Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A2 enzymes

Article abstract of DOI:10.1016/j.bmc.2008.10.046

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA₂ and GVIA iPLA₂) and one human secretory phospholipase (GV

Full text of DOI:10.1016/j.bmc.2008.10.046

Bioorganic & Medicinal Chemistry 16 (2008) 10257–10269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of natural and non-natural amino acid-based
amide and 2-oxoamide inhibitors of human phospholipase A2 enzymes
Georgia Antonopoulou a, Efrosini Barbayianni a, Victoria Magrioti a, Naomi Cotton b, Daren Stephens b,
a,
Violetta Constantinou-Kokotou c, 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, University of California, San Diego, La Jolla, CA 92093-0601, USA
c Chemical Laboratories, Agricultural University of Athens, Athens 11855, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthe-
sized. Their activity on two human intracellular phospholipases (GIVA cPLA2 and GVIA iPLA2) and one
human secretory phospholipase (GV sPLA2) was evaluated. We show that an amide based on (R)-c-nor-
Received 21 July 2008
Accepted 21 October 2008
Available online 1 November 2008
leucine is a highly selective inhibitor of GV sPLA2.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Amides
Amino acids
Inhibitors
2-Oxoamides
Phospholipase A2
1. Introduction
the animal model of multiple sclerosis,8 while it was reported that
cytosolic phospholipase A2-deficient mice were resistant to exper-
imental autoimmune encephalomyelitis.9 While the exact physio-
logical roles remain in question, multiple studies confirm the
primacy of GIVA cPLA2 in lipid mediator production. Consequently,
much effort has been focused on the design, synthesis and charac-
terization of GIVA cPLA2 inhibitors as potential agents for experi-
mental exploration as well as treatment of various inflammatory
conditions.
The role of the GVIA iPLA2 in the inflammatory process is still
unclear, and it has not been a target for the development of novel
medicines up to now.10 This enzyme appears to be the primary
PLA2 for basal metabolic functions within the cell.
It has been shown that in macrophages and other cells, GIVA
cPLA2 and secretory phospholipase A2 work together to release ara-
chidonic acid.11–15 Group GV sPLA2 was identified at the DNA-level
in humans,16 and subsequently expressed and charactized.17 Sev-
eral experiments suggested that GV sPLA2 has a role in amplifying
the action of GIVA cPLA2 in supplying arachidonic acid for eicosa-
noid production.18 In addition, GV sPLA2 has cellular functions,
independent of its ability to provide arachidonic acid, that include
regulation of phagocytosis and foam cell formation.19 Thus, this en-
The phospholipase A2 (PLA2) superfamily consists of a broad
range of structurally distinct enzymes that catalyze the hydrolysis
of the sn-2 ester bond of phospholipids. The PLA2 superfamily has
been systematically grouped based on the source (i.e. organism)
and primary sequence.1–3 In mammals, some of these enzymes
are particularly interesting anti-inflammatory drug targets, in part
due to their ability to liberate arachidonic acid for subsequent
eicosanoid biosynthesis.1–4 Specifically in human macrophages,
three of these groups are known to play significant roles in im-
mune response: cytosolic Ca2+-dependent PLA2 (GIVA cPLA2), cyto-
solic Ca2+-independent PLA2 (GVIA iPLA2) and Ca2+-dependent
secretory PLA2 (GV sPLA2).
GIVA cPLA2 is an attractive target for drug development since it
is the rate-limiting provider of arachidonic acid and lysophospho-
lipids that are subsequently converted into prostaglandins, leuko-
trienes and PAF, respectively.5 Various studies on transgenic mice
lacking GIVA cPLA2 showed a 90% reduction in the production of
prostaglandins and leukotrienes.6,7 Recently, Kalyvas and David
demonstrated that GIVA cPLA2 plays an important role in the
pathogenesis of experimental autoimmune encephalomyelitis,
zyme was recently proposed to have
a unique role in
atherosclerosis.19b
* 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).
Kokotos and Magrioti recently reviewed many synthetic inhib-
itors of GIVA cPLA2 and GVIA iPLA2.20 Since then, new N-benzydryl
indole inhibitors21–23 as well as 1-indol-1-yl-propan-2-ones24 have
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.046

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G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
been reported. Our recent efforts are focused on the development
of a novel class of GIVA cPLA2 inhibitors, initially designed to con-
tain 2-oxoamide functionality and a free carboxyl group.25–32 2-
ate: (a) the distance between the ester group and the 2-oxoamide
functionality, and (b) the nature of the ester group. For structure-
activity relationship (SAR) studies, we evaluated a variety of amino
Oxoamides based on
non-natural amino acids
c
-aminobutyric acid (1a, Fig. 1) and the
-norleucine or d-norleucine (1b and 1c,
acid and ester substitutions such as glycine, b-alanine, c-aminobu-
c
tyric acid, d-aminovaleric acid, and p-aminobenzoic acid as well as
methyl, ethyl, and tert-butyl esters. The esters of amino acids 5a–l
were coupled with 2-hydroxy-hexadecanoic acid (6) using 1-(3-
Fig. 1) are potent inhibitors of GIVA cPLA2 presenting in-vivo
anti-inflammatory and analgesic activity.26,30 We have also dem-
onstrated that ethyl 4-[(2-oxohexadecanoyl)amino]butanoate
(1d, Fig. 1) inhibits both GIVA cPLA2 and GVIA iPLA2 and is the first
systemically bioavailable compound with a significant affinity for
GIVA cPLA2, which produces potent hyperalgesia.28 In-vivo, intra-
thecal and systemic administration of inhibitor 1d blocked carra-
geenan hyperalgesia.28 Recently, a large number of lipophilic 2-
oxoamides have been tested for inhibition of GV sPLA2.30 Only
one compound (2, Fig. 1) significantly inhibited GV sPLA2 with an
XI(50) of 0.035 mole fraction. However, compound 2 inhibited both
GIVA cPLA2 and GVIA iPLA2, in addition to GV sPLA2, with no statis-
tical preference.
dimethylaminopropyl)-3-ethyl carbodiimide as
a condensing
agent in the presence of 1-hydroxybenzotriazole (Scheme 1). The
2-hydroxyamides 7a–l were oxidized to target compounds 8a–l
by treatment with Dess–Martin reagent.38
Three 2-oxoamide esters based on
c-leucine, c-norleucine and
c-alanine were prepared starting from the corresponding amino
alcohols 9a–c (Scheme 2) according to procedures we described
previously.26,30 Oxidation by the NaOCl/AcNH-TEMPO method39,40
followed by Wittig reaction with methyl or tert-butyl (triphenyl-
phosphoranylidene)acetate led to
a,b-unsaturated esters 10a–c.
Oxidation of compounds 11a–c by the Dess–Martin method pro-
Known inhibitors of secreted PLA2 enzyme groups have been re-
viewed.33 Among them, non-phospholipid amide inhibitors based
on non-natural amino acids, such as compounds 334 and 435
(Fig. 1), presented interesting inhibition of pancreatic and non-
pancreatic GI and GII sPLA2. Inhibitor 4 has been reported to pro-
tect rat small intestine from ischaemia and reperfusion injury36
and trinitrobenzene sulfonic acid-induced colitis.37
The selective inhibition of the each PLA2 class is very important
to understand enzyme-specific roles in cells and in-vivo. Therefore,
we synthesized a variety of 2-oxoamide esters and related amides
based on natural and non-natural amino acids and we evaluated
their activity on GIVA cPLA2, GV s PLA2 and GVIA iPLA2. While most
of these compounds exhibited inhibitory properties for all three
duced the target compounds 12a–c. To prepare 2-oxoamide esters
based on
e-norleucine, Boc-L-norleucinol (9b) was oxidized to
aldehyde and directly reacted with triethyl phosphonocrotonate41
(Scheme 3). Catalytic hydrogenation of compound 13, removal of
the Boc group by HCl/Et2O and coupling with 2-hydroxy-hexadeca-
noic acid produced compound 16. The unsaturated 2-hydroxya-
mide 15 was prepared by similar reaction. Both compounds 15
and 16 were oxidized to the corresponding 2-oxoamides 17 and
18 by the Dess–Martin method. 2-Oxoamide 19 containing a free
carboxyl group was also prepared.
Ketone and ether analogues of the in-vivo active inhibitor 1d
were prepared as described in Scheme 4. Treatment of Boc-GABA
with n-BuLi led to ketone 22 in moderate yield. Etherification of
compound 21 under phase-transfer conditions produced ether
23. Both compounds 22 and 23 were deprotected, coupled and oxi-
dized to obtain the target derivatives 26 and 27.
PLA2 classes, we demonstrate that an amide based on (R)-c-norleu-
cine is a highly selective inhibitor of GV sPLA2.
The known inhibitors 3 and 4 (Fig. 1) of GI and GII sPLA2 are
amides consisting of a 7-phenylheptanoyl acyl residue and a
2. Results
non-natural c-amino acid bearing a side chain with two aromatic
2.1. Design and synthesis of inhibitors
groups. We decided to synthesize 2-oxoamides containing the 7-
phenylheptanoyl residue instead of the long aliphatic chain. The
synthesis procedure of 2-oxoamides 37a,b and amides 32a,b,
based on methyl ester of glycine and tert-butyl ester of b-alanine,
is depicted in Scheme 5.
Our previous work has shown that 2-oxoamides containing a
free carboxyl group are selective inhibitors of GIVA cPLA2, not
affecting GVIA iPLA2 and GV sPLA2.26,29,30 On the contrary, methyl
or ethyl esters of 2-oxoamides may inhibit both GIVA cPLA2 and
GVIA iPLA2.29 In addition, the 2-oxoamide that contains a methyl
ester group (2) inhibits all the three forms: GIVA cPLA2, GVIA iPLA2
and GV sPLA2.30 In this study, we decided to systematically evalu-
In addition, we synthesized a
taining a 7-phenylheptanoyl chain. Boc-
pared according to the literature,42,43 was oxidized to aldehyde
c-norleucine-based amide con-
D
-norleucinol (38), pre-
O
O
O
O
H
N
H
N
OR2
O
( )n
( )9
( )13
H
N
O
R1
O
O
( )7
1a, R1 = H, R2 = H, n = 1
2
O
1b, R1 =(CH2)3CH3, R2 = H, n = 1
1c, R1 =(CH2)3CH3, R2 = H, n = 2
1d, R1 = H, R2 = Et, n = 1 (AX048)
O
S
O
O
( )6
N
H
OH
H
O
( )6
N
H
H
O
3
OH
4
Fig. 1. Amino acid-based inhibitors of phospholipase A2 enzymes.

G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
10259
O
OH
OH
( )13
O
a
H
N
H2N
OH
+
( )13
X
OR
X
OR
O
6
O
5a-l
7a-l
5,7,8
X
R
O
O
H
N
b
a
b
c
d
e
f
g
h
i
CH2
Me
X
OR
( )13
CH2
CH2
Et
O
But
Me
Et
(CH2)2
(CH2)2
(CH2)2
(CH2)3
(CH2)3
(CH2)4
(CH2)4
(CH2)4
C6H4
8a-l
But
Bn
All
Me
Et
j
k
l
But
Et
Scheme 1. Reagents: (a) WSCI, HOBt, Et3N, CH2Cl2; (b) Dess–Martin periodinane, CH2Cl2.
O
PHN
PHN
b, c, d (for 11a,b) b,d (for 11c)
OR2
a
OH
R1
R1
9a-c
10a-c
OH
O
O
O
H
N
H
N
e
OR2
( )13
OR2
( )13
R1
O
R1
12a-c
O
11a-c
R1
i-Bu
n-Bu
Me
R2
Me
Me
But
P
9-12
a
b
c
Boc
Boc
Z
Scheme 2. Reagents and conditions: (a) i—NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, ꢀ5 °C; ii—Ph3P@CHCOOR2, THF, reflux; (b) H2, 10% Pd/C, EtOH; (c) 4 N
HCl/Et2O; (d) CH3(CH2)13CH(OH)COOH, WSCI, HOBt, Et3N, CH2Cl2; (e) Dess–Martin periodinane, CH2Cl2.
and treated with methyl or ethyl (phosphoranylidene)acetate to
produce compounds 39a,b (Scheme 6). After hydrogenation and
removal of Boc group, coupling with 7-phenylheptanoic acid35
gave amides 41a,b. The target compound 42 was obtained after
saponification of either 41a or 41b. Its enantiomer 43 was synthe-
sized following similar reactions.
(8i–k) (Table 1) display potent inhibition of GIVA cPLA2 or GVIA
iPLA2. tert-Butyl esters (8c, 8f, and 8k) are better inhibitors than
the corresponding methyl and ethyl esters. Benzyl ester (8g) and
allyl ester (8h) analogues of inhibitor 1d were found to be weak
inhibitors of GIVA cPLA2 and GVIA iPLA2, indicating that the ethyl
ester of GABA leads to better in-vitro results.28
The esters of the 2-oxoamides based on
c-leucine (12a), c-nor-
2.2. In-vitro inhibition of GIVA cPLA2, GVIA iPLA2 and GV sPLA2
leucine (12b) and -alanine (12c) are good inhibitors of GIVA cPLA2
c
(Table 2). They also weakly inhibit GVIA iPLA2. Derivative 8l (Table
2) based on the aromatic amino acid seems to selectively inhibit
GIVA cPLA2, without affecting GVIA iPLA2 activity. 2-Oxoamide
Twenty-six new derivatives including 2-oxoamides and amides
were tested for inhibition of human GIVA cPLA2, GVIA iPLA2 and GV
sPLA2 using previously described mixed micelle-based as-
says.26,29,30 The relative degrees of inhibition are presented in
Tables 1–3 as either percent inhibition or XI(50) values. Initially,
the percent of inhibition for each PLA2 enzyme at 0.091 mole
fraction of each inhibitor was determined, and XI(50) values were
estimated for compounds that displayed greater than 90%
inhibition. Each XI(50) value is derived from the mole fraction of
the inhibitor in the total substrate interface required to inhibit
the enzyme by 50%.
ethyl esters based on e-norleucine (saturated 18 or unsaturated
17, Table 2) are weak inhibitors of both enzymes. It should be no-
ticed that compound 19 (Table 2) containing a free carboxyl group
is a selective inhibitor of GIVA cPLA2, without affecting the activity
of GVIA iPLA2. This observation is in full agreement with our previ-
ous report that 2-oxoamides based on
c-amino acids are selective
inhibitors of GIVA cPLA2.29,30
Among the 2-oxoamides based on esters of
acids, two derivatives (12b and 12c, Table 2) based on esters of
-norleucine and -alanine displayed inhibition of GV sPLA2. How-
ever, compounds 12b and 12c also inhibited GIVA cPLA2 and GVIA
c- and e-amino
None of the methyl, ethyl or tert-butyl esters of 2-oxoamides
based on glycine (8a–c), b-alanine (8d–f), and d-aminovaleric acid
c
c

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G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
O
a, b
BocHN
BocHN
OH
( )2
O
( )2
13
c
d
9b
O
OH
( )13
O
BocHN
H
N
O
O
( )2
O
14
( )2
15
d
e
OH
( )13
O
O
O
O
H
N
H
N
O
( )13
O
O
O
O
( )2
( )2
16
18
17
19
e
O
H
N
f, e
O
O
H
N
( )13
OH
( )13
O
( )2
O
( )2
Scheme 3. Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, ꢀ5 °C; (b) (EtO)2P(@O)CH2CH@CHCOOEt, LiOH, PhCH3, rt; (c) H2, 10% Pd/C,
EtOH; (d) i—4 N HCl/Et2O; ii—CH3(CH2)13CH(OH)COOH, WSCI, HOBt, Et3N, CH2Cl2; (e) Dess–Martin periodinane, CH2Cl2; (f) 1 N NaOH, CH3OH.
O
a
BocHN
BocHN
OH
OH
20
22
21
e
b
O
BocHN
OH
BocHN
OH
O
23
c
c
O
H
H
N
N
( )13
O
O
( )13
O
O
25
24
26
d
d
O
O
O
H
N
H
N
( )13
( )13
O
O
27
Scheme 4. Reagents: (a) i—NMM, ClCO2Et, THF; ii—NaBH4, MeOH; (b) n-BuLi/hexane, THF; (c) i—4 N HCl/Et2O; ii—CH3(CH2)13CH(OH)COOH, WSCI, HOBt, Et3N, CH2Cl2; (d)
Dess–Martin periodinane, CH2Cl2; (e) EtBr, Bu4NHSO4, 50% NaOH, C6H6.
iPLA2, and therefore demonstrated no statistical preference for the
inhibition of the three enzymes.
To characterize the mode of interaction between the ethyl ester
derivative 1d and the active site of GIVA cPLA2, we prepared two
structurally related compounds, a ketone derivative 26 and an
ether derivative 27. In compound 26, the carbonyl group is at the
same position as in the ester derivative 1d, although the alkoxy
group was replaced by an alkyl group. In compound 27, the ethoxy

G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
10261
a
H
OEt
O
O
28
29
b
O
O
c
d
( )n
OEt
f, g
OR
( )6
31
( )6
OH
h, i
N
H
O
e
O
32a,b
30
OH
OH
d
OH
( )6
33
OH
( )6
CN
( )6
34
35
O
OH
O
O
O
j
H
N
32, 36, 37
n
R
H
N
( )n
( )6
OR
( )n
( )6
37a,b
OR
a
b
1
2
Me
But
O
36a,b
O
Scheme 5. Reagents and conditions: (a) BrꢀPh3P+(CH2)5COOEt, NaH, THF; (b) H2, 10% Pd/C, EtOH; (c) 1 N NaOH, dioxane; (d) H2N(CH2)nCOOR, WSCI, HOBt, Et3N, CH2Cl2; (e)
DIBALH, dry Et2O; (f) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, ꢀ5 °C; (g) NaHSO3, KCN, H2O, CH2Cl2; (h) concd HCl; (i) KOH, EtOH/H2O; (j) Dess–Martin
periodinane, CH2Cl2.
O
BocHN
BocHN
f
a, b
BocHN
c
OH
( )2
OR
( )2
39a,b
38
O
O
H
N
d, e
OR
OR
O
( )2
R
( )2
40a,b
41a,b
O
39-41
H
N
a
b
Et
OH
O
Me
( )2
42
Scheme 6. Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO3, EtOAc/PhCH3/H2O 3:3:0.5, ꢀ5 °C; (b) Ph3P@CHCOOR, THF, reflux; (c) H2, 10% Pd/C, EtOH or MeOH; (d)
4 N HCl/Et2O, (e) Ph(CH2)6COOH, WSCI, HOBt, Et3N, CH2Cl2; (f) 1 N NaOH, dioxane.
group of the ester derivative was kept constant, while the carbonyl
group is absent. For synthetic reasons, we prepared a ketone deriv-
ative containing one additional carbon atom in the right chain of
the ketone chain. However, this modification was not expected to
play an essential role in inhibition. Neither compounds 26 or 27
presented interesting inhibitory properties, thereby confirming
the essential inhibitory role of the ester group of compound 1d.
Table 3 summarizes the in-vitro inhibition activities caused by
2-oxoamides and amides containing the 7-phenylheptanoyl chain
instead of the long aliphatic chain. Comparing 37a with 8a, it
seems that such a replacement of the long chain led to decreased
activity for all the enzymes, as observed in the case of glycine
methyl ester. However, similar activities were observed for 37b
and 8b. The corresponding amides 32a and 32b were either totally
inactive or very weak inhibitors. Interestingly, amide 42 based on
(R)-c-norleucine is a potent and selective GV sPLA2 inhibitor, not
affecting at all the activities of GIVA cPLA2 and GVIA iPLA2. The
dose-response curve for the inhibition of GV sPLA2 by amide 42
is shown in Fig. 2, and a XI(50) value 0.003 0.0004 was calculated.
The configuration of the substituted amino acid is very important,
since the amide 43 based on (S)-c-norleucine was inactive for all
the three PLA2 enzymes.
3. Discussion
We previously demonstrated that 2-oxoamide 1d, which is
based on the ethyl ester of -aminobutyric acid, inhibits the GIVA
c
cPLA2 and GVIA iPLA2 and exhibits very interesting antihyperalge-
sic activity.28 The tert-butyl ester variant of compound 1d (AX057)
also inhibits both enzymes.28 In addition, 2-oxoamides based on

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G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
Table 1
Table 2
Inhibition of PLA2 by 2-oxoamide esters
Inhibition of PLA2 by 2-oxoamides based on esters of c- and e-amino acids and
related compounds
Entry Structure
GIVA
cPLA2
GVIA
iPLA2
GV
sPLA2
Entry Structure
GIVA
cPLA2
GVIA
iPLA2
GV
sPLA2
O
O
O
O
H
N
O
O
O
8a
8b
8c
8d
8e
8f
62
1
45 13
62 13
53 13
N.D.
52
62
82
48
59
72
4
H
N
( )13
O
O
O
( )13
O
O
12a
83
4
65
78
9
65
86
9
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
O
74
1
2
5
8
9
8
7
H
N
( )13
( )13
12b
87 13
9
2
6
4
O
( )2
O
H
N
80
( )13
O
O
H
N
12c
85
64
5
4
72
87
( )13
O
O
H
N
O
O
O
N.D.
44
( )13
O
O
O
O
H
N
O
O
O
( )13
O
17
74
5
5
52
7
O
H
N
O
2
2
1
2
51 13
54 13
N.D.
( )2
( )2
( )2
( )13
O
H
N
O
H
N
( )13
O
18
62 12
66
N.D.
N.D.
O
O
79
( )13
O
O
O
H
N
H
N
8g
8h
8i
35
58 12
( )13
OH
19
85
87
4
1
N.D.
( )13
O
O
O
O
O
H
N
O
H
N
53
63
5
38
42
47
8
8
6
( )13
( )13
8l
N.D.
78
8
O
O
O
H
N
O
O
O
O
O
N.D.
31
N.D.
( )13
O
O
O
O
H
N
26
32
5
N.D.
51
N.D.
O
( )13
H
N
8j
2
2
35 12
62 13
O
( )13
O
H
N
27
62 14
2
64 12
O
( )13
O
H
N
8k
74
72 10
O
( )13
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).
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).
The 2-oxoamide compounds are inhibitors of various lipolytic
enzymes (pancreatic and gastric lipases,44–48 GIVA cPLA2,25,26,29,30
GVIA iPLA229) that utilize a serine residue as the nucleophile in
their catalytic mechanism. In the cases of such serine esterases, it
has been proposed that the serine hydroxyl group interacts with
the activated carbonyl group of the 2-oxoamide functionality.
The small, secreted human GV sPLA2, as well as other sPLA2 en-
zymes, utilize a histidine residue in the catalytic site to coordinate
a water molecule for hydrolysis at the sn-2 ester bond of phospho-
lipids. It is likely that the long chain 2-oxoamides able to inhibit GV
sPLA2 resemble the transition state, explaining its high-affinity for
the active site of GV sPLA2.
methyl esters of c-aminobutyric acid or a,b-unsaturated c-norleu-
cine are able to inhibit the same enzymes.29 From the results of Ta-
ble 1, we show that 2-oxoamide esters with varying distance
between the 2-oxoamide functionality and the ester group (one
to four carbon atoms) do not present any significant inhibition of
GIVA cPLA2 or GVIA iPLA2. Interestingly, the introduction of a
side-chain to the non-natural amino acids (Table 2) increases the
potency of methyl or tert-butyl esters of
and -alanine (12a–c) for GIVA cPLA2; but display rather weak
inhibition of GVIA iPLA2. Even so, increasing the spacer length to
five carbon atoms, as seen in the ethyl ester of an -amino acid var-
iant (17), leads to inhibition of both GIVA cPLA2 and GVIA iPLA2.
c-leucine, c-norleucine,
c
e
We demonstrate that an amide based on a non-natural amino
acid with a short linear aliphatic side chain (42) is a potent and

G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
10263
Table 3
Inhibition of PLA2 by 2-oxoamide and amide containing 7-phenylheptanoyl chain
Entry
Structure
GIVA cPLA2
GVIA iPLA2
31 10
GV sPLA2
O
O
O
O
O
H
N
37a
54
4
9
30
2
9
O
O
O
O
H
N
O
37b
32a
32b
82
81 10
69
O
O
N.D.
36
N.D.
N.D.
66
H
N
O
3
57 12
9
N
H
O
O
H
N
42
43
N.D.
N.D.
N.D.
N.D.
95
OH
OH
XI(50) 0.003 0.0004
O
O
( )2
O
H
N
N.D.
( )2
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).
selective inhibitor of GV sPLA2. Comparing the present results with
our previous studies, we conclude that a 2-oxoamide based on (S)-
VG analytical ZAB-SE instrument. Electron spray ionization (ESI)
mass spectra were recorded on a Finnigan, Surveyor MSQ Plus
spectrometer. All amino acid derivatives were purchased from Flu-
ka Chemical Co. TLC plates (silica gel 60 F254) and silica gel 60 (70–
230 or 230–400 mesh) for column chromatography were pur-
chased from Merck. Visualization of spots was effected with UV
light and/or phosphomolybdic acid and/or ninhydrin, both in EtOH
stain. THF, toluene, and Et2O were dried by standard procedures
and stored over molecular sieves or Na. All other solvents and
chemicals were reagent grade and used without further
purification.
c
-norleucine, containing a long aliphatic chain, is a potent and
selective inhibitor of GIVA cPLA2,26 while a simple amide based
on (R)- -norleucine, containing a 7-phenylheptanoyl chain, is a po-
c
tent and selective inhibitor of GV sPLA2.
4. Conclusions
In conclusion, we synthesized a variety of 2-oxoamides and
amides based on various amino acids and studied their activity
on two human intracellular phospholipases (GIVA cPLA2 and GVIA
iPLA2) and one human secretory phospholipase (GV sPLA2). In sum-
mary, compounds with (S) configuration containing the 2-oxoa-
mide functionality and a long fatty acyl chain provide selective
inhibition of GIVA cPLA2, while amide functionality in combination
with the 7-phenylheptanoyl chain and the (R) configuration lead to
selective inhibition of GV sPLA2. Since both of these PLA2s have
been shown to contribute to the production of arachidonic acid,
selective 2-oxoamide and amide inhibitors will be valuable tools
for studies in cells and in-vivo.
5.2. Synthesis of 2-oxoamide Inhibitors
5.2.1. Coupling of 2-hydroxy-acids with amino components
To a stirred solution of 2-hydroxy-acid (1.0 mmol) and the ami-
no component (1.0 mmol) in CH2Cl2 (10 mL), Et3N (3.1 mL,
2.2 mmol) and subsequently 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide (WSCI) (0.21 g, 1.1 mmol) and 1-hydroxybenzotria-
zole (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 rt. 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/MeOH as eluent.
5. Experimental
5.1. General procedures
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. Fast
atom bombardment (FAB) mass spectra were recorded using a
5.2.1.1. Methyl 2-(2-hydroxyhexadecanamido)acetate (7a).
Yield 82%; White solid; mp 116–118 °C; 1Y NMR (200 MHz, CDCl3)
d 7.02 (t, J = 4.8 Hz, 1H), 4.20–4.14 (m, 1H), 4.10–4.06 (m, 2H), 3.77
(s, 3H), 2.78 (br, 1H), 1.94–1.56 (m, 2H), 1.26 (br s, 24H), 0.89 (t,

10264
G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
2H), 2.83 (br, 1H), 2.44 (t, J = 5.8 Hz, 2H), 1.88–1.51 (m, 2H), 1.44
(s, 9H), 1.23 (br s, 24H), 0.86 (t, J = 6.6 Hz, 3H); 13C NMR
(50 MHz, CDCl3) d 173.8, 171.7, 81.2, 72.0, 35.1, 35.0, 34.7, 31.9,
29.6, 29.5, 29.4, 29.3, 28.1, 24.8, 22.7, 14.1. Anal. Calcd for
C23H45NO4: C, 69.13; H, 11.35; N, 3.51. Found: C, 69.01; H, 11.48;
N, 3.44.
5.2.1.7. Benzyl 4-(2-hydroxyhexadecanamido)butanoate (7g).
Yield 64%; White solid; mp 89–91 °C; 1Y NMR (200 MHz, CDCl3)
d 7.36 (m, 5H), 6.72 (t, J = 5.8 Hz, 1H), 5.13 (s, 2H), 4.09–4.03 (m,
1H), 3.37–3.28 (m, 2H), 2.85 (br, 1H), 2.42 (t, J = 7 Hz, 2H), 1.95–
1.50 (m, 4H), 1.26 (br s, 24H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR
(50 MHz, CDCl3) d 174.1, 173.1, 135.7, 128.6, 128.3, 128.2, 72.1,
66.4, 38.4, 34.9, 31.9, 31.6, 29.7, 29.62, 29.58, 29.5, 29.4, 29.3,
25.0, 24.6, 22.7, 14.1. Anal. Calcd for C27H45NO4: C, 72.44; H,
10.13; N, 3.13. Found: C, 72.61; H, 9.95; N, 3.23.
5.2.1.8. Allyl 4-(2-hydroxyhexadecanamido)butanoate (7h).
Yield 71%; White solid; mp 91–93 °C; 1Y NMR (200 MHz, CDCl3)
d 6.71 (t, J = 5.4 Hz, 1H), 6.02–5.82 (m, 1H), 5.36–5.22 (m, 2H),
4.61–4.57 (m, 2H), 4.12–4.06 (m, 1H), 3.39–3.29 (m, 2H), 2.59
(br, 1H), 2.40 (t, J = 7.2 Hz, 2H), 1.95–1.52 (m, 4H), 1.25 (br s,
24H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.0,
173.0, 132.0, 118.4, 72.1, 65.3, 38.5, 34.9, 31.9, 31.6, 29.7, 29.6,
29.5, 29.4, 29.3, 25.0, 24.6, 22.7, 14.1. Anal. Calcd for C23H43NO4:
C, 69.48; H, 10.90; N, 3.52. Found: C, 69.62; H, 10.75; N, 3.57.
Fig. 2. Inhibition curve for amide 42 in a mixed-micelle assay with human GV
sPLA2. Non-linear regression (hyperbolic) estimated
a
XI(50) value of
0.003 0.0004. Compound 42 did not inhibit GIVA cPLA2 and GVIA iPLA2 at
0.091 mole fraction.
J = 6.8 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.2, 170.3, 72.2, 52.4,
40.8, 34.8, 31.9, 29.6, 29.5, 29.4, 29.3, 24.9, 22.7, 14.1. Anal. Calcd
for C19H37NO4: C, 66.43; H, 10.86; N, 4.08. Found: C, 66.65; H,
10.71; N, 4.13.
5.2.1.9. Methyl 5-(2-hydroxyhexadecanamido)pentanoate (7i).
Yield 66%; White solid; mp 105–107 °C; 1Y NMR (200 MHz, CDCl3)
d 6.73 (t, J = 5.8 Hz, 1H), 4.10–4.04 (m, 1H), 3.66 (s, 3H), 3.32–3.22
(m, 2H), 3.17 (br, 1H), 2.34 (t, J = 7 Hz, 2H), 1.79–1.51 (m, 6H), 1.24
(br s, 24H), 0.87 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.2,
174.0, 72.1, 51.6, 38.5, 34.9, 33.4, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3,
28.9, 25.0, 22.6, 22.0, 14.1. Anal. Calcd for C22H43NO4: C, 68.53; H,
11.24; N, 3.63. Found: C, 68.77; H, 11.13; N, 3.58.
5.2.1.2. Ethyl 2-(2-hydroxyhexadecanamido)acetate (7b). Yield
75%; White solid; mp 115–117 °C; 1Y NMR (200 MHz, CDCl3) d
6.98 (m, 1H), 4.29–4.07 (m, 5H), 1.93–1.53 (m, 2H), 1.26 (br s,
27H), 0.89 (t, J = 5.8 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.2,
169.9, 72.2, 61.6, 40.9, 34.8, 31.9, 29.6, 29.5, 29.4, 29.3, 24.9,
22.7, 14.1. Anal. Calcd for C20H39NO4: C, 67.19; H, 10.99; N, 3.92.
Found: C, 67.33; H, 10.83; N, 3.99.
5.2.1.3. tert-Butyl 2-(2-hydroxyhexadecanamido)acetate (7c). Yield
77%; White solid; mp 53–56 °C; Y NMR (200 MHz, CDCl3) d 7.03 (t,
1
5.2.1.10. Ethyl 5-(2-hydroxyhexadecanamido)pentanoate (7j).
Yield 68%; White solid; mp 95–97 °C; 1Y NMR (200 MHz, CDCl3)
d 6.69 (t, J = 5.8 Hz, 1H), 4.17–4.06 (m, 3H), 3.33–3.23 (m, 2H),
3.15 (br, 1H), 2.33 (t, J = 7 Hz, 2H), 1.79–1.58 (m, 6H), 1.24 (br s,
27H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.0,
173.5, 72.1, 60.4, 38.5, 34.9, 33.7, 31.9, 29.7, 29.6, 29.5, 29.4,
29.3, 28.9, 25.0, 22.7, 22.0, 14.1. Anal. Calcd for C23H45NO4: C,
69.13; H, 11.35; N, 3.51. Found: C, 68.97; H, 11.59; N, 3.43.
J = 5.2 Hz, 1H), 4.18–4.12 (m, 1H), 3.99–3.93 (m, 2H), 3.15 (br, 1H),
1.89–1.50 (m, 2H), 1.47 (s, 9H), 1.25 (br s, 24H), 0.88 (t, J = 6.8 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 174.3, 169.1, 82.4, 72.2, 41.5, 34.7,
31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.0, 25.0, 22.7, 14.1. Anal. Calcd
for C22H43NO4: C, 68.53; H, 11.24; N, 3.63. Found: C, 68.45; H,
11.37; N, 3.71.
5.2.1.4. Methyl 3-(2-hydroxyhexadecanamido)propanoate(7d). Yield
79%; White solid; mp 111–114 °C; 1Y NMR (200 MHz, CDCl3) d 7.00 (br,
1H), 4.12–4.06 (m, 1H), 3.71 (s, 3H), 3.61–3.51 (m, 2H), 2.86 (br, 1H),
2.57 (t, J = 6 Hz, 2H), 1.86–1.55 (m, 2H), 1.25 (br s, 24H), 0.88 (t,
J = 6.8 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 174.0, 172.9, 72.0, 51.9, 34.9,
34.5, 33.8, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 24.9, 22.7, 14.1. Anal. Calcd
for C20H39NO4: C, 67.19; H, 10.99; N, 3.92. Found: C, 67.03; H, 11.21; N,
3.86.
5.2.1.11. tert-Butyl 5-(2-hydroxyhexadecanamido)pentanoate
(7k). Yield 61%; White solid; mp 60–63 °C; 1Y NMR (200 MHz,
CDCl3) d 6.71 (t, J = 5.6 Hz, 1H), 4.11–4.06 (m, 1H), 3.32–3.23 (m,
2H), 2.24 (t, J = 7 Hz, 2H), 1.81–1.46 (m, 6H), 1.43 (s, 9H), 1.23
(br s, 24H), 0.87 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d
174.1, 173.0, 80.4, 72.1, 38.6, 34.9, 31.9, 29.7, 29.6, 29.5, 29.4,
29.3, 28.9, 28.0, 25.0, 22.7, 22.1, 14.1. Anal. Calcd for C25H49NO4:
C, 70.21; H, 11.55; N, 3.28. Found: C, 70.02; H, 11.68; N, 3.32.
5.2.1.5. Ethyl 3-(2-hydroxyhexadecanamido)propanoate (7e).
Yield 68%; White solid; mp 102–104 °C; 1Y NMR (200 MHz, CDCl3)
d 7.07 (t, J = 6 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 4.09–4.04 (m, 1H),
3.59–3.49 (m, 2H), 3.20 (br, 1H), 2.54 (t, J = 6.2 Hz, 2H), 1.86–1.50
(m, 2H), 1.24 (br s, 27H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR
(50 MHz, CDCl3) d 174.1, 172.4, 72.0, 60.8, 34.8, 34.5, 34.0, 31.9,
29.6, 29.5, 29.4, 29.3, 24.9, 22.6, 14.1, 14.0. Anal. Calcd for
C21H41NO4: C, 67.88; H, 11.12; N, 3.77. Found: C, 67.64; H, 11.34;
N, 3.71.
5.2.1.12. Ethyl 4-(2-hydroxyhexadecanamido)benzoate (7l).
Yield 43%; Oily solid; 1Y NMR (200 MHz, CDCl3) d 8.74 (s, 1H),
8.06 (s, 1H), 7.95–7.33 (m, 3H), 4.37 (q, J = 7 Hz, 2H), 4.28–4.22
(m, 1H), 3.35 (br, 1H), 1.90–1.61 (m, 2H), 1.25 (br s, 27H), 0.88 (t,
J = 5.8 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 172.7, 166.3, 137.5,
131.1, 129.1, 125.4, 124.1, 120.6, 72.6, 61.2, 34.7, 31.9, 29.7, 29.5,
29.4, 29.3, 25.0, 22.6, 14.2, 14.1. Anal. Calcd for C25H41NO4: C,
71.56; H, 9.85; N, 3.34. Found: C, 71.33; H, 9.97; N, 3.42.
5.2.1.6. tert-Butyl 3-(2-hydroxyhexadecanamido)propanoate
(7f). Yield 63%; White solid; mp 95–97 °C; 1Y NMR (200 MHz,
CDCl3) d 6.97 (t, J = 5.8 Hz, 1H), 4.11–4.05 (m, 1H), 3.56–3.46 (m,
5.2.1.13. (4R)-Methyl 4-(2-hydroxyhexadecanamido)-6-meth-
ylheptanoate (11a). Yield 57%; White solid; 1H NMR (200 MHz,
CDCl3) d 6.40–6.20 (m, 1H), 4.20–3.90 (m, 2H), 3.66 (s, 3H), 3.10–

G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
10265
2.90 (m, 1H), 2.35 (t, J = 7.8 Hz, 2H), 1.95- 1.47 (m, 5H), 1.40–1.18
(m, 26H), 0.95-0.81 (m, 9H). Anal. Calcd for C25H49NO4: C, 70.21; H,
11.55; N, 3.28. Found: C, 70.34; H, 11.46; N, 3.22.
142.5, 128.2, 128.1, 125.5, 52.1, 41.0, 36.1, 35.7, 31.1, 28.9, 28.8,
25.3; MS (ESI) m/z (%): 278 (100) [M+H]+; Anal. Calcd for
C16H23NO3: C, 69.29; H, 8.36; N, 5.05. Found: C, 68.92; H, 8.47;
N, 5.11.
5.2.1.14. (4S)-Methyl 4-(2-hydroxyhexadecanamido)octanoate
(11b). Yield 62%; White solid; 1H NMR (200 MHz, CDCl3) d 6.35–
6.15 (m, 1H), 4.15–4.00 (m, 1H), 4.00–3.80 (m, 1H), 3.67 (s, 3H),
2.80–2.70 (m, 1H), 2.35 (t, J = 7.8 Hz, 2H), 1.97–1.42 (m, 6H),
1.38–1.15 (m, 28H), 0.98–0.81 (m, 6H). Anal. Calcd for
C25H49NO4: C, 70.21; H, 11.55; N, 3.28. Found: C, 70.04; H, 11.62;
N, 3.36.
5.2.1.21. tert-Butyl 3-(7-phenylheptanamido)propanoate (32b).
Yield 45%; Oil; 1H NMR (200 MHz, CDCl3) d 7.40–7.23 (m, 5H), 6.18
(br s, 1H), 3.56 (q, 2H), 2.68 (t, J = 7.4 Hz, 2H), 2.53 (t, J = 6.2 Hz,
2H), 2.23 (t, J = 7.0 Hz, 2H), 1.80–1.62 (m, 4H), 1.54 (s, 9H), 1.51–
1.38 (m, 4H); 13C NMR (50 MHz, CDCl3) d 173.0, 172.2, 142.6,
128.3, 128.2, 125.5, 81.0, 36.7, 35.8, 35.0, 34.8, 31.2, 29.0, 28.9,
28.0, 25.6; MS (ESI) m/z (%): 356 (100) [M+Na]+; Anal. Calcd for
C20H31NO3: C, 72.04; H, 9.37; N, 4.20. Found: C, 71.76; H, 9.59; N,
4.36.
5.2.1.15. (4S)-tert-Butyl 4-(2-hydroxyhexadecanamido)pent-
anoate (11c). Yield 58%; White solid; 1H NMR (200 MHz, CDCl3)
d 6.60–6.40 (m, 1H), 4.10–3.90 (m, 2H), 3.15–2.95 (m, 1H), 2.27
(t, J = 7.8 Hz, 2H), 1.98–1.52 (m, 4H), 1.44 (s, 9H), 1.40–1.21 (m,
24H), 1.16 (d, J = 6.6 Hz, 3H), 0.88 (t, J = 6.6 Hz, 3H). Anal. Calcd
for C25H49NO4: C, 70.21; H, 11.55; N, 3.28. Found: C, 70.05; H,
11.74; N, 3.21.
5.2.1.22. Methyl
2-(2-hydroxy-8-phenyloctanamido)acetate
(36a). Yield 41%; White solid; mp 65–67 °C; 1H NMR (200 MHz,
CDCl3) d 7.39–7.25 (m, 6H), 4.27–4.08 (m, 3H), 3.83 (s, 3H), 3.32
(br s, 1H), 2.68 (t, J = 7.4 Hz, 2H), 1.98–1.62 (m, 4H), 1.58–1.35
(m, 6H); 13C NMR (50 MHz, CDCl3) d 174.7, 170.4, 142.7, 128.3,
128.2, 125.5, 72.1, 52.3, 40.7, 35.8, 34.6, 31.3, 29.2, 29.1, 24.8; Anal.
Calcd for C17H25NO4: C, 66.43; H, 8.20; N, 4.56. Found: C, 66.29; H,
8.11; N, 4.61.
5.2.1.16. (2E,4E,6S)-Ethyl 6-(2-hydroxyhexadecanamido)deca-
2,4-dienoate (15). Yield 58%; Waxy solid; 1Y NMR (200 MHz,
CDCl3) d 7.35 (dd, J1 = 15.6 Hz, J2 = 10.8 Hz, 1H), 6.73–6.67 (m,
1H), 6.26 (dd, J1 = 15.2 Hz, J2 = 11 Hz, 1H), 6.12(dd, J1 = 15 Hz,
J2 = 5.8 Hz, 1H), 5.95 (d, J = 15.4 Hz, 1H), 4.73–4.57 (m, 1H), 4.35–
4.24 (m, 3H), 3.11 (d, J = 4.8 Hz, 2/3H), 3.03 (d, J = 4.8 Hz, 1/3H),
1.92–1.58 (m, 4H), 1.29 (br s, 31H), 0.89 (t, J = 6.6 Hz, 6H); 13C
NMR (50 MHz, CDCl3) d 173.2, 167.0, 143.7, 142.5, 128.0, 121.5,
72.2, 60.4, 50.1, 34.9, 34.5, 31.9, 29.7, 29.6, 29.4, 29.3, 27.8, 24.9,
22.7, 22.4, 14.2, 14.1, 13.9. Anal. Calcd for C28H51NO4: C, 72.21;
H, 11.04; N, 3.01. Found: C, 72.34; H, 10.97; N, 2.97.
5.2.1.23. tert-Butyl 3-(2-hydroxy-8-phenyloctanamido)propan-
oate (36b). Yield 32%; White solid; mp 62–64 °C; 1H NMR
(200 MHz, CDCl3) d 7.40–7.23 (m, 5H), 7.13 (t, J = 5.8 Hz, 1H),
4.16 (dd, J1 = 3.6 Hz, J2 = 7.2 Hz, 1H), 3.59 (q, J = 6.4 Hz, 2H), 3.10
(br s, 1H), 2.68 (t, J = 7.4 Hz, 2H), 2.54 (t, J = 6.2 Hz, 2H), 1.95–
1.78 (m, 1H), 1.76–1.61 (m, 3H), 1.54 (s, 9H), 1.42–1.35 (m, 6H);
13C NMR (50 MHz, CDCl3) d 174.0, 171.7, 142.7, 128.3, 128.2,
125.5, 81.2, 71.9, 35.9, 35.2, 34.8, 34.7, 31.4, 29.2, 29.1, 28.0,
24.8; Anal. Calcd for C21H33NO4: C, 69.39; H, 9.15; N, 3.85. Found:
C, 69.28; H, 9.21; N, 3.94.
5.2.1.17. (6S)-Ethyl 6-(2-hydroxyhexadecanamido)decanoate
(16). Yield 55%; White solid; 1Y NMR (200 MHz, CDCl3) d 6.28–
6.18 (m, 1Y), 4.16–4.06 (m, 3H), 3.92–3.89 (m, 1H), 3.11 (d,
J = 4.4 Hz, 2/3H), 2.94 (d, J = 5 Hz, 1/3H), 2.28 (t, J = 7.4 Hz, 2H),
1.93–1.21 (m, 41H), 0.88 (t, J = 6.6 Hz, 6H); 13C NMR (50 MHz,
CDCl3) d 173.9, 173.5, 72.2 (72.0), 60.3, 48.6 (48.8), 35.1, 34.9,
34.8, 34.7, 34.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.0, 25.4, 25.3,
24.9, 24.8, 24.7, 22.7 (22.6), 14.2, 14.1, 14.0. Anal. Calcd for
C28H55NO4: C, 71.59; H, 11.80; N, 2.98. Found: C, 71.28; H, 11.95;
N, 2.94.
5.2.1.24. (R)-Ethyl 4-(7-phenylheptanamido)octanoate (41a).
Yield 89%; White solid; mp 54–56 °C; ½a D
ꢁ
+8.2 (c 1 CHCl3); 1H
NMR (200 MHz, CDCl3) d 7.39–7.23 (m, 5H), 5.88 (d, J = 9.0 Hz,
1H), 4.19 (q, J = 7.0 Hz, 2H), 4.03 (br s, 1H), 2.69 (t, J = 7.4 Hz, 2H),
2.43 (t, J = 7.6 Hz, 2H), 2.24 (t, J = 6.8 Hz, 2H), 1.99–1.87 (m, 2H),
1.83–1.60 (m, 6H), 1.56–1.36 (m, 8H), 1.32 (t, J = 7.4 Hz, 3H), 0.98
(t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 173.5, 172.6, 142.4,
128.1, 128.0, 125.4, 60.2, 48.5, 36.6, 35.6, 34.8, 31.1, 30.9, 29.8,
28.9, 28.8, 27.8, 25.6, 22.3, 14.0, 13.9; MS (ESI) m/z (%): 398
(100) [M+Na]+; Anal. Calcd for C23H37NO3: C, 73.56; H, 9.93; N,
3.73. Found: C, 73.22; H, 10.18; N, 3.79.
5.2.1.18. 2-Hydroxy-N-(4-oxooctyl)hexadecanamide (24). Yield
70%; White solid; mp 122–124 °C; 1Y NMR (200 MHz, CDCl3) d
6.73 (t, J = 5.8 Hz, 1H), 4.22–4.06 (m, 1H), 3.34–3.28 (m, 2H),
2.56–2.40 (m, 4H), 1.97–1.73 (m, 3H), 1.73–1.20 (m, 30H), 0.88
(t, J = 6.6 Hz, 6H); 13C NMR (50 MHz, CDCl3) d 211.2, 174.0, 72.1,
42.7, 40.0, 38.7, 34.9, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 25.9, 25.0,
23.3, 22.7, 22.3, 14.1, 13.8. Anal. Calcd for C24H47NO3: C, 72.49;
H, 11.91; N, 3.52. Found: C, 72.38; H, 11.99; N, 3.56.
5.2.1.25. (R)-Methyl 4-(7-phenylheptanamido)octanoate (41b).
Yield 70%; White solid; mp 62–64 °C; ½a D
ꢁ
+9.3 (c 1 CHCl3); 1H NMR
(200 MHz, CDCl3) d 7.40–7.23 (m, 5H), 5.45 (d, J = 8.2 Hz, 1H), 4.12–
3.95 (m, 1H), 3.74 (s, 3H), 2.69 (t, J = 7.0 Hz, 2H), 2.43 (t, J = 7.4 Hz,
2H), 2.23 (t, J = 7.4 Hz, 2H), 2.05–1.85 (m, 1H), 1.79–1.60 (m, 5H),
1.55–1.36 (m, 10H), 0.97 (t, J = 6.2 Hz, 3H); 13C NMR (50 MHz,
CDCl3) d 174.2, 172.7, 142.6, 128.3, 128.2, 125.5, 51.6, 48.8, 36.8,
35.8, 31.2, 30.8, 30.0, 29.1, 28.9, 28.0, 25.7, 22.5, 13.9; Anal. Calcd
for C22H35NO3: C, 73.09; H, 9.76; N, 3.87. Found: C, 72.77; H,
9.97; N, 3.95.
5.2.1.19. N-(4-Ethoxybutyl)-2-hydroxyhexadecanamide (25).
Yield 67%; White solid; mp 92–95 °C; 1Y NMR (200 MHz, CDCl3)
d 6.75 (t, J = 5.8 Hz, 1H), 4.10–4.02 (m, 1H), 3.52–3.26 (m, 7H),
1.95–1.50 (m, 6H), 1.25 (br s, 27H), 0.87 (t, J = 6.6 Hz, 3H); 13C
NMR (50 MHz, CDCl3) d 174.1, 72.0, 70.1, 66.2, 38.8, 35.0, 31.9,
29.7, 29.6, 29.5, 29.4, 29.3, 27.1, 26.4, 25.0, 22.6, 15.1, 14.1. Anal.
Calcd for C22H45NO3: C, 71.11; H, 12.21; N, 3.77. Found: C, 71.24;
H, 12.15; N, 3.72.
5.2.2. Oxidation of 2-hydroxy-amides
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 rt. The organic solution
was washed with 10% aqueous NaHCO3, dried over Na2SO4 and
the organic solvent was evaporated under reduced pressure. The
residue was purified by column-chromatography using CHCl3 as
eluent.
5.2.1.20. Methyl 2-(7-phenylheptanamido)acetate (32a). Yield
45%; White solid; mp 48–50 °C; 1H NMR (200 MHz, CDCl3) d
7.40–7.21 (m, 5H), 6.30 (b, 1H), 4.11 (d, J = 5.6 Hz, 2H), 3.83 (s,
3H), 2.69 (t, J = 7.2 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.81–1.62 (m,
4H), 1.55–1.38 (m, 4H); 13C NMR (50 MHz, CDCl3) d 173.3, 170.4,

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G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
5.2.2.1. Methyl 2-(2-oxohexadecanamido)acetate (8a). Yield
91%; White solid; mp 79–81 °C; 1H NMR (200 MHz, CDCl3) d
7.42–7.36 (m, 1H), 4.09 (d, J = 5.8 Hz, 2H), 3.79 (s, 3H), 2.91 (t,
J = 7.8 Hz, 2H), 1.68–1.52 (m, 2H), 1.25 (br s, 22H), 0.88 (t, J = 6.6
Hz, 3H); 13C NMR (50 MHz, CDCl3) d 198.2, 169.3, 160.2, 52.5,
40.9, 36.7, 31.9, 29.62, 29.56, 29.4, 29.33, 29.30, 29.0, 23.1, 22.7,
14.1; MS (ESI): m/z (%): 364 (51) [M+Na]+; Anal. Calcd for
C19H35NO4: C, 66.83; H, 10.33; N, 4.10. Found: C, 66.92; H, 10.25;
N, 4.04.
29.3, 29.0, 24.4, 23.1, 22.7, 14.1; MS (ESI): m/z (%): 468 (100)
[M+Na]+; Anal. Calcd for C27H43NO4: C, 72.77; H, 9.73; N, 3.14.
Found: C, 72.86; H, 9.57; N, 3.23.
5.2.2.8. Allyl 4-(2-oxohexadecanamido)butanoate (8h). Yield
83%; White solid; mp 60–62 °C; 1H NMR (200 MHz, CDCl3) d
7.10 (t, J = 6.2 Hz, 1H), 6.02–5.82 (m, 1H), 5.37–5.22 (m, 2H),
4.61–4.58 (m, 2H), 3.41–3.31 (m, 2H), 2.91 (t, J = 7.6 Hz, 2H),
2.40 (t, J = 7.2 Hz, 2H), 2.05–1.84 (m, 2H), 1.64–1.56 (m, 2H),
1.26 (br s, 22H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (50 MHz, CDCl3)
d 199.2, 172.5, 160.3, 132.0, 118.5, 65.3, 38.6, 36.7, 31.9, 31.5,
29.6, 29.5, 29.4, 29.3, 29.0, 24.4, 23.2, 22.7, 14.1; MS (ESI): m/z
(%): 418 (100) [M+Na]+; Anal. Calcd for C23H41NO4: C, 69.83; H,
10.45; N, 3.54. Found: C, 69.72; H, 10.52; N, 3.47.
5.2.2.2. Ethyl 2-(2-oxohexadecanamido)acetate (8b). Yield 84%;
White solid; mp 66–68 °C; 1H NMR (200 MHz, CDCl3) d 7.10–6.91
(m, 1H), 4.22 (q, J = 7 Hz, 2H), 4.07 (d, J = 5.4 Hz, 2H), 2.90 (t,
J = 7.2 Hz, 2H), 1.89–1.56 (m, 4H), 1.53–1.08 (br s, 23H), 0.86 (t,
J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 198.2, 168.8, 160.2,
61.7, 41.0, 36.7, 31.9, 29.6, 29.5, 29.4, 29.30, 29.26, 29.0, 23.1,
22.6, 14.1; MS (ESI): m/z (%): 378 (100) [M+Na] +, 356 (5)
[M+H]+; Anal. Calcd for C20H37NO4: C, 67.57; H, 10.49; N, 3.94.
Found: C, 67.71; H, 10.42; N, 3.89.
5.2.2.9. Methyl 5-(2-oxohexadecanamido)pentanoate (8i). Yield
93%; White solid; mp 68–70 °C; 1H NMR (200 MHz, CDCl3) d 7.03
(t, J = 6.2 Hz, 1H), 3.68 (s, 3H), 3.36–3.27 (m, 2H), 2.91 (t, J = 7.2
Hz, 2H), 2.35 (t, J = 6.6 Hz, 2H), 1.70–1.56 (m, 6H), 1.25 (br s,
22H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 199.3,
173.7, 160.2, 51.6, 38.8, 36.7, 33.4, 31.9, 29.6, 29.5, 29.4, 29.3,
29.0, 28.6, 23.1, 22.7, 22.0, 14.1; MS (ESI): m/z (%): 406 (100)
[M+Na]+; Anal. Calcd for C22H41NO4: C, 68.89; H, 10.77; N, 3.65.
Found: C, 68.97; H, 10.71; N, 3.62.
5.2.2.3. tert-Butyl 2-(2-oxohexadecanamido)acetate (8c). Yield
81%; White solid; mp 54–56 °C; 1H NMR (200 MHz, CDCl3) d
7.43–7.32 (m, 1H), 3.97 (d, J = 5.6 Hz, 2H), 2.91 (t, J = 7.4 Hz, 2H),
1.65–1.52 (m, 2H), 1.48 (s, 9 H), 1.25 (br s, 22H), 0.88 (t,
J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 198.3, 167.9, 160.1,
82.7, 41.7, 36.7, 31.9, 29.63, 29.57, 29.4, 29.34, 29.30, 29.0, 28.0,
23.1, 22.7, 14.1; MS (ESI): m/z (%): 406 (78) [M+Na]+; Anal. Calcd
for C22H41NO4: C, 68.89; Y, 10.77; N, 3.65. Found: C, 68.95; Y,
10.69; N, 3.72.
5.2.2.10. Ethyl 5-(2-oxohexadecanamido)pentanoate (8j). Yield
87%; White solid; mp 59–61 °C; 1H NMR (200 MHz, CDCl3) d 7.03
(t, J = 6.2 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.36–3.26 (m, 2 H), 2.91
(t, J = 7.4 Hz, 2H), 2.33 (t, J = 7 Hz, 2H), 1.75–1.56 (m, 6H), 1.25
(m, 25H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (50 MHz, CDCl3) d
199.3, 173.3, 160.2, 60.4, 38.8, 36.7, 33.7, 31.9, 29.62, 29.57, 29.4,
29.31, 29.33, 29.0, 28.6, 23.1, 22.7, 22.0, 14.2, 14.1; MS (FAB): m/
z (%): 398 (100) [M+H]+; Anal. Calcd for C23H43NO4: C, 69.48; H,
10.90; N, 3.52. Found: C, 69.34; H, 10.97; N, 3.58.
5.2.2.4. Methyl 3-(2-oxohexadecanamido)propanoate (8d).
Yield 74%; White solid; mp 83–86 °C; 1H NMR (200 MHz, CDCl3)
d 7.45–7.33 (m, 1H), 3.71 (s, 3H), 3.62–3.53 (m, 2H), 2.89 (t,
J = 6.6 Hz, 2H), 2.58 (t, J = 6.2 Hz, 2H), 1.62–1.52 (m, 2H), 1.25 (br
s, 22H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 198.9,
172.2, 160.2, 51.9, 36.7, 34.7, 33.5, 31.9, 29.61, 29.56, 29.4, 29.30,
29.32, 29.0, 23.1, 22.7, 14.1; MS (ESI): m/z (%): 378 (100)
[M+Na]+, 356 (9) [M+H]+; Anal. Calcd for C20H37NO4: C, 67.57; H,
10.49; N, 3.94. Found: C, 67.46; H, 10.53; N, 3.98.
5.2.2.11. tert-Butyl 5-(2-oxohexadecanamido)pentanoate (8k).
Yield 77%; White solid; mp 48–50 °C; 1H NMR (200 MHz, CDCl3) d
7.04 (t, J = 6.2 Hz, 1 H), 3.34–3.24 (m, 2 H), 2.89 (t, J = 7.4 Hz, 2 H),
2.23 (t, J = 6.6 Hz, 2 H), 1.65–1.48 (m, 6 H), 1.43 (s, 9 H), 1.23 (br
s, 22 H), 0.86 (t, J = 7 Hz, 3 H); 13C NMR (50 MHz, CDCl3) d 199.3,
172.6, 160.1, 80.3, 38.9, 36.7, 34.8, 31.9, 29.6, 29.5, 29.4, 39.3,
29.0, 28.6, 28.0, 23.1, 22.6, 22.1, 14.1; MS (ESI): m/z (%): 448
(100) [M+Na]+; Anal. Calcd for C25H47NO4: C, 70.54; H, 11.13; N,
3.29. Found: C, 70.65; H, 11.05; N, 3.21.
5.2.2.5. Ethyl 3-(2-oxohexadecanamido)propanoate (8e). Yield
83%; White solid; mp 60–62 °C; 1H NMR (200 MHz, CDCl3) d 7.42 (t,
J = 6.2 Hz, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.61–3.72 (m, 2H), 2.89 (t,
J = 7.4 Hz, 2H), 2.56 (t, J = 6.2 Hz, 2H), 1.78–1.18 (m, 27H), 0.87 (t,
J = 6.4 Hz, 3H); 13C NMR (50 MHz, CDCl3) d 198.9, 171.8, 160.1, 60.9,
36.7, 34.7, 33.7, 31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 23.1, 22.7, 14.1;
MS (FAB): m/z (%): 370 (100) [M+H]+; Anal. Calcd for C21H39NO4: C,
68.25; H, 10.64; N, 3.79. Found: C, 68.17; H, 10.61; N, 3.84.
5.2.2.12. Ethyl 4-(2-oxohexadecanamido)benzoate (8l). Yield
84%; White solid; mp 86–88 °C; 1H NMR (200 MHz, CDCl3) d 8.87
(s, 1 H), 8.19–7.43 (m, 4 H), 4.39 (q, J = 7.0 Hz, 2 H), 3.02 (t,
J = 7.2 Hz, 2 H), 1.78–1.12 (m, 27 H), 0.88 (t, J = 6.6 Hz, 3 H); 13C
NMR (50 MHz, CDCl3) d 199.2, 165.9, 157.7, 136.5, 131.5, 129.3,
126.2, 123.9, 120.6, 61.2, 36.3, 31.9, 29.63, 29.57, 29.4, 29.34,
29.30, 29.1, 23.3, 22.7, 14.3, 14.1; MS (FAB): m/z (%): 418 (100)
[M+H]+; Anal. Calcd for C25H39NO4: C, 71.91; H, 9.41; N, 3.35.
Found: C, 71.74; H, 9.57; N, 3.29.
5.2.2.6. tert-Butyl 3-(2-oxohexadecanamido)propanoate (8f).
Yield 75%; White solid; mp 44–45 °C; 1H NMR (200 MHz, CDCl3)
d 7.43 (t, J = 6.2 Hz, 1H), 3.57–3.48 (m, 2H), 2.90 (t, J = 7.4 Hz,
2H), 2.47 (t, J = 5.8 Hz, 2H), 1.62–1.55 (m, 2H), 1.45 (s, 9H), 1.24
(br s, 22H), 0.87 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3) d
199.0, 171.0, 160.0, 81.4, 36.7, 34.8, 31.9, 29.61, 29.55, 29.4, 29.3,
29.0, 28.0, 23.1, 22.7, 14.1; MS (ESI): m/z (%): 420 (100) [M+Na]+;
Anal. Calcd for C23H43NO4: C, 69.48; H, 10.90; N, 3.52. Found: C,
69.41; H, 10.84; N, 3.62.
5.2.2.13. (R)-Methyl 6-methyl-4-(2-oxohexadecanamido)hept-
anoate (12a). Yield 76%; Yellowish solid; mp 41–43 °C; ½aꢁD ꢀ9.0
(c 1.0, CHCl3); 1H NMR (200 MHz, CDCl3) d 6.75–6.60 (m, 1 H),
4.10–3.85 (m, 1 H), 3.66 (s, 3 H), 2.91 (t, J = 7.4 Hz, 2 H), 2.32 (t,
J = 7.4 Hz, 2 H), 1.95–1.40 (m, 5 H), 1.40–1.18 (m, 24 H), 0.95–
0.81 (m, 9 H). Anal. Calcd for C25H47NO4: C, 70.54; H, 11.13; N,
3.29. Found: C, 70.39; H, 11.35; N, 3.38.
5.2.2.7. Benzyl 4-(2-oxohexadecanamido)butanoate (8g). Yield
91%; White solid; mp 68–70 °C; 1H NMR (200 MHz, CDCl3) d
7.42–7.29 (m, 5H), 7.11 (t, J = 5.8 Hz, 1H), 5.13 (s, 2H), 3.40–3.30
(m, 2H), 2.90 (t, J = 7.4 Hz, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.98–1.84
(m, 2H), 1.68–1.53 (m, 2H), 1.26 (br s, 22H), 0.89 (t, J = 6.2 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 199.2, 172.7, 160.3, 135.7,
128.6, 128.31, 128.25, 66.5, 38.6, 36.7, 31.9, 31.5, 29.6, 29.5, 29.4,
5.2.2.14. (S)-Methyl 4-(2-oxohexadecanamido)octanoate (12b).
Yield 92%; White solid; mp 56–58 °C; ½a D
ꢁ
–4.6 (c 1.0, CHCl3); 1H
NMR (200 MHz, CDCl3) d 6.80–6.60 (m, 1 H), 4.20–3.80 (m, 1 H),

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3.66 (s, 3 H), 2.91 (t, J = 7.4 Hz, 2 H), 2.35 (t, J = 7.4 Hz, 2 H), 1.97–
1.42 (m, 6 H), 1.38–1.15 (m, 26 H), 0.98-0.81 (m, 6 H); 13C NMR
(50 MHz, CDCl3) d 199.5, 173.6, 160.0, 51.7, 49.3, 36.8, 34.8, 31.9,
30.7, 29.9, 29.6, 29.5, 29.4, 29.3, 29.1, 27.9, 23.2, 22.7, 22.4, 14.1,
13.9. Anal. Calcd for C25H47NO4: C, 70.54; H, 11.13; N, 3.29. Found:
C, 70.25; H, 11.32; N, 3.25.
(t, J = 6.4 Hz, 1 H), 3.52–3.25 (m, 6 H), 2.90 (t, J = 7.8 Hz, 2 H),
1.78–1.48 (m, 6 H), 1.40–1.15 (m, 25 H), 0.87 (t, J = 6.6 Hz, 3 H);
13C NMR (50 MHz, CDCl3) d 199.4, 160.2, 69.9, 66.2, 39.0, 36.7,
31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 27.1, 26.2, 23.1, 22.6, 15.1, 14.1;
MS (FAB): m/z (%): 370 (100) [M+H]+; Anal. Calcd for C22H43NO3:
C, 71.50; H, 11.73; N, 3.79. Found: C, 71.43; H, 11.65; N, 3.91.
5.2.2.15. (S)-tert-Butyl 4-(2-oxohexadecanamido)pentanoate
(12c). Yield 95%; White solid; mp 52–53 °C; ½aꢁD ꢀ1.6 (c 1.0, CHCl3);
1H NMR (200 MHz, CDCl3) d 6.95–6.80 (m, 1 H), 4.05–3.80 (m, 1 H),
2.85 (t, J = 7.4 Hz, 2 H), 2.25 (t, J = 7.4 Hz, 2 H), 1.85–1.65 (m, 2 H),
1.65–1.45 (m, 2 H), 1.40 (s, 9 H), 1.40–1.10 (m, 25 H), 0.88 (t,
J = 6.6 Hz, 3 H); 13C NMR (50 MHz, CDCl3) d 199.3, 172.3, 159.6,
80.4, 45.2, 36.6, 32.1, 31.8, 31.3, 29.5, 29.4, 29.3, 29.2, 29.0, 28.0,
23.1, 22.6, 20.6, 14.0. Anal. Calcd for C25H47NO4: C, 70.54; H, 11.13;
N, 3.29. Found: C, 70.38; H, 11.25; N, 3.21.
5.2.2.21. Methyl 2-(2-oxo-8-phenyloctanamido)acetate (37a).
Yield 82%; Oily solid; 1H NMR (200 MHz, CDCl3) d 7.55–7.42 (m,
1 H), 7.40–7.23 (m, 5 H), 4.16 (d, J = 5.4 Hz, 2 H), 3.86 (s, 3 H),
3.00 (t, J = 6.8 Hz, 2 H), 2.69 (t, J = 7.2 Hz, 2 H), 1.80–1.62 (m, 4 H),
1.55–1.35 (m, 4 H); 13C NMR (50 MHz, CDCl3) d 198.1, 169.2,
160.2, 142.6, 128.3, 128.2, 125.6, 52.5, 40.8, 36.6, 35.8, 31.2, 28.9,
28.8, 23.0; MS (ESI) m/z (%): 328 (100) [M+Na]+; Anal. Calcd for
C17H23NO4: C, 66.86; H, 7.59; N, 4.59. Found: C, 66.48; H, 7.78;
N, 4.65.
5.2.2.16. (S,2E,4E)-Ethyl 6-(2-oxohexadecanamido)deca-2,4-
5.2.2.22. tert-Butyl 3-(2-oxo-8-phenyloctanamido)propanoate
(37b). Yield 83%; Oil; 1H NMR (200 MHz, CDCl3) d 7.58–7.42 (m,
1 H), 7.40–7.22 (m, 5 H), 3.61 (q, J = 6.2 Hz, 2 H), 2.99 (t,
J = 7.0 Hz, 2 H), 2.68 (t, J = 7.4 Hz, 2 H), 2.56 (t, J = 6.2 Hz, 2 H),
1.79–1.60 (m, 4 H), 1.54 (s, 9 H), 1.49–1.40 (m, 4 H); 13C NMR
(50 MHz, CDCl3) d 198.9, 171.0, 160.0, 142.6, 128.3, 128.2, 125.5,
81.3, 36.6, 35.8, 34.8, 34.7, 31.2, 28.9, 28.8, 28.0, 23.0; MS (ESI)
m/z (%): 384 (100) [M+Na]+; Anal. Calcd for C21H31NO4: C, 69.78;
H, 8.64; N, 3.87. Found: C, 69.40; H, 8.89; N, 3.94.
dienoate (17). Yield 91%; White solid; mp 53–55 °C; ½aꢁD ꢀ11.2
(c 0.5 CHCl3); 1H NMR (200 MHz, CDCl3) d 7.22 (dd, J1 = 15.2 Hz,
J2 = 10.8 Hz, 1 H), 6.95 (d, J = 8.8 Hz, 1 H), 6.25 (dd, J1 = 15.2 Hz,
J2 = 10.6 Hz, 1 H), 6.00 (dd, J1 = 15.2 Hz, J2 = 6.2 Hz, 1 H), 5.86 (d,
J = 15.8 Hz, 1 H), 4.74 (m, 1 H), 4.19 (q, J = 7.2 Hz, 2 H), 2.91 (t,
J = 7.4 Hz, 2 H), 1.73–1.44 (m, 4 H), 1.28 (br s, 29 H), 0.88 (m,
6 H); 13C NMR (50 MHz, CDCl3) d 199.3, 166.7, 159.4, 143.3,
141.0, 128.7, 122.0, 60.3, 50.9, 36.7, 34.3, 31.9, 29.6, 29.5, 29.4,
29.3, 29.0, 27.7, 23.1, 22.6, 22.3, 14.2, 14.1, 13.9; MS (FAB): m/z
(%): 464 (37) [M+H]+; Anal. Calcd for C28H49NO4: C, 72.53; H,
10.65; N, 3.02. Found: C, 72.28; H, 10.74; N, 3.09.
5.2.3. (R,E)-Ethyl 4-(tert-butoxycarbonyl)oct-2-enoate (39a)
To a solution of 38 (0.22 g, 1.00 mmol) in a mixture of tolu-
ene–EtOAc (6 mL), a solution of NaBr (0.11 g, 1.05 mmol) in
water (0.5 mL) was added, followed by AcNH-TEMPO (2.2 mg,
0.010 mmol). To the resulting biphasic system, which was cooled
at ꢀ5 °C, an aqueous solution of 0.35 M NaOCl (3.14 mL,
1.10 mmol) containing NaHCO3 (0.25 g, 3 mmol) was added drop-
wise while stirring vigorously at ꢀ5 °C over a period of 1 h. After
the mixture had been stirred for a further 15 min at 0 °C, EtOAc
(6 mL) and H2O (2 mL) were added. The aqueous layer was sepa-
rated and washed with EtOAc (4 mL). The combined organic lay-
ers were washed consecutively with 5% aqueous citric acid
(6 mL) containing KI (0.04 g), 10% aqueous Na2S2O3 (6 mL), and
brine and dried over Na2SO4. The solvents were evaporated under
reduced pressure, and the residue was used immediately in the
next step without any purification. To the solution of the N-pro-
5.2.2.17. (S)-Ethyl 6-(2-oxohexadecanamido)decanoate (18).
Yield 93%; White solid; mp 47–49 °C; ½a D
ꢁ
ꢀ1.6 (c 0.5 CHCl3); 1H
NMR (200 MHz, CDCl3)
d
6.66 (d, J = 9.6 Hz, 1 H), 4.11 (q,
J = 7.2 Hz, 2 H), 4.00–3.81 (m, 1 H), 2.92 (t, J = 7.4 Hz, 2 H), 2.28 (t,
J = 7.4 Hz, 2 H), 1.80–1.21 (m, 39 H), 0.88 (t, J = 6.2 Hz, 6 H); 13C
NMR (50 MHz, CDCl3) d 199.7, 173.5, 159.8, 60.2, 49.4, 36.7, 34.7,
34.1, 31.9, 29.6, 29.4, 29.3, 29.1, 27.9, 25.4, 24.7, 23.2, 22.7, 22.5,
14.2, 14.1, 13.9; MS (FAB): m/z (%): 468 (87) [M+H]+; Anal. Calcd
for C28H53NO4: C, 71.90; H, 11.42; N, 2.99. Found: C, 71.77; H,
11.51; N, 3.08.
5.2.2.18. (S,2E,4E)-6-(2-Oxohexadecanamido)deca-2,4-dienoic
acid (19). Saponification of 15 took place before oxidation. Yield
53%; White solid; mp 91–93 °C; ½a D
ꢁ
ꢀ35.1 (c 1 CHCl3); 1H NMR
tected a-amino aldehyde in dry THF (5 mL), Ph3P@CHCOOEt
(200 MHz, CDCl3) d 7.20 (dd, J1 = 15.2 Hz, J2 = 10.8 Hz, 1 H), 6.85
(d, J = 9 Hz, 1 H), 6.18 (dd, J1 = 15.2 Hz, J2 = 11 Hz, 1 H), 5.92 (dd,
J1 = 15.4 Hz, J2 = 6.2 Hz, 1 H), 5.77 (d, J = 15.4 Hz, 1 H), 4.57–4.43
(m, 1 H), 2.82 (t, J = 7.2 Hz, 2 H), 1.76–1.43 (m, 4 H), 1.14 (br s,
26 H), 0.88 (m, 6 H); 13C NMR (50 MHz, CDCl3) d 199.3, 171.8,
159.5, 145.7, 142.3, 128.4, 121.0, 50.9, 36.7, 34.3, 31.9, 29.6, 29.5,
29.4, 29.3, 29.0, 27.7, 23.1, 22.6, 22.3, 14.1, 13.9; MS (ESI): m/z
(%): 434 (100) [MꢀH]ꢀ; Anal. Calcd for C26H45NO4: C, 71.68; H,
10.41; N, 3.22. Found: C, 71.51; H, 10.52; N, 3.25.
(1.1 mmol) was added and the reaction mixture was refluxed
for 1 h. Saturated aqueous NH4Cl (4 mL) was added and extracted
with Et2O (3 ꢂ 5 mL). The combined organic phases were washed
with brine and dried (Na2SO4). The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography [EtOAc–petroleum ether (bp 40–60 °C) 2:8]. Yield
80% (0.23 g); White solid; mp 48-50 °C; ½aꢁD þ 10:1 (c 1, CHCl3);
1H NMR (200 MHz, CDCl3) d 6.84 (dd, J1 = 5.6 Hz, J2 = 15.8 Hz,
1 H), 5.90 (dd, J1 = 1.4 Hz, J2 = 15.8 Hz, 1 H), 4.52 (m, 1 H), 4.38–
4.05 (m, 3 H), 1.80–1.10 (m, 18 H), 0.89 (t, J = 6.2 Hz, 3 H); 13C
NMR (50 MHz, CDCl3) d 166.4, 155.1, 148.6, 120.5, 79.7, 60.4,
51.4, 34.3, 28.3, 27.7, 22.4, 14.2, 13.9; Anal. Calcd for
C15H27NO4: C, 63.13; H, 9.54; N, 4.91. Found: C, 63.02; H, 9.69;
N, 4.85.
5.2.2.19. 2-Oxo-N-(4-oxooctyl)hexadecanamide (26). Yield 79%;
White solid; mp 73–76 °C; 1H NMR (200 MHz, CDCl3) d 7.09 (t,
J = 6.4 Hz, 1 H), 3.35–3.25 (m, 2 H), 2.90 (t, J = 7.2 Hz, 2 H), 2.50–
2.36 (m, 4 H), 1.98–1.76 (m, 2 H), 1.67–1.45 (m, 4 H), 1.25 (br s,
24 H), 0.88 (t, J = 6.6 Hz, 6 H); 13C NMR (50 MHz, CDCl3) d 210.3,
199.2, 160.3, 42.6, 39.7, 38.8, 36.7, 31.9, 29.6, 29.5, 29.4, 29.3,
29.0, 25.9, 23.2, 23.1, 22.7, 22.3, 14.1, 13.8; MS (FAB): m/z (%):
396 (100) [M+H]+; Anal. Calcd for C24H45NO3: C, 72.86; H, 11.46;
N, 3.54. Found: C, 72.74; H, 11.51; N, 3.59.
5.2.4. General method for catalytic hydrogenation
To a solution of the substrate (1.00 mmol) in MeOH (10 mL)
(through which N2 had been passed for 5 min), 10% Pd/C catalyst
(10 mg, 0.01 mmol) was added. The reaction mixture was stirred
under H2 atmosphere overnight at room temperature. The catalyst
was removed by filtration through a pad of Celite, and the organic
solvent was evaporated under reduced pressure.
5.2.2.20. N-(4-Ethoxybutyl)-2-oxohexadecanamide (27). Yield
88%; White solid; mp 55–57 °C; 1H NMR (200 MHz, CDCl3) d 7.19

10268
G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
5.2.4.1. (S)-Ethyl 6-(tert-butoxycarbonylamino)decanoate (14).
Yield 89%; Oil; 1Y NMR (200 MHz, CDCl3) d 4.26 (d, J = 9.2 Hz, 1 H),
4.10 (q, J = 7 Hz, 2 H), 3.64–3.41 (m, 1 H), 2.28 (t, J = 7.4 Hz, 2 H),
1.71–1.16 (m, 24 H), 0.87 (t, J = 6.2 Hz, 3 H); 13C NMR (50 MHz,
CDCl3) d 173.7, 155.7, 78.8, 60.1, 50.4, 35.2, 34.2, 28.3, 28.0, 25.4,
24.9, 22.6, 14.2, 14.0. Anal. Calcd for C17H33NO4: C, 64.73; H,
10.54; N, 4.44. Found: C, 64.69; H, 10.61; N, 4.46.
by column-chromatography using CHCl3 as eluent. Yield 75%;
Oil; 1H NMR (200 MHz, CDCl3) d 7.40–7.22 (m, 5 H), 4.58–4.35
(m, 1 H), 3.90–3.78 (m, 1 H), 2.69 (t, J = 6.6 Hz, 2 H), 1.95–1.80
(m, 2 H), 1.80–1.60 (m, 2 H), 1.60–1.40 (m, 6 H); 13C NMR
(50 MHz, CDCl3) d 142.4, 128.2, 128.1, 125.5, 120.0, 60.9, 35.7,
34.8, 31.1, 28.8, 28.6, 24.3; Anal. Calcd for C14H19NO: C, 77.38; H,
8.81; N, 6.45. Found: C, 77.11; H, 8.95; N, 6.37.
5.2.4.2. (R)-Ethyl 4-(tert-butoxycarbonyl)octanoate (40a). Yield
5.2.8. 2-Hydroxy-8-phenyloctanoic acid (35)
99%; White solid; mp 32–34 °C; ½a D
ꢁ
+3.4 (c 1 CHCl3); 1H NMR
A solution of 34 (0.36 g, 1.7 mmol) in concd HCl (4.2 mL) was
stirred overnight at room temperature. Then, water was added
and the aqueous solution was extracted with CHCl3 (3ꢂ 5 mL).
The combined organic layers were washed with brine and dried
over Na2SO4. The organic solvent was evaporated under reduced
pressure and the resulting 2-hydroxy-8-phenyloctanamide was
precipitated using Et2O.
(200 MHz, CDCl3) d 4.29 (d, J = 9.2 Hz, 1 H), 4.13 (q, J = 6.4 Hz,
2 H), 3.62–3.45 (m, 1 H), 2.36 (t, J = 7.6 Hz, 2 H) 1.89–1.78 (m,
2 H), 1.76–1.58 (m, 2 H), 1.43 (s, 9 H), 1.40–1.21 (m, 7 H), 0.88 (t,
J = 6.4 Hz, 3 H); 13C NMR (50 MHz, CDCl3) d 173.7, 155.7, 79.0,
60.4, 50.3, 35.5, 31.1, 30.5, 28.4, 28.0, 22.5, 14.2, 14.0; Anal. Calcd
for C15H29NO4: C, 62.69; H, 10.17; N, 4.87. Found: C, 62.55; H,
10.23; N, 4.95.
To
a solution of 2-hydroxy-8-phenyloctanamide (0.32 g,
1.3 mmol) in a mixture of EtOH/water (2:1, 10 mL), KOH (0.75 g,
13.4 mmol) was added and the reaction mixture was refluxed for
4 h. After cooling, EtOH was removed under reduced pressure,
water was added and the aqueous solution was acidified with con-
cd H2SO4 until pH 1, followed by extraction with Et2O (3ꢂ 5 mL).
The combined organic layers were washed with brine and dried
over Na2SO4. The organic solvent was evaporated under reduced
pressure and the residue was purified by recrystallization using
CH2Cl2/petroleum ether. Yield 85%; White solid; mp 84–86 °C; 1H
NMR (200 MHz, CDCl3) d 7.35–7.24 (m, 5 H), 4.40–4.25 (m, 1 H),
2.68 (t, J = 6.6 Hz, 2 H), 2.00–1.82 (m, 2 H), 1.80–1.58 (m, 3 H),
1.58–1.15 (m, 6 H); 13C NMR (50 MHz, CDCl3) d 179.5, 142.6,
128.4, 128.2, 125.6, 70.2, 35.9, 34.1, 29.0, 24.6; Anal. Calcd for
C14H20O3: C, 71.16; H, 8.53. Found: C, 70.92; H, 8.84.
5.2.5. tert-Butyl 4-oxooctylcarbamate (22)
To
a stirred solution of Boc-c-aminobutyric acid (0.40 g,
2.0 mmol) in dry THF (2 mL), cooled at ꢀ78 °C, a solution of n-BuLi
in hexane (3 mL, 2.5 M) was added dropwise. After completion of
the addition, the reaction mixture was allowed to warm to room
temperature and stirring was continued for 4 h. Then, saturated
NH4Cl (5 mL) and EtOAc (10 mL) were added. The aqueous layer
was separated and extracted with EtOAc (2ꢂ 10 mL). The com-
bined organic layers were dried over Na2SO4, the organic solvent
was evaporated under reduced pressure and the residue was puri-
fied by column-chromatography using CHCl3 as eluent. Yield 46%;
White solid; mp 36–38 °C; 1Y NMR (200 MHz, CDCl3) d 4.68 (br,
1 H), 3.13–3.03 (m, 2 H), 2.45–2.33 (m, 4 H), 1.87–1.65 (m, 2 H),
1.55–1.23 (m, 13 H), 0.86 (t, J = 7.2 Hz, 3 H); 13C NMR (50 MHz,
CDCl3) d 210.7, 156.0, 79.0, 42.5, 40.0, 39.7, 28.3, 25.8, 23.9, 22.2,
13.7. Anal. Calcd for C13H25NO3: C, 64.16; H, 10.36; N, 5.76. Found:
C, 64.10; H, 10.42; N, 5.71.
5.2.9. (R)-4-(7-Phenylheptanamido)octanoic acid (42)
To a stirred solution of 41a (or 41b) (2.00 mmol) in a mixture of
dioxane/H2O (9:1, 20 mL), 1 N NaOH (2.2 mL, 2.2 mmol) was
added, and the mixture was stirred for 12 h at room temperature.
The organic solvent was evaporated under reduced pressure, and
H2O (10 mL) was added. The aqueous layer was washed with
EtOAc, acidified with 1 N HCl, and extracted with EtOAc (3ꢂ
12 mL). The combined organic layers were washed with brine,
dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified after recrystallization [EtOAc/petroleum ether
5.2.6. tert-Butyl 4-ethoxybutylcarbamate (23)
To a stirred solution of tert-butyl 4-hydroxybutylcarbamate
(0.38 g, 2.0 mmol) and ethyl bromide (0.65 g, 6 mmol) in ben-
zene (2 mL) at 10 °C, aq 50% NaOH (2 mL) and phase transfer
catalyst Bu4NHSO4 (0.08 g, 0.3 mmol) were added. The reaction
mixture was vigorously stirred for 4 h at 10 °C and overnight
at room temperature. The aqueous layer was separated and ex-
tracted with EtOAc (2ꢂ 5 mL). The combined organic layers were
washed with brine and dried over Na2SO4. The organic solvent
was evaporated under reduced pressure and the residue was
purified by column-chromatography using CHCl3 as eluent. Yield
95%; Oil; 1Y NMR (200 MHz, CDCl3) d 4.72 (br, 1 H), 3.46–3.34
(m, 4 H), 3.19–3.06 (m, 2 H), 1.60–1.43 (m, 4 H), 1.38 (s, 9 H),
1.14 (t, J = 7.4 Hz, 3 H); 13C NMR (50 MHz, CDCl3) d 155.9, 78.7,
70.1, 66.0, 40.3, 28.3, 27.0, 26.8, 15.0. Anal. Calcd for
C11H23NO3: C, 60.80; H, 10.67; N, 6.45. Found: C, 60.55; H,
10.81; N, 6.32.
(bp 40–60 °C)]. Yield 90%; White solid; mp 73–75 °C; ½aꢁD ꢀ5.7 (c 1
CHCl3); 1H NMR (200 MHz, CDCl3) d 11.10 (br s, 1 H), 7.39–7.23 (m,
5 H), 5.86 (d, J = 9.0 Hz, 1 H), 4.10–3.94 (m, 1 H), 2.68 (t, J = 7.4 Hz,
2 H), 2.46 (t, J = 7.4 Hz, 2 H), 2.26 (t, J = 7.4 Hz, 2 H), 2.04–1.87 (m,
1 H), 1.82–1.57 (m, 5 H), 1.57–1.09 (m, 10 H), 0.97 (t, J = 6.4 Hz,
3 H); 13C NMR (50 MHz, CDCl3) d 177.4, 173.9, 142.5, 128.3,
128.1, 125.5, 49.0, 36.6, 35.8, 34.9, 31.2, 31.1, 30.2, 29.0, 28.8,
28.0, 25.7, 22.4, 13.9; MS (ESI) m/z (%): 370 (100) [M+Na]+; Anal.
Calcd for C21H33NO3: C, 72.58; H, 9.57; N, 4.03. Found: C, 72.26;
H, 9.74; N, 4.11.
Compounds 10a–c,26 13,30 29,49 30,50 31,35 33,51 38,52 39b,26
40b26 have been described previously.
5.2.7. 2-Hydroxy-8-phenyloctanenitrile (34)
To a stirred solution of 33 (0.45 g, 2.4 mmol) in CH2Cl2 (3 mL), a
solution of NaHSO3 (0.37 g, 3.5 mmol) in water (0.6 mL) was added
and the mixture was vigorously stirred for 30 min at room temper-
ature. The organic solvent was evaporated under reduced pressure,
water was added and the mixture was cooled at 0 °C. Then, a solu-
tion of KCN (0.23 g, 3.5 mmol) in water (0.6 mL) was added drop-
wise over a period of 3.5 h and then the mixture was left under
stirring overnight at room temperature. The aqueous suspension
was extracted with CH2Cl2 (2ꢂ 5 mL). The combined organic layers
were washed with brine and dried over Na2SO4. The solvent was
evaporated under reduced pressure and the residue was purified
5.3. In-vitro PLA2 assays
Phospholipase A2 activity was determined using the previously
described modified Dole assay25 with buffer and substrate condi-
tions optimized for each enzyme as described previously:25,26,29,30
(i) GIVA cPLA2 substrate mixed-micelles were composed of 400
Triton X-100, 97 M PAPC, 1.8 M PIP2
M 14C-labeled PAPC, and 3
in buffer containing 100 mM HEPES pH 7.5, 90 M CaCl2, 2 mM
DTT and 0.1 mg/ml BSA; (ii) GVI iPLA2 substrate mixed-micelles
were composed of 400 Triton X-100, 99 DPPC, and
1.5
14C-labeled DPPC in buffer containing 200 mM HEPES pH
lM
l
l
l
l
lM
lM
l
M

G. Antonopoulou et al. / Bioorg. Med. Chem. 16 (2008) 10257–10269
10269
20. Magrioti, V.; Kokotos, G. Anti-Inflamm. Anti-Allergy Agents Med. Chem. 2006, 5,
189.
21. McKew, J. C.; Foley, M. A.; Thakker, P.; Behnke, M. L.; Lovering, F. E.; Sum, F.-W.;
Tam, S.; Wu, K.; Shem, M. W. H.; Zhang, W.; Gonzalez, M.; Liu, S.; Mahadevan,
A.; Sard, H. J. Med. Chem. 2006, 49, 135.
22. Lee, K. L.; Foley, M. A.; Chen, L. R.; Behnke, M. L.; Lovering, F. E.; Kirincich, S. J.;
Wang, W. H.; Shim, J.; Tam, S.; Shen, M. W. H.; Khor, S. P.; Xu, X.; Goodwin, D.
G.; Ramarao, M. K.; Nickerson-Nutter, C.; Donahue, F.; Ku, M. S.; Clark, J. D.;
Mckew, J. C. J. Med. Chem. 2007, 50, 1380.
7.0, 1 mM ATP, 2 mM DTT and 0.1 mg/ml BSA; and (iii) GV sPLA2
substrate mixed-micelles were composed of 400 M Triton X-
100, 99 M DPPC, and 1.5
14C-labeled DPPC in buffer contain-
l
l
lM
ing 50 mM Tris pH 8.0 and 5 mM CaCl2.
5.4. In-vitro PLA2 inhibition studies
23. Lee, K. L.; Behnke, M. L.; Foley, M. A.; Chen, L.; Wang, W.; Vargas, R.; Nunez, J.;
Tam, S.; Mollova, N.; Xu, X.; Shen, M. W. H.; Ramarao, M. K.; Goodwin, D. G.;
Nickerson-Nutter, C. L.; Abraham, W. M.; Williams, C.; Clark, J. D.; McKew, J. C.
Bioorg. Med. Chem. 2008, 16, 1345.
24. Ludwig, J.; Bovens, S.; Brauch, C.; Elfringhoff, A. S.; Lehr, M. J. Med. Chem. 2006,
49, 2611.
25. Kokotos, G.; Kotsovolou, S.; Six, D. A.; Constantinou-Kokotou, V.; Beltzner, C. C.;
Dennis, E. A. J. Med. Chem. 2002, 45, 2891.
26. 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. J.
Med. Chem. 2004, 47, 3615.
27. Constantinou-Kokotou, V.; Peristeraki, A.; Kokotos, C. G.; Six, D. A.; Dennis, E. A.
J. Pept. Sci. 2005, 11, 431.
28. Yaksh, T. L.; Kokotos, G.; Svensson, C. I.; Stephens, D.; Kokotos, C. G.;
Fitzsimmons, B.; Hadjipavlou-Litina, D.; Hua, X.-Y.; Dennis, E. A. J. Pharmacol.
Exp. Ther. 2006, 316, 466.
29. Stephens, D.; Barbayianni, E.; Constantinou-Kokotou, V.; Peristeraki, A.; Six, D.
A.; Cooper, J.; Harkewicz, R.; Deems, R. A.; Dennis, E. A.; Kokotos, G. J. Med.
Chem. 2006, 49, 2821.
Initial screening of compounds at 0.091 mole fraction inhibitor
in mixed-micelles was carried out. We considered compounds dis-
playing 25% or less inhibition to have no inhibitory effect (desig-
nated N.D.). We report average percent inhibition (and standard
error, n = 3) for compounds displaying more than 25% and less than
90% enzyme inhibition. If percent inhibition was greater than 90%,
we determined its XI(50) by plotting percent inhibition vs. inhibi-
tor molar fraction (7 points; typically 0.005–0.091 mole fraction).
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.
Acknowledgements
30. Six, D. A.; Barbayianni, E.; Loukas, V.; Constantinou-Kokotou, V.; Hadjipavlou-
Litina, D.; Stephens, D.; Wong, A. C.; Magrioti, V.; Moutevelis-Minakakis, P.;
Baker, S.; Dennis, E. A.; Kokotos, G. J. Med. Chem. 2007, 50, 4222.
31. Moutevelis-Minakakis, P.; Neokosmidi, A.; Filippakou, M.; Stephens, D.;
Dennis, E. A.; Kokotos, G. J. Pept. Sci. 2007, 13, 634.
32. Antonopoulou, G.; Magrioti, V.; Stephens, D.; Constantinou-Kokotou, V.;
Dennis, E. A.; Kokotos, G. J. Pept. Sci. 2008, 14, 1111.
33. Reid, R. C. Curr. Med. Chem. 2005, 12, 3011.
The project is co-funded by the European Social Fund and Na-
tional Resources (EPEAEK II) (G.K.) and by NIH Grant GM 20,501
(E.A.D).
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