Chemistry around imidazopyrazine and ibuprofen: Discovery of novel fatty acid amide hydrolase (FAAH) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.04.040

Based on the imidazo-[1,2-a]-pyrazin-3-(7H)-one scaffold, a dual action prodrug has been designed for combining antioxidant and anti-inflammatory activities, possibly unmasked upon oxidation. The construction of the target-molecule requires two building blocks, namely a 2-amino-1,4-pyrazine and a 2-ketoaldehyde. Attempts to synthesize the 2-ketoaldehyde (5a) derived from ibuprofen failed, but led to the corresponding 2-ketoaldoxime (7a) which could not be condensed with the pyrazine synthons. However, a model compound, i.e. phenylglyoxal aldoxime, reacted well under microwave activation to furnish novel imidazo[1,2-a]-pyrazine-3-(7H)-imine derivatives (18a,b). These heterobicycles behave as antioxidants by inhibiting the lipid peroxidation, and one compound (18b) is endowed with a significant anti-inflammatory effect in a cellular test. Unexpectedly, all the synthetic intermediates derived from ibuprofen are good inhibitors of FAAH, the most active compound (4a) featuring the 1,3-dithian-2-yl motif.

Full text of DOI:10.1016/j.ejmech.2010.04.040

European Journal of Medicinal Chemistry 45 (2010) 3564e3574
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chemistry around imidazopyrazine and ibuprofen: Discovery of novel fatty acid
amide hydrolase (FAAH) inhibitors
Frédéric De Wael a, Giulio G. Muccioli b, Didier M. Lambert c, Thérèse Sergent d, Yves-Jacques Schneider d,
,
Jean-François Rees e, Jacqueline Marchand-Brynaert a
*
a Institute of Condensed Matter and Nanoscience (IMCN), Unité de chimie organique et médicinale, Université catholique de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve,
Belgium
b Louvain Drug Research Institute, Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, Université catholique de Louvain, Avenue Mounier 72.30, B-1200 Bruxelles, Belgium
c Louvain Drug Research Institute, Unité de chimie pharmaceutique et de radiopharmacie, Université catholique de Louvain, Avenue Mounier 73.40, B-1200 Bruxelles, Belgium
d Instistut des Sciences de la vie, Académie universitaire Louvain, Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium
e Instistut des Sciences de la vie, Unité de Biochimie, Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the imidazo-[1,2-a]-pyrazin-3-(7H)-one scaffold, a dual action prodrug has been designed for
combining antioxidant and anti-inflammatory activities, possibly unmasked upon oxidation. The
construction of the target-molecule requires two building blocks, namely a 2-amino-1,4-pyrazine and
a 2-ketoaldehyde. Attempts to synthesize the 2-ketoaldehyde (5a) derived from ibuprofen failed, but led
to the corresponding 2-ketoaldoxime (7a) which could not be condensed with the pyrazine synthons.
However, a model compound, i.e. phenylglyoxal aldoxime, reacted well under microwave activation to
furnish novel imidazo[1,2-a]-pyrazine-3-(7H)-imine derivatives (18a,b). These heterobicycles behave as
antioxidants by inhibiting the lipid peroxidation, and one compound (18b) is endowed with a significant
anti-inflammatory effect in a cellular test. Unexpectedly, all the synthetic intermediates derived from
ibuprofen are good inhibitors of FAAH, the most active compound (4a) featuring the 1,3-dithian-2-yl
motif.
Received 18 March 2010
Received in revised form
26 April 2010
Accepted 30 April 2010
Available online 11 May 2010
Keywords:
Antioxidant
Anti-inflammatory
FAAH inhibitor
2-Ketoaldehyde
Aminopyrazine
Ibuprofen
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
substituent R. Indeed, upon oxidation, this fragment will be released
as the ReCO2H molecule D and a lot of nonsteroidal anti-inflam-
The search of dual action drugs is a field of growing interest in
medicinal chemistry [1,2]. In the particular case of dual action pro-
drugs, two entities featuring synergic activities are covalently
linked. This bond can be chemically or enzymatically transformed in
vivo, leading to the simultaneous release of two active molecules at
the same site. In the course of previous works dedicated to antiox-
idant compounds belonging to the coelenteramine and coelenter-
azine families [3,4], we have disclosed an oxidative degradation
pathway of imidazopyrazinones A (mother antioxidant) producing
a 2-amino-1,4-pyrazine C (daughter antioxidant) and a carboxylic
acid D. This route occurs in parallel to the oxidative degradation into
matory drugs (NSAID) are in fact carboxylic acids [6]. Moreover, the
advantages of molecules combining antioxidant and anti-inflam-
matory activities have been already demonstrated in the case of
modified NSAIDs such as ibuprofen [7,8].
Hence our target-molecule is the so-called “ibuprofen prodrug”
shown in Fig. 2, where R0, R00 could be simply H, or preferably
a phenolic group for increasing the antioxidant activity [3,4].
Classically, the synthesis of imidazo-[1,2-a]-pyrazin-3-(7H)-one (E)
makes use of two building blocks, namely the a-ketoaldehyde (F)
and the aminopyrazine (C) (Fig. 2). Their condensation is performed
in aqueous acidic solutions [9,10].
2-amido-1,4-pyrazine
B
producing light (chemiluminescence
In this article, we describe our persistent attempts to prepare
pathway) (Fig. 1) [5]. This offers a unique opportunity to possibly
combine in the samemolecule Aan antioxidantactivity with an anti-
inflammatory activity by choosing adequately the nature of the
the
sponding oxime was made available, we have also examined the
condensation of -ketoaldoximes with 2-amino-1,4-pyrazines.
Unexpected bicycles have been obtained featuring modest anti-
oxidant and anti-inflammatory activities. Finally, series of
a-ketoaldehyde F derived from ibuprofen. Since the corre-
a
a
synthetic intermediates (toward the target F), endowed with poor
anti-inflammatory activity, has been evaluated for another activity
* Corresponding author.
E-mail address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.040

F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
3565
R
O
H
N
Route II
2, H2O
Route I
O2
N
N
NH2
R'
N
N
R
R
OH
O
N
N
+
O
O
R"
CO2
R"
R'
R"
N
H
R'
CO2, hv
B
A
C
D
Fig. 1. Two pathways of oxidative degradation of imidazopyrazinone A (R ¼ Me, Ph). Route I is preferred in lipophilic media and route II is preferred in hydrophilic media [5]. A is the
“mother anti-oxidant”; B is devoid of antioxidative properties; C is the “daughter anti-oxidant” (R00 ¼ C6H4-pOH) [3,4].
related to the endocannabinoid system. This led to the unexpected
discovery of novel “hits” for the inhibition of fatty acid amide
hydrolase (FAAH).
ammonium nitrate) in CH3CN-borate buffer, NBS (N-bromosucci-
nimide) in MeOH or wet acetone, NBS/TMP in CH3CNeH2O,
Selectfluor in CH3CN, MCPBA (m-chloroperbenzoic acid) then
H3Oþ, Meerwein salt or MeTf in CH3CN, then H2O, etc. Similar
disappointing results were recorded for the deprotection of analogs
4b and 4c (Scheme 1). It is worth noting that the hydrolysis of
2. Results and discussion
a
-carbonyl(dithio)acetal derivatives has not been yet described in
2.1. Synthesis of the a-ketoaldehyde synthon (F)
the literature.
The second route (Scheme 2) was based on the a-nitroketone 6a
Racemic ibuprofen 1a was used as starting material. Indeed, this
anti-inflammatory drug is given as the (R) þ (S) mixture because in
vivo, the inactive (R)-isomer is enzymatically transformed into the
active (S)-isomer [11]. Several approaches toward our target
as key intermediate [21], easily obtained by reaction of the acti-
vated acid 2a with nitromethane in basic conditions. The conver-
sion of a nitromethylene moiety into a carbonyl group is known as
the Nef reaction; beside the traditional use of very strong acids,
smooth conditions have been developed, making this reaction
compatible with a large variety of functionalities [22]. However, to
a
-ketoaldehyde were considered. Aryl
obtained by oxidation of methylketones and diazoketones, or from
-(
0)-(di)haloketones [12e15]. Few methods are dedicated to
benzyl -ketoaldehydes [9,10,16] and none to the -(methyl)benzyl
analogs.
a-ketoaldehydes are usually
a
a
our knowledge, the Nef reaction was never applied to
a-nitro-
a
a
methylketones. The precursor 6a was submitted to a set of repre-
sentative conditions, namely: 10% aq. H2SO4 in alcohol, oxone in
acetone, SnCl2/thiophenol in EtOHeH2O, NaNO2/DBU in AcOH,
TiCl3 reduction, etc. The desired compound 5a was not obtained,
but the oxime intermediate 7a could be isolated after reduction
with tin chloride [23,24]. Attempts to further hydrolyze 7a into 5a
In a first route (Scheme 1), we prepared the 1,3-dithianyl
precursor 4a by substitution of the corresponding Weinreb amide
3a with 1,3-dithiane anion [17]. Previously, ibuprofen 1a was acti-
vated into 2a with N-(methanesulfonyl)benzotriazole [18,19].
Unfortunately, all attempts to “hydrolyze” the dithiane moiety into
an aldehyde function failed: neither compound 5a (or 50a) nor the
corresponding hydrate or dialkyl acetal were identified in the crude
mixtures. Typical conditions investigated were [20]: Hg(ClO4)2/
CaCO3 in alcohol, HgO/CaCO3 in wet acetone, CAN (cerium
were unsuccessful (SnCl2, NaHCO3, (L)-tartric acid, NaHSO3, H2O).
Lastly, we turned to the method using a
a
-bromomethylketone
as key intermediate (Scheme 3) [25]. Sodium salt of ibuprofen was
transformed in acid chloride 8a with oxalyl chloride, and then
O2
H2O
HO
O
(i)
D (Ibuprofen)
CO2
O
N
N
NH2
R'
N
N
R"
R"
N
H
R'
Ibuprofen
Prodrug
C (Aminopyrazine)
E
(ii)
H2O
O
OH
OH
H2O
O
O
F
Fig. 2. Design of potential dual action prodrug endowed with antioxidant and anti-inflammatory activities. Route (i) shows the expected oxidative degradation liberating ibuprofen
and an aminopyrazine antioxidant (R00 ¼ C6H4-pOH). Route (ii) shows the prodrug E synthesis from the aminopyrazine C and the
a-ketoaldehyde synthon F.

3566
F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
R1
R1
N
N
i
ii
Cl
[9a + HCl]
OH
1a
N
i
O
(sodium salt)
O
O
R2
1a
R2
8a: 92%
1
2
: R = Me, R = iBu
1b: R1 = Me, R2 = H
1c: R1 = H, R2 = H
2a
: 97%
iii
N
N
X
O
O
R1
S
R1 Me
9a
10a
, X = Br: 25%
N
S
iii
ii
OMe
11a, X = Cl: 26%
O
iv
O
R2
R2
12a, X = ONO2, 96%
3a: 61%
3b: 60%
4a: 57%
4b
: 55%
3c
: 64%
4c: 50%
NMe2
R1
v
vi
5'c
N
R1
O
R1
O
O
R2
iv
O
13a
(non isolated)
O
OH
R2
R2
Reagents and conditions: (i) (COCl)2, ether, 0 °C to 20 °C, 4 h; (ii) CH2N2,
ether, 20 °C, 2 h; (iii) HBr 47%, AcOH, 20 °C, 1 h; (iv) AgNO3, CH3CN,
20 °C, 40 h; (v) pyridine, then nitrosoaniline, NaOHaq; (vi) 10% H2SO4.
5
5'
Reagents and conditions: (i) BtMs, Et3N, THF, reflux, 17 h; (ii)
S
MeONHMe.HCl, Et3N, THF, 20 °C, 17 h; (iii)
-78 °C, 4 h; (iv) hydrolysis (see text).
, THF,
Li
Scheme 3. Third synthetic approach toward 5.
S
applied to phenylacetyl chloride (R1 ¼ R2 ¼ H) resulted in the
known 2-hydroxy-3-phenyl-2-propenal 50c (tautomer of 5c) [29].
This positive control confirmed that the reactivity of 2-methyl-
arylacetyl is quite different from the unsubstituted arylacetyl, and
we abandoned any effort toward the synthesis of ibuprofen-derived
aldehyde 5a (or 50a).
Scheme 1. First synthetic approach toward ketoaldehyde 5.
reacted with diazomethane in ether solution. The diazo interme-
diate 9a was treated in situ with HBr in acetic acid. Two products
were formed in almost equivalent amounts (NMR of the crude
mixture): the expected
a-bromomethylketone 10a and the a-
The ibuprofen derivatives (2e4; 6e8 and 10e12) have been
purified and characterized as usual (see Experimental protocols).
Typical NMR data are collected in Table 1.
chloromethylketone 11a resulting from the reaction of 9a with HCl
formed during its synthesis. The halogenated compounds were
separated by column chromatography with difficulty. The bromo-
ketone 10a, but not the chloroketone 11a, furnished the nitrate 12a
by substitution with silver nitrate. Unfortunately, nitrite elimina-
tion under basic conditions (AcONa 10% in DMSO) [10,26] failed to
2.2. Condensation of the aminopyrazine synthon (C) with
a-ketoaldoximes
deliver pure
a-ketoaldehyde 5a. We recovered an untractable
mixture, showing only a weak signal at 9.21 ppm (for the aldehyde
proton) in 1H NMR. Treatment of 10a with pyridine (reflux, CH2Cl2),
followed by N,N0-dimethyl-nitrosoaniline in the presence of aq.
NaOH gave a nitrone intermediate [27,28] (13a) which acid
hydrolysis (10% H2SO4) also furnished an untractable mixture.
Nevertheless, the same sequences of reactions (see Scheme 3)
Since we succeeded to obtain the oxime derivative 7a instead of
the target-aldehyde 5a (see Scheme 2), we decided to examine the
reaction of -ketoaldoximes with 2-amino-1,4-pyrazines under
a
acidic conditions, as used to synthesize imidazopyrazinones.
The experimental conditions were first established considering
simple systems, namely the commercial aminopyrazine 14a
(R0 ¼ R00 ¼ H), phenylglyoxal hydrate (15) and phenylglyoxal
aldoxime (16) (Scheme 4). Under the classical conditions (HClaq
(4 equiv.), EtOH, reflux overnight [30,31]), the condensations of 14a
with 15 and 16 led respectively to the bicycles 17a and 18a, in 70%
and 40% yields. The chemical structure of 18, which is different
from the expected one, will be discussed later on. Similarly, the
reactions of 2-amino-5-p-(hydroxy)phenyl-1,4-pyrazine (14b [32])
with 15 and 16 furnished the imidazopyrazine derivatives 17b and
18b, in 66% and 15% yields respectively. We could optimize the
production of the novel heterocycles 18 by using the pre-formed
hydrochloride salts of the aminopyrazines 14 as starting materials
and an excess of 16, and by activating the condensation under
microwave (MW) heating. Thus a 1:2 mixture 14$HCl:16 in ethanol
was heated at 80 ꢀC for 4 h in an MW oven (Scheme 4); compounds
18a and 18b are now recovered in about 60% and 30% yield. The
same protocol applied also readily to the synthesis of the known
imidazopyrazinones 17a,b [31,33] (Scheme 4). Unfortunately, we
N
N
i
O
N
N
O
O
O
2a
6a
: 69%
OH
N
ii
iii
5a
[
]
O
7a: 40%
Reagents and conditions: (i) CH3NO2, tBuOK, DMSO, 10 °C to 20 °C, 4 h; (ii)
SnCl2.H2O, PhSH, Et3N, CH3CN, 20 °C, 1 h; (iii) hydrolysis (see text).
Scheme 2. Second synthetic approach toward 5.

F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
3567
Table 1
Typical NMR data of ibuprofen derivatives.
Entry
1H NMR (ppm)
13C NMR (ppm)
3
2
H-2
H-3
J2,3 (Hz)
R
C-1
C-2
C-3
R
1
R
O
iBu
1
2a
5.40 (q)
1.74 (d)
7.1
7.48 (dd); 7.63 (dd); 8.08 (d); 8.29 (d)
173.9
44.7
18.9
114.9; 120.4; 126.4;
131.7; 131.8; 146.5
32.6; 61.4
2
3
3a
4a
4.10 (q)
4.19 (q)
1.42 (d)
1.44 (d)
7.0
6.9
3.16 (s); 3.40 (s)
1.99 (m); 2.09 (m); 2.50 (m); 2.59 (m);
3.04 (m); 3.51 (m)
175.9
203.1
41.9
44.6
19.8
18.7
25.4; 26.1; 26.3
4
5
6
7
8
9
6a
7a
8a
10a
11a
12a
3.80 (q)
4.55 (q)
4.09 (q)
4.10 (q)
3.89 (q)
3.78 (q)
1.48 (d)
1.43 (d)
1.59 (d)
1.43 (d)
1.43 (d)
1.45 (d)
6.9
7.1
7.0
6.9
6.9
7.0
5.12 (dAB, 14.7 Hz); 5.20 (dAB, 14.7 Hz)
7.50 (s)
/
3.78 (dAB, 12.7 Hz); 3.92 (dAB, 12.7 Hz)
4.02 (dAB, 15.4 Hz); 4.09 (dAB, 15.4 Hz)
4.82 (dAB, 17.5 Hz); 4.91 (dAB, 17.5 Hz)
196.6
198.8
176.0
202.0
202.3
201.6
51.8
47.0
57.4
49.8
49.9
49.8
17.2
18.0
19.0
17.8
17.7
17.0
82.1
149.1
/
33.5
47.4
73.0
were unable to identify any product from the reaction of amino-
pyrazine 14a with the -ketoaldoxime 7a derived from ibuprofen,
Attempts to crystallize other salts (oxalate, fumarate, nitrate,
perchlorate, tetrafluoroborate) also failed: small crystals formed
but they flopped under filtration. Lastly, we tried to derivatize the
heterocycles 18a,b by silylation, alkylation or acylation reactions.
Under the usual conditions of such reactions, only untractable
mixtures were recovered. In the absence of X-ray data (for 18 or
derivatives), the proposed imidazopyrazinimine structures 18a,
b (see Scheme 4, X ¼ H) could not be definitively established, but
they are fitting at best with all the spectral data. Their formation
implies a reductive step, somewhere in the reaction cascade, for
explaining the formal loss of one oxygen atom. The reducing agent
is actually unknown (auto-redox system in the presence of air [34],
aldoxime in excess,.).
The alleged imidazopyrazinimines (18 with X ¼ H) have been
evaluated for their capacity to inhibit the lipid peroxidation, by
using a standard test [35]. Briefly, a micellar solution of linoleic acid
is incubated with AAPH (2,20-azo-bis-(2-aminopropane)dihydro-
chloride) as free-radical generator. In the presence of air, the
substrate radicalar oxidation produces conjugated dienes, which
can be monitored at 234 nm as a function of time. Added antioxi-
a
using a number of experimental conditions (14a$HCl, EtOH, 80 ꢀC,
18 h; dioxane-EtOH, 85 ꢀC, 24 h; dioxane-H2O, 100 ꢀC, 70 h;
dioxane-H2O þ 2 equiv. HClaq, 100 ꢀC, 24 h; EtOH, 80 ꢀC, MW, 4 h;
dioxane-H2O,100 ꢀC, MW, 5 h). Under mild conditions, the reagents
are mainly recovered. In the presence of an excess of HCl, the
aldoxime 7a was degraded and under MW activation, the starting
materials were accompanied with increasing amounts of degra-
dation products in function of the reaction time.
The structure of the heterobicycles 18a,b resulting from the
condensation of 14a,b with the commercial a-ketoaldoxime 16 is
very intriguing. Formation of either the imidazopyrazinoxime
(structure 18 with X ¼OH) or the imidazopyrazinone (structure 17
resulting from the in situ oxime hydrolysis) was expected. But both
the 1H and 13C NMR data collected for compounds 18a,b show
tenuous differences as compared to the corresponding known
imidazopyrazinones 17a,b [4] (see Table 2). From the HRMS data,
the related oximes (see Scheme 4, structure 18 with X ¼OH) can be
excluded: m/e ¼ 211.0983 (18a) for M þ 1 corresponding to the
formula C12H10N4, and m/e ¼ 303.1244 (18b) for M þ 1 corre-
sponding to the formula C18H14N4O. Also the IR and UV spectra of 17
and 18 are different (see Experimental protocols). We tried to
obtain crystals for X-ray diffraction analysis, but without success:
18a,b (hydrochloride salts) precipitated as amorphous powders.
dants delay this oxidation process. At 10 mM, the lag-time induced
by 18a and 18b is respectively of 92 min and 140 min; in the same
test, the reference values of 17a and 17b are 142 min and 133 min.
The imidazopyrazinimines (18) behave thus as antioxidant
reagents, like the known imidazopyrazinones (17). This could
confirm the structural similarity between 17 and 18. However, 18
are devoid of the chemiluminescence properties characteristic of
coelenterazine-like compounds (results not shown).
O
OH
OH
N
NH2.HCl
+
2
3
i
2.3. Biological evaluations
N.HCl
N
5
6
R"
N
R'
9
O
Antioxidant agents could be possibly endowed with anti-
inflammatory properties [36]. Indeed, since the reactive oxygen
species (ROS) exhibit a pro-inflammatory activity, their capture by
8
R'
14
15
R"
N
H
a
, R' = R" = H
b, R' = H; R" =
17
OH
Table 2
OH
X
Comparative NMR data (7$10ꢁ2 M, CD3OD).a
N
N
2
3
i
+
17a
8.23 (d)
7.64 (d)
8.79 (s)
138.5
146.7
130.0
18a
8.30 (d)
7.64 (d)
8.79 (s)
141.9
137.4
133.4
17b
8.42 (s)
/
18b
8.50 (s)
/
N.HCl
9
N
5
6
H-5
H-6
H-8
C-2
C-3
C-9
O
8
R"
N
R'
8.89 (s)
8.79 (s)
16
H
128.6b
147.8b
129.4b
140.2
136.6
132.7
18
(X = H)
14 15 16
= 1:2
Conditions: (i) EtOH, 80 °C, MW, 4 h; ratio of reagents
:
,
1H NMR at 500 MHz and 13C NMR at 125 MHz;
d
values in ppm; d ¼ doublet and
a
Scheme 4. Condensation of aminopyrazine hydrochloride with
a
-ketoaldehyde
s ¼ singlet.
b
hydrate and
a
-ketoaldoxime under microwave heating.
Spectrum recorded in DMSO-d6.

3568
F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
an antioxidant agent should lead to a decrease of the amount of
pro-inflammatory species, and therefore, to the inflammation
abatement. Accordingly, both the imidazopyrazine and ibuprofen
derivatives have been assayed in a cellular test for anti-inflamma-
tory activity based on Caco-2 cells as a widely used model of the
human intestinal barrier [37,38]. Briefly, the secretion of inter-
leukin-8 (IL-8), a pro-inflammatory cytokine, upon stimulation by
IL-1b
, was measured in the presence of the tested molecule at
50
mM. Results are expressed in % vs. the control (no product) and
EGCG ((ꢁ)-epigallocatechin-3-gallate) was considered as the anti-
inflammatory active reference [39]. The molecules were tested 2 or
3 times, each in triplicate. Despite the rather poor reproducibility
inherent to such a cellular assay, the Fig. 3A shows that compounds
17b and 18b, two phenol-substituted imidazopyrazine derivatives,
significantly decreased the secretion of IL-8, considered as a hall-
mark of the intestinal inflammation, to respectively 65% (p < 0.05)
and 56% (p < 0.01) of the control value. Nevertheless, they were less
efficient than EGCG (15%, p < 0.001). The compound 18b was still
Fig. 4. Doseeresponse curves.
Accordingly, this enzyme may be a useful therapeutic target for
the treatment of certain inflammatory states. Recently, our
laboratory became interested in the discovery of novel FAAH
inhibitors [43], so we decided to evaluate the series of ibuprofen
derivatives herein synthesized (see Schemes 1e3), using our in-
house expertise.
active at 5 mM (75%, data not shown). In the same assay realized
with ibuprofen and its derivatives (Fig. 3B), ibuprofen (sodium salt,
1a) appears modestly active, but its derivatives display either
a significant anti-inflammatory effect, i.e. 3a (55%, p < 0.01) > 11a
(70%, p < 0.05) > 2a (76%, p < 0.05), or pro-inflammatory effect, i.e.
7a (290%, p < 0.05).
The production of fatty acid amides (FAAs) is increased under
some inflammation conditions [40]. FAAs are endogenous
compounds interacting with the cannabinoid receptors, a class of
receptors involved in a great number of physiological effects,
among which the dampening of inflammation [41,42]. The rate of
FAAs in vivo is controlled by a hydrolytic enzyme, i.e. FAAH (fatty
acid amide hydrolase), responsible of their metabolism.
It is now well established that ibuprofen inhibits the metabolism
of anandamine, an endogenous cannabimimetic agent [44,45]. Both
enantiomers of ibuprofen, similarly to other NSAIDs (ketorolac,
flurbiprofen, indomethacin and niflumic acid) [46e48], behave as
good inhibitors of FAAH, the enzyme processing anandamide into
arachidonic acid and ethanolamine [49,50]; a pH dependent activity
has been established (pI50 of (R)-Ibu. ¼ 4.26 ꢂ 0.07 at pH 6 and
3.69 ꢂ 0.04 at pH 8, against rat brain FAAH) [46]. The clinical effects
of NSAIDs result from the inhibition of cylcooxygenase enzymes
(COX-1 and COX-2), causing gastrointestinal toxicity as injurious
side effect [6]. Derivatives of ibuprofen such as esters and amides
have been shown to retain the analgesic activity of the parent acid,
while reducing the gastric mucosal damages [51]. Interestingly,
amide derivatives of ibuprofen featuring pyridinyl heterocycles are
also excellent inhibitors of FAAH [52].
The activity of the novel ibuprofen derivatives was assayed
according to a protocol already validated in our group for testing,
Table 3
pI50 values of racemic ibuprofen derivatives (from three independent experiments).
Entry
pI50
R
O
Cmpd
R
hFAAH
hMGL
1
2
1a
eOꢁ Naþ
3.42 ꢂ 0.03
n.d
N
N
N
2a
4.56 ꢂ 0.03
3.71 ꢂ 0.12
3
4
3a
4a
eN(Me)OMe
4.19 ꢂ 0.02
5.86 ꢂ 0.03
3.19 ꢂ 0.10
S
n.d
S
O
N
Fig. 3. Anti-inflammatory effect of imidazopyrazine (A) and ibuprofen (B) derivatives.
Intestinal Caco-2 cells were concomitantly incubated, for 24 h, with the pro-inflam-
5
6a
4.45 ꢂ 0.02
3.31 ꢂ 0.05
O
matory stimulus IL-1
b
at 25 ng/ml in the absence (control) or presence of tested
M, was used as an anti-inflammatory reference. IL-8
OH
N
compound at 50 M. EGCG, at 50
m
m
6
7
7a
4.25 ꢂ 0.04
5.45 ꢂ 0.05
n.d
was then quantified in the extracellular media by ELISA assay. Results are expressed as
the percentage of IL-8 vs the control. Data represent means ꢂ S.E.M. from two or three
independent experiments, each with three replicates per condition. *, **, *** indicate,
respectively, p < 0.05, p < 0.01, p < 0.001 as compared to the control condition.
11a
3.70 ꢂ 0.10
Cl

F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
3569
Table 4
not be applied because the required ibuprofen-synthon (F) was
synthetically inaccessible. However during our Holy Grail quest,
we discovered a novel imidazopyrazine framework, presumably
the imine compounds 18 (X ¼ H) (and not the corresponding
oximes (X ¼ OH)). The p-hydroxyphenyl derivative 18b is
endowed with antioxidant and anti-inflammatory activities and
appears even more active than the related imidazopyrazinone
17b. Nevertheless, this result is rather difficult to exploit further
due to the uncertainty about the structural assignment and the
high synthetic challenge.
Ibuprofen (1a) showed only a poor anti-inflammatory effect on
the IL-8 secretion by the inflamed intestinal cells. This can be
explained as the anti-inflammatory mechanism of NSAIDs impli-
cates another target of the inflammation cascade i.e. the inhibition
of pro-inflammatory enzymes, the cyclooxygenases (COX-1 and
COX-2) [56]. However, some ibuprofen derivatives with masked
acid function (2a, 3a) exhibited a significant activity.
pI50 values of 1,3-dithiane derivatives and references (from three independent
experiments).
Entry
pI50 (hFAAH)
R1
R
O
R2
Cmpd
R1
R2
R
1
2
1a
1b
Me
Me
i-Bu
H
OH
OH
3.55 [48]
2.91 ꢂ 0.04
S
3
4
4a
4b
Me
Me
i-Bu
5.86 ꢂ 0.03
4.00 ꢂ 0.03
S
S
H
S
S
Knowing that the cannabinoid system can contribute to the
pharmacological effects of NSAIDs [52], we evaluated the
ibuprofen derivatives against FAAH. The loss of activity of this
enzyme has been shown to provide beneficial effects in models of
inflammation due to the increased levels of N-acyl-ethanolamines
[57]. Interestingly, we disclosed novel FAAH inhibitors among the
series of ibuprofen derivatives synthesized as precursors of the
synthon F. In particular, the best FAAH inhibitor 4a is more active
than N-(6-(methyl)pyridin-2-yl)-ibuprofen-amide (Table 4, entry
6, Ibu-am5) recently selected by Fowler et al. [52] as “lead” to
explore the potential analgesic effect of compounds combining
COX- and FAAH-inhibitory properties. Even if 4a is devoid of anti-
inflammatory action in our cellular test, its unique activity against
FAAH is of interest. Indeed, the recognized therapeutic promise of
FAAH inhibition for the treatment of anxiety, eating and sleep
disorders, among others [50], nowadays, stimulates the research of
novel inhibitors by structure-based design or high-throughput
screening [58]. Our present work provides an unexpected entry in
this field with the 1,3-dithian-2-yl ketone derived from ibuprofen
as a novel “hit”.
5
6
4c
H
H
4.33 ꢂ 0.06
S
H
N
Ibu-am5
Me
i-Bu
5.08 ꢂ 0.08 [52]
N
Me
among other templates, substrate analogues, lipophilic b-lactams
(azetidin-2-ones), and heterocycle-based inhibitors [43,53e55].
The assay is a competitive hydrolytic experiment using the radio-
labelled substrate (anandamide ¼ arachidonoylethanolamide, AEA)
to measure FAAH activity. Briefly, recombinant human FAAH in
buffer (pH 7.4) is added to glass tubes that contain either the drug
in DMSO or DMSO alone (control). Hydrolysis is initiated by adding
[3H]-AEA solution and the tubes are incubated at 37 ꢀC. Tubes
containing buffer only are used as controls for chemical hydrolysis
(blank) and this value is systematically subtracted. Reactions are
stopped by adding ice-cold MeOH/CHCl3, the radiolabelled etha-
nolamine is extracted and the radioactivity is measured by liquid
scintillation counting (LSC). The selectivity vs. monoacylglycerol
lipase (MGL) was similarly assayed, using the [3H]-labelled 2-
oleoylglycerol (2-OG) as competitive substrate [43,53e55].
4. Experimental protocols
The inhibition results expressed as FAAH activity (% of control) vs.
drug concentration are shown in Fig. 4. From those experimental
dose-response curves, the pI50 (i.e. ꢁlog IC50) values were calculated
(Table 3). The results clearly show that all tested ibuprofen deriva-
tives are more active than the parent carboxylate (sodium salt) and
behave as selective inhibitors of FAAH vs. MGL, another hydrolytic
enzyme of the endocannabinoid system. We recorded the following
decreasing order of activity: 4a (entry 4, R ¼ 1,3-dithian-2-yl),
11a (entry 7, R ¼ chloromethyl), 2a (entry 2, R ¼ benzotriazolyl),
6a (entry 5, R ¼ nitromethyl), 7a (entry 6, R ¼ N-(hydroxy)imino), 3a
(entry 3, R ¼ N-(methoxy)methylamino), and 1a (entry 1, ibuprofen
from commercial source). The most active compound 4a (1-(1,3-
dithian-2-yl)-2-(4-isobutylphenyl)-propan-1-one) was compared
to related dithiane derivatives featuring fewer alkyl substituents,
namely 4b (1-(1,3-dithian-2-yl)-2-phenylpropan-1-one) and 4c (1-
(1,3-dithian-2-yl)-2-phenylethan-1-one). We found that the
ibuprofen core significantly enhances the activity compared to the
simple benzyl derivatives (Table 4, compare entry 3 with entries 4
and 5). The same effect was visible when comparing ibuprofen 1a
(acid, entry 1) with 2-methyl-phenylacetic acid (1b, entry 3).
4.1. Materials and methods
Anhydrous solvents were provided by Fluka. All reagents were
obtained from AldricheFluka, Acros or ROCC and used as received.
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded
on a BRUCKER AVANCE-300 spectrometer. 1H (500 MHz) and 13C
(125 MHz) NMR spectra were recorded on a BRUCKER AM-500
spectrometer. The attributions were established by homonuclear
and heteronuclear correlations or by selective decoupling
experiments. Chemical shifts are reported as
d values (in ppm)
downfield from TMS for 1H and 13C or from CFCl3 for 19F. The
mass spectra were obtained using a Finnigan-MAT TSQ-7000
instrument (FAB, EI and CI modes) or an LCQ Finnigan-MAT LCQ
instrument (APCI and ESI modes). HRMS analyses were obtained
at the Université Mons-Hainaut, Mass spectroscopy laboratory,
Pr. Flammang or at the University College of London (UK),
chemistry department, Dr. L. D. Harris laboratory. UV spectra
were recorded on a UV-vis-NIR Varian-Cary spectrophotometer
(l given in nm). IR spectra were determined using a Shimadzu
FTIR-8400S apparatus. Thin-layer chromatography was carried
out on silica gel 60 plates F254 (Merck, 0.2 mm); product visu-
3. Conclusion
alization was effected with UV light (
l
¼ 254 nm), KMnO4 or I2.
For flash chromatography we used Merck silica gel 60 of
230e400 mesh ASTM. Melting points were determined on an
electro-thermal apparatus BUCHI Melting Point B-540.
Our idea to construct an original ibuprofen prodrug based on
the in situ oxidation of an imidazopyrazinone precursor (E) could

3570
F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
4.2. Organic chemistry
under Ar atmosphere at 0 ꢀC. The mixture was stirred 1 h at RT. The
mixture was diluted with water (5 mL) and acidified with HCl 2 M
(5 mL). The two layers were separated, the organic layer was
washed with NaHCO3 (sat.) and brine, dried over MgSO4, filtered
and concentrated under vacuum. The residue was purified by flash
chromatography (cyclohexane/ethyl acetate 5:1) and 740 mg (64%)
of yellow oil were obtained.
4.2.1. 1-(Benzotriazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (2a)
To a solution of ibuprofen (398 mg, 1.93 mmol) in dry THF (4 mL,
2 mL/mmol) were added BtMs (420 mg, 2.13 mmol) and TEA
(300 mL, 2.14 mmol) under Ar atmosphere. The solution was heated
overnight to reflux. The mixture was cooled to RT and evaporated to
dryness. The residuewas dissolved in ethyl acetate, washed with HCl
0.33 M, Na2CO3 (sat.), brine, dried over MgSO4, filtered and evapo-
rated under vacuum. We obtained 573 mg (97%) of a white solid.
Rf 0.73 (hexane/AcOEt 5:2); m.p. 52e55 ꢀC; 1H NMR (300 MHz,
Rf 0.17 (cyclohexane/ethyl acetate 5:1); 1H NMR (300 MHz,
CDCl3)
(75 MHz, CDCl3)
FT-IR (NaCl, cmꢁ1
d
3.18 (s, 3H), 3.59 (s, 3H), 3.77 (s, 2H), 7.29 (m, 5H); 13C NMR
d
32.2, 39.4, 61.2, 127.1, 128.9, 129.7, 134.8, 172.2;
)
n 2939 (m), 1645 (s), 1455 (w), 1246 (w), 1187
CDCl3)
d
0.86 (d, 3J ¼ 6.6 Hz, 6H),1.74 (d, 3J ¼ 7.1 Hz, 3H),1.80 (m, 1H),
(w), 1032 (w), 730 (w); MS (APCI) m/z: 180 [M þ H]þ, 91 [C7H7]þ.
2.40 (d, 3J ¼ 7.2 Hz, 2H), 5.40 (q, 3J ¼ 7.1 Hz, 1H), 7.09 (d, J ¼ 8.1 Hz,
2H), 7.41 (d, 3J ¼ 8.1 Hz, 2H), 7.48 (dd, 3J ¼ 7.2 Hz, 3J ¼ 8.2 Hz,1H), 7.63
(dd, 3J ¼ 7.2 Hz, 3J ¼ 8.2 Hz, 1H), 8.08 (d, 3J ¼ 8.2 Hz, 1H), 8.29 (d,
3
4.2.5. 1-(1,3-Dithian-2-yl)-2-(4-isobutylphenyl)-propan-1-one (4a)
To a solution of 1,3-dithiane (135 mg,1.12 mmol) in dry THF (2 mL,
3J ¼ 8.2 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d
18.9, 22.7, 30.4, 44.7,
ꢂ 2 mL/mmol) was added dropwise n-BuLi (440
mL, 2.5 M in hexane)
45.3, 114.9, 120.4, 126.4, 128.1, 129.9, 131.7, 131.6, 136.7, 141.4, 146.5,
at ꢁ35 ꢀC under Ar atmosphere. The mixture was stirred vigorously
for 1 h between ꢁ30 ꢀC and ꢁ20 ꢀC and was added dropwise to
173.9; FT-IR (ATR-SeZn, cmꢁ1
) n 2954 (s), 1734 (s), 1450(m), 1380 (s),
1166 (m), 948 (s), 750 (m); MS (APCI) m/z: 308 [M þ H]þ, 120
a
solution of N-methoxy-N-methyl-2-(4-isobutylphenyl)prop-
[Bt þ H]þ; HRMS calcd for C19H21N3ONa: 330.1582, found: 330.1571.
anamide (245 mg, 0.98 mmol) in dry THF (3 mL) at ꢁ78 ꢀC under Ar
atmosphere. The mixture was warmed to RT in 4 h and NH4Cl (sat.,
5 mL) was added to quench the reaction. The mixture was extracted
with diethyl ether (3 ꢃ 10 mL), the organic layers were combined,
dried over MgSO4, filtered and concentrated under vacuum. The
residue was purified by flash chromatography (hexane/ethyl acetate
10:1) and 174 mg (57%) of yellow oil were obtained.
4.2.2. N-Methoxy-N-methyl-2-(4-isobutylphenyl)propanamide
(3a)
To a solution of N-methoxy-N-methylamine hydrochloride
(350 mg, 3.59 mmol) and TEA (500
mL, 3.56 mmol) in dry THF
(5 mL) was added 1-(benzotriazol-1-yl)-2-(4-isobutylphenyl)
propan-1-one (1 g, 3.25 mmol) under Ar atmosphere. The mixture
was stirred overnight at RT, then evaporated to dryness and the
residue was diluted in ethyl acetate (25 mL). The organic layer was
washed with HCl (0.33 M), Na2CO3 (sat.), brine, dried over MgSO4,
filtered and concentrated under vacuum. The residue was purified
by flash chromatography (hexane/ethyl acetate 7:1) and 491 mg
(61%) of pale yellow oil were obtained.
Rf 0.75 (hexane/ethyl acetate 10:1); 1H NMR (300 MHz, CDCl3)
d
0.88 (d, 3J ¼ 6.6 Hz, 6H), 1.44 (d, 3J ¼ 6.9 Hz, 3H), 1.84 (m, 1H), 1.99
(m, 1H), 2.09 (m, 1H), 2.44 (d, 3J ¼ 7.1 Hz, 2H), 2.50 and 2.59 (m, 2H),
3.04 (ddd, 3J ¼ 14.0 Hz, 3J ¼ 11.5 Hz, 3J ¼ 2.6 Hz, 1H), 3.51 (ddd,
3J ¼ 13.8 Hz, 3J ¼ 11.8 Hz, 3J ¼ 2.9 Hz, 1H), 4.19 (s, 1H), 4.19 (q,
3J ¼ 6.9 Hz, 1H), 7.09 (d, 3J ¼ 8.3 Hz, 2H), 7.19 (d, 3J ¼ 8.3 Hz, 2H); 13
C
NMR (75 MHz, CDCl3)
d 18.7, 22.7, 25.4, 26.1, 26.3, 30.4, 44.6, 45.3,
Rf 0.54 (hexane/ethyl acetate 5:2); 1H NMR (300 MHz, CDCl3)
49.7, 128.0, 130.1, 137.4, 141.2, 203.1; FT-IR (ATR-SeZn, cmꢁ1
) n 2956
d
0.88 (d, 3J ¼ 6.6 Hz, 6H), 1.42 (d, 3J ¼ 7.0 Hz, 3H), 1.83 (m, 1H), 2.43
(s), 1709 (s), 1423 (s), 1242 (m), 1029 (s), 848 (w); MS (ESI) m/z: 347
[M þ K]þ, 331 [M þ Na]þ, 309 [M þ H]þ; HRMS calcd for
C17H24ONaS2: 331.1166, found: 331.1155.
(d, 3J ¼ 7.2 Hz, 2H), 3.16 (s, 3H), 3.40 (s, 3H), 4.10 (q, 3J ¼ 7.0 Hz, 1H),
7.07 (d, 3J ¼ 8.1 Hz, 2H), 7.20 (d, 3J ¼ 8.1 Hz, 2H); 13C NMR (125 MHz,
CDCl3)
d 19.8, 22.6, 30.4, 32.6, 41.9, 45.3, 61.4, 127.6, 129.6, 139.4,
140.4, 175.9; FT-IR (ATR-SeZn, cmꢁ1
)
n
2954 (s), 1660 (s), 1510 (m),
4.2.6. 1-(1,3-Dithian-2-yl)-2-phenylpropan-1-one (4b)
The protocol described for 4a was applied.
1462 (s), 1379 (s), 1173 (m), 1119 (m), 1069 (m), 987 (s), 848 (m), 806
(w), 773 (w); MS (ESI) m/z: 521 [2M þ Na]þ, 272 [M þ Na]þ, 250
[M þ H]þ, 161 [M ꢁ C3H6NO2]þ; HRMS calcd for C15H24NO2:
250.1807, found: 250.1814.
Yield: 55%; Rf 0.23 (cyclohexane/ethyl acetate 95:5); 1H NMR
(300 MHz, CDCl3)
d
1.46 (d, 3J ¼ 6.9 Hz, 3H), 2.02 (m, 2H), 2.53 (m,
2H), 3.04 (ddd, 3J ¼ 14.4 Hz, 3J ¼ 11.4 Hz, 3J ¼ 2.1 Hz, 1H), 3.48 (ddd,
3J ¼ 14.4 Hz, 3J ¼ 11.2 Hz, 3J ¼ 2.7 Hz, 1H), 4.18 (s, 1H), 4.23 (q,
4.2.3. N-Methoxy-N-methyl-2-phenylpropionamide (3b)
3J ¼ 6.9 Hz, 1H), 7.27 (m, 5H); 13C NMR (125 MHz, CDCl3)
d 18.3,
To a solution of 2-phenylpropionic acid (2.14 mL, 14.3 mmol) in
dry THF (60 mL) were added dropwise TEA (6.3 mL, 45.2 mmol) and
mesylchloride(1.3 mL,16.5 mmol)underAratmosphereat0 ꢀC.After
10 min, N-methoxy-N-methyl ammonium chloride (2.25 g,
23.1 mmol) was added and the mixture was stirred 1 h at RT. The
mixture was diluted with water (30 mL) and diethyl ether (30 mL).
The two layerswere separatedand the aqueous layerwasextracted (2
ꢃ) with diethyl ether. The combined organic layers werewashed with
HCl (1 M), NaOH (1 M) and brine, dried over MgSO4, filtered and
concentrated under vacuum to obtain 1.653 g (60%) of yellow oil.
25.0, 25.7, 25.9, 44.4, 49.8, 127.3, 127.9, 128.9, 139.9, 202.6; FT-IR
(NaCl, cmꢁ1
) n 2929 (m), 1708 (s), 1493 (m), 1452 (m), 1423 (m),
1261 (w), 1066 (w), 1028 (m), 912 (w), 742 (w), 700 (s); MS (APCI,
positive mode) m/z: 253 [M þ H]þ, 217, 215; MS (APCI, negative
mode) m/z: 251 [M ꢁ H]ꢁ, 209, 179, 175.
4.2.7. 1-(1,3-Dithian-2-yl)-2-phenylethan-1-one (4c)
The protocol described for 4a was applied.
Yield: 50%; Rf 0.32 (cyclohexane/ethyl acetate 95:5); 1H NMR
(300 MHz, CDCl3)
d 2.02 (m, 2H), 2.57 (m, 2H), 3.24 (ddd,
1H NMR (300 MHz, CDCl3)
d
1.44 (d, 3J ¼ 6.9 Hz, 3H), 3.16 (s, 3H),
2J ¼ 14.3 Hz, 3J ¼ 11.1 Hz, 3J ¼ 2.7 Hz, 2H), 3.96 (s, 2H), 4.26 (s, 1H),
3.40 (s, 3H), 4.12 (m, 1H), 7.29 (m, 5H); 13C NMR (125 MHz, CDCl3)
7.27 (m, 5H); 13C NMR (125 MHz, CDCl3)
d 25.0, 25.8, 45.3, 46.7,
d
19.9, 32.7, 42.3, 61.4, 127.0, 127.9, 128.9, 142.2, 177.3; FT-IR (NaCl,
127.1, 128.6, 129.4, 133.6, 199.8; FT-IR (NaCl, cmꢁ1
) n 2916 (m), 1708
cmꢁ1
)
n
2935 (m), 1662 (s), 1452 (m), 1380 (m), 1176 (w), 986 (m),
(s), 1496 (w), 1425 (w), 1315 (m), 1257 (m), 1049 (m), 754 (m), 704
754 (w), 700 (m); MS (ESI) m/z: 194 [M þ H]þ.
(s); MS (APCI) m/z: 239 [M þ H]þ, 163 [C6H11OS2]þ, 149 [C5H9OS2]þ.
4.2.4. N-Methoxy-N-methyl-2-phenylacetamide (3c)
4.2.8. 3-(4-Isobutylphenyl)-1-nitrobutan-2-one (6a)
To a solution of 2-phenylacetyl chloride (870
dry CH2Cl2 (10 mL) were added N-methoxy-N-methyl ammonium
chloride (690 mg, 7.07 mmol) and dropwise TEA (2 mL, 14.3 mmol)
m
L, 6.45 mmol) in
To a vigorously stirred solution of nitromethane (36 mL,
0.66 mmol) in dry DMSO (3.5 mL, 5 mL/mmol) at 10 ꢀC was added
t-BuOK (164 mg, 1.46 mmol) under Ar atmosphere. The mixture

F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
3571
was raised to RT in 10 min. Then, a solution of 1-(benzotriazol-1-
yl)-2-(4-isobutylphenyl)propan-1-one (204 mg, 0.66 mmol) in dry
DMSO (3.5 mL, 5 mL/mmol) was added dropwise to the reaction
mixture. After 4 h of stirring at RT, the mixture was poured into
water (15 mL, ꢂ 4 ꢃ 5 mL/mmol), acidified with acetic acid 10% and
extracted with ethyl acetate (3 ꢃ15 mL). Organic layers were
combined, washed with brine (3 ꢃ 40 mL), dried over MgSO4,
filtered and concentrated under vacuum. After purification by flash
chromatography (hexane/ethyl acetate 10:1), 122 mg (69%) of
a yellow oil were obtained.
with ethyl acetate (3 ꢃ 30 mL), the combined organic layers were
washed with water, brine then dried over MgSO4, filtered and
concentrated under vacuum. The residue was purified by flash
chromatography (hexane/diethylether 97:3) to obtain 352 mg (25%)
of 3-(4-isobutylphenyl)-1-bromobutan-2-one as a colourless oil and
243 mg (26%) of 3-(4-isobutylphenyl)-1-chlorobutan-2-one as a col-
ourless oil (CAUTION: protect from light for storage).
10a: Rf 0.24 (hexane/diethylether 97:3); 1H NMR (300 MHz,
CDCl3)
d
0.90 (d, 3J ¼ 6.6 Hz, 6H), 1.43 (d, 3J ¼ 6.9 Hz, 3H), 1.85 (m,
1H), 2.45 (d, 3J ¼ 7.2 Hz, 2H), 3.78 (d, 2J ¼ 12.7 Hz, 1H), 3.92 (d,
2J ¼ 12.7 Hz, 1H), 4.10 (q, 3J ¼ 6.9 Hz, 1H), 7.12 (s, 4H); 13C NMR
Rf 0.15 (hexane/ethyl acetate 10:1); 1H NMR (500 MHz, CDCl3)
d
0.90 (d, 3J ¼ 6.6 Hz, 6H), 1.48 (d, 3J ¼ 6.9 Hz, 3H), 1.85 (m, 1H), 2.47
(75 MHz, CDCl3) d 17.8, 22.5, 30.3, 33.5, 45.1, 49.8, 127.7, 130.1, 136.8,
(d, 3J ¼ 7.2 Hz, 2H), 3.80 (q, 3J ¼ 6.9 Hz, 1H), 5.12 (d, 2J ¼ 14.7 Hz,1H),
5.20 (d, 2J ¼ 14.7 Hz, 1H), 7.10 (d, 3J ¼ 8.1 Hz, 2H), 7.16 (d, 3J ¼ 8.1 Hz,
141.4, 202.0; FT-IR (ATR-SeZn, cmꢁ1
) n 2953 (s), 1732 (s), 1651 (w),
1510 (m), 1464 (m), 1454 (m), 1385 (m), 1282 (w), 1167 (m), 1018 (s),
2H); 13C NMR (125 MHz, CDCl3)
d
17.2, 22.6, 30.4, 45.3, 51.8, 82.1,
848 (s), 796 (m); MS (CI/CH4) m/z: 285 [M (81Br) þ H]þ, 283 [M
127.9, 130.7, 135.2, 142.3, 196.6; FT-IR (ATR-SeZn, cmꢁ1
)
n
2956 (s),
(
(
79Br) þ H]þ, 161 [M ꢁ C2H2OBr]þ; MS (ESI) m/z: 590 [2 ꢃ M
81Br) þ Na]þ, 588 [M (81Br) þ M (79Br) þ Na]þ, 586 [2 ꢃ M (79Br) þ
1735 (s), 1562 (s), 1382 (m), 1035 (w), 671 (w); MS (APCI) m/z: 248
[M ꢁ H]ꢁ; HRMS calcd for C14H19NO3Na: 272.1263, found: 272.1259.
Na]þ, 307 [M (81Br) þ Na]þ, 305 [M (79Br) þ Na]þ; HRMS calcd for
C14H19ONaBr: 305.0517, found: 305.0517.
4.2.9. 1-(N-Hydroxyimino)-3-(4-isobutylphenyl)butan-2-one (7a)
11a: Rf 0.19 (hexane/diethylether 97:3); 1H NMR (300 MHz,
To a stirred solution of SnCl2.H2O (230 mg, 1.21 mmol), PhSH
CDCl3)
d
0.90 (d, 3J ¼ 6.6 Hz, 6H), 1.43 (d, 3J ¼ 6.9 Hz, 3H), 1.85 (m,
(370
mL, 3.59 mmol) and NEt3 (510
mL, 3.61 mmol) in acetonitrile
1H), 2.45 (d, 3J ¼ 7.2 Hz, 2H), 3.98 (q, 3J ¼ 6.9 Hz, 1H), 4.02 (d,
(4 mL) was added a solution of 3-(4-isobutylphenyl)-1-nitrobutan-
2-one (200 mg, 0.80 mmol) in 1 mL of acetonitrile. After 1 h the
solvent was removed under reduced pressure, water (10 mL) was
added and the resulting solution was extracted with diethyl ether
(3 ꢃ 10 mL). The combined organic layers were washed with
NaHCO3 (sat.), brine, dried over MgSO4, filtered and concentrated
under reduced pressure. The residue was purified by flash chro-
matography (hexane/ethyl acetate 10:1); 75 mg (40%) of yellow oil
were obtained.
2J ¼ 15.4 Hz, 1H), 4.09 (d, 2J ¼ 15.4 Hz, 1H), 7.12 (s, 4H); 13C NMR
(75 MHz, CDCl3) d 17.7, 22.5, 30.3, 45.1, 47.4, 49.9, 127.7, 130.1, 136.6,
141.4, 202.3; FT-IR (ATR-SeZn, cmꢁ1
) n 2955 (s), 1732 (s), 1510 (m),
1464 (m), 1454 (m), 1382 (m), 1333 (w), 1284 (w), 1166 (m), 1111
(m),1064 (m),1020 (m), 848 (s), 796 (s), 781 (m); MS (FAB) m/z: 240
[M (37Cl)]ꢄþ, 238 [M (35Cl)]ꢄþ, 161 [M ꢁ C2H2OCl]þ; HRMS calcd for
C14H19ONaCl: 261.1022, found: 261.1020.
4.2.12. 3-(4-Isobutylphenyl)-1-nitrate-butan-2-one (12a)
Rf 0.22 (hexane/ethyl acetate 10:1); 1H NMR (300 MHz, CDCl3)
To a solution of 3-(4-isobutylphenyl)-1-bromobutan-2-one
d
0.88 (d, 3J ¼ 6.6 Hz, 6H), 1.43 (d, 3J ¼ 7.1 Hz, 3H), 1.83 (m, 1H), 2.42
(268 mg, 0.95 mmol) in dry acetonitrile (5 mL) was added AgNO3
(340 mg, 2.0 mmol) at RT and the mixture was stirred for 40 h. The
mixture was filtered on a flash silice pad and concentrated under
vacuum to obtain 241 mg (96%) of yellow oil.
(d, 3J ¼ 7.2 Hz, 2H), 4.55 (q, 3J ¼ 7.1 Hz, 1H), 7.07 (d, 3J ¼ 8.1 Hz, 2H),
7.14 (d, 3J ¼ 8.1 Hz, 2H), 7.50 (s,1H); 13C NMR (75 MHz, CDCl3)
d 18.0,
22.7, 30.4, 45.3, 47.0, 128.1, 129.8, 137.3, 140.9, 149.1, 198.8; FT-IR
(ATR-SeZn, cmꢁ1
)
n
3315 (m), 2952 (s), 1674 (s), 1604 (w), 1510 (m),
1H NMR (300 MHz, CDCl3)
d
0.89 (d, 3J ¼ 6.6 Hz, 6H), 1.45 (d,
1456 (s), 1382 (w), 1267 (w), 1169 (m), 1069 (m), 1004 (s), 927 (w),
849 (m), 796 (w), 767 (w), 690 (m); MS (APCI) m/z: 234 [M þ H]þ,
216 [M ꢁ OH]þ, 201 [M ꢁ OH ꢁ CH3]þ, 161 [M ꢁ C2H2NO2]þ; HRMS
calcd for C14H19NO2Na: 256.1313, found: 256.1307.
3J ¼ 7.0 Hz, 3H), 1.85 (m, 1H), 2.46 (d, 3J ¼ 7.2 Hz, 2H), 3.78 (q,
3J ¼ 7.0 Hz, 1H), 4.82 (d, 2J ¼ 17.5 Hz, 1H), 4.91 (d, 2J ¼ 17.5 Hz, 1H),
7.13 (s, 4H); 13C NMR (75 MHz, CDCl3)
d
17.0, 22.5, 30.3, 45.1, 49.8,
2955
73.0, 127.7, 130.1, 135.8, 141.7, 201.6; FT-IR (ATR-SeZn, cmꢁ1
) n
(s), 1736 (s), 1645 (s), 1512 (m), 1454 (m), 1404 (m), 1365 (m), 1283
(s), 1130 (w), 1058 (w), 1008 (m), 970 (m), 941 (w), 847 (s), 800 (m),
752 (w); MS (CI/CH4) m/z: 265 [M]ꢄꢁ, 218 [M ꢁ HNO2]ꢁ, 161
[M ꢁ C2H2OBr]þ; MS (ESI) m/z: 288 [M þ Na]þ; HRMS calcd for
C14H19NO4Na: 288.1212, found: 288.1210.
4.2.10. 2-(4-Isobutyl-phenyl)-propionyl chloride (8a)
To a suspension of ibuprofen sodium salt (1.47 g, 6.4 mmol) in
dry diethyl ether (20 mL) was added dropwise oxalyl chloride
(605 m
L, 7.05 mmol) at 0 ꢀC under Ar atmosphere and the mixture
was stirred for 4 h. The mixture was filtered, concentrated under
vacuum, diluted with hexane, filtered again and evaporated under
vacuum to obtain 1.32 g (92%) of colourless liquid.
4.2.13. 1-Bromo-3-phenylpropan-2-one (10c)
To a solution of 2-phenylacetyl chloride (670 mL, 5.06 mmol) in
1H NMR (500 MHz, CDCl3)
d
0.90 (d, 6H),1.59 (d, 3H),1.86 (m,1H),
2.47 (d, 2H), 4.09 (q, 1H), 7.15 (d, 2H), 7.20 (d, 2H); 13C NMR (75 MHz,
CDCl3) 19.0, 22.7, 30.4, 45.3, 57.4, 127.9, 130.1, 134.8, 142.1, 176.0.
diethyl ether (15 mL) at 0 ꢀC was added a diethyl ether solution of
diazomethane until acyl chloride was perfectly consumed. After
removing the solvent under vacuum, acetic acid (6 mL) was added.
The solution was cooled to 0 ꢀC and aqueous HBr (47%, 1 mL) was
added dropwise slowly. The mixture was allowed to warm to RT
and was stirred for 1 h. Then the solution was poured into cold
water (10 mL), neutralized with Na2CO3 to pH 7. The solution was
extracted with ethyl acetate (3 ꢃ 30 mL), the combined organic
layers were washed with water, brine then dried over MgSO4,
filtered and concentrated under vacuum to obtain 818 mg of
a colourless oil. As the NMR spectrum showed only two products,
1-bromo-3-phenylpropan-2-one as major product (w90%) and 1-
chloro-3-phenylpropan-2-one as minor product (<10%), the crude
material was used without any purification.
d
4.2.11. 3-(4-Isobutylphenyl)-1-bromo- and 1-chloro-butan-2-one
(10a) and (11a)
To a solution of 2-(4-isobutyl-phenyl)-propionyl chloride (1.32 g,
5.87 mmol) in diethyl ether (50 mL) at 0 ꢀC was added dropwise
a diethyl ether solution of diazomethane (see Arndt, F. Organic
Synthesis Coll.1943, 2,165) until acyl chloridewas perfectlyconsumed
(disappearance of the yellow colour of CH2N2 solution). After
removing the solvent under vacuum, acetic acid (6 mL) was added.
The solution was cooled to 0 ꢀC and aqueous HBr (47%, 1.3 mL) was
added dropwise slowly. The mixture was allowed to warm to RT and
was stirred for 1 h. Then the solution was poured into cold water
(10 mL), neutralized with Na2CO3 to pH 7. The solution was extracted
10c: Yield: 69%; Rf 0.41 (hexane/diethylether 97:3); 1H NMR
(300 MHz, CDCl3) d C
3.92 (s, 2H), 3.95 (s, 2H), 7.22e7.38 (m, 5H); 13

3572
F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
NMR (75 MHz, CDCl3)
d
33.4, 46.7, 127.4, 129.4, 128.9, 133.1, 199.3;
NMR (125 MHz, DMSO-d6)
128.4, 128.5, 128.6, 129.2, 129.4, 132.5, 147.8, 158.5; 13C NMR
(75 MHz, CD3OD) 111.9, 117.2, 123.7, 128.2, 128.8, 129.5, 129.9,
130.2, 131.2, 133.0, 137.2, 161.2 (two quaternary carbons not detec-
ted); FT-IR (ATR-SeZn, cmꢁ1
3149 (m), 2964 (m), 1662 (m), 1608
d 108.1, 115.8, 122.7, 126.1, 127.5, 127.6,
FT-IR (NaCl, cmꢁ1
)
n
3059 (m), 3030 (m), 2936 (s), 1725 (s), 1495
(m), 1391 (m), 1181 (m), 1045 (s), 739 (m); MS (CI/CH4) m/z: 215 [M
d
(
81Br) þ H]þ, 213 [M (79Br) þ H]þ.
11c: Yield: 8%; Rf 0.36 (hexane/diethylether 97:3); 1H NMR
) n
(300 MHz, CDCl3)
d
3.90 (s, 2H), 4.12 (s, 2H), 7.22e7.38 (m, 5H); 13
C
(s), 1516 (s), 1448 (m), 1346 (w), 1218 (s), 1155 (s), 1016 (w), 920 (w),
NMR (75 MHz, CDCl3)
d
45.1, 52.2, 127.4, 128.7, 129.9, 132.2, 194.1;
837 (m), 769 (m), 690 (w); MS (ESI) m/z: 304 [M þ H]þ.
MS (CI/CH4) m/z: 171 [M (37Cl) þ H]þ, 169 [M (35Cl) þ H]þ.
4.2.17. 2-Phenylimidazo[1,2-a]pyrazin-3(7H)-imine (18a)
4.2.14. 2-Hydroxy-3-phenyl-2-propenal (50c)
A solutionof2-aminopyrazine chlorohydrate(134 mg,1.02 mmol)
and phenylglyoxal aldoxime (315 mg, 2.11 mmol) in ethanol (4 mL)
was heated (at 80 ꢀC) in a Mw oven at 150 W for 5 min, then at 75 W
overnight. Thereactionmixturewasconcentratedundervacuum. The
product was precipitated by adding diethyl ether to the residue dis-
solved in a minimum of methanol. After two precipitations, 159 mg
(64%) of a dark red solid were obtained.
A
solution of 1-bromo-3-phenylpropan-2-one (810 mg,
3.8 mmol) in DCM (40 mL) was refluxed in the presence of pyridine
(1.3 mL) under Ar atmosphere for 2 h. Concentration of the solution
under vacuum afforded the pyridinium salt as a solid. The solid was
dissolved in water (40 mL) and N,N-dimethyl-4-nitrosoaniline
(600 mg, 4.00 mmol) was added at 0 ꢀC followed by 1 M aqueous
NaOH (4 mL). The resulting suspension was allowed to reach RT and
stirred for 2 h with sonicating occasionally. The suspension was
filtered and the solid was suspended into 10% aqueous H2SO4 (50 mL)
at 0 ꢀC. The suspension was warmed to RT and stirred 2 h with soni-
cating occasionally. The suspension was extracted with diethyl ether
(2 ꢃ 50 mL) and the combined organic layers were washed with
water (2ꢃ)andbrine.ThesolutionwasdriedwithNa2SO4, filteredand
concentrated under vacuum to obtain 347 mg (62%) of a brown solid.
M.p.: >225 ꢀC, decomposition; 1H NMR (300 MHz, CD3OD)
d 7.51
(m, 3H), 7.65 (d, 3J ¼ 5.6 Hz, 1H), 7.93 (d, 3J ¼ 7.8 Hz, 2H), 8.30 (d,
3J ¼ 5.6 Hz,1H), 8.79 (s, 1H); 13C NMR (75 MHz, CD3OD)
d 116.1,119.2,
128.6, 130.2, 130.8, 131.8, 132.6, 133.4, 137.4, 141.9; FT-IR (ATR-SeZn,
cmꢁ1
) n 3120 (s),1633 (s), 1556 (m),1467 (s),1404 (s), 1296 (w),1261
(w),1217 (m),1130 (w),1060 (s),1022 (w), 905 (w), 775 (m), 696 (m);
MS (ESI) m/z: 211 [M þ H]þ; HRMS calcd for C12H11N4: 211.0984,
found: 211.0983; UVevis (MeOH, 10ꢁ4 M): lm (nm) 429, 269.
1H NMR (300 MHz, CDCl3)
d 6.18 (s,1H), 6.60 (s, 1H, OH), 7.40 (m,
3H), 7.85 (d, 3J ¼ 7.8 Hz, 1H), 9.25 (s, 1H); 13C NMR (75 MHz, CDCl3)
4.2.18. 2-Phenyl-6-p-hydroxyphenylimidazo[1,2-a]pyrazin-3(7H)-
imine (18b)
d
122.9, 128.9, 129.5, 130.6, 133.7, 148.8, 188.4; MS (APCI, positive
mode) m/z: 181, 167, 149 [M þ H]þ, 131 [M ꢁ OH]þ, 120 [M ꢁ CO]þ;
MS (APCI, negative mode) m/z: 179,147 [M ꢁ H]ꢁ,117; MS (ESI) m/z:
181, 167, 149 [M þ H]þ.
To a solution of 2-amino-5-(p-hydroxyphenyl)-1,4-pyrazine
(181 mg, 0.97 mmol) and phenylglyoxal aldoxime (301 mg,
2.02 mmol) in ethanol (4 mL) was added concentrated HCl (36%,
310 m
L) and the mixture was heated (at 80 ꢀC) in a Mw oven at 150 W
4.2.15. 2-Phenylimidazo[1,2-a]pyrazin-3-(7H)-one (17a)
for 5 min, then at 100 W for 4 h. The reaction mixture was concen-
trated under vacuum. The product was precipitated by addition of
diethyl ether totheresiduedissolved in aminimumofmethanol.After
two precipitations, 98 mg (30%) of a dark red solid were obtained.
2-Aminopyrazine hydrochloride (14a) was prepared from
commercial 2-aminopyrazine (solution in ether) and 2 M HCl (5
eq.). The precipitated salt was filtered off and washed with ether. A
solution of 2-aminopyrazine hydrochloride (133 mg, 1.02 mmol)
and phenylglyoxal (308 mg, 2.02 mmol) in ethanol (4 mL) was
heated (at 80 ꢀC) in Microwave (Mw) oven at 150 W for 5 min, then
at 75 W of 4 h. After concentration under vacuum, the residue was
dissolved in methanol (0.5 mL) and the product was precipitated by
addition of diethyl ether to furnish 165 mg of a dark red solid (66%).
M.p.: >250 ꢀC, decomposition; 1H NMR (300 MHz, CD3OD)
d 6.98
(d, 3J ¼ 8.7 Hz, 2H), 7.49 (t, 3J ¼ 7.5 Hz,1H), 7.55 (t, 3J ¼ 7.5 Hz, 2H), 7.68
(d, 3J ¼ 8.7 Hz, 2H), 7.93 (d, 3J ¼ 7.5 Hz, 2H), 8.50 (s, 1H), 8.79 (s, 1H);
13C NMR (125 MHz, CD3OD)
d 111.6, 117.3, 123.2, 128.6, 129.5, 130.3,
130.9, 131.8, 132.0, 132.7, 134.5, 136.6, 140.2, 161.2; FT-IR (ATR-SeZn,
cmꢁ1
)
n
2927 (s), 2854 (m), 1652 (m), 1612 (m), 1452 (m), 1252 (m),
1H NMR (500 MHz, CD3OD)
d
7.49 (m, 3H), 7.64 (d, 3J ¼ 5.6 Hz,1H),
1111 (s), 1056 (m), 837 (s), 779 (m), 698 (m); MS (ESI, positive mode)
m/z:303[M þ H]þ;MS(ESI,negativemode)m/z:301[Mꢁ H]ꢁ;HRMS
calcd for C18H15N4O: 303.1246, found: 303.1244.
8.23 (d, 3J ¼ 5.6 Hz,1H), 8.25 (d, 3J ¼ 6.9 Hz, 2H), 8.79 (s,1H); 13C NMR
(125 MHz, CD3OD) d116.1,119.4,128.2,129.9,130.0,130.6,130.9,132.0,
138.5, 146.7; FT-IR (ATR-SeZn, cmꢁ1
) n 3096 (s), 2923 (s), 1668 (m),
1573 (s),1497 (s),1448 (m),1367 (w),1288 (w),1230 (s),1124 (s),1088
(w), 1070 (w), 1000 (w), 906 (m), 775 (s), 696 (s); MS (ESI) m/z: 212
[M þ H]þ; HRMS calcd for C12H9N3ONa: 234.0643, found: 234.0638;
UVevis (MeOH, 10ꢁ4 M): lm (nm) 466, 287.
4.3. Evaluations
4.3.1. Inhibition of lipid peroxidation
A
micellar solution of linoleic acid (4 mM) containing
Tweenꢁ20 as surfactant in phosphate buffer (50 mM, pH 7.4) and
EDTA (0.1 mM) is incubated at 37 ꢀC with 2 mM AAPH (2,20-azo-bis
(2-amidinopropane)-dihydrochloride) as the free-radical gener-
ator. The production of conjugated dienes by the peroxidation of
linoleate is monitored continuously at 234 nm using a wavelength
tuneable microplate spectrophotometer reader (SpectraMAX 190,
Molecular Devices). Antioxidant efficiency of the tested compound
was evaluated by the lag phase duration until onset of peroxidation.
Tested compounds were initially dissolved in ethanol (10ꢁ2M) then
in phosphate buffer (50 mM, pH 7.4); they were assayed at final
4.2.16. 2-Phenyl-6-p-hydroxyphenylimidazo[1,2-a]pyrazin-3(7H)-
one (17b)
To a stirred solution of 2-amino-5-(p-hydroxyphenyl)-1,4-pyr-
azine (180 mg, 0.96 mmol) and phenylglyoxal (306 mg, 1.97 mmol)
in ethanol (3 mL) was added concentrated HCl (36%) (310 mL) and
the mixture was heated at reflux overnight. The reaction mixture
was concentrated under vacuum. The product was precipitated by
addition of diethyl ether to the residue dissolved in a minimum of
methanol. After two precipitations, 225 mg (69%) of a dark red solid
were obtained.
concentrations of 1.25, 2.5, 5, 10 and 20 mM.
1H NMR (500 MHz, DMSO-d6)
d
6.89 (d, 3J ¼ 8.6 Hz, 2H), 7.36 (t,
3J ¼ 7.9 Hz, 1H), 7.47 (t, 3J ¼ 7.9 Hz, 2H), 7.65 (d, 3J ¼ 8.6 Hz, 2H), 7.99
(s, 1H), 8.38 (d, 3J ¼ 7.9 Hz, 2H), 8.41 (s, 1H); 1H NMR (300 MHz,
4.3.2. Anti-inflammatory assay
The human intestinal Caco-2 cells (ATCC, Rockville, MD), used
between passages 30 and 50 were routinely grown in DMEM
containing 4.5 g/l glucose, 25 mM Hepes, 10% (v/v) fetal calf
CD3OD)
d
6.94 (d, 3J ¼ 8.4 Hz, 2H), 7.49e7.55 (m, 3H), 7.71 (d,
3J ¼ 8.4 Hz, 2H), 8.12 (d, 3J ¼ 7.2 Hz, 2H), 8.42 (s, 1H), 8.89 (s, 1H); 13
C

F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
3573
[9] M. Isobe, T. Fujii, M. Kuse, K. Miyamoto, K. Koga, 19F-dehydrocoelenterazine as
probe to investigate the active site of symplectin. Tetrahedron 58 (2002)
2117e2126.
[10] M. Mosrin, T. Bresser, P. Knochel, Regio and chemoselective multiple func-
tionalization of chloropyrazine derivatives. Application to the synthesis of
coelenterazine. Org. Lett. 11 (2009) 3406e3409.
[11] C. Reichel, R. Brugger, H. Bang, G. Geisslinger, K. Brune, Molecular cloning and
expression of a 2-arylpropionyl-coenzyme A epimerase: a key enzyme in the
inversion metabolism of ibuprofen. Mol. Pharm. 51 (1997) 576e582.
[12] M. Tiecco, L. Testaferri, M. Tingoli, D. Bartoli, Selenium-catalyzed conversion of
serum (FCS) (Hyclone Perbio-Sciences, Erembodegem, BE), 2% (v/
v) -glutamine 200 mM and 1% (v/v) nonessential amino acids
(NEAA) (Invitrogen, Carlsbad, CA). For the experiments, cells
were seeded at a density of 40 ꢃ 103 cells/cm2 in 96-well plates
(Nunc, Roskilde, DK) and cultivated until 7 days post-confluence
with culture medium changes three times per week. The cells
L
were incubated with the pro-inflammatory stimulus, IL-1
25 ng/ml in DMEM containing 0.5% (v/v) FCS, alone (control) or in
presence of the tested compound at 50 M. After 24 h treatment,
b at
methyl ketones into a-keto acetals. J. Org. Chem. 55 (1990) 4523e4528.
m
[13] M.N. Deshpande, S. Wehrli, M. Jawdosiuk, J.T. Guy Jr., D.W. Bennett, J.M. Cook,
Formation of the tetracyclo[5.4.2.02,6.02,9]tridecane ring system by a novel
transannular aldolization reaction. J. Org. Chem. 51 (1986) 2436e2444.
[14] R. Verhé, D. Courtheyn, N. De Kimpe, L. De Buyck, N. Schamp, Preparation of
1,1-dialkoxy-2-alkanones. Synthesis 8 (1982) 667e670.
the extracellular media were collected and processed for IL-8
quantification with the Human IL-8 ELISA kit (BD Biosciences
Pharmingen, San Diego, CA), according to the manufacturer’s
instructions.
[15] N. De Kimpe, N. Schamp, Reactivity of N-2-(1,1-dichloroalkylidene)cyclo-
hexylamines. II, Bull. Soc. Chim. Belg 83 (1974) 507e514.
[16] M. Kuse, M. Isobe, Synthesis of 13C-dehydrocoelenterazine and NMR studies
on the bioluminescence of a Symplectoteuthis model. Tetrahedron 56 (2000)
2629e2639.
4.3.3. FAAH and MGL inhibition
Recombinant human FAAH (in 165
mL of TriseHCl, 100 mM, pH
[17] M. Keenan, K. Jones, F. Hibbert, Highly efficient and flexible total synthesis of
coelenterazine. Chem. Comm. (1997) 323e324.
7.4) was added on ice to glass tubes (5
mg protein/tube) containing
either drugs or DMSO (10
m
L). Hydrolysis was initiated by adding
[18] A.R. Katritzky, H. Yang, S. Zhang, M. Wang, An efficient conversion of
carboxylic acids into Weinreb amides. ARKIVOC 11 (2002) 39e44.
[19] A.R. Katritzky, H.Y. He, K. Suzuki, N-Acylbenzotriazoles: neutral acylating
reagents for the preparation of primary, secondary, and tertiary amides. J. Org.
Chem. 65 (2000) 8210e8213.
[20] P.J. Kocienski, Protecting Groups, third ed. Georg Thieme Verlag, Stuttgart,
2004, pp. 77e96.
[21] A.R. Katritzky, A.A.A. Abdel-Fattah, A.V. Gromova, R. Witek, P.J. Steel, a-Nitro
ketonesynthesis usingN-acylbenzotriazoles. J. Org. Chem. 70 (2005)9211e9214.
[22] R. Ballini, M. Petrini, Recent synthetic developments in the nitro to carbonyl
conversion (Nef reaction). Tetrahedron 60 (2004) 1017e1047.
[23] F. Urpi, J. Vilarrasa, New synthetic tricks. A novel one-pot procedure for the
conversion of primary nitro groups into aldehydes. Tetrahedron Lett. 31
(1990) 7499e7500.
[24] M. Bartra, P. Romea, F. Urpi, J. Vilarrasa, A fast procedure for the reduction of
azides and nitro compounds based on the reducing ability of Sn(SR)3-species.
Tetrathedron 46 (1990) 587e594.
25
m
L of [3H]-anandamide (50,000 dpm, 2
m
M final concentration) in
TriseHCl containing 0.1% BSA and tubes were incubated for 10 min
at 37 ꢀC in a shaking water bath. Reactions were stopped by rapidly
placing the tubes on ice and adding 400
(1:1 v/v). Following centrifugation (1700 g, 5 min, 4 ꢀC) the [3H]-
ethanolamine in the aqueous layer was recovered (200 L) and
counted by liquid scintillation. Blanks (controls for chemical
hydrolysis) were prepared (buffer instead of FAAH) and the values
systematically subtracted. MGL activity was assayed in TriseHCl pH
8, according to a similar protocol and using [3H]-oleoylglycerol
mL of ice-cold MeOHeCHCl3
m
(50,000 dpm, 10 mM final concentration) as substrate.
4.3.4. Data analysis
[25] F.-Q. Chen, T. Hirano, Y. Hashizume, Y. Ohmiya, M. Ohashi, Synthesis and
Results were expressed as means ꢂ S.E.M. Statistical analysis
was performed using one-way analysis of variance (ANOVA)
coupled with Scheffe’s post-hoc test. The computer program was
Systat 5.2.1 (Systat Inc., Evanston, IL). Results with a two-sided p
value < 0.05 were considered statistically significant.
preliminary chemi- and bio-luminescence studies of
coelenterazine analog with a trifluoromethyl diazirine group. J. Chem. Soc.
Chem. Commun. 20 (1994) 2405e2406.
a novel photolabile
[26] F.-Q. Chen, J.L. Zheng, T. Hirano, H. Niwa, Y. Ohmiya, M. Ohashi, A potential
photoaffinity probe for labeling the active site of aequorin: a photolabile
coelenterazine analog with a trifluoromethyldiazirine group. J. Chem. Soc.
Perkin Trans. 1: Org. Bio-Organic Chem. 17 (1995) 2129e2134.
[27] S.K. Moitra, A.N. Ganguli, N.N. Chakravarti, R.N. Adhya, New synthetic method
Acknowledgements
for aromatic
a-oxo aldehydes: FriedeleCrafts reaction. Tetrahedron Lett.
(1971) 199e200.
[28] M. Kuse, I. Doi, N. Kondo, Y. Kageyama, M. Isobe, Synthesis of azide-fluoro-
dehydrocoelenterazine analog as a photoaffinity-labeling probe and photol-
ysis of azide-fluoro-coelenterazine. Tetrahedron 61 (2005) 5754e5762.
This work was supported by UCL, F.R.I.A.-FNRS (fellowship to
F. D. W.) and by F.R.S.-FNRS (Belgium). J. M.-B. is senior research
associate of F.R.S-FNRS.
[29] K. Layachi, I. Ariès-Gautron, M. Guerro, A. Robert, Synthesis of
alized ketone intermediates for -enol aldehydes by ring opening of
-hydroxymethyl epoxides. Tetrahedron 48 (1992) 1585e1594.
a
,
a
0-function-
-cyano
a
a
a
References
[30] I. Devillers, B. de Wergifosse, M.-P. Bruneau, B. Tinant, J.-P. Declercq,
R. Touillaux, J.-F. Rees, J. Marchand-Brynaert, Synthesis, structural character-
ization and antioxidative properties of aminopyrazine and imidazolopyrazine
[1] J.B. Bremner, J.I. Ambrus, S. Samosorn, Dual action-based approaches to
antibacterial agents. Curr. Med. Chem. 14 (2007) 1459e1477.
[2] B. Meunier, Hybrid molecules with a dual mode of action: dream or reality?
Acc. Chem. Res. 41 (2008) 69e77.
[3] M.L.N. Dubuisson, J.-F. Rees, J. Marchand-Brynaert, Discovery and validation of
a new family of antioxidants: the aminopyrazine derivatives. Mini-Rev. Med.
Chem. 4 (2004) 421e435.
[4] M. Dubuisson, J.-F. Rees, J. Marchand-Brynaert, Coelenterazine (marine
bioluminescent substrate): a source of inspiration for the discovery of novel
antioxidants. Drug Develop. Indust. Pharm. 31 (2005) 827e849.
[5] F. De Wael, P. Jeanjot, C. Moens, A. Cordi, E. Bouskela, J.-F. Rees, J. Marchand-
Brynaert, In vitro and in vivo studies of 6,8-(diaryl)imidazo[1,2-a]pyrazin-3
(7H)-ones as new antioxidants. Bioorg. Med. Chem. 17 (2009) 4336e4344.
[6] A. Burke, E. Smyth, G.A. Fitzgerald, Goodman and Gilman’s, the Pharmaco-
logical Basis of Therapeutics, eleventh ed. Mc Graw-Hill, New York, 2006, pp.
671e716.
derivatives. J. Chem. Soc. Perkin Trans. 2: Phys. Org. Chem.
7 (1999)
1481e1487.
[31] I. Devillers, G. Dive, C. De Tollenaere, B. Falmagne, B. de Wergifosse, J.-F. Rees,
J. Marchand-Brynaert, Imidazolopyrazinones as potential antioxidants. Bioorg.
Med. Chem. Lett. 11 (2001) 2305e2309.
[32] P. Jeanjot, F. Bruyneel, A. Arrault, S. Gharbi, J.-F. Cavalier, A. Abels,
C. Marchand, R. Touillaux, J.-F. Rees, J. Marchand-Brynaert, N-(Alkyl)-2-
amino-1,4-pyrazine derivatives: synthesis and antioxidative properties of 3-
and 3,5-p-hydroxyphenyl-substituted compounds. Synthesis (2003)
513e522.
[33] T. Hirano, S. Nishibushi, M. Yoneda, K. Tsujimoto, M. Ohashi, Mass spectro-
metric studies on chemiluminescence of coelenterate luciferin analogs.
Tetrahedron 49 (1993) 9267e9276.
[34] S. Inouye, S. Sasaki, Imidazole-assisted catalysis of luminescence reaction in
blue fluorescent protein from the photoprotein aequorin. Biochem. Biophys.
Res. Comm. 354 (2007) 650e655.
[35] M. Burton, C. De Tollenaere, J.F. Cavalier, C. Marchand, F. Dussart, J. Marchand-
Brynaert, J.F. Rees, Synthesis, structure-activity relationship and in vitro
evaluation of coelenterazine and coelenteramine derivatives as inhibitors of
lipid peroxidation. Free Rad. Res. 37 (2003) 145e158.
[36] I. Rahman, S. Biswas, P. Kirkham, Regulation of inflammation and redox sig-
nalling by dietary polyphenols. Biochem. Pharmacol. 72 (2006) 1439e1452.
[37] E. Le Ferrec, C. Chesne, P. Artusson, D. Brayden, G. Fabre, P. Gires, F. Guillou,
M. Rousset, W. Rubas, M.-L. Scarino, In vitro models of the intestinal barrier.
ATLA 29 (2001) 649e668.
[7] D. Galanakis, A.P. Kourounakis, K.C. Tsiakitzis, C. Doulgkeris, E.A. Rekka,
A. Gavalas, C. Kravaritou, C. Charitos, P.N. Kourounakis, Synthesis and phar-
macological evaluation of amide conjugates of NSAIDs with
L-cysteine ethyl
ester, combining potent antiinflammatory and antioxidant properties with
significantly reduced gastrointestinal toxicity. Bioorg. Med. Chem. Lett. 14
(2004) 3639e3643.
[8] A.P. Kourounakis, D. Galanakis, K. Tsiakitzis, E.A. Rekka, P.N. Kourounakis,
Synthesis and pharmacological evaluation of novel derivatives of anti-
inflammatory drugs with increased antioxidant and anti-inflammatory
activities. Drug Dev. Res. 47 (1999) 9e16.

3574
F. De Wael et al. / European Journal of Medicinal Chemistry 45 (2010) 3564e3574
[38] P. Shah, V. Jogani, T. Bagchi, A. Misra, Role of Caco-2 cell monolayers in
prediction of intestinal drug absorption. Biotechnol. Prog. 22 (2006) 186e198.
[39] B. Romier, J. Van de Walle, A. During, Y. Larondelle, Y.J. Schneider, Modulation
[49] M. Seierstad, J.G. Breitenbucher, Discovery and development of fatty acid
amide hydrolase (FAAH) inhibitors. J. Med. Chem. 51 (2008) 7327e7343.
[50] S. Pillarisetti, C.W. Alexander, I. Khanna, Pain and beyond: fatty acid amides
and fatty acid amide hydrolase inhibitors in cardiovascular and metabolic
diseases. Drug Discov. Today 14 (2009) 1098e1111.
[51] M.T. Cocco, C. Congiu, V. Onnis, M. Morelli, O. Cauli, Synthesis of ibuprofen
heterocyclic amides and investigation of their analgesic and toxicological
properties. Eur. J. Med. Chem. 38 (2003) 513e518.
of signaling nuclear factor-kB activation pathway by polyphenols in human
intestinal Caco-2 cells. Br. J. Nutr. 100 (2008) 542e551.
[40] L. De Petrocellis, D. Melck, T. Bisogno, V. Di Marzo, Endocannabinoids and fatty
acid amides in cancer, inflammation and related disorders. Chem. Phys. Lipids
108 (2000) 191e207.
[41] R.G. Pertwee, Cannabinoid pharmacology: the first 66 years. Br. J. Pharmacol.
147 (2006) S163eS171.
[42] A.J. Brown, Review: novel cannabinoid receptors. Br. J. Parmacol. 152 (2007)
567e575.
[52] S. Holt, B. Paylor, L. Boldrup, K. Alajakku, S. Vandevoorde, A. Sundström, M.
T. Cocco, V. Onnis, C.J. Fowler, Inhibition of fatty acid amide hydrolase, a key
endocannabinoid metabolizing enzyme, by analogues of ibuprofen and
indomethacin. Eur. J. Pharmacol. 565 (2007) 26e36.
[43] M. Feledziak, C. Michaux, A. Urbach, G. Labar, G.G. Muccioli, D.M. Lambert,
[53] G.G. Muccioli, N. Fazio, G.K.E. Scriba, W. Poppitz, F. Cannata, J.H. Poupaert,
J. Wouters, D.M. Lambert, Substituted 2-thioxoimidazolidin-4-ones and imi-
dazolidine-2,4-diones as fatty acid amide hydrolase inhibitors templates. J.
Med. Chem. 49 (2006) 417e425.
[54] S. Vandevoorde, K.O. Jonsson, C.J. Fowler, D.M. Lambert, Modifications of the
ethanolamine head in N-palmitoylethanolamine: synthesis and evaluation of
new agents interfering with the metabolism of anandamide. J. Med. Chem. 46
(2003) 1440e1448.
J. Marchand-Brynaert,
b-Lactams derived from a carbapenem chiron are
selective inhibitors of human fatty acid amide hydrolase versus human
monoacylglycerol lipase. J. Med. Chem. 52 (2009) 7054e7068.
[44] C.J. Fowler, G. Tiger, A. Stenström, Ibuprofen inhibits rat brain deamidation of
anandamide at pharmacologically relevant concentrations. Mode of inhibition
and structureeactivity relationship. J. Pharmacol. Exper. Ther. 283 (1997)
729e734.
[45] C.J. Fowler, A. Stenström, G. Tiger, Ibuprofen inhibits the metabolism of the
endogenous cannabimimetic agent anandamide. Pharmacol. Toxicol. 80
(1997) 103e107.
[46] C.J. Fowler, U. Janson, R.M. Johnson, G. Wahlström, A. Stenström, A. Norström,
G. Tiger, Application of a thermodynamic model to the prediction of phase
separations in freeze-concentrated formulations for protein lyophilization.
Arch. Biochem. Biophys. 362 (1999) 191e196.
[47] C.J. Fowler, S. Holt, G. Tiger, Acidic nonsteroidal anti-inflammatory drugs
inhibit rat brain fatty acid amide hydrolase in a pH-dependent manner. J.
Enzym. Inhib. Med. Chem. 18 (2003) 55e58.
[48] S. Holt, J. Nilsson, R. Omeir, G. Tiger, C.J. Fowler, Effects of pH on the inhibition
of fatty acid amidohydrolase by ibuprofen. Br. J. Pharmacol. 133 (2001)
513e520.
[55] G. Labar, C. Bauvois, G.G. Muccioli, J. Wouters, D.M. Lambert, Disulfiram is an
inhibitor of human purified monoacylglycerol lipase, the enzyme regulating
2-arachidonoylglycerol signaling. ChemBioChem. 8 (2007) 1293e1297.
[56] K. Higuchi, E. Umegaki, T. Watanabe, Y. Yoda, E. Morita, M. Murano, S. Tokioka,
T. Arakawa, Present status and strategy of NSAIDs-induced small bowel injury.
J. Gastroenterol. 44 (2009) 879e888.
[57] A. Jayamanne, R. Greenwood, V.A. Mitchell, S. Aslan, D. Piomelli, C.
W. Vaughan, Action of the FAAH inhibitor URB597 in neuropathic and
inflammatory chronic pain models. Br. J. Pharmacol. 147 (2006) 281e288.
[58] X. Wang, K. Sarris, K. Kage, D. Zhang, S.P. Brown, T. Kolasa, C. Surowy, O.F. El
Kouhen, S.W. Muchmore, J.D. Bioni, A.O. Stewart, Synthesis and evaluation of
benzothiazole-based analogues as novel, potent, and selective fatty acid
amide hydrolase inhibitors. J. Med. Chem. 52 (2009) 170e180.