Synthesis, biological evaluation, and docking analysis of a novel family of 1-methyl-1H-pyrrole-2,5-diones as highly potent and selective cyclooxygenase-2 (COX-2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.02.074

As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives. New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them, compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE₂ IC₅₀ = 8.7 nM, COX-2 IC₅₀ = 6.0 nM; COX-2 selectivity index (SI) = >168] than celecoxib. Molecular docking experiments were further performed against COX-2 and COX-1 isozymes to determine their probable binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully complementary theoretical support for the obtained experimental biological data.

Full text of DOI:10.1016/j.bmcl.2014.02.074

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1958–1962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, and docking analysis of a novel
family of 1-methyl-1H-pyrrole-2,5-diones as highly potent
and selective cyclooxygenase-2 (COX-2) inhibitors
Kyung Ju Kim a, Min Ji Choi a, Ji-Sun Shin b, Minju Kim a, Hye-Eun Choi b, Seoung Mook Kang a, Jae Ho Jin a,
Kyung-Tae Lee b, , 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 Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, 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:
As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical
synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives.
New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE2 production in
RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them,
compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE2 IC50 = 8.7 -
nM, COX-2 IC50 = 6.0 nM; COX-2 selectivity index (SI) = >168] than celecoxib. Molecular docking
experiments were further performed against COX-2 and COX-1 isozymes to determine their probable
binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has
stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully
complementary theoretical support for the obtained experimental biological data.
Received 27 September 2013
Revised 15 February 2014
Accepted 28 February 2014
Available online 12 March 2014
Keywords:
Inflammation
Prostaglandin E2
Cyclooxygenase-2
1-Methyl-1H-pyrrole-2,5-dione
Molecular docking study
Ó 2014 Elsevier Ltd. All rights reserved.
Cyclooxygenase (COX) is an enzyme that is responsible for
formation of important biological mediators called prostanoids,
including prostaglandins, prostacyclin and thromboxane. Three
COX isoenzymes are known: COX-1, COX-2, and COX-3 which is
a splice variant of COX-1.1 COX-1 is considered a constitutive en-
zyme, being found in most mammalian cells. On the other hand,
COX-2, undetectable in normal tissues, and induced during inflam-
mation, hypoxia, and Wnt-signalling, is present in many cancers.2
Classical non-steroidal anti-inflammatory drugs (NSAIDs) such as
aspirin block both COX-1 and COX-2. When the COX-1 is inhibited,
inflammation is reduced, but the protection of the lining of the
stomach is also lost. This can cause stomach upset as well as
ulceration and bleeding from the stomach and even the intestines.3
Therefore, selective COX-2 inhibitors such as celecoxib and
rofecoxib had been developed and prescribed.4 As COX-2 is usually
specific to inflamed tissue, there is much less gastric irritation
associated with COX-2 inhibition together with the decreased risk
of peptic ulceration.5 However, coxib drugs such as rofecoxib (Vio-
xxÒ) and valdecoxib (BextraÒ) were withdrawn from the market in
2004 and 2005, respectively, because they excessively increased
the risk of heart attacks and strokes with long term use.6 Celecoxib
(CelebrexÒ) is the only COX-2 inhibitor available in the United
States. On the other hand, some studies have suggested that rofec-
oxib’s adverse cardiac events may not be a class effect but rather an
intrinsic chemical property related to its metabolism.7 For this rea-
son, novel scaffolds with selective COX-2 inhibitory activity need
to be found and evaluated for anti-inflammatory effect.
As an initial attempt to discover novel COX-2 inhibitor with
selectivity and safety profile, we have recently reported that 1H-
furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives including
compound 7a from our compound library showed an good inhibi-
tory activity against LPS-induced PGE2 production in RAW 264.7
macrophages via a moderate potent and selective COX-2 inhibition
(Fig. 1)8–12 In continuation of our ongoing research work directed
towards the development of selective COX-2 inhibitors, we have
focused on the modification of 1H-pyrrole-2,5-dione ring as the
template. A group of pyrrole-2,5-dione derivatives, employing fo-
cused substitution at the N-position of parent ring and the C-4
positions of one side ring, were synthesized (Fig. 1). The COX-1/
COX-2 inhibitory activities of this new class of compounds were
determined and the plausible binding interactions of new com-
pounds within the COX-1/COX-2 active sites were explored using
molecular docking studies.
The synthetic methodology used to synthesize the target
compounds 7b–c and 9a–d is shown in Scheme 1. As a general
⇑
Corresponding authors. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail addresses: ktlee@khu.ac.kr (K.-T. Lee), ljy@khu.ac.kr (J.Y. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.02.074
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

K. J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1958–1962
1959
Figure 1. Discovery of 1-methyl-1H-pyrrole-2,5-dione (9d: MPO-0029) as highly potent and selective COX-2 inhibitor.
Scheme 1. Reagents and conditions: (a) AlCl3, ClCOCO2Et, DCM, 0 °C, 3 h, 55–80%; (b) 2 N NaOH, H2O, rt, 4 h, 50–60%; (c) ClSO3H, 0 °C to rt, 24 h, 91%; (d) (i) (COCl)2, DMF,
DCM, rt, 17 h; (ii) NH3 (gas), CH3CN, rt, 24 h, 35%; (e) NaH, DMSO, rt, 3 h, 23–43%; (f) NH4OH, MeOH, rt, 24 h, 56%; (g) (i) Ac2O, reflux, 24 h; (ii) CH3NH2, MeOH, rt, 24 h, 21–
31%.
synthetic procedure, the Friedel–Crafts acylation of mono-substi-
tuted-benzene 1a–d with ethyl chlorooxoacetate afforded ethyl
para-substituted-phenyloxoacetate 2a–d in 55–80% yield, which
was hydrolyzed with 2 N NaOH to provide para-substituted-phen-
yloxoacetic acid (3a–d) in 50–60% yield. For the other coupling
part, the chlorosulfonylation of phenylacetic acid 4 with chlorosul-
fonic acid gave para-chlorosulfonylphenylacetic acid 5 in 91% yield.
The treatment of compound 5 with oxalyl chloride was followed by
an addition of ammonia gas (NH3) to provide para-amin-
osulfonylphenylacetamide 6 (35%), which was condensed with
the above compound 2b–d in the presence of sodium hydride
(NaH) in dry DMSO to furnish 1H-pyrrole-2,5-dione 7b–d (23–
43%; isolated yields), respectively. In the case of 1-methy-1H-pyr-
role-2,5-dione 9a–d, compound 5 was treated with ammonium
hydroxide (NH4OH) to provide para-aminosulfonylphenylacetic
acid 8 (56%). The condensation of compound 8 with the above
compound 3a–d under acetic anhydride reflux condition provided
1H-furan-2,5-dione (the structure is not shown here) as an inter-
mediate. Without further purification, this intermediate was
directly treated with methylamine (CH3NH2) to furnish the target
products 9a–d (21–31%; isolated yields).
Initially, we examined the cytotoxicity of synthetic compounds
in RAW 264.7 cells in the presence of LPS using MTT assays: RAW
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.13 None of compound affected the viabilities of RAW
264.7 cells at concentrations above 10 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
cytotoxicity.
The inhibitory activity of COX-2 catalyzed PGE2 production
from LPS-induced RAW 264.7 cells was performed according to
the published procedure using NS-398 as a positive control.14 In
addition, the inhibitory activities against ovine COX-1 and human
recombinant COX-2 were also carried out using an enzyme immu-
noassay (EIA) kit (kit catalog number 560101, Cayman Chemical,
Ann Arbor, MI, USA) for synthetic compounds showing IC50 value
of single unit nM against PGE2 production.15 Potent and selective
COX-2 inhibitor, celecoxib was used as a reference compound in

1960
K. J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1958–1962
Table 1
Inhibition of PGE2 production, in vitro COX-1 and COX-2 inhibition, COX-2 selectivity index (SI), and Eintermolecular data
O
H2N
O
S
O
N
R1
O
R2
R1
R2
Cell viability (
lM)
IC50 (lM)
COX-2 SIc
Eintermolecular (kcal/mol)
COX-1 (1EQG) COX-2 (3LN1)
a
d
Compound
b
PGE2
COX-1
COX-2
7a
7b
7c
7d
9a
9b
9c
H
H
H
H
CH3
CH3
CH3
CH3
H
>10
>10
>10
>10
>10
>10
>10
>10
0.61
0.87
0.45
4.7
127
9.1
1.5
1.2
26
0.036
0.085
0.053
0.006
1.8
0.072
(0.070)g
30
14
À124
À127
À119
À124
À130
À131
À136
À129
À116
À122
À162
À168
À173
À167
À166
À172
À178
À171
À136
À179
CH3
OCH3
Cl
>100
>100
>100
>1.0
>1.0
>1.0
>1.0
220
>67
>83
>3.8
>28
>12
>19
>168
122
>14
(110)g
0.87
H
0.011
CH3
OCH3
Cl
0.0078
0.0031
0.0087
0.0070
0.0087
9d (MPO-0029)
NS-398e
Celecoxibf
>1.0
(7.7)g
26
Ibuprofenh
0.86
À106
À108
a
b
c
IC50 value is the compound concentration required to produce 50% inhibition of PGE2 production, COX-1 and COX-2 enzymatic activity, respectively.
LPS-induced PGE2 production.
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Calculated energy of intermolecular interactions during docking in the COX-1 (PDB code: 1EQG) and COX-2 (PDB code: 3LN1) active site.
Positive control for PGE2 production inhibition.
d
e
f
Positive control as selective COX-2 inhibitor.
g
h
Reported data (Kaur, J. et al. Bioorg. Med. Chem. Lett. 2012, 22, 2154).16
Positive control as non-selective COX-2 inhibitor.
the COX activity assay. All experiments were carried out at least
three times and the data of bioassays were summarized in Table 1.
As shown in Table 1, all compounds except 7d displayed good
inhibitory potencies against LPS-induced PGE2 production in the
submicromolar into nanomolar range. Specifically, 1-methyl-1H-
pyrrole-2,5-dione series 9a–d (IC50 = 3.1–11 nM range) showed
more potent inhibition against PGE2 production than 1H-pyrrole-
assay, meanwhile, celecoxib showed high COX-2 inhibitory po-
tency with IC50 value of 72 nM and high selectivity for COX-2/1
(>14), which are in agreement with previously reported data
(IC50 = 70 nM, COX-2 SI = 110) in the literature.16 The relative pro-
file with respect to the R2-substituent was Cl (9d: IC50 = 6.0 nM,
COX-2 SI = >168) >> H (9a: IC50 = 36 nM, COX-2 SI = >28) > OCH3
(9c: IC50 = 53 nM, COX-2 SI = >19) > CH3 (9b: IC50 = 85 nM, COX-2
SI = >12) as shown in Figure 2(b and c). This is somewhat in
contrast to PGE2-related data wherein compound 9c (PGE2
IC50 = 3.1 nM, COX-2 IC50 = 53 nM) showed more inhibitory effect
on LPS-induced PGE2 production than compound 9d (PGE2
IC50 = 8.7 nM, COX-2 IC50 = 6.0 nM). As multiple enzymes are in-
volved in PGE2 biosynthesis, including phospholipases, cyclooxy-
genases (COX), 5-lipoxygenase, and the PGE2 synthases (PGES),17
this present results suggest that some of these 1H-pyrrole-2,5-
dione derivatives could inhibit the activity of these enzymes based
on the recent literatures.18–20 Nonetheless, both R1 = CH3 and
R2 = Cl groups were shown to be important pharmacophores to
confer COX-2 selectivity and potency. In the viewpoint of overall
2,5-dione series 7a–d (IC50 = 0.45–4.7 lM range), meaning that
R1 substituent (that is, N-CH3) was a major determinant of PGE2
production inhibitory potency. The relative profile with respect
to R2 substituent was found to be OCH3 (7c) > H (7a) > CH3
(7b) >> Cl (7d) in 7 series (R1 = H), whereas the relative profile of
R2 substituent was OCH3 (9c) > CH3 (9b) P Cl (9d) > H (9a) within
9 series (R1 = CH3). Taken together, 9c (R1 = CH3 and R2 = OCH3;
IC50 = 3.1 nM) was most strong and ca. 2.8-fold more potent than
celecoxib (IC50 = 8.7 nM) for LPS-induced PGE2 production inhibi-
tion in RAW 264.7 cells. Compound 9d (IC50 = 8.7 nM) was found
to be almost comparable to celecoxib as shown in Figure 2(a).
Therefore, this finding implies that N-CH3 (R1) group was shown
to be important pharmacophore to confer PGE2 production inhibi-
tory potency again.
respects (PGE2 IC50
, COX-2 IC50, and COX-2 SI), therefore,
compound 9d (MPO-0029) was identified to be more potent and
selective COX-2 inhibitor than celecoxib.
In vitro COX-1/COX-2 isozyme inhibition studies on compound
7 series (R1 = H) showed that all compounds were more potent
To gain insight into the plausible mode of interaction(s) of syn-
thetic compounds within the COX-1 and COX-2 isozymes, molecu-
lar modeling (docking) experiments were performed using X-ray
crystal structure data for COX-1 (PDB code: 1EQG)21 and COX-2
(PDB code: 3LN1)22 using Molegro Virtual Docker (MVD)
2013.6.0 for Windows.23 The energy associated with intermolecu-
lar interactions (Eintermolecular) obtained upon computational analy-
sis (docking) for compounds within the COX-1 and COX-2 active
site is summarized in Table 1. The docking results of NS-398, cele-
coxib, and ibuprofen were also inserted for comparison. First of all,
all compounds occupy the same binding site as that of celecoxib in
COX-2 structure. The obvious interaction differences between
COX-1 and COX-2 structures appropriately support the experimen-
tal high selectivity indexes for COX-2/COX-1 in Table 1, when
inhibitors of COX-2 (IC50 = 1.2–26
(IC50 = >100
M). The relative profile with respect to the R2-substi-
tuent was OCH3 (7c) > CH3 (7b) > H (7a) >> Cl (7d). Among this ser-
ies, compound 7c (PGE2 IC50 = 0.45 M, COX-2 IC50 = 1.2 M, and
COX-2 selectivity index = >83) was identified as potent and selec-
tive COX-2 inhibitor compared to ibuprofen (COX-2 IC50 = 30 M,
lM
range) than COX-1
l
l
l
l
COX-2 selectivity index = 0.87). On the other hand, compound 9
series (R1 = CH3) displayed very high inhibitory potency towards
COX-2 (IC50 = 6.0–85 nM range) but they exhibited little inhibition
against COX-1 at even higher 1.0 lM concentration, thus the high-
er COX-2 selectivity. This structure–activity data acquired showed
that the N-CH3 (R1) group was a determinant of both COX-2 inhib-
itory potency and COX-2 selectivity. In our enzyme inhibitory

K. J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1958–1962
1961
Figure 2. (a) Inhibitory effects of compound 9d (MPO-0029) on LPS-induced PGE2 production using NS-398 as a positive control; (b) effects of compound 9d (MPO-0029) on
COX-2 enzyme activity using Dup697 (10
control.
lM) as a positive control; (c) effects of compound 9d (MPO-0029) on COX-1 enzyme activity using SC-560 (1 lM) as a positive
Figure 3. Overlap of stick forms of compound 9d (MPO-0029, red color) and celecoxib (white color) showing hydrogen bond interactions (blue dotted lines) of compound 9d
(MPO-0029) with amino acids (green color) in the active site of COX-2 (PDB code: 3LN1).
compared to that of ibuprofen as nonselective inhibitor. The com-
parison of Eintermolecular for compounds 9a–d (À166 to À178 kcal/
mol) with that of NS-398 (À136 kcal/mol) supports the appreciable
interaction of compounds 9a–d within the COX-2 active site. These
favorable entry and binding interactions of compounds 9a–d with-
in the COX-2 active site are consistent with their experimentally
observed potent COX-2 inhibition. Compounds 7c, 9d, and cele-
coxib showed potent selectivity to COX-2 with obvious differences
in the interaction energy (54, 42 and 57 kcal/mol), respectively.
Among them, docking result for most potent and selective
compound 9d was provided. Compound 9d assumes a favorable
orientation within the COX-2 binding site (Fig. 3). The overlap of
docking conformation of compound 9d with co-crystallized cele-
coxib with COX-2 (PDB code: 3LN1) shows that compound 9d
can bind into the active site of COX-2 enzyme in almost the same
fashion as celecoxib. Two oxygen atoms of SO2NH2 moiety in

1962
K. J. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1958–1962
11. Pham, V. C.; Shin, J. S.; Choi, M. J.; Kim, T. W.; Lee, K.; Kim, K. J.; Huh, G.; Kim, J.;
Choo, D. J.; Lee, K.-T.; Lee, J. Y. Bull. Korean Chem. Soc. 2012, 33, 721.
12. Pham, V. C.; Choi, M. J.; Kim, T. W.; Kim, J.; Choo, D. J.; Lee, K.-T.; Lee, J. Y. Bull.
Korean Chem. Soc. 2012, 33, 305.
13. Won, J. H.; Im, H. T.; Kim, Y. H.; Yun, K. J.; Park, H. J.; Choi, J. W.; Lee, K. T. Br. J.
Pharmacol. 2006, 148, 216.
14. Mossman, T. J. Immunol. Methods 1983, 65, 55. The RAW 264.7 macrophage cell
line was obtained from the Korea Cell Line Bank (Seoul, Korea). Cells were
grown at 37 °C in DMEM supplemented with 10% FBS, penicillin (100 units/ml),
compound 9d show three hydrogen bonds with Arg499 (3.54 Å),
Phe504 (3.16 Å), and Ile503 (3.54 Å). Two hydrogen atoms of its
SO2NH2 moiety show two hydrogen bonds with Gln178 (3.11 Å)
and Leu338 (2.07 Å). More importantly, one carbonyl oxygen of
pyrrole-2,5-dione ring shows the additional hydrogen bond with
OH of Tyr341 (3.42 Å), which can provide rationale for higher
potency and selectivity of compound 9d against COX-2.
and streptomycin sulfate (100 lg/ml) in a humidified 5% CO2 atmosphere. Cells
In conclusion, new 1H-methyl-pyrrole-2,5-dione derivatives
were synthesized for evaluation as COX-2 inhibitors. Within them,
compound 9d (MPO-0029)24 exhibited potent both COX-2 inhibi-
tory activity and selectivity (PGE2 IC50 = 8.7 nM, COX-2 IC50 = 6.0 -
nM; COX-2 selectivity index (SI) = >168). Molecular docking
studies indicate appreciable binding interaction of compound 9d
within the COX-2 active site that is enhanced by two C@O groups
of pyrrole-2,5-dione ring, which supports that compound 9d inter-
acts with COX-2 enzyme more efficiently than celecoxib. Therefore,
this compound can be further evaluated for in vivo anti-inflamma-
tory activity using animal model.
were incubated with the tested samples at increasing concentrations or
positive chemical (NS-398) and then stimulated with LPS 10 ng/ml for the
indicated time. PGE2 concentration in the medium was quantified using EIA
kits (R&D Systems, Minneapolis, MN)..
15. The COX activity assay directly measures PGF2
a produced by SnCl2 reduction
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 values were obtained in the absence of compound, and
COX-1 and COX-2 enzymes were inactivated by boiling enzyme for 3 min.
COX-1 or COX-2 enzyme was mixed with different concentrations 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. SC-560 (1 M) and Dup697 (10 M) were used as a
positive COX-1 and COX-2 inhibitor, respectively.
16. Kaur, J.; Bhardwaj, A.; Huang, Z.; Knaus, E. E. Bioorg. Med. Chem. Lett. 2012, 22,
2154.
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education (NRF-2013R1A1A2058915).
17. Samuelsson, B.; Morgenstern, R.; Jakobsson, P. J. Pharmacol. Rev. 2007, 59, 207.
18. Ghatak, S.; Vyas, A.; Misra, S.; O’Brien, P.; Zambre, A.; Fresco, V. M.; Markwald,
R. R.; Swamy, K. V.; Afrasiabi, Z.; Choudhury, A.; Khetmalas, M.; Padhye, S.
Bioorg. Med. Chem. Lett. 2014, 24, 317.
19. Leone, S.; Ottani, A.; Bertolini, A. Curr. Top. Med. Chem. 2007, 7, 265.
20. Elkady, M.; Nieß, R.; Schaible, A. M.; Bauer, J.; Luderer, S.; Ambrosi, G.; Werz,
O.; Laufer, S. A. J. Med. Chem. 2012, 55, 8958.
References and notes
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DMSO-d6)
d 7.86 (2H, d, J = 8.4 Hz), 7.56 (2H, d, J = 8.4 Hz), 7.53 (2H, d,
J = 8.4 Hz), 7.45 (2H, br s), 7.41 (2H, d, J = 8.4 Hz), 3.04 (3H, s); 13C NMR
(100 MHz, DMSO-d6) d 171.35, 171.29, 145.82, 138.13, 137.06, 136.39, 133.39,
132.58, 131.36, 129.64, 128.37, 127.05, 23.24; Low MS (ESÀ): m/z 375.60
[MÀH]À; HRMS (FAB+): m/z estimated for C17H14ClN2O4S [M+H]+: 377.0363,
found 377.0364.