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European Journal of Medicinal Chemistry 58 (2012) 452e463

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European Journal of Medicinal Chemistry


journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Analysis of the discriminative inhibition of mammalian digestive lipases


by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones
Vanessa Point a, K.V.P. Pavan Kumar b, Sylvain Marc b, Vincent Delorme a, b, Goetz Parsiegla a,
Sawsan Amara a, Frdric Carrire a, Grard Buono b, Frdric Fotiadu b, Stphane Canaan a,
Julien Leclaire b, Jean-Franois Cavalier a, *
a
b

CNRS e Aix-Marseille Universit e Enzymologie Interfaciale et Physiologie de la Lipolyse e UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
CNRS e UMR 7313, Ecole Centrale Marseille e Aix-Marseille Universit, Equipe Chirosciences, Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 4 June 2012
Received in revised form
5 September 2012
Accepted 21 October 2012
Available online 29 October 2012

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e. MmPPOX and
MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human
(HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipaserelated proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts
(PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the
FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared
however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities
obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of
DGL, we demonstrated that the phenoxy group plays a key role in specic molecular interactions within
the lipases active site. The absence of this group in the case of MPOX, as well as its connectivity to the
neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory
efciency of these oxadiazolones and leads to a signicant gain in selectivity towards the lipases tested.
The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone
derivatives could also provide useful leads for the development of novel and more discriminative
inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual
lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal
lipolysis process.
2012 Elsevier Masson SAS. All rights reserved.

Keywords:
Enzyme
Enzyme inhibition
Lipases
Oxadiazolone
In silico molecular docking

1. Introduction
Lipase inhibitors have a wide range of applications in the elds
of pharmaceutics and medicine. During the last decade, the use of
digestive lipase inhibitors to reduce the adsorption of dietary fat

Abbreviations: AchE, acetylcholinesterase; DAG, diacylglycerol; DGL, dog gastric


lipase; FAAH, fatty acid amide hydrolase; FFA, free fatty acid; GPLRP2, guinea pig
pancreatic lipase-related protein 2; hCEH, human carboxyl ester hydrolase; HGL,
human gastric lipase; HPL, human pancreatic lipase; HPLRP2, human pancreatic
lipase-related protein 2; HSL, hormone-sensitive lipase; MAG, monoacylglycerol;
PMF, peptide mass ngerprint; PPE, porcine pancreatic extracts; PPL, porcine
pancreatic lipase; rhMGL, recombinant human monoacylglycerol lipase; TAG, triacylglycerol; TC4, tributyrin; xI, inhibitor molar excess related to 1 mol of enzyme;
xI50, inhibitor molar excess leading to 50% lipase inhibition.
* Corresponding author. Tel.: 33 491164093; fax: 33 491715857.
E-mail address: jfcavalier@imm.cnrs.fr (J.-F. Cavalier).
0223-5234/$ e see front matter 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.040

has become the main pharmacological approach adopted for the


treatment of obesity [1]. The FDA-approved anti-obesity drug
Orlistat (marketed with prescription since 1998 by Roche under the
trade name of Xenical; and more recently over-the-counter by
GlaxoSmithKline under the name of Alli) illustrates this approach
(Fig. 1). This drug is an active site-directed inhibitor that reacts with
the nucleophilic serine residue of the catalytic triad of digestive
lipases [2e4], namely, human gastric (HGL) and pancreatic (HPL)
lipases, human pancreatic lipase-related protein 2 (HPLRP2) and
human carboxyl-ester hydrolase (hCEH, also known as bile-salt
stimulated lipase, BSSL). By covalently blocking the lipases active
site, Orlistat inhibits the hydrolysis of dietary triacylglycerols
(TAGs) and reduces the subsequent intestinal absorption of the
lipolysis products, i.e. monoacylglycerols (MAGs) and free fatty
acids (FFAs). Orlistat is also known to inhibit various lipases and
some other enzymes like fatty acid synthase [5] with great potency,

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

453

Fig. 1. Structure of Orlistat and preparation of MmPPOX (1c), MpPPOX (2c) and MPOX (3c). Reagents and conditions: i) NaNO2, HCl, 0  C; ii) SnCl2, HCl, 0  C, 73e81%; iii)
MeOCOCl, pyridine, 0  C to RT, 68e78%; iv) ClCO2CCl3, CHCl2, pyridine, 0  C to RT, 70e80%.

providing this compound with potential applications in the treatment of type II diabetes [6] and Alzheimers disease [7], as well as in
anti-tumoral [5,8] and anti-mycobacterial [9,10] activities. Similar
b-lactone metabolites exhibiting inhibitory properties towards
lipase and other esterases have also been identied from various
microbial sources [11]. However, these compounds never reached
similar levels of inhibition to those exerted by Orlistat [12,13].
Among the wide range of lipolytic enzyme inhibitors, 3-aryl-5methoxy-1,3,4-oxadiazol-2(3H)-one compounds have been reported to show tunable inhibitory properties, depending on the
nature of the N-3 substituent used. Oxadiazolones with aliphatic
substituents at position N-3 were rst reported by Huang et al. [14] to
be efcient inhibitors of housey head acetylcholinesterase (AchE)
as well as potent insecticides. In the early 2000s, high-throughput
screening strategies were developed at Sano-Aventis Pharma for
identifying various classes of hormone sensitive lipase (HSL) inhibitors [15]. This research led to the identication of 3-phenyl oxadiazolone derivatives (e.g. CAY10499 from Cayman Chemical, and
MmPPOX formerly known as compound 7600) due to their high
in vitro inhibitory activity on partially puried HSL from rat epididymal adipose tissue [16]. Further tests showed that the process of
HSL inhibition by MmPPOX was partially reversible, and the mechanism possibly underlying the reaction between this inhibitor and
the enzymes catalytic serine residue has been described [17,18]. It
was also noted that apart from the members of the HSL family, no
other carboxylester hydrolases (including esterases such as AchE and
pig liver esterase; and lipases such as HPL, Thermomyces lanuginosus
lipase and Bacillus subtilis LipA lipase) were inhibited by this
compound under the experimental conditions tested [17]. Contradictory results were however reported at the same time in a SanoAventis Pharmas patent, showing that the same oxadiazolone
molecules could inhibit the lipase activity from porcine pancreas
crude extracts [19]. It was assumed in this work that porcine
pancreatic lipase (PPL) could be inhibited by oxadiazolones. More
recently, similar N-3 substituted oxadiazolones developed for
HSL inhibition have been described as reversible inhibitors of
endocannabinoid-hydrolyzing enzymes, namely fatty acid amide
hydrolase (FAAH) and recombinant human monoacylglycerol lipase
(rhMGL) [20,21], both involved in several physiological processes of
pharmaceutical interest such as pain and inammation [22].
In this context, designing and synthesizing more selective
inhibitors of the various animal and microbial lipases is not only of
fundamental value for understanding the function and catalytic
mechanism of these lipases, but could also provide better drug

candidates for the specic treatment of various diseases. In order to


better understand the inhibition of mammalian digestive lipases by
oxadiazolone core compounds and shed more light on the molecular interactions at the active site, we investigated in the present
study the in vitro lipase inhibition and selectivity of three 5methoxy-N-3-phenyl substituted-1,3,4-oxadiazol-2(3H)-ones (i.e.
MmPPOX, MpPPOX and MPOX; Fig. 1) towards the main lipolytic
enzymes present in the gastrointestinal tract and involved in the
digestion of triacylglycerols (TAGs), diacylglycerols (DAGs), monoacylglycerols (MAGs), phospholipids (PLA1 activity) and galactolipids. HPL, PPL, dog gastric lipase (DGL), HPLRP2, guinea pig
pancreatic lipase-related protein 2 (GPLRP2) and hCEH, available at
our laboratory, were then used as model enzymes with distinct
substrate specicities.
DGL provides a good model for HGL. These two lipases have
85.7% amino acid sequence identity, superimposable 3-D structures
[23,24] and similar activities when simulating in vitro the gastrointestinal lipolysis [25]. HPLRP2 and GPLRP2 belong to the
pancreatic lipase gene family and they share 65% and 63% amino
acid identity with HPL, respectively [4,26]. The lid domain
controlling the access to the active site is however missing in
GPLRP2 and the catalytic serine is therefore always accessible to the
solvent [26,27]. In HPLRP2, a full length lid domain is observed as in
HPL, but its unusual open conformation observed in the absence of
amphiphiles, reveals a large active site capable of accommodating
more hydrophilic substrates than HPL [28]. Indeed, both of these
PLRP2 enzymes exhibit lipase, phospholipase A1 and galactolipase
activities [4,29,30] and their main physiological function is the
hydrolysis of dietary galactolipids [4,31] and phospholipids [26].
Finally, hCEH is a non-specic esterase [32,33] which contributes to
overall gastrointestinal lipolytic activity by hydrolyzing a broad
range of substrates, including cholesterol esters [34], phospholipids
and acylglycerols [35], as well as galactolipids [30].
The comparative inhibition of these digestive lipases by the
three oxadiazolone derivatives carrying distinct substituents, as
well as by the well-known lipase inhibitor Orlistat, could therefore
yield interesting data in terms of selectivity.
2. Results
2.1. Chemistry
The synthesis of the 5-methoxy-N-3-phenyl substituted-1,3,4oxadiazol-2(3H)-one derivatives, namely MmPPOX, MpPPOX and

454

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

MPOX, was carried out (Fig. 1) in three successive steps. The


cyclization reaction was performed from the corresponding
hydrazinecarboxylate derivatives (1b, 2b, 3b) and diphosgene, by
optimizing reported procedures [16,21,36]. These three compounds
were further used to investigate the role of the oxadiazolone
moiety in the inhibition of various mammalian digestive lipases,
with the perspective of establishing a rst structureeactivity relationship (SAR) within this family.
2.2. Lipase inhibition by MmPPOX, MpPPOX, MPOX and Orlistat

Table 2
Inhibition levels (%) of HPL, HPLRP2, GPLRP2, DGL, hCEH, PPL and PPE after a 30-min
incubation period with each inhibitor.a
Lipolytic enzymes

xI

Lipase inhibition (%)


MmPPOX

MpPPOX

MPOX

Orlistat

HPL

100
400
100
400
2
20
2
20
2
20
2
20
400

26.3
42.7
85.1
87.7
63.7
81.2
80.1
90.4
10.9
81.9
54.5
84.7
89.7

NI
20.0
72.9
87.1
66.0
88.8
13.3
23.6
12.4
92.7
77.1
78.6
90.6

NI
NI
10.7
35.8
49.9
92.7
77.6
86.0
1.4
6.6
71.6
81.3
34.1

77.2
85.0
85.5
90.9
83.6
86.3
82.7
87.3
51.5
56.3
80.1
83.6
93.1

PPL
HPLRP2
GPLRP2

The pH-stat technique [37] was used to quantify the inhibitory


power, dened here as the inhibitor molar excess leading to 50%
lipase inhibition (xI50) [38], of these three oxadiazolone derivatives.
With each lipase, linear kinetics corresponding to the FFAs released
(mmol) vs. time (min) were obtained in the presence and absence of
inhibitor. The slope (U mmol/min) was measured by applying
linear regression (r2 > 0.99) in order to assess the residual activity
of each lipase after incubation with each compound. Signicant
differences were observed not only among the three oxadiazolone
derivatives inhibiting the same lipase, but also between the
inhibitory potency of each oxadiazolone compound with each of
the six lipases. The weakest inhibition was observed with HPL,
which was inhibited by only 42.7% and 20.0% by MmPPOX and
MpPPOX, respectively, at a very high inhibitor molar excess
(xI 400), while MPOX had no effect on HPL under the same
experimental conditions (Tables 1 and 2). In contrast, opposite
results were obtained with PPL. At the same xI value of 400, both
MmPPOX (xI50 26.5) and MpPPOX (xI50 57.6) nearly abolished
PPL activity after 30 min of incubation period (Fig. 2 and Tables 1
and 2), while MPOX reduced the activity of PPL by only 35.8%.
The same levels of inhibition (Table 2) were also reached when
using crude porcine pancreatic extracts (PPE), thus supporting the
previous results obtained by Sano-Aventis Pharma [19].
Regarding the other four lipases tested, HPLRP2, GPLRP2, DGL
and hCEH were strongly inactivated by MmPPOX with very low xI50
values of 0.62, 0.77, 8.33 and 1.48, respectively. hCEH and HPLRP2
(Supplementary data Fig. S1) exhibited almost no chemopreference
between MpPPOX (xI50 0.93 and 1.04, respectively) and MPOX
(xI50 0.77 and 2.27, respectively), while GPLRP2 and DGL
discriminated strongly between these two compounds (Tables 1
and 2 and Fig. 3A and B). Indeed, MPOX efciently inhibited
GPLRP2 (xI50 0.74), whereas this compound had almost no effect
on DGL activity (6.6% inhibition at xI 20). Conversely, MpPPOX
was found to be a potent inhibitor of DGL (xI50 7.12), but reduced
the activity of GPLRP2 by only 23.6% at a high molar excess (xI 20).
The inuence of the incubation time on the levels of inhibition
of PPL, HPLRP2, GPLRP2, DGL and hCEH was further investigated
(Table 1 and Fig. 3C and D). The residual activity of the ve enzymes
decreased rapidly and reached a plateau value after approximately
30 min of incubation. The half-inactivation times (t) leading

DGL
hCEH
PPE
a

Results are expressed as mean values of at least two independent assays (CV
% < 5.0%). NI no inhibition.

to 50% lipase inhibition were then determined from these inhibition curves (Table 1). The t values were found to increase
concomitantly with the decrease in the inhibitory power, as shown
in the case of PPL: MmPPOX (t 1.69 min) > MpPPOX
(t 1.84 min); HPLRP2: MmPPOX (t 0.78 min) > MpPPOX
(t 2.06 min) > MPOX (t 14.6 min) and hCEH: MPOX
(t 1.00 min) > MpPPOX (t 1.43 min) > MmPPOX
(t 1.55 min). With GPLRP2, although MmPPOX and MPOX
displayed almost the same xI50 value, the inhibition rate with
MmPPOX (t 0.46 min) was nearly 22 times faster than that
observed with MPOX (t 9.97 min) (Fig. 3C). A similar pattern of
behaviour was also observed with MmPPOX (t 1.88 min) and
MpPPOX (t 9.31 min) acting on DGL (Fig. 3D).
In order to compare the inhibitory activity of these oxadiazolones with that of Orlistat, the same kinetic parameters were also
measured with this inhibitor and each of the lipases tested. The xI50
and t values obtained with PPL (2.76; 1.66 min), HPLRP2 (0.77;
0.55 min), GPLRP2 (0.53; 0.40 min), DGL (5.60; 0.59 min) and hCEH
(0.78; 0.52 min) as well as the lipase inhibition rates, which are
presented in Tables 1 and 2, were found to be of the same order
of magnitude as those reached with the three oxadiazolone
compounds.
2.3. Mass spectrometry analysis
To investigate whether these oxadiazolones inhibit HPLRP2,
GPLRP2, DGL and hCEH by forming a covalent bond with the
catalytic site, mass spectrometry analyses were conducted on
inhibited enzymes. MmPPOX was used in these experiments
because it efciently inhibits all these four lipases. After preincubating each lipolytic enzyme for 30 min at 25  C with

Table 1
Half-inactivation time (t) and xI50 values of MmPPOX, MpPPOX, MPOX and Orlistat measured on digestive lipases.a
Lipolytic enzymes

HPL
PPL
HPLRP2
GPLRP2
DGL
hCEH

xI50

t (min)

MmPPOX

MpPPOX

MPOX

Orlistat

MmPPOX

MpPPOX

MPOX

Orlistat

>400
26.5
0.62
0.77
8.33
1.48

>400
57.6
1.04
>40
7.12
0.93

NI
>400
2.27
0.74
>40
0.77

18.4
2.76
0.77
0.53
5.60
0.78

>120
1.69
0.78
0.46
1.88
0.90

>120
1.84
2.06
>60
9.31
1.43

NI
>60
14.6
9.97
>60
1.00

1.64
1.66
0.55
0.40
0.59
0.52

a
The half-inactivation time (t) and the xI50 values were determined as described in Experimental section. The inhibitor molar excess (xI) used for the kinetic determinations of t was: 100, 5, 2, 20 and 4 for HPL & PPL, HPLRP2, GPLRP2, DGL and hCEH, respectively. Results are expressed as mean values of at least two independent assays (CV
% < 5.0%). NI no inhibition.

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

Fig. 2. Effects of increasing molar excess (xI) of MmPPOX on the rates of hydrolysis of
TC4 emulsions by PPL (-) and HPL (,). Each enzyme was pre-incubated at various
inhibitor molar excess for 30 min at 25  C. Kinetic assays were performed as described
in Experimental section. Results are expressed as mean values of at least two independent assays (CV% < 5.0%).

MmPPOX at a molar excess of xI 40, no further residual lipolytic


activity could be detected. A sample of this incubation medium was
then analyzed by performing MALDI-TOF mass spectrometry. The
m/z mass values measured are presented in Table 3. In the presence
of MmPPOX (Mw, 284.3 Da), a clearly visible shift in the molecular
mass of each inhibited lipase was observed, thereby reecting the
covalent bonding of the oxadiazolone compound to the lipases.
However, in order to conrm that MmPPOX bound specically to
the catalytic serine residue, and thus to exclude any noncompetitive covalent inhibition, tryptic digestion of the DGLMmPPOX adduct was performed. The peptide mass ngerprint
(PMF) [39] obtained was then analyzed by performing MALDI-TOF
mass spectrometry. The peptide L147-K168 (LHYVGHSQGTTIGFIAFSTNPK) containing the catalytic Ser153 residue was detected at
a molecular mass of 2375.5 Da with the lipase alone (Fig. 4A). By
contrast, when DGL was incubated with MmPPOX, a mass increase
of 284.0 Da was observed in the PMF spectrum for the peptide
containing the active site serine (Fig. 4B), which conrmed that
a covalent bond had been formed between the oxadiazolone
compound and the catalytic serine residue of DGL.
2.4. Molecular docking
In order to shed more light on the lipase binding interactions of
the oxadiazolone derivatives and to compare them with Orlistat, in
silico molecular docking was performed using the validated
molecular dock open-source program AutoDock Vina [40]. We used
as receptors the reported crystal structures of HPL (PDB: 1LPB [41]),
DGL (1K8Q [24]), PPL (PDB: 1ETH [42]), GPLRP2 (PDB: 1GPL [26])
and hCEH (PDB: 1F6W [43]) which were all determined at a resolution < 2.80 
A (nal R-factor  21%). In each of these 3-D structures,
the lipase active site was found to be fully accessible thus enabling
the docking experiments with the four inhibitors. Moreover, the use
of the open conformations of HPL and DGL in complex with
a phosphonate inhibitor covalently bound to their respective catalytic serine residue stand for reference models to check the reliability of the obtained docked positions. Accordingly, these
receptors appeared as optimal structures for in silico docking of
lipase inhibitors and allowed to establish potential molecular
interactions with the oxadiazolone compounds at the respective
lipase active site. By contrast, HPLRP2 was not included in these

455

simulations, since the crystallographic data available so far (PDB:


2OXE and 2PVS [28]) only present a partially open structure which is
unsuitable for in silico docking. With each of the compounds tested,
the top-scoring docking position (i.e. lowest energy complex) was
a productive one, and the calculated distance between the catalytic
serine and the reactive carbon atom of the inhibitors carbonyl group
was shorter than a conventional H-bond (2.7 
A).
In silico docking experiments conducted on Orlistat with HPL,
GPLPR2, DGL and hCEH suggested that the overall shape adopted by
this inhibitor was that of a fork (Fig. 5AeC and Supplementary data
Fig. S2 and S3). In each lipase, the docked Orlistat was located at
the bottom of the active site crevice with the N-a-formyl-D-leucine
amino-acid chain roughly perpendicular to the hexyl and undecyl
chains. In the case of DGL, it is noteworthy that this N-a-formyl-Dleucine side-chain is located in the same pocket initially occupied
by the b-octylglucoside molecule in the inhibited-DGL X-ray
structure [24]. In each of the docking experiments performed with
the ve lipases, the phenoxyphenyl group of the top scoring
productive orientations (i.e. 1st rank) of the docked MmPPOX and
the hexyl chain of docked Orlistat were found to occupy the same
hydrophobic pocket. Moreover with all ve lipases, the results of
the docking experiments showed that the reactive carbonyl group
of the docked MmPPOX may be perfectly superimposed with the
reactive lactone ring of the docked Orlistat (Fig. 5AeC and
Supplementary data Fig. S2 and S3). Within the active site of HPL
and DGL, a high level of concordance between the most favourable
docked conformations of both inhibitors and the crystal structures
of the covalently bound phosphonate was also observed
(Supplementary data Fig. S4). In particular, the overlay of this
phosphonate complex with the respective reactive carbonyl functions of both the lactone and the oxadiazolone rings suggests that
these sp2 carbonyl atoms may be stabilized by the residues forming
the oxyanion hole in HPL (Phe77 and Leu153) and DGL (Gly154 and
Leu67). However, with HPL the docking experiments could not
discriminate between productive and non-productive orientation
of MmPPOX (Fig. 5D). In this docked non-productive binding mode,
the oxadiazolone ring was therefore found to be too far away to
react with the catalytic serine (d[Ser152-Og/C(carb)]  4.2 
A).
The in silico docking experiments also provided some clues for
the different inhibitory activities observed experimentally between
MpPPOX and MPOX towards GPLRP2 and DGL, respectively. In
GPLRP2, the highest-scoring productive docking positions of MPOX
and MmPPOX were perfectly superimposable, which is in agreement with the similar xI50 values obtained for these two molecules.
Conversely, the presence of the phenoxy group in the para position
as in the case of MpPPOX (Fig. 5E) led to two equally scoring
concurrent and opposite binding modes corresponding to
productive and non-productive orientations, respectively. These
two in silico docking modes observed might be a possible explanation for the lower in vitro inhibitory activity experimentally
displayed by MpPPOX towards GPLRP2. In DGL, a highly discriminating effect related to the substitution of the phenyl ring was
observed. The phenoxy group located in both meta or para position
ts exactly the hydrophobic pocket opposite the catalytic Ser153
residue (Fig. 5C and F), resulting in productive binding. In contrast,
the absence of this phenoxy substituent in MPOX induces the
formation of a high scoring non-productive orientation adopted by
this oxadiazolone ring. Accordingly, the sp2 carbon atom of the
carbonyl group in MPOX is found in an unfavorable position thus
preventing the occurrence of any reactions with the catalytic serine
(Fig. 5F), which is in good agreement with the experimental kinetic
data obtained with this compound acting on DGL (Tables 1 and 2
and Fig. 3B). All these collected results enable to propose preliminary structural basis for the selective inhibition of gastric lipase by
oxadiazolone derivatives (Fig. 6).

456

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

Fig. 3. Residual activities of GPLRP2 (upper panels) and DGL (lower panels) measured on TC4 emulsion using the pH-stat technique. Effects of increasing molar excess (xI) of
compounds MmPPOX (-), MpPPOX (,), MPOX (;) and Orlistat (A) on the rates of hydrolysis of TC4 by (A) GPLRP2 and (B) DGL. Each enzyme was pre-incubated at various
inhibitor molar excess for 30 min at 25  C. (C) GPLRP2 and (D) DGL residual activities measured as a function of the incubation time at a constant inhibitor molar excess (xI 2 with
GPLRP2; xI 20 with DGL). Kinetic assays were performed as described in Experimental section. Results are expressed as mean values of at least two independent assays (CV
% < 5.0%).

3. Discussion
We report here the rst and extensive comparative study
focussing on the activity and selectivity of three oxadiazolone
inhibitors, MmPPOX, MpPPOX and MPOX, towards each of the
digestive enzymes involved in gastrointestinal lipolysis. The results
obtained allowed to establish that these compounds are potent
inhibitors of lipases belonging to the acid lipase (DGL) and
pancreatic lipase (PPL, HPLRP2, GPLRP2) families, as well as human
carboxyl ester hydrolase (hCEH). These are new ndings, since
oxadiazolone compounds were previously assumed to exhibit some
selectivity only for the members of the HSL family [17] and
endocannabinoid-hydrolyzing enzymes [20,21]. The inhibition of
PPL conrms however the inhibitory effects of oxadiazolones
observed with crude PPE [19]; although PPL inhibition was only
supposed at that time and not demonstrated with the pure enzyme.
Like Orlistat, oxadiazolones can therefore inhibit enzymes from the
main lipase families.
The ability of each of the oxadiazolone compounds to inhibit the
catalytic activity of the aforementioned lipases was assessed in

terms of the xI50 and t values, which correspond to the inhibitor


molar excess and the half-inactivation time leading to 50% lipase
inhibition, respectively (Table 1). Experimental data obtained with
soluble enzymes acting on insoluble substrates have to be analyzed
and processed with care, because the partitioning of the enzyme
between the aqueous phase and the substrate interface must be
taken into account. In most experimental set-ups, the enzyme
activity and the partitioning process cannot be measured

Table 3
MALDI-TOF mass spectrometry analysis of inhibited and non-inhibited lipases by
MmPPOX (Mw, 284.3 Da).a
Lipolytic
enzymes

m/z Native
lipase ( )

m/z Inhibited
lipase ( )

Mass
modications (

HPLRP2
GPLRP2
DGL
hCEH

52,874
47,884
48,509
84,595

53,165
48,190
48,794
84,890

291
306
285
295

Da
Da
Da
Da

Da
Da
Da
Da

Da
Da
Da
Da

a
Molecular masses were measured to within a mean accuracy of 0.05% of the
total molecular mass of the sample.

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

457

Fig. 4. Peptide mass ngerprint spectra of the digested DGL, before (upper panels, A) and after (lower panels, B) a 30-min incubation period with MmPPOX at a molar ratio of
xI 40. Zoom on the region of interest. Left panels, region of the unmodied isotopic peptide L147-K168 (LHYVGHSQGTTIGFIAFSTNPK) containing the catalytic Ser153. Right panels,
region in which a new isotopic peptide was expected to occur, resulting from the covalent binding of compound MmPPOX to the catalytic serine. Mass shifts were calculated as the
difference between the experimental m/z of the modied peptide and the theoretical m/z of the unmodied peptide. The expected exact mass shift is 284.08 Da.

simultaneously. In consequence, the MichaeliseMenteneHenri


model no longer applies [44] and the Km, Ki and IC50 values
which are often estimated for lipases and expressed in terms of
volume concentrations are irrelevant when insoluble substrates are
involved. In view of this specic mechanism of action, the use of
more appropriate kinetic constants such as the xI50 [38] and t [45]
values is therefore recommended. Thereby, a xI50 value of 0.5 is
synonymous with a 1:1 stoichiometric ratio between the inhibitor
and the lipase, and is therefore the highest level of inhibitory
activity that can be achieved.
In addition to these kinetic parameters, in silico molecular
docking provided some additional explanations for the biochemical
experimental results obtained. The molecular docking obtained
with HPL showed the existence of a non-productive orientation of
MmPPOX at the rst rank (i.e.: lower energy conformation). In this
binding mode, the oxadiazolone core of MmPPOX would be buried
at the bottom of the active site crevice opposite Ser152 (Fig. 5D);
thus preventing any reaction with this catalytic serine in line with
the fact that HPL was slightly inhibited by this compound (Fig. 2,
Table 1 and [17]). The in silico docking results also revealed the
existence of a second productive orientation of this inhibitor.
Interestingly, in this productive position the oxadiazolone moiety
of MmPPOX and the lactone ring of Orlistat are almost superimposed (Fig. 5A). These ndings suggest that MmPPOX possesses
all the features required to react with the catalytic Ser152 residue at
the active site of HPL and thus to inhibit this lipase. Contrasting
results were however obtained during the in vitro inhibition of HPL
and of the closely related enzyme PPL. Whereas the extremely low
inhibition observed with HPL suggests that the major orientation
adopted by MmPPOX in HPL active site is the non-productive one,
the complete inhibition of PPL (Fig. 2) is in agreement with the
single productive position of MmPPOX ranked rst by molecular
docking (Supplementary data Fig. S2). Interestingly, we also found
out that the lipase activity of PPE was fully inhibited by MmPPOX
(Table 2). PPE, also known as pancreatin or pancrelipase, is a crude
extract of porcine pancreatic enzymes containing the classical
pancreatic lipase, PPL, but also porcine CEH that displays some
lipase activity depending on substrate and experimental conditions
[30,46]. Consequently, activity measurements and inhibition tests
using PPE may involve these two lipolytic enzymes [47,48]. Our

results showing a complete inhibition of PPE lipase activity strongly


support an efcient inhibition by MmPPOX of porcine CEH contained in PPE, in addition to that of PPL. We have shown that HPL
and PPL differ in their reactivity towards the oxadiazolone derivatives, although these enzymes are the closest ones within the
pancreatic lipase gene family [49]. These ndings suggest that PPL
and PPE may be not suitable for screening lipase inhibitors with
a view of inhibiting human pancreatic lipase. At this stage, in silico
molecular docking did not allow, however, to explain such
a difference in the reactivity of the two pancreatic lipases towards
oxadiazolone molecules. Further experiments with a larger set of
inhibitors and a more detailed structural analysis are needed to
conrm whether PPL and HPL are not equivalent enzymes.
On the other hand, treating HPLRP2, GPLRP2, DGL and hCEH
with MmPPOX resulted in the indisputable and total inhibition of
these lipolytic enzymes, even at low xI values (Table 2). MALDI-TOF
mass spectrometry analysis (Table 3) and tryptic-peptide mass
ngerprint (Fig. 4) conrmed that a covalent 1:1 complex was
formed through a linkage between the catalytic serine residue of
these lipases and the inhibitor. The combination of the docking
results with the experimental inhibition data therefore highlights
the importance of structural discrimination between productive
and non-productive enzyme/inhibitor interactions as a prerequisite in the inhibition process.
The most noteworthy results obtained here focus on the strong
inhibition of HPLRP2, GPLRP2, DGL and hCEH observed, and the fact
that these inhibitory effects reach similar or even higher levels than
those achieved with Orlistat (Tables 1 and 2, Fig. 3 and
Supplementary data Fig. S1). More specically, the xI50 values
exhibited by both MmPPOX and MpPPOX towards DGL are of the
same order of magnitude as those obtained with Orlistat. Concerning the kinetics of inhibition, Orlistat reacts nearly 3.2e15.8
times faster on DGL than MmPPOX and MpPPOX, respectively.
However, treating DGL with these two oxadiazolone inhibitors
results in the complete inactivation of this lipase, while in the case
of Orlistat a plateau of around 40% residual activity is reached (i.e.
60% inhibition e Fig. 3B). In this case, in silico molecular docking
experiments conducted on MmPPOX, MpPPOX and Orlistat with
DGL and the analysis of the most productive respective orientations
did not allow to explain such a difference in reactivity between

458

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

Fig. 5. Left panels: in silico molecular docking of Orlistat (yellow) and MmPPOX (green) into crystallographic structures of (A) HPL, (B) GPLRP2 and (C) DGL in a van der Waals
surface representation. Hydrophobic residues (alanine, leucine, isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline and methionine) are highlighted in white. Right
panels: superimposition of the top-scoring docking positions of: (D) MmPPOX (green and light blue) in HPL; (E) MpPPOX (blue and salmon) in GPLRP2; and (F) MPOX (pale green)
and MpPPOX (blue) in DGL active sites, respectively. Each inhibitor is represented in atom colour-code sticks (nitrogen, blue; oxygen, red); and the catalytic serine residues in each
protein are coloured in magenta. Structures were drawn with PyMOL Molecular Graphics System (Version 1.3, Schrdinger, LLC), using the following PDB les: 1LPB, 1GPL and 1K8Q,
for HPL, GPLRP2 and DGL, respectively. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

MmPPOX/MpPPOX and Orlistat towards this lipase. In this


context, these results seem to indicate that the most important
issue here is the solubilization of each molecule in buffer; which
surely affects their activity by the way the inhibitor is presented to
the enzyme. The inhibitory power of Orlistat towards pancreatic
and gastric lipases is known to increase upon adding bile salts
above their critical micelle concentration [50]. The dependence of
lipase inhibition on the presence of bile salts in the incubation

medium has been attributed to the formation of mixed micelles


composed of Orlistat and bile salts [51]. This micellar solubilization
may increase the availability of this amphiphilic inhibitor in solution and thus improve its inhibitory efciency. On the other hand,
we have observed that DGL was inactivated by MmPPOX and
MpPPOX even in the absence of bile salts in the incubation medium
(65e75% inhibition e Supplementary data Fig. S5). These ndings
tend to show that contrary to Orlistat, these inhibitors can be

Fig. 6. Proposed structural basis for the selective inhibition of DGL by 3-phenyl substituted oxadiazolone derivatives.

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

efciently solubilized without the presence of bile salts. The role of


bile salts is however not restricted to the inhibitor solubilization:
their presence allows the lid opening in HPL, giving access to the
active site when the enzyme is in solution [52]. The results obtained
here with oxadiazolones suggest that these molecules might also
induce and stabilize the open conformation of the lid. The addition
of bile salts also increases the rate of inhibition rate to 90e98%.
Based on the present results and those obtained in previous
studies [17,21], oxadiazolone derivatives can be highly reactive
towards various serine hydrolases including several lipases,
whereas the inhibitor MmPPOX was assumed until recently to be
a selective inhibitor of the HSL family members [17,18]. Contrary to
Orlistat, which is considered to be a non-selective inhibitor due to
its capacity to inhibit all known lipases, the experimental kinetic
data obtained on these oxadiazolone inhibitors with GPLRP2 and
DGL (Tables 1 and 2 and Fig. 3) highlight the fact that the presence
of the phenoxy group and its localization on the aromatic ring can
be exploited to introduce strong selectivity during the inhibition
process. Indeed these two lipolytic enzymes highly discriminate
between MpPPOX and MPOX (Fig. 3). In good agreement with
these ndings, the results of the molecular docking experiments
suggest that distinct modes of interaction of each compound with
these two lipases may occur. Regarding the interactions with
GPLRP2, a simple regioisomerism of the phenoxy group from the
meta to para position strongly impairs the rate of inhibition, as
shown by the results obtained with MpPPOX vs. MmPPOX or
MPOX (Table 1 and Fig. 5E). With DGL, the presence of the phenoxy
moiety seems to be essential for the correct orientation of the
oxadiazolone structure in the lipases active site (Figs. 5C, F and 6),
and thus to exert an efcient and potent inhibition (Table 1).
Accordingly, oxadiazolone inhibitors can become more selective of
either GPLRP2 or DGL by changing the nature and positioning of the
N-3-phenyl substituent.
To our knowledge, this is the rst example of a signicant
change in the selectivity of a lipase inhibitor due to a minor
molecular modication in terms of substituent pattern. In addition,
it is worth mentioning that selective inhibitors of gastric lipase
have never been described so far. All the lipase inhibitors studied to
date, including phosphonates [53,54], 2-oxo amide triacylglycerol
analogues [55,56] or triuoromethyl ketones [57], have been found
to exhibit non-selective inhibitory activities towards lipolytic
enzymes. More importantly, the signicant levels of selectivity
towards digestive lipases reached with oxadiazolone inhibitors
open new paths for studying gastrointestinal lipolysis and the
individual roles of the various lipases found in the gastrointestinal
tract. With Orlistat, all the main digestive lipases are inhibited
when the drug is orally administered [1]. With oxadiazolones
specically designed to block gastric lipase, the contribution of this
enzyme to fat digestion processes could be investigated in order to
determine how the lipolysis products generated in the stomach can
stimulate biliary and pancreatic secretions, as well as the subsequent action of pancreatic lipase in the intestine [58,59]. A similar
approach could be applied to HPLRP2 and hCEH, which, like HPL,
are produced by the human exocrine pancreas and present in
pancreatic juice [29]. Whereas HPL is highly specic for TAGs and
DAGs, HPLRP2 and hCEH display a specicity for substrates forming
mixed micelles in the gastrointestinal tract such as galactolipids,
phospholipids and MAGs [4,29,30]. These enzymes can act synergistically [60], and a selective inhibitor of HPLRP2 and/or hCEH
would be particularly useful for studying and discriminating their
respective contribution to the overall lipolysis process without
impacting the action of pancreatic lipase. Other new applications
for lipase inhibitors could also be envisaged. In particular, the loss of
gastric lipolysis could slow down the overall lipolytic process and
trigger satiety mechanisms via the so-called ileal brake [61,62]. This

459

could possibly provide an indirect approach to the treatment of


obesity based on the reduction of food intake [63,64].
4. Conclusion
The inhibitory effects of oxadiazolones on gastric lipase, PLRP2s
and hCEH were found to be similar to those of Orlistat on the same
enzymes in terms of potency, but they were more selective. We
have highlighted different effects of these inhibitors on the human
pancreatic lipase, HPL that was poorly inhibited, while the related
porcine enzyme, PPL, was found to be fully inactivated. The in silico
molecular docking experiments conducted on HPL, DGL and hCEH
provide some possible ways of improving and optimizing the
chemical structure of oxadiazolone inhibitors in order to enhance
their selectivity towards these enzymes. Based on the results obtained in this study, we plan to design, synthesize and assess
a second generation of more efcient and selective lipase inhibitors
based on the oxadiazolone backbone. These inhibitors could be
used for a better understanding of individual lipase function, as
well as for drug development aiming at modulating the whole
gastrointestinal lipolysis process.
5. Experimental section
5.1. Chemistry
5.1.1. General methods of synthesis
Commercially available starting materials were purchased from
SigmaeAldrich (St Quentin Fallavier, France) and used without
further purication. All reactions were carried out under scrupulously dry conditions; and all solvents were puried according to
usual methods [65]. Analytical thin-layer chromatography (TLC)
was carried out on Merck silica gel F254 (60 
A, 40e63 mm, 230e
400 mesh) precoated aluminium sheets, and the following detection methods were used: UV lamp (254 nm) and PMA: dipped into
a solution containing 5% phosphomolybdic acid in absolute
ethanol, and heated on a hot plate. Flash chromatography separations were performed using Merck silica gel 60 (230e400 mesh)
according to the protocol of Still et al. [66]. 1H and 13C NMR
spectra were recorded on BRUKER Avance 200 spectrometer
operating at 200 MHz for 1H and 50 MHz for 13C. Chemical shifts are
reported in ppm on the d scale from an internal standard of solvent
(CDCl3, d 7.27 ppm for 1H NMR and 77.23 ppm in 13C NMR).
Coupling constant(s) (J) in hertz. HPLC-MS analysis were performed
using a Waters 600 pump, a C18 reversed phase column Waters
XBridge C18 (4.6 mm  100 mm, 3.5 mm particle size) at room
temperature coupled with diode-array detection (Waters 2998
DAD detectors) and on-line mass spectrometry (Waters 3100 single
quadrupole spectrometer (SQ) in ESI-MS or APCI-MS mode).
Infrared spectra were recorded on a Thermo-Nicolet IR 200 spectrophotometer. Melting points were registered using a Bchi B-545.
Syntheses of the oxadiazolone derivatives described in the
following paragraphs were performed by using reported procedures [16,21,36]. The chemical characterizations (Mp, IR, NMR,
HPLC-MS) of nal pure molecules (i.e. MPOX, MmPPOX and
MpPPOX) were in agreement with the literature data for these
three compounds [16,21].
5.1.2. Preparation of compounds 1a and 2a
5.1.2.1. (3-Phenoxyphenyl)hydrazine hydrochloride (1a) [16,21,36].
To a solution of 3-phenoxyaniline (7 g, 37.7 mmol, 1 equiv.) in conc.
HCl (23.2 mL) was added dropwise an ice-cold solution of sodium
nitrite (2.86 g, 41.5 mmol, 1.1 equiv.) in ater (14 mL) over a period of
30 min at 0  C. To the reaction mixture a solution of tin(II) chloride
dihydrate (25.57 g, 113.1 mmol, 3 equiv.) in conc. HCl (20 mL) was

460

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

added dropwise during 50 min. After stirring at 0  C for 1 h, the


reaction mixture was basied with cold 50% NaOH (30 mL). The
mixture was further diluted with water (34 mL), another portion of
50% NaOH (17 mL) and then water (100 mL). The reaction mixture
was extracted with ether (3  100 mL) and the combined organic
phases were washed with brine, dried over Na2SO4, and then
ltered. The ltrate was acidied by adding an anhydrous solution
of 2 M HCl in ether (35 mL). The formed precipitate was collected
and dried under reduced pressure to give title compound as a light
brown solid (6.11 g, 68%). Analytical data for 1a: mp 186e188  C; IR
(neat) n 3174e2847 (NeH); For NMR analysis, the free hydrazine
was released by action of 50% NaOH solution. Rf (AcOEt/hexane 1:9,
v/v) 0.43; 1H NMR (200 MHz, CDCl3) d (ppm) 7.38e7.29 (m, 2H), 7.17
(app t, J 7.4 Hz, 1H), 7.09e6.98 (m, 3H), 6.58e6.52 (m, 1H), 6.51e
6.48 (m, 1H), 6.44 (m, 1H), 5.34e5.07 (m, 1H), 3.63e3.45 (m, 2H);
13
C NMR (50 MHz, CDCl3) d (ppm) 158.4 (s), 157.1 (s), 152.9 (s),
130.1 (d), 129.6 (2 d), 123.1 (d), 118.9 (2 d), 109.4 (d), 107.0 (d),
102.5 (d).
5.1.2.2. (4-Phenoxyphenyl)hydrazine hydrochloride (2a) [67].
To a solution of 4-phenoxyaniline (2 g, 10.8 mmol, 1 equiv.) in
conc. HCl (22 mL) was added dropwise an ice-cold solution of
sodium nitrite (1.49 g, 21.6 mmol, 2.0 equiv.) in water (7.6 mL)
over 15 min whilst maintaining the temperature below 10  C. The
reaction was then cooled to 0  C and stirred for 1 h. To the
reaction mixture, sulfamic acid (1.05 g, 10.8 mmol, 1 equiv.) was
added portion wise over a 20 min period. Then a solution of tin(II)
chloride dihydrate (9.78 g, 43.2 mmol, 4 equiv.) in conc. HCl
(8.2 mL) was added drop wise over a 20 min period whilst
maintaining the temperature below 15  C. The reaction mixture
was stirred at 0  C for 2 h and basied to pH 14 with 5 M NaOH
(aq) whilst maintaining the reaction temperature below 30  C.
The reaction mixture was then rapidly extracted with methylene
chloride (2  100 mL). The combined organic phases were dried
over Na2SO4, ltered and the solvent removed in vacuo. (4-Phenoxyphenyl)hydrazine which is unstable was converted to its
hydrochloride by using a solution of 2 M HCl in Et2O (20 mL). The
formed precipitate was collected and dried under reduced pressure to give title compound as a pale brown solid (1.2 g, 44%).
Analytical data for 2a: mp decomp. 173e176  C; IR (neat) n 3174e
2903 (NeH). For NMR analysis, the free hydrazine was released by
action of 50% NaOH solution. Rf (AcOEt/hexanes 1/9) 0.35; 1H
NMR (200 MHz, CDCl3) d (ppm) 7.41e7.19 (m, 2H), 7.15e6.86 (m,
5H), 6.87e6.75 (m, 2H), 4.05 (s, 3H); 13C NMR (50 MHz, CDCl3)
d (ppm) 158.9 (s), 149.4 (s), 147.9 (s), 129.7 (2 d), 122.3 (d), 121.0
(2 d), 117.5 (2 d), 113.5 (2 d).
5.1.3. Synthesis of compounds 1b, 2b and 3b [16,21,36]
5.1.3.1. Methyl 2-(3-phenoxyphenyl) hydrazinecarboxylate (1b).
Methyl chloroformate (1.43 mL, 18.5 mmol, 1.1 equiv.) was
cautiously added dropwise to a solution of 3a (4 g, 16.8 mmol,
1 equiv.) in dry pyridine (2.73 mL, 33.7 mmol) and methylene
chloride (20 mL) at 0  C under nitrogen atmosphere. The reaction
mixture was stirred 15 min at 0  C and 30 min at room temperature,
and then diluted with 20 mL water. The product was extracted with
ether (100 mL). The organic layer was separated and concentrated
in vacuo to a volume of approximately 10 mL. The product began to
crystallize upon leaving the concentrated solution at 4  C for a few
hours. The crystals were collected by ltration, washed with ether,
and dried under vacuo to give the title compound as a light yellow
solid (3.6 g, 76%). Analytical data for 1b: mp 122e124  C; IR (neat) n
3337 (NeH), 1724 (C]O), 1215 (CeO); Rf (AcOEt/hexane 3:7, v/v)
0.35; 1H NMR (200 MHz, CDCl3) d 7.38e7.27 (m, 2H), 7.22e7.05 (m,
2H), 7.05e6.97 (m, 2H), 6.60e6.45 (m, 4H), 5.77 (br s, 1H), 3.74 (s,
3H); 13C NMR (50 MHz, CDCl3) d 158.3 (s), 157.6 (s), 156.9 (s), 149.6

(s), 130.2 (d), 129.6 (2 d), 123.2 (d), 119.0 (2 d), 110.8 (d), 107.7 (d),
103.5 (d), 52.8 (q).
5.1.3.2. Methyl
2-(4-phenoxyphenyl)hydrazinecarboxylate
(2b).
Prepared from 2a and methyl chloroformate applying a similar
method as described for the preparation of 1b. Light brown solid
(1.5 g, 78%). Analytical data for 2b: mp 102e104  C; IR (neat) n
3223 (NeH), 1722 (C]O), 1218 (CeO). Rf (AcOEt/hexane 3:7, v/v)
0.27; 1H NMR (200 MHz, CDCl3) d (ppm) 7.40e7.27 (m, 4H), 7.12e
7.03 (m, 1H), 7.03e6.94 (m, 4H), 6.53 (br s, 1H), 3.78 (s, 3H); 13C
NMR (50 MHz, CDCl3) d (ppm) 158.3 (s), 157.7 (s), 150.7 (s), 144.0
(s), 129.5 (2 d), 122.4 (d), 120.6 (2 d), 117.6 (2 d), 114.4 (br d),
52.8 (s).
5.1.3.3. Methyl 2-phenylhydrazinecarboxylate (3b). Prepared from
commercial phenylhydrazine 3a and methyl chloroformate
applying similar method described for the preparation of 1b. Light
yellow solid (3 g, 70%). Analytical data for 3b: mp 116e118  C; IR
(neat) n 3225 (NeH), 1721 (C]O), 1235 (CeO); Rf (AcOEt/hexane
2:8, v/v) 0.25; 1H NMR (200 MHz, CDCl3) d (ppm) 7.31e7.15 (m, 2H),
6.98e6.72 (m, 3H), 6.48 (br s, 1H), 5.73 (br s, 1H), 3.76 (s, 3H); 13C
NMR (50 MHz, CDCl3) d (ppm) 157.7 (s), 147.9 (s), 129.1 (2 d), 120.9
(d), 112.9 (d), 52.8 (s).
5.1.4. Synthesis of compounds 1c, 2c and 3c [16,21,36]
5.1.4.1. 5-Methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one
(1c MmPPOX). To a cold solution of 1b (1 g, 3.87 mmol, 1 equiv.)
in dry methylene chloride (3 mL) dry pyridine (0.63 mL, 7.74 mmol,
2 equiv.) was added dropwise, and the mixture was stirred 30 min
a 0  C. A solution of diphosgene in methylene chloride (0.51 mL,
4.25 mmol, 1.1 equiv.) was added dropwise over a period of 15 min
while cooling in an ice-salt bath under argon atmosphere. After the
addition was complete the reaction mixture was brought to room
temperature and stirred overnight. The reaction mixture was
washed with water, and the organic phase dried (Na2SO4) and
evaporated under reduced pressure. Crystallization in petroleum
ether gave the title compound as yellow crystals (824 mg, 75%).
Analytical data for 1c: mp 104e106  C; IR (neat) n 1797 (C]O), 1659
(C]N); Rf (AcOEt/hexane 1/9) 0.45; 1H NMR (200 MHz, CDCl3)
d (ppm) 7.59 (ddd, J 8.2, 2.0, 0.9 Hz, 1H), 7.50 (t, J 2.2 Hz, 1H),
7.42e7.29 (m, 3H), 7.18e7.08 (m, 1H), 7.08e6.99 (m, 2H), 6.83 (ddd,
J 8.2, 2.3, 0.9 Hz, 1H), 4.09 (s, 3H); 13C NMR (50 MHz, CDCl3)
d (ppm) 157.9 (s), 156.7 (s), 155.8 (s), 148.1 (s), 137.5 (s), 130.2 (d),
129.8 (d), 123.6 (d), 119.0 (d), 115.6 (d), 112.5 (d), 108.9 (d), 57.7 (q);
HPLC-MS: 285 (M 1).
5.1.4.2. 5-Methoxy-3-(4-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one
(2c MpPPOX). Prepared from 2b applying a similar method as
described for the preparation of 1c. Brown solid (490 mg, 70%).
Analytical data for 2c: mp 112e114  C; IR (neat) n 1785 (C]O), 1681
(C]N); Rf (AcOEt/hexane 1:9, v/v) 0.37; 1H NMR (200 MHz, CDCl3)
d (ppm) 7.80e7.69 (m, 2H), 7.41e7.29 (m, 2H), 7.19e6.96 (m, 3H),
4.10 (s, 3H); 13C NMR (50 MHz, CDCl3) d (ppm) 157.1 (s), 155.8 (s),
154.8 (s), 148.3 (s), 131.6 (s), 129.8 (d), 123.4 (d), 119.8 (d), 119.4 (d),
118.7 (d), 57.7 (q); HPLC-MS: 285 (M 1).
5.1.4.3. 5-Methoxy-3-phenyl-1,3,4-oxadiazol-2(3H)-one (3c MPOX).
Prepared from 3b applying a similar method as described for
the preparation of 1c. Yellow crystals (146 mg, 80%). Analytical
data for 3c: mp 97e99  C; IR (neat) n 1782 (C]O), 1662 (C]N);
Rf (AcOEt/hexane 1/9) 0.48; 1H NMR (200 MHz, CDCl3) d (ppm)
7.88e7.72 (m, 2H), 7.48e7.35 (m, 2H), 7.26e7.14 (m, 1H), 4.11 (s,
3H); 13C NMR (50 MHz, CDCl3) d (ppm) 155.7 (s), 148.2 (s), 136.1
(s), 129.0 (d), 125.4 (d), 117.8 (d), 57.6 (q); HPLC-MS: 193
(M 1).

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

5.2. In vitro biological evaluation


5.2.1. Reagents
Orlistat, Glyceryl tributyrate (tributyrin, TC4), bovine serum
albumin (BSA), sodium taurodeoxycholate (NaTDC), calcium chloride dihydrate (CaCl2, 2H2O), benzamidine and 4-morpholineethanesulfonic acid (MES) were purchased from SigmaeAldriche
Fluka Chimie (St-Quentin-Fallavier, France). Sodium chloride
(NaCl) and Trise(hydroxymethyl)-aminomethane (Tris) were
purchased from VWR International (Fontenay-sous-Bois, France)
and from Euromedex (Mundolsheim, France), respectively. All
organic solvents were purchased from Carlo Erba Reactifs-SDS (Val
de Reuil, France) and were of HPLC grade. For protein digestion,
trypsin and chymotrypsin of sequencing grade were purchased
from SigmaeAldricheFluka Chimie (St-Quentin-Fallavier, France)
and Roche Diagnostics (Meylan, France), respectively.
5.2.2. Enzymes
All lipases were produced and puried to homogeneity.
Recombinant human pancreatic lipase (HPL) and pancreatic lipaserelated protein 2 (HPLRP2) were produced in the yeast Pichia pastoris and puried as described previously [4,29,52]. Recombinant
guinea pig pancreatic lipase-related protein 2 (GPLRP2) was
expressed in Aspergillus orizae and puried according to Ref. [27].
Recombinant dog gastric lipase (DGL) was produced in transgenic
maize by Meristem Therapeutics (Clermont-Ferrand, France) [68]
and puried as described previously [24]. Native human pancreatic carboxylic ester hydrolases (hCEH) was puried to homogeneity
from pancreatic juice according to the method previously described
[69]. The native porcine pancreatic lipase (PPL) was puried from
delipidated pancreas powder according to Rovery et al. [70]. Porcine
pancreatic extracts (PPE), also named pancreatin (P7545; 8  USP
grade) was purchased from SigmaeAldricheFluka Chimie (StQuentin-Fallavier, France). Porcine pancreatic colipase was puried
from lipid-free porcine pancreatic powder (Laboratoire Industriel
de Biologie e Soisy-sous-Montmorency, France) [71].
5.2.3. Lipase activity measurements using the pH-stat technique
Enzymatic activity was assayed at 37  C by measuring the
amount of free fatty acid (FFA) released from a mechanically stirred
tributyrin (TC4) emulsion, using 0.1 N NaOH with a pH-stat (Metrohm 718 STAT Titrino, Switzerland) adjusted to a xed end point
value [37]. The TC4 emulsions were formed by mixing 0.5 mL TC4
with 14.5 mL buffer solution. The activity of HPL, PPL or PPE was
determined using the standard assay solution for pancreatic lipase:
0.3 mM TriseHCl (pH 8.0), 150 mM NaCl, 2 mM CaCl2, and 4 mM
NaTDC [72], in the presence of colipase added at a molar excess
[73]. The assay solution for pancreatic lipase-related protein 2 was
1 mM TriseHCl (pH 8.0), 150 mM NaCl, 5 mM CaCl2 and either
0.5 mM or 2 mM NaTDC in the case of HPLRP2 [30] or GPLRP2 [27],
respectively. In the case of DGL, the assay solution was 150 mM
NaCl, 2 mM NaTDC, 2 mM BSA at pH 5.5 [23]. The assay solution used
with hCEH was 1 mM TriseHCl (pH 8.0), 100 mM NaCl, 5 mM CaCl2,
containing 1 mM NaTDC [30]. All experiments were performed at
least in duplicate. Activities were expressed as international units:
1 U 1 mmole FFA released per minute. The specic activities of
HPL, PPL, GPLRP2, HPLRP2, DGL and hCEH, expressed in U per mg of
pure enzyme, were found to be 8021  79, 10232  359, 2270  33,
1275  32, 340  21 and 318  7 U/mg, respectively.
5.2.4. Inhibition assays
The lipase-inhibitor pre-incubation method was used to test in
aqueous medium and in the absence of substrate, the possible
direct reactions between lipases and inhibitors [37]. Stock solutions
(5 mg/mL) of each inhibitor were prepared in dimethyl sulfoxide

461

(DMSO). The following lipases stock solutions were used: 3.2 mg/
mL PPE in 10 mM MES (pH 7.0), 150 mM NaCl; 0.2 mg/mL HPL in
10 mM MES (pH 7.0), 150 mM NaCl; 0.2 mg/mL PPL in 5 mM Tris (pH
8.0), 100 mM NaCl; 0.2 mg/mL HPLRP2 in 1 mM TriseHCl (pH 8.0),
150 mM NaCl, 5 mM CaCl2; 0.5 mg/mL GPLRP2 in 20 mM Tris (pH
8.0), 1 mM Benzamidine; 0.1 mg/mL DGL in 10 mM MES (pH 6.0),
150 mM NaCl; and 1 mg/mL hCEH in 10 mM MES (pH 6.5), 150 mM
NaCl. These enzyme solutions were used for enzyme-inhibitor preincubation for 30 min at 25  C at various inhibitor molar excess (xI).
Experiments with HPL or PPL were performed in the presence of
a ve-fold molar excess of colipase vs. pancreatic lipase. The presence of 4 mM NaTDC in the incubation medium was required for
the PPL, HPL, DGL and PPE inhibition assays [74]. Regarding the
inhibition by Orlistat, 2 mM NaTDC was used when HPLRP2 and
GPLRP2 were tested. The nal enzyme concentration in the incubation medium was constant and equal to: HPL or PPL y0.6 mM;
HPLRP2 y3.7 mM; GPLRP2 y10.0 mM; DGL y1.9 mM; hCEH
y14.1 mM; and PPE y23.0 mg/mL. During the inhibition experiments, samples of the incubation medium were collected and
injected into the pH-stat vessel in order to measure the residual
lipase activity at various times, and at various inhibitor molar
excess. The variation in the residual lipase activity was then used to
determine the molar excess of the inhibitor which reduced the
enzyme activity to 50% of its initial value (xI50) [38]. In addition, the
enzymes were pre-incubated at 25  C with each inhibitor at a xed
molar excess (xI 100 with HPL or PPL; xI 5 with HPLRP2; xI 2
with GPLRP2; xI 20 with DGL; and xI 4 with hCEH) for 1 h; and
the residual enzyme activity was measured at various times in
order to determine the half-inactivation time (t) corresponding to
50% residual enzyme activity [45]. The xI values used in these latter
experiments were chosen so that the average residual activities of
PPL, HPLRP2, GPLRP2, DGL and hCEH obtained after 30 min of
incubation were in the 15e20% range. In each case, control experiments were performed in the absence of inhibitor but with the
same volume of DMSO. It is worth noting that DMSO at a nal
volume concentration of less than 10% has no effect on the enzyme
activity.
5.2.5. Protein separation and digestion prior to MALDI-TOF mass
spectrometry analysis
Protein in-gel digestion was performed with sequencing grade
trypsin or chymotrypsin, in line with the manufacturers recommendations. Briey, bands were excised, placed in a clean Eppendorf tube and cut into small pieces with a sharp, clean scalpel blade.
All operations were performed at 37  C unless otherwise stated. Gel
pieces were destained by incubating them with 30% ethanol in
water and vortexed for 30 min. The supernatant was discarded and
the process repeated until complete discolouration had occurred.
The gel pieces were washed three times with 50% acetonitrile in
100 mM ammonium bicarbonate (pH 8.0) buffer for 10 min.
Disulde bridges were reduced with 10 mM dithiothreitol (DTT) in
100 mM ammonium bicarbonate (pH 8.0) buffer for 45 min at 56  C.
After removing the supernatant, gel pieces were treated with
55 mM iodoacetamide (IAA) in 100 mM ammonium bicarbonate
(pH 8.0) buffer for 30 min at room temperature in the dark.
Supernatants were discarded and gel pieces were washed twice
using 50% acetonitrile in 100 mM ammonium bicarbonate (pH 8.0)
buffer and dried in a SpeedVac. Dried samples were incubated with
10 mg/mL trypsin or chymotrypsin solution (20 mL) in 25 mM
ammonium bicarbonate (pH 8.0) buffer for 45 min in an ice bath.
Then, 25 mM ammonium bicarbonate (pH 8.0) buffer (50 mL) was
added and the sample was incubated overnight at 37  C for
digestion. Peptides were extracted several times with 0.1% TFA in
water and acetonitrile, pooled together and concentrated in
a SpeedVac for MALDI-TOF analysis.

462

V. Point et al. / European Journal of Medicinal Chemistry 58 (2012) 452e463

5.2.6. Matrix-assisted laser desorption ionization time-of-ight


(MALDI-TOF) mass spectrometry analysis
Enzymes were pre-incubated for 30 min at 25  C with
MmPPOX (7 mM in DMSO) at a xI molar excess of 40. Blank
experiments were performed in the absence of compound
MmPPOX. A saturated solution of a-cyano-4-hydroxycinnamic
acid in acidied water (0.1% triuoroacetic acid, TFA) and acetonitrile (30:70, v/v) was used as a matrix. Prior to the direct mass
analysis of the entire lipases, aliquots of 1 mL of native and
inhibited lipase solutions (corresponding to 20e100 pmol of
protein) were mixed with 1.0 mL of a saturated solution of sinapinic acid in acidied water (0.1% TFA) and acetonitrile (60:40, v/v)
and spotted onto the target. Samples were left to air dry at room
temperature. MALDI-TOF analyses were performed on a Bruker
Microex II mass spectrometer (Daltonik, Deutchland). Mass
spectra were acquired in the positive ion mode, using the
FlexAnalysis software program (Bruker, Daltonik, Deutchland).
Protein identications were carried out using the MASCOT
version 2.2.0 search engine (Matrix Science, London, UK) and the
NCBI protein database. Theoretical and experimental peptide
masses were obtained using the BioTools software program
(Bruker, Daltonik, Deutchland).
5.2.7. Molecular docking
In silico molecular docking of lipase inhibitors present in the
active site of lipases was performed with the Autodock Vina
program [40]. The PyMOL Molecular Graphics System (version 1.3,
Schrdinger, LLC) was used as working environment with an inhouse version of the AutoDock/Vina PyMOL plugin [75]. The Xray crystallographic structures of HPL, PPL, DGL, GPLRP2 and hCEH
(PDB entry codes: 1LPB e 2.46 
A resolution [41]; 1ETH e 2.80 
A

resolution [42]; 1K8Q e 2.70 A resolution [24]; 1GPL e 2.01 
A
resolution [26] and 1F6W e 2.30 
A resolution [43], respectively)
available at the Protein Data Bank were used as receptors. The
amino acid positions had to be re-numbered in the HPL and
GPLRP2 pdb les to make them usable as inputs to the Autodock
Vina program. Docking runs were performed after replacing the
catalytic serine by a glycine residue to enable the ligand to adopt
a suitable position corresponding to the pre-bound intermediate
before the nucleophilic attack in the lipase active site. The box size
used for the various receptors was chosen to t the whole active
site cleft and to allow non constructive binding positions. The
model structure le was generated for each inhibitor molecule, and
its geometry was rened using the Avogadro open-source program
(Version 1.0.1. http://avogadro.openmolecules.net/). At each
molecular docking run, the Autodock Vina program gives the
Cartesian positions of the docked compound as well as a scoring
table with a ranking.
Acknowledgements
Vincent Delorme was funded by PhD fellowships from the
Ministre de lEnseignement Suprieur et de la Recherche. This
work and the post-doctoral employment of Dr. K.V.P. Pavan
Kumar and Dr. Sylvain Marc were funded by the Agence Nationale
de la Recherche in the framework of the PHELIN project (ANR PCV
2007e184840). The post-doctoral employment of Dr. Sawsan
Amara was supported by Agence Nationale de la Recherche in the
framework of the GALACTOLIPASE project (ANR-09-CP2D-06-01).
Authors would like to thank Dr. R. Lebrun, S. Lignon and R. Puppo
(at the Proteomics platform of the Institut de Microbiologie de la
Mditerrane, Marseille, France) for their precious help and
support with the mass spectrometry experiments. The authors
also wish to thank Dr. Jessica Blanc for revising the English
manuscript.

Appendix A. Supplementary data


Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.ejmech.2012.
10.040.
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