Toward the discovery of new agents able to inhibit the expression of microsomal prostaglandin e synthase-1 enzyme as promising tools in drug development

Article abstract of DOI:10.1111/j.1747-0285.2010.00984.x

In our recent studies, we focused our attention on the synthesis of several γ-hydroxybutenolides designed on the basis of petrosaspongiolide M 1 (PM) structure that has been recognized to potently inhibit the inflammatory process through the selective PLA

Full text of DOI:10.1111/j.1747-0285.2010.00984.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00984.x
Chem Biol Drug Des 2010; 76: 17–24
Research Article
Toward the Discovery of New Agents Able
to Inhibit the Expression of Microsomal
Prostaglandin E Synthase-1 Enzyme as
Promising Tools in Drug Development
1
2
In the last few years, we have been involved in the discovery of
new lead candidates suitable for the development of promising
anti-inflammatory agents. Our research work was based on a sest-
erterpene marine metabolite, petrosaspongiolide M (PM) 1, which
was proved to potently inhibit the human synovial PLA2 type IIA
enzyme, responsible for the triggering of the inflammation pathway
(1). Encouraged by its interesting biological profile, PM (Figure 1)
was selected by our research group for extensive investigations in
terms of chemical, synthetical, and biological aspects. In fact, com-
putational methods allowed us to propose molecular details of its
mode of interaction with the target enzyme (2). The high densely
functionalized scaffold, represented by the c-hydroxybutenolide moi-
ety, forced us to find efficient synthetic strategies to generate
focused collections of simplified analogs of the parent molecule
suitable for a structure–activity relationship study. Despite we did
not find for the synthetic molecules satisfying level of activity
toward our selected target, PLA2 enzyme, nevertheless we had the
chance to discover a very interesting product, the benzothiophene
c-hydroxybutenolide 2, which has been proved to be a potent and
selective modulator of mPGES-1 expression (3,4). This downstream
PG synthesizing enzyme, glutathione dependent, is of inducible type
and that is one of the reasons why it has established itself as a
novel drug target in the areas of inflammation (5,6), atherosclerosis
(7), stroke (8), and cancer (9–11). On the basis of these premises,
we decided to rely on some well-reasoned structural changes of
the basic molecule 2, in the attempt to improve its pharmacological
behavior. For this purpose, we synthesized a series of analogs clo-
sely related with the leader compound 2, whose biological activity
was also investigated (Figure 2).
´
Rosa De Simone , Rosa M. Andres ,
Maurizio Aquino1, Ines Bruno1,*, Marı´a D.
Guerrero2, Marı´a C. Terencio2, Miguel
Paya2 and Raffaele Riccio1
1Dipartimento di Scienze Farmaceutiche, Universitꢀ degli Studi di
Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA) Italy
2Departamento de Farmacologꢁa, Facultad de Farmacia, Universidad
de Valencia, Av. Vicente Andrꢂs Estellꢂs s ⁄ n, 46100 Burjasot,
Valencia, Spain
*Corresponding author: Ines Bruno, brunoin@unisa.it
In our recent studies, we focused our attention on
the synthesis of several c-hydroxybutenolides
designed on the basis of petrosaspongiolide M 1
(PM) structure that has been recognized to potently
inhibit the inflammatory process through the selec-
tive PLA2 enzyme inhibition. By means of a combina-
tion of computational methods and efficient
synthetic strategies, we generated small collections
of PM modified analogs to identify new potent PLA2
inhibitors, suitable for clinical development. In the
course of the biological screening of our compounds,
we discovered a potent and selective inhibitor of
mPGES-1 expression, the benzothiophene c-hydroxy-
butenolide 2, which so far represents the only prod-
uct, together with resveratrol, able to reduce PGE2
production through the selective downregulation of
mPGES-1 enzyme. In consideration that microsomal
prostaglandin E synthase 1 (mPGES-1) is one of the
most strategic target involved both in inflammation
and in carcinogenesis processes, we decided to
explore the biological effects of some structural
changes of the c-hydroxybutenolide 2, hoping to
improve its biological profile. This optimization pro-
cess led to the identification of three strictly corre-
lated compounds 14g, 16g, and 18 with higher
inhibitory potency on PGE2 production on mouse
macrophage cell line RAW264.7 through the selec-
tive modulation of mPGES-1 enzyme expression.
Methods and Materials
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, dichloromethane (DCM) and tetrahydrofuran (THF) were dis-
tilled from CaH2 immediately prior to use. Water was degassed
under vacuum (10 mbar). All reagents were used from commercial
sources without any further purification. Organic extracts were dried
over anhydrous Na2SO4. Reactions were monitored on silica gel
60 F254 (Merck) plates and visualized with potassium permanga-
nate, cerium sulfate or ninhydrin and under UV (k = 254 nm,
Key words: COX-2, mPGES-1, PGE2, c-Hydroxybutenolide derivatives
Received 28 October 2009, revised 31 March 2010 and accepted for
publication 2 April 2010
17

De Simone et al.
[5,6,8,11,12,14,15(n)-3H] PGE2 and [9,10-3H]oleic acid were pur-
chased from Amersham Biosciences (Barcelona, Spain). The rest of
reagents were from Sigma (St. Louis, MO, USA). Escherichia coli
strain CECT 101 was a gift from Professor Uruburu, Department of
Microbiology, University of Valencia, Spain.
OAc
O
H
S
Br
H
O
O
HO
HO
O
O
1
2
Synthesis of 3,4-dibromo-5-(2-methoxy-
ethoxymethoxy)-5H-furan-2-one (4)
Figure 1: Petrosaspongiolide M (1) and 4-benzo[b]thiophen-2-yl-
3-bromo-5-hydroxy-5H-furan-2-one (2).
Mucobromic acid 3 (100 mg, 0.387 mmol) was dissolved in 10 mL
of dry dichloromethane, and MEM-Cl (66 lL, 0.581 mmol) was
added to the solution. Diisopropylethylamine (101 lL, 0.581 mmol)
was added dropwise over a period of 15 min. After 4 h, the reac-
tion mixture was quenched with 20 mL of HCl 1 M. The aqueous
layer was extracted with dichloromethane (3 · 30 mL), and the
organics were dried with Na2SO4, filtered and concentrated in
vacuo affording dark oil. The crude oil was purified by flash chro-
matography (10% diethyl ether ⁄ hexane) to give 4 (115 mg, 86%
365 nm). Flash column chromatography was performed using Merck
60 ⁄ 230–400 mesh silica gel. Analytical and semi-preparative
reverse-phase HPLC purifications were performed on a Waters
instrument using Jupiter C-18 column (250 · 4.60 mm, 5 lm, 300
ꢀ; 250 · 10.00 mm, 10 lm, 300 ꢀ, 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 ꢀ; Method B: Jupiter C-4,
250 · 4.60 mm, 5 lm, 300 ꢀ). Reaction yields refer to chromato-
graphically 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 and 600 MHz and 75 and 150 MHz, respectively. Chemical
shifts are expressed in parts per million (ppm) on the delta (d)
scale. The solvent peak was used as internal reference: for 1H
NMR CDCl3 = 7.26 ppm; for 13C NMR: CDCl3 = 77.0 ppm. Multi-
plicities are reported as follows: s, singlet; d, doublet; t, triplet;
m, multiplet; dd, doublet of doublets. High-resolution mass spec-
tra (HRMS) were recorded on a Q ⁄ TOF Premier WATERS (Milford,
MA, USA) mass spectrometer using an electrospray ion source
(ESI-MS).
1
yield): H NMR d (300 MHz; CDCl3): d 6.10 (1H, s, OCHO), 5.20 (1H,
d, J = 7 Hz, OCH HO), 4.87 (1H, d, J = 7 Hz, OCHH O), 3.79 (1H, m,
OCH HCH2O), 3.40 (3H, s, OCH3), 3.60 (1H, m, OCHH CH2O), 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).
Esterification of boronic acid 7 and 8
The boronic acids 7 and 8 (0.667 mmol) were dissolved in 6 mL of
ethyl acetate, and under stirring, pinacol (0.667 mmol) was added.
After 4 h, the reaction was stopped adding anhydrous Na2SO4 (1 g)
and CaCl2 (1 g). The mixture was filtered and concentrated in vacuo
(yield: 90% of 9 and 92% of 10).
5''
6''
4''
3''
F
O
N
H
N
1''
2''
3'
4'
H3CO
2'
O
Br
Br
HN
Br
Br
Br
Br
O
5'
1'
6'
O
HO
HO
O
HO
O
HO
O
HO
O
O
O
O
O
O
O
HO
6b
6a
6d
6c
6e
6f
O
H3CO
H
S
S
S
O
HO
O
HO
O
O
HO
O
O
HO
O
O
O
O
O
O
O
O
O
O
O
16j
16i
16h
16g
15g
14g
OCH3
HO
H
S
S
O
O
O
O
O
HO
O
HO
O
O
O
O
HO
O
HO
O
O
HO
O
O
O
18
16k
17g
17h
17j
17k
17i
Figure 2: Collection of synthesized compounds.
18
Chem Biol Drug Des 2010; 76: 17–24

Inhibitors of Microsomal Prostaglandin E Synthase-1 Expression
Reductive amination: general procedure
The organics were dried over Na2SO4, filtered and concentrated
Under inert atmosphere (N2), 1 equiv. of boronic ester 9 or 10 was
dissolved in anhydrous methanol (1 mL ⁄ 0.18 mmol of ester). The
mixture was kept under stirring at room temperature; anhydrous
amine 11 or 12 (4 equiv.), ZnCl2 (0.5 equiv.), and NaCNBH3 (1
equiv.) were added. After 4 h, when the reagents disappeared, the
reaction was stopped and 10 mL of an aqueous solution of NaOH
0.1 M was added. After concentration of methanol in vacuo, the
aqueous phase was extracted with ethyl acetate (3 · 10 mL) and
the organics were dried over Na2SO4, filtered and concentrated in
vacuo.
in vacuo.
3-Bromo-5-hydroxy-4-(1H-indol-2-yl)-5H-furan-2-
one (6a)
Yield: 90%; 1H-NMR d (300 MHz; CDCl3): 9.25 (1H, s, NH), 7.72
(1H, d, H-8¢), 7.46 (2H, m, H-3¢, H-7¢), 7.38 (1H, t, H-6¢), 7.20 (1H, d,
H-5¢), 6.48 (1H, s, OCHOH); 13C-NMR d (75 MHz; CDCl3): 166.7,
149.1, 139.6, 135.5, 134.8, 129.1, 124.0, 124.4, 124.3, 122.9, 112.0,
98.1; HRMS calcd. for C12H7BrNO3: [M - H]) 293.1 and 291.1 (1:1);
found 293.1 and 291.1 (1:1).
The obtained oil was purified on silica gel by flash chromatography
(10% ethyl acetate ⁄ n-hexane; yield: 79% of e).
4-Benzofuran-2-yl-3-bromo-5-hydroxy-5H-furan-2-
one (6b)
BOC2O protection of amine 13
Yield: 88%; 1H-NMR d (300 MHz; CDCl3): 7.74 (1H, s, H-3¢), 7.73
(1H, d, H-8¢), 7.59 (1H, d, H-5¢), 7.47 (1H, t, H-7¢), 7.35 (1H, t, H-6¢),
6.55 (1H, s, OCHOH); 13C-NMR d (75 MHz; CDCl3): 166.8, 151.1,
141.2, 137.0, 135.8, 130.8, 126.2, 125.8, 124.6, 124.1, 113.2, 99.1;
HRMS calcd. for C12H6BrO4: [M - H]) 294.1 and 292.1 (1:1); found
294.1 and 292.1 (1:1).
At 0 ꢀC, 1 equiv. of compound 13 was dissolved in a mixture of tri-
ethylamine ⁄ methanol 1:7 (v ⁄ v). The reaction was stirred for 10 min,
and then a solution of BOC2O (1.5 equiv.) was added dropwise.
After 1 h, the ice bath was removed and the reaction was kept on
stirring at room temperature overnight. Finally, the reaction material
was first concentrated in vacuo and then dissolved in 20 mL of di-
chloromethane and 20 mL of distilled water. The aqueous phase
was extracted with dichloromethane (3 · 20 mL); the organics were
dried over Na2SO4, filtered and concentrated in vacuo (yield: 57%
of N-BOC-f).
3-Bromo-5-hydroxy-4-styryl-5H-furan-2-one (6c)
1
Yield: 89%; H-NMR d (300 MHz; CDCl3): 7.59 (3H, m, H-3¢, H-4¢,
H-5¢), 7.42 (2H, d, H-2¢, H-6¢), 7.38 (1H, d, H-1), 6.98 (1H, d, H-2),
6.34 (1H, s, OCHOH); 13C-NMR d (75 MHz; CDCl3): 166.3, 156.3,
136.2, 130.5, 130.4, 130.2, 129.7, 128.2, 128.1, 127.1, 107.1, 103.8;
HRMS calcd. for C12H8BrO3: [M - H]) 280.1 and 278.1 (1:1); found
280.1 and 278.1 (1:1).
Microwave-assisted Suzuki coupling: general
procedure
Compound 4 (1 equiv.), the boronic ester (e or N-BOC-f) or boro-
nic acid a-d (1.5 equiv.), Pd(dppf)2Cl2 (0.03 equiv.), TBAB (0.5
equiv.), and CsF (4 equiv.) were placed in a CEM Discover vial.
Water (500 lL) and THF (500 lL) were added under argon. The
mixture was irradiated for 3–6 min, setting the power at 200 W,
the temperature at 120 ꢀC, the pressure at 250 psi, and the Power
Max ON. After diluting (10 mL) with dichloromethane (DCM), 10 mL
of an aqueous solution of HCl 1 N was added. The aqueous layer
was extracted with DCM (3 · 10 mL). The organics were then dried
over Na2SO4, filtered and concentrated in vacuo. The crude was
purified by flash chromatography (from 10% diethyl ether ⁄ n-hexane
to 40% diethyl ether ⁄ n-hexane), yield: 40–60%.
3-Bromo-4-[2-(4-fluoro-phenyl)-vinyl]-5-hydroxy-
5H-furan-2-one (6d)
Yield: 88%; H-NMR d (300 MHz; CDCl3): 7.58 (2H, d, H-3¢, H-5¢),
7.36 (1H, d, H-1), 7.11 (2H, d, H-2¢, H-6¢), 6.90 (1H, s, H-2), 6.34
(1H, s, OCHOH); 13C-NMR d (75 MHz; CDCl3): 166.5, 103.8, 156.7,
107.6, 127.9, 130.8, 132.5, 130.1, 117.1, 163.5; HRMS calcd. for
C12H7BrFO3: [M - H]) 298.1 and 296.1 (1:1); found 298.1 and 296.1
(1:1).
1
3-Bromo-5-hydroxy-4-(3-morpholin-4-ylmethyl-
phenyl)-5H-furan-2-one (6e)
1
General procedure for MEM cleavage
Yield: 93%; H-NMR d (600 MHz; MeOD): 8.12 (1H, d, H-6¢), 8.10
The protected compounds 5a-e and N-BOC-f were dissolved in
a solution of trifluoroacetic acid (95%), triisopropylsilan (2.5%), and
water (2.5%). The mixture was stirred at room temperature for
1.5 h and concentrated in vacuo at the end of the reaction.
(1H, d, H-2¢), 7.73–7.68 (2H, d, H-3¢, H-5¢), 6.62 (1H, s, OCHOH),
4.46 (2H, s, -CH2N), 3.99–3.81 (4H, m, O-(CH2)2), 2.99 (2H, m, N-
CH2), 2.85 (2H, m, N-CH2); 13C-NMR (150 MHz, MeOD): 166.6,
155.7, 138.1, 133.2, 130.8, 130.4, 129.7, 129.3, 110.4, 98.8, 69.2,
69.1, 56.6, 56.4, 51.3; ESI-MS calcd. for C15H17BrNO4: [M+H]+ 354.0
and 356.0 (1:1); found 354.1 and 356.1 (1:1).
General procedure for acetylation of 16g
Compound 16g was dissolved in dichloromethane, the mixture
was stirred at room temperature and then acetic anhydride (5
equiv.) and diisopropylethylamine (5 equiv.) were added. After
2 h, the mixture was diluted with 10 mL of HCl 1 N and the
aqueous phase was extracted with dichloromethane (3 · 10 mL).
4-[3-(Benzylamino-methyl)-4-methoxy-phenyl]-3-
bromo-5-hydroxy-5H-furan-2-one (6f)
Yield: 97%; H-NMR d (300 MHz; MeOD): 8.20 (1H, d, H-6¢), 8.05
1
(1H, s, H-2¢), 7.49 (5H, m, H-2¢', H-3¢', H-4¢', H-5¢', H-6¢'), 7.27 (1H,
Chem Biol Drug Des 2010; 76: 17–24
19

De Simone et al.
d, H-5¢), 6.55 (1H, s, OCHOH), 4.27–4.25 (4H, s, -CH2NCH2), 3.97
(3H, s, OCH3); 13C-NMR (150 MHz, MeOD): 167.5, 160.3, 155.6,
134.3, 132.7, 132.3, 130.9, 130.6, 129.2, 128.6, 125.5, 124.9, 112.3,
119.5, 111.1, 107.4, 98.4, 55.5, 53.7; ESI-MS calcd. for
C19H19BrNO4: [M + H]+ 374.0 and 376.0 (1:1); found 374.0 and
376.0 (1:1).
mPGES-1 (1 ⁄ 200). Finally, membranes were incubated with peroxi-
dase-conjugated goat anti-rabbit IgG (1 ⁄ 10,000). The immunoreac-
tive bands were visualized using an enhanced chemiluminescence
system (Amersham Biosciences).
Culture of murine macrophage RAW 264.7 cell
line
Acetic acid 3-benzo[b]thiophen-2-yl-5-oxo-2,5-
dihydro-furan-2-yl ester (18)
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 containing 2 mM L-glu-
tamine, 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 resuspended at a
concentration of 1.5 · 106 cells ⁄ mL.
Yield: 81%; 1H-NMR d (300 MHz, CDCl3): 7.86 (1H, d, H-8¢), 7.50
(1H, s, H-3¢), 7.44 (3H, m, H-5¢, H-6¢, H-7¢), 6.35 (1H, s, OCHOC-
OCH3), 2.23 (3H, s, CH3); 13C-NMR d (75 MHz, CDCl3): 170.5, 170.1,
157.1, 141.1 108.5, 139.3, 132.8, 128.6, 127.7, 125.4, 125.2, 122.3,
115.2, 22.1; HRMS calcd. for C14H9O4S: [M - H]) 273.3, found
273.3.
PGE2 production in RAW 264.7 macrophages
RAW 264.7 macrophages (1.5 · 106 cells ⁄ mL) were co-incubated 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 lL of test compound dissolved in ethanol or 2.0 lL of etha-
nol (control group). PGE2 levels were determined in culture superna-
tants by radioimmunoassay (31). The mitochondrial-dependent
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) to formazan (32) was used to assess the possible cyto-
toxic effects of compounds.
Assay of sPLA2
sPLA2 activity was assayed using [3H]oleate-labeled membranes of
E. coli, following a modification of the method of Franson et al.
(29,30). E. coli strain CECT 101 was grown for 6–8 h at 37 /C in
the presence of 5 lCi ⁄ mL [3H]oleic acid (specific activity 10 Ci ⁄ m-
mol) until the end of the logarithmic phase. After centrifugation at
1800 g for 10 min at 4 /C, the membranes were washed, resus-
pended in phosphate-buffered saline (PBS), and autoclaved for 30–
45 min. At least 95% of the radioactivity was incorporated into the
phospholipid fraction. Human recombinant synovial (group IIA sPLA2)
enzyme was used as source of sPLA2. Enzyme was diluted in 10 lL
of 100 mM Tris–HCl, 1 mM CaCl2 buffer, pH 7.5, and preincubated
at 37 /C for 5 min with 2.5 lL of test compound dissolved in etha-
nol or 2.5 lL of ethanol (control group) to get a final volume of
250 lL. Incubation proceeded for 15 min in the presence of 20 lL
of [3H]oleic-E. coli membranes and was terminated by addition of
100 lL ice-cold solution of 0.25% bovine serum albumin (BSA) solu-
tion in saline to a final concentration of 0.07% (w ⁄ v). After centri-
fugation at 2500 g for 10 min at 4 /C, the radioactivity (cpm) in the
supernatants was determined by liquid scintillation counting.
Statistical analysis
The results are presented as means € SEM; n represents the num-
ber 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 analysis of vari-
ance (ANOVA) followed by Dunnett's t-test for multiple comparisons.
Significance was assumed at a p value of 0.05 or less.
Results and Discussion
Western blot assay of COX-2 and mPGES-1
Chemistry
Cellular lysates from RAW 264.7 (murine macrophages,
1.5 · 106 cells ⁄ mL) incubated for 18 h with LPS (1 lg ⁄ mL) were
obtained with lysis buffer A [10 mM N-(2-hydroxyethyl)piperazine-
N'-ethanesulfonic acid (HEPES), pH 8.0, 1 mM ethylenediaminetetra-
acetic acid, 1 mM ethylene glycol bis(b-aminoethyl ether)-N,N,N',
N'- tetraacetic acid (EGTA), 10 mM KCl, 1 mM dithiothreitol, 5 mM
NaF, 1 mM Na3VO4, 10 mM Na2MoO4, 1 lg ⁄ mL leupeptin,
0.1 lg ⁄ mL aprotinin and 0.5 mM phenylmethanesulfonyl fluoride].
Following centrifugation (10 000 g, 15 min, 4 ꢀC), supernatant pro-
tein was determined by the Bradford method with BSA as standard.
COX-2 or mPGES-1 protein expression was studied in the total frac-
tion 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 (0.02M pH
7.0)-Tween 20 (0.1%), containing 3% (w ⁄ v) nonfat milk and incu-
bated with specific polyclonal antibody against COX-2 (1 ⁄ 1000) or
Considering that compound 2, our target molecule, consists of a 3-
bromo 4-substituted hydroxybutenolide, we decided to start with
the substitution of the benzothiophene appendage with bioisosteric
moieties, such as indole and benzofurane units 6a and 6b (see
chemistry section); as a result, we observed a clear loss of activity,
especially for benzofurane derivative (Table 1). Then, we took
into consideration another small array of 4-differently substituted
3-bromo-hydroxybutenolides, 6c-f. Among these, compounds 6e
and 6f belong to a small collection of products generated by Ludi
(12–14) in the course of our previous medicinal chemistry project
focused on the discovery of potential PLA2 inhibitors. On the con-
trary, 6c and 6d were conceived to mimic the same structural moi-
ety present in resveratrol, which has recently emerged as a potent
modulator of mPGES-1 expression (15). Unfortunately, we did not
observe an activity increase with respect to the lead compound 2
for any of the previous molecules. Afterward, we decided to investi-
gate the role played by the bromine atom, and for this purpose, we
20
Chem Biol Drug Des 2010; 76: 17–24

Inhibitors of Microsomal Prostaglandin E Synthase-1 Expression
Table 1: Inhibitory activity and cytotoxic effect of the c-hydroxy-
butenolide derivatives at 10 lM on the production of PGE2 in LPS-
stimulated RAW 264.7 cells.
compound 16g, the most active one so far obtained, the protection
of OH either with acetyl 18 or with THP group 14g to verify
whether the masked aldehyde was crucial for the activity, as it was
proved to be for the inhibition mechanism of the other known natu-
ral butenolides (2,17,18). Compounds 18 and 14g at last displayed
a higher potency in inhibiting the expression of mPGES-1, in com-
parison with the reference structure 2, which is of relevance in
consideration that only very few compounds are known to affect
the level of mPGES-1 enzyme, so that the availability of pharmaco-
logically active molecules capable of selective modulate mPGES-1
expression may provide new tools for better therapeutic approach.
Compound 10 lM
% Inhibition
IC50 (lM)
% Toxicity
6a
6b
81.6 € 4.7**
46.7 € 6.1**
45.1 € 9.7**
46.5 € 11.1**
61.7 € 8.1**
72.2 € 9.6**
100.0 € 0.0**
85.1 € 3.5**
87.1 € 3.9**
<20.0
76.0 € 5.7**
<20.0
<20.0
65.9 € 8.1**
<20.0
n.d.
4.20
n.d.
n.d.
n.d.
7.30
4.80
0.85
3.40
1.25
n.d.
3.17
n.d.
n.d.
4.46
n.d.
n.d.
n.d.
n.d.
0.79
1.80
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
28.9 € 3.0**
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
89.9 € 0.2**
<5.0
<5.0
6c
6d
6e
6f
14g
15g
16g
16h
16i
16j
16k
17g
17h
17i
17j
17k
18
For the synthesis of compounds 6a-f, according to Scheme 1, we
first protected the mucobromic acid 3 with methoxy-ethoxy-methyl-
chloride (MEM-Cl) (19) and then we connected the MEM-protected
butenolide 4 with the boronic acids R-B(OH)2 or their respective
pinacolic esters (20), using the Suzuki coupling reaction (21), previ-
ously optimized by us (22). To speed up the reaction, we took
advantages of microwaves heating strategy (23), which gave
compounds 5a-f in good yields, together with some by-products,
mainly consisting of bis-coupling and homo-coupling adducts.
Finally, the removal of MEM protecting group afforded the desired
compounds 6a-f.
<20.0
<20.0
100.0 € 0.0**
72.2 € 5.7**
<5.0
<5.0
2
Results show means € SEM (n = 6). Statistical significances: **p < 0.01,
with respect to the LPS-stimulated control group. PGE2 (non-stimulated
cells = 0.6 € 0.2 ng ⁄ mL; LPS-stimulated cells = 16.0 € 1.6 ng ⁄ mL). n.d. =
not determined.
All the boronic partners utilized for the synthesis of 6a-f are com-
mercially available except for e and N-BOC-f. These last were
obtained, according to Scheme 2, through a convergent synthetic
approach. Based on this, we first assembled the molecular append-
ages by a reductive amination between the pinacolic esters 9 and
10 with the amines 11 and 12, respectively, and then we per-
formed the Suzuki coupling to connect these fragments to the bute-
nolide scaffold 4.
examined the effects of two arrays of regioisomeric debrominated
c-hydroxybutenolides 16g-k and 17g-k, respectively, previously
synthesized by us, in the frame of a project focused on a synthetic
implementation task and re-synthesized, for the present purpose,
using the same strategy reported by us, the photooxidation of
3-bromofuran with singlet oxygen in the presence of a suitable
base (16). In the case of 16g, the debrominated 2, we observed a
moderate activity increase. On the contrary, the other 4-substituted
butenolides 16h-k (except 16i, which showed a discrete potency)
were found completely inactive. The only consideration emerged
actually from these preliminary results is that the only structural
change proved to be effective for the activity was the elimination
of bromine atom from the C-3 position of the lead compound 2. In
line with these findings, the other array of regioisomeric compounds
17g-k, all presenting substitutions on C-3 position of the scaffold,
did not give satisfactory results. Finally, we decided to perform on
The two regioisomeric arrays of compounds 16g-k and 17g-k
(Figure 2), respectively, were obtained utilizing the optimized proto-
col of photooxidation reaction performed on 3-bromofuran in the
presence of a suitable base, as previously reported by us (16).
Finally, the acetylation of compound 16g furnished the desired
compound 18 (Scheme 3).
Biology
Enzymatic inhibition of sPLA2, mainly type IIA sPLA2, is a pharmaco-
logical approach that can modulate the availability of arachidonic
R
Br
R
Br
Br
Br
Br
MEMO
Br
i
ii
iii
O
OH
R-B(OH)2
or
MEMO
O
HO
O
O
O
O
6a-f
O
4
H O
R-BO2C2(CH3)4
5a-e and N-BOC-f
3
F
Scheme 1: Synthesis of
compounds 6a-f. aReagents and
conditions: (i) MEM-Cl, DIPEA,
CH2Cl2, rt, 4h; (ii) Pd(dppf)2Cl2,
TBAB, CsF, THF ⁄ H2O 1:1, MW,
120ꢀC, 3-6 min; (iii) TFA (95%), TIS
(2.5%), H2O (2.5%).
N
O
b
H
c
a
d
R
H3CO
O
H
N
N
f
e
Chem Biol Drug Des 2010; 76: 17–24
21

De Simone et al.
N
ZnCl2
NaCNBH3
O
OH
B
O
O
B
O
O
B
H
OH
OH
O
H
e
O
O
NH
O
O
7
8
9
11
Pinacol
EtOAc
H
N
N
O
OH
B
ZnCl2
NaCNBH3
O
O
H3CO
H3CO
B
BOC2O
H
O
H
TEA/CH3OH
O
NH2
B
O
H3CO
B
H3CO
O
10
13
O
12
N-BOC-f
Scheme 2: Synthesis of appendages e and N-BOC-f.
LPS
(CH3CO)2O
S
2
16g
18
14g
Dx
S
DIPEA
CH2Cl2
O
B
C
1 µM 10 µM 1 µM 10 µM 1 µM 10 µM 1 µM
1 µM
O
O
HO
O
O
O
16g
mPGES-1
18
COX-2
Scheme 3: Acetylation of compound 16g.
β-actin
Figure 3: Effect of c-hydroxybutenolide derivatives on mPGES-1
and COX-2 expression in LPS-stimulated RAW 264.7 cells. The fig-
ure is representative of two similar experiments. B: normal cells. C:
LPS-stimulated cells. Dx. dexamethasone.
acid and consequently the production of PGE2 and the inflammatory
process (24). Among the nineteen new c-hydroxybutenolide deriva-
tives tested at 10 lM, under the same experimental conditions, on
sPLA2 belonging to the group IIA (human synovial recombinant), only
compound 6e exerted a weak inhibitory profile (30.4%). All the
other c-hydroxybutenolide derivatives tested were devoid of signifi-
cant inhibition (data not shown) against this pro-inflammatory
enzyme, unlike did the reference inhibitor LY311727 (96.3%) used
as a reference tool (25).
whereas dexamethasone, as expected, reduced the expression of
both inducible proteins.
COX-2 and mPGES-1 are the main inducible enzymes responsible
for PGE2 synthesis (26), and both of them are downregulated by
glucocorticoids in various cells (27,28). The use of Non Steroidal
Anti-Inflammatory Drugs (NSADs) and glucocorticoids is a mainstay
of anti-inflammatory therapy. Inhibition of PGE2 formation by COX-2
inhibitors is effective in ameliorating symptoms of inflammation.
However, the cardiovascular side effects associated with COX-2
inhibitors have limited their use. The concomitant increased cardio-
vascular safety observed in mPGES-1-deficient animals compared
with COX-2 inhibition under similar conditions makes mPGES-1 an
attractive target for development of a new class of therapeutic
agents. The selective pharmacological profile exerted by these com-
pounds could be of great interest to discover new promising drugs,
as well as to provide pharmacological tools to discern the role of
mPGES-1 and COX-2 in a great variety of inflammatory disorders.
We determined the effect of the nineteen c-hydroxybutenolide
derivatives on PGE2 production on mouse macrophage cell line
RAW264.7 stimulated with LPS (Table 1).
After 18-h stimulation, compounds 2, 6a, 6e, 6f, 14g, 15g, 16g,
16i, 17g, and 18 were able to inhibit PGE2 production with a per-
centage of inhibition higher than 50% at 10 lM, showing IC50 val-
ues in the micromolar range. Only compounds 18, 14g, and 16g
exerted an inhibitory potency higher than the leader compound 2.
This profile is especially relevant for compounds 18 and 14g, the
two protected derivatives of the debrominated 2. On the other
hand, all the derivatives except 17i, which was discarded, were
devoid of significant cytotoxic effects on RAW264.7 at concentra-
tions up to 10 lM, as assessed by the mitochondrial-dependent
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to formazan (Table 1). Compound 14g showed a slight cyto-
toxic effect that disappeared at lower concentrations.
Acknowledgments
Western blot analysis for mPGES-1 and COX-2 proteins using 18-h
LPS-stimulated RAW 264.7 cells (Figure 3) shows clearly that com-
pounds 18, 14g, and 16g and the leader compound 2 inhibit
mPGES-1 expression, without any effect on COX-2 expression,
Financial support by the University of Salerno and by Ministero
dell'Istruzione, dell'Universitꢁ e della Ricerca (MIUR), PRIN-06 is
gratefully acknowledged. M.D.G. was the recipient of a research
fellowship from FPU program (AP 20041633) of Spanish Ministerio
22
Chem Biol Drug Des 2010; 76: 17–24

Inhibitors of Microsomal Prostaglandin E Synthase-1 Expression
de Educaciꢂn y Ciencia. This work was supported in part by Grant
FIS PI051659 from Spanish Instituto de Salud Carlos III.
15. Candelario-Jalil E., de Oliveira A.C., Graf S., Bhatia H.S., Hull
M., Munoz E., Fiebich B.L. (2007) Resveratrol potently reduces
prostaglandin E2 production and free radical formation in lipo-
polysaccharide-activated primary rat microglia. J Neuroinflamma-
tion;4:25.
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