Synthesis and PGE2 production inhibition of 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.11.067

3,4-Diphenyl-substituted 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives were synthesized and evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells. Both 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione rings as main scaffolds were easily obtained using one of three synthetic methods. Among the compounds investigated, 1H-3-(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) showed a strong inhibitory activity (IC₅₀ = 0.61 μM) of PGE₂ production.

Full text of DOI:10.1016/j.bmcl.2009.11.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 734–737
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and PGE2 production inhibition of 1H-furan-2,5-dione
and 1H-pyrrole-2,5-dione derivatives
Jong Taik Moon a, Ji Young Jeon a, Hang Ah Park a, Young-Soo Noh b, Kyung-Tae Lee b, Jungahn Kim a,
Dong Joon Choo a, Jae Yeol Lee a,
*
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b Kyung Hee East–West Pharmaceutical Research Institute and Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic
of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2009
Revised 15 October 2009
Accepted 13 November 2009
Available online 11 December 2009
3,4-Diphenyl-substituted 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives were synthesized
and evaluated for the inhibitory activities on LPS-induced PGE2 production in RAW 264.7 macrophage
cells. Both 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione rings as main scaffolds were easily obtained
using one of three synthetic methods. Among the compounds investigated, 1H-3-(4-sulfamoylphenyl)-
4-phenyl-pyrrole-2,5-dione (6l) showed a strong inhibitory activity (IC50 = 0.61 lM) of PGE2 production.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PGE2 production
Anti-inflammatory
RAW 264.7 macrophage cells
1H-Furan-2,5-dione
1H-Pyrrole-2,5-dione
New generations of anti-inflammatory drugs have been devel-
oped to enhance the anti-inflammatory and analgesic activities of
classic nonsteroidal anti-inflammatory drugs (NSAIDs), and to re-
duce the adverse effects of these agents. Selective COX-2 inhibitors
are viewed enthusiastically because they match traditional NSAIDs
in terms of efficacy, but circumvent constitutively active COX-1
and are comparatively free of stomach-associated complications.
Diarylheterocycles, and other central ring pharmacophore tem-
plates, have been extensively studied as selective COX-2 inhibi-
tors.1 All these tricyclic molecules have 1,2-diaryl substitutions
on their central hetero- or carbocyclic ring systems. The recent
withdrawal of the selective COX-2 inhibitors rofecoxib and valdec-
oxib because of their adverse cardiovascular side effects demon-
strates the need to identify new scaffolds with COX-2 inhibitory
activity, but without the side effects of known agents.2 PGE2 has
long been considered the principal prostaglandin of acute inflam-
mation and of chronic diseases such as rheumatoid arthritis3 and
inflammatory bowel disease.4 Macrophages play particularly
important roles in inflammation because they produce many pro-
inflammatory molecules such as PGE2. Therefore, the pharmaco-
logical interference of PGE2 production has been postulated as a
means of alleviating a number of disease states mediated by exces-
sive and/or protracted macrophage activation. As an attempt to
discover novel compound with potent anti-inflammatory activity,
therefore, we have synthesized 1H-furan-2,5-dione and 1H-pyr-
role-2,5-dione derivatives and evaluated their inhibitory activities
against LPS-induced PGE2 production in RAW 264.7 macrophages.
The synthetic procedures and reaction conditions for 1H-furan-
2,5-dione and 1H-pyrrole-2,5-dione derivatives are shown in
Schemes 1 and 2. As a general synthetic procedure, the condensa-
tion reaction of benzoylformic acid (3) with phenylacetic acid (4)
under acetic anhydride reflux condition provided 1H-furan-2,5-
dione (5).5 Meanwhile, some commercially unavailable benzoylfor-
mic acids (3) were prepared by Friedel–Crafts acylation and subse-
quent NaOH hydrolysis.6,7 1H-Furan-2,5-dione (5) was easily
converted into 1H-pyrrole-2,5-dione (6) on treatment with hexam-
ethyldisilazane (HMDS) in MeOH/DMF solution.8 For the struc-
ture–activity relationship (SAR) study, deprotection of
compounds 6 containing methoxy group with BBr3 gave 1H-pyr-
role-2,5-diones (6f–6h and 6m) containing free hydroxy groups9
and also the oxidation of 1H-furan-2,5-dione (5) containing thio-
methoxy group with m-CPBA (1 or 2 equiv) gave 1H-furan-2,5-
dione (5e, 5g, 5h, 5j, and 5k) containing methanesulfinyl or meth-
anesulfonyl groups as shown in Scheme 1. As another synthetic
procedure for 1H-pyrrole-2,5-diones (6), phenylacetic acid (4)
was converted into phenylacetamide (7), which was condensed
with ethyl 4-methanesulfanylbenzoylformate (2) and NaH to
* Corresponding author. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail address: ljy@khu.ac.kr (J.Y. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.067

J. T. Moon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 734–737
735
Method 1
(O)n
S
Me
R1
O
1
O
R2
a
O
R3
d
R1
O
R4
O
OEt
5e, g, h, j, k
NH
O
R1
R2
R3
e (R1 = SMe)
2
O
b
R1
R4
6a-h, n, o
O
O
O
d
R2
R3
OH
O
R2
R3
c
OH
+
O
O
R1
R4
R4
3
4
5a-d, f, i, l, m
Scheme 1. Reagents and conditions: (a) AlCl3, ClCOCO2Et, DCM, 0 °C; (b) 2 N NaOH, H2O, rt; (c) Ac2O, reflux; (d) (i) HMDS, MeOH, DMF, rt; (ii) BBr3, DCM, À78 °C to rt; (e) m-
CPBA (1 or 2 equiv), DCM, À20 °C.
(O)n
Method 2
S
S
Me
Me
O
O
OH
NH2
NH
NH
2 (R1 = SMe)
a
c
O
O
b
R3
R3
O
O
R3
R3
4
7
6i, m, p
6j, k, q, r
O
S
Method 3
H2N
O
O
OH
OH
NH2
O
2 (R1 = H)
d
e
NH
O
S
O
O
O
b
R3
S
O
Cl
H2N
O
O
4 (R3 = H)
8
9
6l
Scheme 2. Reagents and conditions: (a) (i) SOCl2, DMF, rt to reflux; (ii) NH4OH, rt; (b) NaH, THF, 0 °C; (c) m-CPBA, DCM, À20 °C; (d) ClSO3H, 0 °C to rt; (e) (i) SOCl2, DMF, rt; (ii)
NH3 (g), CH3CN, rt.
afford 1H-pyrrole-2,5-diones (6i, 6m, and 6n) as shown in method
2 of Scheme 2. In the case of 1H-pyrrole-2,5-dione containing 4-
sulfamoylphenyl group (6l), 4-chlorosulfonylphenylacetic acid (8)
was prepared from the reaction of phenylacetic acid (4) with
ClSO3H, and then treated with SOCl2 and subsequent NH3 gas in
CH3CN to provide 4-sulfamoylphenylacetamide (9), which was
condensed with ethyl benzoylformate (2) and NaH to yield 1H-3-
(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) as shown in
method 3 of Scheme 2. The synthesized compounds were purified
by flash column chromatography and analyzed for the structures
based on 1H NMR and 13C NMR spectra.
264.7 cells were plated at a density of 105 cells/well in 96-well
plates. To determine the appropriate concentration not toxic to
cells, cytotoxicity studies were performed 24 h after treating cells
with various concentrations of synthetic compounds. Viabilities
were determined using colorimetric MTT assays as described pre-
viously.10 These compounds did not affect the viabilities of RAW
264.7 cells at concentrations up to 100 lM in the presence of LPS
over 24 h (Table 1), indicating that their suppressive effects on
PGE2 production could not be attributable to non-specific cytotox-
icity. The inhibitory activity of COX-2 catalyzed PGE2 production
from LPS-induced RAW 264.7 cells was performed according to
the published procedure:11 The RAW 264.7 macrophage cell line
was obtained from the Korea Cell Line Bank (Seoul, Korea). Cells
Initially, we examined the cytotoxicity of synthetic compounds
in RAW 264.7 cells in the presence of LPS using MTT assays: RAW

736
J. T. Moon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 734–737
were grown at 37 °C in DMEM supplemented with 10% FBS, peni-
cillin (100 units/ml), and streptomycin sulfate (100 g/ml) in a
humidified 5% CO2 atmosphere. Cells were incubated with the
moiety in ring A (5d, 5f, and 5i) were generally more active than
those with sulfoxide or sulfone moiety (5e, 5g, and 5j) except for
5h and 5k. Among this series, 1H-furan-2,5-dione with 4-metha-
nesulfanyl group (5d) showed good inhibitory activity
l
tested samples at increasing concentrations or positive control
chemical (NS398) and then stimulated with LPS 1
l
g/ml for the
(IC50 = 3.58 lM). With respect to a series of 1H-pyrrole-2,5-dione
indicated time. PGE2 concentration in the medium was quantified
using EIA kits (R&D Systems, Minneapolis, MN). In addition, the
inhibitory activity against COX-2 was also measured for com-
derivatives (6a–6r), we could find a general structure–activity rela-
tionship (SAR) when compared to 1H-furan-2,5-dione series (5). In
the case of compounds without sulfur-substituent (6a–6h) in ring
A, compounds having appropriate hydrophobic or hydrophilic moi-
ety (6d and 6g) revealed considerable PGE2 inhibitory activities.
For compounds with sulfur-substituent (6i–6k and 6m–6r) in ring
A, compounds with sulfoxide moiety (6j, 6n, and 6q) showed re-
duced bioactivity compared to those with sulfide or sulfone moiety
(6i, 6k, 6m, 6p, and 6r) except for 6o. The introduction of hydroxyl
group at 4-position in ring B led to the increased inhibitory activity
(6i vs 6m) but the introduction of the fluoride atom at 4-position in
ring B showed the decreased bioactivity (6i–6k vs 6p–6r) regard-
less of the oxidation state of sulfur atom. Among all compounds
pounds showing less than IC50 of PGE2 production = 10 lM and
then Dup-697 was used as a positive control COX-2 inhibitor.12
All experiments were carried out at least three times and the data
of bioassays were summarized in Table 1.
As shown in Table1, both 1H-furan-2,5-dione and 1H-pyrrole-
2,5-dione derivatives showed the broad inhibitory spectrum
against PGE2 production. With respect to a series of 1H-furan-
2,5-dione derivatives (5a–5m), we could not find a clear struc-
ture–activity relationship (SAR) based on the substituents of phe-
nyl rings. However, 1H-furan-2,5-dione derivatives with sulfide
Table 1
Inhibition of PGE2 production from LPS-induced RAW 264.7 cells by synthetic 1H-furan-2,5-dione (5a–5m) and 1H-pyrrole-2,5-dione (6a–6r) derivatives
R1
R1
O
O
A
A
O
NH
R2
R3
R2
R3
B
B
O
O
R4
R4
5a-m
6a-r
Compound
R1
R2
R3
R4
Cell viability
IC50 M)
Inhibition of PGE2 production
Inhibition of COX-2 activity
IC50 M)
(
l
IC50
(lM)
(l
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
H
H
H
SMe
S(O)2Me
SMe
S(O)Me
S(O)2Me
SMe
S(O)Me
S(O)2Me
SMe
SMe
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
OH
H
H
H
H
H
H
OCH2O
OMe
OMe
H
328.68
257.40
312.56
194.77
265.89
333.94
261.90
261.82
266.08
283.66
271.24
249.75
276.61
269.90
259.63
305.41
195.36
150.55
158.01
206.59
261.45
151.88
303.22
268.74
158.18
124.74
212.89
171.20
121.22
242.04
260.76
7.13
18.52
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
71.29
37.10
3.58
15.42
30.46
68.75
4.41
12.60
23.75
Inactive
5.71
78.48
27.94
9.95
131.53
7.96
25.08
13.83
2.69
18.45
H
OAc
OAc
OAc
OMe
OMe
OMe
NHAc
NAc2
H
16.63
20.11
H
OMe
OCH2O
OMe
H
OH
OH
H
H
H
H
OH
OH
OH
F
F
F
OMe
OH
OH
OH
H
H
H
H
H
H
H
H
H
16.04
17.51
16.34
H
SMe
8.19
4.73
21.52
2.71
0.61
S(O)Me
S(O)2Me
S(O)2NH2
SMe
S(O)Me
S(O)2Me
SMe
17.50
15.62
21.01
6m
6n
6o
6p
6q
6r
0.84
98.12
Inactive
25.50
45.20
10.76
4.80
H
H
H
H
S(O)Me
S(O)2Me
H
NS398a
Dup-697b
0.17
a
Positive control compound for assay of PGE2 production inhibition.
Positive control compound for assay of COX-2 inhibition.
b

J. T. Moon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 734–737
737
tested, 1H-pyrrole-2,5-dione (6l) containing 4-sulfamoyl group at
4-position in ring A showed the strongest inhibitory activity
(IC50 = 0.61 M) of PGE2 production. This result indicated that the
sulfamoyl group at 4-position in ring A plays very an important
role on the inhibition of PGE2 production. In addition, compound
6l showed the highest therapeutic index (TI = cytotoxicity/
PGE2 = 259.3) although the TI data were not inserted in Table 1.
Therefore, this preliminary result indicated that the inhibition
of PGE2 production by 1H-pyrrole-2,5-dione (6l) was not associ-
ated with its cytotoxicity. With respect to activity against COX-2
References and notes
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l
enzyme, however, all compounds having less than IC50 = 10
showed the similar inhibitory activity range (IC50 = 16.62–
21.01 M) against COX-2 enzyme regardless of IC50 values of
lM
l
PGE2 production. Thus, this result means that further experiments
should be performed to evaluate the effect of 1H-pyrrole-2,5-dione
compound on PGE2 production and to investigate the related
mechanism involved in RAW 264.7 cells, and thus the mechanism
study is currently under the progress.
In summary, we prepared 1H-furan-2,5-dione and 1H-pyrrole-
2,5-dione derivatives, and evaluated their inhibitory activities on
PGE2 production from LPS-induced RAW264.7 cells. We found that
1H-3-(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) pos-
sessed strong inhibitory activity compared to that of NS398, a po-
sitive control. Further SARs study on the A ring and B ring is
currently under progress as well as a mechanism study.
11. Mossman, T. J. Immnol. Methods 1983, 65, 55.
12. The COX-2 activity assay directly measures PGF2 produced by SnCl2 reduction
a
of COX-derived PGH2. The prostanoid product is quantified via enzyme
immunoassay (EIA) using a broadly specific antibody that binds to all the
major prostaglandin compounds using COX Inhibitor Screening Assay
(Cayman, MI). Briefly, control value was obtained in the absence of
compound. COX-2 enzyme was mixed with different concentration of each
tested compound and heme, and incubated for 10 min at 37 °C. The reaction
was initiated by adding arachidonic acid and all tubes were incubated for
another 2 min at 37 °C. Dup-697 was used as
inhibitor.
a positive control COX-2
Acknowledgment
This research was supported by the Kyung Hee University Re-
search Fund in 2006 (KHU-20060396).