Development of a second generation of inhibitors of microsomal prostaglandin E synthase 1 expression bearing the γ-hydroxybutenolide scaffold

Article abstract of DOI:10.1016/j.bmc.2008.08.001

Petrosaspongiolide M (PM, 1), a marine sesterterpene metabolite bearing the γ-hydroxybutenolide scaffold and displaying a potent inhibitory activity toward PLA₂ enzyme, was selected by us as an attractive target in order to explore its mechanis

Full text of DOI:10.1016/j.bmc.2008.08.001

Bioorganic & Medicinal Chemistry 16 (2008) 9056–9064
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a second generation of inhibitors of microsomal prostaglandin
E synthase 1 expression bearing the c-hydroxybutenolide scaffold
b
b
a
Maurizio Aquino a, , Maria D. Guerrero b, , Ines Bruno a, , María C. Terencio , Miguel Paya , Raffaele Riccio
*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano, SA, Italy
b Departamento de Farmacología, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andrés Estellés s/n, 46100 Burjasot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 February 2008
Revised 31 July 2008
Accepted 1 August 2008
Available online 6 August 2008
Petrosaspongiolide M (PM, 1), a marine sesterterpene metabolite bearing the
c
-hydroxybutenolide scaf-
fold and displaying a potent inhibitory activity toward PLA2 enzyme, was selected by us as an attractive
target in order to explore its mechanism of action at molecular level. In the course of our investigations
we decided to synthetically modify the parent compound to clarify the structural determinants respon-
sible for the activity; in fact, very recently, our research group reported the synthesis and the pharmaco-
logical properties of a first collection of PM analogues generated by Ludi approach. The synthesized
compounds showed a poor or moderate activity toward PLA2 enzymes, nevertheless we discovered a
potent and selective modulator of the expression of microsomal prostaglandin E synthase 1 (mPGES-
1), an enzyme highly involved in the inflammatory response, which represents an interesting target
for the development of a new class of anti-inflammatory agents. In this paper we report the synthesis
Dedicated to the memory of Professor Luigi
Gomez-Paloma
Keywords:
c
-Hydroxybutenolide derivatives
of a further collection of nine analogues, having the same scaffold of PM, the
c-hydroxybutenolide,
sPLA2
mPGES-1, COX-2
and bearing, as side chain, more complex aromatic portions, in substitution of the sesterterpene moiety.
Their pharmacological behavior against PLA2 enzymes as well as to modulate the expression of inducible
cyclooxygenase 2 (COX-2) and mPGES-1 enzymes is also described.
Ó 2008 Published by Elsevier Ltd.
1. Introduction
an interesting selective modulation on the expression of micro-
somal prostaglandin E synthase 1 (mPGES-1), a key enzyme in-
Continuing our studies on bioactive natural compounds, in the
recent years we focused our attention on a marine metabolite,
the PM (Table 1 and 1), possessing relevant anti-inflammatory
properties due to its ability of irreversibly blocking PLA2, the en-
zyme promoting the arachidonic acid cascade.1 Its mechanism of
volved in the prostanoids production and located downstream
along the arachidonic acid cascade.3 This serendipitous discovery
can be considered of great interest as mPGES-1 represents a poten-
tial target for the development of new therapeutics useful in the
treatment of several diseases.4 Encouraged by this finding we
decided to undertake the synthesis of a further collection of unnat-
ural variants of PM, bearing complex aromatic portions linked to
the butenolide scaffold. As experienced for our previous com-
pounds, also in this case to design the new analogues, we decided
to take advantage from Ludi approach,5 a computational method
which has been proven to be fairly predictive of the affinity prop-
erties of potential ligands. Ludi was set in order to construct new
promising binders, starting from the known parent compound
PM (1) and replacing, the sesterterpene moiety of the natural prod-
action, in which the
c-hydroxybutenolide moiety is highly in-
volved, was explored in great detail through Mass Spectrometry
analysis and Molecular Modeling calculation and, finally, a 3D
model of interaction of this natural ligand with its biological target
was also proposed.2 In consideration of the prominent role played
by
c-hydroxybutenolide scaffold in the inhibition event, we
decided to synthesize small collections of analogues having the
same pharmacophore and different molecular fragments linked
on it, in substitution of sesterterpene moiety, in order to explore
some structure–activity relationships useful for the discovery of
new binders with higher affinity for the target enzyme. In our
recent paper, dealing with the synthesis of a first collection of
uct, with suitable molecular fragments; the 3-bromo c-hydroxy-
butenolide, which resulted accepted by Ludi, was selected as
scaffold, in order to simplify the synthetic approach. This choice
was also made in consideration that in our parallel findings on
PM analogues we found that the compounds lacking in the bro-
mine atom on C-3 position of the scaffold displayed almost the
same pharmacological behavior of the 3-bromo analogues.
In this paper, we describe in detail the synthesis and the phar-
macological profile of the new collection of products.
c-hydroxybutenolide
analogues,
we
showed
that
the
benzothiophene -hydroxybutenolide (Table 1, BTH) displayed
c
* Corresponding author. Tel.: +39 089 969743; fax: +39 089 969702.
E-mail address: brunoin@unisa.it (I. Bruno).
These authors contributed equally to the paper.
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.08.001

M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
9057
Table 1
Fragments suggested by Ludi and their calculated scores (L.S. = Ludi score)
S
Br
Br
Br
HO
O
HO
O
O
HO
O
O
OAc
H
O
BTH
L. S. = 830
L. S. = 960
O
L. S. = 705
O
H
O
HO
O
S
Petrosaspongiolide M (PM, 1)
Br
Br
Br
HO
O
HO
O
O
O
HO
O
O
L. S. = 820
L. S. = 874
L. S. = 850
I
2. Results and discussion
2.1. Chemistry
Br
HO
Br
Ar-B(OH)2
+
O
B(OR)2
O
2
5
4
As mentioned before, to design our collection of compounds
based on the suggestions coming from Ludi program which, start-
ing from the 3D structure of the PM-bvPLA2 complex,2 generated a
series of analogues of the parent compound (Table 1), according to
our simplification plan.
Protection
Br
A
Suzuki coupling
Ar
B
Br
MEM-O
O
O
B(OR)2
3
In particular, our intention was to leave the pharmacophore c-
8
hydroxybutenolide scaffold quite unchanged, nevertheless, in or-
der to simplify the synthetic strategy we preferred, at least, to
use the 3-bromo substituted butenolide, which allowed us to use
the commercially available mucobromic acid ( Figs. 1 and 2) as
starting material6; more incisive modifications were provided for
the molecular recognition domain of PM, in fact the sesterterpene
skeleton was decided to be replaced by several molecular frag-
ments selected from the database connected to the software. From
a structural point of view, the new generation of compounds, re-
spect to the former ones recently reported,3 possesses more com-
plex aromatic fragments linked to the hydroxybutenolide
scaffold, and that required a change of the synthetic approach
adopted before. Based on the retro synthetic analysis (Fig. 1) and
considering the two C–C disconnections indicated with broken
lines, we decided to adopt a convergent synthetic approach (Fig.
2), in which, we first assembled separately the two aromatic build-
ing blocks 4 and 5, through an organometallic catalyzed Suzuki
cross coupling reactions,7 and then we connected the generated
Suzuki
coupling
C
Ar
Ar
Br
Br
Deprotection
D
HO
O
MEM-O
O
O
7
O
6
Figure 2. Synthetic strategy adopted for the synthesis of PM analogues.
aromatic portion 8 to the Mem protected8 mucobromic acid 3,
through a second Suzuki reaction. Thus, a final deprotection step
afforded our products.
According to this scheme, to search for the best experimental
conditions, we utilized, as pilot reaction, the coupling of benzothy-
R
Pd(PPh3)4, Na2CO3
H2O, PhMe, 80 ºC
O
I
+
R-B(OH)2
B
O
B
Ar-B(OH)2
Suzuki
O
11-15
O
+
Ar
9
3-iodo
coupling
I
11a-15a
Suzuki
10 4-iodo
Br
HO
Br
coupling
Br
B(OR)2
n-Bu
O
O
O
S
+
HO
O
O
R
X
Br
Mucobromic acid
2
PM analogue
11
12
14
15
RO
O
13
O
Figure 1. Retrosynthetic scheme.
Scheme 1. Parallel synthesis of the aromatic building blocks.

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M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
ophene boronic acid 11 with the 3-iodophenyl boronic ester (9,
Scheme 1), obtained from the related commercial acid,9 in order
to decrease its reactivity as nucleophile and to force the reaction
toward the chemoselective formation of adduct 11a.
From these preliminary trials, we had the opportunity to refine
the conditions of the reaction (Table 2, entry 1) and, once we found
the more efficient synthetic procedure, we applied it for the reac-
tion between the selected boronic acids and the two aromatic pina-
colic esters 9 and 10; hence a parallel protocol was followed, by
using the ASW1000 Chemspeed apparatus, to generate a small col-
lection of organoboronic esters (Table 2).
of the crude solution obtained from the previous step containing a
mixture of the unreacted boronic acid and the bis aromatic adduct.
In any case we decided to select these by-products for pharma-
cological screening in order to gain more detail about structural
requirements essential for the inhibition of the expression of
mPGES-1.
2.2. Biology
Enzymatic inhibition of sPLA2, mainly type IIA sPLA2, is a phar-
macological approach that can modulate the availability of arachi-
donic acid and consequently the production of PGE2.12 The enzyme
concentration in serum tissues is correlated with diverse severity
The next stage consisted in the Suzuki coupling between these
complex aromatic portions (11a–15a) and the Mem protected 3-
bromo-c-hydroxybutenolide scaffold (3), obtaining the coupling
in several inflammatory pathologies such rheumatoid arthritis13
septic shock14, and psoriasis.15
,
adducts 16a–24a. Even in this case, we optimized the experimental
conditions to improve the reaction yields, and, in order to speed up
the reactions, we took advantage from microwave heating (Scheme
2).10,11 The last step, consisting in the removal of the protecting
Mem group, using AlCl3 in CH2Cl2,3 afforded our products in satis-
factory yield (16b–24b, Scheme 2).
The new
c-hydroxybutenolide derivatives have been tested at
10
lM (Table 3), under the same experimental conditions, on four
different sPLA2 belonging to the groups IA (Naja naja venom), IB
(porcine pancreatic enzyme), IIA (human synovial recombinant),
and III (bee venom enzyme).
What we found, even in this case, was the formation, together
with the desired monosubstituted c-hydroxybutenolide analogues
LY311727, a well-known inhibitor of group IIA sPLA2, was used
as a reference tool.16
(16b–20b, Scheme 2), of lower amounts of bis-substituted adducts
as major by-products. Differently from what we experienced in the
synthesis of the first collection of compounds, in this case we ob-
tained two types of bis-adducts: the symmetric ones (21b and
23b, Scheme 2) and the mixed substituted ones (22b and 24b,
Scheme 2), these last due to the use, in the second Suzuki process,
None of the c-hydroxybutenolide derivatives was able to inhibit
preferentially human synovial sPLA2, with inhibition percentages
higher than 50%, as it did with the reference inhibitor LY311727.
The effect of the c-hydroxybutenolide derivatives on PGE2 pro-
duction on mouse macrophage cell line RAW 264.7 stimulated
with LPS was determined (Table 4).
Table 2
Experimental conditions for the synthesis of the aromatic building blocks
Entry
Boronic ester
Boronic acid
Pd(PPh3)4
1 mol%
Na2CO3 acq.
2.4 equiv
Time (h)
Product (yield, %)
S
1
9 (1 equiv)
(1.2 equiv)
6
(89%)
(92%)
(93%)
S
B(OH)2
11
B(OR)2
11a
2
3
4
5
9 (1 equiv)
9 (1 equiv)
10 (1 equiv)
10 (1 equiv)
(1.2 equiv)
1 mol%
1 mol%
1 mol%
1 mol%
2.4 equiv
2.4 equiv
2.4 equiv
2.4 equiv
6.5
6.2
7
B(OH)2
12
12a
n-Bu
B(OR)2
B(OR)2
n-Bu
(1.2 equiv)
B(OH)2
13
13a
O
O
(1.2 equiv)
(87%)
14
B(OH)2
14a
B(OR)2
(1.2 equiv)
6.5
(95%)
B(OH)2
15
15a
B(OR)2

M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
9059
R
R
R
Br
MEM-O
Br
R'
Br
Pd(dppf)Cl2, TBAB
+
+
CsF, THF/H2O
200W, 120 ºC
5 min
O
MEM-O
O
MEM-O
21a-24a
O
O
3
O
O
B(OR)2
11a-15a
16a-20a
16b-20b
AlCl3
DCM
21b-24b
O-Ph
n-Bu
Ph
S
Br
Br
Br
O
Br
O
Br
HO
O
HO
O
HO
O
O
HO
HO
O
O
O
20b
O
17b
18b
19b
16b
O-Ph
O-Ph
n-Bu
n-Bu
S
S
HO
O
O
HO
O
HO
O
HO
O
O
22b
O
23b
O
24b
21b
Scheme 2. Microwave-assisted synthesis of PM analogues.
Table 3
molar range. On the other hand, all the derivatives except 20b,
which was discarded, were devoid of significant cytotoxic effects
on RAW 264.7 at concentrations up to 10 lM, as assessed by the
Inhibitory activity of the c-hydroxybutenolide derivatives at 10 lM on different sPLA2
belonging to the groups IIA, IA (Naja naja venom), IB (porcine pancreatic enzyme), and
III (bee venom enzyme)
mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan (Table 4).
Western blot analysis for COX-2 and mPGES-1 proteins using
18 h LPS-stimulated RAW 264.7 cells (Fig. 3) shows clearly that
compounds 17b, 18b, 21b, 22b, and 24b inhibit dose-dependently
mPGES-1 expression, without any effect on COX-2 expression,
whereas dexamethasone, as expected, reduced the expression of
both inducible proteins.
Compound
(10 lM)
GroupIIA-
sPLA2 % I
GroupIA-
sPLA2 % I
GroupIB-
sPLA2 % I
GroupIII-
sPLA2 % I
16b
17b
18b
19b
20b
21b
22b
23b
24b
LY
30.0 5.2**
12.1 2.6
0.0 0.0
2.4 1.5
0.0 0.0
2.4 1.1
4.4 2.6
10.6 2.2
14.8 1.0
96.3 1.7**
11.7 3.4
0.0 0.0
0.0 0.0
5.8 5.3
0.0 0.0
3.6 2.2
2.6 1.8
16.6 2.9
2.0 1.3
7.9 5.6
28.6 4.3**
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
8.3 0.9
0.0 0.0
10.2 7.9
0.0 0.0
36.9 11.0**
13.9 4.2
0.0 0.0
3.5 1.8
5.3 3.0
0.0 0.0
4.0 1.8
2.8 1.3
6.0 4.5
2.8 1.9
2.4 1.8
The three enzymes mainly involved in the biosynthesis of PGE2
are PLA2, COX-2, and mPGES-1.17 Due to the great interest evoked
by mPGES-1 enzyme, as an attractive target for the development of
a new class of anti-inflammatory drugs, these c-hydroxybutenolide
Results show means SEM (n = 6). Statistical significances: **p < 0.01, with respect
to the corresponding enzyme control group (IIA sPLA2 = 12129 384 cpm; IA
sPLA2 = 10973 350 cpm; IB sPLA2 = 8008 47 cpm; III sPLA2 = 14854 1054 cpm).
Enzyme control group contains the vehicle (ethanol 1%).
derivativesby inhibitingPGE2 productionthroughtheselectiveinhi-
bition of mPGES-1 expression, can be considered interesting candi-
dates. The obtained biological results, however, do not allow us yet
to draw a clear structure–activity relationship, in any case we can
make few observations, waiting to gather more data which can illu-
minate the structural aspects involved in the biological behavior. For
example, altogether, the present compounds, except the case of
compound 16b, show an increased activity compared to the former
collection of analogues,3 probably depending on the presence of
more bulky aromatic substituents on the C-4 position of the scaffold;
this hypothesis seems to be strengthened by the unexpected obser-
vation that the reaction by-products bis-substituted hydroxybute-
nolides (21b and 24b), possessing a further bulky appendage on
the scaffold, displayed an interesting activity. Notably the 23b ana-
logue, belonging to the same series of products, is completely inac-
tive; probably the presence of an oxygen atom in the side chain is
detrimental for the activity, as, also the related monosubstituted
compound, 19b, proved to be totally ineffective. In any case, to put
these results under the right perspective, we have to consider them
as a useful basis for the structural optimization process and, further-
more, this selective pharmacological profile could be of great inter-
est to discover new promising anti-inflammatory agents, as well as
to discern the role of mPGES-1 in a great variety of inflammatory
pathologies.
Table 4
Inhibitory activity and cytotoxic effect of the
c-hydroxybutenolide derivatives at
10 lM on the production of PGE2 in LPS-stimulated RAW 264.7 cells
Compound (10
lM)
% Inhibition
IC50
n.d.
3.61 (2.3–6.8)
2.24 (1.2–3.4)
n.d.
(
l
M)
% Toxicity
16b
17b
18b
19b
20b
21b
22b
23b
24b
BTH
33.6 13.7
73.1 4.1**
62.2 4.3**
51.0 14.0**
66.8 4.7**
69.2 8.4**
58.7 6.9**
22.2 10.3
79.6 4.7**
72.2 5.7**
0.0
1.2
5.5
0.0
n.d.
18.6**
4.1
3.23 (1.0–6.7)
4.59 (3.5–6.4)
n.d.
0.84 (0.4–1.4)
1.80 (0.5–3.8)
0.0
0.0
8.6
0.0
Results show means SEM (n = 6). Statistical significances: **p < 0.01, with respect
to the LPS-stimulated control group (contains the vehicle ethanol 1%). PGE2 (non-
stimulated cells = 0.6 0.2 ng/ml; LPS-stimulated cells = 16.0 1.6 ng/ml). n.d., not
determined.
After 18 h stimulation, compounds 17b, 18b, 20b, 21b, 22b, and
24b were able to inhibit PGE2 production with a percentage of inhi-
bition higher than 50% at 10 lM, showing IC50 values in the micro-

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M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
LPS
17b
18b
21b
Dx
B
C
1µM
10µM 1µM 10µM
1µM 10µM
10µM
mPGES-1
48.7 100
71.8
52
89.7
42
43.2 35.6
54
β-actin
LPS
22b
24b
Dx
B
C
1µM 10µM 1µM 10µM
10µM
mPGES-1
42
100 100
26.7 82.2 26.7
22.2
β-actin
LPS
10 µM
B
C
17b 18b 21b 22b 24b Dx
COX-2
16.8
100 101.1 114.7 113.7 112.6 115.8 73.7
β-actin
Figure 3. Effect of c-hydroxybutenolides on COX-2 and mPGES-1 expression in LPS-stimulated RAW 264.7 cells. The figure is representative of two similar experiments. B:
normal cells. C: LPS-stimulated cells. Dx, dexamethasone.
or cerium sulfate and under UV (k = 254, 365 nm). Flash column
chromatography was performed using Merck 60/230-400 mesh sil-
ica gel. Analytical and semipreparative reverse phase HPLC purifi-
cations were performed on a Waters instrument using Jupiter C-18
3. Experimental
3.1. Ludi design
column (250 ꢀ 4.60 mm, 5
l
m, 300 Å; 250 ꢀ 10.00 mm, 10
lm,
The Ludi module of Insight II (Accelrys, San Diego, CA, USA) was
used for the in silico design. Computation was performed on a Sil-
icon Graphics Indigo 2 workstation equipped with a R10000 pro-
cessor. The 3D complex structure bvPLA2 (1POC)-PM4 was
imported into the graphic modeling program Insight II; the tetracy-
clic portion of PM was deleted from the active site of the above-
mentioned adduct, whereas the tridimensional coordinates of the
300 Å, respectively). Purity grade of final products was determined
on a Agilent 1100 HPLC using two different analytical reverse
phase columns (Method A: Jupiter C-18, 250 ꢀ 4.60 mm, 5
lm,
300 Å; Method B: Jupiter C-4, 250 ꢀ 4.60 mm, 5 m, 300 Å). Paral-
l
lel reactions were performed on Chemspeed Automated Synthesis
Workstation ASW1000. Reaction yields refer to chromatographi-
cally and spectroscopically pure products. Proton detected (1H,
HMBC, HSQC) and carbon detected NMR spectra were recorded
on Bruker instruments of Avance series operating at 300, 400,
and 600 MHz and 75, 100, and 150 MHz, respectively. Chemical
shifts are expressed in parts per million (ppm) on the delta (d)
scale. In 1H NMR chemical shift description, Roman numbers in
superscript (I, II, and III) are referred to the aromatic rings (first,
second, third) linked in sequence to the scaffold; when letters
are present, A protons belong to aromatic rings in C-4 position
and B protons in C3 position. The solvent peak was used as internal
reference: for 1H NMR CDCl3 = 7.26 ppm; for 13C NMR:
CDCl3 = 77.0 ppm. Multiplicities are reported as follows: s, singlet;
d, doublet; t, triplet; q, quartet; quint, quintet; sext, sextet; m, mul-
tiplet; dd, doublet of doublets; dt, doublet of triplets; b, broad.
High resolution mass spectra (HRMS) were recorded on a Q/TOF
Premier WATERS (Milford, MA, USA) mass spectrometer using an
electrospray ion source (ESI-MS).
c-hydroxybutenolide ring were kept unaltered. On this dataset,
Ludi performed a database screening on the User_link_library (pro-
vided by Accelerys version 1998 and 2000.2) to select appropriate
aromatic and heteroaromatic fragments to replace the sesterter-
pene skeleton of PM by linking the
c-hydroxybutenolide ring.
The value of the maximum RMS deviation was fixed at 0.4 Å, the
lipo weight was set at 10, the H bond weight was set at 1, and
the value of the minimum separation was definitively 3.00. The
other parameters were used as standard default. For each frag-
ment, the Ludi score was calculated by means of the scoring func-
tion mentioned as energy estimate_3.
3.2. General methods
All water and air sensitive reactions were carried out under an
inert atmosphere (N2) in oven- or flame-dried glassware. Ethyl ace-
tate, methylene chloride, and toluene were distilled from CaH2
immediately prior to use. Water was degassed under vacuum
(10 mbar). All reagents were used from commercial sources with-
out any further purification. Organic extracts were dried over
anhydrous Na2SO4. Reactions were monitored on silica gel 60
F254 (Merck) plates and visualized with potassium permanganate
3.2.1. 3,4-Dibromo-5-(2-methoxy-ethoxymethoxy)-5H-furan-2-
one (3)
To a solution of mucobromic acid (2) (100 mg, 0.387 mmol) in
10 mL of dry dichloromethane, MEM-Cl (66 ll, 0.581 mmol) was

M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
9061
added. Diisopropylethylamine (101
l
l, 0.581 mmol) was added
The mixture was irradiated for 5 min, setting the power to
200 W, the temperature to 120 °C, the pressure to 250 psi and
the Power Max ON. After diluting (10 mL) with DCM, 10 mL of an
aqueous solution of HCl 1 N was added. The aqueous layer was ex-
tracted with DCM (3ꢀ 15 mL). The organics were then dried over
Na2SO4, filtered, and concentrated. The crude was purified by flash
chromatography (9:1 hexane/diethyl ether to 6:4 hexane/diethyl
ether).
dropwise over a period of 15 min. After 4 h, the reaction mixture
was quenched with 20 mL of HCl 1 M. The aqueous layer was ex-
tracted with dichloromethane (2 ꢀ 30 mL) and the organics were
dried, filtered, and concentrated in vacuo to leave dark oil. The
crude oil was purified by flash chromatography (10% diethyl
ether/hexane) to give
3 d
(115 mg, 86% yield): 1H NMR
(300 MHz; CDCl3): d 6.10 (1H, s, H-5), 5.20 (1H, d, J = 7 Hz, OCHHO),
4.87 (1H, d, J = 7 Hz, OCHHO), 3.79 (1H, m, OCHHCH2O), 3.40 (3H, s,
OCH3), 3.60 (1H, m, OCHHCH2O), 3.54 (2H, dd, OCH2CH2O); 13C
NMR d (75 MHz; CDCl3): d 167.4, 144.2, 119.1, 100.4, 95.2, 72.1,
69.0, 59.8; HRMS calcd for C8H11Br2O5: [M+H]+ 344.8973,
346.8953, 348.8932 (ratio 1:2:1); found [M+H]+ 344.8942,
346.8925, 348.8918 (ratio 1:2:1).
3.2.7. 4-(3-Benzo[b]thiophen-3-yl-phenyl)-3-bromo-5-(2-
methoxy-ethoxymethoxy)-5H-furan-2-one (16a)
Yield: 68%; 1H NMR d (300 MHz; CDCl3): 8.07 (1H, s, H-2I), 7.93
(2H, d, J = 6.9 Hz, H-8II, H-4I), 7.87 (1H, m, H-5II), 7.75 (1H, d, H-6I),
7.64 (1H, t, H-5I), 7.48 (1H, s, H-2II), 7.41 (2H, m, H-6II, H-7II), 6.64
(1H, s, H-5), 5.17 (1H, d, J = 7 Hz, OCHHO), 4.85 (1H, d, J = 7 Hz, OCH-
HO), 3.73–3.64 (2H, m, OCH2CH2O), 3.60–3.52 (2H, m, OCH2CH2O),
3.23 (3H, s, OCH3); HRMS calcd for C22H20BrO5S: [M+H]+ 475.0215,
477.0194 (ratio 1:1); found 475.0208, 477.0190 (ratio 1:1).
3.2.2. General procedure for the esterification of the boronic
acids
To a solution of 3- or 4-iodophenylboronic acid (100 mg,
0.403 mmol) in 6 mL of dry ethyl acetate, 2,3-dimethylbutane-
2,3-diol (47.5 mg, 0.403 mmol) was added, upon stirring and under
argon. The mixture was then stirred at rt for 4 h. After dilution with
ethyl acetate were added 1 g of Na2SO4 and 1 g of CaCl2 in order to
remove water. The mixture was finally filtered and concentrated
on the rotavapor. The crude did not require any further
purification.
3.2.8. 3-Bromo-5-(2-methoxy-ethoxymethoxy)-4-
[1,10;40,100]terphenyl-4-yl-5H-furan-2-one (17a)
Yield: 81%; 1H NMR d (300 MHz; CDCl3): 8.03 (2H, d, J = 8.4 Hz,
H-2I, H-6I), 7.79 (2H, d, J = 8.4 Hz, H-3I, H-5I), 7.70–7.64 (4H, m, H-
2II, H-3II, H-5II H-6II), 7.51–7.46 (4H, m, H-2III, H-3III, H-5III H-6III),
7.39 (1H, t, H-4III), 6.63 (1H, s, H-5), 5.21 (1H, d, J = 7 Hz, OCHHO),
4.91 (1H, d, J = 7 Hz, OCHHO), 3.89–3.84 (1H, m, OCHHCH2O), 3.72–
3.66 (1H, m, OCHHCH2O), 3.59 (2H, t, J = 8.4 Hz, OCH2CH2O), 3.43
(3H, s, OCH3); HRMS calcd for C26H24BrO5: [M+H]+ 495.0807,
497.0787 (ratio 1:1); found 495.0817, 497.0793 (ratio 1:1).
3.2.3. 2-(4-Iodo-phenyl)-4,4,5,5-tetramethyl-
[1,3,2]dioxaborolane (9)
Yield: 90%; 1H NMR d (300 MHz; CDCl3): 7.72 (2H, d, J = 8.2 Hz,
H2-3), 7.51 (2H, d, J = 8.2 Hz, H2-2), 1.33 (12H, s, (CH3)4); 13C NMR d
(75 MHz; CDCl3): 137.4, 130.2, 127.1, 97.0, 88.4, 21.6; HRMS calcd
for C12H17BIO2: [M+H]+ 331.0361; found 331.0355.
3.2.9. 3-Bromo-4-(40-butyl-biphenyl-4-yl)-5-(2-methoxy-
ethoxymethoxy)-5H-furan-2-one (18a)
Yield: 72%; 1H NMR d (300 MHz; CDCl3): 7.95 (2H, d, J = 8.7 Hz,
H-2I, H-6I), 7.70 (2H, d, J = 8.7 Hz, H-3I, H-5I), 7.48 (2H, d, J = 8.4 Hz,
H-2II, H-6II), 7.26 (2H, d, J = 8.4 Hz, H-3II, H-5II), 6.61 (1H, s, H-5),
5.19 (1H, d, J = 7 Hz, OCHHO), 4.90 (1H, d, J = 7 Hz, OCHHO),
3.86–3.78 (1H, m, OCHHCH2O), 3.70–3.62 (1H, m, OCHHCH2O),
3.57 (2H, t, OCH2CH2O), 3.41 (3H, s, OCH3), 2.64 (2H, t, J = 7.8 Hz,
PhCH2), 1.58 (2H, quint., J1 = 7.5 Hz, J3 = 15.4 Hz, PhCH2CH2), 1.31
(2H, sest., J1 = 7.14 Hz and J3 = 14.5 Hz, CH2CH3), 0.92 (3H, t,
J = 7.6 Hz, CH3); HRMS calcd for C24H28BrO5: [M+H]+ 475.1120,
477.1100 (ratio 1:1); found 475.1114, 477.1108 (ratio 1:1).
3.2.4. 2-(3-Iodo-phenyl)-4,4,5,5-tetramethyl-
[1,3,2]dioxaborolane (10)
Yield: 97%; 1H NMR d (300 MHz; CDCl3): 8.16 (1H, s, H-2), 7.76
(2H, d, J = 6.3 Hz, H-4, H-6), 7.10 (1H, t, J = 7.6 Hz, H-5); 13C NMR d
(75 MHz; CDCl3): 137.4, 130.2, 127.1, 97.0, 88.4, 21.6; HRMS calcd
for C12H17BIO2: [M+H]+ 331.0361; found 331.0358.
3.2.5. General procedure for the ChemspeedÒ assisted coupling
of the aromatic building blocks
All the automatic sequences were programmed through the
ChemspeedÒ software. The boronic acids (1.2 equiv, see Table 2)
were manually introduced, under argon, in five reactors (12 mL
vol). The stock solutions were then prepared : 600 mg (1.82 mmol)
of 4-iodophenylboronic ester in 6 mL of toluene; 400 mg
(1.21 mmol) of 3-iodophenylboronic ester in 4 mL of toluene;
35 mg (0.052 mmol) of Pd(PPh3)4 in 5 mL of toluene; 10 mL of an
aqueous solution 0.73 M of Na2CO3 (Table 2). All the stock solu-
tions were then sequentially added, in equal proportions, to the
six reactors respecting the order presented in Table. 2. The reactors
were then stirred at 900 rpm setting the temperature to 80 °C.
Every 2 h TLC controls collecting small aliquots of the mixtures
were programmed. Time reactions varied in function of the sub-
strates (Table. 2). The workup was automatically performed adding
4 mL of an aqueous solution of HCl 1 N then extracting with tolu-
ene (2ꢀ 4 mL). The organics were then dried over Na2SO4, filtered
and concentrated. The crudes were directly used in the next step.
3.2.10. 3-Bromo-5-(2-methoxy-ethoxymethoxy)-4-(40-
phenoxy-biphenyl-3-yl)-5H-furan-2-one (19a)
Yield: 74%; 1H NMR d (600 MHz; CDCl3): 8.14 (1H, s, H-2I), 7.82
(1H, d, J = 7.8 Hz, H-4I), 7.69 (1H, d, J = 7.8 Hz, H-6I), 7.55 (1H, t,
J = 7.8 Hz, H-5I), 7.53 (2H, d, J = 8.4 Hz, H-2II, H-6II), 7.34 (2H, t,
J = 7.8 Hz, H-3III, H-5 III), 7.11 (1H, t, J = 7.8 Hz, H-4III), 7.08 (2H, d,
J = 8.4 Hz, H-3II, H-5II), 7.06 (2H, d, J = 7.8 Hz, H-2 III, H-6III), 6.58
(1H, s, H-5), 5.15 (1H, d, J = 7 Hz, OCHHO), 4.86 (1H, d, J = 7 Hz, OCH-
HO), 3.70–3.62 (2H, m, OCH2CH2O), 3.56–3.48 (2H, t, OCH2CH2O),
3.25 (3H, s, OCH3); HRMS calcd for C26H24BrO6: [M+H]+ 511.0756,
513.0736 (ratio 1:1); found 511.0746, 513.0730 (ratio 1:1).
3.2.11. 3-Bromo-5-(2-methoxy-ethoxymethoxy)-4-(3-
naphthalen-1-yl-phenyl)-5H-furan-2-one (20a)
Yield: 66%; 1H NMR d (300 MHz; CDCl3): 8.09 (1H, s, H-2I), 8.05
(1H, s, H-1II), 7.78 (1H, d, H-4I), 7.75 (1H, d, H-3II), 7.69 (1H, d, H-6I),
7.50 (1H, d, H-4II), 7.49–7.46 (2H, m, H-7II, H-8II), 7.44 (1H, t, H-5I),
7.41–7.40 (1H, m, H-6II), 7.38–7.36 (1H, m, H-9II), 6.75 (1H, s, H-5),
5.29 (1H, d, J = 7 Hz, OCHHO), 4.94 (1H, d, J = 7 Hz, OCHHO), 3.83–
3.71 (1H, m, OCHHCH2O), 3.67–3.55 (1H, m, OCHHCH2O), 3.41 (2H,
m, OCH2CH2O), 3.25 (3H, s, OCH3); HRMS calcd for C24H22BrO5:
[M+H]+ 469.0651, 471.0630 (ratio 1:1); found 469.0645,
471.0641 (ratio 1:1).
3.2.6. Microwave-assisted Suzuki coupling: general procedure
In a CEM Discover vial were placed 3,4-dibromo-5-(2-methoxy-
ethoxymethoxy)-5H-furan-2-one (1 equiv), the boronic ester
(1.5 equiv), Pd(dppf)Cl2 (0.03 equiv), TBAB (1 equiv), and CsF
(4 equiv). All the reactions were performed on 100 mg scale of fur-
an-2-one. Under Ar were added water (500 ll) and THF (500 ll).

9062
M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
3.2.12. 3,4-Bis-(3-benzo[b]thiophen-3-yl-phenyl)-5-(2-
methoxy-ethoxymethoxy)-5H-furan-2-one (21a)
3.2.18. 3-Bromo-5-hydroxy-4-[1,10;40,100]terphenyl-4-yl-5H-
furan-2-one (17b)
Yield: 20%; 1H NMR d (300 MHz; CDCl3): 7.87 (2H, dd, H-5IIA, H-
5IIB), 7.68–7.64 (6H, m, H-4IA, H-5IA, H-2IIA, H-6IIA, H-7IIA, H-8IIA),
7.62–7.53 (6H, m, H-4IB, H-5IB, H-2IIB, H-6IIB, H-7IIB, H-8IIB), 7.37–
7.29 (4H, m, H-6IA, 6IB, H-2IA, H-2IB), 6.64 (1H, s, H-5), 5.24 (1H, d,
J = 7 Hz, OCHHO), 4.84 (1H, d, J = 7 Hz, OCHHO), 3.73–3.50 (2H, m,
OCH2CH2O), 3.33 (2H, t, OCH2CH2O), 3.25 (3H, s, OCH3); HRMS
calcd for C36H29O5S2: [M+H]+ 605.1456; found 605.1464.
Yield: 77%; 1H NMR d (600 MHz; CDCl3): 8.14 (2H, d, J = 8.4 Hz,
H-2I, H-6I), 7.82 (2H, d, J = 8.4 Hz, H-3I, H-5I), 7.67–7.64 (4H, m, H-
2II, H-3II, H-5II H-6II), 7.50–7.48 (4H, m, H-2III, H-3III, H-5III H-6III),
7.40 (1H, t, H-4III), 6.59 (1H, s, H-5); 13C NMR d (150 MHz; CDCl3):
167.5, 155.2, 145.3, 145.0, 142.5, 141.8, 139.7, 130.6, 130.5, 130.2,
130.0, 129.7, 129.4, 129.3, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4,
111.6, 98.9; HRMS calcd for
C
22H15BrO3: [M+H]+ 407.0283,
409.0262 (ratio 1:1); found 407.0266, 409.0250 (ratio 1:1).
3.2.13. 4-(40-Butyl-biphenyl-4-yl)-3-(4-butyl-phenyl)-5-(2-
methoxy-ethoxymethoxy)-5H-furan-2-one (22a)
3.2.19. 3-Bromo-4-(40-butyl-biphenyl-4-yl)-5-hydroxy-5H-
furan-2-one (18b)
Yield: 16%; 1H NMR d (300 MHz; CDCl3): 7.59–7.56 (4H, m, H-
2IA, H-3IA, H-5IA, H-6IA), 7.54–7.50 (4H, m, H-2IIA, H-3IIA, H-5IIA,
H-6IIA), 7.27–7.25 (4H, dd, H-2IB, H-6IB), 6.60 (1H, s, H-5), 5.25
(1H, d, J = 7 Hz, OCHHO), 4.93 (1H, d, J = 7 Hz, OCHHO), 3.85–3.78
(1H, m, OCHHCH2O), 3.72–3.65 (1H, m, OCHHCH2O), 3.58 (2H, t,
OCH2CH2O), 3.42 (3H, s, OCH3), 2.65 (4H, m, (PhCH2)2), 1.63 (4H,
m, (PhCH2CH2)2), 1.38 (4H, m, (CH2CH3)2), 0.94 (6H, m, (CH2CH3)2);
HRMS calcd for C30H41O5: [M+H]+ 529.2954; found 529.2948.
Yield: 70%; 1H NMR d (300 MHz; CDCl3): 8.09 (2H, d, J = 8.7 Hz,
H-2I, H-6I), 7.74 (2H, d, J = 8.7 Hz, H-3I, H-5I), 7.56 (2H, d, J = 8.4 Hz,
H-2II, H-6II), 7.30 (2H, d, J = 8.4 Hz, H-3II, H-5II), 6.54 (1H, s, H-5),
2.64 (2H, t, J = 7.8 Hz, PhCH2), 1.58 (2H, quint., J1 = 7.5 Hz,
J3 = 15.4 Hz, PhCH2CH2), 1.31 (2H, sest., J1 = 7.14 Hz and
J3 = 14.5 Hz, CH2CH3), 0.92 (3H, t, J = 7.6 Hz, CH2CH3); 13C NMR d
(75 MHz; CDCl3): 167.5, 154.7, 143.3, 143.1, 136.6, 136.4, 129.2,
129.0, 128.3, 128.0, 127.3, 127.1, 126.9, 126.2, 109.7, 98.1, 37.6,
33.4, 22.5, 14.0; HRMS calcd for C20H19BrO3: [M+H]+ 387.0518,
389.0497 (ratio 1:1); found 387.0528, 387.0485 (ratio 1:1).
3.2.14. 5-(2-Methoxy-ethoxymethoxy)-3,4-bis-(40-phenoxy-
biphenyl-3-yl)-5H-furan-2-one (23a)
Yield: 10%; 1H NMR d (600 MHz; CDCl3): 7.69 (2H, d, J = 7.8 Hz,
H-4IA, H-4IB), 7.58 (2H, d, J = 7.8 Hz, H-6IA, H-6IB), 7.44–7.42 (3H, d,
J = 8.4 Hz, H-2IIB, H-5IB, H-6IIB), 7.33–7.30 (6H, m, H-2IA, H-3IIIA, H-
5IIIA, H-2IB, H-3IIIB, H-5IIIB), 7.12 (2H, dt, H-4IIIA, H-4IIIB), 6.98 (4H,
d, H-3IIA, H-5IIA, H-3IIB, H-5IIB), 6.86 (4H, d, H-2IIIA, H-6IIIA, H-2IIIB,
H-6IIIB), 6.65 (1H, s, H-5), 5.12 (1H, d, J = 7 Hz, OCHHO), 4.80 (1H, d,
J = 7 Hz, OCHHO), 3.80–3.70 (2H, m, OCH2CH2O), 3.50–3.42 (2H, t,
OCH2CH2O), 3.25 (3H, s, OCH3); HRMS calcd for C44H37O7: [M+H]+
677.2534; found 677.2528.
3.2.20. 3-Bromo-5-hydroxy-4-(40-phenoxy-biphenyl-3-yl)-5H-
furan-2-one (19b)
Yield: 93%; 1H NMR d (600 MHz; CDCl3): 8.21 (1H, s, H-2I), 7.93
(1H, d, J = 7.8 Hz, H-4I), 7.75 (1H, d, J = 7.8 Hz, H-6I), 7.60 (1H, t,
J = 7.8 Hz, H-5I), 7.57 (2H, d, J = 8.4 Hz, H-2II, H-6II), 7.39 (2H, t,
J = 7.8 Hz, H-3III, H-5III), 7.16 (1H, t, J = 7.8 Hz, H-4III), 7.12 (2H, d,
J = 8.4 Hz, H-3II, H-5II), 7.09 (2H, d, J = 7.8 Hz, H-2III, H-6III), 6.58
(1H, s, H-5) 13C NMR d (150 MHz; CDCl3): 166.6, 157.8, 157.0,
155.2, 141.8, 135.2, 134.6, 134.3, 130.4, 130.2, 129.7, 129.6,
129.4, 129.3, 128.7, 127.8, 127.3, 123.9, 119.5, 119.3, 111.3, 98.1;
HRMS calcd for C22H16BrO4: [M+H]+ 423.0232, 425.0211 (ratio
1:1); found 423.0224, 425.0204 (ratio 1:1).
3.2.15. 5-(2-Methoxy-ethoxymethoxy)-3-naphthalen-2-yl-4-(3-
naphthalen-2-yl-phenyl)-5H-furan-2-one (24a)
Yield: 14%; 1H NMR d (300 MHz; CDCl3): 8.18 (1H, s, H-1IIA),
8.17 (1H, s, H-1IB), 7.90 (2H, m, H-9IIA, H-9IB), 7.87–7.83 (4H, m,
H-3IIA, H-4IIA, H-3IB H-4IB), 7.81 (1H, d, H-4IA), 7.79 (1H, d, H-
6IA), 7.72 (2H, m, H-6IIA, H-6IB), 7.67 (1H, s, H-2IA), 7.53–7.51
(4H, m, H-7IIA, H-8IIA, H-7IB H-8IB), 7.48 (1H, t, H-5IA), 6.75
(1H, s, H-5), 5.29 (1H, d, J = 7 Hz, OCHHO), 4.94 (1H, d, J = 7 Hz,
OCHHO), 3.83–3.72 (1H, m, OCHHCH2O), 3.67–3.58 (1H, m,
OCHHCH2O), 3.41 (2H, t, J = 8.4 Hz, OCH2CH2O), 3.28 (3H, s,
OCH3); HRMS calcd for C34H28O5: [M+H]+ 516.1937; found
516.1921.
3.2.21. 3-Bromo-5-(2-methoxy-ethoxymethoxy)-4-(3-
naphthalen-1-yl-phenyl)-5H-furan-2-one (20b)
Yield: 80%; 1H NMR d (300 MHz; CDCl3): 8.16 (1H, s, H-2I), 8.12
(1H, s, H-1II), 7.82 (1H, d, H-4I), 7.80 (1H, d, H-3II), 7.70 (1H, d, H-6I),
7.54 (1H, d, H-4II), 7.53–7.50 (2H, m, H-7II, H-8II), 7.51 (1H, t, H-5I),
7.43–7.42 (1H, m, H-6II), 7.41–7.39 (1H, m, H-9II), 6.68 (1H, s, H-5);
13C NMR d (75 MHz; CDCl3): 170.1, 154.7, 140.4, 137.1, 135.0,
133.5, 130.0, 129.6, 129.4, 129.3, 129.0, 128.5, 127.8, 127.1,
126.0, 125.8, 125.7, 124.9, 110.4, 97.0; HRMS calcd for C20H14BrO3:
[M+H]+ 381.0126, 383.0106 (ratio 1:1); found 381.0112, 383.0120
(ratio 1:1).
3.2.16. General procedure for MEM cleavage
To a suspension of AlCl3 (5 equiv) in dry DCM (3 mL), at 0 °C and
upon stirring, the protected hydroxybutenolide in 2 mL of dry DCM
was added dropwise. The mixture was stirred at 0 °C for 5 h. The
mixture was then washed with aqueous NaHCO3 sat. (5 mL) and
then with brine (5 mL). The aqueous layers were extracted with
DCM (3ꢀ 10 mL). The organics were then dried over Na2SO4, fil-
tered, and concentrated.
3.2.22. 3,4-Bis-(3-benzo[b]thiophen-3-yl-phenyl)-5-hydroxy-
5H-furan-2-one (21b)
Yield: 88%; 1H NMR d (300 MHz; CDCl3): 7.88 (2H, dd, H-5IIA, H-
5IIB), 7.71–7.66 (6H, m, H-4IA, H-5IA, H-2IIA, H-6IIA, H-7IIA, H-8IIA),
7.54–7.61 (6H, m, H-4IB, H-5IB, H-2IIB, H-6IIB, H-7IIB, H-8IIB), 7.35
(1H, d, H-6IA), 7.32 (1H, d, H-6IB), 7.31 (1H, s, H-2IA), 7.28 (1H, s, H-
2IB), 6.60 (1H, s, H-5); 13C NMR d (150 MHz; CDCl3): 170.1, 156.1,
141.3, 141.0, 140.6, 140.4, 138.5, 138.4, 137.0 136.9, 135.8, 135.7,
131.8, 131.6, 129.5, 129.4, 128.8, 128.7, 127.6, 127.3, 125.4, 125.0,
124.8, 124.7, 124.6, 124.5, 123.0, 122.8, 122.7, 122.2, 122.0, 96.3;
HRMS calcd for C32H22O3S2: [M+H]+ 517.0932; found 517.0922.
3.2.17. 4-(3-Benzo[b]thiophen-3-yl-phenyl)-3-bromo-5-
hydroxy-5H-furan-2-one (16b)
Yield: 74%; 1H NMR d (600 MHz; CDCl3): 8.23 (1H, s, H-2I), 8.03
(1H, d, H-4I), 7.96 (2H, dd, H-8II), 7.93 (1H, dd, H-5II), 7.78 (1H, d, H-
6I), 7.66 (1H, t, H-5I), 7.50 (1H, s, H-2II), 7.45–7.43 (2H, m, H-6II, H-
7II), 6.58 (1H, s, H-5); 13C NMR d (150 MHz; CDCl3): 166.8, 154.7,
141.1, 140.4, 138.7, 136.9, 135.7, 131.8, 129.7, 128.7, 127.6,
125.0, 124.8, 124.6, 123.2, 122.5, 111.3, 96.5; HRMS calcd for
3.2.23. 4-(40-Butyl-biphenyl-4-yl)-3-(4-butyl-phenyl)-5-
hydroxy-5H-furan-2-one (22b)
18H12BrO3S: [M+H]+ 386.9691, 388.9670 (ratio 1:1); found
386.9684, 388.9662 (ratio 1:1).
Yield: 80%. 1H NMR d (300 MHz; CDCl3): 7.59–7.56 (4H, m, H-
2IA, H-3IA, H-5IA, H-6IA), 7.54–7.50 (4H, m, H-2IIA, H-3IIA, H-5IIA,
C

M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
9063
H-6IIA), 7.27–7.25 (4H, dd, H-2IB, H-6IB), 6.60 (1H, s, H-5), 2.65
(4H, m, (PhCH2)2), 1.63 (4H, m, (PhCH2CH2)2), 1.38 (4H, m,
CH2CH3)2), 0.94 (6H, m, CH2CH3)2); 13C NMR d (75 MHz; CDCl3):
169.9, 154.5, 140.4, 137.6, 137.1, 134.5, 134.1, 133.9, 133.0,
132.7, 130.6, 129.5, 129.2, 128.6, 128.3, 128.1, 127.9, 127.8,
127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 126.5, 126.2, 126.0,
125.3, 124.8, 96.6; HRMS calcd for C30H32O3: [M+H]+ 440.2351;
found 440.2342.
2500g for 10 min at 4 °C, the radioactivity (cpm) in the superna-
tants was determined by liquid scintillation counting.
3.5. Western blot assay of COX-2 and mPGES-1
Cellular lysates from RAW 264.7 (murine macrophages,
1.5 ꢀ 106 cells/mL) incubated for 18 h with LPS (1
lg/mL) were ob-
tained with lysis buffer A [10 mM N-(2-hydroxyethyl)piperazine-
N0-ethanesulfonic acid (HEPES), pH 8.0, 1 mM ethylenediaminetet-
raacetic acid (EDTA), 1 mM ethylene glycol bis(b-aminoethyl
ether)-N,N,N0,N0-tetraacetic acid (EGTA), 10 mM KCl, 1 mM dithio-
3.2.24. 5-Hydroxy-3,4-bis-(40-phenoxy-biphenyl-3-yl)-5H-
furan-2-one (23b)
Yield: 86%; 1H NMR d (300 MHz; CDCl3): 7.71 (2H, d, J = 7.8 Hz,
H-4IA, H-4IB), 7.62 (2H, d, J = 7.8 Hz, H-6IA, H-6IB), 7.43–7.45 (3H, d,
J = 8.4 Hz, H-2IIB, H-5IB, H-6IIB), 7.34–7.36 (6H, m, H-2IA, H-3IIIA, H-
5IIIA, H-2IB, H-3IIIB, H-5IIIB), 7.17 (2H, dt, H-4IIIA, H-4IIIB), 7.00 (4H,
d, H-3IIA, H-5IIA, H-3IIB, H-5IIB), 6.92 (4H, d, H-2IIIA, H-6IIIA, H-2IIIB,
H-6IIIB), 6.60 (1H, s, H-5); 13C NMR d (75 MHz; CDCl3): 170.2, 157.0,
156.9, 156.4, 156.3, 141.6, 141.5, 135.9, 135.8, 134.8, 134.7, 134.3,
134.2, 133.7, 133.6, 132.6, 132.2, 130.8, 130.5, 129.6, 129.5, 129.3,
129.2, 128.9, 128.7, 127.8, 127.7, 127.5, 127.4, 126.9, 126.7, 126.3,
125.9, 125.6, 125.5, 124.7, 124.5, 119.6, 119.3, 96.2; HRMS calcd for
threitol, 5 mM NaF, 1 mM Na3VO4, 10 mM Na2MoO4, 1
lg/mL leu-
peptin, 0.1 g/mL aprotinin, and 0.5 mM phenylmethanesulfonyl
l
fluoride). Following centrifugation (10,000g, 15 min, 4 °C), super-
natant protein was determined by the Bradford method with BSA
as standard. COX-2 or mPGES-1 protein expression was studied
in the total fraction or microsomal fractions, respectively. Equal
amounts of protein (50 lg for both COX-2 and mPGES-1) were
loaded on SDS–15% PAGE and transferred onto poly(vinylidene
difluoride) membranes for 90 min at 125 mA. Membranes were
blocked in PBS (0.02 M, pH 7.0)-Tween 20 (0.1%), containing 3%
(w/v) nonfat milk, and incubated with specific polyclonal antibody
against COX-2 (1/1000) or mPGES-1 (1/200). Finally, membranes
were incubated with peroxidase-conjugated goat anti-rabbit IgG
(1/10,000). The immunoreactive bands were visualized using an
enhanced chemiluminescence system (Amersham Biosciences,
Barcelona, Spain).
C
40H28O5: [M+H]+ 588.1937; found 588.1921.
3.2.25. 5-Hydroxy-3-naphthalen-2-yl-4-(3-naphthalen-2-yl-
phenyl)-5H-furan-2-one (24b)
Yield: 81%; 1H NMR d (300 MHz; CDCl3): 8.18 (1H, s, H-1IIA),
8.17 (1H, s, H-1IB), 7.90 (2H, m, H-9IIA, H-9IB), 7.87–7.83 (4H, m,
H-3IIA, H-4IIA, H-3IB H-4IB), 7.81 (1H, d, H-4IA), 7.79 (1H, d, H-
6IA), 7.72 (2H, m, H-6IIA, H-6IB), 7.67 (1H, s, H-2IA), 7.53–7.51
(4H, m, H-7IIA, H-8IIA, H-7IB H-8IB), 7.48 (1H, t, H-5IA), 6.69 (1H,
s, H-5); 13C NMR d (75 MHz; CDCl3): 169.9, 154.5, 140.4, 137.6,
137.1, 134.5, 134.1, 133.9, 133.0, 132.7, 130.6, 129.5, 129.2,
128.6, 128.3, 128.1, 127.9, 127.8, 127.6, 127.5, 127.4, 127.3,
127.2, 127.1, 126.5, 126.2, 126.0, 125.3, 124.8, 96.6; HRMS calcd
for C30H20O3: [M+H]+ 428.1412; found 428.1402.
3.6. Culture of murine macrophage RAW 264.7 cell line
The mouse macrophage cell line RAW 264.7 (Cell Collection,
Department of Animal Cell Culture, C.S.I.C., Madrid, Spain) was cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM) containing
2 mM L-glutamine, 100 U/mL penicillin, 100 lg/mL streptomycin,
and 10% fetal bovine serum. Cultures were maintained at 37 °C in
5% CO2 (air/CO2 95:5) humidified incubator. Cells were resus-
pended at a concentration of 1.5 ꢀ 106 cells/mL.
3.3. Materials
[5,6,8,11,12,14,15(n)-3H] PGE2 and [9,10-3H]oleic acid were
purchased from Amersham Biosciences (Barcelona, Spain).
LY311727 was a gift from Lilly Corporate Center (Indianapolis,
IN). The rest of reagents were from Sigma (St. Louis, MO). Esche-
richia coli strain CECT 101 was a gift from Professor Uruburu,
Department of Microbiology, University of Valencia, Spain.
3.7. PGE2. production in RAW 264.7 macrophages
RAW 264.7 macrophages (1.5 ꢀ 106 cells/mL) were co-incu-
bated in 96-well culture plate (200 ll) with 1 lg/mL of E. coli
[serotype 0111:B4] lipopolysaccharide (LPS) at 37 °C for 20 h in
the presence of 2.0 l of test compound dissolved in ethanol or
2.0
l
l
l of ethanol (control group). PGE2 levels were determined in
3.4. Assay of sPLA2
culture supernatants by radioimmunoassay.20 The mitochondrial-
dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) to formazan21 was used to assess
the possible cytotoxic effects of compounds.
sPLA2 activity was assayed using [3H]oleate labeled membranes
of E. coli, following a modification of the method of Franson and
co-workers.18,19 E. coli strain CECT 101 was grown for 6–8 h at
37 °C in the presence of 5
l
Ci/mL [3H]oleic acid (specific activity
3.8. Statistical analysis
10 Ci/mmol) until the end of the logarithmic phase. After centrifu-
gation at 1800g for 10 min at 4 °C, the membranes were washed,
resuspended in phosphate-buffered saline (PBS), and autocleaved
for 30–45 min. At least 95% of the radioactivity was incorporated
into the phospholipid fraction. N. naja venom (Group IA sPLA2),
porcine pancreatic (Group IB sPLA2), human recombinant synovial
(Group IIA sPLA2), and bee venom (Group III sPLA2) enzymes were
The results are presented as means SEM; n represents the
number of experiments. Inhibitory concentration 50% (IC50) values
were calculated from at least four significant concentrations
(n = 6). The level of statistical significance was determined by anal-
ysis of variance (ANOVA) followed by Dunnett’s t-test for multiple
comparisons. Significance was assumed at a p value of 0.05 or less.
used as sources of sPLA2. Enzymes were diluted in 10
Tris–HCl, 1 mM CaCl2 buffer, pH 7.5, and preincubated at 37 °C for
5 min with 2.5 l of test compound dissolved in ethanol or 2.5 l of
ethanol (control group) to get a final volume of 250 l. Incubation
proceeded for 15 min in the presence of 20
l of [3H]oleic-E. coli
membranes and was terminated by addition of 100 l ice-cold
ll of 100 mM
Acknowledgments
l
l
l
Financial support by the University of Salerno and MIUR (Rome)
is gratefully acknowledged. M.D.G. was the recipient of a research
fellowship from FPU program (AP 20041633) of Spanish Ministerio
de Educación y Ciencia. This work was supported in part by Grant
FIS PI051659 from Spanish Instituto de Salud Carlos III.
l
l
solution of 0.25% bovine serum albumin (BSA) solution in saline
to a final concentration of 0.07% (w/v). After centrifugation at

9064
M. Aquino et al. / Bioorg. Med. Chem. 16 (2008) 9056–9064
11. Aquino, M.; Bruno, I.; Riccio, R.; Gomez-Paloma, L. Org. Lett. 2006, 8, 4831.
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