Inhibition of secretory phospholipase A2. 1-Design, synthesis and structure-activity relationship studies starting from 4- tetradecyloxybenzamidine to obtain specific inhibitors of group II sPLA 2s

Article abstract of DOI:10.1016/j.ejmech.2005.03.027

Starting from 4-tetradecyloxybenzamidine (PMS815), a non-specific inhibitor of GI and GII PLA₂s, we report in this work the discovery of the specificity through design, synthesis and structure-activity relationships studies of different kinds o

Full text of DOI:10.1016/j.ejmech.2005.03.027

European Journal of Medicinal Chemistry 40 (2005) 850–861
www.elsevier.com/locate/ejmech
Original article
Inhibition of secretory phospholipase A2. 1-Design, synthesis
and structure–activity relationship studies starting from
4-tetradecyloxybenzamidine to obtain specific inhibitors
of group II sPLA2s
Léon Assogba a, Azali Ahamada-Himidi a, Nadia Meddad-Bel Habich a, Darina Aoun a,
Latifa Boukli a, France Massicot a,b, Carine M. Mounier c, Jack Huet a, Aazdine Lamouri a,
Jean-Edouard Ombetta a, Jean-Jacques Godfroid a, Chang-Zhi Dong a, Françoise Heymans a,*
a Unité de Pharmacochimie Moléculaire et Systèmes Membranaires (EA2381), Laboratoire de Pharmacochimie Moléculaire, Université Paris 7 -
Denis-Diderot, case 7066, 2, place-Jussieu, 75251 Paris cedex 5, France
b Laboratoire de toxicologie, Université Paris V, UFR de Pharmacie, 4, avenue de l’Observatoire, 75006 Paris, France
c Unités des Venins, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris cedex 15, France
Received 20 July 2004; received in revised form 1 March 2005; accepted 7 March 2005
Available online 09 August 2005
Abstract
Starting from 4-tetradecyloxybenzamidine (PMS815), a non-specific inhibitor of GI and GII PLA2s, we report in this work the discovery
of the specificity through design, synthesis and structure–activity relationships studies of different kinds of PMS815 derivatives. The leading
compound, 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one (9b, PMS1062) exhibits a micromolar IC50 towards three group
II PLA2s, while inactive towards four group I and one group III enzymes in two in vitro enzymatic assay conditions. It is also able to block the
PLA2-II activities induced by LPS and IL-6 in HepG2 cell line and no cytotoxicity is observed when PMS1062 is tested up to a concentration
of 100 µM in two different cell lines (A549 and LLC-PK1).
© 2005 Elsevier SAS. All rights reserved.
Keywords: sPLA2 inhibitors; Oxadiazolone; Structure–activity relationships; Cytotoxicity
1. Introduction
PLA2s) or secretory (sPLA2s). According to the similarity of
their polypeptide sequence, sPLA2s have been classified ini-
tially into three groups [1]: group I (PLA2-I) including PLA2s
from mammalian pancreatic secretions and Elapidae ven-
oms; group II (PLA2-II) comprising PLA2s from Crotalidae
and Viperidae venoms as well as the mammalian non-
pancreatic secretory PLA2 and group III (PLA2-III) from bee
and lizard venoms. Thanks to the success of genomics, a great
number of mammalian sPLA2s have been recently identified
and cloned [2–5]. Among the 10 human sPLA2s known up to
date [2], it remains true that the most studied are pancreatic
or group IB PLA2 (PLA2-IB) and non-pancreatic or group
IIA PLA2 (PLA2-IIA), especially in regards to their associa-
tion with various inflammatory diseases [6].
Phospholipases A2 (PLA2s) are ubiquitous enzymes that
catalyze the hydrolysis of the ester bond at the sn-2 position
of glycerophospholipids. The fatty-acid released, such as
arachidonic acid (AA), may be enzymatically metabolized
into potent pro-inflammatory mediators known as eicosanoids
(prostaglandins, leucotrienes and thromboxanes), while the
lyso-phospholipid, other product of the PLA2 catalyzed reac-
tion in the case of lyso-platelet-activating-factor (lyso-PAF),
may be converted by the PAF-acetyltransferase into PAF,
another well-known pro-inflammatory mediator.
The PLA2 family encompasses two classes of enzymes,
either intracellular (cytosolic PLA2s and Ca2+-independent
PLA2-IB is a digestive enzyme synthesized by pancreatic
acinar cells as a proenzyme, which is secreted through the
* Corresponding author. Tel.: +33 1 44 27 56 26; fax: +33 1 44 27 56 41.
E-mail address: heymans@ccr.jussieu.fr (F. Heymans).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.027

L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
851
Scheme 1
.
pancreatic duct to the lumen of the small intestine and cleaved
by trypsin into enzymatically active form [7]. In acute pan-
creatitis, characterized by destruction of pancreatic tissue,
PLA2-IB is released into the circulation, but its role in pan-
creatic or any other tissue damages is still hypothetical [6].
PLA2-IIA activity is shown to be highly elevated in many
inflammatory tissues such as rheumatoid arthritic joints [8],
psoriatic skin [9], and serum of patients suffering from pan-
creatitis [10] or gram negative shock [11]. Furthermore, its
concentration increases in blood plasma in generalized inflam-
matory response resulting from infections, chronic inflam-
matory diseases, acute pancreatitis, trauma and surgical opera-
tions [6]. Even if PLA2-IIA shows no preference for any fatty-
acid at the sn-2 position, in particular arachidonic acid [12],
its expression seems related to the potentiation and perpetu-
ation of inflammatory reactions [13]. In addition, this expres-
sion is inducible in a wide variety of cells and tissues, such as
macrophages [14], as well as hepatoma-derived cells in vitro
[15] and hepatocytes in vivo [16], when they are stimulated
by LDL, LPS, cytokines such as interleukin-6 (IL-6), tumor
necrosis factor (TNF), interleukin-1 (IL-1) etc.
The crystal structures of sPLA2-IB and -IIA, free or com-
plexed with a substrate analogue, have shown that several con-
served features at the active site are defined by the presence
of a hydrophobic channel lined on one side by the N-terminal
helix [17]. This hydrophobic channel binds a single phospho-
lipid molecule and then hydrolyzes it following interfacial
recognition between the enzyme and the aggregated sub-
strate. This structural similarity shows how much it is diffi-
cult to obtain inhibitors specific of one particular enzyme.
Due to the involvement of lipid mediators in diverse patho-
logical processes, the suppression of their production has long
been considered as therapeutical strategies. In a previous
paper, we have demonstrated that compounds as simple as
4-alkoxybenzamidines could inhibit secretory PLA2 in a com-
petitive manner [18]. Particularly, PMS815 (Scheme 1, n = 0)
showed interesting IC50 of 3 and 5 µM, respectively, for group
I bovine pancreatic PLA2 and group II PLA2 from rabbit plate-
let lysate.
In order to clarify the mode of action of such kind of com-
pounds and find specific inhibitors of group II PLA2, several
structural modifications shown in Scheme 1 were investi-
gated. Different substitutions were performed on the sp2 and
the sp3 nitrogens of the amidine and their influence on the
inhibitory potency studied. Our SAR study was also enlarged
by using various heterocycles as imidazoline, oxazolidine,
imidazole, tetrazole and oxadiazolone. This led us to the dis-
covery of some specific inhibitors of group II PLA2 versus
group I and III enzymes. The leading compound, 4,5-dihydro-
3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one (9b,
PMS1062) exhibits a micromolar IC50 towards a number of
group II PLA2s, while inactive at 100 µM, the highest con-
centration used, towards different group I and group III
enzymes in two in vitro enzymatic assay conditions, one using
an aggregated and the other using a monomeric substrate. It
was also shown to be able to block the PLA2-II activities
induced by LPS and IL-6 in HepG2 cell line. Moreover, no
cytotoxicity was observed when PMS1062 was tested up to a
concentration of 100 µM in two different cell lines (A549 and
LLC-PK1). Furthermore, in a carrageenan-induced oedema
model, although completely inactive when administrated per
os, likely due to its high lipophilicity, PMS1062 was a little
less active than indomethacin, when i.p. administrated. These
results suggest that PMS1062 can act likely as a PLA2-II

852
L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
Scheme 2
.
inhibitor on the acute inflammation model in which the
involvement of PLA2-II has been established, whereas, its
high lipophilic character could prevent it from being well bio-
distributed per os. This will be further addressed in our fol-
lowing manuscripts.
derivative 8 was synthesized from 5 using the method of Rob-
ertson et al. [23] with aminoacetaldehyde diethylacetal. The
oxadiazolone moiety of 9a (PMS 1075) and 9b (PMS1062)
was obtained by reaction of the amidoxime 3a, b with
2-ethylhexyl chloroformate and reflux of the intermediate in
xylene, according to the method of Kohara et al. [24]. Imida-
zoline derivatives 11a, b, 12a, b and 13a, b, were synthe-
sized following the method of Rondu et al. [25] starting from
the corresponding esters 10a, b and using trimethylalumi-
num and substituted (or not) ethylene diamine (EDA) (Scheme
2). These last esters were also used in the preparation of the
oxazolines 17a, b (Scheme 3): the acids 14a, b, resulting from
the hydrolysis of the esters 10a, b, were condensed with etha-
nolamine by action of N,N’-dicyclohexylcarbodiimide (DCC)
to give 15a, b, of which the alcohol function was converted
into chloride by thionyl chloride to give 16a, b. Intramolecu-
lar cyclization of 16a, b occurred in alkaline conditions to
afford 17a, b.
2. Chemistry
The majority of the compounds reported in this work were
prepared as depicted in Scheme 1. 1b was synthesized using
a similar protocol as described previously to prepare 1a [18],
while NaOH was used instead of NaH to obtain the corre-
sponding phenolate, and then treated with NH2Al(CH3)Cl,
freshly prepared from trimethylaluminum and ammonium
chloride according to the method developed to obtain
PMS815 [19], to afford 2. The nitriles 1a, b were converted
into the amidoximes 3a, b and acetylamidoxime 4 following
a method adapted from Diana et al. [20], while the dimethy-
lamidine 6 needed the treatment of the intermediate iminoet-
her hydrochloride 5 with dimethylamine according to McFar-
land et al. [21]. The tetrazole ring analogues 7a, b were
prepared from 1a, b by the method of Harfenist et al. [22],
with NaN3 and ammonium chloride while the imidazole
3. Results and discussion
We have previously published that compounds as simple
as 4-alkoxybenzamidines could be potent sPLA2 inhibitors
Scheme 3
.

L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
853
Table 1a
capacity to inhibit the sPLA2s activities, confirming the results
obtained with its homologue 3a. However, when two meth-
yls were used to substitute the so-called sp3 nitrogen of 2, the
compound 6, obtained through the intermediate 5, showed an
enhanced inhibitory effect on both group I and II PLA2s (two
or three times). These results indicate that the presence of a
more basic nitrogen at this position due to the dimethylation,
to which a protonation can happen at neutral pH used in our
in vitro enzymatic assay, seems to be favorable for the anti-
sPLA2 activity.
Inhibition of the enzymatic activity of porcine pancreatic PLA2 (GIB) and
PLA2 from rabbit platelet lysates (GIIA) by amidine analogues with the IC50
values determined using spectrofluorimetric assay
IC50 (µM)a
Compound
Z
GIB
GIIA
1a (PMS815)
3a
4
11a
12a
13a
17a
7a
2.8 0.5
> 100
2.5 0.5
> 100
> 100
> 100
1.8 0.1
2.0 0.2
2.4 0.1
> 100
1.9 0.2
2.1 0.3
2.3 0.5
> 100
3.2. Cyclic analogues of PMS815 (Table 1a,b)
> 100
46 1
PMS 1075
LY311727 [28]
a Mean S.D.
> 100
11
1
Given that the above structural modifications only im-
proved faintly the anti-PLA2 activity, but not at all the selec-
tivity, we have diversified our SAR study by using different
heterocycles in the place of the amidine function. Based on
the fact that the N,N-dimethylamidine analogues did not sig-
nificantly affect the activity, the amidine motif was firstly inte-
grated in an imidazoline cycle (compounds 11a, b). This
modification induced no amelioration of the selectivity and
the inhibition potency remained almost the same as that of
the linear analogues. None of both properties was influenced
by the C or N-methylation of the imidazoline ring (com-
pounds 12a, b and 13a, b). These results are in agreement
with those related to 6, since the imidazoline, substituted or
not, remains basic and thus protonable in the enzymatic assay
conditions. This protonation is likely important for the for-
mation of a hydrogen bond between the His48 of the enzyme
and the inhibitors, as proposed previously [19].
Along with this argument, the imidazole derivative (com-
pound 8), although containing the amidine motif and possess-
ing almost the same lipophilicity, was shown to be inactive
against both enzymes in the spectrofluorimetric assay, as its
pKa is very close to 7. The fact that the oxazoline derivatives
(compounds 17a, b) were neither active, implies that the inte-
gration of the amidine motif would be necessary to the estab-
lishment of important interactions between the inhibitors and
certain residues of the enzymes.
8.0 0.1
0.47 0.04
and the leader of the series possessed an alkyl chain of 14 car-
bons (compound 1a or PMS815, Scheme 1) [19]. In this work,
the amidine function of PMS815 was substituted or replaced
by different heterocycles for the purpose to improve the inhibi-
tory activity, as well as to reach a specific inhibition of sPLA2-
IIA versus sPLA2-IB.
3.1. Non-cyclic analogues of PMS815 (Table 1a)
When the hydrogen on the so-called sp2 nitrogen of
PMS815 was replaced by a hydroxy or an acetoxy group, 3a
and 4 thus obtained lost the ability to inhibit both PLA2-I and
-II activities. This could be explained by either the decrease
of 1–2 units of log P due to the introduction of the OH func-
tion (compound 3a), or suppression of the interactions
between this hydrogen and certain residues of the enzyme
(compound 4).
As shown in Table 1b, insertion of a methylene group
between the amidine function and the phenyl moiety (com-
pound 2) did not induce a significant improvement of the
inhibitory activity on both group I and II enzymes. The
N-hydroxylation of 2 led to compound 3b, which lost its
Table 1b
Tetrazole derivatives 7a, b were the first compounds which
showed a little selectivity towards PLA2-IIA from rabbit plate-
let lysates versus PLA2-IB, although their activity was much
weaker than the amidine and the imidazoline counterparts.
When submitted to the in vitro spectrofluorimetric enzy-
matic assay, 7a (Table 1a) was completely inactive at 100 µM,
the highest concentration tested, against porcine pancreatic
PLA2 (GIB), while possessed an IC50 of 46.2 µM against
PLA2 from rabbit platelet lysates (GIIA). Similarly, 7b was
twofold more potent against GIIA PLA2 than GIB PLA2
(Table 1b).
Inhibition of the enzymatic activity of porcine pancreatic PLA2 (GIB) and
PLA2 from rabbit platelet lysates (GIIA) by amidine homologue derivatives
with the IC50 values determined using spectrofluorimetric assay
IC50 (µM)a
Compound
n
0
1
1
1
1
1
1
1
1
1
1
Z
Group IB
2.8 0.5
1.9 0.3
> 100
Group IIA
2.5 0.5
1.7 0.5
> 100
PMS815
2
3b
6
11b
12b
13b
8
0.9 0.4
1.3 0.5
1.4 0.4
1.9 0.3
> 100
1.3 0.2
1.5 0.3
1.4 0.7
1.7 0.5
> 100
3.3. Replacement of the tetrazole by an oxadiazolone ring:
towards PLA2-II specificity
17b
7b
9b (PMS1062)
> 100
> 100
70
2
34
1
> 100
4.0 0.9
LY311727 [28]
a Mean S.D.
8.0 0.1
0.47 0.04
The first results obtained with the tetrazole ring, bioisos-
tere of carboxylic acid function, encouraged us to investigate

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L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
Table 2
Inhibition by PMS1062 of the enzymatic activity of different sPLA2s in the in vitro fluorimetric (FA) and spectrophotometric (SA) assays
Group I Group II
bGIB RPL
hGIB
FA-IC50 (µM)a > 100
SA-IC50 (µM)a > 100
pGIB
> 100
> 100
NNA
> 100
> 100
hGIIA
CB
> 100
> 100
3.4 0.4
0.40 0.06
4.0 0.9
ND
ND
0.10 0.01
Group I: human pancreatic (hGIB), porcine pancreatic (pGIB), bovine pancreatic (bGIB) and N. naja atra of Elapidae venom (NNA); group II: human
non-pancreatic (hGIIA), rabbit platelet lysates (RPL) and C. durissus terrifucus venom (CB).
a Mean S.D.
other heterocycles possessing an acid character. Oxadiaz-
olone was chosen because it was described to be an indis-
pensable component in a great number of bioactive mol-
ecules, such as anti-5-lipoxygenase and anti-cyclooxygenase
agents [26], antagonists of angiotensine II receptor [24] and
an anti-thrombotic prodrug [27].
assay (no effect on porcine pancreatic PLA2 at 10 µM). This
could be due to the difference of the two assay systems and in
particular to the side-specific effect of the inhibitor on the
substrate vesicles in the case of our fluorimetric assay
(Table 2).
Indeed, 9a (PMS1075), in which the oxadiazolone was
linked directly to the phenyl group, shows a much better selec-
tivity and activity than 7a corresponding to the tetrazole ana-
logue (Table 1a). No activity was detected at 100 µM of this
inhibitor when PLA2-I was used in the fluorimetric assay con-
ditions, however, the IC50 of PMS1075 was four-times smaller
than that of 7a, when PLA2-II was used (Table 1a). More
interestingly, when the homologue 9b (PMS1062) was sub-
mitted to the same assay conditions, we found that the activ-
ity and the selectivity were more advantageously enhanced
(Table 1b). Apparently, this gain of both the properties is
related to the flexibility increase between the oxadiazolone
and the phenyl rings, since the conjugation between these
rings in the case of 9a is disrupted by the insertion of a meth-
ylene group in the case of 9b.
To confirm the selectivity of 9b against GIIA PLA2 versus
other groups of sPLA2s, the study was enlarged to various
group I and II PLA2s, as well as group III PLA2 of different
species. For all the other PLA2s-I tested, including human
pancreatic, bovine pancreatic PLA2s and that from the venom
of Naja naja atra of Elapidae, PMS1062 at 100 µM had not
any detectable effect on their enzymatic activity in our fluo-
rimetric conditions as observed in the case of porcine pancre-
atic PLA2. The same observation was noted when the assay
was carried out with a group III PLA2 from the bee venom of
Apis mellifera. By contrast, a similar IC50 of micromolar mag-
nitude (Table 2) was found in this fluorimetric assay for all
the other group II PLA2s used, including human secretory
non-pancreatic one and that from the venom of Crotalus duris-
sus terrificus (basic sub-unit of the crotoxine, CB).
3.4. Spectrophotometric competition assay using
a monomeric 1,2-di-n-hexanoyl-1,2-dithio-sn-glycero-3-
phosphoglycerol, lithium salt (thio-PG) substrate and spe-
cific inhibition of group II PLA2s by PMS1062
One compound could appear as an inhibitor by interfering
simply with the organized substrate, which has been shown
to be preferred by interfacial activating PLA2s, and thus pre-
venting it from enzymatic action or leading to a sub-estimation
of a real inhibition. On the other hand, an inhibition could
occur in an irreversible manner, if one molecule reacted with
one of the important residues at the active site of an enzyme.
In the latter case, the molecule would be in general excluded
from a therapeutical application. In consequence, these fea-
tures needed to be addressed through elucidating the mecha-
nism of action of PMS1062.
Spectrophotometric competition assay was therefore devel-
oped and the substrate in this assay was maintained under the
critical micellar concentration (CMC). The substrate, 1,2-di-
n-hexanoyl-1,2-dithio-sn-glycero-3-phosphoglycerol, lithium
salt (thio-PG), was prepared in our laboratory and the CMC
was determined to be about 1 mM (data to be published else-
where). It has been observed in this assay conditions that CB
and human recombinant (group II) PLA2s could be inhibited
by PMS1062 with a sub-micromolar IC50 (Table 2), whereas
neither N. naja atra, bovine, porcine and human pancreatic
PLA2s (group I), nor bee venom PLA2 (group III) were sen-
sible to PMS1062, up to a concentration of 100 µM (Table 2).
These results provided solid evidence that PMS1062 is a spe-
cific and active site-targeted inhibitor of group II PLA2s. The
difference between the IC50 values obtained by the fluorimet-
ric and the spectrophotometric assays could be explained by
the different assay conditions, and probably also by a sub-
estimation in the fluorimetric conditions.
To verify the reliability of our fluorimetric assay, one of
the specific hGIIA PLA2 inhibitors of Lilly Company,
LY311727 [28], was tested in the same conditions as for our
compounds. It exhibited effectively a more potent inhibitory
activity towards hGIIA PLA2 with an IC50 of 0.47 µM than
towards porcine pancreatic PLA2 with an IC50 of 8 µM
(Table 1a,b). Our inhibition data of this compound against
hGIIA PLA2 is well consistent with its apparent KB of 0.27 µM
against the same enzyme obtained in a tissue-based assay [29],
although its specificity is observed less in our fluorimetric
assay conditions than reported previously using the same
The reversibility of this inhibition was demonstrated by a
dilution experience. When a preformed human group II PLA2-
PMS1062 complex was diluted, a total recovery of the PLA2
activity was observed.
The above interesting activities of PMS1062 in our in vitro
enzymatic assays encouraged us to study its pharmacologi-
cal properties using different cell lines and a carrageenan-

L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
855
4. Conclusion
Starting from a non-specific inhibitor of group I and II
PLA2s, PMS815, we describe in this work how a rational
SAR study led us towards the specific inhibition of sPLA2-II.
Using three groups of sPLA2s of different species and two in
vitro enzymatic assay conditions, we have demonstrated that
the leading compound, PMS1062 is an active site-targeted
and specific inhibitor of group II PLA2s and in addition, this
inhibition seems to be reversible. Furthermore, biological
studies using cellular or animal models provide evidences that
this kind of compounds could be of therapeutical interests in
the treatment of inflammation by targeting specifically group
II sPLA2s. More detailed SAR studies will be described in
the following manuscripts and we are actually to enlarge our
specificity determination to recently reported human group
III, V and X sPLA2s.
Fig. 1. Effects of PMS1062 on sPLA2 activity in HebG2 cells, stimulated
with LPS and IL6 (MIX). ##: p < 0.01 compared to control; ** P < 0.01 com-
pared to MIX.
induced oedema model. As reported [16], stimulation with
LPS + Il-6 (MIX) induced an increase of sPLA2 activity in
HepG2 cells, by sevenfold in our experimental conditions
compared to the control. However, co-treatment with MIX
plus PMS1062 (50–0.1 µM, n = 6) resulted in a decrease of
this activity by 84–45% with an IC50 of 0.5 µM (Fig. 1). We
observed that 50 µM of PMS1062 could completely sup-
press the LPS and IL-6 induced sPLA2 activity in HepG2 cells.
Cytotoxicity of PMS1062 was assessed using MTT and
neutral red assays. As can be seen in Table 3, cell viability is
not altered even at 100 µM, the highest dose used in both
A549 and LLC-PK1 cell lines, indicating that PMS1062 is
poorly cytotoxic. Moreover, the effect of PMS1062 on the
release of two marker enzymes, c-GT and NAG, which is
directly correlated to tubular cell damages, was assessed using
LLC-PK1 cells. Since PMS1062 has no effect on this release
at all (data not shown), it seems to be non-nephrotoxic and
not cytotoxic for LLC-PK1 cells.
Anti-inflammatory property of PMS1062 has also been
evaluated on acute response where mammalian PLA2-II is
involved, such as in carrageenan-induced oedema formation.
When i.p. administrated, PMS1062 was able to reduce the
oedema formation in the rear rat paw by 30% at a dose of
30 mg kg–1 (results not shown), while indomethacin, a well-
known anti-inflammatory agent, by 40% at a dose of
10 mg kg–1 in the same experimental conditions. However,
when administrated per os, this inhibition disappeared com-
pletely. These results suggest that PMS1062 can act likely as
a PLA2-II inhibitor on the acute inflammation model in which
the involvement of PLA2-II has been established, whereas,
its high lipophilic character could prevent it from being well
biodistributed per os. This will be further addressed in our
following manuscripts.
5. Experimental section
5.1. Chemistry
All chemicals were of reagent quality and were used with-
out further purification. The purity of each compound was
checked by thin-layer chromatography on TLC plastic sheets
(silica gel 60F 254, layer thickness 0.2 mm) from Merck.
Column chromatography purification was carried out on silica
gel 60 (particle size 0.063–0.200 mm) from Merck without
any special treatment. All melting points were determined in
a digital melting point apparatus (Electrothermal) and are
uncorrected. The structures of all compounds were con-
firmed by IR and 1H NMR spectra. IR spectra were obtained
with anATI Mattson GENESIS SERIES FTIR infrared spec-
1
trometer, and H NMR spectra were recorded in CDCl3 or
DMSO-d6 on a Bruker AC 200 spectrometer using hexam-
ethyldisiloxane (HMDS) as an internal standard. Chemical
shifts are reported in parts per million (d) and coupling con-
stants expressed in Hz. Elemental analyses were carried out
for C, H and N by the Service Régional de Microanalyse de
l’Université Paris 6 and are within 0.4% of theoretical val-
ues.
5.1.1. 4-Tetradecyloxyphenylacetonitrile (1b)
To an ice-bath cooled solution of 4-hydroxybenzyl cya-
nide (6.00 g, 45.1 mmol) in absolute EtOH (30 ml) was added
dropwise a solution of NaOH (1.90 g, 47.5 mmol) in EtOH
(20 ml). After stirred for 15 min, the solution was evaporated
to dryness and the residue, taken up in N,N-dimethyl-
formamide (DMF, 30 ml), was added dropwise to a solution
of 1-bromotetradecane (13 g, 47.5 mmol) in DMF (200 ml)
at 0 °C. After stirred at room temperature for 4 h, the mixture
was diluted with water, extracted with EtOAc. The organic
phases were dried over MgSO4, filtered and evaporated. Puri-
fication on a silica gel column (ether/spirit) yielded 1b (7.1 g,
48%) as a white solid: m.p. 68 °C; Rf 0.81 (MeOH/CH2Cl2,
Table 3
Effects of PMS1062 on cell viability expressed by IC50 (µM), determined
(n = 6) at the end of 24 or 48 h incubation by MTT and Neutral Red methods
using A549 and LLC-PK1 cell lines (see Section 5)
MTT
48 h
Neutral Red
48 h
24 h
24 h
A549
> 100
> 100
> 100
> 100
> 100
> 100
> 100
LLC-PK1
> 100

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L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
5:95, v/v); IR (KBr, cm−1) 2245 (CN), 1597 (C=C); 1H NMR
(CDCl3) 7.15, 6.89 (2d, 4H, J = 8.70, Har), 3.86 (t, 2H,
J = 6.52, CH2O), 3.59 (s, 2H, CH2CN), 1.70 (m, 2H,
CH2CH2O), 1.36–1.19 (m, 22H, CH2), 0.81 (t, 3H, J = 6.37,
CH3).
(100 ml) was stirred at room temperature for 2 h. Salts were
eliminated by washing with H2O and the organic layer was
dried (MgSO4), filtered and evaporated to dryness. Crystalli-
zation in ether/CH2Cl2 (2:1, v/v) yielded 4 as white crystals
(0.43 g, 43%): m.p. 140 °C; Rf 0.12 (MeOH/CH2Cl2, 5:95,
v/v); IR (KBr, cm−1) 3426, 3320 (N–H), 1737 (C=O), 1625
(C=N); 1H NMR (CDCl3) 7.12, 6.79 (2 d, 4H, J = 8.49, Har),
4.57 (s, 2H, NH2), 3.86 (t, 2H, J = 6.50, CH2O), 3.45 (s, 2H,
CH2CN), 2.10 (s, 3H, CH3CO), 1.67 (qt, 2H, J = 6.90,
CH2CH2O), 1.19 (s, 22H, CH2), 0.82 (t, 3H, J = 6.56, CH3).
Anal. (C24H40N2O3) C, H, N.
5.1.2. 4-Tetradecyloxyphenylacetamidine hydrochloride (2)
To a suspension of ammonium chloride (4.82 g,
90.0 mmol) in dry benzene (90 ml) at 5 °C was added drop-
wise a solution of trimethylaluminum in toluene (2.0 M,
45 ml, 90 mmol). The mixture was stirred at room tempera-
ture for 2 h and then added to a stirred solution of 1b (15.0 g,
41.4 mmol) in dry toluene (150 ml). After heated to reflux for
6 h at 110 °C under argon atmosphere, the solution was poured
on to a stirred suspension of silica gel (120 g) in chloroform
(600 ml) and stirred for a further 5 min at room temperature.
The silica gel was filtered and washed with chloroform to
eliminate the unreacted nitrile 1b. The amidine hydrochlo-
ride 2 was then recovered by methanol as colorless crystals
after evaporation of the solvent (6.32 g, 40%): m.p. 88 °C; Rf
0.23 (MeOH/CHCl3/NH4OH, 20:80:02, v/v/v); IR (KBr,
5.1.6. 4-Tetradecyloxyphenylethoxyacetimine
hydrochloride (5)
A solution of 1b (2.4 g, 7.3 mmol) in absolute EtOH
(60 ml) was chilled in an ice-bath and saturated with dry HCl
gas. The solution was then standed at –18 °C to give 5 as
white crystals (2.1 g, 70%). The title compound was used
immediately in the next step upon formation due to its insta-
bility.
1
cm−1) 3196 (N–H), 1688 (C=N), 1613 (C=C); H NMR
5.1.7. N,N-Dimethyl-4-tetradecyloxybenzylamidine
hydrochloride (6)
(DMSO-d6) 8.95 (br s, 4H, NH), 7.32, 6.84 (2 d, 4H, J = 8.31,
Har), 3.87 (t, 2H, J = 6.00, CH2O), 3.57 (s, 2H, CH2CN), 1.61
(qt, 2H, J = 6.38, CH2CH2O), 1.18 (s, 22H, CH2), 0.79 (t,
3H, J = 6.37, CH3). Anal. (C22H39ClN2O) C, H, N.
To an ice-chilled and stirred solution of Me2NH (0.20 g,
4.4 mmol) in MeOH (25 ml), the iminoether hydrochloride 5
(1.5 g, 3.6 mmol) was added portionwise. After stirred over-
night at room temperature, the solution was evaporated to
dryness and the residue prepurified by crystallization in
MeOH/CH2Cl2/ether (1:1:1, v/v/v).A silica gel column chro-
matography using 5% MeOH in CH2Cl2 as eluent yielded 6
as white crystals (1.0 g, 75%): m.p. 149 °C; Rf 0.42
(MeOH/CH2Cl2, 20:80, v/v); IR (KBr, cm−1) 3160 (N–H),
1611 (C=N); 1H NMR (CDCl3) 10.08 (s, 1H, NH), 9.60 (s,
1H, NH), 7.14, 6.78 (2d, 4H, J = 8.60, Har), 4.00 (s, 2H,
CH2Ph), 3.85 (t, 2H, J = 6.46, CH2O), 3.28 (s, 3H, CH3N),
3.00 (s, 3H, CH3N), 1.70 (qt, 2H, J = 6.70, CH2CH2O), 1.19
(s, 22H, CH2), 0.82 (t, 3H, J = 6.64, CH3).Anal. (C24H42N2O)
C, H, N.
5.1.3. 4-Tetradecyloxyphenylacetamidoxime (3b)
A mixture of 1b (5.00 g, 15.2 mmol), K2CO3 (11.0 g,
79.7 mmol) and hydroxylamine hydrochloride (5.3 g,
76 mmol) in absolute EtOH (60 ml) was heated to reflux for
18 h. The suspension was filtered and the solid washed with
hot absolute EtOH. The filtrate was concentrated in vacuo
and the residue purified on a silica gel column using CH2Cl2
as eluent to give 3b as white crystals (4.0 g, 74%): m.p.
102 °C; Rf 0.18 (MeOH/CH2Cl2, 5:95, v/v); IR (KBr, cm−1)
3456 (N–H), 3150 (O–H), 1656 (C=N), 1610 (C=C); 1H NMR
(CDCl3) 9.41 (br s, 1H, D2O exchange, OH), 7.13, 6.77 (2d,
4H, J = 8.62, Har), 4.41 (s, 2H, NH2), 3.85 (t, 2H, J = 6.52,
CH2O), 3.55 (s, 2H, CH2CN), 1.68 (qt, 2H, J = 6.70,
CH2CH2O), 1.19 (s, 22H, CH2), 0.81(s, 3H, J = 6.56, CH3).
Anal. (C22H38N2O2) C, H, N.
5.1.8. 2-(4-Tetradecyloxyphenyl)-1H-tetrazole (7a)
A mixture of 1a (3.00 g, 9.52 mmol), NaN3 (0.80 g,
12.5 mmol) and NH4Cl (0.67 g, 12.5 mmol) in DMF (50 ml)
was stirred at 100 °C for 12 h. The solvent was evaporated
under reduced pressure and the residue partitioned in an aque-
ous NaOH solution (20%) and ether. The aqueous phase was
poured onto 1 N HCl and the precipitate purified by silica gel
chromatography using 2% MeOH in CHCl3 as eluent to yield
7a as a white solid (1.00 g, 29%): m.p. 134 °C; Rf 0.29
(MeOH/CHCl3, 1:9, v/v); IR (KBr, cm−1) 3379 (N–H), 1614
5.1.4. 4-Tetradecyloxybenzylamidoxime (3a)
4-Tetradecyloxybenzylamidoxime (3a) was prepared in
76% yield following the same procedure as for 3b but start-
ing from 4-tetradecyloxybenzonitrile 1a: m.p. 110 °C; Rf 0.17
(MeOH/CH2Cl2, 5:95, v/v); IR (KBr, cm−1) 3449, 3348
1
(N–H), 3240 (O–H), 1653 (C=N), 1612 (C=C); H NMR
1
(CDCl3) 9.44 (br s, 1H, D2O exchange, OH), 7.57, 6.89 (2 d,
4H, J = 8.68, Har), 5.71 (s, 2H, NH2), 3.94 (t, 2H, J = 6.32,
CH2O), 1.69 (m, 2H, CH2CH2O), 1.23 (s, 22H, CH2), 0.83
(t, 3H, J = 6.56, CH3). Anal. (C21H36N2O2) C, H, N.
(C=N); H NMR (DMSO-d6) 7.96, 7.13 (2d, 4H, J = 8.60,
Har), 4.00 (t, 2H, J = 6.40, CH2O), 1.73 (qt, 2H, J = 7.40,
CH2CH2O), 1.23 (m, 22H, CH2), 0.84 (t, 3H, J = 6.00, CH3).
Anal. (C21H34N4O·HCl) C, H, N.
5.1.5. O-Acetyl-4-tetradecyloxyphenylacetamidoxime (4)
A mixture of 3b (0.90 g, 2.48 mmol), acetic anhydride
(0.30 g, 2.94 mmol) and Et3N (0.30 g, 2.97 mmol) in CH2Cl2
5.1.9. 2-(4-Tetradecyloxybenzyl)-1H-tetrazole (7b)
2-(4-Tetradecyloxybenzyl)-1H-tetrazole (7b) was obtained
in 30% yield following the same procedure as for 7a but start-

L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
857
ing from 1b and treatment of the hydrochloride salt by sodium
5.1.13. Methyl 4-tetradecyloxybenzoate (10a)
hydrogen carbonate: m.p. 68 °C; Rf 0.47 (MeOH/CHCl3, 1:9,
Methyl 4-tetradecyloxybenzoate (10a) was obtained in
93% yield using the same protocol as for 1b, but starting from
methyl 4-hydroxybenzoate and with a reaction time of 7 h, as
white crystals (MeOH, 4 °C): m.p. 59.0–60.5 °C; Rf 0.52
(ether/spirit, 10:90, v/v); IR (KBr, cm−1) 1723 (C=O), 1609
(C=C); 1H NMR (CDCl3) 7.88, 6.80 (2d, 4H, J = 8.84, Har),
3.90 (t, 2H, J = 6.51, CH2O), 3.78 (s, 3H, CH3O), 1.70 (dt,
2H, J = 7.09, CH2CH2O), 1.18 (s, 22H, CH2), 0.79 (t, 3H,
J = 6.05, CH3).
1
v/v); IR (KBr, cm−1) 3434 (N–H), 1613 (C=N); H NMR
(CDCl3) 9.50 (br s, 1H, D2O exchange, NH), 7.16, 6.86
(2d, 4H, J = 8.53, Har), 4.18 (s, 2H, CH2Ph), 3.89 (t, 2H,
J = 6.38, CH2O), 1.66 (qt, 2H, J = 6.26, CH2CH2O), 1.22 (m,
22H, CH2), 0.83 (t, 3H, J = 5.90, CH3). Anal. (C22H36N4O)
C, H, N.
5.1.10. 2-(4-Tetradecyloxybenzyl)-1H-imidazole (8)
A mixture of 5 (2.00 g, 4.86 mmol) and aminoacetalde-
hyde diethyl acetal (0.64 g, 4.47 mmol) in absolute EtOH
(70 ml) was stirred at room temperature for 72 h.After evapo-
ration of the solvent, the residue was taken up in 2 N HCl
(30 ml) and heated at 90 °C for 1 h. The solution was then
neutralized with an aqueous NaOH solution (20%) and the
precipitate, dissolved in CH2Cl2, was washed with water. The
organic layer was dried over MgSO4 and concentrated to dry-
ness. The residue was crystallized in CH2Cl2/ether (1:5, v/v)
to yield 8 as white crystals (0.70 g, 40%): m.p. 131 °C; Rf
0.38 (MeOH/CHCl3, 10:90, v/v); IR (KBr, cm−1) 3445 (N–H),
1611 (C=N), 1584 (C=C); 1H NMR (CDCl3) 9.50 (br s, 1H,
NH), 7.00, 6.77 (2 d, 4H, J = 8.53, Har), 6.86 (s, 2H, CHN),
3.95 (s, 2H, CH2Ph), 3.84 (t, 2H, J = 6.54, CH2O), 1.69 (qt,
2H, J = 6.52, CH2CH2O), 1.19 (s, 22H, CH2), 0.81 (t, 3H,
J = 5.94, CH3). Anal. (C24H38N2O•1/2H2O) C, H, N.
5.1.14. Methyl 4-tetradecyloxyphenylacetate (10b)
Methyl 4-tetradecyloxyphenylacetate (10b) was obtained
in 93% yield using the same protocol as for 1b, but starting
from methyl 4-hydroxyphenylacetate, as white crystals
(MeOH): m.p. 37 °C; Rf 0.64 (ether/spirit, 10:90, v/v); IR
1
(KBr, cm−1) 1720 (C=O), 1614 (C=C); H NMR (CDCl3)
7.11, 6.78 (2d, 4H, J = 8.42, Har), 3.86 (t, 2H, J = 6.49,
CH2O), 3.61 (s, 3H, OCH3), 3.49 (s, 2H, CH2Ph), 1.69 (m,
2H, CH2CH2O), 1.19 (s, 22H, CH2), 0.81 (t, 3H, J = 6.29,
CH3).
5.1.15. 4,5-Dihydro-2-(4-tetradecyloxybenzyl)-1H-imida-
zole (11b)
In a cooled (0 °C) three-necked round-bottom flask
equipped with a condenser and a dropping funnel under argon
atmosphere were introduced successively, using a syringe,
dry toluene (100 ml), trimethylaluminum (AlMe3, 2.0 M in
toluene, 40 ml, 80 mmol), ethylenediamine (EDA, 1.80 g,
30 mmol) and finally a solution of 10b (5.00 g, 13.8 mmol) in
dry toluene (30 ml). The mixture was heated to reflux for 4 h
and the excess of AlMe3 was hydrolyzed with an aqueous
NaOH solution (20%, 100 ml). The salts were filtered and
the filtrate concentrated to dryness. The residue was then dis-
solved in CHCl3, washed with water, dried over MgSO4 and
filtered. After removal of the solvent, the product was chro-
matographied on a silica gel column using firstly 2%, then
10% MeOH in CH2Cl2 as eluents to yield 11b as white crys-
tals (1.50 g, 30%): m.p. 103 °C; Rf 0.24 (MeOH/
CHCl3/NH4OH, 20:80:2, v/v/v); IR (KBr, cm–1) 3173 (N–H),
1610 (C=N); 1H NMR (CDCl3) 7.10, 6.78 (2d, 4H, J = 8.77,
Har), 3.85 (t, 2H, J = 6.49, CH2O), 3.65 (s, 1H, NH), 3.47 (s,
4H, CH2N), 3.46 (s, 2H, CH2CN), 1.72 (m, 2H, CH2CH2O),
1.19 (s, 22H, CH2), 0.81 (t, 3H, J = 6.14, CH3). Anal.
(C24H40N2O•1/2H2O) C, H, N.
5.1.11. 4,5-Dihydro-3-(4-tetradecyloxyphenyl)-4H-1,2,4-
oxadiazol-5-one 9a (PMS 1075)
A mixture of 3a (0.50 g, 1.38 mmol) and pyridine (0.14 g,
1.88 mmol) in DMF (10 ml) was stirred 5 min at 0 °C.
2-Ethylhexyl chloroformate (0.27 g, 1.40 mmol) was added
dropwise and the solution was stirred for 45 min at 0 °C.
Then the mixture was diluted with water (40 ml) and extracted
with EtOAc (2 × 40 ml). The combined organic phases were
dried over MgSO4, filtered and concentrated under reduced
pressure. The residue was heated to reflux for 2 h in toluene
(50 ml) and concentrated. Crystallization at room tempera-
ture with 30% EtOAc in chloroforme provided 9a as color-
less crystals (0.22 g, 43%): m.p. 151 °C; Rf 0.30
(MeOH/CH2Cl2, 5:95, v/v); IR (KBr, cm−1) 3301 (N–H), 1813
(C=O), 1615 (C=N); 1H NMR (CDCl3) 9.70 (br s, 1H, NH),
7.72, 7.00 (2d, 4H, J = 8.83, Har) 4.00 (t, 2H, J = 6.40, CH2O),
1.71 (m, 2H, CH2CH2O), 1.23 (m, 22H, CH2), 0.82 (t, 3H,
J = 6.62, CH3). Anal. (C22H34N2O3) C, H, N.
5.1.12. 4,5-Dihydro-3-(4-tetradecyloxybenzyl)-4H-1,2,4-
oxadiazol-5-one (9b, PMS1062)
5.1.16. 4,5-Dihydro-2-(4-tetradecyloxyphenyl)-1H-imida-
zole (11a)
4,5-Dihydro-3-(4-tetradecyloxybenzyl)-4H-1,2,4-oxa-
diazol-5-one (9b, PMS1062) was obtained in 61% yield fol-
lowing the same procedure as for 9a, but starting from 2b, as
beige crystals: m.p. 123–124 °C; IR (KBr, cm−1) 3116 (NH),
4,5-Dihydro-2-(4-tetradecyloxyphenyl)-1H-imidazole
(11a) was obtained in 41% yield following the same proce-
dure as for 11b but starting from 10a: m.p. 144 °C; Rf 0.40
(MeOH/CHCl3/NH4OH, 20:80:2, v/v/v); IR (KBr, cm–1) 3334
(N–H), 1610 (C=N), 1561 (C=C); 1H NMR of 11a hydrochlo-
ride (DMSO-d6) 10.39 (s, 2H, D2O exchange, NH), 7.92, 7.12
(2d, 4H, J = 8.82, Har), 4.00 (t, 2H, J = 6.62, CH2O), 3.90 (s,
1
1839 (C=O), 1728 (C=N); H NMR (CDCl3) 7.10, 6.79
(2d, 2H, J = 8.68, Har), 3.85 (t, 2H, J = 6.53, CH2O), 3.72
(s, 2H, CH2Ph), 1.70 (dt, 2H, J = 6.87, CH2CH2O), 1.20 (m,
22H, CH2), 0.81 (t, 3H, J = 6.44, CH3). Anal. (C23H36N2O3)
C, H, N.

858
L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
4H, CH2N), 1.65 (m, 2H, CH2CH2O), 1.17 (s, 22H, CH2),
0.79 (t, 3H, J = 6.45, CH3).Anal. (C23H37N2O•3/4H2O) C, H,
N.
heated to reflux for 4 h. The solution was then acidified and
the solvent removed under reduced pressure. The residue was
taken up in CH2Cl2 and washed with H2O, dried over Na2SO4.
The title compound was obtained upon evaporation of the
solvent, followed by a recrystallization in ethanol as pale yel-
low crystals (1.8 g, 65%): m.p. 70 °C; Rf 0.15
(MeOH/CH2Cl2, 2:98, v/v); IR (KBr, cm−1) 3033 (OH), 1738
(C=O), 1584 (C=C); 1H NMR (CDCl3) 8.70 (br s, 1H, OH),
7.12, 6.78 (2d, 4H, J = 8.15, Har), 3.86 (t, 2H, J = 6.54,
CH2O), 3.50 (s, 2H, CH2Ph), 1.70 (m, 2H, CH2CH2O), 1.19
(m, 22H, CH2), 0.81 (t, 3H, J = 6.08, CH3).Anal. (C22H36O3)
C, H, N.
5.1.17. 4,5-Dihydro-4-methyl-2-(4-tetradecyloxybenzyl)-
1H-imidazole (12b)
4,5-Dihydro-4-methyl-2-(4-tetradecyloxybenzyl)-1H-
imidazole (12b) was obtained in 32% yield following the same
procedure as for 11b but using 1,2-diaminopropane: m.p.
59 °C; Rf 0.32 (MeOH/CHCl3/NH4OH, 20:80:2, v/v/v); IR
1
(KBr, cm–1) 3125 (N–H), 1605 (C=N); H NMR (CDCl3)
7.10, 6.77 (2d, 4H, J = 8.60, Har), 3.86 (m, 3H, CH2O and
CHN), 3.63 (t, 1H, J = 10.00, CH2N), 3.46 (s, 2H, CH2Ph),
3.00 (m, 2H, D2O exchange, CH2N and NH), 1.70 (qt, 2H,
J = 6.74, CH2CH2O), 1.18 (m, 22H, CH2), 0.82 (t, 3H,
J = 6.60, CH3). Anal. (C25H42N2O•H2O) C, H, N.
5.1.22. 4-Tetradecyloxybenzoic acid (14a)
4-Tetradecyloxybenzoic acid (14a) was obtained quanti-
tatively following the same procedure as for 14b but starting
from 10a and without purification by recrystallization: m.p.
98.2–99.6 °C, Rf 0.43 (MeOH/CH2Cl2, 5:95, v/v); IR (KBr,
cm−1) 3173 (OH), 1649 (C=O); 1H NMR (CDCl3) 7.98, 6.86
(2d, 4H, J = 8.90, Har), 3.96 (t, 2H, J = 6.52, CH2O), 1.74
(dt, 2H, J = 6.87, CH2CH2O), 1.20 (s, 22H, CH2), 0.81 (t,
3H, J = 6.40, CH3). Anal. (C21H34O3) C, H, N.
5.1.18. 4,5-Dihydro-4-methyl-2-(4-tetradecyloxyphenyl)-
1H-imidazole (12a)
4,5-Dihydro-4-methyl-2-(4-tetradecyloxyphenyl)-1H-
imidazole (12a) was obtained in 35% yield following the same
procedure as for 12b but starting from 10a and 1,2-
diaminopropane: m.p. 71 °C; Rf 0.33 (MeOH/
CHCl3/NH4OH, 20:80:2, v/v/v); IR (KBr, cm−1) 3203 (N–H),
1601 (C=N); 1H NMR (CDCl3) 7.63, 6.82 (2d, 4H, J = 8.72,
Har), 3.93 (m, 5H, CH2O, CHN and CH2N), 3.27 (br s, 1H,
NH), 1.71 (qt, 2H, J = 7.62, CH2CH2O), 1.19 (s, 22H, CH2),
0.81 (t, 3H, J = 6.66, CH3). Anal. (C24H40N2O•1/2H2O) C,
H, N.
5.1.23. N-(2-Hydroxyethyl)-4-tetradecyloxyphenylaceta-
mide (15b)
Ethanolamine (38.5 µl, 0.54 mmol) and 14b (200 mg,
0.57 mmol) were stirred in DMF (10 ml) and EtOAc (10 ml)
in the presence of N,N′-dicyclohexylcarbodiimide (DCC,
119 mg, 0.58 mmol) and Et3N (121 µl, 0.86 mmol) over-
night. The reaction mixture was diluted with EtOAc (40 ml)
and washed with 2 N HCl (15 ml). The aqueous phase was
extracted once again with EtOAc (40 ml) and the combined
organic phases were washed with H2O, 10% NaHCO3, H2O
and brine. After dried over MgSO4, the solution was evapo-
rated to dryness and the residue was purified by silica gel
column chromatography (MeOH/CH2Cl2) to afford 15b as a
white solid (187 mg, 83%): m.p. 105.2–107.2 °C; Rf 0.5
(MeOH/CHCl3, 1:9, v/v); IR (KBr, cm−1) 3268 (OH), 3094
5.1.19. 4,5-Dihydro-1-methyl-2-(4-tetradecyloxybenzyl)-
1H-imidazole (13b)
4,5-Dihydro-1-methyl-2-(4-tetradecyloxybenzyl)-1H-
imidazole (13b) was obtained in 36% yield following the same
procedure as for 11b but using N-methylethylenediamine: m.p.
75 °C; Rf 0.44 (MeOH/CHCl3/NH4OH, 20:80:2, v/v/v); IR
(KBr, cm−1) 1619 (C=N); 1H NMR (CDCl3) 7.10, 6.76 (2d,
4H, J = 8.54, Har), 3.85 (t, 2H, J = 6.50, CH2O), 3.62 (t, 2H,
J = 9.57, CH2N), 3.47 (s, 2H, CH2CN), 3.20 (t, 2H, J = 9.57,
CH2N), 2.62 (s, 3H, CH3N), 1.67 (qt, 2H, J = 6.56,
CH2CH2O), 1.19 (s, 22H, CH2), 0.81 (t, 3H, J = 6.00, CH3).
Anal. (C25H42N2O•5/4H2O) C, H, N.
1
(NH), 1634 (C=O); H NMR (CDCl3) 7.09, 6.80 (2d, 4H,
J = 6.22, Har), 5.90 (br s, 1H, NH), 3.87 (t, 2H, J = 6.28,
CH2OPh), 3.59 (t, 2H, J = 4.74, CH2OH), 3.46 (s, 2H,
CH2Ph), 3.31 (t, 2H, J = 4.74, CH2N), 1.75 (m, 2H,
CH2CH2OPh), 1.19 (m, 22H, CH2), 0.81 (t, 3H, J = 5.83,
CH3).
5.1.20. 4,5-Dihydro-1-methyl-2-(4-tetradecyloxyphenyl)-
1H-imidazole (13a)
4,5-Dihydro-1-methyl-2-(4-tetradecyloxyphenyl)-1H-
imidazole (13a) was obtained in 39% yield following the same
procedure as for 13b but starting from 10a: m.p. 47 °C; Rf
0.37 (MeOH/CHCl3/NH4OH, 20:80:2, v/v/v); IR (KBr, cm−1)
1614 (C=N); 1H NMR (CDCl3) 7.42, 6.83 (2d, 4H, J = 8.55,
Har), 3.90 (t, 2H, J = 6.50, CH2O), 3.78 (t, 2H, J = 9.53,
CH2N), 3.37 (t, 2H, J = 9.53, CH2N), 2.74 (s, 3H, CH3N),
1.73 (qt, 2H, J = 6.64, CH2CH2O), 1.19 (s, 22H, CH2), 0.81
(t, 3H, J = 6.00, CH3). Anal. (C24H40N2O•3/5 H2O) C, H, N.
5.1.24. N-(2-Hydroxyethyl)-4-tetradecyloxybenzamide
(15a)
N-(2-Hydroxyethyl)-4-tetradecyloxybenzamide (15a) was
obtained in 89% yield using the same procedure as for 15b
from 14a: m.p. 91.3–92.7 °C; Rf 0.5 (MeOH/CHCl3, 1:9, v/v);
1
IR (KBr, cm−1) 3303 (OH, NH), 1636 (C=O); H NMR
(CDCl3) 7.66, 6.82 (2d, 4H, J = 6.84, Har), 6.63 (t, 1H,
J = 5.00, NH), 3.90 (t, 2H, J = 7.36, CH2O), 3.74 (t, 2H,
J = 4.64, CH2OH), 3.52 (m, 2H, CH2N), 3.00 (br s, 1H, D2O
exchange, OH), 1.75 (qt, 2H, J = 7.20, CH2CH2O), 1.19 (s,
22H, CH2), 0.81 (t, 3H, J = 6.00, CH3).
5.1.21. 4-Tetradecyloxyphenylacetic acid (14b)
To a solution of 10b (3.0 g, 8.0 mmol) in methanol (25 ml)
was added 20% NaOH solution (10 ml) and the mixture was

L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
859
5.1.25. N-(2-Chloroethyl)-4-tetradecyloxybenzamide (16a)
5.2. Biology
To a solution of 15a (6.00 g, 15.9 mmol) in CHCl3 (40 ml)
at 0 °C was added dropwise thionyl chloride (2.0 ml,
27.4 mmol) in CHCl3 (40 ml) and the mixture was stirred at
room temperature overnight. It was then washed with 1 M
Na2CO3, dried over MgSO4, filtered and concentrated. The
residue was purified on a silica gel column using CHCl3 as
eluent to afford 16a as a yellow solid (3.8 g, 60%): m.p. 108.4–
109.9 °C; Rf 0.42 (CHCl3); IR (KBr, cm−1) 3305 (N–H), 1632
Bovine and porcine pancreatic (GIB) PLA2s and fatty-
acid free bovine serum albumin (BSA) were purchased from
Sigma (St. Louis, MO, USA). Human recombinant GIB PLA2
was a kind gift from the late Dr. H.Verheij and human recom-
binant GIIA PLA2, basic sub-unit of the GIIA sPLA2 from
C. durissus terrificus venom (CB), GIB sPLA2 from N. naja
atra venom (NNA) and bee venom sPLA2 (GIII) were gifts
from Dr. C. Bon (Institut Pasteur, Paris, France). Rabbit plate-
let lysates containing a GII sPLA2 were prepared as pub-
lished previously [12]. The fluorescent substrate for PLA2
assay, 1-hexadecanoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-
phosphoglycerol, ammonium salt (b-py-C10-PG) was from
Molecular Probes (Eugene, OR). LY311727 was a kind gift
from Lilly Company (Lilly Research Laboratories, Eli Lilly
and Company, IN).
1
(C=O), 1608 (C=C); H NMR(CDCl3) 7.65, 6.87 (2d, 4H,
J = 8.66, Har), 6.51 (s, 1H, NH), 3.92 (t, 2H, J = 6.46, CH2O),
3.7 (m, 4H, Cl(CH2)2N), 1.72 (qt, 2H, J = 7.00, CH2CH2O),
1.19 (m, 22H, CH2), 0.81 (t, 3H, J = 5.92, CH3).
5.1.26. N-(2-Chloroethyl)-4-tetradecycloxyphenylaceta-
mide (16b)
N-(2-Chloroethyl)-4-tetradecycloxyphenylacetamide (16b)
was obtained in 30% yield using the same procedure as for
16a from 15b: m.p. 112.4–113.3 °C; Rf 0.42 (CHCl3); IR
5.2.1. Spectrofluorimetric PLA2 assay
1
(KBr, cm−1) 3275 (N–H), 1641 (C=O); H NMR(CDCl3)
PLA2 activity was evaluated by the method of Radvanyi et
al. [30] using the fluorescent phospholipid analogue, b-py-
C10-PG as the substrate. This assay is specific for secretory
PLA2, cytosolic PLA2 being inactive on substrates with a
pyrene group at the sn-2 position. Bovine pancreatic PLA2 or
PLA2 from rabbit platelet lysate were used to test the effect
of the various inhibitors. In a total volume of 1 ml, the stan-
dard reaction medium contained 50 mM Tris–HCl (pH 7.5),
100 mM NaCl, 1 mM ethyleneglycol-bis(2-aminoethylether)-
N,N,N′,N′-tetraacetic acid (EGTA), 2 µM substrate, fatty-
acid free BSA solution in water (15 µM or 0.1%) and 5 nM of
pancreatic PLA2 or 10 µl of platelet lysate (0.4 µg of pro-
teins). The fluorescence of the enzymatic reaction medium
(blank) was recorded for 1 min with a spectrofluorimeter SFM
25 (Kontron Instruments) equipped with a Xenon lamp. The
reaction was then initiated by addition of CaCl2 (10 mM, final
concentration). The increase in fluorescence was continu-
ously recorded for 2 min and PLA2 activity was calculated as
described by Radvanyi et al. [30]. When used, the solution of
inhibitor in ethanol was added to the reaction medium after
introduction of BSA. The activity was expressed in micro-
moles of fluorescent b-py-C10-PG hydrolyzed per min and
per mg of bovine pancreatic PLA2, or in nmol of fluorescent
b-py-C10-PG hydrolyzed per min per 109 cells, in the case of
rabbit platelet lysate. The standard error of the mean of three
independent experiments was less than 10%. This allowed
the determination of the IC50 values (concentration of inhibi-
tor producing 50% inhibition) of each compound.
7.16, 6.84 (2d, 4H, J = 6.98, Har), 4.55 (s, 1H, NH), 3.90 (t,
2H, J = 6.46, CH2O), 3.56 (m, 2H, ClCH2), 3.47 (m, 2H,
CH2N), 3.32 (s, 2H, CH2Ph), 1.72 (qt, 2H, J = 7.00,
CH2CH2O), 1.19 (m, 22H, CH2), 0.81 (t, 3H, J = 5.92, CH3).
5.1.27. 4,5-Dihydro-2-(4-tetradecyloxyphenyl)-1,3-oxazole
(17a)
Sodium hydroxide pellets (0.52 g, 13.0 mmol) were dis-
solved in absolute ethanol (20 ml) and a solution of 16a
(5.00 g, 12.6 mmol) in the same solvent (30 ml) was added
dropwise. The mixture was stirred at 50 °C overnight. The
salts were filtered and the filtrate was evaporated in vacuo.
The residue was taken up in chloroform and washed with
water. The organic phase was then dried over MgSO4, fil-
tered and concentrated to yield 17a as bright crystals (3.63 g,
80%): m.p. 56 °C; Rf 0.44 (MeOH/CHCl3, 5:95, v/v); IR
1
(KBr, cm−1) 1649 (C=N), 1611 (C=C); H NMR (CDCl3)
7.79, 6.85 (2d, 4H, J = 8.86, Har), 4.33 (t, 2H, J = 7.76,
CH2CH2N), 3.96 (t, 2H, J = 7.76, CH2N), 3.91 (t, 2H,
J = 6.46, CH2OPh), 1.68 (qt, 2H, J = 5.46, CH2CH2O), 1.19
(m, 22H, CH2), 0.81 (t, 3H, J = 6.40, CH3). Anal.
(C23H37NO2) C, H, N.
5.1.28. 4,5-Dihydro-2-(4-tetradecyloxybenzyl)-1,3-oxazole
(17b)
4,5-Dihydro-2-(4-tetradecyloxybenzyl)-1,3-oxazole (17b)
was obtained in 25% yield following the same procedure as
for 17a but starting from 16b: m.p. 48 °C; Rf 0.37
(MeOH/CHCl3, 5:95, v/v); IR (KBr, cm−1) 1671 (C=N), 1586
(C=C); 1H NMR (CDCl3) 7.12, 6.76 (2 d, 4H, J = 8.57, Har),
4.13 (t, 2H, J = 9.62, CH2CH2N), 3.84 (t, 2H, J = 6.50,
CH2OPh), 3.73 (t, 2H, J = 9.62, CH2N), 3.46 (s, 2H, CH2Ph),
1.65 (qt, 2H, J = 6.72, CH2CH2O), 1.18 (s, 22H, CH2), 0.80
(t, 3H, J = 6.40, CH3). Anal. (C24H39NO2•6/5H2O) C, H, N.
5.2.2. Spectrophotometric UV assay
Bovine pancreatic PLA2 (group I), human recombinant
PLA2 (group II), or C. durissus terrificus venom enzyme
(group II) were used to test the potency of various inhibitors.
Hydrolysis of thiophospholipid-PG was followed by allow-
ing the released lysothiophospholipid to react with 5,5′-
dithiobis(2-nitrobenzoic acid) (DTNB) and monitoring the
absorbance at 412 nm using a spectrophotometer UV Unikon

860
L. Assogba et al. / European Journal of Medicinal Chemistry 40 (2005) 850–861
810 Kontron equipped with a recorder. The buffer used con-
tains 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 1 mM EGTA.
Fresh stock of DTNB (10 mM) was prepared in ethanol.
Thiophospholipid-PG was dissolved in distilled water at the
appropriate concentrations to cover submicellar test condi-
tions. The final concentration of calcium chloride was of
5 mM. Briefly, 500 µl of the buffer solution was placed in a
polystyrene cuvette, followed by addition of 10 mM DTNB
(5 µl), 10 mM substrate (10 µl), 5 µl of ethanol (or appropri-
ate concentrations of the inhibitors), 100 to 300 ng of one of
the enzymes (in 5 µl of the same buffer) and finally 0.5 M
calcium solution (5 µl). TNB anion absorbance was recorded
at 412 nm every 30 s for 20 min to calculate residual activity.
in the absence or in the presence of PMS1062 (0.1–50 µM,
so-called MIX + PMS1062). sPLA2 activity in the superna-
tant was assessed as previously described. Protein concentra-
tion was determined by the BCA method (Sigma-Aldrich),
using albumin bovine as a standard.
5.2.7. Statistical analysis
Results were given as the mean of triplicate values of enzy-
matic activities standard deviation (S.D.). Statistical sig-
nificance was assessed by the Mann–Whitney non-parametric
free sample test with P ≤ 0.05.
Acknowledgments
5.2.3. Cell cultures
This work was supported by grant from the CNRS and the
INSERM on French 2000–2002 national program “Molé-
cules et Cibles Thérapeutiques”. Darina Aoun is a recipient
of a grant from the CNRS of Lebanon.
LLC-PK1 cells (American Type Culture Collection, CRL-
1392, ICN Biochemicals, passage 200–220) and human epi-
thelial cell line A549 (European Collection of Cell Culture,
Sigma-Aldrich) were, respectively, grown in DMEM or in
RPMI 1640 supplemented with 10% fetal calf serum (FCS,
Biowhittaker), 1% penicillin (5000 IU ml–1), 5000 mg ml–1
streptomycin and 1% glutamine (complete DMEM or RPMI)
under a 5% CO2–95% O2 atmosphere at 37 °C. Cells were
seeded in 96-well plates at a density of 2.0 × 105 cells per ml
DMEM or RPMI (100 µl per well) for viability studies or in
6-well dishes (2 ml per well) for the measurement of the
c-glutamyl-transferase (c-GT) release and the sPLA2 activ-
ity in the culture medium.
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