was added
at 0 °C. Palmitoyl chloride (302 mg, 1.1 mmol) and 1 N NaOH
(1.1 mL, 1.1 mmol) were added, and the mixture was stirred
for 3 h at room temperature. After acidification with 1 N HCl,
the white solid formed was filtered and recrystallized (MeOH).
1
Yield 58%; white solid; mp 104-105 °C; H NMR (CD3OD) δ
7.84 (1H, m, NH), 3.30 (2H, m, CH2NH), 2.39 (2H, t, J ) 7
Hz, CH2COOH), 2.18 (2H, t, J ) 7 Hz, CH2CONH) 1.87 (2H,
m, CH2CH2COOH), 1.56 (2H, m, CH2CH2CO), 1.25 (24H, m,
12 × CH2), 0.88 (3H, t, J ) 7 Hz, CH3); 13C NMR δ 177.0, 174.6,
38.9, 35.9, 31.9, 30.9, 29.6, 29.3, 29.0, 24.2, 22.5, 14.1.
Com p ou n d s 13, 14a -c, a n d 15 were synthesized from
compounds 11a -d by procedures E and F.
Ben zyl (E,4S)-4-[(ter t-b u t oxyca r b on yl)a m in o]oct -2-
en oa te (13): yield 65%; colorless oil; [R]D -9.6 (c 1, CHCl3);
1H NMR δ 7.36 (5H, m, C6H5), 6.90 (1H, dd, J 1 ) 15.8 Hz, J 2
) 5.4 Hz, CHdCHCOO), 5.96 (1H, dd, J 1 ) 15.8 Hz, J 2 ) 1.4
Hz, CHdCHCOO), 5.18 (2H, s, CH2C6H5), 4.51 (1H, d, J ) 7.8
Hz, NH), 4.28 (1H, m, CHNH), 1.62-1.17 [15H, m, 3 × CH2,
C(CH3)3], 0.90 (3H, t, J ) 6.0 Hz, CH3); 13C NMR δ 166.4, 155.4,
149.6, 136.1, 128.7, 128.5, 120.4, 79.9, 66.5, 51.7, 34.6, 28.6,
28.0, 22.6, 14.1; MS (FAB) m/z (%) 348 (5) [M + H+].
Met h yl (E,4S)-4-[(ter t-b u t oxyca r b on yl)a m in o]oct -2-
en oa te (14a ): yield 75%; pale-yellow oil; [R]D -9.8 (c 0.8, CH3-
1
Cl); H NMR δ 6.83 (1H, dd, J 1 ) 15.4 Hz, J 2 ) 5.2 Hz, CHd
CHCOO), 5.89 (1H, dd, J 1 ) 15.4 Hz, J 2 )1.8 Hz, CHd
CHCOO), 4.64 (1H, d, J ) 8.0 Hz, NH), 4.24 (1H, m, CHNH),
3.70 (3H, s, OCH3), 1.53 (2H, m, CH2CHNH), 1.41 [9H, s,
C(CH3)3], 1.38-1.21 (4H, m, CH2CH2), 0.87 (3H, t, J ) 6.6 Hz,
CH3); 13C NMR δ 166.6, 155.0, 148.9, 119.9, 79.4, 51.4, 34.1,
28.1, 27.6, 22.2, 13.7.
Meth yl (E,4S)-4-[(ter t-bu toxyca r bon yl)a m in o]-5-p h e-
n ylp en t-2-en oa te (14b): yield 82%; white solid; mp 73-74
°C; [R]D +4.5 (c 1.0, CH3Cl); 1H NMR δ 7.20 (m, 5H, C6H5),
6.90 (1H, dd, J 1 ) 15.4 Hz, J 2 ) 4.8 Hz, CHdCHCOO), 5.87
(1H, dd, J 1 ) 15.4 Hz, J 2 ) 1.4 Hz, CHdCHCOO), 4.49-4.65
(2H, m, NH, CH), 3.70 (3H, m, OCH3), 2.89 (2H, m, CH2), 1.40
[s, 9H, C(CH3)3]; 13C NMR δ 166.5, 154.9, 147.7, 136.3, 129.5,
129.2, 128.7, 128.5, 126.9, 120.6, 79.8, 52.3, 51.6, 40.7, 28.3.
Meth yl 4-[(2-h yd r oxyh exa d eca n oyl)a m in o]bu ta n oa te
Meth yl (E,4S)-4-[(ter t-bu toxyca r bon yl)a m in o]-6-m eth -
ylh ep t-2-en oa te (14c): yield 85%; colorless oil; [R]D -17.0 (c
1
(10b): yield 67%; white solid; mp 90-91 °C; H NMR δ 6.79
(1H, t, J ) 7 Hz, NH), 4.05 (1H, m, CH), 3.67 (3H, s, COOCH3),
3.27 (3H, m, CH2NH, OH), 2.39 (2H, t, J ) 7 Hz, CH2-
COOCH3), 1.83 (4H, m, CH2CH2COOCH3, CH2CH), 1.65-1.20
(24H, m, 12 × CH2), 0.83 (3H, t, J ) 7 Hz, CH3); 13C NMR δ
174.3, 173.8, 72.2, 51.7, 38.4, 31.9, 31.3, 29.6, 24.9, 22.6, 14.1.
AX005 a n d AX006 were synthesized from compounds 10a ,b
by procedures B and C.
1
1.0, CH3Cl); H NMR δ 6.80 (1H, dd, J 1 ) 15.4 Hz, J 2 ) 4.8
Hz, CHdCHCOO), 5.87 (1H, dd, J 1 ) 15.4 Hz, J 2 ) 1.4 Hz,
CHdCHCOO), 4.57 (1H, d, J ) 8.6 Hz, NH), 4.30 (1H, m, CH),
3.70 (3H, m, OCH3), 1.67 (2H, m, CH2CH), 1.20-1.45 [m, 10H,
CH(CH3)2, C(CH3)3], 0.90 [6H, d, J ) 6.6 Hz, CH(CH3)2]; 13C
NMR δ 166.8, 155.0, 149.2, 119.8, 79.6, 51.5, 49.6, 43.6, 28.3,
24.6, 22.6, 22.1.
4-[(2-Oxoocta n oyl)a m in o]bu ta n oic a cid (AX005): yield
68%; white solid; mp 79-80 °C; 1H NMR δ 7.22 (1H, m, NH),
3.38 (2H, m, CH2NH), 2.91 (2H, t, J ) 7 Hz, CH2CO), 2.39
(2H, t, J ) 7 Hz, CH2COOH), 1.88 (2H, m, CH2CH2COOH),
1.58 (2H, m, CH2CH2CO), 1.25 (6H, m, 3 × CH2), 0.83 (3H, t,
J ) 7 Hz, CH3); 13C NMR δ 199.2, 178.4, 160.4, 38.5, 36.7,
31.5, 31.2, 28.6, 24.1, 22.4, 13.9.
ter t-Bu tyl (E,4S)-4-[(ben zyloxyca r bon yl)a m in o]p en t-
2-en oa te (15): yield 31%; white solid; [R]D -11.7 (c 1.8, CH3-
Cl); 1H NMR δ 7.34 (5H, m, C6H5), 6.76 (1H, dd, J 1 ) 15.6 Hz,
J 2 ) 5.0 Hz, CHdCHCOO), 5.73 (1H, dd, J 1 ) 15.6 Hz, J 2
)
1.4 Hz, CHdCHCOO), 5.10 (2H, s, OCH2), 4.96 (1H, d, J )
8.0 Hz, NH), 4.42 (1H, m, CHNH), 1.47 [s, 9H, C(CH3)3], 1.26
(3H, d, J ) 6.8 Hz, CH3); 13C NMR δ 165.5, 155.4, 147.5, 136.2,
128.4, 128.1, 122.0, 80.5, 66.8, 47.4, 28.0, 27.9, 20.2.
4-[(2-Oxoh exa d eca n oyl)a m in o]bu ta n oic a cid (AX006):
1
yield 63%; white solid; mp 113-114 °C; H NMR δ 7.20 (1H,
Ben zyl (E,4S)-4-[(2-Hyd r oxyd od eca n oyl)a m in o]oct-2-
en oa te (16). Compound 16 was prepared from compound 13
by procedure G (using 4 N HCl in Et2O instead of 4 N HCl in
m, NH), 3.36 (2H, m, CH2NH), 2.90 (2H, t, J ) 7 Hz, CH2CO),
2.39 (2H, t, J ) 7 Hz, CH2COOH), 1.87 (2H, m, CH2CH2-
COOH), 1.56 (2H, m, CH2CH2CO), 1.25 (22H, m, 11 × CH2),
0.83 (3H, t, J ) 7 Hz, CH3); 13C NMR δ 199.2, 178.3, 160.4,
38.5, 36.4, 31.9, 30.9, 29.6, 29.3, 29.0, 24.2, 22.6, 14.1.
1
MeOH) followed by procedure A. Yield 75%; colorless oil; H
NMR δ 7.35 (5H, m, C6H5), 6.90 (1H, dd, J 1 ) 15.6 Hz, J 2
5.4 Hz, CHdCHCOO), 6.65 (1H, m, NH), 5.92 (1H, dd, J 1
)
)
Meth yl 4-[(2-Oxoh exa d eca n oyl)a m in o]bu ta n oa te (AX-
010). AX010 was synthesized from 10b by procedure D. Yield
85%; white solid; mp 70-71 °C; 1H NMR δ 7.18 (1H, m, NH),
3.67 (3H, s, COOCH3), 3.45 (2H, m, CH2NH), 2.90 (2H, t, J )
15.6 Hz, J 2 ) 1.8 Hz, CHdCHCOO), 5.16 (2H, s, CH2C6H5),
4.62 (1H, m, CHNH), 4.13 (1H, m, CH), 1.90-1.11 (24H, m,
12 × CH2), 0.88 (3H, t, J ) 6.0 Hz, CH3); 13C NMR δ 173.4,

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3625
166.0, 148.4, 135.7, 128.6, 128.3, 120.5, 72.2, 66.4, 49.5, 34.9,
34.7, 33.9, 31.9, 29.5, 29.3, 27.7, 25.0, 24.9, 22.6, 22.4, 22.3,
14.1, 13.8.
Com p ou n d s 17a -e were synthesized from compounds
14a -c by procedures H, G, and A.
mp 112-113 °C; [R]D -0.7 (c 0.7, CH3Cl); 1H NMR δ 6.93 (1H,
d, J ) 9 Hz, NH), 4.03 (1H, m, CH), 2.91 (2H, t, J ) 7.8 Hz,
CH2COCO), 2.39 (2H, m, CH2COOH), 1.85 (2H, m, CH2), 1.59
(2H, m, CH2), 1.25 (23H, m, 10 × CH2, CH3), 0.88 (3H, t, J )
6.7 Hz, CH3); 13C NMR δ 199.4, 178.3, 159.7, 45.1, 36.7, 31.9,
31.3, 30.7, 29.6, 29.4, 29.3, 29.0, 23.1, 22.7, 20.7,14.1; MS (FAB)
m/z (%) 356 (100) [M+ + H].
Met h yl (4S)-4-[(2-h yd r oxyp en t a d eca n oyl)a m in o]oc-
1
ta n oa te (17a ): yield 75%; yellow oil; H NMR δ 6.39 (1H, m,
AX012, AX013, AX016, AX017, a n d AX024 were synthe-
sized from compounds 17a -e by procedures B and C.
(4R)-6-Meth yl-4-[(2-oxopen tadecan oyl)am in o]h eptan o-
ic a cid (AX012): yield 67%; white solid; mp 58-59 °C; [R]D
-9.0 (c 1.28 CH3Cl); 1H NMR δ 6.95 (1H, d, J ) 9.2 Hz, NH),
4.00 (1H, m, CH), 2.91 (2H, t, J ) 7.8 Hz, CH2COCO), 2.30
(2H, m, CH2COOH), 1.91 [3H, m, CH2, CH(CH3)2], 1.59 (4H,
m, 2 × CH2), 1.25 (20H, m, 10 × CH2), 0.91 (9H, t, J ) 6.8 Hz,
3 × CH3); 13C NMR δ 199.3, 178.8, 160.0, 47.3, 44.1, 36.8, 31.8,
31.1, 30.4, 29.5, 29.3, 29.2, 28.9, 24.7, 23.0, 22.9, 22.5, 21.9,
13.9; MS (FAB) m/z (%) 398 (100) [M+ + H].
(4R)-6-Met h yl-4-[(2-oxod od eca n oyl)a m in o]h ep t a n oic
a cid (AX013): yield 83%; white solid; mp 55-56 °C; [R]D -9.5
(c 1.3 CH3Cl); 1H NMR δ 6.83 (1H, d, J ) 9 Hz, NH), 4.02
(1H, m, CH), 2.91 (2H, t, J ) 7.8 Hz, CH2COCO), 2.36 (2H, m,
CH2COOH), 1.91 [3H, m, CH2, CH(CH3)2], 1.59 (4H, m, 2 ×
CH2), 1.25 (14H, m, 7 × CH2), 0.91 (9H, t, J ) 6.8 Hz, 3 ×
CH3); 13C NMR δ 199.4, 178.4, 160.1, 47.4, 44.3, 36.8, 31.8,
30.7, 30.4, 29.5, 29.4, 29.3, 29.2, 29.0, 24.9, 23.2, 22.9, 22.6,
22.0, 14.1; MS (FAB) m/z (%) 400 (100) [M+ + 2Na - 1], 378
(46) [M+ + Na].
NH), 4.08 (2H, m, 2 × CH), 3.66 (3H, s, OCH3), 2.95 (1H, br,
OH), 2.34 (2H, m, CH2COO), 1.97-1.42 (6H, m, 3 × CH2),
1.38-1.15 (26H, m, 13 × CH2), 0.98-0.81 (6H, m, 2 × CH3);
13C NMR δ 174.2, 173.8, 72.1, 51.7, 48.7, 35.0, 31.9, 30.8, 30.2,
29.6, 29.5, 29.4, 29.3, 27.9, 25.0, 22.6, 22.5, 14.0, 13.9.
Meth yl (4R)-4-[(2-h yd r oxyocta n oyl)a m in o]-5-p h en yl-
1
p en ta n oa te 17b): yield 80%; white solid; mp 75-77 °C; H
NMR δ 7.23 (5H, m, C6H5), 6.31 (1H, d, J ) 8.4 Hz, NH), 4.20
(1H, m, CH), 4.01 (1H, m, CH), 3.65 (3H, s, OCH3), 2.82-2.64
(3H, m, CH2C6H5, OH), 2.35 (2H, m, CH2COO), 2.02-1.15
(12H, m, 6 × CH2), 0.87 (3H, t, J ) 6.6 Hz, CH3); 13C NMR δ
174.0, 173.6, 137.4, 129.2, 128.6, 126.5, 72.0, 51.7, 49.7, 41.4,
35.0, 31.6, 30.9, 29.0, 24.8, 22.5, 14.0.
Met h yl (4R)-4-[(2-h yd r oxyp en t a d eca n oyl)a m in o]-5-
p h en ylp en ta n oa te (17c): yield 86%; white solid; mp 53-55
°C; 1H NMR δ 7.23 (5H, m, C6H5), 6.47 (1H, m, NH), 4.20 (1H,
m, CH), 4.01 (1H, m, CH), 3.65 (3H, s, OCH3), 2.90 (1H, br,
OH), 2.81 (2H, m, CH2C6H5), 2.35 (2H, m, CH2COO), 2.02-
1.42 (4H, m, 2 × CH2), 1.38-1.12 (22H, m, 11 × CH2), 0.88
(3H, t, J ) 6.6 Hz, CH3); 13C NMR δ 174.0, 173.8, 137.4, 129.3,
128.4, 126.6, 72.1, 51.7, 49.7, 41.3, 34.9, 31.9, 30.9, 29.6, 29.5,
29.4, 29.3, 29.0, 24.9, 22.6, 14.1.
(4R)-4-[(2-Oxopen tadecan oyl)am in o]-5-ph en ylpen tan o-
ic a cid (AX016): yield 82%; white solid; mp 91-92 °C; [R]D
-6.1 (c 0.8, CH3Cl); 1H NMR δ 7.33-7.14 (5H, m, C6H5), 6.97
(1H, d, J ) 9 Hz, NH), 4.20 (1H, m, CH), 2.86 (4H, m, CH2C6H5,
CH2COCO), 2.37 (2H, t, J ) 7.2 Hz, CH2COOH), 1.95 (1H, m,
CHH), 1.81 (1H, m, CHH), 1.56 (2H, m, CH2), 1.26 (20H, m,
10 × CH2), 0.89 (3H, t, J ) 6.8 Hz, CH3); 13C NMR δ 199.2,
178.0, 160.0, 136.9, 129.2, 128.6, 126.8, 50.3, 41.1, 36.7, 31.9,
30.7, 29.6, 29.4, 29.3, 29.0, 28.9, 23.1, 22.7, 14.1; MS (FAB)
m/z (%) 476 (65) [M+ + 2Na - 1], 454 (30) [M+ + Na].
(4R)-4-[(2-Oxooctan oyl)am in o]-5-ph en ylpen tan oic acid
(AX017): yield 75%; pale-yellow solid; mp 94-96 °C; [R]D -8.7
(c 0.24, CH3Cl); 1H NMR δ 7.38-7.14 (5H, m, C6H5), 6.95 (1H,
d, J ) 9 Hz, NH), 4.20 (1H, m, CH), 2.86 (4H, m, CH2C6H5,
CH2COCO), 2.37 (2H, t, J ) 7.4 Hz, CH2COO), 1.97 (1H, m,
CHH), 1.78 (1H, m, CHH), 1.57 (2H, m, CH2), 1.26 (6H, m, 3
× CH2), 0.87 (3H, t, J ) 6.8 Hz, CH3); 13C NMR δ 199.2, 177.6,
160.0, 136.9, 128.7, 128.1, 127.0, 50.3, 41.1, 36.7, 31.5, 28.7,
26.3, 22.4, 14.0; MS (FAB) m/z (%) 334 (57) [M+ + H].
(4S)-4-[(2-Oxop en t a n od eca n oyl)a m in o]oct a n oic a cid
(AX024): yield 56%; white solid; mp 74-75 °C; [R]D -1.6 (c
0.6, CH3Cl); 1H NMR δ 6.85 (1H, d, J ) 9.8 Hz, NH), 3.93
(1H, m, CH), 2.92 (2H, t, J ) 7.4 Hz, CH2COCO), 2.37 (2H, t,
J ) 7.1 Hz, CH2COO), 2.03-1.44 (6H, m, 3 × CH2), 1.42-
1.17 (24H, m, 12 × CH2), 0.91-0.83 (6H, t, J ) 6.3 Hz, 2 ×
CH3); 13C NMR δ 199.4, 178.1, 160.1, 49.3, 36.8, 34.7, 31.9,
30.8, 30.0, 29.6, 29.4, 29.3, 29.0, 28.0, 23.2, 22.7, 22.4, 14.1,
13.9; MS (FAB) m/z (%) 398 (100) [M+ + H].
Meth yl (4R)-4-[(2-Hyd r oxyd od eca n oyl)a m in o]-6-m eth -
ylh ep ta n oa te (17d ): yield 65%; yellow oil; 1H NMR δ 6.35
(1H, m, NH), 4.04 (2H, m, 2 × CH), 3.65 (3H, s, OCH3), 3.22
(1H, br, OH), 2.34 (2H, t, J ) 7.6 Hz, CH2COO), 1.47-1.95
(5H, m, 2 × CH2, CH), 1.40-1.18 (18H, m, 9 × CH2), 0.95-
0.78 (9H, m, 3 × CH3); 13C NMR δ 174.1, 173.8, 72.0, 51.6,
46.8, 46.6, 44.7, 44.5, 34.9, 31.9, 30.8, 30.6, 29.6, 29.5, 29.4,
25.1, 24.9, 23.0, 22.6, 22.0, 14.0.
Met h yl (4R)-4-[(2-h yd r oxyp en t a d eca n oyl)a m in o]-6-
1
m eth ylh ep ta n oa te (17e): yield 63%; yellow oil; H NMR δ
6.37 (1H, m, NH), 4.04 (2H, m, 2 × CH), 3.65 (3H, s, OCH3),
3.15 (1H, br, OH), 2.34 (2H, t, J ) 7.8 Hz, CH2COO), 1.95-
1.47 (5H, m, 2 × CH2, CH), 1.40-1.18 (24H, m, 12 × CH2),
0.95-0.81 (9H, m, 3 × CH3); 13C NMR δ 174.0, 173.8, 72.1,
51.6, 46.8, 46.6, 44.7, 44.5, 34.9, 31.9, 30.8, 30.6, 29.6, 29.5,
29.4, 29.3, 25.0, 24.9, 23.0, 22.6, 22.1, 14.0.
ter t-Bu tyl (4S)-4-[(2-Hyd r oxyp en ta d eca n oyl)a m in o]-
p en ta n oa te (18). Compound 18 was synthesized from com-
pound 15 by procedure H (using EtOH instead of MeOH)
followed by procedure A (using 2.2 mmol of Et3N instead of
1
4.4 mmol of Et3N). Yield 92%; wax; H NMR δ 6.48 (1H, m,
NH), 4.12-3.90 (2H, m, 2 × CH), 3.05 (1H, br, OH), 2.27 (2H,
t, J ) 7.8 Hz, CH2CO2), 1.98-1.52 (4H, m, 2 × CH2), 1.44 [9H,
s, C(CH3)3], 1.40-1.21 (22H, m, 11 × CH2), 1.16 (3H, d, J )
6.6 Hz, CH3), 0.88 (3H, t, J ) 6.6 Hz, CH3); 13C NMR δ 173.5,
173.0, 80.6, 72.0, 44.8, 35.0, 32.3, 31.9, 31.6, 29.6, 29.5, 29.4,
29.3, 28.0, 24.9, 22.6, 21.0, 14.1.
AX018 a n d AX019 were synthesized from Boc-L-prolinol by
procedures E-H and A-C.
(4S)-4-[(2-Oxododecan oyl)am in o]octan oic Acid (AX007).
AX007 was synthesized from compound 16 by procedure H
(using EtOH instead MeOH) followed by procedure C. Yield
(S)-3-[1-(2-Oxop en t a d eca n oyl)p yr r olid in -2-yl]p r op i-
on ic a cid (AX018): Pale-yellow solid; mp 55-56 °C; [R]D -42.7
(c 1.0, CH3Cl); 1H NMR δ 4.21 (1H, m, CH), 3.54 (2H, m, CH2-
NH), 2.84 (2H, m, CH2COCO), 2.35 (2H, m, CH2COOH), 2.01-
1.41 (8H, m, 4 × CH2), 1.25 (20H, m, 10 × CH2), 0.86 (3H, t,
J ) 6.6 Hz, CH3); 13C NMR δ 201.0, 177.9, 163.6, 57.1, 47.2,
39.2, 31.8, 31.1, 29.6, 29.4, 29.3, 29.1, 29.0, 28.4, 24.2, 22.9,
22.6, 14.1; MS (FAB) m/z (%) 426 (100) [M+ + 2Na - 1], 404
(27) [M+ + Na].
1
57%; white solid; mp 50-52 °C; [R]D -1.8 (c 0.5, CH3Cl); H
NMR δ 6.86 (1H, d, J ) 9.4 Hz, NH), 3.90 (1H, m, CHNH),
2.90 (2H, t, J ) 7.8 Hz, CH2COCO), 2.35 (2H, t, J ) 6.3 Hz,
CH2COO), 2.01-1.05 (24H, m, 12 × CH2), 0.87 (3H, t, J ) 6.0
Hz, CH3); 13C NMR δ 199.4, 178.5, 160.1, 49.3, 36.8, 34.7, 31.8,
30.7, 29.8, 29.5, 29.3, 29.2, 29.0, 27.9, 23.1, 22.6, 22.4, 14.1,
13.9; MS (FAB) m/z (%) 378 (35) [M+ + Na], 356 (45) [M+
H].
+
(S)-3-[1-(2-Oxod od eca n oyl)p yr r olid in -2-yl]p r op ion ic
1
AX008 was synthesized in a similar way to AX007. The
spectroscopic data were identical to those obtained for the (S)-
enantiomer. [R]D -1.6 (c 0.5, CH3Cl).
(4S)-4-[(2-Oxop en t a d eca n oyl)a m in o]p en t a n oic Acid
(AX014). AX014 was synthesized from compound 18 by
procedure D followed by procedure I. Yield 63%; white solid;
a cid (AX019): yellow oil; [R]D -37.7 (c 0.5, CH3Cl); H NMR
δ 4.21 (1H, m, CH), 3.54 (2H, m, CH2NH), 2.84 (2H, m, CH2-
COCO), 2.35 (2H, m, CH2COOH), 2.01-1.41 (8H, m, 4 × CH2),
1.25 (14H, m, 7 × CH2), 0.86 (3H, t, J ) 7 Hz, CH3); 13C NMR
δ 201.0, 177.6, 164.1, 57.1, 47.2, 39.3, 31.9, 31.2, 29.5, 29.4,
29.3, 29.1, 28.6, 24.2, 22.9, 22.6, 14.1.

3626 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-d ecyloxyp r o-
p a n oic Acid (20). To a solution of 19 (20 g, 100 mmol) in DMF
(500 mL), NaH (8.2 g, 220 mmol) was added at 0 °C.
Iododecane (24 mL, 110 mmol) was added when the H2 release
had finished. The reaction mixture was stirred for 5 h at room
temperature. The organic solvent was evaporated under
reduced pressure, and the product was extracted with brine.
The product was purified by column chromatography (CHCl3,
CHCl3-MeOH, 9:1). Yield 68%; oil; [R]D +5.5 (c 1.75, CHCl3);
1H NMR δ 4.88 (H, m, OCOΝΗ), 4.23 (1H, m, CHNH), 3.69
(2H, d, CHCH2O, J ) 13.6 Hz), 3.43 (2H, t, OCH2 J ) 4.8),
1.52-1.21 (25H, m, 8 × CH2C(CH3)3), 0.88 (3H, t, J ) 6.6 Hz,
CH3); 13C NMR δ 176.1, 154.7, 79.0, 71.3, 54.0, 36.4, 31.2, 28.9,
28.6, 27.7, 27.6, 24.4, 22.4, 22.0, 14.0.
J . P h osp h olip a se A2 Activity Assa ys. Phospholipase A2
assays were performed as described in the Supporting Infor-
mation of our initial study.17 Unless otherwise indicated, the
phospholipid/detergent mixed micelles were composed of sub-
strate, PAPC, PIP2, and the detergent TX-100 at a ratio of 97:
3:400 (µM), respectively. Where indicated, the mole fraction
of each of the 2-oxoamide compounds was varied within
otherwise identical phospholipid substrate micelles and was
plotted against the percent inhibition of GIVA PLA2 activity
relative to a control with dimethyl sulfoxide (DMSO) present.
K. Cell Ma in ten a n ce. P388D1 murine macrophages were
maintained at 37 °C in a humidified 10% CO2 atmosphere.
Cells were grown in Iscove’s modified Dulbecco’s medium
(IMDM) with 10% fetal calf serum (HyClone Labs, Provo, UT),
100 units/mL penicillin, and 100 mg/mL streptomycin (Invit-
rogen, Carlsbad, CA). Cells were routinely passaged every 2-3
days when the cells reached 75-80% confluence. Cells used
for stimulation were plated at a density of 106 cells per well
in standard 12-well tissue culture plates and were allowed to
adhere overnight.
L. Cell Stim u la tion . The standard regimen for treatment
of the P338D1 cells with either LPS alone or LPS combined
with PAF has been previously described.37-40 Briefly cells were
washed twice with starvation media (IMDM containing 0.2%
fetal calf serum, 100 units/mL penicillin, and 100 mg/mL
streptomycin) and then incubated with 1 mL of starvation
media for 1 h. Cells were then exposed to either 100 ng/mL
LPS for 18 h or 200 ng/mL LPS for 1 h. Cells treated with 200
ng/mL LPS were then exposed to 100 nM PAF for 15 min. At
the end of the stimulation, the medium was removed from the
cells, cleared of cellular debris by centrifugation, and analyzed
for PGE2 levels by enzyme-linked immunoassay (Cayman
Chemical, Ann Arbor, MI). Inhibitor compounds were dissolved
in DMSO and diluted into starvation media prior to addition
to cells; the DMSO concentration was kept below 0.5% v/v in
all studies. All inhibitors were added 30 min prior to stimula-
tion.
M. In h ibition of Ca r r a geen a n -In d u ced Ed em a . Edema
was induced in the right hind paw of Fisher 344 rats (150-
200 g) by the intradermal injection of 0.1 mL of 2% w/v
carrageenan (K100, commercially available) in sterilized saline
into the right footpad.41 Both sexes were used. Pregnant
females were excluded. The animals, bred in our laboratory,
were housed under standard conditions and received a diet of
commercial food pellets and water ad libitum. During the
experiment period, food, but not water, was withheld. These
studies were in accordance with recognized guidelines on
animal experimentation (Guidelines for the Care and Use of
Laboratory Animals, published by the Greek Goverment 160/
1991, based on EU regulations 86/609).
The tested compounds were suspended in a few drops of
Tween 80, ground in a mortar before use, diluted dropwise
with water to the appropriate concentrations, and were given
intraperitoneally (ip) at the same time as the carrageenin. The
rats were euthanized 3.5 h after carrageenin injection. The
experiment was repeated twice for each compound (two groups
of five animals) at four different concentrations. The difference
between the weight of the injected and uninjected paws was
calculated for each animal. The change in paw weight was
compared with that in control animals (injected with water)
and expressed as a percent inhibition of the edema (CPE %
values). Indomethacin in 0.01 mmol/kg (47%) was adminis-
tered as a standard comparative drug. Values for CPE % are
the mean from two different experiments with a standard error
of the mean less than 10%. Statistical studies were made with
Student’s t-test.
N. An a lgesic Scr een . The writhing reflex was used. To a
group of seven Fischer rats (150-200 g) compounds tested for
anti-inflammatory activity were administered ip with a dose
equivalent to the ED50 30 min prior to the ip administration
of 1 mL/100 g of body weight of 0.6% acetic acid.42 Sodium
acetylsalicylate was administered ip as a standard drug. After
5 min, the number of stretches characterized by repeated
contractions of the abdominal musculature accompanied by
(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-(d ecyloxy)p r o-
p a n ol (21). To a stirred solution of 20 (0.34 g, 1.0 mmol) in
dry THF (5 mL) at -10 °C, NMM (0.11 mL, 1.0 mmol) was
added, followed by ClCOOEt (0.096 mL, 1.0 mmol). After 10
min, NaBH4 (0.11 g, 3.0 mmol) was added in one portion.
MeOH (10 mL) was then added dropwise to the mixture over
a period of 10 min at 0 °C. The solution was stirred for an
additional 10 min and then neutralized with 1 Μ KHSO4. The
organic solvents were evaporated under reduced pressure, and
the product was extracted with EtOAc (3 × 7 mL). The organic
phase was washed with 1 Μ KHSO4 (5 mL), Η2Ã (10 mL), 5%
NaHCO3 (5 mL), and Η2Ã (10 mL) and dried, and the solvent
was evaporated under reduced pressure. The residue was
purified by crystallization with Et2O/petroleum ether. Yield
75%; oil; [R]D +6.4 (c 1, CHCl3); 1H NMR δ 4.21 (1H, m,
OCONH), 3.82-3.25 (7H, m, 2 × CH2O, CH2OH, CΗNH),
1.69-1.08 (25H, m, 8 × CH2, C(CH3)3), 0.88 (3H, t, J ) 6.6
Hz, CH3); 13C NMR δ 156.2, 79.6, 71.8, 64.4, 51.4, 31.8, 29.4,
29.3, 28.4, 28.3, 26.0, 22.6, 14.1; MS (FAB): m/z (%) 332 (45)
[Μ+ + H].
Ben zyl (E,4R)-4-[(ter t-Bu t oxyca r b on yl)a m in o]-5-d ec-
yloxyp en t-2-en oa te (22). Compound 22 was synthesized
from compound 21 by procedures E and F. Yield 52%; oil; [R]D
-2.3 (c 1, AcOEt); 1H NMR δ 7.37 (5H, C6H5), 6,97 (1H, dd, J
) 14.6 Hz, J ) 4.2 Hz, CHdCHCOOBn), 6.03 (1H, dd, J )
14.6 Hz, J ) 1.4 Hz, CHdCHCOOBn), 4.18 (s, 2H, CH2C6H5),
4.00 (1H, d, J ) 7.0 Hz, OCONH), 4.45 (1H, m, CHNH), 3.51
(2H, d, CHCH2O, J ) 4.4 Hz), 3.41 (2H, t, CH2CH2O, J ) 6,4
Hz), 1.62-1.16 (25H, m, 8’CH2., C(CH3)3), 0.9 (3H, t, J ) 6.0
Hz, CH3); 13C NMR δ 166.0, 154.8, 146.9, 139.8, 128.3, 126.8,
121.4, 79.6, 71.6, 66.2, 51.4, 31.8, 29.4, 29.2, 28.4, 28.3, 26.0,
22.6, 14.1.
Ben zyl (E,4R)-5-(Decyloxy)-4-[(2-h yd r oxyd od eca n oyl)-
a m in o]p en t-2-en oa te (23). Compound 23 was prepared from
22 by procedure G (using 4 N HCl in Et2O instead of 4 N HCl
in MeOH) followed by procedure A. Yield 65%; oil; 1H NMR δ
7.36 (5H, C6H5),7.02-6.90 (2H, m, CONH, CHdCHCOOBn),
6.00 (1H, d, J ) 14.6 Hz, CHdCHCOOBn), 4.17 (2H, s,
CH2C6H5), 4.81 (1H, m, CHNH), 4.14 (1H, m, CHOH), 3.54
(2H, d, CHCH2O, J ) 4.0 Hz), 3.40 (2H, t, CH2CH2O, J ) 6,2
Hz), 1.81-1.08 (34H, m, 17 × CH2), 0.88 (3H, t, J ) 6.0 Hz,
CH3); 13C NMR δ 173.4, 157.6, 146.0, 134.7, 128.4, 128.3, 121.9,
72.2, 71.7, 71.4, 66.6, 49.6, 31.8, 29.6, 29.4, 29.2, 24.9, 22.6,
14.1; MS (FAB) m/z (%) 560 (20) [Μ+ + H].
(4R)-5-(Decyloxy)-4-[(2-oxododecan oyl)am in o]pen tan o-
ic Acid (AX027). AX027 was synthesized from compound 23
by procedure H (using EtOH instead MeOH) followed by
procedure C. Yield 46%; white solid; mp 54-56 °C; [R]D -16.2
1
(c 0.5, CHCl3); H NMR δ 7.24 (1H, d, CONH, J ) 9.,4 Hz),
4.08 (1H, m, CHNH), 3.42 (4H, m, 2’CH2O), 2.90 (2H, t, CH2-
COCO, J ) 7.8 Hz), 2.39 (2H, t, CH2COOH, J ) 7.0 Hz), 1.99
(2H, m, CH2CH2COOH), 1.58 (4H, m, CH2CH2COCO, CH2-
CH2O), 1.26 (28H, m, 14 × CH2), 0.88 (3H, t, J ) 6,0 Hz, CH3);
13C NMR δ 199.0, 178.4, 160.0, 71.6, 69.7, 48.7, 36.8, 31.8, 29.6,
29.4, 29.3, 29.0, 26.0, 23.1, 22.6, 14.1; MS (FAB) m/z (%) 470
(15) [Μ+ + H].
AX026 was synthesized starting from D-serine in a similar
way as AX027. The spectroscopic data were identical to those
obtained for the (S)-enantiomer. [R]D +15.6 (c 0.5, CH3Cl).

Inhibition of Phospholipase A2 by 2-Oxoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3627
(8) Ghomashchi, F.; Loo, R.; Balsinde, J .; Bartoli, F.; Apitz-Castro,
R.; Clark, J . D.; Dennis, E. A.; Gelb, M. H. Trifluoro-
methyl ketones and methyl fluorophosphonates as inhibitors of
group IV and VI phospholipases A2: structure-function studies
with vesicle, micelle, and membrane assays. Biochim. Biophys.
Acta 1999, 1420, 45-56.
extension of the hind limbs was counted every 5 min for the
next 30 min. The total number of writhes exhibited by each
animal in the test group was recorded and compared to that
of a vehicle-treated control group. The percent analgesic
activity (aa) was calculated as
(9) Bonventre, J . V.; Huang, Z.; Taheri, M. R.; O’Leary, E.; Li, E.;
Moskowitz, M. A.; Sapirstein, A. Reduced fertility and postis-
chaemic brain injury in mice deficient in cytosolic phospholipase
A2. Nature 1997, 390, 622-625.
n - n′
n
% aa )
× 100
(10) Klivenyi, P.; Beal, M. F.; Ferrante, R. J .; Andreassen, O. A.;
Wermer, M.; Chin, M. R.; Bonventre, J . V. Mice deficient in
group IV cytosolic phospholipase A2 are resistant to MPTP
neurotoxicity. J . Neurochem. 1998, 71, 2634-2637.
where n is the average number of writhes of the control group
and n′ is the average number of writhes of the test group.
(11) Uozumi, N.; Kume, K.; Nagase, T.; Nakatani, N.; Ishii, S.;
Tashiro, F.; Komagata, Y.; Maki, K.; Ikuta, K.; Ouchi, Y.;
Miyazaki, J .; Shimizu, T. Role of cytosolic phospholipase A2 in
allergic response and parturition. Nature 1997, 390, 618-622.
(12) Fujishima, H.; Sanchez Mejia, R. O.; Bingham, C. O., III; Lam,
B. K.; Sapirstein, A.; Bonventre, J . V.; Austen, K. F.; Arm, J . P.
Cytosolic phospholipase A2 is essential for both the immediate
and the delayed phases of eicosanoid generation in mouse bone
marrow-derived mast cells. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 4803-4807.
Ack n ow led gm en t. We thank Dr. Ruth Kramer of
Lilly Research for a generous gift of human Group IVA
PLA2. We thank Dr. C. Hansch (Biobyte Corp.) for
providing the C-QSAR program. This work was sup-
ported by NIH Grants GM 20501 and GM 064611
(E.A.D.). D.A.S. was supported by NIH Training Grant
DK07202.
(13) Uozumi, N.; Shimizu, T. Roles for cytosolic phospholipase A2R
as revealed by gene-targeted mice. Prostaglandins Other Lipid
Mediators 2002, 68-69, 59-69.
(14) Nagase, T.; Uozumi, N.; Ishii, S.; Kume, K.; Izumi, T.; Ouchi,
Y.; Shimizu, T. Acute lung injury by sepsis and acid aspiration:
a key role for cytosolic phospholipase A2. Nat. Immunol. 2000,
1, 42-46.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
results for the compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
Ap p en d ix
(15) Song, H.; Lim, H.; Paria, B. C.; Matsumoto, H.; Swift, L. L.;
Morrow, J .; Bonventre, J . V.; Dey, S. K. Cytosolic phospholipase
A2R is crucial for “on-time” embryo implantation that directs
subsequent development. Development 2002, 129, 2879-2889.
(16) Serhan, C. N. Preventing injury from within, using selective
cPLA2 inhibitors. Nat. Immunol. 2000, 1, 13-15.
(17) Kokotos, G.; Kotsovolou, S.; Six, D. A.; Constantinou-Kokotou,
V.; Beltzner, C. C.; Dennis, E. A. Novel 2-oxoamide inhibitors
of Group IVA phospholipase A2. J . Med. Chem. 2002, 45, 2891-
2893.
(18) Street, I. P.; Lin, H.-K.; Laliberte, F.; Ghomaschi, F.; Wang, Z.;
Perrier, H.; Tremblay, N. M.; Huang, Z.; Weeck, P. K.; Gelb, M.
H. Slow- and tight-binding inhibitors of the 85-kDa human
phospholipase A2. Biochemistry 1993, 32, 5935-5940.
(19) Trimble, L. A.; Street, I. P.; Perrier, H.; Tremblay, N. M.; Weech,
P. K.; Berstein, M. A. NMR structural studies of the tight
complex between a trifluoromethyl ketone inhibitor and the 85-
kDa human phospholipase A2. Biochemistry 1993, 32, 12560-
12565.
(20) Huang, Z.; Payette, P.; Abdullah, K.; Cromlish, W. A.; Kennedy,
B. P. Functional identification of the active-site nucleophile of
the human 85-kDa cytosolic phospholipase A2. Biochemistry
1996, 35, 3712-3721.
Abbr eviation s. AA, arachidonic acid; AATFK, arachi-
donoyl trifluoromethyl ketone; AcNH-TEMPO, 4-acet-
amido-2,2,6,6-tetramethylpiperidine-1-yloxy free radi-
cal; Bn, benzyl; Boc, tert-butoxycarbonyl; DMF, N,N′-
dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc,
ethyl acetate; GABA, γ-aminobutyric acid; GIVA PLA2,
human Group IVA cytosolic phospholipase A2; HOBt,
1-hydroxybenzotriazole; IMDM, Iscove’s modified Dul-
becco’s medium; ip, intraperitoneally; LPS, lipopolysac-
charide; MAFP, methylarachidonoyl fluorophosphonate;
NMM, N-methylmorpholine; PAF, platelet activating
factor; PAPC, 1-palmitoyl-2-arachidonoylphosphatidyl-
choline; PDC, pyridinium dichromate; PGE2, prostag-
landin E2; PIP2, phosphatidylinositol 4,5-bisphosphate;
PLA2, phospholipase A2; THF, tetrahydrofuran; TX-100,
Triton X-100; WSCI, 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide; Z, benzyloxycarbonyl.
(21) Conde-Frieboes, K.; Reynolds, L. J .; Lio, Y.-C.; Hale, M. R.;
Wasserman, H. H.; Dennis, E. A. Activated ketones as inhibitors
of intracellular Ca2+-dependent and Ca2+-independent phospho-
lipase A2. J . Am. Chem. Soc. 1996, 118, 5519-5525.
(22) Removed in proofs.
(23) Lehr, M. Structure-activity relationships of (4-acylpyrrol-2-yl)-
alkanoic acids as inhibitors of the cytosolic phospholipase A2:
variation of the substitutents in positions 1, 3, and 5. J . Med.
Chem. 1997, 40, 3381-3392.
Refer en ces
(1) Six, D. A.; Dennis, E. A. The expanding superfamily of phos-
pholipase A2 enzymes: classification and characterization. Bio-
chim. Biophys. Acta 2000, 1488, 1-19.
(2) Pickard, R. T.; Chiou, X. G.; Strifler, B. A.; DeFelippis, M. R.;
Hyslop, P. A.; Tebbe, A. L.; Yee, Y. K.; Reynolds, L. J .; Dennis,
E. A.; Kramer, R. M.; Sharp, J . D. Identification of essential
residues for the catalytic function of 85-kDa cytosolic phospho-
lipase A2. Probing the role of histidine, aspartic acid, cysteine,
and arginine. J . Biol. Chem. 1996, 271, 19225-19231.
(3) Dessen, A.; Tang, J .; Schmidt, H.; Stahl, M.; Clark, J . D.; Seehra,
J .; Somers, W. S. Crystal structure of human cytosolic phospho-
lipase A2 reveals a novel topology and catalytic mechanism. Cell
1999, 97, 349-360.
(24) Lehr, M.; Klimt, M.; Elfringhoff, A. S. Novel 3-dodecanoylindone-
2-carboxylic acid inhibitors of cytosolic phospholipase A2. Bioorg.
Med. Chem. Lett. 2001, 11, 2569-2572.
(25) Seno, K.; Okuno, T.; Nishi, K.; Murakami, Y.; Watanabe, F.;
Matsuura, T.; Wada, M.; Fujii, Y.; Yamada, M.; Ogawa, T.;
Okada, T.; Hashizume, H.; Kii, M.; Hara, S.-I.; Hagishita, S.;
Nakamoto, S.; Yamada, K.; Chikazawa, Y.; Ueno, M.; Teshirogi,
I.; Ono, T.; Ohtani, M. Pyrrolidine inhibitors of human cytosolic
phospholipase A2. J . Med. Chem. 2000, 43, 1041-1044.
(26) Seno, K.; Okuno, T.; Nichi, K.; Murakami, Y.; Yamada, K.;
Nakamoto, K.; Ono, T. Pyrrolidine inhibitors of human cytosolic
phospholipase A2. Part 2: Synthesis of potent and crystallized
4-triphenylmethylthio derivative “pyrrophenone”. Bioorg. Med.
Chem. Lett. 2001, 11, 587-590.
(27) (a) Chiou, A.; Markidis, T.; Constantinou-Kokotou, V.; Verger,
R.; Kokotos, G. Synthesis and study of a lipolytic R-keto amide
inhibitor of pancreatic lipase. Org. Lett., 2000, 2, 347-350. (b)
Chiou, A.; Verger, R.; Kokotos, G. Synthetic routes and lipase-
inhibiting activity of long-chain R-keto amides. Lipids 2001, 36,
535-542.
(4) Larsson, P. K.; Claesson, H. E.; Kennedy, B. P. Multiple splice
variants of the human calcium-independent phospholipase A2
and their effect on enzyme activity. J . Biol. Chem. 1998, 273,
207-214.
(5) Ackermann, E. J .; Conde-Frieboes, K.; Dennis, E. A. Inhibition
of macrophage Ca2+-independent phospholipase A2 by bromoenol
lactone and trifluoromethyl ketones. J . Biol. Chem. 1995, 270,
445-450.
(6) Conde-Frieboes, K.; Reynolds, L. J .; Lio, Y.; Hale, M.; Wasser-
man, H. H.; Dennis, E. A. Activated ketones as inhibitors of
intracellular Ca2+-dependent and Ca2+-independent phospholi-
pase A2. J . Am. Chem. Soc. 1996, 118, 5519-5525.
(7) Lio, Y.; Reynolds, L. J .; Balsinde, J .; Dennis, E. A. Irreversible
inhibition of Ca2+-independent phospholipase A2 by methyl
arachidonyl fluorophosphonate. Biochim. Biophys. Acta 1996,
1302, 55-60.

3628 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Kokotos et al.
activity. J . Biol. Chem. 2003, 278, 23842-23850.
(36) Beaton, H. G.; Bennion, C.; Connolly, S.; Cook, A. R.; Gens-
mantel, N. P.; Hallam, C.; Hardy, K.; Hitchin, B.; J ackson, C.
J .; Robinson, D. H. The discovery of a new nonphospholipid
inhibitors of the secretory phospholipases A2. J . Med. Chem.
1994, 37, 557-559.
(37) Balsinde, J .; Barbour, S. E.; Bianco, I. D.; Dennis, E. A.
Arachidonic acid mobilization in P388D1 macrophages is con-
trolled by two distinct Ca2+-dependent phospholipase A2 en-
zymes. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11060-11064.
(38) Shinohara, H.; Balboa, M. A.; J ohnson, C. A.; Balsinde, J .;
Dennis, E. A. Regulation of delayed prostaglandin production
in activated P388D1 macrophages by group IV cytosolic and
group V secretory phospholipase A2s. J . Biol. Chem. 1999, 274,
12263-12268.
(39) Balsinde, J .; Shinohara, H.; Lefkowitz, L. J .; J ohnson, C. A.;
Balboa, M. A.; Dennis, E. A. Group V phospholipase A2 depend-
ent induction of cyclooxygenase-2 in macrophages. J . Biol. Chem.
1999, 274, 25967-25970.
(28) (a) Kokotos, G.; Verger, R.; Chiou, A. Synthesis of 2-oxo amide
triacylglycerol analogues and study of their inhibition effect on
pancreatic and gastric lipases. Chem.sEur. J . 2000, 6, 4211-
4217. (b) Kotsovolou, S.; Chiou, A.; Verger, R.; Kokotos, G. Bis-
2-oxo amide triacylglycerol analogues: a novel class of potent
human gastric lipase inhibitors. J . Org. Chem. 2001, 66, 962-
967.
(29) Connolly, S.; Bennion, C.; Botterell, S.; Croshaw, P. J .; Hallam,
C.; Hardy, K.; Hartopp, P.; J ackson, C. G.; King, S. J .; Lawrence,
L.; Mete, A.; Murray, D.; Robinson, D. H.; Smith, G. M.; Stein,
L.; Walters, I.; Wells, E.; Withnall, W. J . Design and synthesis
of a novel and potent series of inhibitors of cytosolic phospho-
lipase A2 based on a 1,3-disubstituted propan-2-one skeleton.
J . Med. Chem. 2002, 45, 1348-1362.
(30) Sheehan, J . C.; Cruickshank, P. A.; Boshart, G. L. A convenient
synthesis of water-soluble carbodiimides. J . Org. Chem. 1961,
26, 2525-2528.
(31) (a) Leanna, M. R.; Sowin, T. J .; Morton, H. E. Synthesis of
R-amino and R-alkoxy aldehydes via oxammonium oxidation.
Tetrahedron Lett. 1992, 33, 5029-5032. (b) Ma, Z.; Bobbitt, J .
M. Organic oxoammonium salts: a new convenient method for
the oxidation of alcohols to aldehydes and amines. J . Org. Chem.
1991, 56, 6110-6114.
(32) Kokotos, G. A convenient one-pot conversion of N-protected
amino acids and peptides into alcohols. Synthesis 1990, 299-
301.
(33) Kokotos, G.; Noula, C. Selective one-pot conversion of carboxylic
acids into alcohols. J . Org. Chem. 1996, 61, 6994-6996.
(34) Mosior, M.; Six, D. A.; Dennis, E. A. Group IV cytosolic
phospholipase A2 binds with high affinity and specificity to
phosphatidylinositol 4,5-bisphosphate resulting in dramatic
increases in activity. J . Biol. Chem. 1998, 273, 2184-2191.
(35) Six, D. A.; Dennis, E. A. Essential Ca2+-independent role of the
Group IVA cytosolic phospholipase A2 C2 domain for interfacial
(40) Balsinde, J .; Dennis, E. A. Distinct roles in signal transduction
for each of the phospholipase A2 enzymes present in P388D1
macrophages. J . Biol. Chem. 1996, 271, 6758-6765.
(41) Kontogiorgis, C.; Hadjipavlou-Litina, D. Biological evaluation
of several coumarin derivatives designed as possible anti-
inflammatory/antioxidant agents. J . Enzyme Inhib. Med. Chem.
2003, 18, 63-69.
(42) Hadjipavlou-Litina, D.; Rekka, E.; Hadjipetrou-Kourounakis, L.;
Kourounakis, P. N. Synthesis and biological studies of some
novel anti-inflammatory aryl-hydroxy-amino-ketones. Eur. J .
Med. Chem. 1991, 26, 85-90.
J M030485C