Inhibition of secreted phospholipase A2. 4-Glycerol derivatives of 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one with broad activities

Article abstract of DOI:10.1021/jm060082n

Secreted phospholipases A₂ (sPLA₂s) have been reported to play an important role in various inflammatory conditions and thus represent an attractive therapeutic target. Previous SAR studies from our laboratory have revealed certain important features of our recently discovered specific hGIIA sPLA₂ inhibitors, and we report here the synthesis and biological activities of glycerol-containing derivatives of our lead compound III (Figure 1). Efficient and selective synthesis methods have been developed to make glycerol trisubstituted by different groups on desired positions. In terms of biological activities, the best compounds (A3, A6, and A15) are more active than III (Figure 1), as potent as Me-Indoxam, an sPLA₂s inhibitor of reference, against hGIIA, hGV, and hGX sPLA₂s and at least 10 times less active toward the GIB enzymes in two in vitro assay systems. By synthesis of enantiopure (S)-A6, we demonstrated that no important improvement of the inhibitory potency could be achieved by this approach. Furthermore, the results show that the global lipophilicity is likely responsible for the anti-PLA ₂ activity and two oxadiazolone moieties seem too big to be accommodated by the active site of the hGIIA enzyme.

Full text of DOI:10.1021/jm060082n

1618
J. Med. Chem. 2007, 50, 1618-1626
Inhibition of Secreted Phospholipase A2. 4-Glycerol Derivatives of
4,5-Dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one with Broad Activities
Mohamed Touaibia,† Atime´ Djimde´,† Fei Cao,† Eric Boilard,‡ Sofiane Bezzine,‡ Ge´rard Lambeau,‡ Catherine Redeuilh,†
Aazdine Lamouri,† France Massicot,†,§ Franc¸ois Chau,† Chang-Zhi Dong,† and Franc¸oise Heymans*,†
Unite´ de Pharmacochimie Mole´culaire et Syste`mes Membranaires (EA2381), UniVersite´ Paris7-Denis Diderot, case 7066, 2 place Jussieu,
75251 Paris, Cedex 05, France, Institut de Pharmacologie Mole´culaire et Cellulaire, UMR 6097 CNRS et UniVersite´ de Nice-Sophia Antipolis,
660 route des Lucioles, Sophia Antipolis, Valbonne 06560, France, and Laboratoire de Toxicologie, Faculte´ des Sciences Pharmaceutiques et
Biologiques, 4 aVenue de l’ObserVatoire, 75270 Paris, Cedex 06, France
ReceiVed January 25, 2006
Secreted phospholipases A2 (sPLA2s) have been reported to play an important role in various inflammatory
conditions and thus represent an attractive therapeutic target. Previous SAR studies from our laboratory
have revealed certain important features of our recently discovered specific hGIIA sPLA2 inhibitors, and
we report here the synthesis and biological activities of glycerol-containing derivatives of our lead compound
III (Figure 1). Efficient and selective synthesis methods have been developed to make glycerol trisubstituted
by different groups on desired positions. In terms of biological activities, the best compounds (A3, A6, and
A15) are more active than III (Figure 1), as potent as Me-Indoxam, an sPLA2s inhibitor of reference, against
hGIIA, hGV, and hGX sPLA2s and at least 10 times less active toward the GIB enzymes in two in Vitro
assay systems. By synthesis of enantiopure (S)-A6, we demonstrated that no important improvement of the
inhibitory potency could be achieved by this approach. Furthermore, the results show that the global
lipophilicity is likely responsible for the anti-PLA2 activity and two oxadiazolone moieties seem too big to
be accommodated by the active site of the hGIIA enzyme.
Introduction
shown recently that exogenously added group V or group X
sPLA2s to mammalian cells leads to arachidonic acid (AA)
release,20-22 which is also observed in mammalian cells trans-
fected with groups IIA and X sPLA2s.23 Data from GV PLA2-
null mice have provided clear evidence of a role for GV PLA2
in regulating eicosanoid generation in response to an acute innate
stimulus of the immune system in ViVo,24 suggesting a potential
function of this enzyme in innate immunity. Moreover, GV
PLA2 is detected in human atherosclerotic aortic lesions and
aortic root sections from apolipoprotein E-deficient mice.25
Similarly, GX PLA2 is detected in foam cell lesions in the
arterial intima of high fat-fed apolipoprotein E-deficient mice.26
Therefore, modification of plasma lipoproteins by GV and GX
PLA2s may be more relevant than that by GIIA PLA2 to the
pathogenesis of atherosclerosis. Recent studies have indicated
the presence of various types of cross-talk between sPLA2 and
cPLA2R resulting in effective arachidonic acid (AA) release.
By working on cPLA2R-deficient mice and by using specific
sPLA2 and cPLA2R inhibitors, GX PLA2 has been found to
induce AA release and eicosanoid production without activation
of cPLA2R.27
The various distribution of each group of mammalian sPLA2s
in healthy or inflamed tissues14-17,28 suggests that these enzymes
play different roles in physiological and pathophysiological
conditions. It is thus important to develop selective inhibitors
of each sPLA2 isozyme and inhibitors with broad spectrum
activities to all inflammatory-related sPLA2s to study the
physiopathological functions of each sPLA2 under different
biological conditions. Many GIIA sPLA2 inhibitors have been
reported, and to date the most potent ones are those described
by Lilly and Shionogi research laboratories.29-32 Previous work
from our laboratory (Figure 1) has shown that molecules as
simple as the amidine I33 and the piperazine derivative II34 are
fairly potent inhibitors of GIIA sPLA2. They also inhibit the
enzymatic activity of GIB PLA2 which is known to play a
Phospholipases A2 (PLA2, phosphatidyl 2-acylhydrolase, EC
3.1.1.4) are involved in various physiopathological processes
by promoting the release of free fatty acids and lyso-phospho-
lipids from membrane glycero-phospholipids. The implication
of certain PLA2s in inflammation has been demonstrated by
their ability to regulate in the inflammatory reaction the amount
of arachidonic acid (AA), precursor for the formation of
prostaglandins, leukotrienes, and thromboxanes, three of the
most powerful lipid inflammatory mediators. Meanwhile, the
lyso-phospholipids released in the enzymatic reaction result in
certain settings in the formation of platelet-activating factor
(PAF), another potent mediator of inflammation.
To date, 12 groups of PLA2s (group I or GI to group XII or
GXII) are known and classified mainly into two categories:
intracellular PLA2s, including cytosolic PLA2s (cPLA2s) and
Ca2+-independent PLA2s (iPLA2s), and secreted PLA2s (sPLA2s)
of which a great number have been cloned and expressed in
the past decade and the characterization remains to be an active
area.1,2 Until recently, the increased serum PLA2 activity has
been attributed to hGIIA enzyme in different human inflam-
matory diseases,3 such as septic shock,4 rhumatoid arthritis,5,6
Crohn’s disease,7 acute pancreatitis,8 or partly to hGIIA enzyme
in psoriasis9 and adult respiratory distress syndrome.10 However,
a large body of evidence indicates that the group V and group
X sPLA2s also contribute to the production of various inflam-
matory mediators in mammalian cells, as well as in tissues
pretreated with pro-inflammatory agents or isolated from patients
suffering from various inflammatory diseases.11-20 It has been
* To whom correspondence should be addressed. Telephone: 33-(0)1-
44-27-56-26, Fax: 33-(0)1-44-27-56-41, E-mail: heymans@ccr.jussieu.fr.
† Unite´ de Pharmacochimie Mole´culaire et Syste`mes Membranaires
(EA2381).
‡ Institut de Pharmacologie Mole´culaire et Cellulaire.
§ Laboratoire de Toxicologie.
10.1021/jm060082n CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/03/2007

Inhibition of Secreted Phospholipase A2
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1619
Figure 1. Previous sPLA2s inhibitors discovered in our laboratory.
critical role in digestion, indicating that the inhibition is not
enzyme-specific. An extensive structure-activity relationship
study has led to the identification of the lead compound III
(Figure 1), which is more specific for hGIIA than GIB sPLA2.
One of the key elements of this specificity appears to be the
presence of the oxadiazolone cycle.35
As shown in Scheme 2, the nitrile group of the intermediates
2a, 3a, 5a-12a, 2d, and 3d was converted into the amidoxime
function with NH2OH, formed in situ from its HCl salt and K2-
CO3. Treatment of these amidoximes 13-24 with phenyl
chloroformate yielded the corresponding carbonates which, after
heating to reflux in toluene, provided the final oxadiazolone
derivatives A1-A12. The trityl group in A6 was removed by
acid hydrolysis to afford A13.
Following Wheeler and Wilson’s method and starting from
the commercially available 2,3-dibromo-1-propanol and 1,3-
dibromo-2-propanol (Scheme 3), compounds 25a, 25b and the
precursors of 28a, 28-30b were obtained in good yields and
next substituted under the conditions as described above to
afford 26a, 26b, 28a, 28-30b. The esters 26b, 28-30b were
subsequently converted into the nitrile homologues 26e, 28-
30e. The diamidoximes 27a, 27e, 31a, 31e, 32e and the
monoamidoximes 33-34 resulted from hydroxylamination of
the corresponding dinitrile compounds in the same manner as
for 13-24. Their transformation into the carbonate intermediates
and then the ring closure were performed using the same
procedure as above to yield the target molecules, B1-B2, C1-
C3, and A14-A15
We have also reported that when the chain length in III
(Figure 1) was shortened, some new interactions should be
created by introducing new structural elements to maintain the
activity and the specificity. The design has been assisted by
molecular modeling.36 On the other hand, when the C14 chain
in III (Figure 1) was split up to two equal fragments and
assembled in the same molecule by disubstitution, the activity
decreased significantly unless a long chain such as C14 was
present as one of both the substituents.37 These results prompted
us to examine the importance of the hydrophobic part of III
(Figure 1) in a different series. Given that the glycerol backbone
is a natural structural element in the native substrates of PLA2s,
we designed and synthesized a series of glycerol derivatives of
III (Figure 1) through efficient and selective alkylations of the
glycerol backbone. In these pseudosubstrate analogues, the
hydrophilic head of the substrates was replaced by an oxadia-
zolone ring and different alkyl chains were attached to the two
other positions of glycerol. Their biological activities were
evaluated in two different in Vitro assay systems using group
IB, IIA, V, and X sPLA2s. The best inhibitor was found to be
highly active at sub-micromolar concentrations against hGIIA,
hGV, and hGX sPLA2s, and about 10 times less potent toward
the GIB enzymes. Me-Indoxam, a sPLA2 inhibitor of reference,
displays comparable activities in the same assay conditions.
Chemistry. In this work, we needed O-dihomo or O-dihet-
eroalkyl-(4-cyanophenyl)glycerols and O-dihomo or O-dihet-
eroalkyl-(4-cyanomethylphenyl)glycerols as key intermediates.
Wheeler and Willson38 have reported that phenol can be
condensed with 3-chloro-1,2-propanediol to provide 1-O-
phenylglycerol in a 64% yield. The same procedure was thus
applied to our syntheses (Scheme 1), and the compounds 1a-d
were obtained with comparable or better yields: 60% for 1a,
65% for 1b,39 quantitative for 1c, and 60% for 1d. Dihomoal-
kylation of 1a-c using different alkyl bromides led to com-
pounds 2a-c and 3a-c (Scheme 1).
Results and Discussion
We have recently reported a series of hGIIA sPLA2 specific
inhibitors, and the specificity appears to be related to the
presence of an oxadiazolone ring as the hydrophilic head, which
in its anionic form should play a role of Ca2+ ligand at the active
site of the enzyme.35,37 On the other hand, the potency of an
inhibitor is largely dependent on the nature and the disposition
of the hydrophobic group(s).36,37 While the optimal linear chain
Scheme 1a
Direct dialkylation of 1d under the same conditions as for
1a-c failed, since a simultaneous alkylation of the alpha CH2
of the nitrile function occurred in high proportion (90%).
Consequently, a new synthetic pathway was designed. The
alcohols 2e, 3e were prepared either by reduction of the esters
2b, 3b or acid cleavage of the acetal function of 2c, 3c in the
presence of pyridinium p-toluenesulfonate (PPTS). Bromination
of 2e, 3e, followed by a substitution reaction using sodium
cyanide gave the corresponding nitriles, 2d, 3d in 60% yield.
Dissymmetrical substitution strategy leading to 6a-12a is also
outlined in Scheme 1. The primary hydroxyl group of 1a was
selectively blocked with a trityl group (4a), and the remaining
secondary hydroxyl group was alkylated after removal of the
proton by NaH, using the corresponding bromide to yield 5a.
Acid-catalyzed hydrolysis of the trityl group was then carried
out, leading to the free alcohol function from which the ethers
6a-12a were formed with the suitable alkyl bromides as above.
a Reagents and conditions: (a) p-HOPhY, EtONa, EtOH, rt; (b) (i) NaH,
DMF, rt, (ii) R1Br ()R2Br); (c) 2b or 3b, LiAlH4, dry Et2O, rt; (d) 2c or
3c, PPTS, EtOH, 50 °C; (e) 15% HBr in acetic acid, 0 °C; (f) NaCN, DMF,
rt; (g) Ph3CCl, Py, cat. DMAP, CH2Cl2, rt; (h) (i) NaH, DMF, rt, (ii) R2Br;
(i) 1 N HCl, THF, rt; (j) NaH, DMF, rt, (ii) R1Br.

1620 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Touaibia et al.
Scheme 2a
a Reagents and conditions: (a) NH2OH HCl, K2CO3, absol EtOH, reflux; (b) PhOCOCl, Et3N, CH2Cl2, 0 °C; (c) toluene, reflux; (d) 2 N HCl, THF, rt.
Scheme 3a
a
Reagents and conditions: (a) p-HOPhY, EtONa, EtOH, rt; (b) (i) NaH, DMF, rt, (ii) RBr; (c) LiAlH4, THF, rt; (d) (i) 15% HBr in acetic acid, 0 °C;
(ii) NaCN, DMF, rt; (e) NH2OH HCl, K2CO3, absol EtOH, reflux; (f) PhOCOCl, Et3N, CH2Cl2, 0 °C; (g) toluene, reflux.
length is composed of 14 carbons (C14),37 a much shorter chain
(C5) is needed when it is terminated by an aromatic group, such
as an indole ring.36 According to our docking study, the indole
ring can generate new π-π or cation-π interactions with certain
residues of hGIIA sPLA2, which is likely to compensate for
the loss of Van der Waals forces between the inhibitor and the
enzyme induced by the cutoff of a part of the C14 chain.36 To
better understand the mechanism of action and more deeply
evaluate the importance of the hydrophobic part for the activity
and selectivity, we describe here the synthesis and structure-
activity relationship (SAR) study of a series of glycerol
derivatives of III (Figure 1).
various groups. In regard to the other members of the A series,
either the two same alkyl groups (A9-A12) or C18 at the
2-position (A15) were used.
All the glycerol derivatives A1-A15 were first submitted to
an in Vitro fluorimetric assay (see Experimental Section) to
determine their inhibitory potency and selectivity against human
group IIA PLA2 (hPLA2) versus porcine group IB (pPLA2). The
reference compound used in this assay condition is one of the
specific and active site targeted hGIIA PLA2 inhibitors of Lilly
Laboratories, LY311727, as previously reported.37
The results in Table 1 show that the presence of a free alcohol
function at the 1-position of glycerol is not favorable to the
biological activity. A13 displays a weak activity, even though
it presents a lipophilicity close to that of III (Figure 1),35 our
home reference compound (log P ) 7). This implies that, as in
the natural substrates, a certain length of alkyl chain is necessary
at this position to establish supplemental Van der Waals
On the basis of our earlier results,37 a C14 chain was
maintained at the 2-position of glycerol in 10 compounds of
the A series (A1-A8, A13-A14) and oxadiazolone attached
to the 3-position through a phenyl or benzyl as linkers. The
OH function at the 1-position was either free or substituted with

Inhibition of Secreted Phospholipase A2
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1621
Table 1. Inhibition of the Enzymatic Activity of Porcine Pancreatic PLA2 (pGIB) and Human Group IIA PLA2 (hGIIA) by Glycerol Derivatives of III
(Figure 1), Determined by Using the Fluorimetric Assay
IC50 (µM)
compd
n
R1
R2
pGIB
>40
>100
>100
>25
2.2 ( 0.2
>50
>50
hGIIA
log Pa
A1
A2
A3
A4
A5
A6
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
1
C2H5
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C14H29
n-C14H29
C6H5(CH2) 4
n-C7H15
n-C7H15
n-C9H19
n-C14H29
n-C14H29
n-C18H37
29.8 ( 0.3
8.3 ( 0.2
0.28 ( 0.02
14.8 ( 0.2
0.29 ( 0.03
0.9 ( 0.1
0.33 ( 0.12
0.28 ( 0.02
2.2 ( 0.1
76.7 ( 0.3
50.9 ( 0.1
11.6 ( 0.3
2.7 ( 0.1
56.0 ( 0.4
1.1 ( 0.1
0.15 ( 0.03
4.0 ( 0.9
0.47
7.34
8.72
11.5
8.48
10.0
11.8
11.8
10.1
8.56
6.51
6.30
7.12
9.18
6.59
8.94
11.0
7.08
n-C5H11
n-C10H21
C6H5CH2
C6H5(CH2)4
(C6H5)3C
(S)-A6
(C6H5)3C
A7
A8
A9
A10
A11
A12
A13
A14
(C6H5)2CH
m-MeOC6H4CH 2
C6H5(CH2)4
n-C7H15
n-C7H15
n-C9H19
5.6 ( 0.1
39 ( 1
>100
>100
>100
>100
H
>100
p-NCCH2C6H4
p-NCCH2C6H4
9.6 ( 0.1
36.6 ( 0.4
>100
A15
III (Figure 1)35,37
LY31172737
8.0
a Calculated using the Rekker’s hydrophobic fragmental constants.
interactions with the hydrophobic side chains of certain sPLA2s
residues. Indeed, alkylating this OH leads to an increasing
activity and selectivity as a function of the chain length.
Attaching an ethyl group to this position provides compound
A1, twice as active as the nonsubstituted A13 (Table 1). Further
increase was observed when a pentyl group was used (A2) and
the activity is particularly enhanced with decyl (A3), 100 times
more active than A1. The difference can be explained by the
increase of the global molecular lipophilicity of 4.1 units of
log P and by the creation of new hydrophobic interactions
between this alkyl chain and the corresponding residues of
hGIIA PLA2. It is important to note that the selectivity of these
compounds toward hGIIA versus GIB enzyme also increases
as a function of the chain length. This increase in selectivity
suggests that the presence of decyl in A3 seems unable to create
sufficient interactions with the porcine GIB PLA2 enzyme (see
A5 and A7 below), as it does in the case of hGIIA PLA2, since
it still remains inactive at 100 µM, the highest concentration
tested against this enzyme.
The substitution at the 1-position by arylalkyl groups in the
compounds A4-A7 was performed in order to study possible
steric, electronic, and/or hydrophobic effects induced by these
groups. As shown in Table 1, a benzyl group at the 1-position
(A4) leads to an activity equivalent to that of the isolipophilic
A2. However, separating the phenyl from the oxygen by three
more methylenes gives the compound A5 which is about 50
times more active than A4. Unfortunately, the selectivity
decreases significantly in parallel. Replacing the benzyl moiety
in A4 by a diphenylmethyl (A7) leads to a scenario similar to
that of A5. Taken together, these results and those described
above for the linear chains suggest that the hydrophobic pocket
of the hGIIA enzyme, well-known to naturally accommodate
the lipophilic part of substrates, is big enough in depth and in
volume and well distanced from the active site to tolerate both
kinds of molecules. However, it should not be the case for the
GIB enzyme, since the decrease in selectivity is only observed
when arylalkyl groups are used, implying that some new
interactions are likely created in this case and consequently
increase the affinity of the inhibitors to the GIB enzyme.
Interestingly, when a trityl group was used, the selectivity was
restored almost completely (A6). The steric hindrance induced
by the third phenyl compared to A7 seems responsible for this
recovery of selectivity, and the potency depends on both the
steric hindrance and the global lipophilicity.
To complete this approach, we undertook to incorporate
substituted phenyl groups onto the glycerol backbone. Introduc-
ing m-methoxybenzyl to the 3-position of glycerol (A8)
increased the anti-PLA2 activity by 7 fold, as compared to the
isolipophilic benzyl derivative (A4), indicating that the presence
of a methoxy group at this position is favorable to the binding
of the inhibitor to the hGIIA enzyme. In compound A14, two
structural modifications were performed: (i) insertion of a
methylene between the oxadiazolone and the phenyl and (ii)
direct connection of the phenyl group to the 3-position of
glycerol with a p-cyanomethyl substitution. These modifications
had little influence on activity but decreased selectivity com-
pared to A8. Interestingly, an elongation by four methylenes of
the alkyl chain at the 2-position of glycerol (A15) improved
the activity by a factor of 7, in agreement with our earlier
observations,37 as well as the selectivity. The latter is a specific
feature for the glycerol derivative A15.
It has been frequently reported that one enantiomer could be
much more potent than the corresponding racemates. In order
to check out if it can happen to this series of compounds, the
(S)-isomer of A6 was synthesized and tested under the same
conditions. Unfortunately, no important improvement of the
inhibitory potency was achieved, as it was found only a little
more active toward hGIIA PLA2 than the racemate with an IC50
of 0.33 ( 0.12 µM against 0.9 ( 0.1 µM for the latter in our
fluorimetric assay (Table 1). Given this fact and the structure
similarity of all final compounds in the A series, no synthesis
of other enantiopure compounds was performed.
Another type of modification in the A series was to use two
identical chains for both the substituents R1 and R2. The
compound A10 in which two heptyls are connected to the 1-
and 2-positions of glycerol showed much less activity, compared
with III (Figure 1), as already observed in our previous work
in which two C7 chains are connected to the different positions

1622 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Touaibia et al.
Table 2. Inhibition of the Enzymatic Activity of Human Group IB
(hGIB), Group IIA (hGIIA), Group V (hGV) and Group X (hGX)
PLA2s by Selected Glycerol Derivatives of III (Figure 1), Determined
by Using the Radiometric Assay
Table 3. Influence of Two Oxadiazolone-Containing Moieties at 2- and
3-Positions of the Glycerol Backbone on the Activity and Specificity,
Determined by Using the Fluorimetric Assay
IC50 (µM)
compd
A3
A4
A5
A6
A7
A14
A15
Me-IDX
hGIB
10
>100
15
5.75
18
7.6
hGIIA
hGV
0.55
>100
hGX
log Pa
1
>100
26
1
14
12
1.25
5.3
5
0.9
4.57
11.5
8.48
10.0
11.8
10.1
8.94
11.0
ND
22
0.66
2
1.73
1
2
5
7.6
1.15
0.18
IC50 (µM)
compd
n
R
pGIB
hGIIA
log Pa
8.7
3.3
B1
B2
0
1
n-C14H29
n-C14H29
63.6 ( 0.4
57.8 ( 0.2
21.0 ( 0.5
3.1 ( 0.1
7.05
7.64
0.27
a Calculated using the Rekker’s hydrophobic fragmental constants
a Calculated using the Rekker’s hydrophobic fragmental constants.
of the phenyl group.37 When both the heptyl groups were
replaced by 4-phenylbutyl (A9), the potency is even worse,
implying that the presence of the phenyl groups is likely not to
induce supplemental interactions with hGIIA enzyme. The
difference between A11 and A10 can be explained by the
insertion of a methylene between the phenyl and oxadiazolone
moieties. The 5-fold gain in potency probably results from a
more optimal length spacer between the Ca2+ ligand and the
hydrophobic pockets of the enzyme, a higher lipophilicity, and
flexibility due to the disruption of the conjugation between both
the two rings. No longer linker was introduced, since we found
earlier in a different series of III (Figure 1) derivatives that
two methylenes did not make the inhibitor more active than
one methylene.37 The fact that A12 is a stronger inhibitor than
A11 is probably due to the increase of two units of log P caused
by the presence of longer chains.
Table 4. Influence of Two Oxadiazolone-Containing Moieties at 1- and
3-Positions of the Glycerol Backbone on the Activity and Specificity,
Determined by Using the Fluorimetric Assay
IC50 (µM)
compd
n
R
pGIB
>100
39.9 ( 0.1
>100
hGIIA
log Pa
C1
C2
C3
0
1
1
n-C14H29
n-C14H29
n-C5H11
47.7 ( 0.2
2.1 ( 0.1
30.5 ( 0.5
7.05
7.64
2.97
a Calculated using the Rekker’s hydrophobic fragmental constants.
In order to check the influence of relative positions of the
three moieties on the glycerol backbone to the activity and
selectivity, the position isomer of A11, 1,3-O-di-n-heptyl-2-O-
[4-(4,5-dihydro-5-oxo-1,2,4-4H-oxadiazol-3-yl)methylphenyl]-
glycerol, was prepared and tested (data not shown). No
difference in terms of activity and selectivity was observed
between A11 and this position isomer, suggesting that this factor
is not important.
the inhibitors and the membrane substrate used in the radiometric
assay, leading to an underestimation of the inhibitory potency,
as often observed in similar works. It is worthwhile to emphasize
that the best compounds (A3, A6, and A10) were found to be
the same in both the cases. In addition, they possess a good
inhibitory activity, not only against hGIIA PLA2 but also against
hGV and hGX enzymes. Although they are a little less active
than Me-Indoxam to inhibit hGIIA and hGV PLA2s, our
inhibitors appear quite potent against hGX enzyme and possess
the same profile as Me-Indoxam for the different groups of
sPLA2s. Taking into account all of these results, certain
compounds of this series are interesting enough to be further
investigated and may represent useful tools to study physiologi-
cal and pathological roles of various secreted PLA2s.
Among the synthetic hGIIA PLA2 inhibitors reported, Lilly
laboratories have shown that the inhibitory activity of a molecule
can be greatly enhanced when a second calcium ligand is
introduced and well positioned in their indole derivatives.29-31
On the basis of our previous results,36-37 we argued that the
oxadiazolone (pKa ) 5.5) should be present in anionic form at
neutral pH, which, by chelating the calcium ion at the catalytic
center of the enzyme, could play a critical role for the activity
and the selectivity of the III (Figure 1) derivatives. To improve
the potency of our inhibitors, two oxadiazolone heterocycles
were connected to either the 2,3- (B1 and B2) or 1,3-positions
(C1-C3) of glycerol. The length of the unique alkyl chain, as
well as the linker between the phenyl and the oxadiazolone,
was also varied to study their influences. The results are listed
in Table 3 and Table 4 due to the difference of the general
structures.
A radiometric assay (see Experimental Section) was then used
in this work, not only to confirm the results obtained with the
fluorimetric assay, but also to analyze the effects of our
inhibitors on other groups of sPLA2s, such as hGV and hGX
enzymes, since they have been reported to be both emerging
inflammatory-type enzymes11-20 and seem to be more relevant
than hGIIA PLA2 to certain pathogeneses.24-27 Moreover, to
make the data more relevant, hGIB PLA2 instead of porcine
GIB PLA2 was employed in this assay system. A preliminary
screening (data not shown) using a single concentration of 10
µM led to the selection of the glycerol derivatives listed in Table
2 for their IC50 determination in this new assay. Me-Indoxam
(Me-IDX), a well-known reference compound,40 was tested
under the same conditions for comparison. Some differences,
especially in term of the IC50 values of the inhibitors toward
hGIIA PLA2 and the hGIIA versus GIB selectivity, have been
observed between the results obtained from the two assay
systems. It is likely due to the difference of the GIB enzymes
used on one hand and the assay conditions on the other hand.
In fact, the GIB PLA2 used in the fluorimetric assay is of porcine
pancreatic source, while that used in the second assay is human
recombinant enzyme. It is not really surprising to observe a
difference of IC50 values, bearing in mind this detail, neither in
consequence of the hGIIA versus GIB selectivity. Meanwhile,
taking into account the high lipophilicity of the tested com-
pounds, some nonspecific interactions could happen between
Compared with the compound A1 of equivalent lipophilicity
in the previous series, B1 (Table 3) shows the same activity
but a worse selectivity, suggesting that doubling the oxadi-

Inhibition of Secreted Phospholipase A2
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1623
Molecular Probes (Leiden, Netherlands). Me-Indoxam is a kind gift
from Dr. M. H. Gelb (University of Washington, Seattle, WA).
(()-1-O-(4-Cyanophenyl)glycerol (1a). To absolute ethanol
(100 mL) was added sodium (1.15 g, 50.0 mmol) piece by piece,
and the mixture was heated to reflux till complete disappearance
of the sodium. 4-Cyanophenol (5.0 g, 42 mmol) was then added
gradually through the condenser, followed by freshly distilled
3-chloro-1,2-propanediol (5.10 g, 46.2 mmol) dropwise. The
mixture was heated to reflux for further 1 h and the solvent
evaporated. The residue was dissolved in water and extracted with
ether. The organic layer was dried (MgSO4), filtered, and concen-
trated in Vacuo to afford an oil which was purified on silica gel
column chromatography (MeOH/CH2Cl2, 5:95, v/v) to give 1a (4.9
g, 60% yield) as a white solid: mp 97-99 °C (litt. 95-98 °C);43
1H NMR (CDC13) δ 7.52 (d, 2H, J ) 8.6, Har), 6.93 (d, 2H, J )
8.6, Har), 3.99 (br s, 2H, OH), 3.72-3.40 (m, 5H, CH2O, CH).
(S)-1a was obtained according to the same procedure from (S)-
azolone heterocycle leads to no improvement in the activity.
However, introducing a methylene group as in the A series has
a beneficial effect since compound B2 is seven times more
active and selective than B1.
The same scenario was observed for the compounds with two
oxadiazolone rings connected to 1,3-positions of the glycerol
backbone (Table 4). It means that the position of the second
oxadiazolone ring has little influence on the activity and the
selectivity. By contrast, a sufficient chain length is required to
maintain the biological properties, since compound C3, more
hydrophilic than C2, is obviously less active.
Taken together, the inhibition data of the compounds B and
C seem to indicate that the active site of hGIIA enzyme could
not accommodate two calcium ligands as big as two oxadi-
azolones.
3-chloro-1,2-propanediol: [R]20 ) -4.13 c 5.11 (CH3COCH3).
D
Conclusion
(()-1-O-[4-(Tetrahydropyran-2-yloxymethyl)phenyl]glycer-
ol (1c). Several drops of concentrated HCl (36%) were added
dropwise to a mixture of 3,4-dihydro-2H-pyran (12.9 g, 0.153 mol)
and 4-hydroxybenzyl alcohol (20.0 g, 0.160 mol) in anhydrous THF
(150 mL) at 0 °C. The reaction mixture was stirred at room
temperature overnight and then reduced to dryness. The residue
was taken up in ether, washed with H2O to neutral pH, and dried
over MgSO4. After filtration, the solvent was evaporated and the
crude product chromatographed (Et2O/petroleum ether, 1:4, v/v)
to provide 4-(tetrahydropyran-2-yloxymethyl)phenol44 as a colorless
oil (18.5 g, 58% yield). It was then used to prepare the compound
1c (quantitative yield) as a colorless oil, following the same
procedure as for 1a, after purification with silica gel column
chromatography using MeOH/CH2Cl2 (2:98, v/v) as eluent: 1H
NMR (CDCl3) δ 7.18 (d, 2H, J ) 8.7, Har), 6.78 (d, 2H, J ) 8.6,
Har), 4.63 (d, 1H, J ) 11.6, OCH2C6H4), 4.60 (t, 1H, J ) 3.3,
OCHO), 4.35 (d, 1H, J ) 11.6, OCH2C6H4), 3.99-3.41 (m, 9H,
CH2OH, CHOH, CH2OC6H4, CH2CH2O), 1.75-1.47 (m, 6H, CH2-
CH2CH2).
(()-3-O-(4-Cyanophenyl)-1,2-O-di-n-heptylglycerol (2a). A
solution of 1a (2.00 g, 10.4 mmol) in dry DMF (25 mL) was added
dropwise to a suspension of NaH (60% dispersion in mineral oil,
1.24 g, 31.0 mmol) in dry DMF (150 mL). The mixture was stirred
at room temperature for 1 h, and 1-bromoheptane (5.56 g, 26.0
mmol) was then added dropwise. After being stirred at room
temperature for 12h, the reaction mixture was diluted with water
(100 mL) and extracted with ether. The combined organic layers
were dried (MgSO4) and filtered, and the solvent was evaporated
under reduced pressure to give an oil which was purified by silica
gel column chromatography (Et2O/petroleum ether, 1:4, v/v) to
afford 2a (2.2 g, 55% yield) as a colorless oil: 1H NMR (CDCl3)
δ 7.50 (d, 2H, J ) 8.7, Har), 6.69 (d, 2H, J ) 8.7, Har), 4.13-3.95
(m, 2H, CH2OC6H4), 3.73-3.69 (m, 1H, CH), 3.57-3.49 (m, 4H,
CH2OC7, CHOCH2C6), 3.39 (t, 2H, J ) 6.5, OCH2C6), 1.50-1.47
(m, 4H, OCH2CH2C5), 1.20-1.09 (m, 16H, CH2), 0.80-0.77 (m,
6H, CH3).
In this work, we have developed an efficient and selective
method to anchor different alkyl and/or arylalkyl groups on the
glycerol backbone. In consequence, a certain number of glycerol
derivatives of III (Figure 1) encompassing one or two oxadi-
azolone rings were synthesized, and their in Vitro inhibitory
potencies toward different secreted PLA2s, including GIB,
hGIIA, hGV, and hGX were evaluated in two distinct in Vitro
assay conditions. Several of them (A3, A6, and A15) are active
with sub-micromolar IC50 values and selective against all
inflammatory-type sPLA2s tested (GIIA, GV, GX) versus GIB
enzyme. By synthesis of enantiopure (S)-A6, we demonstrated
that no important improvement of the inhibitory potency could
be achieved by this approach. Apparently, the global lipophilicity
plays a determinant role in their anti-PLA2 activity, while the
presence of aromatic group(s) induces a negative influence on
selectivity. On the other hand, our results with compounds B
and C suggest that the active site of the GIIA enzyme would
not be large enough to accommodate two oxadiazolone moieties.
These SAR data, associated with our earlier results, prompted
us to design novel compounds for which the synthesis and
inhibitory profiles are now under investigation.
Experimental Section
Chemistry. All materials were obtained from commercial
suppliers and used without further purification or purified as
described. Thin layer chromatography was performed on TLC
plastic sheets of silica gel 60F254 (layer thickness 0.2 mm) from
Merck. Column chromatography purification was carried out on
silica gel 60 (70-230 mesh ASTM, Merck). All melting points
were determined on a digital melting point apparatus (Electrother-
mal) and are uncorrected. The structures of all compounds were
confirmed by IR and NMR spectra. IR spectra were obtained in
paraffin oil with an ATI Mattson Genesis Series FTIR spectrometer.
1H and 13C NMR spectra were recorded on a Bruker AC 200
spectrometer using hexamethyldisiloxane (HMDS) as an internal
standard. Chemical shifts are given in ppm, coupling constant in
Hertz, and peak multiplicities are assigned as follows: s for singlet,
d for doublet, t for triplet, br s for broad singlet, and m for multiplet.
Rotary powers were determined with a Jasco P-1010 polarimeter.
Elemental analyses were obtained from the “Service re´gional de
microanalyse” (Universite´ Pierre et Marie Curie, Paris, France) and
were within (0.4% of theoretical values.
(()-3-O-(4-Cyanophenyl)-1-O-triphenylmethylglycerol (4a).
To a solution of 1a (10.0 g, 52.0 mmol) in CH2Cl2 (300 mL) were
added triethylamine (20.0 mL, 142 mmol), trityl chloride (16.5 g,
59.0 mmol), and 4-(N,N-dimethylamino)pyridine (DMAP, 0.2 g).
The mixture was stirred at room temperature for 48 h and then
washed with water to neutral pH. The organic phase was dried over
MgSO4 and the solvent removed under reduced pressure. The crude
product was purified by silica gel chromatography (pure CH2Cl2)
to afford 4a (13.5 g, 60% yield) as a viscous oil: 1H NMR (CDCl3)
δ 7.48 (d, 2H, J ) 8.6, Har), 7.42-7.28 (m, 6H, Har), 7.24-7.10
(m, 9H, Har), 6.80 (d, 2H, J ) 8.6, Har), 4.04-3.96 (m, 3H, CH2-
OPh, CH), 3.27 (d, 2H, J ) 4.9, CH2O), 2.48 (s, 1H, OH).
(S)-4a was obtained according to the same procedure from (S)-
Biological Materials. Porcine pancreatic sPLA2 and fatty acid-
free bovine serum albumin (BSA) (fraction V) were purchased from
Sigma (Paris, France), and hGIIA sPLA2 was synthesized as
previously reported.41 HGIB, hGV, and hGX sPLA2s were ex-
pressed and purified as described earlier.23,42 The fluorescent
substrate for PLA2 assay, 1-hexadecanoyl-2-(10-pyrenyldecanoyl)-
sn-glycero-3-phosphoglycerol (â-pyC-10-PG), was obtained from
1a: [R]20 ) -5.82 c 4.35 (CHCl3).
D
(()-3-O-(4-Cyanophenyl)-2-O-tetradecyl-1-O-triphenylmeth-
ylglycerol (5a). Compound 5a was prepared according to the same
procedure as for 2a from 4a in 70% yield as a viscous oil after

1624 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Touaibia et al.
purification by rapid silica gel column chromatography (Et2O/
petroleum ether, 5:95, v/v): 1H NMR (CDCl3) δ 7.47 (d, 2H, J )
8.9, Har), 7.43-7.34 (m, 6H, Har), 7.24-7.16 (m, 9H, Har), 6.80
(d, 2H, J ) 8.9, Har), 4.10-3.97 (m, 2H, CH2OPh), 3.68-3.63
(m, 1H, CH), 3.49-3.46 (m, 2H, OCH2C13), 3.20 (d, 2H, J ) 5.2,
CH2OCPh3), 1.50-1.42 (m, 2H, OCH2CH2), 1.18-1.04 (m, 22H,
CH2), 0.77 (t, 3H, J ) 5.5, CH3).
colorless oil: 1H NMR (CDCl3) δ 7.15 (d, 2H, J ) 8.7, Har), 6.80
(d, 2H, J ) 8.7, Har), 4.00 (m, 2H, CH2OC6H4), 3.70 (m, 1H, CH),
3.60 (s, 2H, CH2CN), 3.50 (m, 4H, CHOCH2C6, CH2OC7), 3.40
(t, 2H, J ) 6.6, OCH2C6), 1.50 (m, 4H, OCH2CH2C5), 1.20 (m,
16H, CH2), 0.80 (t, 6H, J ) 5.5, CH3).
General Procedure to Synthesize the Amidoximes 13-24.
Each of the nitriles 2a, 3a, 5a-12a, 2d, and 3d, hydroxylamine
hydrochloride (5 equiv), and K2CO3 (5.5 equiv) were heated to
reflux in absolute ethanol for 18 h. The salts were filtered, and the
filtrate was evaporated under reduced pressure and then purified
by column chromatography to give the corresponding amidoximes.
(()-1-O-Ethyl-3-O-[4-(N-hydroxylamidino)phenyl]-2-O-tet-
radecylglycerol (13). Compound 13 was obtained as a viscous oil
from 6a in 90% yield after chromatography using MeOH/CH2Cl2
(1:99, v/v) as eluent: 1H NMR (CDCl3) δ 7.50 (d, 2H, J ) 8.5,
Har), 6.80 (d, 2H, J ) 8.5, Har), 4.79 (br s, 2H, NH2), 4.09-3.92
(m, 2H, CH2OC6H4), 3.71-3.66 (m, 1H, CH), 3.58-3.39 (m, 6H,
OCH2C13, CH2OEt, OCH2Me), 1.52 (m, 2H, CH2CH2C12), 1.19 (m,
22H, CH2), 1.13 (t, 3H, J ) 7.0, OCH2CH3), 0.81 (t, 3H, J ) 6.2,
CH2CH2CH3).
General Procedure to Synthesize Oxadiazolones A1-A13.
Phenyl chloroformate (1.1 equiv) was added to a solution of
amidoxime (1.0 equiv) in CHCl3 in the presence of triethylamine
(1.5 equiv) at 0 °C. Upon completion, the solution was stirred for
1 h, washed with water to neutral pH, and dried over MgSO4. The
solvent was removed under reduced pressure, and the carbonate,
dissolved in toluene, was heated to reflux for 18 h. After evaporation
of the solvent, the residue was chromatographed to yield pure
oxadiazolone derivative.
(S)-5a was obtained according to the same procedure from (S)-
4a: [R]20 ) -5.29 c 4.61 (CHCl3).
D
General Procedure to Prepare 6a-12a. Compound 5a (10
mmol) was heated to reflux in THF (100 mL) in the presence of
concentrated HCl (2 mL) for 1 h. The solvent was removed and
the residue taken up into ether, washed with H2O to neutral pH,
and dried over MgSO4. The crude product was purified by silica
gel column chromatography to provide 1-O-(4-cyanophenyl)-2-O-
tetradecylglycerol as a white solid in 90% yield: mp 67-69 °C;
1H NMR (CDCl3) δ 7.50 (d, 2H, J ) 8.9, Har), 6.85 (d, 2H, J )
8.9, Har), 4.05 (d, 2H, J ) 4.6, CH2OPh), 3.90-3.40 (m, 5H, CH2-
OH, CHOC14, OCH2C13), 2.10 (s, 1H, OH), 1.60-1.40 (m, 2H,
OCH2CH2C12), 1.18 (m, 22H, CH2), 0.80 (t, 3H, J ) 6.1, CH3).
The above alcohol was used to prepare 6a-12a according to
the same procedure as described for 2a using the corresponding
alkyl bromides. The crude products were purified by silica gel
column chromatography using ether/petroleum ether (2:8, v/v) as
eluent to give pure 6a-12a as a colorless oil.
(()-3-O-(4-Cyanophenyl)-1-O-ethyl-2-O-tetradecylglycerol (6a).
70% yield: 1H NMR (CDCl3) δ 7.51 (d, 2H, J ) 8.8, Har), 6.80
(d, 2H, J ) 8.8, Har), 4,14-3.96 (m, 2H, CH2OC6H4), 3.74-3.60
(m, 1H, CH), 3.58-3.41 (m, 6H, OCH2), 1.44-1.53 (m, 2H,
OCH2CH2C12), 1.19 (m, 22H, CH2), 1.13 (t, 3H, J ) 7.0,
OCH2CH3), 0.81 (t, 3H, J ) 6.5, CH3).
(()-3-O-(4-Hydroxymethylphenyl)-1,2-O-di-n-heptylglycer-
ol (2e). Method A: A solution of 2b (2.00 g, 4.58 mmol) in dry
ether (20 mL) was added dropwise to a suspension of LiAlH4 (0.35
g, 9.2 mmol) in dry ether (50 mL), and the reaction was stirred at
room temperature for further 1 h. The excess of LiAlH4 was
neutralized by slow addition of NaOH (20%, 20 mL) to the mixture,
and the precipitate was then filtered. The aqueous layer was
extracted three times with ether, and the combined organic layers
were washed with water. After being dried over MgSO4, the solution
was concentrated to dryness to provide pure 2e as a white solid in
quantitative yield.
(()-3-O-[4-(4,5-Dihydro-5-oxo-1,2,4-4H-oxadiazol-3-yl)phen-
yl]-2-O-tetradecyl-1-O-triphenylmethylglycerol (A6). In 55%
yield as a white solid using MeOH/CH2Cl2 (2:98, v/v) for the
1
chromatography: mp 109-111 °C; H NMR (CDC13) δ 7.60 (d,
2H, J ) 8.8, Har), 7.40-7.36 (m, 6H, Har), 7.36-7.22 (m, 9H, Har),
6.90 (d, 2H, J ) 8.9, Har), 4.15-4.00 (m, 2H, CH2OC6H4), 3.72-
3.67 (m, 1H, CH), 3.48 (t, 2H, J ) 6.5, OCH2C13), 3.20 (d, 2H, J
) 5.1, CH2OCPh3), 1.49-1.46 (m, 2H, OCH2CH2C12), 1.28-1,18
(m, 22H, CH2), 0.80 (t, 3H, J ) 6.4, CH3). Anal. (C44H54N2O5):
C, H, N.
(S)-A6 was obtained according to the same procedure from (S)-
5a: [R]20 ) -6.20 c 3.00 (CHCl3).
D
Fluorimetric Assay. PLA2 activity is evaluated by the method
of Radvanyi et al.45 using the fluorescent phospholipid analogue
â-pyC-10-PG as the substrate. This assay is specific for secretory
PLA2, cytosolic PLA2 being inactive on substrate with a pyrene
group at the sn-2 position.46 In a total volume of 1 mL, the standard
medium contains 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM
EGTA, 2 µM substrate, 0.1% fatty acid free BSA solution in water,
and 1 ng of hGIIA PLA2 or 60 ng of pPLA2. The fluorescence (λex
) 342 nm and λem ) 388 nm) of the enzymatic reaction medium
(blank) is recorded for 1 min with a spectrofluorimeter LS50
(Perkin-Elmer). The reaction is then initiated by addition of CaCl2
(10 mM, final concentration). The increase in fluorescence is
continuously recorded for 2 min, and PLA2 activity is calculated
as described by Radvanyi et al.45 The inhibitor in ethanol (or
DMSO) was added to the reaction medium after introduction of
BSA. The activity is expressed in micromoles of fluorescent â-pyC-
10-PG hydrolyzed per min and per mg of PLA2. This allows the
determination of the IC50 values (concentration of inhibitors
producing 50% inhibition) of each compound.
Method B: Pyridinium p-toluenesulfonate (PPTS, 0.52 g, 2.1
mmol) was added to a solution of 2c (2.00 g, 4.18 mmol) in ethanol
(50 mL), and the mixture was stirred at 60 °C for 12 h. After
dilution with water, it was then extracted three times with ether.
The organic layers were washed with water, dried (MgSO4), and
concentrated to provide pure 2e as a white solid in quantitative
1
yield: mp 66-68 °C; H NMR (CDCl3) δ 7.20 (d, 2H, J ) 8.7,
Har), 6.80 (d, 2H, J ) 8.7, Har), 4.50 (s, 2H, CH2OH), 4.10-3.90
(m, 2H, CH2OC6H4), 3.70 (m, 1H, CH), 3.60-3.45 (m, 4H,
CHOCH2C6, CH2OC7), 3.40 (t, 2H, J ) 6.6, OCH2C6), 1.70 (br s,
1 H, OH), 1.60-1.40 (m, 4H, OCH2CH2C5), 1.40-1.10 (m, 16H,
CH2), 0.80 (t, 6H, J ) 5.7, CH3).
(()-3-O-(4-Cyanomethylphenyl)-1,2-O-di-n-heptylglycerol (2d).
The compound 2e (4.00 g, 10.0 mmol) in glacial acetic acid (60
mL) was added to 33% HBr in CH3CO2H (50 mL) at 0 °C, and
the solution was stirred at room temperature for 30 min. It was
then poured onto ice-water (500 g), and the precipitate was
recovered by filtration. The solid was dissolved in Et2O (200 mL)
and washed with H2O to neutral pH. The organic layer was dried
over MgSO4 and concentrated to dryness to provide the corre-
sponding bromide in quantitative yield. It was used without further
purification.
The bromide derivative (2.00 g, 4.37 mmol) and NaCN (0.25 g,
5.6 mmol) in dry DMF (25 mL) were stirred at room temperature
for 12 h. The mixture was diluted with water (100 mL) and extracted
several times with ether. The organic layers were dried (MgSO4),
and the solvent was evaporated under reduced pressure to give an
oil which was purified by silica gel column chromatography (ether/
petroleum ether, 1:9, v/v) to afford 1.2 g (70% yield) of 2d as a
Radiometric Assay.42 Preparation of autoclaved and [3H]oleate-
labeled E. coli membranes and sPLA2 assays were performed
essentially as described previously.42 Briefly, sPLA2 assays were
performed at 25 °C in a total volume of 100 µL consisting of 140
mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM CaCl2, 0.1% bovine
serum albumin, and 100 000 dpm of [3H]oleate-labeled E. coli
membranes. Incubation times and sample volumes were adjusted
to ensure hydrolysis rates within the linear range of enzymatic
assays. Typically, 3-10 µL of solution containing different sPLA2s
were incubated for 30-60 min at 20 °C to measure sPLA2 activity.
Reaction mixtures were stopped by adding 300 µL of 0.1 M EDTA,

Inhibition of Secreted Phospholipase A2
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1625
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pH 8.0, and 1% fatty acid-free bovine serum albumin. After
centrifugation at 10 000g for 3 min, 300 µL of supernatant
containing hydrolyzed phospholipids was counted. Control incuba-
tions in the absence of added sPLA2 were carried out in parallel
and used to calculate specific hydrolysis.
Acknowledgment. We thank Dr. M. H. Gelb for advices
and fruitful discussions. This work was supported by grants from
the CNRS and the INSERM on French 2000-2002 national
program “Mole´cules et Cibles The´rapeutiques” (to F.H.) and
by CNRS, the University of Nice-Sophia Antipolis, and the
“Association pour la recherche sur le Cancer” (to G.L.).
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Supporting Information Available: Routine experimental
procedures and spectroscopic data, as well as elemental analysis
data, of the test compounds. This material is available free of charge
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