2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides

Article abstract of DOI:10.1016/j.bmc.2009.03.069

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A₂ (GIVA cPLA₂ and GVIA iPLA₂) and one human secretory phospholipase A

Full text of DOI:10.1016/j.bmc.2009.03.069

Bioorganic & Medicinal Chemistry 17 (2009) 4833–4843
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Oxoamide inhibitors of phospholipase A2 activity and cellular
arachidonate release based on dipeptides and pseudodipeptides
Efrosini Barbayianni a, Daren Stephens b, Andrej Grkovich b, Victoria Magrioti a, Yuan-Hao Hsu b,
a,
Panagiotis Dolatzas a, Dimitrios Kalogiannidis a, Edward A. Dennis b, , George Kokotos
*
*
a Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece
b Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0601, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities
towards two human intracellular phospholipases A2 (GIVA cPLA2 and GVIA iPLA2) and one human secre-
tory phospholipase A2 (GV sPLA2) were evaluated. Derivatives containing a free carboxyl group are selec-
tive GIVA cPLA2 inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-
oxoamide, which preferentially inhibits GVIA iPLA2. The effect of 2-oxoamides on the generation of ara-
chidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA2
inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC50
Received 23 December 2008
Accepted 21 March 2009
Available online 3 May 2009
Keywords:
Dipeptides
Inhibitors
2-Oxoamides
Phospholipase A2
Pseudodipeptides
value of 2 lM.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
oratories have developed a novel class of GIVA cPLA2 inhibitors
designed to contain the 2-oxoamide functionality and a free car-
Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the
sn-2 ester bond of glycerophospholipids producing free fatty acids
and lysophospholipids.1,2 The PLA2 superfamily currently consists
of 15 groups and many subgroups of which a number of enzymes
differ in primary sequence, structure and catalytic mechanism.1
However, the three predominant types of phospholipase A2
(PLA2) found in human tissues are the cytosolic (such as the GIVA
cPLA2), the secreted (such as the GV sPLA2) and the calcium-
independent (such as the GVIA iPLA2) enzymes. GIVA cPLA2
preferentially hydrolyzes membrane phospholipids containing ara-
chidonic acid (AA), which is converted to a variety of proinflamma-
tory eicosanoids.3 Therefore, inhibiting AA release is of great
therapeutic relevance for the development of new anti-inflamma-
tory drugs. In many cases the activity of GIVA cPLA2 has been
shown to be dependent on or linked to the activity of sPLA2.4–6
GVIA iPLA2 appears to be the primary phospholipase for basal met-
abolic functions within the cell, and perhaps has additional func-
tions in specific cell types; however its role in inflammation is
still unclear.1,2,7,8
boxyl group.12–20 2-Oxoamides based on
c
-aminobutyric acid
(compound AX006, Fig. 1) and the non-natural amino acids -(S)-
c
norleucine or d-(S)-norleucine (compounds AX062 and AX109,
Fig. 1) are potent inhibitors of GIVA cPLA2 presenting in vivo
anti-inflammatory and analgesic activity.12,13,17 In addition, the
2-oxoamide ethyl ester derivative AX048 (Fig. 1), which in vitro
inhibits both GIVA cPLA2 and GVIA iPLA2, presents a potent anti-
hyperalgesic effect.15 Most recently, we have reported that an
amide based on
c-(R)-norleucine is a selective inhibitor of GV
sPLA2.20
To extend our studies on the inhibition of phospholipase A2 by
2-oxoamides, we synthesized a variety of 2-oxoamides based on
dipeptides and pseudodipeptides and we studied their in vitro
activity on three human PLA2 classes: GIVA cPLA2, GVIA iPLA2
and GV sPLA2. Furthermore, to further understand the role and
specificity of 2-oxoamide inhibitors in cells, we studied the effect
of various 2-oxoamides on arachidonic acid release from RAW
264.7 macrophages.
The various classes of intracellular and extracellular PLA2 inhib-
itors have been summarized in recent review articles.9–11 Our lab-
2. Results and discussion
2.1. Design of inhibitors
* Corresponding authors. Tel.: +1 858 534 3055; fax: +1 858 534 7390 (E.A.D.);
tel.: +30 210 727 4462; fax: +30 210 727 4761 (G.K.).
E-mail addresses: edennis@ucsd.edu (E.A. Dennis), gkokotos@chem.uoa.gr (G.
Kokotos).
Dipeptides are considered to be d-amino acid analogues, be-
cause of the distance between the N- and C-terminal groups
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.069

4834
E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
addition, using amide bond isosters is a convenient way to eluci-
O
O
O
O
H
N
H
N
OH
date the role of the amide unit in protein–ligand interactions and
thus, to understand the structure–biological activity relationship.
The methylene ether group constitutes an interesting amide bond
( )13
( )13
OH
OH
O
O
( )2
O
O
surrogate, since the calculated Cia ꢀ Caiþ1 distance of
w[CH2O]
AX006
AX109
pseudodipeptides (3.7 Å) is almost the same as that in a dipeptide
(3.8 Å).22 In the past, ether pseudopeptides have been studied
such as renin23 and cAMP-dependent protein kinase24 inhibitors,
as agonists of substance P,25 and as analogues of the antidiuretic
drug desmopressin.26
O
H
N
O
O
H
N
OR2
( )13
( )13
N
H
R1
O
( )2
O
R1 = (CH2)3CH3, CH(CH3)2
R2 = H, Me, Et, But
AX062
2.2. Synthesis of inhibitors
N-Protected L-norleucine (1a) and L-valine (1b) were coupled
O
O
H
N
with methyl and ethyl glycinate using 1-(3-dimethylaminopro-
pyl)-3-ethyl carbodiimide (WSCI)27 as a condensing agent in the
presence of 1-hydroxybenzotriazole (HOBt) (Scheme 1). Removal
of the Boc group, followed by coupling with 2-hydroxyhexadeca-
noic acid yielded 2-hydroxyamides 3a–c, which were oxidized
either by the Dess–Martin method28 or by the NaOCl/AcNH-TEMPO
method29,30 to the target compounds 4a–c. Following similar reac-
tions, tert-butyl ester derivatives 8a,b were prepared as depicted in
Scheme 2, starting from Z-protected norleucine and valine. 2-
Oxoamides 9a,b, containing a free carboxyl group, were obtained
by treatment of 8a,b with trifluoroacetic acid.
O
H
( )13
( )13
OEt
N
OR2
O
O
AX048
O
O
( )2
R = H, Et, But
Figure 1. Known 2-oxoamide inhibitors and inhibitors designed for this work.
(Fig. 1). Ether pseudopeptide derivatives may be also considered
d-amino acid derivatives. Thus, in the present work we chose
norleucine and -valine as starting materials to synthesize a series
of derivatives that contain the 2-oxoamide functionality and an
amide group or ether group to replace the two methylenes of
the d-amino acid derivative. The replacement of the peptide bond
with a suitable surrogate can increase the stability of peptides to-
wards enzymatic hydrolysis, prolong the half-time of peptide ac-
tion and improve the transport of peptides into cells.21 In
L-
The synthesis of pseudopeptide derivatives is described in
L
Schemes 3 and 4. Boc-
L-norleucinol (10), obtained by reduction
of Boc-
L
-norleucine,31,32 reacted with ethyl bromoacetate using so-
dium hydride in the presence of 18-crown-622 to produce ether 11
(Scheme 3). Removal of Boc group, followed by coupling and oxida-
tion yielded 2-oxoamide 13. Pseudodipeptide derivatives 17 and
18 were synthesized by similar procedures (Scheme 4). However,
for the synthesis of tert-butyl ester derivative the reaction of Z-L-
O
O
OH
( )13
O
H
N
a
b, c
OR2
OR2
BocHN
BocHN
OH
N
H
N
H
R1
R1
O
O
R1
O
2a-c
1a,b
3a-c
R1
R2
1 4
-
a
b
c
O
O
H
N
OR2
n-Bu Me
i-Pr Me
n-Bu Et
d or e
( )13
N
H
O
R1
O
4a-c
Scheme 1. Reagents and conditions: (a) HClꢁH-Gly-OR2, Et3N, WSCI, HOBt, CH2Cl2; (b) 4 N HCl/Et2O; (c) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (d) Dess–Martin
reagent, CH2Cl2; (e) NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, 0 °C.
O
O
OH
( )13
O
H
N
a
b, c
ZHN
ZHN
O
O
OH
N
H
N
H
R
R
O
O
R
O
6a,b
7a,b
5a,b
O
O
O
O
H
N
f
5 9
a
b
H
N
-
R
n-Bu
i-Pr
d or e
OH
O
( )13
N
H
( )13
N
H
O
R
O
O
R
O
8a,b
9a,b
Scheme 2. Reagents and conditions: (a) HClꢁH-Gly-OBut, Et3N, WSCI, HOBt, CH2Cl2; (b) H2, 10% Pd/C, EtOH; (c) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (d) Dess–
Martin reagent, CH2Cl2; (e) NaOCl, AcNH-TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, 0 °C; (f) 50% TFA/CH2Cl2.

E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4835
OH
H
N
a
b, c
BocHN
10
BocHN
11
O
O
OH
( )2
O
( )13
O
O
O
O
( )2
( )2
12
O
H
N
O
d
( )13
O
O
O
13
( )2
Scheme 3. Reagents: (a) BrCH2COOEt, NaH, 18-crown-6, THF; (b) 4 N HCl/Et2O; (c) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (d) Dess–Martin reagent, CH2Cl2.
OH
H
( )13
ZHN
ZHN
O
a
b, c
N
O
OH
( )2
O
O
O
O
O
( )2
( )2
14
15
16
O
O
H
N
H
N
e
d
OH
O
( )13
( )13
O
O
O
O
O
O
( )2
( )2
17
18
Scheme 4. Reagents: (a) BrCH2COOBut, Bu4NHSO4, 50% NaOH, C6H6; (b) H2, 10% Pd/C, EtOH; (c) CH3(CH2)13CHOHCOOH, Et3N, WSCI, HOBt, CH2Cl2; (d) Dess–Martin reagent,
CH2Cl2; (e) 50% TFA/CH2Cl2.
norleucinol with tert-butyl bromoacetate was carried out under
comparable to that of the reference 2-oxoamide inhibitor AX006
[XI(50) 0.024].15 Interestingly, both ethyl ester 13 and tert-butyl es-
ter 17 preferentially inhibited GVIA iPLA2. In particular, derivative
13 is a potent inhibitor of GVIA iPLA2 with a XI(50) value of 0.017,
while high mole fraction (0.091) of the inhibitor caused only 52%
inhibition of GIVA cPLA2.
Among the compounds tested in this study, only two esters
(compounds 8b and 13) were able to reduce GV sPLA2 activity
(around 80% inhibition at 0.091 mole fraction). GV sPLA2 utilizes
a catalytic histidine to activate a water molecule as the nucleophile
in phospholipid hydrolysis. Although there is no serine nucleophile
in GV sPLA2, the 2-oxoamides may resemble the substrate phos-
pholipids or the transition state such that they would bind to the
GV sPLA2 active site and inhibit the enzyme.
phase transfer conditions.33,34
2.3. In vitro inhibition of GIVA cPLA2, GVIA iPLA2 and GV sPLA2
All the inhibitors synthesized in this work were tested for inhi-
bition of GIVA cPLA2, GVIA iPLA2 and GV sPLA2 using mixed micel-
lar assays. Details for the assays have been published
previously.12,13,16,17 The results of the inhibition are presented in
Table 1 using either percent inhibition or XI(50) values. Initially,
the percent inhibition of each PLA2 at 0.091 mole fraction of inhib-
itor was determined. XI(50) values were estimated when the per-
cent inhibition was higher than 90%. The XI(50) is the mole
fraction of the inhibitor in the total substrate interface required
to inhibit the enzyme by 50%. Data for the reference 2-oxoamide
Both the intracellular enzymes GIVA cPLA2 and GVIA iPLA2
share the same catalytic mechanism using active site serine as
the nucleophilic residue. Thus, compounds designed to inhibit
GIVA cPLA2, may show cross reactivity with GVIA iPLA2. Very inter-
estingly, ethyl and tert-butyl esters 4c, 8a, 13 and 17, based on Nle-
Gly or the corresponding ether pseudodipeptide preferentially
inhibit GVIA iPLA2. Up to now, we have shown that 2-oxoamide es-
ters may inhibit both GIVA cPLA2 and GVIA iPLA2 either with sim-
ilar potency or with a preference for GIVA cPLA2.16 However, we
identified for the first time in the course of the present work sev-
eral 2-oxoamides, such as 8a or 13, which show higher inhibition
against GVIA iPLA2 than GIVA cPLA2. The dose-response curves of
the inhibition of GVIA iPLA2 by these inhibitors are shown in
Figure 2. It seems that the replacement of the amino acid unit of a
2-oxoamide inhibitor by a dipeptide unit may shift the selectivity
in favour of GVIA iPLA2. Since there is a lack of potent and selective
inhibitors for GVIA iPLA2,35 the findings of the present study may
help in designing 2-oxoamides presenting selectivity for this
particular PLA2.
inhibitors AX006 and AX048 are included in Table
comparison.
1 for
Dipeptide-based 2-oxoamides 9a and 9b containing a free car-
boxyl group inhibited GIVA cPLA2 without affecting the activity
of the other intracellular enzyme GVIA iPLA2. This observation is
in full agreement with our previous report, that amino acid-based
2-oxoamides containing a free carboxyl group are selective inhibi-
tors of GIVA cPLA2.16,17
Methyl ester 4a based on dipeptide Nle-Gly and tert-butyl ester
8b based on dipeptide Val-Gly, inhibited both GIVA cPLA2 and GVIA
iPLA2, showing a small preference for GIVA cPLA2. The methyl ester
4b based on dipeptide Val-Gly lost the activity against both GIVA
cPLA2 and GVIA iPLA2. To the contrary, the tert-butyl ester 8a
and the ethyl ester 4c, both based on Nle-Gly dipeptide presented
potent inhibition of GVIA iPLA2 with XI(50) values of 0.011 and
0.020, respectively. At the same time, both 8a and 4c inhibited very
weakly GIVA cPLA2 and GV sPLA2.
A bioisosteric replacement of the amide bond in compounds 4c,
8a and 9a by an ether group, resulted to the structurally related
compounds 13, 17 and 18. Pseudodipeptide derivative 18, selec-
tively inhibited GIVA cPLA2, as expected, due to the free carboxyl
group. This derivative is the most potent inhibitor of GIVA cPLA2
[XI(50) 0.017] identified in the present work and its potency is
2.4. Ex vivo inhibition of cellular arachidonic acid release
Arachidonic acid (AA) is the ideal metabolic indicator of GIVA
cPLA2 activity, since it is the product of GIVA cPLA2-mediated

4836
E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
Table 1
Inhibition of PLA2 by 2-oxoamides based on dipeptides and pseudodipeptidesa
Compound
Structure
GIVA cPLA2
GVIA iPLA2
% Inhibition
GV sPLA2
% Inhibition
XI(50)
% Inhibition
XI(50)
XI(50)
O
O
H
N
O
( )13
N
H
( )2
4a
0.021 0.009
0.045 0.018
65
O
O
O
O
O
H
N
O
O
4b
4c
25
73
N.D.a
N.D.a
( )13
N
H
O
O
O
O
O
H
N
( )13
N
H
0.020 0.002
0.011 0.001
0.044 0.025
63
( )2
O
O
H
N
O
( )13
N
H
( )2
8a
8b
9a
9b
13
72
59
O
O
O
O
H
N
O
0.028 0.015
0.035 0.014
0.061 0.017
78
( )13
N
H
O
O
O
O
O
O
H
N
OH
( )13
N.D.a
N.D.a
N.D.a
81
N
H
( )2
O
O
H
N
N.D.a
OH
O
( )13
N
H
O
O
O
O
O
H
N
( )13
O
52
44
0.017 0.002
( )2
O
H
N
O
( )13
O
17
18
81
57
O
O
O
O
( )2
O
H
N
OH
( )13
O
0.017 0.002
N.D.a
N.D.a
( )2

E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4837
Table 1 (continued)
Compound
Structure
GIVA cPLA2
GVIA iPLA2
GV sPLA2
% Inhibition
XI(50)
% Inhibition
XI(50)
% Inhibition
N.D.a,b
XI(50)
O
O
O
H
N
AX006
0.024 0.015b
0.022 0.009b
( )13
OH
O
O
H
N
AX048
0.027 0.009b
( )13
OEt
O
a
Average percent inhibition and standard error (n = 3) reported for each compound at 0.091 mole fraction. N.D. signifies compounds with less than 25% inhibition (or no
detectable inhibition).
b
Data taken from Ref. 15.
8a
a
b
13
100
80
60
40
20
0
100
80
60
40
20
0
0.00
0.02
0.04
0.06
0.08
0.10
0.00
0.02
0.04
0.06
0.08
0.10
Inhibitor Mole Fraction
Inhibitor Mole Fraction
Figure 2. Dose–response curves for 2-oxoamide inhibitors of GVIA iPLA2. Inhibition of the activity of human GVIA iPLA2 by inhibitors (a) 8a and (b) 13 was tested on mixed-
micelles containing 100 M PAPC and 400 M Triton X-100. Inhibition curves were generated by Graphpad Prism using a non-linear regression (one-site binding model—
l
l
hyperbola) to calculate the reported XI(50) values.
catalysis. In addition to some of the new 2-oxoamides based on
dipeptides and pseudodipeptides that were synthesized in this
work, a series of potent 2-oxoamide in vitro inhibitors of GIVA
cPLA2, previously reported by us,12,13,16,17,19,20 were further tested
in a cellular ex vivo system to investigate whether they reduced
cellular AA production. For this purpose, RAW 264.7 macrophages
were preincubated with 25 lM concentrations of the inhibitor
prior to treatment of the cells with Kdo2-Lipid A. Subsequently,
AA release was quantitated from the supernatants.
The results of our studies for 21 2-oxoamides are summarized in
Table 2. Note that some of the compounds led to an actual increase
or activation of AA release which is not unusual in this kind of assay
due to detergent or other non-specific effects. Those compounds
giving an activation of AA release were not evaluated further. Com-
pounds 18, 19,12 20,12 25, 26, 3016 and 35,17 which contain a free
carboxyl group and are potent and selective inhibitors of GIVA
cPLA2, induce a significant reduction of AA release. Derivatives 26
ered as a d-amino acid analogue, lead to a considerable AA reduction
in cells. Both molecules are strong inhibitors of GIVA cPLA2.
Although production of arachidonic acid can be catalyzed by
other phospholipases as well, such as GVIA iPLA2 and GV sPLA2,
most of the non-selective inhibitors of the three phospholipases
tested here, did not show a significant inhibitory effect on AA re-
lease. Compounds 23,17 24,15 27,20 29,16 3120 and 36,20 carry an
esterified carboxyl group. In compound 32,19 the ester group has
been replaced by a sulfonamide group. They all inhibit GIVA cPLA2
to a high extent, though not selectively, since they inhibit also both
GVIA iPLA2 and GV sPLA2.
Dipeptide inhibitor 8b reduced the cellular release of AA by 30%,
while derivative 18 reduced AA production by 68%. Interestingly,
the 2-oxoamide inhibitor 18, which contains a free carboxyl group,
leads to more than double the AA release inhibition, indicating the
importance of selectivity of an inhibitor against GIVA cPLA2.
and 30, based on
c-norleucine, cause the highest inhibition percent-
2.5. IC50 values for arachidonic acid inhibition
age in AA release. Both strongly inhibit GIVA cPLA2. Comparison be-
tween compounds 20 and 26 indicates the importance of the long
Dose–response curves were measured for the seven synthetic
inhibitors that reduced AA release the most (see Table 2) in order
to calculate the IC50 values. All of these contained free carboxylic
acids and were specific for GIVA cPLA2. The IC50 values for these se-
ven compounds are summarized in Table 3. The individual dose–
response curve for the inhibition of AA release from RAW 264.7
cells stimulated with Kdo2-Lipid A in the presence of 18 is depicted
aliphatic chain. Compound 19, based on
c-aminobutyric acid and
compound 25, based on -leucine, cause significant inhibition,
c
though to a lesser extent, of AA production. Moderate inhibitors of
GIVA cPLA2 result to a lower inhibition of AA release (compounds
21,17 2820). Compound 35,17 a d-norleucine derivative, and com-
pound 18, based on an pseudodipeptide, which also can be consid-

4838
E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
Table 2 (continued)
Table 2
Effect of 2-oxoamide inhibitors on Kdo2-Lipid A stimulated AA release from RAW
264.7 macrophages
Compound Structure
AA release inhibition
(%)
Compound
Structure
AA release inhibition
(%)
O
O
O
H
N
OH
O
( )13
O
O
O
30
74
8
H
N
O
19
52
56
5
6
( )2
OH
OH
( )13
O
O
H
N
O
O
O
H
N
31
32
33
34
35
36
37
18
Activation
Activation
( )13
( )9
20
21
22
O
O
O
( )2
O
O
O
O
O
H
N
S
O
N
O
( )13
H
N
O
O
OH
( )9
27
6
O
( )2
H
N
( )13
16
11
59
6
6
4
O
( )2
H
N
OH
( )12
Activation
O
O
O
( )2
H
N
O
( )13
O
O
O
O
O
O
H
N
O
( )9
O
H
N
23
24
25
26
27
28
8b
29
Activation
Activation
O
OH
( )13
( )7
O
O
N
H
( )2
O
H
N
O
O
( )13
H
N
O
O
O
Activation
( )13
O
O
O
O
O
O
H
N
OH
OH
O
( )13
62
5
O
O
H
N
30
68
2
4
O
( )13
O
O
O
H
N
O
( )13
H
N
79 0.5
OH
O
O
O
O
( )13
( )2
O
( )2
O
H
N
( )13
Activation
( )2
O
O
H
N
in Figure 3. Inhibitors 19, 25, 26 and 30 all display IC50 values of
approximately 25 M whereas 20, 35 and 18 display values of
10, 7 and 2 M, respectively. Interestingly, inhibitors 35 and 18,
which contain the carboxylic acid functional group spaced at the
d position relative to the 2-oxoamide moiety, demonstrated the
l
OH
( )13
6
10
2
l
( )2
O
O
lowest IC50 values at 7 and 2
linked inhibitors, 19, 20, 25, 26 and 30, all displayed higher IC50
values at 25, 10, 25, 25 and 25 M. These data suggest that inhib-
itors with d structural spacing display more effective AA release
inhibition than structural spacing within this inflammatory cel-
lM, respectively. In contrast, the five
H
N
O
c
30
N
( )13
l
H
O
O
O
O
c
O
O
H
N
lular model. Of course, this specificity may relate to the ability of
the compound to be taken up by the cell and other factors, but
these results demonstrate that the oxoamides are able to inhibit
within the cellular milieu.
( )13
Activation
( )2

E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4839
Table 3
cPLA2 inhibitors and also preferentially inhibit AA release intracel-
lularly. 2-Oxoamides ethyl and tert-butyl esters, based on the
dipeptide Nle-Gly or the corresponding ether pseudodipeptide
preferentially inhibit the other major intracellular enzyme GVIA
iPLA2. Three of them are potent inhibitors of GVIA iPLA2 with
XI(50) values between 0.011 and 0.020. We demonstrated that
selective GIVA cPLA2 inhibitors inhibit AA release in RAW 264.7
macrophages and a 2-oxoamide based on a pseudodipeptide inhib-
IC50 values for the inhibition of arachidonic acid release
Compound
Structure
IC50
25
(lM)
O
O
O
H
N
19
OH
OH
( )13
O
its AA release with an IC50 value of 2 lM.
O
O
H
N
( )9
20
25
26
30
35
18
10
25
25
25
7
4. Experimental
4.1. General
O
O
O
O
O
O
( )2
O
O
O
H
N
OH
OH
OH
Melting points were determined on a Buchi 530 apparatus and
are uncorrected. Specific rotations were measured at 25 °C on a
Perkin–Elmer 343 polarimeter using a 10 cm cell. NMR spectra
were recorded on a Varian Mercury (200 MHz) spectrometer. All
amino acid derivatives were purchased from Fluka Chemical Co.
Electron spray ionization (ESI) mass spectra were recorded on a
Finnigan, Surveyor MSQ Plus spectrometer. TLC plates (Silica Gel
60 F254) and Silica Gel 60 (70–230 or 230–400 mesh) for column
chromatography were purchased from Merck. Visualization of
spots was effected with UV light and/or phosphomolybdic acid
and/or ninhydrin, both in EtOH stain. THF was dried by standard
procedures and stored over molecular sieves or Na. All other sol-
vents and chemicals were reagent grade and used without further
purification. Inhibitors 25 and 26 were prepared according to pro-
cedures described in the literature.13,17
( )13
O
H
N
( )13
( )2
( )2
( )2
O
H
N
( )13
O
H
N
OH
( )13
O
O
4.2. Synthesis of 2-oxoamide inhibitors
O
4.2.1. General method for the coupling of 2-hydroxy-
hexadecanoic acid with amino components
H
N
OH
O
( )13
2
To a stirred solution of 2-hydroxy-hexadecanoic acid (0.27 g,
1.0 mmol) and hydrochloride amino component (1.0 mmol) in
CH2Cl2 (10 mL), Et3N (0.3 mL, 2.2 mmol) and subsequently 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (WSCI)
(0.21 g, 1.1 mmol) and 1-hydroxybenzotriazole (HOBt) (0.14 g,
1.0 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 evap-
orated under reduced pressure. The residue was purified by col-
umn-chromatography using CHCl3–CHCl3/MeOH 99:1 as eluent.
( )2
4.2.1.1. Methyl 2-((S)-2-(2-hydroxyhexadecanamido)hexanami-
do)acetate (mixture of diastereomers) (3a). Yield 68%; white so-
lid; mp 78–80 °C; 1H NMR (200 MHz, CDCl3) d 7.53–7.45 (m, 1H),
7.40 (d, J = 8.6 Hz, 1H), 4.59–4.43 (m, 1H), 4.15–4.06 (m,1H),
4.02–3.94 (m, 2H), 3.71 (s, 3H), 1.89–1.53 (m, 4H), 1.23 (br s,
28H), 0.98–0.80 (m, 6H); 13C NMR (50 MHz, CDCl3) d 175.1
(174.8), 172.7 (172.5), 170.1 (170.0), 72.0 (71.9), 52.4 (52.7),
52.2, 41.1, 34.8 (34.6), 32.0 (31.8), 29.6, 29.5, 29.3, 27.6, 27.5,
25.0, 22.6, 22.3, 22.2, 14.0, 13.8. Anal. Calcd for C25H48N2O5: C,
65.75; H, 10.59; N, 6.13. Found: C, 65.49; H, 10.78; N, 6.01.
Figure 3. Dose–response curve for the inhibition of AA release from RAW 264.7
cells by inhibitor 18. RAW 264.7 cells were preincubated with the indicated
concentrations of 18 prior to stimulation with Kdo2-Lipid A and AA quantitation.
The data is graphically expressed in terms of percent inhibition of AA release as a
function of the concentration of 18. The curve is a fit of the data to a non-linear
regression function using the sigma plot program. Based on the maximal inhibition
4.2.1.2. Methyl 2-((S)-2-(2-hydroxyhexadecanamido)-3-methyl-
butanamido)acetate (mixture of diastereomers) (3b). Yield 73%;
mp 109–111 °C; 1H NMR (200 MHz, CDCl3) d 7.34–7.15 (m, 2H),
4.43–4.27 (m, 1H), 4.10–3.96 (m, 3H), 3.75 (s, 3H), 2.24–2.08 (m,
1H), 1.87–1.63 (m, 2H), 1.26 (br s, 24H), 1.05–0.91 (m, 6H), 0.88
(t, J = 6.6 Hz, 3H). Anal. Calcd for C24H46N2O5: C, 65.12; H, 10.47;
N, 6.33. Found: C, 64.98; H, 10.68; N, 6.18.
observed in this assay, half maximal inhibition occurs at about 2 lM of 18.
3. Conclusions
In conclusion, we have synthesized new 2-oxoamides based on
dipeptides and pseudodipeptides and we have confirmed that
derivatives containing a free carboxyl group are selective GIVA

4840
E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4.2.1.3. Ethyl 2-((S)-2-(2-hydroxyhexadecanamido)hexanami-
do)acetate (mixture of diastereomers) (3c). Yield 64%; white so-
lid; mp 77–79 °C; 1H NMR (200 MHz, CDCl3) d 7.31–7.07 (m, 2H),
4.59–4.42 (m, 1H), 4.25–4.06 (m, 3H), 4.02–3.95 (m, 2H), 1.97–
1.51 (m, 4H), 1.24 (br s, 31H), 0.97–0.80 (m, 6H); 13C NMR
(50 MHz, CDCl3) d 174.9 (174.5), 172.4 (172.1), 169.7 (169.5),
72.1 (72.0), 61.5, 52.5 (52.7), 41.3, 34.6 (34.8), 31.9, 29.7, 29.4,
29.3, 27.6, 25.0, 22.7, 22.3, 14.1, 13.9. Anal. Calcd for C26H50N2O5:
C, 66.35; H, 10.71; N, 5.95. Found: C, 66.19; H, 10.99; N, 5.89.
4.2.2.1.
(S)-Methyl 2-(2-(2-oxohexadecanamido)hexanami-
do)acetate (4a). Yield 85%; white solid; mp 65–67 °C;
[a]
D = ꢀ21.8 (c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.50 (d,
J = 8.4 Hz, 1H), 6.74 (t, J = 5.4 Hz, 1H), 4.49–4.37 (m, 1H), 4.17–
3.95 (m, 2H), 3.75 (s, 3H), 2.89 (t, J = 7.6 Hz, 2H), 2.05–1.47 (m,
4H), 1.24 (br s, 26H), 0.97–0.81 (m, 6H); 13C NMR (50 MHz, CDCl3)
d 198.3, 170.9, 170.0, 160.0, 53.0, 52.4, 41.1, 36.8, 31.9, 31.8, 29.6,
29.5, 29.4, 29.3, 29.0, 27.4, 23.0, 22.6, 22.3, 14.1, 13.8; MS (ESI): m/z
(%): 477 (77) [M+Na]+. Anal. Calcd for C25H46N2O5: C, 66.04; H,
10.20; N, 6.16. Found: C, 66.19; H, 10.13; N, 6.21.
4.2.1.4. tert-Butyl 2-((S)-2-(2-hydroxyhexadecanamido)hexa-
namido)acetate (mixture of diastereomers) (7a). Yield 69%;
white solid; mp 50–52 °C; 1H NMR (200 MHz, CDCl3) d 7.37–7.26
(m, 1H), 7.23–7.08 (m, 1H), 4.58–4.45 (m, 1H), 4.15–4.03 (m,
1H), 3.97–3.81 (m, 2H), 1.98–1.52 (m, 4H), 1.44 (s, 9H), 1.24 (br
s, 28H), 0.98–0.79 (m, 6H); 13C NMR (50 MHz, CDCl3) d 174.9
(174.6), 172.4 (172.1), 168.6 (168.7), 82.2, 72.1 (72.0), 52.9
(52.5), 42.0, 34.8 (34.6), 32.1, 31.8, 29.6, 29.5, 29.3, 27.9, 27.6,
27.5, 25.0, 22.6, 22.3, 22.2, 14.0, 13.8. Anal. Calcd for
C28H54N2O5: C, 67.43; H, 10.91; N, 5.62. Found: C, 67.28; H,
11.08; N, 5.47.
4.2.2.2. (S)-Ethyl 2-(2-(2-oxohexadecanamido)hexanamido)ace-
tate (4c). Yield 76%; white solid; mp 63–65 °C; [
a]
D = ꢀ20.8 (c 0.5
CHCl3); 1H NMR (200 MHz, CDCl3) d 7.51 (d, J = 8.4 Hz, 1H), 6.76 (t,
J = 5.2, 1H), 4.50–4.36 (m, 1H), 4.19 (q, J = 7 Hz, 2H), 4.12–3.91 (m,
2H), 2.88 (t, J = 7.4 Hz, 2H), 2.05–1.45 (m, 4H), 1.26 (br s, 29H),
0.95–0.80 (m, 6H); 13C NMR (50 MHz, CDCl3) d 198.3, 170.9,
169.5, 160.1, 61.6, 53.0, 41.3, 36.8, 32.0, 31.9, 29.6, 29.5, 29.4,
29.3, 29.0, 27.5, 23.0, 22.6, 22.3, 14.1, 13.8; MS (ESI): m/z (%):
491 (100) [M+Na]+, 469 (55) [M+]+. Anal. Calcd for C26H48N2O5:
C, 66.63; H, 10.32; N, 5.98. Found: C, 66.58; H, 10.39; N, 5.91.
4.2.1.5. tert-Butyl 2-((S)-2-(2-hydroxyhexadecanamido)-3-methyl-
butanamido)acetate (mixture of diastereomers) (7b). Yield 81%;
white solid; mp 81–83 °C; 1H NMR (200 MHz, CDCl3) d 7.25 (d,
J = 8.8 Hz, ½H), 7.16 (d, J = 8.8 Hz, ½H), 6.96 (t, J = 4.8 Hz, ½H), 6.88
(t, J = 4.8 Hz, ½H), 4.34–4.22 (m, 1H), 4.18–4.03 (m, 1H), 3.92–3.81
(m, 2H), 2.23–2.05 (m, 1H), 1.84–1.48 (m, 2H), 1.42 (s, 9H), 1.21 (br
s, 24H), 0.93 (t, J = 5.8 Hz, 6H), 0.84 (t, J = 6.6 Hz, 3H); 13C (50 MHz,
CDCl3) NMR d 174.7 (175.0), 171.7 (171.4), 168.8 (168.6), 82.4, 72.3
(72.0), 58.1 (57.9), 42.0, 34.9 (34.6), 31.9, 30.8 (30.7), 29.7, 29.6,
29.4, 29.3, 28.0, 25.0, 22.6, 19.3, 18.1, 14.1. Anal. Calcd for
C27H52N2O5: C, 66.90; H, 10.81; N, 5.78. Found: C, 66.65; H, 10.98;
H, 5.62.
4.2.2.3. (S)-tert-Butyl 2-(2-(2-oxohexadecanamido)hexanami-
do)acetate (8a). Yield 85%; white solid; mp 38–39 °C;
[a]
D = ꢀ17.2 (c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.47 (d,
J = 8.4 Hz, 1H), 6.57 (t, J = 5 Hz, 1H), 4.48–4.35 (m, 1H), 4.03–3.80
(m, 2H), 2.89 (t, J = 8 Hz, 2H), 2.02–1.52 (m, 4H), 1.45 (s, 9H),
1.24 (br s, 26H), 0.97–0.79 (m, 6H); 13C NMR (50 MHz, CDCl3) d
198.3, 170.7, 168.5, 160.1, 82.4, 53.1, 42.0, 36.8, 32.0, 31.9, 29.6,
29.5, 29.4, 29.3, 29.0, 28.0, 27.5, 23.1, 22.6, 22.3, 14.1, 13.8; MS
(ESI): m/z (%): 519 (100) [M+Na]+, 497 (32) [M+H]+. Anal. Calcd
for C28H52N2O5: C, 67.70; H, 10.55; N, 5.64. Found: C, 67.58; H,
10.73; N, 5.58.
4.2.2.4. (S)-Ethyl 2-(2-(2-oxohexadecanamido)hexyloxy)acetate
4.2.1.6. Ethyl
2-((S)-2-(2-hydroxyhexadecanamido)hexyloxy)
(13). Yield 88%; white solid; mp 41–43 °C; [
a]
D = ꢀ8.4 (c 0.5
CHCl3); 1H NMR (200 MHz, CDCl3) d 7.29 (d, J = 9.6 Hz, 1H), 4.21
(q, J = 7.4 Hz, 2H), 4.06 (s, 2H), 4.05–3.91 (m, 1H), 3.64 (dd,
J1 = 9.6 Hz, J2 = 4.0 Hz, 1H), 3.52 (dd, J1 = 9.4 Hz, J2 = 3.8 Hz, 1H),
2.90 (t, J = 6.6 Hz, 2H), 1.71–1.48 (m, 4H), 1.24 (br s, 29H), 0.96–
0.80 (m, 6H); 13C NMR (50 MHz, CDCl3) d 199.2, 170.3, 159.9,
72.5, 68.2, 60.9, 49.4, 36.8, 31.9, 30.9, 29.6, 29.5, 29.4, 29.3, 29.0,
28.0, 23.1, 22.6, 22.4, 14.1, 13.9; MS (ESI): m/z (%): 478 (100)
[M+Na]+. Anal. Calcd for C26H49NO5: C, 68.53; H, 10.84; N, 3.07.
Found: C, 68.65; H, 10.71; N, 3.12.
acetate (mixture of diastereomers) (12). Yield 69%; waxy white
solid; 1H NMR (200 MHz, CDCl3) d 7.03–6.92 (m, 1H), 4.25–3.95
(m, 6H), 3.65–3.42 (m, 2H), 1.83–1.53 (m, 4H), 1.25 (br s, 31H),
0.95–1.79 (m, 6H); 13C NMR (50 MHz, CDCl3) d 174.2 (173.9),
170.8 (170.7), 73.2 (73.1), 72.2 (71.8), 68.0 (68.1), 61.0, 48.5
(48.8), 34.9 (34.7), 31.8, 31.0, 29.6, 29.5, 29.4, 29.3, 28.1, 24.9,
24.8, 22.6, 22.5, 14.0, 13.9. Anal. Calcd for C26H51NO5: C, 68.23;
H, 11.23; N, 3.06. Found: C, 68.04; H, 11.34; N, 2.91.
4.2.1.7. tert-Butyl 2-((S)-2-(2-hydroxyhexadecanamido)hexyl-
oxy)acetate (mixture of diastereomers) (16). Yield 77%; oil; 1H
NMR (200 MHz, CDCl3) d 7.05 (d, J = 8.8 Hz, 1H), 4.17–3.96 (m,
2H), 3.93 (s, 2H), 3.73 (br s, 1H), 3.57 (dd, J1 = 9.6 Hz, J2 = 3.6 Hz,
1H), 3.45 (dd, J1 = 8.8 Hz, J2 = 3.6 Hz, 1H), 1.90–1.55 (m, 4H),
1.45 (s, 9H), 1.23 (br s, 28H), 0.98–1.78 (m, 6H); 13C NMR
(50 MHz, CDCl3) d 174.2 (174.0), 170.1, 82.0, 73.2, 72.2 (71.7),
68.4 (68.6), 48.7 (48.9), 34.7 (35.0), 31.9, 31.0, 29.6, 29.5, 29.3,
28.2, 28.0, 24.9, 24.8, 22.6, 22.5, 14.0, 13.9. Anal. Calcd for
C28H55NO5: C, 69.23; H, 11.41; N, 2.88. Found: C, 69.01; H,
11.59; N, 2.73.
4.2.2.5. (S)-tert-Butyl 2-(2-(2-oxohexadecanamido)hexyloxy)
acetate (17). Yield 93%; white solid; low mp; [
a
]
D = ꢀ10.8 (c 0.5
CHCl3); 1H NMR (200 MHz, CDCl3) d 7.26 (d, J = 8.8 Hz, 1H), 4.03–
3.85 (m, 3H), 3.61 (dd, J1 = 9.4 Hz, J2 = 4.0 Hz, 1H), 3.49 (dd,
J1 = 9.4 Hz, J2 = 4.0 Hz, 1H), 2.89 (t, J = 7.2 Hz, 2H), 1.69–1.48 (m,
4H), 1.45 (s, 9H), 1.23 (br s, 26H), 0.96–0.79 (m, 6H); 13C NMR
(50 MHz, CDCl3) d 199.2, 169.4, 160.0, 81.7, 72.4, 68.7, 49.4, 36.7,
31.8, 31.0, 29.6, 29.5, 29.4, 29.3, 29.0, 28.0, 23.1, 22.6, 22.4, 14.0,
13.9; MS (ESI): m/z (%): 506 (57) [M+Na]+. Anal. Calcd for
C28H53NO5: C, 69.52; H, 11.04; N, 2.90. Found: C, 69.57; H, 11.08;
N, 2.83.
4.2.2. General method for the oxidation of 2-hydroxy-amides.
Method A
4.2.3. General method for the oxidation of 2-hydroxy-amides.
Method B
To a solution of 2-hydroxy-amide (1 mmol) in dry CH2Cl2
(10 mL) Dess–Martin periodinane was added (0.64 g, 1.5 mmol)
and the mixture was stirred for 1 h at room temperature. The or-
ganic solution was washed with 10% aqueous NaHCO3, dried over
Na2SO4 and the organic solvent was evaporated under reduced
pressure. The residue was purified by column-chromatography
using CHCl3 as eluent.
To a solution of 2-hydroxy-amide (1.0 mmol) in a mixture of
toluene (3 mL) and EtOAc (3 mL) a solution of NaBr (0.11 g,
1.1 mmol) in water (0.5 mL) was added followed by AcNH-TEMPO
(2.2 mg, 0.01 mmol). To the resulting biphasic system, which was
cooled at 0 °C, an aqueous solution of 0.35 M NaOCl (3.1 mL,

E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4841
1.1 mmol) containing NaHCO3 (0.25 g, 3 mmol) was added drop-
wise under vigorous stirring, at 0 °C over a period of 1 h. After
the mixture had been stirred for a further 15 min at 0 °C, EtOAc
(10 mL) and H2O (10 mL) were added. The aqueous layer was sep-
arated and washed with EtOAc (20 mL). The combined organic lay-
ers were washed consecutively with 5% aqueous citric acid (10 mL)
containing KI (0.04 g), 10% aqueous Na2S2O3 (10 mL), and brine and
dried over Na2SO4. The solvents were evaporated under reduced
pressure and the residue was purified by column-chromatography
using CHCl3 as eluent.
4.2.4.3. (S)-2-(2-(2-Oxohexadecanamido)hexyloxy)acetic acid
(18). Yield 88%; white solid; mp 64–66 °C; [
a]
D = ꢀ5.2 (c 0.5
CHCl3); 1H NMR (200 MHz, CDCl3) d 7.32 (d, J = 8.8 Hz, 1H),
4.14 (s, 2H), 4.07–3.95 (m, 1H), 3.66 (dd, J1 = 9.6 Hz, J2 = 4.8 Hz,
1H), 3.55 (dd, J1 = 9.6 Hz, J2 = 3.6 Hz, 1H), 2.90 (t, J = 7.4 Hz,
2H), 1.75–1.46 (m, 4H), 1.25 (br s, 26H), 0.98–0.80 (m, 6H);
13C NMR (50 MHz, CDCl3) d 199.1, 174.6, 160.2, 72.9, 67.8,
49.5, 36.8, 31.9, 30.8, 29.6, 29.4, 29.3, 29.0, 28.0, 23.2, 22.6,
22.4, 14.1, 13.9; MS (ESI): m/z (%): 450 (100) [M+Na]+. Anal.
Calcd for C24H45NO5: C, 67.41, H, 10.61, N, 3.28. Found: C,
67.59, H, 10.64, N, 3.14.
4.2.3.1. (S)-Methyl 2-(3-methyl-2-(2-oxohexadecanamido)buta-
namido)acetate (4b). Yield 73%; white solid; mp 118–119 °C;
4.2.5. General method for the synthesis of dipeptides
The dipeptides were prepared following the general method
4.2.1.
[
a]
D = ꢀ20.9 (c 1 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.46 (d,
J = 9.2 Hz, 1H), 6.53–6.37 (m, 1H), 4.24 (dd, J1 = 6.6 Hz, J2 = 9.2 Hz,
1H), 4.05 (dd, J1 = 5.2, J2 = 9.6 Hz, 2H), 3.77 (s, 3H), 2.91 (t,
J = 7.4 Hz, 2H), 2.22 (m, 1H), 1.61 (m, 2H), 1.26 (br s, 22H), 0.98
(t, J = 5.8 Hz, 6H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3)
d 198.3, 170.9, 170.0, 160.2, 58.6, 52.5, 41.1, 36.8, 31.9, 31.0, 29.6,
29.4, 29.3, 29.0, 23.1, 22.7, 19.2, 18.0, 14.1. Anal. Calcd for
C24H44N2O5: C, 65.42; H, 10.07; N, 6.36. Found: C, 65.55; H, 9.97;
N, 6.41.
4.2.5.1. (S)-Methyl 2-(2-(tert-butoxycarbonylamino)hexanami-
do)acetate (2a)38,39. Yield 65%; white solid; mp 95–96 °C;
[
a
]
D = ꢀ17.8 (c 1 CHCl3); 1H NMR (200 MHz, CDCl3) d 6.85–6.73
(m, 1H), 5.12 (d, J = 8.2 Hz, 1H), 4.22–4.07 (m, 1H), 4.03 (d,
J = 5.6 Hz, 2H), 3.74 (s, 3H), 1.93–1.72 (m, 1H), 1.69–1.48 (m,
1H), 1.43 (s, 9H), 1.39–1.21 (m, 4H), 0.88 (t, J = 6.6 Hz, 3H); 13C
NMR (50 MHz, CDCl3) d 172.6, 170.1, 155.7, 80.0, 54.4, 52.3,
41.0, 32.2, 28.2, 27.6, 22.3, 13.9.
4.2.3.2. (S)-tert-Butyl 2-(3-methyl-2-(2-oxohexadecanamido)
butanamido)acetate (8b). Yield 90%; white solid; mp 58–61 °C;
[
a]
D = ꢀ17.2 (c 1 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.50 (d,
4.2.5.2. (S)-Methyl 2-(2-(tert-butoxycarbonylamino)-3-methyl-
butanamido)acetate (2b). Yield 73%; white solid; mp 104–106 °C;
J = 8.8 Hz, 1H), 6.59–6.45 (m, 1H), 4.34–3.18 (m, 1H), 4.01 (dd,
J1 = 18.2 Hz, J2 = 5.4 Hz, 1H), 3.85 (dd, J1 = 18.2 Hz, J2 = 4.8 Hz, 1H),
2.89 (t, J = 6.8 Hz, 2H), 2.28–2.19 (m, 1H), 1.64–1.48 (m, 2H), 1.45
(s, 9H), 1.24 (br s, 22H), 0.96 (t, J = 5.8 Hz, 6H), 0.87 (t, J = 6.6 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 198.3, 170.1, 168.5, 160.2, 82.4,
58.5, 41.9, 36.8, 31.9, 31.1, 29.6, 29.5, 29.4, 29.3, 29.0, 28.0, 23.1,
22.6, 19.2, 18.0, 14.1; MS (FAB): m/z (%): 483 (24) [M+H]+. Anal.
Calcd for C27H50N2O5: C, 67.18; H, 10.44; N, 5.80. Found: C,
67.31; H, 10.39; N, 5.68.
[
a
]
D = ꢀ15.7 (c 1 CHCl3); 1H NMR (200 MHz, CDCl3) d 6.64 (m, 1H),
5.09 (d, J = 7.6 Hz, 1H), 4.06–3.96 (m, 3H), 3.76 (s, 3H), 2.19 (m, 1H),
1.42 (s, 9H), 0.99 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H); 13C NMR
(50 MHz, CDCl3) d 171.9, 170.1, 155.7, 80.0, 59.7, 52.3, 41.0, 30.8,
28.2, 19.2, 18.0. Anal. Calcd for C13H24N2O5: C, 54.15; H, 8.39; N,
9.72. Found: C, 53.92; H, 8.52; N, 9.61.
4.2.5.3. (S)-Ethyl 2-(2-(tert-butoxycarbonylamino)hexanamido)
acetate (2c)40. Yield 69%; oil; [
a
]
D = ꢀ14.8 (c 1 CHCl3); 1H NMR
4.2.4. General method for the cleavage of tert-butyl protecting
group
(200 MHz, CDCl3) d 7.00–6.85 (m, 1H), 5.23 (d, J = 7.2 Hz, 1H),
4.22–4.05 (m, 3H), 4.02–3.93 (m, 2H), 1.91–1.69 (m, 1H), 1.66–
1.45 (m, 1H), 1.40 (s, 9H), 1.35–1.18 (m, 7H), 0.86 (t, J = 6.6 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 172.7, 169.6, 155.6, 79.8, 61.3,
54.3, 41.1, 32.3, 28.2, 27.5, 22.3, 14.0, 13.8.
A solution of the tert-butyl ester derivative (1 mmol) in 50%
TFA/CH2Cl2 (10 mL) was stirred for 1 h at room temperature. The
organic solvent was evaporated under reduced pressure. The resi-
due was purified by recrystallization [EtOAc/petroleum ether (bp
40–60 °C)].
4.2.5.4. (S)-tert-Butyl 2-(2-(benzyloxycarbonylamino)hexanam-
ido)acetate (6a). Yield 64%; white solid; mp 80–82 °C; [
a
]
D = ꢀ8.4
4.2.4.1. (S)-2-(2-(2-Oxohexadecanamido)hexanamido)acetic acid
(9a). Yield 61%; colorless oil; 1H NMR (200 MHz, CDCl3) d 7.66 (d,
J = 8.8 Hz, 1H), 7.20–7.05 (m, 1H), 4.60–4.43 (m, 1H), 4.06 (d,
J = 3.6 Hz, 2H), 2.88 (t, J = 3.8 Hz, 2H), 1.83–1.59 (m, 4H), 1.25 (br
s, 26H), 0.89 (t, J = 8.0 Hz, 6H); 13C NMR (50 MHz, CDCl3) d 198.1,
172.6, 171.4, 160.1, 53.0, 41.4, 36.9, 32.1, 31.9, 29.6, 29.4, 29.3,
29.0, 27.5, 23.0, 22.7, 22.3, 14.1, 13.8; MS (ESI): m/z (%): 442 (100)
[M+H]+. Anal. Calcd for C24H44N2O5: C, 65.42; H, 10.07; N, 6.36.
Found: C, 65.19; H, 10.32; N, 6.25.
(c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.41–7.26 (m, 5H), 6.62–
6.49 (m, 1H), 5.41 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H), 4.29–4.11 (m,
1H), 3.92 (d, J = 4.6 Hz, 2H), 1.98–1.77 (m, 1H), 1.75–1.59 (m,
1H), 1.47 (s, 9H), 1.42–1.21 (m, 4H), 0.89 (t, J = 7 Hz, 3H); 13C
NMR (50 MHz, CDCl3) d 171.9, 168.7, 156.1, 136.2, 128.5, 128.1,
128.0, 82.3, 67.0, 54.9, 42.0, 32.4, 28.0, 27.5, 22.3, 13.8. Anal. Calcd
for C20H30N2O5: C, 63.47; H, 7.99; N, 7.40. Found: C, 63.28; H, 8.15;
H, 7.28.
4.2.4.2.
(S)-2-(3-Methyl-2-(2-oxohexadecanamido)butanami-
4.2.5.5. (S)-tert-Butyl 2-(2-(benzyloxycarbonylamino)-3-methyl
-butanamido)acetate (6b). Yield 81%; white solid; mp 139–
141 °C; [a]
7.42–7.28 (m, 5H), 6.47–6.35 (m, 1H), 5.39 (d, J = 8.6 Hz, 1H),
5.12 (s, 2H), 4.11–4.00 (m, 1H), 4.99–3.83 (m, 2H), 2.28–2.05 (m,
1H), 1.47 (s, 9H), 0.99 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H);
13C NMR (50 MHz, CDCl3) d 171.2, 168.6, 156.0, 136.1, 128.5,
128.2, 128.0, 82.4, 67.1, 60.3, 42.0, 31.0, 28.0, 19.2, 17.6. Anal. Calcd
for C19H28N2O5: C, 62.62; H, 7.74; N, 7.69. Found: C, 62.39; H, 7.91;
N, 7.55.
do)acetic acid (9b). Yield 68%; white solid; mp 87–89 °C;
D = ꢀ1.8 (c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.71 (d,
D = ꢀ6.8 (c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d
[
a]
J = 9 Hz, 1H), 7.21–7.06 (m, 1H), 4.45–4.34 (m, 1H), 4.18–4.01
(m, 2H), 3.03–2.72 (m, 2H), 2.25–2.04 (m, 1H), 1.68–1.45 (m,
2H), 1.25 (br s, 22H), 0.97 (t, J = 5.8 Hz, 6H), 0.88 (t, J = 6.6 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 198.0, 172.3, 170.9, 160.4,
58.6, 41.3, 36.9, 31.9, 31.1, 29.6, 29.4, 29.3, 29.0, 23.0, 22.7,
19.1, 18.1, 14.1; MS (ESI): m/z (%): 425 (100) [MꢀH]ꢀ. Anal. Calcd
for C23H42N2O5: C, 64.76; H, 9.92; N, 6.57. Found: C, 64.65; H,
9.87; N, 6.63.

4842
E. Barbayianni et al. / Bioorg. Med. Chem. 17 (2009) 4833–4843
4.2.6. (S)-Ethyl 2-(2-(tert-butoxycarbonylamino)
hexyloxy)acetate (11)
error, n = 3) for compounds displaying more than 25% and less than
90% enzyme inhibition. If the percent inhibition was greater than
90%, we determined its XI(50) by plotting percent inhibition versus
inhibitor molar fraction (7 points; typically 0.005–0.091 mole frac-
tion). Inhibition curves were modeled in Graphpad Prism using
either a linear (x, y intercept = 0) or non-linear regression (one-site
binding model—hyperbola, BMAX = 100) to calculate the reported
XI(50) and associated error values.
To as stirred solution of Boc-L-Nle-ol (0.50 g, 1.9 mmol) in dry
THF (15 mL), cooled at 0 °C under nitrogen, NaH (0.05 g, 2.1 mmol)
was added. The reaction mixture was stirred for 45 min at 0 °C, fol-
lowed by addition of 18-crown-6 (0.25 g, 0.9 mmol) and subse-
quently a solution of ethyl bromoacetate (0.47 g, 2.8 mmol) in
dry THF (4 mL) was added dropwise at 0 °C. The reaction mixture
was stirred for 1 h at 0 °C and overnight at room temperature.
The organic solvent was evaporated under reduced pressure and
the residue was purified by column-chromatography using CHCl3
4.5. Inhibition of arachidonic acid release. RAW 264.7 cell
culture and AA quantitation
as eluent. Yield 55%; oil; [
a
]
D = ꢀ13.9 (c 1 CHCl3); 1H NMR
(200 MHz, CDCl3) d 4.85–4.78 (m, 1H), 4.17 (q, J = 7.4 Hz, 2H),
4.03 (s, 2H), 3.72–3.58 (m, 1H), 3.56–3.41 (m, 2H), 1.62–1.21 (m,
18H), 0.85 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 170.3,
155.6, 78.9, 73.3, 68.4, 60.7, 50.3, 31.5, 28.3, 28.1, 22.5, 14.1,
13.9. Anal. Calcd for C15H29NO5: C, 59.38; H, 9.63; N, 4.62. Found:
C, 59.16; H, 9.82; N, 4.51.
RAW 264.7 macrophages were maintained in a humidified
atmosphere at 37 °C with 5% CO2, as described elsewhere.36 The
cells were cultured in DMEM media that was supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin,
100 lg/ml streptomycin, and non-essential amino acids. The cells
were plated at a confluency of 2 ꢂ 106 cells per well in 6 well tis-
sue culture plates and allowed to adhere overnight. Inhibitors
were added to the medium 30 min before Kdo2-Lipid A was
added and the supernatants were collected at 1 h following stim-
ulation. Subsequently, AA was extracted from the supernatants
and quantitated by HPLC-MS, as described elsewhere.36,37 AA re-
lease levels were normalized to pmol AA release per million cells
though DNA quantitation using the Broad Range DNA Quant-Kit
(Invitrogen). IC50 values were determined by estimating the con-
centration of inhibitor required for half maximal inhibition of the
maximum inhibition observed with a given inhibitor.
4.2.7. (S)-tert-Butyl 2-(2-(benzyloxycarbonylamino)
hexyloxy)acetate (15)
To a stirred solution of Z-L-Nle-ol (0.20 g, 0.8 mmol) in benzene
(0.8 mL), tert-butyl bromoacetate (0.47 g, 2.4 mmol) and subse-
quently aq NaOH 50% (0.8 mL) and the phase transfer catalyst
Bu4NHSO4 (0.07 g, 0.2 mmol) were added. The reaction mixture
was vigorously stirred for 2 h. EtOAc (10 mL) and H2O (10 mL)
were added and the aqueous layer was separated and extracted
with EtOAc (2 ꢂ 10 mL). The combined organic layers were washed
with brine and dried over Na2SO4. The organic solvent was evapo-
rated under reduced pressure and the residue was purified by col-
umn-chromatography using petroleum ether (bp 40–60 °C)/EtOAc
Acknowledgments
8:2 as eluent. Yield 74%; oil; [
a]
D = ꢀ12.2 (c 1 CHCl3); 1H NMR
The project is co-funded by the European Social Fund and Na-
tional Resources (EPEAEK II) (G.K.) and by NIH Grants GM 20,501,
GM 64,611, and U54 GM069338 (E.A.D).
(200 MHz, CDCl3) d 7.34–7.22 (m, 5H), 5.30 (d, J = 8 Hz, 1H), 5.06
(s, 2H), 3.91 (s, 2H), 3.80–3.62 (m, 1H), 3.55 (dd, J1 = 9.0 Hz,
J2 = 4.0 Hz, 1H), 3.46 (dd, J1 = 9.2 Hz, J2 = 4.2 Hz, 1H), 1.62–1.45
(m, 2H), 1.43 (s, 9H), 1.37–1.21 (m, 4H), 0.85 (t, J = 6.8 Hz, 3H);
13C NMR (50 MHz, CDCl3) d 169.5, 156.0, 136.6, 128.3, 128.0,
127.8, 81.5, 73.0, 68.7, 66.3, 50.9, 31.4, 28.0, 27.9, 22.4, 13.8. Anal.
Calcd for C20H31NO5: C, 65.73; H, 8.55; N, 3.83. Found: C, 65.52; H,
8.71; H, 3.70.
References and notes
1. Schaloske, R. H.; Dennis, E. A. Biochim. Biophys. Acta 2006, 1761, 1246.
2. Kudo, I.; Murakami, M. Prostag. Oth. Lipid M. 2002, 68–69, 3.
3. Leslie, C. C. Prostag. Leukot. Essent. Fatty Acids 2004, 70, 373.
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J. P. J. Biol. Chem. 2004, 279, 16488.
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Phospholipase A2 activity was determined using the previously
described modified Dole assay12 with buffer and substrate condi-
tions optimized for each enzyme as described previously:12,13,16,17
(i) GIVA cPLA2 substrate mixed-micelles were composed of
400
and 3
90 M CaCl2, 2 mM DTT and 0.1 mg/ml BSA; (ii) GVI iPLA2 sub-
strate mixed-micelles were composed of either (a) 400 M Triton
X-100, 99 M DPPC and 1.5
14C-labelled DPPC in buffer con-
taining 200 mM HEPES pH 7.0, 1 mM ATP, 2 mM DTT and
0.1 mg/ml BSA or (b) 400 M Triton X-100, 98.3 M PAPC and
1.7
14C-labelled PAPC in buffer containing 100 mM HEPES
pH 7.5, 2 mM ATP and 4 mM DTT and (iii) GV sPLA2 substrate
mixed-micelles were composed of 400 M Triton X-100, 99
DPPC and 1.5
14C-labelled DPPC in buffer containing 50 mM
lM Triton X-100, 97
lM PAPC, 1.8 lM
14C-labelled PAPC
lM PIP2 in buffer containing 100 mM HEPES pH 7.5,
l
l
l
lM
l
l
lM
l
lM
lM
Tris pH 8.0 and 5 mM CaCl2.
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playing 25% or less inhibition to have no inhibitory affect (desig-
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