Structure-activity relationship of 2-oxoamide inhibition of group IVA cytosolic phospholipase A2 and group V secreted phospholipase A 2

Article abstract of DOI:10.1021/jm0613673

The Group IVA cytosolic phospholipase A₂ (GIVA cPLA₂) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and

Full text of DOI:10.1021/jm0613673

4222
J. Med. Chem. 2007, 50, 4222-4235
Structure-Activity Relationship of 2-Oxoamide Inhibition of Group IVA Cytosolic
Phospholipase A2 and Group V Secreted Phospholipase A2
David A. Six,‡,# Efrosini Barbayianni,†,# Vassilios Loukas,† Violetta Constantinou-Kokotou,§ Dimitra Hadjipavlou-Litina,|
Daren Stephens,‡ Alan C. Wong,‡ Victoria Magrioti,† Panagiota Moutevelis-Minakakis,† Sharon F. Baker,‡
Edward A. Dennis,*,‡ and George Kokotos*,†
Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, MC 0601, UniVersity of California, San
Diego, La Jolla, California 92093-0601, Laboratory of Organic Chemistry, Department of Chemistry, UniVersity of Athens, Panepistimiopolis,
Athens 15771, Greece, 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 NoVember 27, 2006
The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production
of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-
based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from δ- and γ-amino
acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly
developed 2-oxoamides as well as those previously described have now been tested with the human Group
V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only
one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2
inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were
also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well
below the control nonsteroidal anti-inflammatory drugs.
Introduction
messenger in inflammation as well as other pathophysiological
processes.7,8,11
Group IVA cytosolic phospholipase A2 (GIVA cPLA2 a) cata-
lyzes the preferential release of arachidonic acid (AA) from
intracellular membrane phospholipids.1,2 GIVA cPLA2 is one
of a large number of mammalian PLA2 enzymes that share the
ability to hydrolyze the central (sn-2) fatty acyl chain from sub-
strate phospholipids.1,3 GIVA cPLA2 is an R/â hydrolase that
utilizes a unique catalytic dyad of Ser-Asp, with the Ser hydroxyl
positioned for nucleophilic attack on the substrate ester.4,5 The
resulting acyl-enzyme intermediate is rapidly hydrolyzed to
yield a free fatty acid and a lysophospholipid. The two most
important products of GIVA cPLA2 action are AA and lyso-
platelet-activating factor (lyso-PAF).6-8 AA and its many eicos-
anoid metabolites are critical second messengers in inflamma-
tion, pain, and many pathophysiological processes.6-10 Lyso-
PAF is acetylated to form PAF, another important second
During times of inflammation, GIVA cPLA2 inhibition would
simultaneously block the up-regulated production of the AA
and lyso-PAF metabolites.8,11,12 The evidence for the key
pathophysiological roles of GIVA cPLA2 has come with detailed
experimentation of the GIVA cPLA2 knockout mouse6,7,13,14 as
well as confirmatory findings with existing inhibitors or
antisense techniques that reduce GIVA cPLA2 activity in vitro,
ex vivo, and in vivo.3,8,15 Together, this body of evidence
suggests that GIVA cPLA2 is a good target for the development
of anti-inflammatory agents.12,16,17
There are several clear paralogues of GIVA cPLA2 in mam-
malian genomes, but the expression of these paralogues is more
restricted and the functions are limited or unknown.3,14 The mem-
bers of Group VI PLA2 and GIVA cPLA2 share distant sequence
similarity in the active site residues.3,18,19 The Group VIA cal-
cium-independent phospholipase A2 (GVIA iPLA2) is the best-
studied of this group and plays a key role in membrane homeo-
stasis in many systems.20,21 The other major class of PLA2
enzymes includes the disulfide-rich small, secreted sPLA2s and
is characterized by a catalytic His/Asp dyad and catalytic Ca2+.1,3
These enzymes are present in a variety of tissues and, because
they are secreted, can act in a transcellular manner.22,23 While
the active sites of diverse sPLA2 paralogues are well con-
served, the specific membrane targets and activities vary
widely.24 A well-studied, simple example of this class is the
human Group V secreted phospholipase A2 (GV sPLA2).24,25
GV sPLA2 is able to bind to and hydrolyze phosphatidylcholine-
containing membranes and to release AA.26,27 In many cases
the activity of GV sPLA2 depends on or is linked to the activity
of GIVA cPLA2.3,28
* To whom correspondence should be addressed. For E.A.D.: (phone)
858-534-3055; (fax) 858-534-7390; (e-mail) edennis@ucsd.edu. For G.K.:
(phone) (30210) 7274462; (fax) (30210) 7274761; (e-mail) gkokotos@
chem.uoa.gr.
‡ University of California, San Diego.
# These authors contributed equally to this work.
† University of Athens.
§ Agricultural University of Athens.
| Aristotelian University of Thessaloniki.
a Abbreviations: AA, arachidonic acid; Boc, N-(tert-butoxycarbonyl);
ClogP, calculated log of the octanol/water partition coefficient; DMAP,
4-dimethylaminopyridine; DMSO, dimethyl sulfoxide; DPPC, 1-palmitoyl-
2-[14C]palmitoylphosphatidylcholine; DTT, dithiothreitol; EDTA, ethyl-
enediaminetetraacetic acid; EtOAc, ethyl acetate; Et2O, diethyl ether; Et3N;
triethylamine; GIVA cPLA2, Group IVA cytosolic phospholipase A2; GV
sPLA2, Group V secreted phospholipase A2; GVIA iPLA2, Group VIA
calcium-independent phospholipase A2; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HOBt, 1-hydroxybenzotriazole; KHMDS,
potassium bis(trimethylsilyl)amide; NSAIDs, nonsteroidal anti-inflammatory
drugs; PAF, platelet-activating factor; PAPC, 1-palmitoyl-2-[14C]arachi-
donoylphosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
TEMPO, 2,2,6,6-tetramethylpiperidine-1-yloxy free radical; THF, tetrahy-
drofuran; TLC, thin-layer chromatography; Tris-HCl, tris (hydroxy-
methyl)aminomethane HCl; WSCI, 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide; Z, benzyloxycarbonyl.
A recently identified class of inhibitors of human GIVA
cPLA2 and GVIA iPLA2 are the 2-oxoamides.10,19,29-31 The
2-oxoamides were designed as phospholipid substrate analogues
taking advantage of the noncleavable, electrophilic 2-oxoamide
moiety. The 2-oxoamide inhibitors of GIVA cPLA2 are char-
10.1021/jm0613673 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/02/2007

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4223
Scheme 1. General Structure of 2-Oxoamide Inhibitors
various 2-oxoamides containing a carbamate or an amide group
is depicted in Schemes 6-9.
In Vitro Inhibition of GIVA cPLA2. Variable Carboxy
Linker
Previous results have shown that a carboxy group in the
2-oxoamide structure is necessary to inhibit GIVA cPLA2 in
many 2-oxoamides,29,30 but more recently a carboxymethyl ester
analogue was shown to inhibit GIVA cPLA2 with significant
potency.19 The polar carboxy or carboxymethyl ester group
presumably acts as a mimic of the polar head group of a natural
substrate phospholipid. For a natural substrate phospholipid, the
number of bonds between the scissile sn-2 ester bond and the
polar head group matches the number of bonds between the
electrophilic 2-oxoamide and the polar group of a γ-amino
butyric acid-based 2-oxoamide (compound 35) or a γ-norleu-
cine-based 2-oxoamide (compound 36). The R1 norleucine side
chain of compound 36 gives the compound approximately 2-fold
greater potency than the unsubstituted compound 35.29,30 To
investigate whether the γ-spacing between the 2-oxoamide
moiety and the free carboxyl is optimal, several R-, â-, δ-, and
ꢀ-amino acid variants of the γ-norleucine-containing 2-oxoamide
(36) were synthesized and tested for inhibitor potency. The
results, summarized in Table 1, show that the best inhibition is
found with the δ and γ variants. Shorter and longer spacers to
the carboxy group result in weaker inhibition of GIVA cPLA2.
An R-amino acid variant, compound 3, was a very poor inhibitor
(approximately 10-fold less potent than 36). A â-norleucine
compound, 14, was a moderate inhibitor at approximately 3-fold
less potent than 36. Two δ-norleucine variants (17a and 17b)
were at least as potent as the γ-norleucine variants, compounds
36-38. However, the ꢀ-amino acid variants, 10a and 10b, were
less potent than the γ-amino acid variants, indicating an ideal
distance with the δ- or γ-norleucine variants. The appearance
of a polar group in all potent 2-oxoamide inhibitors and the
phospholipid-like γ- or δ-spacing between the polar group and
the 2-oxamide moiety are consistent with occupation of the
phospholipid substrate binding site of GIVA cPLA2.
acterized by a central 2-oxoamide moiety (Scheme 1) that is
presumably acting as an electrophilic target for the nucleophilic
active site serine.29,30 Coming off the distal carbonyl is a
hydrophobic R2 group that likely binds the substrate fatty acyl
chain binding site.29,30 Attached to the amide nitrogen is a
terminal charged or polar carboxy group that is likely interacting
with the substrate polar head group binding site.29,30 Branching
off the linker to the polar carboxy is the R1 side chain that
introduces stereoselective enhancement of potency to the
2-oxoamide inhibitors of GIVA cPLA2.29,30 Because many
inhibitors of GIVA cPLA2 also inhibit GVIA iPLA2, the
2-oxoamide compounds were tested on GVIA iPLA2.19 None
of the potent GIVA cPLA2 inhibitors inhibited GVIA iPLA2,
but several uncharged 2-oxoamides did inhibit GVIA iPLA2.19
Several 2-oxoamides have been shown to have systemic anti-
inflammatory, analgesic, and/or antihyperalgesic properties in
animal models.10,30
We have now explored the structure-activity relationship of
2-oxoamides and GIVA cPLA2 inhibition showing definitively
that δ- and γ-amino acid-based 2-oxoamides contain the optimal
spacing between the 2-oxoamide moiety and the polar carboxy
group.32 In addition, the R1 side chain was optimized. All of
the newly developed 2-oxoamides as well as those previously
described have now been tested with the human GV sPLA2 and
GVIA iPLA2, demonstrating the specificity of the most potent
GIVA cPLA2 inhibitors. Two of these specific GIVA cPLA2
inhibitors were found to have notably improved therapeutic
effects in animal models of pain and inflammation above and
beyond the control nonsteroidal anti-inflammatory drugs
(NSAIDs).
Chemical Synthesis of 2-Oxoamides
The synthesis of norleucine- and glutamate-based inhibitors
was carried out using methods previously described29,30 and is
depicted in Scheme 2. The AX series was initially described in
ref 30 and subsequently in U.S. Patent Application 20050148549.
For the synthesis of the ꢀ-norleucine-based inhibitors, N-(tert-
butoxycarbonyl)norleucinol 733 was oxidized to the aldehyde
by NaOCl/2,2,6,6-tetramethylpiperidine-1-yloxy free radical
(TEMPO),34,35 which was directly reacted with triethyl phospho-
nocrotonate (Scheme 3).36 Catalytic hydrogenation of compound
8, removal of the N-(tert)-butoxycarbonyl (Boc) group, and
coupling with 2-hydroxy acids produced compounds 9a,b.
Saponification, followed by oxidation with NaOCl/TEMPO, led
to inhibitors 10a (AX023) and 10b (AX025).
For the synthesis of â- and δ-norleucine inhibitors, the
conversion of a phenyl group to a carboxylic acid was the key
reaction. Boc-D-phenylalaninol (11)37,38 was oxidized to the
aldehyde and treated with propanylidene phosphorane to produce
compound 12 (Scheme 4). Removal of the protecting group and
coupling with 2-hydroxypentadecanoic acid, followed by hy-
drogenation, led to compound 13. The phenyl group of 13 was
converted to a carboxylic acid by oxidation with NaIO4, RuCl3
in CH3CN, ethyl acetate (EtOAc), H2O (1:1:8).39,40 Under these
conditions the 2-hydroxyamide group was simultaneously
converted into the 2-oxoamide group, leading to compound 14
(AX021). Compounds 17a (AX022) and 17b (AX109) were
prepared by similar reactions (Scheme 5). The synthesis of
In Vitro Inhibition of GIVA cPLA2. Side Chain Variation
GIVA cPLA2 has a strong preference for unsaturated fatty
acids such as AA at the sn-2 position of its substrate phospho-
lipids.41 This preference has also been reported for fatty alkyl
trifluoromethyl ketone inhibitors.42,43 However, the preference
for unsaturated radyl chains in the substrates and inhibitors of
GIVA cPLA2 is not absolute. In addition to its PLA2 activity,
GIVA cPLA2 also possesses comparable lysophospholipase
activity toward substrates with saturated fatty acyl chains.44-46
Inhibitors of GIVA cPLA2 containing saturated radyl chains
include trifluoromethyl ketone-based inhibitors,43 tricarbonyl-
based inhibitors,47 and 2-oxoamide-based inhibitors.19 Com-
pound 39 has a R2 oleate-based radyl chain in place of the
saturated chain of compound 35. As shown in Tables 1 and 2,
there is no significant difference in potency between compound
35 and compound 39. This suggests that there is no preference
for unsaturated chains in the 2-oxoamide inhbitors of GIVA
cPLA2. Interestingly, the methyl ester variant (40) of compound
39 has comparable potency. This marks just the second
uncharged 2-oxoamide to inhibit GIVA cPLA2 with any
potency. The first uncharged 2-oxoamide inhibitor of GIVA
cPLA2 (compound 11 from reference 19) is also a methyl ester
variant of a free carboxy-containing GIVA cPLA2 inhibitor.19
Previous studies have indicated that 2-oxoamide inhibitors
of GIVA cPLA2 had stereospecific enhancements of potency

4224 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
Scheme 2. Synthesis of R-Norleucine- and Glutamic Acid-Based Inhibitorsa
a Reagents and conditions: (a) HCl, H-L-Nle-OCH3, WSCI, HOBt, Et3N, CH2Cl2; (b) 1 N NaOH, dioxane/H2O (9:1), then HCl; (c) NaOCl, TEMPO,
NaBr, NaHCO3, EtOAc/PhCH3/H2O (3:3:0.5), 0 °C, then HCl; (d) H-Glu(OBut)-OBut, WSCI, HOBt, Et3N, CH2Cl2; (e) 50% trifluoroacetic acid/CH2Cl2.
Scheme 3. Synthesis of ꢀ-Norleucine-Based Inhibitorsa
Scheme 5. Synthesis of δ-Norleucine-Based Inhibitorsa
a Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/
PhCH3/H2O (3:3:0.5), -5 °C; (b) Ph3P+(CH2)2PhBr-, KHMDS, -15 °C,
PhCH3; (c) H2, 10% Pd/C; (d) 4 N HCl/Et2O; (e) CH3(CH2)nCHOHCOOH,
WSCI, HOBt, Et3N, CH2Cl2; (f) NaIO4, RuCl3, EtOAc/MeCN/H2O
(1:1:8).
a Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/
PhCH3/H2O (3:3:0.5), -5 °C; (b) (EtO)2P(dO)CH2CHdCHCOOEt, LiOH,
PhCH3, room temp; (c) H2, 10% Pd/C, EtOH; (d) 4 N HCl/THF; (e)
CH3(CH2)nCHOHCOOH, WSCI, HOBt, Et3N, CH2Cl2; (f) 1 N NaOH,
dioxane/H2O (9:1), then HCl; (g) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/
PhCH3/H2O (3:3:0.5), 0 °C, then HCl.
represents the calculated partition coefficient in octanol/water
in a logarithmic scale. ClogP is a measure of hydrophobicity
of particular use in comparisons of related compounds. ClogP
values are calculated for the neutral form of partially ionized
molecules. Compound 22a has a ClogP of 7.7, while 22b and
22c each have a ClogP of 4.5.
Scheme 4. Synthesis of â-Norleucine-Based Inhibitora
Compounds 26 and 31a are enantiomeric compounds that
contain an amide side chain, analogous to the carbamate-
containing 22c and 22b. Both of these compounds are very poor
inhibitors of GIVA cPLA2. The addition of eight extra meth-
ylenes off the end of the R1 amide chain results in compound
31b that does inhibit GIVA cPLA2 with an XI(50) of 0.025 mole
fraction. This modest increase in inhibitory potency may be due
to an increase in hydrophobicity for these compounds from a
ClogP of 2.9 up to a ClogP of 9.3. This same trend is seen for
the carbamates, as described above.
Two other novel 2-oxoamide compounds were synthesized
and tested. Compound 6 contains a second carboxylic acid in
the R1 side chain. This analogue is significantly more hydro-
philic (ClogP ) 3.1) than 35 and has no detectible inhibition
(<10%) at up to 0.091 mole fraction. It is possible that the
second carboxy group increases the solubility of the compound
enough to reduce the fraction partitioning to the substrate
micelles. Compound 34 is a carbamate analogue (containing a
R1 norleucine side chain) that is extremely hydrophobic (ClogP
) 12.1) and gave no inhibition (<10%) of GIVA cPLA2 at up
to 0.091 mole fraction. If the polar carboxy or carboxy ester
groups are interacting with the GIVA cPLA2 phosphate-binding
site, a polar carbamate should be sufficient. However, it is
possible that the eight-carbon extension off the carbamate causes
a conformational change or structural distortion, possibly causing
a change in orientation in the micelle surface, or merely creates
a Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/
PhCH3/H2O (3:3:0.5), -5 °C; (b) Ph3P+(CH2)2CH3Br-, KHMDS, -15 °C,
PhCH3; (c) H2, 10% Pd/C; (d) 4 N HCl/Et2O; (e) CH3(CH2)12CHOHCOOH,
WSCI, HOBt, Et3N, CH2Cl2; (f) NaIO4, RuCl3, EtOAc/MeCN/H2O
(1:1:8).
with small hydrophobic aliphatic chains at the R1 position (the
side-chain).29,30 To further characterize that side chain prefer-
ence, several other γ-substituted 2-oxoamides were synthesized
(22a-c, 26, 31a, and 31b). Compounds 22b and 22c are
enantiomers that contain a carbamate side chain. Neither
compound presents any inhibition of GIVA cPLA2. Interestingly,
the addition of six extra methylenes off the end of the R1
carbamate chain gives compound 22a that does inhibit GIVA
cPLA2, though only 60% at the highest concentration tested
(0.091 mole fraction). In general 2-oxoamides with longer R1
or R2 side chains seem to be relatively poor inhibitors of GIVA
cPLA2 and to lack stereospecificity.30 One explanation for the
inhibition by 22a and not by 22b or 22c is that the short-chain
carbamates 22c and 22b are not as hydrophobic. ClogP

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4225
Scheme 6. Synthesis of Inhibitors Containing a Carbamate Groupa
a Reagents and conditions: (a) CH3(CH2)nNCO, DMAP, CHCl3; (b) (i) 4 N HCl/MeOH; (ii) CH3(CH2)9CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (c)
1 N NaOH, CH3OH; (d) NaOCl, TEMPO, NaBr, NAHCO3, ACOEt/PhCH3/H2O (3:3:0.5), 0 °C, then HCl.
Scheme 7. Synthesis of Inhibitors Containing an Amide Groupa
a Reagents and conditions: (a) C2H5NH2, WSCI, HOBt Et3N, CH2Cl2; (b) (i) 4 N HCl/MeOH; (ii) CH3(CH2)9CHOHCOOH, WSCI, HOBt, Et3N, CH2Cl2;
(c) (i) 1 N NaOH, CH3OH; (ii) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O (3:3:0.5), 0 °C, then HCl.
Scheme 8. Synthesis of Inhibitors Containing an Amide Groupa
a Reagents and conditions: (a) CH3(CH2)mNH2, WSCI, HOBt, Et3N, CH2Cl2; (b) (i) H2, Pd/C; (ii) CH3(CH2)nCHOHCOOH, WSCI, HOBt, Et3N, CH2Cl2;
(c) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/toluene/H2O (3:3:0.5), 0 °C; (d) 50% trifluoroacetic acid/CH2Cl2.
Scheme 9. Synthesis of Norleucinol Carbamate-Based Inhibitora
a Reagents and conditions: (a) CH3(CH2)7NCO, DMAP, CHCl3; (b) (i) 4 N HCl/MeOH; (ii) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (c)
NaOCl, TEMPO, NaBr, NAHCO3, ACOEt/PhCH3/H2O (3:3:0.5), 0 °C.
a steric clash in the active site. We have previously shown that
2-oxoamides based on long-chain â-amino acids are relatively
weak inhibitors of GIVA cPLA2.31
specificity (or specificity has not been determined), are time-
dependent and/or covalent, are membrane-impermeant, or are
too large for pharmaceutical use.48,49
The 2-oxoamide compounds were designed as and are potent
inhibitors of serine esterases like pancreatic lipase and gastric
lipase,50,51 GIVA cPLA2,29 and most recently GVIA iPLA2.19
Previously, activated ketones (hydrated ketones) have been
shown to inhibit both serine proteases that utilize a nucleophilic
Specificity for GIVA cPLA2 vs GV sPLA2 and GVIA
iPLA2
Many inhibitors of GIVA cPLA2 and GVIA iPLA2 have been
developed, though most have the problems that they lack

4226 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
Table 1. Inhibition of GIVA cPLA2 with R- through ꢀ-Amino
Acid-Based 2-Oxoamides
Table 2. Inhibition of GIVA cPLA2 by 2-Oxoamides with Varying Side
Chains
a XI(50) is the concentration of the inhibitor at which 50% inhibition is
detected, with units of mole fraction calculated by taking the number of
moles of inhibitor divided by total number of moles of substrate plus
inhibitor plus detergent. b Data taken from ref 29. c N/A signifies not
applicable because an XI(50) value was determined. d N.D. signifies
inhibition was not potent enough to determine an XI(50). e Data taken from
ref 30.
a Data taken from ref 19. b None signifies no significant inhibition was
detected at the highest concentration tested.
serine and also zinc metalloproteases and aspartylproteases that
utilize an activated nucleophilic water molecule.52 The small,
secreted human GV sPLA2 utilizes a catalytic histidine to
activate a water molecule as the nucleophile in phospholipid
hydrolysis.53 Although there is no serine nucleophile in GV
sPLA2, the 2-oxoamides may resemble the substrate phospho-
lipids or the transition state such that they would bind to the
GV sPLA2 active site and inhibit the enzyme. Additionally, some
potent and selective sPLA2 inhibitors are indole analogues that
contain a primary 2-oxoamide moiety.54,55 Long-running phar-
maceutical research into inhibitors of the sPLA2 paralogues has
resulted in clinical trials for GIIA sPLA2 inhibitors that has
shown no benefit for sepsis, rheumatoid arthritis, or asthma.54-58
Research is ongoing to design potent, specific inhibitors of the
various human sPLA2 paralogues, including the GV sPLA2.25,59-61
To confirm the specificity of the 2-oxoamides, recombinant
human GV sPLA2 was expressed, folded, purified, and tested
against 31 of the published 2-oxoamide compounds.19,29-31 In
addition, the 16 novel compounds described herein were also
tested. These 47 compounds showed varying levels of inhibition
of GV sPLA2. A total of 27 compounds did not significantly
inhibit GV sPLA2 (<14% inhibition at the highest dose tested,
Table 1 in Supporting Information), 19 compounds moderately
inhibited GV sPLA2 (14-50% inhibition, Table 3), and 1
compound significantly inhibited GV sPLA2 with an XI(50) of
0.035 mole fraction (Table 3). All compounds that showed
significant inhibition were maximally tested at 0.091 mole
fraction except that compound 41 was tested at 0.01 mole
fraction, 42 was tested at 0.02 mole fraction, 40 was tested at
0.04 mole fraction, and 43 was tested at 0.05 mole fraction, as
indicated in Table 3.
The 2-oxoamides that showed moderate inhibition of GV
sPLA2 are shown in Table 3. The inhibition of GV sPLA2 by
41 was low (22%), though the maximum concentration tested
was only 0.01 mole fraction. Compound 42 has significant
activity toward GV sPLA2 at a relatively low 0.02 mole fraction
but requires heating to 65 °C to solubilize it in dimethyl

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4227
Table 3. Notable Inhibition of GV sPLA2 by 2-Oxoamides
Table 4. In Vivo Anti-Inflammatory and Analgesic Activity of Selected
2-Oxoamide Inhibitors
a Statistical significance of results was established using the Student’s
t-test, P < 0.001. b Analgesic activity (% analgesis) for the corresponding
ED50 doses.
its low hydrophobicity, unique features among the 2-oxoamides
tested thus far. Compound 31b also poorly, but significantly,
inhibits GV sPLA2 as well as GIVA cPLA2 (Table 2) but not
GVIA iPLA2 (data not shown). Compound 31b contains an
amide in the R1 side chain position. Phospholipid analogues
with an sn-2 amide-linked acyl chain are potent inhibitors of
secreted PLA2 family members;62,63 therefore, it is possible that
the amide and not the 2-oxoamide of 31b is responsible for the
measurable inhibition of GV sPLA2.
Compound 52 uniquely inhibited all three PLA2 enzymes
tested with no statistical preference for GV sPLA2 (Table 3),
GIVA cPLA2 (Table 2), or GVIA iPLA2 (XI(50) ) 0.20 ( 0.09,
data not shown). The inhibition of these diverse enzymes that
utilize widely varying active sites and catalytic mechanisms
suggests there may be an effect on the phospholipid substrate
presentation. It is also possible that this compound most closely
mimics a phospholipid containing two acyl chains and therefore
binds the active site of all three enzymes. None of the other 15
2-oxoamides whose syntheses are described here for the first
time (Schemes 2-9) significantly inhibited GVIA iPLA2 (data
not shown), consistent with a lack of inhibition of GVIA iPLA2
by 2-oxoamides containing a free carboxy group.19
Animal Models of Inflammation and Pain
Previously, several 2-oxoamides that inhibited GIVA cPLA2
were found to have potent anti-inflammatory and analgesic
effects, comparable to those of indomethacin and acetylsalicyl-
ate, respectively.30 These effects are consistent with the role of
GIVA cPLA2 as the rate-limiting provider of the free AA
precursors of inflammatory prostaglandins.6,7 Five additional
2-oxoamides have now been investigated in the same animal
models of inflammation and pain (Table 4). All of the
compounds inhibit GIVA cPLA2 (Tables 1 and 2) with
measurable XI(50) values ranging from 0.008 for compound 38
up to 0.025 for compounds 10a and 31b. Of these five
compounds tested, compounds 39 and 17b presented anti-
inflammatory activity at very low doses, with ED50 values of
0.0001 and 0.00005 mmol/kg, respectively. These dosages are
approximately 2 orders of magnitude lower than the best
2-oxoamides previously tested, including AX006 (compound
a No inhibition (<14% inhibition) found for any other 2-oxoamide
compounds with maximal concentration tested, typically 0.091 mole fraction.
sulfoxide (DMSO) and to integrate it into substrate mixed
micelles. The extreme hydrophobic nature of both 41 and 42
make them unlikely candidates for practical use. Compound 45,
a poor inhibitor of GIVA cPLA2 and GVIA iPLA2 inhibitor,19
also does not strongly inhibit GV sPLA2. Compound 46
significantly inhibits GV sPLA2 but not GIVA cPLA2 or GVIA
iPLA2,19,30 which may be because of its primary amide and/or

4228 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
35) and AX012.30 The control drug indomethacin, tested at 0.01
mmol/kg, gave a comparable reduction in edema to compounds
39 and 17b, which were tested at 100-fold lower dosages.
Moreover, at the ED50 values determined in the inflammation
assay, there is significant analgesis for compounds 39 (53%
analgesic activity) and 17b (71% analgesic activity).
iPLA2 seems to have a housekeeping role in membrane
homeostasis that results in low-level release of free fatty acids
from the cell membranes.21 GV sPLA2 and its many extracellular
paralogues would also remain free to act. GV sPLA2 and other
close paralogues have been shown to release significant amounts
of free fatty acids but generally do not lead to up-regulated
inflammatory eicosanoid production without the activity of
GIVA cPLA2.12,54,55 The potencies of the specific GIVA cPLA2
2-oxoamide inhibitors in animal models of inflammation and
pain both validate the target and provide enticing leads for
development of anti-inflammatory therapeutics.
It is unclear why these two compounds present anti-
inflammatory and analgesic activities at such low dosages. While
compound 39 lacks a R1 side chain, so does compound 35,
which is not nearly as potent.30 Compound 39 is the only
compound tested thus far with an unsaturated linear radyl chain
in the R2 main chain. It is possible that this facilitates delivery
to the cytosol or affects the physiological half-life of the
compound. Compound 17b is the only δ-amino acid-based
2-oxoamide tested, with the γ- and ꢀ-amino acid-based 2-oxo-
amides being significantly less potent (Table 4 and ref 30). We
previously noted a disconnect between compounds with similar
in vitro potency but disparate in vivo potency as seen with
compounds 35 and 36.30
Experimental Section
Synthesis of 2-Oxoamide Compounds. 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) spectrometer. All amino acid derivatives were
purchased from Fluka Chemical Co. Thin layer chromatography
(TLC) plates (silica gel 60 F254) and silica gel 60 (70-230 or 230-
400 mesh) for column chromatography were purchased from Merck.
Visualization of spots was effected with UV light and/or phospho-
molybdic acid and/or ninhydrin, both in EtOH stain. Tetrahydro-
furan (THF), toluene, and diethyl ether (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. The ClogP values were calculated using ChemDraw
Ultra 8.0 (with the embedded C-QSAR program of Biobyte Corp.).
The syntheses of 16 2-oxoamide compounds have been previously
described: compounds 43 (AX005), 35 (AX006), 36 (AX007), 41
(AX003), and 42 (AX004) in ref 41; compounds 37 (AX024), 44
(AX009), 45 (AX010), 46 (AX015), and 47 (AX017) in ref 42;
and compounds 39 (AX040), 40 (AX041), 48 (AX035), 49
(AX036), 50 (AX037), and 51 (AX038) in ref 21. The synthesis
of 38 (AX062) was accomplished in the same fashion as the closely
related compound 37.42 The complete set of structures are shown
in Tables 1-3.
None of the compounds tested have any effect on GVIA
iPLA2 activity (ref 19 and data not shown), and only compounds
10a and 31b present even weak GV sPLA2 inhibition. The
discrepancy between in vitro inhibition and in vivo effects may
reflect differential bioavailability throughout the body, uptake,
intracellular localization, and/or half-life of the compounds. One
additional caveat is that the in vitro inhibition assays were done
with the human GIVA cPLA2, while the animal studies were
done with rats. The human and rat GIVA cPLA2 enzymes are
94% identical at the amino acid level, and both possess the three
known regulated serine phosphorylation sites.1 Besides the 6%
amino acid sequence difference, there is a three-amino acid
C-terminal extension in the rat enzyme. Also, there is no way
to discern the critical post-translational phosphorylation pattern
of the rat GIVA cPLA2. These differences could also explain
the lack of correlation of in vitro and in vivo potency.
In conclusion, potent 2-oxoamide inhibitors of GIVA cPLA2
are γ- and δ-amino acid-based compounds with a free carboxy
group that do not inhibit GVIA iPLA2 or appreciably inhibit
GV sPLA2. Of the 47 2-oxoamides tested with GV sPLA2, only
one compound had appreciable inhibition. Two of the novel
2-oxoamide inhibitors of GIVA cPLA2 have potent anti-
inflammatory and analgesic effects in animal models. Effective
dosages for these two compounds are significantly lower than
those for indomethacin and acetylsalicylate, the control drugs.
These 2-oxoamide compounds specifically inhibited GIVA
cPLA2 and not GVIA iPLA2 or GV sPLA2 when tested in vitro.
Because GIVA cPLA2 action is a rate-limiting step in the
inflammatory cascade,8,64 GIVA cPLA2 is an important target
for the development of safe anti-inflammatory and analgesic
pharmaceuticals.11,65 The overall reduction in stimulated AA
production by selective inhibition of GIVA cPLA2 is particularly
important to avoid an imbalance of one branch of eicosanoid
production over another through substrate shunting, a phenom-
enon seen with the use of NSAIDs.66,67 Alternative anti-
inflammatory targets are particularly relevant given the recently
identified adverse cardiovascular side effects associated with
the cyclooxygenase-2-specific inhibitors and the known gas-
trointestinal and renal side effects of traditional nonsteroidal
anti-inflammatory drugs.66,68 It may be expected that inhibitors
of GIVA cPLA2 would avoid the pitfalls of both nonspecific
cyclooxygenase inhibitors and cyclooxygenase-2-selective in-
hibitors because the action of fatty acid biosynthesis, uptake,
and liberation by other PLA2 enzymes should not be affected
by specific GIVA cPLA2 inhibitors.66,67,69 In particular, GVIA
Coupling of Carboxylic Acids with Amino Components. To
a stirred solution of 2-hydroxy acid (2.0 mmol) and the hydrochlo-
ride amino component (2.0 mmol) in CH2Cl2 (20 mL), triethylamine
(Et3N) (6.2 mL, 4.4 mmol) and subsequently 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide (WSCI) (0.42 g, 2.2 mmol) and
1-hydroxybenzotriazole (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.
(2S)-Methyl 2-(2-Hydroxydodecanamido)hexanoate (2). Yield
1
70%; yellow oil. H NMR: δ 7.02 (1H, m, NH), 4.58 (1H, m,
CH), 4.13 (1H, m, CH), 3.74 (3H, s, OCH3), 3.06 (1H, br, OH),
1.95-1.52 (4H, m, 2 × CH2), 1.50-1.17 (20H, m, 10 × CH2),
0.91-0.72 (6H, m, 2 × CH3). 13C NMR: δ 174.1, 173.2, 72.2,
52.3, 51.5, 34.8, 31.8, 29.5, 29.4, 29.3, 27.3, 24.8, 22.6, 22.2, 14.0,
13.8. Anal. (C19H37NO4) C, H, N.
(2S)-Di-tert-butyl 2-(2-Hydroxydodecanamido)pentanedioate
(4). Yield 96%; oil. 1H NMR: δ 7.08 (1H, d, J ) 7.0 Hz, NHCO),
4.48 (1H, m, CH), 4.11 (1H, m, CH), 2.31 (2H, m, CH2COO),
2.20-1.63 (4H, m, 2 × CH2), 1.46 [9H, s, C(CH3)3], 1.41 [9H, s,
C(CH3)3], 1.25 (16H, br s, 8 × CH2), 0.87 (3H, t, J ) 7.0 Hz,
CH3). 13C NMR: δ 174.1, 172.2, 171.1, 82.4, 80.8, 72.0, 51.9, 34.9,
31.9, 31.5, 29.6, 29.5, 29.3, 28.0, 27.9, 27.6, 25.0, 24.9, 22.6, 14.1.
Anal. (C25H47NO6) C, H, N.
(6S)-Ethyl 6-(2-Hydroxydodecanamido)decanoate (9a). Yield
70%; yellow oil. 1H NMR: δ 6.31 (1H, d, J ) 8.4 Hz, NH), 4.10
(2H, t, J ) 7.0 Hz, OCH2), 4.08 (1H, m, CH), 3.87 (1H, m, CH),

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4229
3.30 (1H, br, OH), 2.27 (2H, t, J ) 7.2 Hz, CH2COO), 1.87-1.14
(33H, m, 15 × CH2, CH3), 0.86 (6H, m, 2 × CH3). 13C NMR: δ
173.7, 173.5, 72.0, 60.2, 48.8, 34.9, 34.1, 31.8, 29.5, 29.4, 29.3,
28.0, 25.3, 24.9, 24.7, 22.6, 22.5, 14.1, 14.0, 13.9. Anal. (C24H47-
NO4) C, H, N.
(6S)-Ethyl 6-(2-Hydroxypentadecanamido)decanoate (9b).
Yield 72%; yellow oil. 1H NMR: δ 6.33 (1H, m, NH), 4.02-4.15
(3H, m, OCH2, CH), 3.88 (1H, m, CH), 3.18 (1H, br, OH), 2.27
(2H, t, J ) 7.4 Hz, CH2COO), 1.87 (1.14 (37H, m, 18 × CH2,
CH3), 0.90-0.81 (6H, m, 2 × CH3). 13C NMR: δ 173.9, 173.5,
72.1, 60.3, 48.8, 34.9, 34.7, 34.1, 31.9, 29.6, 29.5, 29.4, 29.3, 28.0,
25.3, 24.9, 24.8, 22.6, 22.5, 14.1, 14.0, 13.9. Anal. (C27H53NO4)
C, H, N.
Hz, CH3). 13C NMR: δ 173.8, 171.2, 155.7, 79.9, 53.8, 51.8, 34.3,
30.2, 28.2, 28.1, 14.7. Anal. (C13H24N2O5) C, H, N.
(4R)-Methyl 5-(Ethylamino)-4-(2-hydroxydodecanamido)-5-
1
oxopentanoate (25). Yield 67%; white solid; mp 83-85 °C. H
NMR: δ 7.48 (1H, m, CONH), 6.83 (1H, m, CONH), 4.48 (2H,
m, CH), 4.10 (1H, m, CH), 3.68 (3H, s, CH3O), 3.26 (2H, m,
CH2NH), 2.41 (2H, m, CH2COO), 2.17-2.09 (2H, m, CH2), 1.80-
1.62 (2H, m, CH2), 1.25 (16H, br s, 8 × CH2), 1.13 (3H, t, J ) 7.2
Hz, CH3), 0.87 (3H, t, J ) 7.0 Hz, CH3). 13C NMR: δ 177.1, 173.6,
171.2, 72.0, 51.8, 34.7, 31.8, 29.6, 29.4, 29.3, 27.8, 25.1, 22.6, 14.2,
14.1. Anal. (C20H38N2O5) C, H, N.
(S)-tert-Butyl 4-(Benzyloxycarbonyl)-5-(ethylamino)-5-oxo-
pentanoate (28a). Yield 85%; white solid; mp 69-71 °C; [R]D
2-Hydroxy-N-((S)-1-phenylhexan-2-yl)pentadecanamide (13).
1
-5.4 (c 0.5, CHCl3). H NMR: δ 7.35 (5H, m, C6H5), 6.25 (1H,
1
Yield 89%; white solid. H NMR: δ 7.24 (5H, m, C6H5), 6.19
m, NHCO), 5.67 (1H, d, J ) 8.0 Hz, OCONH), 5.10 (2H, s,
CH2C6H5), 4.14 (1H, m, CH), 3.29 (2H, m, CH2NH), 2.36 (2H, m,
CH2COO), 1.96 (2H, m, CH2CH), 1.44 [9H, s, C(CH3)3], 1.13 (3H,
t, J ) 7.4 Hz, CH3). 13C NMR: δ 172.8, 171.0, 156.0, 136.2, 128.5,
128.1, 128.0, 80.9, 66.9, 54.3, 34.4, 31.6, 28.1, 28.0, 14.6. Anal.
(C19H28N2O5) C, H, N.
(1H, m, NH), 4.18 (1H, m, CH), 4.02 (1H, m, CH), 2.55-2.83
(3H, m, CH2C6H5, OH), 1.78-1.47 (4H, m, 2 × CH2), 1.42-1.15
(26H, m, 13 × CH2), 0.91-0.82 (6H, m, 2 × CH3). 13C NMR: δ
173.1, 137.9, 129.4, 128.3, 126.4, 72.0, 49.8, 41.0, 35.0, 33.9, 31.9,
29.6, 29.5, 29.4, 29.3, 28.1, 24.8, 22.7, 22.5, 14.1, 14.0. Anal.
(C27H47NO2) C, H, N.
(S)-tert-Butyl 4-(Benzyloxycarbonyl)-5-(decylamino)-5-oxo-
2-Hydroxy-N-((S)-1-phenyloctan-4-yl)pentadecanamide (16a).
1
pentanoate (28b). Yield 98%; oil; [R]D -3.4 (c 1, CHCl3). H
1
Yield 68%; white solid. H NMR (CDCl3): δ 7.39-7.11 (5H, m,
NMR: δ 7.34 (5H, m, C6H5), 6.40 (1H, m, CONH), 5.75 (1H, d,
J ) 7.2 Hz, OCONH), 5.09 (2H, s, CH2C6H5), 4.19 (1H, m, CH),
3.24 (2H, m, CH2NH), 2.32 (2H, m, CH2COO), 1.96 (4H, m, 2 ×
CH2), 1.44 [9H, s, C(CH3)3], 1.25 (14H, br s, 7 × CH2), 0.88 (3H,
t, J ) 6.6 Hz, CH3). 13C NMR: δ 172.9, 171.0, 156.2, 136.1, 128.5,
128.1, 128.0, 81.0, 66.9, 54.3, 39.5, 31.8, 29.5, 29.4, 29.3, 29.2,
28.2, 28.0, 26.8, 22.6, 14.1. Anal. (C27H44N2O5) C, H, N.
C6H5), 6.28 (1H, d, J ) 9.6 Hz, NH), 4.05 (1H, m, CH), 3.91 (1H,
m, CH), 2.99 (1H, br s, OH), 2.64 (2H, m, CH2C6H5), 1.81-1.02
(34H, m, 17 × CH2), 0.88 (6H, t, J ) 6.6 Hz, 2 × CH3). 13C NMR
(CDCl3): δ 173.7, 142.3, 128.3, 128.2, 125.7, 72.1, 48.6, 35.6, 35.0,
34.9, 31.8, 29.7, 29.6, 29.5, 29.3, 28.0, 27.6, 24.7, 22.7, 22.5, 14.1,
14.0. Anal. (C29H51NO2) C, H, N.
2-Hydroxy-N-((S)-1-phenyloctan-4-yl)hexadecanamide (16b).
Yield 73%; white solid; mp 43-45 °C. 1H NMR (CDCl3): δ 7.32-
7.14 (5H, m, C6H5), 6.25 (1H, d, J ) 9.6 Hz, NH), 4.08 (1H, m,
CH), 3.95 (1H, m, CH), 3.08 (1H, br s, OH), 2.62 (2H, m,
CH2C6H5), 1.89-1.05 (36H, m, 18 × CH2), 0.88 (6H, t, J ) 6.6
Hz, 2 × CH3). 13C NMR (CDCl3): δ 173.4, 142.1, 128.3, 128.2,
125.7, 72.0, 48.7, 35.6, 35.0, 34.8, 31.9, 29.7, 29.6, 29.5, 29.3, 28.0,
27.6, 24.8, 22.6, 22.5, 14.1, 14.0. Anal. (C30H53NO2) C, H, N.
(4S)-tert-Butyl 5-(Ethylamino)-4-(2-hydroxydodecanamido)-
5-oxopentanoate (29a). Yield 55%; oil. 1H NMR: δ 7.45 (1H, d,
J ) 8.6 Hz NHCO), 6.85 (1H, m, NHCO), 4.41 (1H, m, CH), 4.11
(1H, m, CH), 3.25 (2H, m, NHCH2), 2.30 (2H, m, CH2COO), 2.05
(2H, m, CH2), 1.80-1.62 (2H, m, CH2), 1.43 (9H, s, 3 × CH3),
1.29 (16H, br s, 8 × CH2), 1.09 (3H, t, J ) 7.2 Hz, CH3), 0.88
(3H, t, J ) 7.0 Hz, CH3). 13C NMR: δ 174.8, 172.6, 171.1, 80.8,
72.0, 51.9, 34.7, 34.4, 31.8, 31.5, 29.5, 29.4, 29.3, 28.0, 25.1, 22.6,
14.5, 14.0. Anal. (C23H44N2O5) C, H, N.
(4S)-tert-Butyl 5-(Decylamino)-4-(2-hydroxyhexadecanamido)-
5-oxopentanoate (29b). Yield 52%; white solid; mp 73-75 °C.
1H NMR: δ 7.35 (1H, m, CONH), 6.59 (1H, m, CONH), 4.41
(1H, m, CH), 4.11 (1H, m, CH), 3.25 (2H, m, CH2NH), 2.39 (2H,
m, CH2COO), 2.08-1.63 (6H, m, CH2CH, CH2CH2NH, CH2CHOH),
1.45 [9H, s, C(CH3)3], 1.25 (38H, br s, 19 × CH2), 0.88 (6H, t, J
) 6.6 Hz, 2 × CH3). 13C NMR: δ 174.6, 172.9, 170.9, 81.1, 72.0,
52.1, 39.6, 34.8, 31.9, 29.7, 29.5, 29.4, 29.3, 28.0, 27.8, 26.9, 25.0,
22.7, 14.1. Anal. (C35H68N2O5) C, H, N.
(S)-2-(2-Hydroxyhexadecanamido)hexyl Octylcarbamate (33).
Yield 59%; white solid; mp 65-66 °C. 1H NMR: δ 6.62 (1H, m,
NHCO), 4.99 (1H, m, OCONH), 4.12 (3H, m, 2 × CH,
CH2OCONH), 3.12 (2H, m, CH2NH), 1.79 (1H, m, CHHCH),
1.66-1.44 (5H, m, CHHCH, 2 × CH2), 1.25 (28H, br s, 14 ×
CH2), 0.88 (3H, t, J ) 6.6 Hz, CH3). 13C NMR: δ 174.0, 156.8,
72.2, 66.4, 66.1, 48.7, 41.1, 34.9, 34.7, 31.9, 31.8, 31.1, 29.9, 29.7,
29.5, 29.3, 29.2, 27.9, 26.7, 25.0, 22.7, 22.6, 22.5, 14.1, 13.9. Anal.
(C31H62N2O4) C, H, N.
Oxidation of 2-Hydroxyamides. To a solution of 2-hydroxy-
amide (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 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
(4S)-Methyl 4-(2-Hydroxydodecanamido)-5-(octylcarbamoyl-
oxy)pentanoate (20a). Yield 47%; white solid; mp 70-73 °C. 1H
NMR: δ 6.69 (1H, m, CONH), 4.79 (1H, m, OCONH), 4.08 (4H,
m, 2 × CH, CH2OCONH), 3.68 (3H, s, CH3O), 3.17 (2H, m,
CH2NH), 2.40 (2H, m, CH2COO), 1.85 (4H, m, 2 × CH2), 1.49-
1.26 (28H, m, 14 × CH2), 0.88 (6H, t, J ) 6.6 Hz, 2 × CH3). 13
C
NMR: δ 174.5, 173.7, 156.5, 72.1, 66.1, 51.8, 48.1, 41.1 34.7,
31.9, 30.4, 29.8, 29.6, 29.4, 29.3, 29.2, 26.7, 25.0, 22.6, 14.0. Anal.
(C27H52N2O6) C, H, N.
(4S)-Methyl 5-(Ethylcarbamoyloxy)-4-(2-hydroxydodecan-
amido)pentanoate (20b). Yield 61%; white solid; mp 79-81 °C.
1H NMR: δ 6.75 (1H, m, CONH), 4.91 (1H, m, OCONH), 4.11
(4H, m, 2 × CH, CH2OCONH), 3.68 (3H, s, CH3O), 3.21 (2H, m,
CH2NH), 2.40 (2H, m, CH2COO), 1.83 (4H, m, 2 × CH2), 1.60-
1.26 (16H, m, 8 × CH2), 1.18 (3H, t, J ) 6.0 Hz, CH3), 0.88 (3H,
t, J ) 6.0 Hz, CH3). 13C NMR: δ 174.6, 173.7, 156.4, 72.1, 66.1,
51.7, 48.1, 35.1, 34.7, 31.8, 30.5, 30.4, 29.4, 29.3, 26.5, 26.4, 25.1,
25.0, 22.6, 15.0, 14.0. Anal. (C21H40N2O6) C, H, N.
(4R)-Methyl 5-(Ethylcarbamoyloxy)-4-(2-hydroxydodecan-
amido)pentanoate (20c). Yield 58%; white solid; mp 78-80 °C.
1H NMR: δ 6.75 (1H, m, CONH), 4.91 (1H, m, OCONH), 4.10
(4H, m, 2 × CH, CH2OCONH), 3.68 (3H, s, CH3O), 3.21 (2H, m,
CH2NH), 2.40 (2H, m, CH2COO), 1.83 (4H, m, 2 × CH2), 1.62-
1.26 (16H, m, 8 × CH2), 1.18 (3H, t, J ) 7.2 Hz, CH3), 0.88 (3H,
t, J ) 7.2 Hz, CH3). 13C NMR: δ 174.5, 173.8, 156.4, 72.1, 66.1,
51.8, 48.1, 35.9, 34.7, 31.8, 30.4, 29.6, 29.5, 29.3, 26.5, 26.4, 25.1,
25.0, 22.6, 15.1, 14.1.
(R)-Methyl 4-(tert-Butoxycarbonyl)-5-(ethylamino)-5-oxopen-
tanoate (24). Yield 69%; white solid; mp 64-66 °C; [R]D +7.1 (c
1
1, CHCl3). H NMR: δ 6.45 (1H, m, CONH), 5.39 (1H, d, J )
9.0 Hz, OCONH), 4.11 (1H, m, CH), 3.67 (3H, s, CH3O), 3.27
(2H, m, CH2NH), 2.41 (2H, m, CH2COO), 2.12 (1H, m, CHHCH),
1.92 (1H, m, CHHCH), 1.42 [9H, s, (CCH3)3], 1.12 (3H, t, J ) 7.4

4230 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
were evaporated under reduced pressure, and the residue was
purified by column chromatography [EtOAc-petroleum ether (bp
40-60 °C)].
7.03 (1H, m, CONH), 4.63 (1H, m, CH), 3.32 (2H, m, NHCH2),
2.89 (2H, t, J ) 7.4 Hz, CH2COCO), 2.49 (2H, m, CH2COOH),
2.12 (2H, m, CH2), 1.59 (2H, m, CH2), 1.23 (14H, m, 7 × CH2),
1.17 (3H, t, J ) 7.0 Hz, NHCH2CH3), 0.88 (3H, t, J ) 6.6 Hz,
CH3). 13C NMR: δ 197.9, 175.9, 170.1, 160.2, 52.0, 36.8, 34.5,
31.7, 29.7, 29.4, 29.3, 29.2, 29.1, 28.9, 27.6, 22.9, 22.5, 14.3, 14.0.
Anal. (C19H34N2O5) C, H, N.
(S)-2-(2-Oxododecanamido)hexanoic Acid (3). Yield 55%;
1
pale-yellow solid; mp 42-43 °C; [R]D +13.5 (c 0.5, CHCl3). H
NMR: δ 7.41 (1H, d, J ) 9 Hz, NH), 4.52 (1H, m, CH), 2.90
(2H, t, J ) 7.8 Hz, CH2COCO), 2.00-1.56 (6H, m, 3 × CH2),
1.25 (16H, m, 8 × CH2), 0.88 (6H, t, J ) 6.7 Hz, 2 × CH3). 13C
NMR: δ 198.5, 176.4, 159.9, 52.8, 36.8, 31.9, 29.6, 29.4, 29.3,
29.0, 27.4, 23.1, 22.7, 22.3, 14.1, 13.8. MS (FAB), m/z (%): 372
(100) [M + 2Na - H]+. Anal. (C18H33NO4) C, H, N.
(S)-tert-Butyl 5-(Ethylamino)-5-oxo-4-(2-oxododecanamido)-
1
pentanoate (30a). Yield 81%; oil; [R]D -6.4 (c 1, CHCl3). H
NMR: δ 7.69 (1H, d, J ) 9.0 Hz, NHCOCO), 7.42 (1H, m,
CONH), 4.38 (1H, m, CH), 3.29 (2H, m, NHCH2), 2.87 (2H, t, J
) 7.4 Hz, CH2COCO), 2.36 (2H, m, CH2COO), 2.09 (2H, m, CH2),
1.59 (2H, m, CH2), 1.44 [9H, s, C(CH3)3], 1.25 (14H, m, 7 × CH2),
(S)-Di-tert-butyl 2-(2-Oxododecanamido)pentanedioate (5).
1
Yield 93%; oil; [R]D +10.9 (c 1.15, CHCl3). H NMR: δ 7.45
1.18 (3H, t, J ) 7.0 Hz, CH3), 0.88 (3H, t, J ) 6.6 Hz, CH3). 13
C
(1H, d, J ) 9.2 Hz, NH), 4.45 (1H, m, CH), 2.89 (2H, t, J ) 7.4
Hz, CH2COCO), 2.30-1.95 (4H, m, 2 × CH2), 1.60 (2H, m, CH2),
1.48 [9H, s, C(CH3)3], 1.44 [9H, s, C(CH3)3], 1.26 (16H, br s, 8 ×
CH2), 0.88 (3H, t, J ) 7.0 Hz, CH3). 13C NMR: δ 198.4, 171.7,
170.0, 159.9, 82.7, 80.8, 52.1, 36.7, 31.8, 31.3, 29.5, 29.4, 29.3,
29.0, 28.0, 27.9, 27.5, 23.1, 22.6, 14.1. Anal. (C25H45NO6) C, H,
N.
NMR: δ 198.2, 172.5, 169.8, 160.2, 81.1, 52.3, 36.8, 34.5, 31.8,
31.5, 29.5, 29.4, 29.2, 29.0, 28.0, 23.0, 22.6, 14.6, 14.1. Anal.
(C23H42N2O5) C, H, N.
(S)-tert-Butyl
5-(Decylamino)-5-oxo-4-(2-oxohexadecan-
amido)pentanoate (30b). Yield 90%; white solid; mp 43-45 °C;
1
[R]D -6.0 (c 1, CHCl3). H NMR: δ 7.95 (1H, d, J ) 8.0 Hz,
NHCOCO), 6.31 (1H, m, CONH), 4.38 (1H, m, CH), 3.24 (2H,
m, NHCH2), 2.89 (2H, t, J ) 7.4 Hz, CH2COCO), 2.32 (2H, m,
CH2COO), 2.09 (2H, m, CH2), 1.55 (4H, m, 2 × CH2), 1.46 [9H,
s, C(CH3)3], 1.26 (36H, m, 18 × CH2), 0.88 (6H, t, J ) 6.6 Hz, 2
× CH3). 13C NMR: δ 198.2, 172.6, 169.9, 160.2, 81.2, 52.4, 39.7,
36.8, 31.9, 31.8, 31.6, 29.6, 29.5, 29.4, 29.3, 29.2, 29.0, 28.1, 28.0,
26.8, 23.1, 22.7, 14.1. Anal. (C35H66N2O5) C, H, N.
(S)-6-(2-Oxododecanamido)decanoic Acid (10a). Yield 73%;
1
white solid; mp 58-59 °C; [R]D -0.9 (c 2.0, CHCl3). H NMR:
δ 6.72 (1H, d, J ) 9 Hz, NH), 3.85 (1H, m, CH), 2.90 (2H, t, J )
7.8 Hz, CH2COCO), 2.31 (2H, t, J ) 7.4 Hz, CH2COOH), 1.78-
1.23 (28H, m, 14 × CH2), 0.88 (6H, t, J ) 6.8 Hz, 2 × CH3). 13
C
NMR: δ 199.7, 179.3, 159.8, 49.4, 36.8, 34.6, 33.7, 31.8, 29.5,
29.4, 29.3, 29.0, 27.9, 25.3, 24.4, 23.2, 22.6, 22.5, 14.1, 13.9. MS
(FAB), m/z (%): 428 (100) [M + 2Na - H]+, 406 (63) [M +
Na]+. Anal. (C22H41NO4) C, H, N.
(S)-2-(2-Oxohexadecanamido)hexyl Octylcarbamate (34). Yield
1
66%; white solid; mp 73-75 °C; [R]D -22.8 (c 0.5, CHCl3). H
(S)-6-(2-Oxopentadecanamido)decanoic Acid (10b). Yield
80%; pale-yellow solid; mp 63-64 °C; [R]D -0.5 (c 1.0, CHCl3).
1H NMR: δ 6.72 (1H, d, J ) 9.8 Hz, NH), 3.85 (1H, m, CH),
2.93 (2H, t, J ) 7.4 Hz, CH2COCO), 2.34 (2H, t, J ) 7.4 Hz,
CH2COOH), 1.70-1.12 (34H, m, 17 × CH2), 0.91-0.82 (6H, m,
2 × CH3). 13C NMR: δ 199.7, 179.0, 159.8, 49.4, 36.8, 34.6, 33.7,
31.9, 29.6, 29.4, 29.3, 29.0, 28.0, 25.3, 24.4, 23.2, 22.7, 22.5, 14.1,
13.9. MS (FAB), m/z (%): 426 (100) [M + H]+. Anal. (C25H47-
NO4) C, H, N.
(S)-5-(Octylcarbamoyloxy)-4-(2-oxododecanamido)pentano-
ic Acid (22a). Yield 42%; oily solid; [R]D -16.8 (c 0.26, CHCl3).
1H NMR: δ 7.22 (1H, m, NHCOCO), 4.85 (1H, m, OCONH),
4.15 (3H, m, CH, CH2OCONH), 3.15 (2H, m, NHCH2), 2.91 (2H,
t, J ) 7.8 Hz, CH2COCO), 2.40 (2H, m, CH2COOH), 1.90 (2H,
m, CH2), 1.70-1.40 (4H, m, 2 × CH2), 1.27 (24H, m, 12 × CH2),
0.89 (6H, t, J ) 6.8 Hz, 2 × CH3). 13C NMR: δ 198.9, 176.9,
160.2, 156.0, 65.5, 48.6, 41.2, 36.8, 31.9, 31.8, 30.3, 29.8, 29.5,
29.4, 29.3, 29.2, 29.0, 26.7, 26.3, 23.1, 22.6, 14.1. MS (FAB), m/z
(%): 485 (40) [Μ + H]+. Anal. (C26H48N2O6) C, H, N.
(S)-5-(Ethylcarbamoyloxy)-4-(2-oxododecanamido)pentano-
ic Acid (22b). Yield 51%; white solid; mp 70-72 °C; [R]D -25.6
(c 1, CHCl3). 1H NMR: δ 7.25 (1H, m, NHCOCO), 4.85 (1H, m,
OCONH), 4.15 (3H, m, CH2OCONH, CH), 3.20 (2H, m, NHCH2),
2.90 (2H, t, J ) 7.4 Hz, CH2COCO), 2.42 (2H, m, CH2COOH),
1.93 (2H, m, CH2), 1.59 (2H, m, CH2), 1.29 (14H, m, 7 × CH2),
1.14 (3H, t, J ) 7.4 Hz, NHCH2CH3), 0.88 (3H, t, J ) 7.0 Hz,
CH3). 13C NMR: δ 198.8, 177.3, 160.1, 156.0, 65.5, 48.4, 36.7,
35.9, 31.8, 30.3, 29.4, 29.3, 29.2, 29.0, 28.9, 26.2, 23.0, 22.6, 15.0,
14.1. MS (FAB), m/z (%): 423 (50) [Μ + Na]+.
(R)-5-(Ethylcarbamoyloxy)-4-(2-oxododecanamido)pentano-
ic Acid (22c). Yield 56%; white solid; mp 70-72 °C; [R]D +26.1
(c 1, CHCl3). 1H NMR: δ 7.25 (1H, m, NHCOCO), 4.82 (1H, m,
OCONH), 4.19 (3H, m, CH2OCONH, CH), 3.21 (2H, m, NHCH2),
2.90 (2H, t, J ) 7.4 Hz, CH2COCO), 2.42 (2H, m, CH2COOH),
1.98 (2H, m, CH2), 1.59 (2H, m, CH2), 1.29 (14H, m, 7 × CH2),
1.14 (3H, t, J ) 7.4 Hz, NHCH2CH3), 0.88 (3H, t, J ) 7.0 Hz,
CH3). 13C NMR: δ 198.7, 177.0, 160.0, 155.9, 65.4, 48.4, 36.6,
35.8, 31.7, 30.2, 29.4, 29.2, 29.1, 28.9, 26.1, 22.9, 22.5, 15.0, 13.9.
Anal. (C20H36N2O6) C, H, N.
NMR: δ 7.03 (1H, m, NHCOCO), 4.71 (1H, m, OCONH), 4.10
(3H, m, CH2OCONH, CH), 3.16 (2H, m, CH2NH), 2.92 (2H, t, J
) 7.6 Hz, CH2COCO), 1.62-1.51 (6H, m, 3 × CH2), 1.26 (28H,
br s, 14 × CH2), 0.85 (3H, t, J ) 5.6 Hz, CH3). 13C NMR: δ
199.5, 160.1, 156.1, 66.0, 49.3, 41.4, 37.0, 32.1, 32.0, 31.2, 30.1,
29.9, 29.7, 29.6, 29.4, 29.3, 28.1, 27.0, 23.4, 22.9, 22.8, 22.7, 14.3,
14.1. MS (FAB), m/z (%): 525 (20) [Μ + H]+. Anal. (C31H60N2O4)
C, H, N.
(S)-Methyl 5-(Octylcarbamoyloxy)-4-(2-oxododecanamido)-
pentanoate (52). This compound was prepared by oxidation of
compound 20a. Yield 63%; white solid; mp 55-57 °C; [R]D -20.8
1
(c 0.5, CHCl3). H NMR: δ 7.14 (1H, m, NHCOCO), 4.76 (1H,
m, OCONH), 4.12 (3H, m, CH, CH2OCONH), 3.67 (3H, s, CH3O),
3.15 (2H, m, NHCH2), 2.90 (2H, t, J ) 7.2 Hz, COCOCH2), 2.37
(2H, m, CH2COO), 1.90 (2H, m, CH2), 1.59-1.48 (4H, m, 2 ×
CH2), 1.26 (24H, m, 12 × CH2), 0.88 (6H, t, 2 × CH3, J ) 7.0
Hz). 13C NMR: δ 198.9, 173.2, 160.0, 156.0, 65.5, 51.8, 48.5, 41.1,
36.7, 31.8, 31.7, 30.4, 29.8, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 26.7,
26.4, 23.1, 22.6, 14.0. MS (FAB), m/z (%): 499 (45) [Μ + H]+.
Anal. (C27H50N2O6) C, H, N.
Cleavage of the tert-Butyl Protecting Group. A solution of
the compound (1 mmol) in 50% trifluoroacetic acid/CH2Cl2 (10
mL) was stirred for 1 h at room temperature. The organic solvent
was evaporated under reduced pressure. The residue was purified
by recrystallization [EtOAc-petroleum ether (bp 40-60 °C)].
(S)-2-(2-Oxododecanamido)pentanedioic Acid (6). Yield 92%;
1
white solid; mp 97-98 °C; [R]D +24.4 (c 0.5, CHCl3). H NMR:
δ 7.61 (1H, d, J ) 9.2 Hz, NHCOCO), 4.61 (1H, m, CH), 2.91
(2H, t, J ) 7.4 Hz, CH2COCO), 2.54 (2H, m, CH2COOH), 2.31
(2H, m, CH2), 1.61 (2H, m, CH2), 1.26 (14H, m, 7 × CH2), 0.88
(3H, t, J ) 6.6 Hz, CH3). 13C NMR: δ 198.1, 178.7, 176.5, 159.8,
51.2, 36.7, 31.9, 29.5, 29.4, 29.3, 29.0, 26.1, 23.1, 22.7, 14.1. MS
(FAB), m/z (%): 366 (15) [M + Na]+, 344 (65) [Μ + H]+. Anal.
(C17H29NO6) C, H, N.
(S)-5-(Ethylamino)-5-oxo-4-(2-oxododecanamido)pentanoic Acid
(31a). Yield 90%; white solid; mp 103-104 °C; [R]D -14.2 (c
1
0.5, CHCl3). H NMR: δ 8.10 (1H, d, J ) 9.0 Hz, NHCOCO),
7.20 (1H, m, CONH), 4.65 (1H, m, CH), 3.30 (2H, m, NHCH2),
2.87 (2H, t, J ) 7.4 Hz, CH2COCO), 2.45 (2H, m, CH2COOH),
2.11 (2H, m, CH2), 1.57 (2H, m, CH2), 1.25 (14H, m, 7 × CH2),
1.15 (3H, t, J ) 7.0 Hz, NHCH2CH3), 0.87 (3H, t, J ) 6.6 Hz,
CH3). 13C NMR: δ 198.0, 176.2, 170.6, 160.5, 52.1, 37.0, 34,7,
(R)-5-(Ethylamino)-5-oxo-4-(2-oxododecanamido)pentanoic
Acid (26). Yield 57%; white solid; mp 109-112 °C; [R]D +11.6
(c 0.5, CHCl3). 1H NMR: δ 8.02 (1H, d, J ) 9.0 Hz, NHCOCO),

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4231
31.8, 29.8, 29.5, 29.4, 29.3, 29.2, 29.0, 27.5, 23.0, 22.6, 14.4, 14.1.
MS (FAB), m/z (%): 393 (86) [Μ + Νa]+. Anal. (C19H34N2O5) C,
H, N.
reduced pressure. The residue was purified after recrystallization
[EtOAc-petroleum ether (bp 40-60 °C)].
(4S)-4-(2-Hydroxydodecanamido)-5-(octylcarbamoyloxy)pen-
1
tanoic Acid (21a). Yield 69%; white solid; mp 96-98 °C. H
(S)-5-(Decylamino)-5-oxo-4-(2-oxohexadecanamido)pentano-
ic Acid (31b). Yield 59%; white solid; mp 91-93 °C; [R]D -10.2
(c 0.5, CHCl3). 1H NMR: δ 7.95 (1H, d, J ) 9.2 Hz, NHCOCO),
6.99 (1H, m, CONH), 4.67 (1H, m, CH), 3.25 (2H, m, NHCH2),
2.88 (2H, t, J ) 7.2 Hz, CH2COCO), 2.46 (2H, m, CH2COOH),
2.10 (2H, m, CH2), 1.54 (4H, m, 2 × CH2), 1.26 (36H, m, 18 ×
CH2), 0.88 (6H, t, J ) 6.6 Hz, 2 × CH3). 13C NMR: δ 197.8,
175.9, 170.2, 160.3, 52.1, 39.8, 37,1, 31.9, 29.6, 29.5, 29.4, 29.3,
29.0, 27.7, 26.8, 23.0, 22.7, 14.1. MS (FAB), m/z (%): 539 (10)
[Μ + H]+. Anal. (C31H58N2O5) C, H, N.
NMR: δ 7.13 (1H, m, CONH), 5.20 (2/3H, t, J ) 7.0 Hz, OCONH),
5.16 (1/3H, t, J ) 7.0 Hz, OCONH), 4.09 (4H, m, 2 × CH,
CH2OCONH), 3.12 (2H, m, CH2NH), 2.40 (2H, m, CH2COOH),
1.84 (4H, m, 2 × CH2), 1.49-1.26 (28H, m, 14 × CH2), 0.88 (6H,
t, J ) 6.6 Hz, 2 × CH3). 13C NMR: δ 176.6, 175.2, 156.4, 72.2,
48.4, 41.2, 34.6, 31.9, 31.8, 29.7, 29.3, 29.2, 26.8, 22.7, 22.6, 14.0.
Anal. (C26H50N2O6) C, H, N.
(4S)-5-(Ethylcarbamoyloxy)-4-(2-hydroxydodecanamido)pen-
1
tanoic Acid (21b). Yield 99%; white solid; mp 84-86 °C. H
NMR: δ 7.20 (1H, m, CONH), 5.31 (1/2H, t, J ) 7.0 Hz,
OCONH), 5.19 (1/2H, t, J ) 7.0 Hz, OCONH), 4.08 (4H, m, 2 ×
CH, CH2OCONH), 3.10 (2H, m, CH2NH), 2.42 (2H, m, CH2COOH),
1.83 (4H, m, 2 × CH2), 1.60-1.25 (16H, m, 8 × CH2), 1.18 (3H,
t, J ) 6.0 Hz, CH3), 0.88 (3H, t, J ) 6.0 Hz, CH3). 13C NMR: δ
176.8, 175.9, 156.7, 72.1, 66.1, 48.1, 35.9, 34.5, 31.9, 30.9, 30.4,
29.6, 26.2, 25.2, 22.6, 15.0, 14.1. Anal. (C20H38N2O6) C, H, N.
(4R)-5-(Ethylcarbamoyloxy)-4-(2-hydroxydodecanamido)pen-
(4S)-4-(2-Oxohexadecanamido) octanoic Acid (38). Yield 72%;
white solid; mp 67-68 °C; [R]D -1.6 (c 0.5, CHCl3). H NMR:
1
δ 6.86 (1H, d, J ) 9.4 Hz, NH), 3.93 (1H, m, CHNH), 2.92 (2H,
t, J ) 7.8 Hz, CH2COCO), 2.35 (2H, t, J ) 6.3 Hz, CH2COOH),
2.10-1.41 (6H, m, 3 × CH2), 1.40-1.05 (26H, m, 13 × CH2),
0.87 (6H, t, J ) 6.0 Hz, 2 × CH3). 13C NMR: δ 199.4, 178.3,
160.1, 49.3, 36.8, 34.7, 31.9, 30.7, 29.9, 29.6, 29.4, 29.3, 29.1, 29.0,
28.0, 23.2, 22.7, 22.4, 14.1, 13.9. Anal. (C24H45NO4) C, H, N.
1
tanoic Acid (21c). Yield 99%; white solid; mp 74-76 °C. H
Synthesis of Carbamates. To a solution of alcohol (1 mmol)
and 4-dimethylaminopyridine (DMAP) (0.24 g, 2 mmol) in dry
CHCl3 (10 mL) was added dropwise, under nitrogen atmosphere,
the isocyanate component (2.4 mmol), and the mixture was stirred
overnight at room temperature. The organic solution was washed
with aqueous HCl (2.5%) and water. The organic layer was dried
over Na2SO4, and the organic solvent was evaporated under reduced
pressure. The residue was purified by column chromatography
[EtOAc-petroleum ether (bp 40-60 °C)].
NMR: δ 7.22 (1H, m, CONH), 5.40 (2/3H, t, J ) 7.0 Hz, OCONH),
5.22 (1/3H, t, J ) 7.0 Hz, OCONH), 4.11 (4H, m, 2 × CH,
CH2OCONH), 3.18 (2H, m, CH2NH), 2.40 (2H, m, CH2COOH),
1.83 (4H, m, 2 × CH2), 1.60-1.25 (16H, m, 8 × CH2), 1.13 (3H,
t, J ) 7.0 Hz, CH3), 0.88 (3H, t, J ) 7.0 Hz, CH3). 13C NMR: δ
176.8, 175.9, 156.7, 72.1, 66.1, 48.1, 35.9, 34.5, 31.9, 30.9, 30.4,
29.6, 26.2, 25.2, 22.6, 15.0, 14.1.
Cleavage of the Benzyloxycarbonyl (Z) Protecting Group. To
a solution of the compound (1 mmol) in EtOH (10 mL) catalyst
10% Pd/C was added, and the mixture was stirred overnight at room
temperature under H2 atmosphere. The catalyst was removed by
filtration over Celite, and the organic solvent was evaporated under
reduced pressure.
Oxidation of N-Protected Amino Alcohols to Aldehydes. The
procedure described above for the oxidation of 2-hydroxyamides
was followed except that the residue was used immediately in the
next step without any purification.
(S)-Methyl 4-(tert-Butoxycarbonyl)-5-(octylcarbamoyloxy)-
pentanoate (19a). Yield 67%; white solid; mp 64-66 °C; [R]D
1
-16.1 (c 0.5, CHCl3). H NMR: δ 4.68 (2H, m, 2 × OCONH),
4.05 (2H, m, CH2OCONH), 3.85 (1H, m, CH), 3.68 (3H, s, CH3O),
3.14 (2H, m, CH2NH), 2.42 (2H, t, J ) 7.6 Hz, CH2COO), 1.79
(2H, m, CH2CH), 1.44 [9H, s, C(CH3)3], 1.28 (12H, m, 6 × CH2),
0.88 (3H, t, J ) 6.6 Hz, CH3). Anal. (C20H38N2O6) C, H, N.
(S)-Methyl 4-(tert-Butoxycarbonyl)-5-(ethylcarbamoyloxy)-
pentanoate (19b). Yield 72%; white solid; mp 70-71 °C; [R]D
Wittig-Horner-Emmons Reaction. A suspension of the
aldehyde (2 mmol), triethyl 4-phosphonocrotonate (2.2 mmol),
LiOH‚H2O (2.2 mmol), and activated molecular sieves (beads, 4-8
mesh, 1.5 g/mmol aldehyde) in THF (overall 0.1 M aldehyde) was
stirred under a nitrogen atmosphere for 12 h at room temperature.
The reaction mixture was filtered through a pad of Celite, and the
filtrate was evaporated under reduced pressure. The residue was
purified by flash column chromatography [EtOAc-petroleum ether
(bp 40-60 °C), 2:8].
1
-20.5 (c 1, CHCl3). H NMR: δ 4.68 (2H, m, 2 × OCONH),
4.06 (2H, m, CH2OCONH), 3.83 (1H, m, CH), 3.67 (3H, s, CH3O),
3.21 (2H, m, CH2NH), 2.41 (2H, t, J ) 7.8 Hz, CH2COO), 1.80
(2H, m, CH2CH), 1.44 [9H, s, C(CH3)3], 1.13 (3H, t, J ) 7.4 Hz,
CH3). 13C NMR: δ 173.7, 156.2, 155.5, 79.5, 66.3, 51.7, 49.6, 35.9,
30.6, 28.3, 27.0, 15.1. Anal. (C14H26N2O6) C, H, N.
(R)-Methyl 4-(tert-Butoxycarbonyl)-5-(ethylcarbamoyloxy)-
pentanoate (19c). Yield 75%; white solid; mp 70-71 °C; [R]D
1
+20.6 (c 1, CHCl3). H NMR: δ 4.75 (2H, m, 2 × OCONH),
(S,2E,4E)-Ethyl 6-(tert-Butoxycarbonyl)deca-2,4-dienoate (8).
4.06 (2H, m, CH2OCONH), 3.81 (1H, m, CH), 3.67 (3H, s, CH3O),
3.20 (2H, m, CH2NH), 2.40 (2H, t, J ) 7.6 Hz, CH2COO), 1.80
(2H, m, CH2CH), 1.42 [9H, s, C(CH3)3], 1.13 (3H, t, J ) 7.2 Hz,
CH3). 13C NMR: δ 173.7, 156.1, 155.5, 79.4, 66.3, 51.7, 49.6, 35.8,
30.5, 28.3, 27.0, 15.1.
1
Yield 66%; colorless oil; [R]D -5.2 (c 1.2, CHCl3). H NMR: δ
7.24 (1H, dd, J1 ) 15.4 Hz, J2 ) 11.0 Hz, CHdCHCHdCHCOO),
6.25 (1H, dd, J1 ) 15.4 Hz, J2 ) 11.0 Hz, CHdCHCHdCHCOO),
5.97 (1H, dd, J1 ) 15.4 Hz, J2 ) 6.2 Hz, CHdCHCHdCHCOO),
5.85 (1H, d, J ) 15.4 Hz, CHCOO), 4.46 (1H, d, J ) 8.0 Hz,
NH), 4.23-4.06 (3H, m, OCH2, CHNH), 1.56-1.17 [18H, m, CH3,
C(CH3)3, 3 × CH2], 0.91 (3H, t, J ) 6.0 Hz, CH3). 13C NMR: δ
167.0, 155.2, 144.0, 143.4, 127.6, 121.2, 79.6, 60.3, 52.3, 34.8,
28.3, 27.8, 22.4, 14.3, 14.0. Anal. (C17H29NO4) C, H, N.
(S)-(2-tert-Butoxycarbonyl)hexyl Octylcarbamate (32). Yield
1
68%; white solid; mp 43-44 °C; [R]D -20.8 (c 1, CHCl3). H
NMR: δ 4.72 (1H, m, OCONH), 4.61 (1H, m, OCONH), 4.04
(2H, m, CH2O), 3.78 (1H, m, CH), 3.14 (2H, m, CH2NH), 1.44
[11H, m, CH2CH, C(CH3)3], 1.27 (16H, br s, 8 × CH2), 0.88 (6H,
t, J ) 6.6 Hz, 2 × CH3). 13C NMR: δ 156.5, 155.5, 79.2, 66.4,
50.0, 41.0, 31.7, 31.6, 29.9, 29.2, 29.1, 28.3, 27.9, 26.7, 22.6, 22.5,
14.0, 13.9. Anal. (C20H40N2O4) C, H, N.
Wittig Reaction of N-Protected r-Aminoaldehydes with
Nonstabilized Ylides. To a stirred suspension of the phosphonium
salt Br-Ph3P+CH2CH2CH3 or Br-Ph3P+CH2CH2C6H5 (2.40 mmol)
in dry toluene (12 mL), a solution of 0.5 M potassium bis-
(trimethylsilyl)amide (KHMDS) (4.8 mL, 2.40 mmol) in toluene
was added dropwise over a period of 5 min at 0 °C under N2. The
bright-red solution was stirred for another 15 min and cooled to
-78 °C, when the solution of the aldehyde in dry toluene (4 mL)
was instantly added, and the temperature was left to rise from -78
°C to room temperature. The light-yellow mixture was stirred at
room temperature for 20 h. The reaction mixture was quenched
with saturated aqueous NH4Cl (20 mL) and extracted with Et2O (3
× 6 mL). The combined organic phases were washed with brine
Saponification of 2-Hydroxyamides Containing Ester Groups.
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) was added
1 N NaOH (2.2 mL, 2.2 mmol), 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

4232 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
and dried over Na2SO4. The solvent was removed, and the residue
was purified by column chromatography [EtOAc-petroleum ether
(bp 40-60 °C), 1:9].
at 1000g for 15 min at 4 °C, the media were decanted, and the cell
pellet was stored at -20 °C.
GV sPLA2 was purified in a manner analogous to that of the
human group IIA sPLA2.71,72 The cells were thawed and resus-
pended in 100 mL of buffer containing 100 mM tris (hydroxy-
methyl)aminomethane HCl (Tris-HCl), pH 8.0, 5 mM ethylenedi-
amine tetraacetic acid (EDTA), 50 mM NaCl, 0.5 mM phenylmethyl-
sulfonyl fluoride, 0.4% (w/v) deoxycholic acid, and 0.4% (v/v)
Triton X-100. Following 20 min of stirring at 4 °C, the suspension
was disrupted by homogenization (Kinematica Polytron, Brinkmann
Instruments) for 15 min on ice and then centrifuged at 12000g for
10 min at 4 °C. The pellet was collected and resuspended in 100
mL of buffer containing 100 mM Tris-HCl, pH 8.0, 5 mM EDTA,
50 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.8% w/v
deoxycholic acid, and 0.8% v/v Triton X-100. After being stirred
at 4 °C for 20 min, the suspension was again disrupted by
homogenization for 15 min on ice and then centrifuged at 12000g
for 10 min at 4 °C. The pellet was resuspended a third time in the
same buffer but this time containing 1% v/v Triton X-100 and no
deoxycholic acid. This suspension was stirred at room temperature
for 20 min and then centrifuged at 12000g for 10 min at 4 °C,
resulting in a pelleting of the bacterial inclusion bodies containing
GV sPLA2. The inclusion body pellet was washed with 50 mL of
50 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 5 M urea for 30 min
at 4 °C and then collected by centrifugation at 12000g at 4 °C.
The inclusion body pellet was solubilized by stirring overnight at
4 °C in 25 mL of 50 mM Tris-HCl, pH 8.5, 0.3 M sodium sulfite,
and 8 M guanidine HCl. Insoluble material was discarded after
centrifugation at 15000g for 30 min at 4 °C. To this clarified protein
solution, 5 mL of 50 mM 2-nitro-5-(sulfothio)benzoate solution was
added and stirred for 45 min at room temperature.
The sulfonated protein mixture was loaded onto a G25 Sephadex
(Amersham Pharmacia) column (4.5 cm × 45 cm) equilibrated with
25 mM Tris-HCl, pH 8.0, 5 M urea, and 5 mM EDTA. The elution
was monitored spectrophotometrically at 280 nm, and the first major
protein peak was collected (30 mL). This sulfonated protein solution
was then loaded onto a G75 Sephadex (Amersham Pharmacia)
column (4.5 cm × 45 cm) pre-equilibrated with 25 mM Tris-HCl,
pH 8.0, 5 M urea, and 5 mM EDTA. The elution was monitored
spectrophotometrically at 280 nm. The fractions were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), and those containing GV sPLA2 (the second major protein
peak) were pooled (50 mL). GV sPLA2 was precipitated after
dialyzing against deionized water for 8 h and against 0.4% v/v
glacial acetic acid overnight at 4 °C. The precipitated GV sPLA2
was washed twice with 50 mL of deionized water and then
resuspended in 5 mL of 50 mM Tris-HCl, pH 7.5, 5 M guanidine
HCl, and 5 mM EDTA. This concentrated protein solution was
then loaded onto a G75 Sephadex column (4.5 cm × 45 cm) pre-
equilibrated with 50 mM Tris-HCl, pH 7.5, 5 M guanidine HCl,
and 5 mM EDTA, and elution was monitored spectrophotometri-
cally at 280 nm. The fractions were analyzed with SDS-PAGE,
and those containing GV sPLA2 (the major protein peak) were
collected and pooled (40 mL).
(R,Z)-tert-Butyl 1-Phenylhex-3-en-2-ylcarbamate (12). Yield
75%; white solid; mp 56-58 °C; [R]D +2.2 (c 1, CHCl3). 1H
NMR: δ 7.27 (5H, m, C6H5), 5.43 (1H, dt, J1 ) 10.6 Hz, J2 ) 7.2
Hz, CHCHdCH2), 5.23 (1H, dd, J1 ) 10.6 Hz, J2 ) 9.6 Hz,
CHCHdCH2), 4.40-4.72 (2H, m, NH, CH), 2.95 (1H, dd, J1 )
13.2 Hz, J2 ) 5.2 Hz, C6H5CHH), 2.72 (1H, dd, J1 ) 13.2 Hz, J2
) 7.4 Hz, C6H5CHH), 2.10-1.80 (2H, m, CH2CH3), 1.46 [9H, s,
C(CH3)3], 0.82 (3H, t, J ) 7.8 Hz, CH3). 13C NMR: δ 154.7, 137.3,
133.9, 129.3, 128.1, 127.8, 125.9, 78.9, 48.9, 41.8, 28.0, 20.6, 13.5.
Anal. (C17H25NO2) C, H, N.
(S,Z)-tert-Butyl 1-Phenyloct-2-en-4-ylcarbamate (15). Yield
71%; white solid; [R]D +1.9 (c 2.1, CHCl3). 1H NMR: δ 7.26 (5H,
m, C6H5), 5.65 (1H, dt, J1 ) 11.0 Hz, J2 ) 7.4 Hz, CH2CHdCH),
5.31 (1H, dd, J1 ) 11.0 Hz, J2 ) 10.0 Hz, CH2CHdCH), 4.69-
4.40 (2H, m, NH, CH), 3.53 (2H, m, CH2C6H5), 1.61 (2H, m, CH2),
1.45 [9H, s, C(CH3)3], 1.31 (4H, m, 2 × CH2), 0.91 (3H, t, J ) 6.4
Hz, CH3). 13C NMR: δ 154.9, 140.4, 131.6, 130.4, 128.4, 125.9,
79.1, 47.9, 35.8, 34.0, 28.4, 27.8, 22.5, 14.0. Anal. (C19H29NO2)
C, H, N.
Oxidation of a Phenyl Group to a Carboxylic Group. To a
solution of 13 or 16a,b (1.00 mmol) in a mixture of EtOAc-
CH3CN-H2O (1:1:8, 30 mL) were added NaIO4 (6.2 g, 29.0 mmol)
and RuCl3 (10 mg, 0.05 mmol). After being stirred overnight at
room temperature, the mixture was diluted with H2O (25 mL) and
EtOAc (50 mL). The organic layer was separated, washed with
H2O (25 mL), dried over Na2SO4, and filtered through a pad of
silica gel 60 (230-400 mesh) and a short pad of Celite. The filtrate
was evaporated under reduced pressure, and the residue was purified
by column chromatography using EtOAc as the eluent.
(S)-3-(2-Oxopentadecanamido)heptanoic Acid (14). Yield
1
61%; white solid; mp 81-82 °C; [R]D -14.5 (c 0.4, CHCl3). H
NMR: δ 7.31 (1H, d, J ) 9 Hz, NHCOCO), 4.20 (1H, m, CH),
2.91 (2H, t, J ) 7.8 Hz, CH2COCO), 2.61 (2H, d, J ) 5 Hz,
CH2COOH), 1.60 (4H, m, 2 × CH2), 1.25 (24H, m, 12 × CH2),
0.88 (6H, t, J ) 6.8 Hz, 2 × CH3). 13C NMR: δ 199.2, 176.4,
159.7, 46.1, 38.2, 36.8, 33.5, 31.9, 29.6, 29.4, 29.3, 29.0, 28.1, 23.1,
22.7, 22.3, 14.1, 13.9. MS (FAB), m/z (%): 384 (100) [M + H]+.
Anal. (C22H41NO4) C, H, N.
(S)-5-(2-Oxopentadecanamido)nonanoic Acid (17a). Yield
1
69%; white solid; mp 76-77 °C; [R]D +0.9 (c 0.8, CHCl3). H
NMR: δ 6.73 (1H, d, J ) 9 Hz, NHCOCO), 3.87 (1H, m, CH),
2.92 (2H, t, J ) 7.8 Hz, CH2COCO), 2.35 (2H, m, CH2COOH),
1.72-1.46 (8H, m, 4 × CH2), 1.25 (24H, m, 12 × CH2), 0.88 (6H,
t, J ) 6.8 Hz, 2 × CH3). 13C NMR: δ 199.6, 178.3, 159.9, 49.3,
36.8, 34.6, 34.2, 33.4, 31.9, 29.6, 29.4, 29.3, 29.0, 27.9, 23.2, 22.7,
22.5, 20.9, 14.1, 13.9. MS (FAB), m/z (%): 412 (100) [M + H]+.
Anal. (C24H45NO4) C, H, N.
(S)-5-(2-Oxohexadecanamido)nonanoic Acid (17b). Yield 37%;
1
white solid; mp 87-89 °C; [R]D +0.8 (c 0.5, CHCl3). H NMR:
δ 6.75 (1H, d, J ) 9.6 Hz, NHCOCO), 3.87 (1H, m, CH), 2.92
(2H, t, J ) 7.6 Hz, CH2COCO), 2.37 (2H, m, CH2COOH), 1.75-
1.40 (8H, m, 4 × CH2), 1.39-1.20 (26H, br s, 13×CH2), 0.88 (6H,
t, J ) 6.6 Hz, 2 × CH3). 13C NMR: δ 199.6, 178.9, 159.9, 49.3,
36.8, 34.6, 34.2, 33.5, 31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 27.9, 23.1,
22.7, 22.4, 21.0, 14.1, 13.9. MS (FAB), m/z (%): 426 (100) [M +
H]+. Anal. (C25H47NO4) C, H, N.
To 10 mL of this sulfonated GV sPLA2 protein solution, 10 mL
of 50 mM Tris-HCl, pH 8.5, 8 mM reduced glutathione, 7 mM
oxidized glutathione, and 10% glycerol buffer were added dropwise
over 3 h with stirring. Stirring was continued for 20 h at room
temperature. The GV sPLA2 solution was dialyzed against 3
volumes of 4 L of 25 mM Tris-HCl, pH 8.5, 0.2 M guanidine HCl,
and 10% glycerol at 4 °C. The purity of the refolded GV sPLA2
was assessed by SDS-PAGE and judged to be higher than 90%.
Group IVA cPLA2 Activity Assays. GIVA cPLA2 activity was
measured as described previously.29,30,73 Pure, native human GIVA
cPLA2 was a generous gift from Dr. Ruth Kramer of Lilly Research
Laboratories. In brief, the liberation of [14C]-AA from phospholipid-
detergent mixed micelles containing 1-palmitoyl-2-[14C]arachi-
donoyl phosphatidylcholine (PAPC), phosphatidylinositol 4,5-
bisphosphate (PIP2), and Triton X-100 (97:3:400 µM, respectively)
was quantitated. The various 2-oxoamides, dissolved at appropriate
concentrations in DMSO, were added to the mixed micelle solution
Human Group V sPLA2 Expression and Purification. The
wild-type human GV sPLA2 cDNA70 was cloned into the pET-
21a(+) vector using the NdeI and EcoRI restriction sites and
transformed into BL21 (DE3) E. coli. The bacteria were streaked
onto LB/ampicillin (100 µg/mL) plates and grown overnight at 37
°C. Single colonies were picked to innoculate two 25-mL LB/
ampicillin (100 µg/mL) precultures, which were grown at 37 °C
overnight. Each 25 mL preculture was used to innoculate 1 L of
LB/ampicillin (100 µg/mL). Following 4 h of incubation at 37 °C,
protein expression was induced by the addition of isopropyl â-D-
thiogalactopyranoside to a concentration of 400 µM. Following an
additional 3 h of incubation at 37 °C, the cultures were centrifuged

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4233
to give a final DMSO concentration of 1% (v/v). The specific
activity of GIVA cPLA2 was calculated and compared to the control
reaction where only DMSO was added. In addition to any
2-oxamide compounds, the final assay buffer contained 10 ng of
GIVA cPLA2, 100 mM 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES), pH 7.5, 80 µM CaCl2, 0.1 mg/mL fatty
acid-free bovine serum albumin, 2 mM dithiothreitol (DTT), 97
µM PAPC (100 000 cpm/assay), 3 µM PIP2, 400 µM Triton X-100,
and 1% (v/v) DMSO. The reaction was initiated with the addition
of enzyme or buffer control, incubated at 40 °C for 10-60 min,
and quenched, extracted, and anlyzed using the modified Dole
assay.74 Because there are no commercially available inhibitors of
GIVA cPLA2 that possess the same mode of inhibition as the
2-oxoamide inhibitors (fast, reversible), we assayed pyrrophenone,
a well-studied inhibitor of GIVA cPLA2,75 received as a generous
gift from Dr. Kohji Hanasaki (Shionogi Research Laboratories of
Shionogi & Co., Ltd.). While an excellent tool, pyrrophenone is
not itself being pursued as a drug at least in part because of its
large size.76 When the GIVA cPLA2 assay conditions described
above were used, pyrrophenone gave an XI(50) of 0.0005 ( 0.0003
mole fraction. In this specific in vitro assay, the most potent
2-oxoamide inhibitor of GIVA cPLA2 is somewhat less potent with
an XI(50) of 0.003 mole fraction,19 and the most potent newly
described 2-oxoamides have XI(50) values of 0.005 mole fraction
(compounds 17a and 17b).
Group V sPLA2 Activity Assays. GV sPLA2 activity was
measured in an assay similar to the assays for GIVA cPLA2 and
GVIA iPLA2. Briefly, the reaction monitored the release of [14C]-
palmitic acid from phospholipid-detergent mixed micelles contain-
ing 1-palmitoyl-2-[14C]palmitoyl phosphatidylcholine (DPPC) and
Triton X-100 (1:4 ratio). Each assay was performed in 500 µL total
volume made up of 100 µL of 5× substrate solution (20 µL of 10
mM Triton X-100 and 80 µL of assay buffer), 390 µL of assay
buffer, 10 µL of GV sPLA2 solution (1 µL of 40 ng/µL stock and
9 µL of assay buffer), and 5 µL of DMSO or 2-oxoamide in DMSO.
The assay buffer was composed of 50 mM Tris-HCl (pH 8.0) and
5 mM CaCl2. The 5× substrate solution was prepared by drying
down the phospholipids (from organic solvent) with N2. The
appropriate volume of 10 mM Triton X-100 was added, heated,
and vortexed until clear. Then assay buffer was added to make a
5× substrate solution. The final assay buffer contained 40 ng of
GV sPLA2, 50 mM Tris-HCl, pH 8.0, 5 mM CaCl2, 100 µM DPPC
(100 000 cpm/assay), 400 µM Triton X-100, and 1% (v/v) DMSO.
The reaction was initiated and terminated as described for GIVA
cPLA2.
Group VIA iPLA2 Activity Assays. Human GVIA iPLA2 was
expressed, purified, and assayed as described previously.19 The
assay is similar to that described for GV sPLA2. The final assay
buffer was composed of 190 ng of GVIA iPLA2, 10 mM HEPES,
pH 7.5, 5 mM EDTA, 2 mM DTT, 1 mM ATP, 100 µM DPPC,
400 µM Triton X-100, and 1% (v/v) DMSO. The final volume for
each assay was 500 µL and contained 100 000 cpm of DPPC. The
reaction was initiated and terminated as described for GIVA
cPLA2.
Rat-Paw Carrageenan-Induced Edema Assay. The anti-
inflammatory activities of specific 2-oxoamides were determined
using the rat-paw carrageenan-induced edema assay as described
previously.30,77 Briefly, the compounds were given intraperitoneally
at the same time as carrageenan was given by intradermal injection.
After 3.5 h the rats were euthanized. Each compound was tested at
four different concentrations on two groups of five animals. The
weight of the uninjected paw was subtracted from the weight of
the injected paw for each animal. The change in paw weight for
treated animals was compared to the control animals and expressed
as percent inhibition of edema. Values for percent inhibition at each
concentration were an average from two different experiments with
a standard error of the mean that was less than 10%. Statistical
significance of the results was established with the Student’s t-test,
P < 0.001. The ED50 value for each compound was determined
using the values for percent inhibition of edema at the four
concentrations tested. Indomethacin was administered at 0.01 mmol/
kg as a standard comparative drug and gave 47% inhibition of
edema.30
Analgesis Screen. The analgesic properties of specific 2-oxo-
amides were determined using the writhing reflex as described
previously.30,78 Briefly, the compounds were given intraperitoneally
with a dose equivalent to the ED50 (derived from the edema assay)
30 min prior to intraperitoneal administration of acetic acid. The
total number of writhes exhibited by each animal in the test group
was compared to that of the vehicle-treated control group. The
percent decrease in writhes was used as a measure of the analgesic
activity of the compound. Sodium acetylsalicylate was administered
intraperitoneally as a control drug and gave 93.4% analgesis at 1
mmol/kg.30
Acknowledgment. This work was supported by NIH Grant
GM 20,501 and University of California Discovery/AnalgesiX
Grant Bio02-10303 (E.A.D.) and by the University of Athens,
Special Account for Research Grants (G.K.).
Note Added in Proof. A paper by Lee et al. (J. Med. Chem.
2007, 50, 1380-1400) describing the discovery and character-
ization of ecopladib, an indole inhibitor of GIVA cPLA2,
appeared after this mancuscript was reviewed. The paper
confirms that a 2-oxoamide (compound 35) inhibits GIVA
cPLA2 with potency comparable to that of a previously
published indole inhibitor of GIVA cPLA2 that was the lead
compound for their study. The potency of ecopladib in animal
models of edema cannot be directly compared to the results
reported herein because of the differential timing and modes of
administration of the compounds in their studies, but the efficacy
of ecopladib confirms that specific GIVA cPLA2 inhibitors have
significant promise as anti-inflammatory drugs.
Supporting Information Available: Elemental analysis results
for the compounds and a table of the 2-oxoamides that do not inhibit
GV sPLA2. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Six, D. A.; Dennis, E. A. The expanding superfamily of phospholipase
A(2) enzymes: classification and characterization. Biochim. Biophys.
Acta 2000, 1488, 1-19.
(2) Leslie, C. C. Regulation of the specific release of arachidonic acid
by cytosolic phospholipase A2. Prostaglandins, Leukotrienes, Essent.
Fatty Acids 2004, 70, 373-376.
(3) Schaloske, R. H.; Dennis, E. A. The phospholipase A(2) superfamily
and its group numbering system. Biochim. Biophys. Acta 2006, 1761,
1246-1259.
(4) 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 phospholipase A2.
Probing the role of histidine, aspartic acid, cysteine, and arginine. J.
Biol. Chem. 1996, 271, 19225-19231.
(5) Dessen, A.; Tang, J.; Schmidt, H.; Stahl, M.; Clark, J. D.; Seehra,
J.; Somers, W. S. Crystal structure of human cytosolic phospholipase
A2 reveals a novel topology and catalytic mechanism. Cell 1999,
97, 349-360.
(6) Bonventre, J. V.; Huang, Z.; Taheri, M. R.; O’Leary, E.; Li, E.;
Moskowitz, M. A.; Sapirstein, A. Reduced fertility and postischaemic
brain injury in mice deficient in cytosolic phospholipase A2. Nature
1997, 390, 622-625.
(7) 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.
(8) Rubin, B. B.; Downey, G. P.; Koh, A.; Degousee, N.; Ghomashchi,
F.; Nallan, L.; Stefanski, E.; Harkin, D. W.; Sun, C.; Smart, B. P.;
Lindsay, T. F.; Cherepanov, V.; Vachon, E.; Kelvin, D.; Sadilek,
M.; Brown, G. E.; Yaffe, M. B.; Plumb, J.; Grinstein, S.; Glogauer,
M.; Gelb, M. H. Cytosolic phospholipase A2-alpha is necessary for
platelet-activating factor biosynthesis, efficient neutrophil-mediated
bacterial killing, and the innate immune response to pulmonary
infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase
activity. J. Biol. Chem. 2005, 280, 7519-7529.

4234 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Six et al.
(9) Funk, C. D. Prostaglandins and leukotrienes: advances in eicosanoid
biology. Science 2001, 294, 1871-1875.
(30) Kokotos, G.; Six, D. A.; Loukas, V.; Smith, T.; Constantinou-
Kokotou, V.; Hadjipavlou-Litina, D.; Kotsovolou, S.; Chiou, A.;
Beltzner, C. C.; Dennis, E. A. Inhibition of group IVA cytosolic
phospholipase A2 by novel 2-oxoamides in vitro, in cells, and in
vivo. J. Med. Chem. 2004, 47, 3615-3628.
(10) Yaksh, T. L.; Kokotos, G.; Svensson, C. I.; Stephens, D.; Kokotos,
C. G.; Fitzsimmons, B.; Hadjipavlou-Litina, D.; Hua, X. Y.; Dennis,
E. A. Systemic and intrathecal effects of a novel series of phospho-
lipase A2 inhibitors on hyperalgesia and spinal prostaglandin E2
release. J. Pharmacol. Exp. Ther. 2006, 316, 466-475.
(11) Meyer, M. C.; Rastogi, P.; Beckett, C. S.; McHowat, J. Phospholipase
A2 inhibitors as potential anti-inflammatory agents. Curr. Pharm.
Des. 2005, 11, 1301-1312.
(31) Constantinou-Kokotou, V.; Peristeraki, A.; Kokotos, C. G.; Six, D.
A.; Dennis, E. A. Synthesis and activity of 2-oxoamides containing
long chain beta-amino acids. J. Pept. Sci. 2005, 11, 431-435.
(32) Preliminary results from this paper were from the following: (a)
Loukas, V.; Six, D. A.; Constantinou-Kokotou, V.; Moutevelis-
Minakakis, P.; Dennis, E. A.; Kokotos, G. Synthesis of 2-Oxoamides
Based on δ-Amino Acids and Study of Their Activity on Human
GIVA Phospholipase A2. In Peptides 2004, Proceedings of the 3rd
International and 28th European Peptide Symposium, Prague, Czech
Republic, September 5-10, 2004; Flegel, M., Fridkin, M., Gilon,
C. Slaninova, J., Eds.; KENES International: Geneva, Switzerland,
2005; pp 615-616. (b) Kokotos, G.; Loukas, V.; Six, D. A.;
Hadjipavlou-Litina, D.; Kotsovolou, S.; Constantinou-Kokotou, V.;
Dennis, E. A. 2-Oxoamides Based on γ-Amino Acids Inhibit Human
GIVA Phospholipase A2 and Present in Vivo Anti-Inflammatory
Activity. In Peptides 2004, Proceedings of the 3rd International and
28th European Peptide Symposium, Prague, Czech Republic, Sep-
tember 5-10, 2004; Flegel, M., Fridkin, M., Gilon, C., Slaninova,
J., Eds.; KENES International: Geneva, Switzerland, 2005; pp 643-
644. (c) Barbayianni, E.; Six, D. A.; Dennis, E. A.; Kokotos, G.
2-Oxoamides Based on Glutamic acid as Inhibitors of Human
GIVA Phospholipase A2. In 4th Hellenic Forum on BioactiVe
Peptides; Cordopatis, P. A., Manessi-Zoupa, E., Pairas, G., Eds.;
TYPORAMA: Patras, Greece, 2005; pp 247-251.
(33) Kokotos, G.; Constantinou-Kokotou, V.; Noula, C.; Nicolaou, A.;
Gibbons, W. A. Synthesis of lipidic amino acid and dipeptide
inhibitors of human platelet phospholipase A2. Int. J. Pept. Protein
Res. 1996, 48, 160-166.
(34) Leanna, M. R.; Sowin, T. J.; Morton, H. E. Synthesis of alpha-amino
and alpha-alkoxy aldehydes via oxoammonium oxidation. Tetrahe-
dron Lett. 1992, 33, 5029-5032.
(35) Ma, Z.; Bobbitt, J. M. Organic oxoammonium salts: a new convenient
method for the oxidation of alcohols to aldehydes and ketones. J.
Org. Chem. 1991, 56, 6110-6114.
(36) Takacs, J. M.; Jaber, M. R.; Clement, F.; Walters, C. A useful
procedure for the preparation of (E,E)-2,4-dienoates: lithium hydroxide-
promoted dienylation by 4-phosphonocrotonate. J. Org. Chem. 1998,
63, 6757-6760.
(37) Kokotos, G.; Noula, C. Selective one-pot conversion of carboxylic
acids into alcohols. J. Org. Chem. 1996, 61, 6994-6996.
(38) Kokotos, G. A convenient one-pot conversion of N-protected amino-
acids and peptides into alcohols. Synthesis 1990, 299-301.
(39) Nunez, M. T.; Martin, V. S. Efficient oxidation of phenyl groups to
carboxylic-acids with ruthenium tetraoxidesa simple synthesis of
(R)-gamma-caprolactone, the pheromone of trogoderma-granarium.
J. Org. Chem. 1990, 55, 1928-1932.
(40) Moutevelis-Minakakis, P.; Sinanoglou, C.; Loukas, V.; Kokotos, G.
Synthesis of non-natural amino acids based on the ruthenium-
catalysed oxidation of a phenyl group to carboxylic acid. Synthesis
2005, 933-938.
(41) Clark, J. D.; Lin, L. L.; Kriz, R. W.; Ramesha, C. S.; Sultzman, L.
A.; Lin, A. Y.; Milona, N.; Knopf, J. L. A novel arachidonic acid-
selective cytosolic PLA2 contains a Ca(2+)-dependent translocation
domain with homology to PKC and GAP. Cell 1991, 65, 1043-
1051.
(42) Street, I. P.; Lin, H. K.; Laliberte, F.; Ghomashchi, F.; Wang, Z.;
Perrier, H.; Tremblay, N. M.; Huang, Z.; Weech, P. K.; Gelb, M. H.
Slow- and tight-binding inhibitors of the 85-kDa human phospholi-
pase A2. Biochemistry 1993, 32, 5935-5940.
(43) Ghomashchi, F.; Loo, R. W.; Balsinde, J.; Bartoli, F.; Apitz-Castro,
R.; Clark, J. D.; Dennis, E. A.; Gelb, M. H. Trifluoromethyl ketones
and fluoromethyl phosphonates as selective inhibitors of group IV
and VI phospholipases A2: structure-function studies with vesicle,
micelle, and membrane assays. Biochim. Biophys. Acta 1999, 1420,
45-56.
(44) Leslie, C. C. Kinetic properties of a high molecular mass arachi-
donoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase
activity. J. Biol. Chem. 1991, 266, 11366-11371.
(45) Reynolds, L.; Hughes, L.; Louis, A. I.; Kramer, R. A.; Dennis, E.
A. Metal ion and salt effects on the phospholipase A2, lysophospho-
lipase, and transacylase activities of human cytosolic phospholipase
A2. Biochim. Biophys. Acta 1993, 1167, 272-280.
(12) Yedgar, S.; Cohen, Y.; Shoseyov, D. Control of phospholipase A(2)
activities for the treatment of inflammatory conditions. Biochim.
Biophys. Acta 2006, 1761, 1373-1382.
(13) Uozumi, N.; Shimizu, T. Roles for cytosolic phospholipase A2alpha
as revealed by gene-targeted mice. Prostaglandins Other Lipid
Mediators 2002, 68-69, 59-69.
(14) Ghosh, M.; Tucker, D. E.; Burchett, S. A.; Leslie, C. C. Properties
of the group IV phospholipase A(2) family. Prog. Lipid Res. 2006,
45, 487-510.
(15) Ghomashchi, F.; Stewart, A.; Hefner, Y.; Ramanadham, S.; Turk,
J.; Leslie, C. C.; Gelb, M. H. A pyrrolidine-based specific inhibitor
of cytosolic phospholipase A(2)alpha blocks arachidonic acid release
in a variety of mammalian cells. Biochim. Biophys. Acta 2001, 1513,
160-166.
(16) Bellido-Reyes, Y. A.; Akamatsu, H.; Kojima, K.; Arai, H.; Tanaka,
H.; Sunamori, M. Cytosolic phospholipase A2 inhibition attenuates
ischemia-reperfusion injury in an isolated rat lung model. Trans-
plantation 2006, 81, 1700-1707.
(17) Malaviya, R.; Ansell, J.; Hall, L.; Fahmy, M.; Argentieri, R. L.; Olini,
G. C., Jr.; Pereira, D. W.; Sur, R.; Cavender, D. Targeting cytosolic
phospholipase A2 by arachidonyl trifluoromethyl ketone prevents
chronic inflammation in mice. Eur. J. Pharmacol. 2006, 539, 195-
204.
(18) Phillips, R. M.; Six, D. A.; Dennis, E. A.; Ghosh, P. In vivo
phospholipase activity of the Pseudomonas aeruginosa cytotoxin
ExoU and protection of mammalian cells with phospholipase A2
inhibitors. J. Biol. Chem. 2003, 278, 41326-41332.
(19) Stephens, D.; Barbayianni, E.; Constantinou-Kokotou, V.; Peristeraki,
A.; Six, D. A.; Cooper, J.; Harkewicz, R.; Deems, R. A.; Dennis, E.
A.; Kokotos, G. Differential inhibition of group IVA and group VIA
phospholipases A(2) by 2-oxoamides. J. Med. Chem. 2006, 49, 2821-
2828.
(20) Winstead, M. V.; Balsinde, J.; Dennis, E. A. Calcium-independent
phospholipase A(2): structure and function. Biochim. Biophys. Acta
2000, 1488, 28-39.
(21) Balsinde, J.; Balboa, M. A. Cellular regulation and proposed
biological functions of group VIA calcium-independent phospholipase
A(2) in activated cells. Cell. Signalling 2005, 17, 1052-1062.
(22) Masuda, S.; Murakami, M.; Ishikawa, Y.; Ishii, T.; Kudo, I. Diverse
cellular localizations of secretory phospholipase A2 enzymes in
several human tissues. Biochim. Biophys. Acta 2005, 1736, 200-
210.
(23) Wijewickrama, G. T.; Kim, J. H.; Kim, Y. J.; Abraham, A.; Oh, Y.;
Ananthanarayanan, B.; Kwatia, M.; Ackerman, S. J.; Cho, W.
Systematic evaluation of transcellular activities of secretory phos-
pholipases A2. High activity of group V phospholipases A2 to induce
eicosanoid biosynthesis in neighboring inflammatory cells. J. Biol.
Chem. 2006, 281, 10935-10944.
(24) Singer, A. G.; Ghomashchi, F.; Le Calvez, C.; Bollinger, J.; Bezzine,
S.; Rouault, M.; Sadilek, M.; Nguyen, E.; Lazdunski, M.; Lambeau,
G.; Gelb, M. H. Interfacial kinetic and binding properties of the
complete set of human and mouse groups I, II, V, X, and XII secreted
phospholipases A2. J. Biol. Chem. 2002, 277, 48535-48549.
(25) Winget, J. M.; Pan, Y. H.; Bahnson, B. J. The interfacial binding
surface of phospholipase A2s. Biochim. Biophys. Acta 2006, 1761,
1260-1269.
(26) Cho, W. Structure, function, and regulation of group V phospholipase
A(2). Biochim. Biophys. Acta 2000, 1488, 48-58.
(27) Satake, Y.; Diaz, B. L.; Balestrieri, B.; Lam, B. K.; Kanaoka, Y.;
Grusby, M. J.; Arm, J. P. Role of group V phospholipase A2 in
zymosan-induced eicosanoid generation and vascular permeability
revealed by targeted gene disruption. J. Biol. Chem. 2004, 279,
16488-16494.
(28) Shirai, Y.; Balsinde, J.; Dennis, E. A. Localization and functional
interrelationships among cytosolic group IV, secreted group V, and
Ca2+-independent group VI phospholipase A2s in P388D1 macro-
phages using GFP/RFP constructs. Biochim. Biophys. Acta 2005,
1735, 119-129.
(46) Loo, R. W.; Conde-Frieboes, K.; Reynolds, L. J.; Dennis, E. A.
Activation, inhibition, and regiospecificity of the lysophospholipase
activity of the 85 kDa cytosolic group IV phospholipase A2. J. Biol.
Chem. 1997, 272, 19214-19219.
(29) Kokotos, G.; Kotsovolou, S.; Six, D. A.; Constantinou-Kokotou, V.;
Beltzner, C. C.; Dennis, E. A. Novel 2-oxoamide inhibitors of human
group IVA phospholipase A(2). J. Med. Chem. 2002, 45, 2891-
2893.

2-Oxoamide Inhibition of Phospholipases
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4235
(47) Conde-Frieboes, K.; Reynolds, L. J.; Lio, Y.; Hale, M.; Wasserman,
H. H.; Dennis, E. A. Activated ketones as inhibitors of intracellular
Ca2+-dependent and Ca2+-independent phospholipase A2. J. Am.
Chem. Soc. 1996, 118, 5519-5525.
(64) Sapirstein, A.; Bonventre, J. V. Specific physiological roles of
cytosolic phospholipase A(2) as defined by gene knockouts. Biochim.
Biophys. Acta 2000, 1488, 139-148.
(65) Serhan, C. N. Preventing injury from within, using selective cPLA2
inhibitors. Nat. Immunol. 2000, 1, 13-15.
(66) Cicero, A. F.; Derosa, G.; Gaddi, A. Combined lipoxygenase/cyclo-
oxygenase inhibition in the elderly: the example of licofelone. Drugs
Aging 2005, 22, 393-403.
(67) Martel-Pelletier, J.; Lajeunesse, D.; Reboul, P.; Pelletier, J. P.
Therapeutic role of dual inhibitors of 5-LOX and COX, selective
and non-selective non-steroidal anti-inflammatory drugs. Ann. Rheum.
Dis. 2003, 62, 501-509.
(68) Mitchell, J. A.; Warner, T. D. COX isoforms in the cardiovascular
system: understanding the activities of non-steroidal anti-inflamma-
tory drugs. Nat. ReV. Drug DiscoVery 2006, 5, 75-86.
(69) Moreau, M.; Daminet, S.; Martel-Pelletier, J.; Fernandes, J.; Pelletier,
J. P. Superiority of the gastroduodenal safety profile of licofelone
over rofecoxib, a COX-2 selective inhibitor, in dogs. J. Vet.
Pharmacol. Ther. 2005, 28, 81-86.
(70) Chen, Y.; Dennis, E. A. Expression and characterization of human
group V phospholipase A2. Biochim. Biophys. Acta 1998, 1394, 57-
64.
(71) Othman, R.; Baker, S.; Li, Y.; Worrall, A. F.; Wilton, D. C. Human
non-pancreatic (group II) secreted phospholipase A2 expressed from
a synthetic gene in Escherichia coli: characterisation of N-terminal
mutants. Biochim. Biophys. Acta 1996, 1303, 92-102.
(72) Baker, S. F.; Othman, R.; Wilton, D. C. Tryptophan-containing mutant
of human (group IIa) secreted phospholipase A2 has a dramatically
increased ability to hydrolyze phosphatidylcholine vesicles and cell
membranes. Biochemistry 1998, 37, 13203-13211.
(73) Yang, H.-C.; Mosior, M.; Johnson, C. A.; Chen, Y.; Dennis, E. A.
Group-specific assays that distinguish between the four major types
of mammalian phospholipase A2. Anal. Biochem. 1999, 269, 278-
288.
(74) Reynolds, L. J.; Washburn, W. N.; Deems, R. A.; Dennis, E. A. Assay
Strategies and Methods for Phospholipases. In Methods in Enzymol-
ogy; Dennis, E. A., Ed.; Academic Press: Orlando, FL, 1991; Vol.
197, pp 3-23.
(75) Ono, T.; Yamada, K.; Chikazawa, Y.; Ueno, M.; Nakamoto, S.;
Okuno, T.; Seno, K. Characterization of a novel inhibitor of cytosolic
phospholipase A2alpha, pyrrophenone. Biochem. J. 2002, 363, 727-
735.
(76) Seno, K.; Okuno, T.; Nishi, K.; Murakami, Y.; Yamada, K.;
Nakamoto, S.; 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.
(77) 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.
(78) Hadjipavlou-Litina, D.; Rekka, E.; Hadjipetrou-Kourounakis, L.;
Kourounakis, P. Synthesis and biological studies of some novel
antiinflammatory aryl-hydroxy-amino-ketones. Eur. J. Med. Chem.
1991, 26, 85-90.
(48) Magrioti, V.; Kokotos, G. Synthetic inhibitors of group IVA and
group VIA phospholipase A2. Anti-Inflammatory Anti-Allergy Agents
Med. Chem. 2006, 5, 189-203.
(49) McKew, J. C.; Foley, M. A.; Thakker, P.; Behnke, M. L.; Lovering,
F. E.; Sum, F. W.; Tam, S.; Wu, K.; Shen, M. W.; Zhang, W.;
Gonzalez, M.; Liu, S.; Mahadevan, A.; Sard, H.; Khor, S. P.; Clark,
J. D. Inhibition of cytosolic phospholipase A2alpha: hit to lead
optimization. J. Med. Chem. 2006, 49, 135-158.
(50) 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.
(51) 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.
(52) Gelb, M. H.; Svaren, J. P.; Abeles, R. H. Fluoro ketone inhibitors of
hydrolytic enzymes. Biochemistry 1985, 24, 1813-1817.
(53) White, S. P.; Scott, D. L.; Otwinowski, Z.; Gelb, M. H.; Sigler, P.
B. Crystal structure of cobra-venom phospholipase A2 in a complex
with a transition-state analogue. Science 1990, 250, 1560-1563.
(54) Reid, R. C. Inhibitors of secretory phospholipase A2 group IIA. Curr.
Med. Chem. 2005, 12, 3011-3026.
(55) Smart, B. P.; Pan, Y. H.; Weeks, A. K.; Bollinger, J. G.; Bahnson,
B. J.; Gelb, M. H. Inhibition of the complete set of mammalian
secreted phospholipases A(2) by indole analogues: a structure-guided
study. Bioorg. Med. Chem. 2004, 12, 1737-1749.
(56) Zeiher, B. G.; Steingrub, J.; Laterre, P. F.; Dmitrienko, A.; Fukiishi,
Y.; Abraham, E. LY315920NA/S-5920, a selective inhibitor of group
IIA secretory phospholipase A2, fails to improve clinical outcome
for patients with severe sepsis. Crit. Care Med. 2005, 33, 1741-
1748.
(57) Bowton, D. L.; Dmitrienko, A. A.; Israel, E.; Zeiher, B. G.; Sides,
G. D. Impact of a soluble phospholipase A2 inhibitor on inhaled
allergen challenge in subjects with asthma. J. Asthma 2005, 42, 65-
71.
(58) Bradley, J. D.; Dmitrienko, A. A.; Kivitz, A. J.; Gluck, O. S.; Weaver,
A. L.; Wiesenhutter, C.; Myers, S. L.; Sides, G. D. A randomized,
double-blinded, placebo-controlled clinical trial of LY333013, a
selective inhibitor of group II secretory phospholipase A2, in the
treatment of rheumatoid arthritis. J. Rheumatol. 2005, 32, 417-423.
(59) OmPraba, G.; Velmurugan, D. Quantitative structure-activity rela-
tionship (QSAR) analysis of a series of indole analogues as inhibitor
for human group V secretory phospholipase A2. Indian J. Biochem.
Biophys. 2006, 43, 154-159.
(60) Smart, B. P.; Pan, Y. H.; Weeks, A. K.; Bollinger, J. G.; Bahnson,
B. J.; Gelb, M. H. Inhibition of the complete set of mammalian
secreted phospholipases A(2) by indole analogues: a structure-guided
study. Bioorg. Med. Chem. 2004, 12, 1737-1749.
(61) Smart, B. P.; Oslund, R. C.; Walsh, L. A.; Gelb, M. H. The first
potent inhibitor of mammalian group X secreted phospholipase A2:
elucidation of sites for enhanced binding. J. Med. Chem. 2006, 49,
2858-2860.
(62) Yu, L.; Deems, R. A.; Hajdu, J.; Dennis, E. A. The interaction of
phospholipase A2 with phospholipid analogues and inhibitors. J. Biol.
Chem. 1990, 265, 2657-2664.
(63) Yu, L.; Dennis, E. A. Defining the dimensions of the catalytic site
of phospholipase A2 using amide substrate analogues. J. Am. Chem.
Soc. 1992, 114, 8757-8763.
JM0613673