Inhibition of the enzymes in the leukotriene and prostaglandin pathways in inflammation by 3-aryl isocoumarins

Article abstract of DOI:10.1016/j.ejmech.2016.08.066

The biosynthesis of leukotrienes in one of the arachidonic acid pathways and PGE₂in the other by 5-LOX and mPGES1 respectively, play pivotal roles in augmenting inflammatory responses. PGE₂is known to participate in cancer pathologic

Full text of DOI:10.1016/j.ejmech.2016.08.066

European Journal of Medicinal Chemistry 124 (2016) 428e434
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Inhibition of the enzymes in the leukotriene and prostaglandin
pathways in inflammation by 3-aryl isocoumarins
,
,
, *
Meera Ramanan a 1, Shweta Sinha a 1, Kasireddy Sudarshan b, Indrapal Singh Aidhen b
,
,
**
Mukesh Doble a
a Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology, Madras, Tamil Nadu, 600036, India
b Department of Chemistry, Indian Institute of Technology, Madras, Tamil Nadu, 600036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The biosynthesis of leukotrienes in one of the arachidonic acid pathways and PGE2 in the other by 5-LOX
and mPGES1 respectively, play pivotal roles in augmenting inflammatory responses. PGE2 is known to
participate in cancer pathological processes as well. Isocoumarins are natural compounds with a wide
range of biological activities. In this study, 3-aryl isocoumarin derivatives are synthesized and tested
against 5-LOX enzyme in vitro and PGE2 production in HeLa cells. Most of the compounds show high
Received 3 June 2016
Received in revised form
9 August 2016
Accepted 29 August 2016
Available online 30 August 2016
activity, and 1c is identified as a dual inhibitor with an IC50 of 4.6 0.26
LOX and PGE2 production respectively. Another compound 7f, exhibits an IC50 of 12.4 0.14
5-LOX. Further investigations reveal that the mechanism of action of 1c and 7f against 5-LOX is mixed
and competitive modes of action respectively. Thunberginol (7c) exhibits IC50 of 15.8 0.03 M against
m
M and 6.3 0.13
m
M against 5-
m
M against
Keywords:
Inflammation
5-Lipoxygenase
m
PGE2 production. 1c and 7c inhibit the mRNA expression of mPGES1 and COX-2. The study has identified
a novel scaffold, 1c with a dual inhibitory activity which can be further optimized to compete against
Microsomal prostaglandin E2 synthase 1
Isocoumarins
Licofelone which is under clinical trials (with IC50 of 6.0
mM for mPGES1 & 0.2 mM for 5-LOX). To
conclude, 3-aryl isocoumarin derivatives appears as promising tools to fight against inflammatory dis-
eases as well as cancer.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
containing enzyme in the biosynthesis of leukotrienes. It catalyzes
AA to 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE) and further
Inflammation is an important hallmark of many pathological
diseases including rheumatoid arthritis, periodontitis, cancer etc
[1]. It progresses through the prostaglandin (PG) and leukotriene
(LT) pathways starting with the release of arachidonic acid (AA)
from the membrane lipid bilayer [2]. Inhibitors designed to target
selectively the cyclooxygenase-2 (COX-2) enzyme in an attempt to
curb inflammation, resulted in serious side effects including car-
diovascular complications and gastro-intestinal disturbances [3]. In
this regard, 5-lipoxygenase (5-LOX) and microsomal prostaglandin
E2 synthase 1 (mPGES1), the two key downstream enzymes in the
LT & PG pathways respectively, appear to be the best targets in
to LTA4 [4]. Microsomal prostaglandin E2 synthase 1 (mPGES1) is
the enzyme that converts PGH2 to PGE2. PGE2 exerts its effect
through its receptors, EP1eEP4, and is found to be involved in the
etiology of tumorigenesis, cancer survival, Epithelial Mesenchymal
Transition (EMT), regulation of vascular tone, fever and pain [5].
HIF1a which is an important trigger for tumor progression and
angiogenesis is induced by the production of PGE2 [6]. PGE2 also
participates in EMT by aiding the cancer cells to breach and cross
the basement membrane thereby reaching the extracellular matrix
[7]. Inhibition of its synthesis reduces Akt phosphorylation which
leads to the activation of pro-apoptotic molecules and down-
regulation of anti-apoptotic markers in leukaemia [8]. So, inhibition
of PGE2 synthesis is viewed as one of the approaches to limit the
growth and spread of several types of cancers.
combating inflammation. 5-LOX is
a crucial non-heme iron
Several chemical derivatives and natural products are developed
and tested as potential inhibitors against these enzymes. The
identification of dual inhibitors has definite advantage over single
target inhibitor in a complex scenario such as inflammation.
* Corresponding author.
** Corresponding author.
E-mail addresses: isingh@iitm.ac.in (I.S. Aidhen), mukeshd@iitm.ac.in
(M. Doble).
1
Both authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2016.08.066
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
429
Licofelone has been observed to act against multiple targets such as
COX-2, mPGES1, 5-LOX and FLAP [9]. Aminothiazole [10], embelin
[11], -naphthylpirinixic acid scaffold [12] and lonazolac de-
a
rivatives and indomethacin derivatives [13], have been shown to
suppress the activity of both 5-LOX and mPGES1 effectively. Despite
all these reported compounds, none of them could be developed as
a drug due to their pharmacokinetic parameters. Hence, identifying
a potential inhibitor that could act against multiple targets is still
the need of the hour.
Isocoumarins are isomers of coumarins (1,2-benzopyrone)
[14,15]. They are present widely throughout the plant kingdom,
including bergenin (Bergenia ciliate, Shorea leprosula) [15], thun-
berginol (Hydrangea macrophylla) and cajanolactone (Cajanus
cajan) [16,17]. The anti-inflammatory, anti-microbial and anti-
cancer effects of these naturally occurring compounds have
attracted the medicinal chemists to develop several structural de-
rivatives with the aim of increasing their potencies [17].
Isocoumarins are known to act as anti-inflammatory molecules
and they also possess radical scavenging ability [18]. Paepalantine,
an isocoumarin obtained from Paepalanthus bromelioidesis is
demonstrated to have anti-inflammatory activity against colitis
when tested in rat models [19]. Several fluorinated derivatives are
found to exhibit anti-inflammatory activity [20]. Though significant
anti-inflammatory activity of isocoumarins has been documented,
no reports are available that elucidate their mechanism of action
and their ability to act as dual inhibitors.
Isocoumarins have also been reported to exhibit anti-cancer
activity. Cytogenin, an isocoumarin isolated from Streptoverticil-
lium eurocidium exhibits significant anti-cancer activity in mice
models of Ehrlich carcinoma [21]. Synthetically prepared ones
(NM-3) when tested with Cytogenin resulted in synergistic inhi-
bition of angiogenesis in mouse models [22]. Fluorinated and 3,4
dihydroisocoumarins has been found to prevent metastasis in MCF-
7 breast cancer cell line [23].
Scheme 1. Synthesis of 3-aryl isocoumarins.
groups attached to the C3-aryl substitution (hydroxyl in 1c and
methoxy in 7f) seemed to be responsible for the inhibitory activity.
It can be seen from Table 1 that, compound 1c having hydroxyl
groups at R1 and R2 positions exhibits 84.1% inhibition. The pres-
ence of two hydroxyl groups in the ortho position might have
resulted in additional resonance stabilization and o-quinone for-
mation which is responsible for the high activity [26e28]. In
contrast, one hydroxyl group at the R4 position in compound 7c
lowers the activity by half. Compound 1d substituted with one
methoxy and one hydroxyl group at the R1 and R2 positions
respectively shows 78.8% inhibition probably due to the formation
of a methylene dioxy ring by these two groups [29,30]. Inhibition
activity decreased drastically to almost by half (35.6%) when R4 is
substituted with hydroxyl group, as seen in 7d. In compound 7f
(80.3%), incorporation of hydroxyl at R4 decreases activity
marginally (1f ¼ 72.0%). However, presence of one methoxy group
at R5 position in 1h decreases inhibition to half (36.2%).
In this paper, the inhibitory effect of chemically synthesized 3-
aryl isocoumarin derivatives against the enzymes in the leuko-
triene and prostaglandin pathways are investigated including their
mode of action.
2. Chemistry
1d also exhibited appreciable inhibitory activity (78.8% at 50 mM
concentration) because of the presence of OH and OCH3 groups. But
one of the naturally occurring plant isocoumarin synthesized here
in the laboratory, thunberginol (7c), showed moderate 5-LOX in-
All the screened 3-aryl Isocoumarins, 1 and 7, were synthesized
using a new method developed by us earlier [17], which involved
use of a-aryl aminonitriles 2 as acyl anion equivalents. The a-aryl
aminonitriles, 2, on alkylation with bromides, 3 and 4, followed by
hydrolysis afforded the corresponding aryl ketones 5 and 6
respectively. These aryl ketones, on base promoted intramolecular
cyclization, furnished 3-aryl Isocoumarins 1 and 7 respectively
(Scheme 1). The route has been generalized and applied to the
synthesis of thunberginol A (7c) and other 3-aryl isocoumarins
used in the present study.
hibition (only 40.9% at 50
known dual inhibitor Licofelone were tested as positive controls
which exhibited an IC50 of ~1.0 M and 0.5 M respectively [31,32].
mM). Commercial drug, Zileuton and the
m
m
Thus, one can conclude that, for the first set of six compounds
(Table 1) having H in the R4 position, it is important to have OH at R2
position and for the remaining set of eight compounds having OH
in the R4 position, presence of OMe group at R1, R2 and R3 is
necessary but not at R5 position for high 5-LOX inhibitory activity.
3. Result and discussion
3.2. Kinetics of 5-LOX inhibition
3.1. 5-LOX enzyme activity
The mechanism of action of these two most active isocoumarins
(1c and 7f) was determined by measuring the initial rate of product
formation for various concentrations of the substrate (AA) at two
different concentrations of these inhibitors [33]. Lineweaver-Burke
plot (Fig. 1a) indicates that in the presence of 1c, Vmax (0.83 0.06,
0.93 0.11 and 0.64 0.1 nmol/min) and Km values (2.2, 1.38 and
The 5-LOX enzyme activity was determined in a cell free assay
by measuring the conversion of AA to the product, 5(S)-hydro-
peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) which
was measured in terms of OD at l236 [24,25]. Most of the com-
pounds (at 50
Compound 1c was found to be the best with an IC50 of 4.6 0.26
followed by 7f with an IC50 of 12.4 0.14 M. Other compounds
such as 1f and 1d in the series exhibited IC50 of 20.9 0.03 M and
22.8 0.07 M respectively. Compounds that had electron donating
mM) exhibited 5-LOX inhibitory activity (Table 1).
mM
1.17
50
m
M) are different for different concentrations of it (0, 10 and
m
m
M) indicating mixed or non con-competitive inhibition. If the
m
inhibitor has same affinity for both the enzyme and enzyme-
substrate complex then it can be regarded as non-competitive
m

430
M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
Table 1
Inhibition of 5-LOX & mPGES1, antioxidant activity & cellular toxicity of 3-aryl isocoumarins.
Compounds (50
conc.)
m
M
R1
R2
R3
R4 R5
% 5-Lox
% PGE2
% Scavenging of
DPPH SD
MTT assay (% cell
proliferation)
inhibition SDa
inhibition SDb
1a
1b
1c
1d
1e
1f
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH H
OH H
OH H
OH H
OH H
H
H
H
H
H
H
50.0 8.6
34.1 1.1
84.1 3.2
78.8 2.1
65.9 5.3
72.0 1.1
55.3 1.1
36.4 6.4
40.9 10.7
35.6 5.3
59.1 6.4
80.3 4.3
75 5.6
65 7.2
78 2.9
73 5.7
18 7.9
73 2.6
60 7.42
74 3.5
74 3.7
75 9.14
44 9.9
68 6.6
54 5.29
14 3.37
14.0 0.6
17.0 0.3
53.2 3.4
47.8 1.4
20.0 0.6
15.8 2.5
13.4 0.3
12.6 0.3
49.0 3.1
36.2 0.8
17.8 0.3
12.4 0.6
16.6 0.3
15.4 0.3
76.7 3.2
81.4 2.0
78.7 8.1
65.0 7.3
70.5 2.8
75.7 5.6
75.6 1.8
70.6 2.0
68.7 6.5
79.8 3.2
77.4 4.9
80.2 1.1
78.1 3.7
60.5 9.0
OH
OH
OH
OCH3
OCH3 OCH3
H
OH
OH
OH
OCH3
OCH3 OCH3 OH H
H
OH
OCH3
OH
OCH3
H
H
H
H
H
H
H
OH
OCH3
OH
OCH3
H
1g
1h
H
OH OCH3 49.2 1.1
OCH3
OCH3 OCH3 OH OCH3 36.2 12.8
Zileuton
97.0 4.3
50.0 4.8
33.6 4.5
78.0 2.2
Licofelone
71.0 3.3
48.0 9.1c
e
85.0 4.1
Ascorbic acid
e
e
82.2 0.3
e
a
Cell free assay at 50
Cell based assay at 50
m
M compounds concentration.
M compounds concentration in HeLa cells.
Licofelone tested for mPGES1 inhibition at 5 M concentration.
b
c
m
m
inhibition and if the affinities are different then it is termed as a
mixed inhibition. Lineweaver-Burke plot for the compound 7f
(Fig. 1b) indicates that its mechanism of inhibition is competitive
since Vmax values remain constant (i.e 0.87 0.04 nmol/min for
3.4. Radical scavenging activity
Radical scavenging activity of all the compounds is also deter-
mined with ascorbic acid as the positive control [35]. Most of the
compounds possess poor antioxidant activity (Table 1). 1c shows
the highest radical scavenging activity (53%) which also supports
the result that the mechanism of inhibition of 5-LOX by it is through
the disruption of redox cycle and hence may be non-competitive or
mixed inhibition. Compound 7f possesses very low antioxidant
activity (12%) which suggests that inhibition of 5-LOX by it is
through non-redox process, may be competitive type, which is
again supported from the two assays described above.
control, and 0.83 0.02, 0.93 0.1 nmol/min at 10 and 30
mM of 7f
respectively) while Km values increases with increasing concen-
tration of the inhibitor (0.09, 1.2 and 1.45 mM for 0, 10 and 30 mM of
7f respectively).
3.3. Pseudoperoxidase activity
3.5. Inhibition of PGE2 production
5-LOX enzyme in its activated ferric form consumes lipid per-
oxides. In the presence of redox inhibitor, ferric ion of the activated
enzyme gets converted to ferrous form (inactive enzyme). The
ferrous iron consumes lipid peroxides to become ferric thereby
reactivating the enzyme. This is measured in terms of the decrease
in the absorbance at 234 nm wavelength due to the consumption of
13(S)-hydroperoxyoctadecadienoic acid (13(S) HpODE) [34]. The
pseudoperoxidase activity of 5-LOX in the presence of the in-
hibitors, 1c and 7f and standard drug Zilueton are determined
(Fig. 2). In the presence of compound 1c, absorbance decreased by
~35% (indicating considerable degradation of the hydroperoxide
product) suggesting that the inhibition follows the redox mecha-
nism. Positive control Zilueton which is also a redox inhibitor
consumes ~50% of the hydroperoxide substrate. Whereas with
compound 7f, no degradation of the hydroperoxide product is
observed (Fig. 2) indicating a non-redox inhibitory mechanism
These results also corroborate with the findings from L-B plot.
Over expression of COX-2 and mPGES1 results in the increased
levels of PGE2 which is observed in cervical (HeLa) [2], non small
cell lung (A549) [2] and prostate (DU145) cancers [36]. Hence HeLa
cells are selected in this study to test the effectiveness of iso-
coumarins at a concentration of 50 mM. The sequential action of
COX and PGES leads to the production of PGE2, which is released
into the cell culture supernatant and is monitored using an ELISA
kit. Most of the compounds tested at 50
of inhibition with 1c being the best of all (78%) (Table 1). Licofelone,
has an IC50 of 6 0.17. 7f which is seen to inhibit 5-LOX effi-
mM showed varying levels
mM
ciently (80%) exhibits 68% inhibitory potential towards the pro-
duction of PGE2. Bio-mimetically prepared natural product
thunberginol (7c), is observed to reduce the PGE2 levels by 74%.
The presence of hydroxyl group in 7c at the R4 position de-
creases the inhibitory potential marginally when compared to 1c.
As discussed earlier, the presence of two hydroxyl groups may

M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
431
enzymes, one would be able to determine the exact IC50
.
3
2
1
(a)
The structure activity relation for the first set of six compounds
(Table 1) having H in the R4 position indicates that, the presence of
OH in R1 and R2 is beneficiary. For the remaining set of eight
compounds which have OH in the R4 position, the presence of OH in
the R2 position is necessary for high activity.
50 μM
10 μM
control
1/v
3.6. Inhibition of PGE2 production upon addition of arachidonic acid
AA is the direct substrate for COX enzymes [37]. To determine
the effect of exogenously added substrate, AA, the HeLa cells were
challenged with it as per a previously reported protocol [38]. There
-0.5
0.5
1.0
1.5
1/[s]
-1
is a drastic increase in the levels of PGE2 produced in AA (15 mM)
supplemented cells when compared to the cells which did not
receive it. Addition of AA externally is will be taken up by COX
enzyme resulting in excess turnover of PGH2 which is used as the
substrate by the mPGES1 enzyme resulting in increased production
of PGE2.
3
(b)
30 μM
10 μM
control
2
1
1/v
The percentage inhibition of PGE2 which is released into the cell
culture supernatant by those treated with 1c & 7c (50
15 min is 76 & 75% respectively. However, when the cells were
stimulated with 15 M AA for 15 min, following the compound
mM) for
m
treatment, the percentage inhibition dropped down to 44 & 17%
respectively. The amount of PGE2 produced is normalized by sub-
tracting the amount produced by AA treatment alone (no com-
-0.5
0.5
1.0
1.5
pound treatment). Similarly, for Licofelone (5 mM) the percentage
1/[s]
-1
inhibition in the absence and in the presence of AA is 81% and 54.7%
respectively. Even though exogenously added AA is shared by both,
COX-1 as well as COX-2 isoenzymes, it is predominantly taken up by
Fig. 1. (a) Lineweaver-Burk plot for compounds 1c (at 10 and 50 mM concentrations)
and (b) 7f tested at concentrations (10 and 30
V ¼ Velocity of reaction. Control (without inhibitor).
m
M). [S] ¼ Substrate (AA) concentration,
the latter as its expression is considerably induced by TNFa [37].
Moreover, we do not observe any change in the mRNA expression of
COX-1 in 1c and 7c treated cells (Fig. 4a). Hence, the reduction of
PGE2 levels observed would be mainly due to the inhibition of COX-
2 and mPGES1. A specific assay to determine the individual activity
of mPGES1 and COX-2 would give the exact percentage of inhibition
of each of these enzymes by these compounds. (see Fig. 3)
stabilize the compound resulting in improved activity against
mPGES1. Activity of 1d (73%) is comparable to that of 7d (75%). The
presence of OMe and OH substituents in these compounds could
result in the formation of a methylene dioxy ring which may be
responsible for the efficient inhibition of PGE2 production [29]. The
IC50 values for both these compounds when tested with HeLa cells
3.7. Inhibition of mRNA expression of PG enzymes
were 6.3 0.13 and 15.84 0.03
other compounds which exhibited more than 70% inhibition were
also determined and found to be 9.3 0.07 M (1f), 9.5 0.11
(1a), 7.1 0.20 M (7b), 9.7 0.10 M (1e) and 11.3 0.03 M (7e).
mM respectively. The IC50 values of
The reduction in PGE2 levels could be due to, either the inhibi-
tion of mPGES1 alone or the upstream enzymes namely COX-2/
COX-1. Previous studies have shown that compounds that target
FLAP such as MK886 is able to inhibit mPGES1 also, as both of them
belongs to the same MAPEG family [39]. Hence real time PCR
analysis was carried out to determine the mRNA expression of
m
mM
m
m
m
HeLa cells express both the pro-inflammatory enzymes, COX-2 and
mPGES1, and hence if these compounds are tested with pure
Fig. 3. Effect of compounds on PGE2 production in the presence of exogenous AA. HeLa
cells were treated with 50 ng/ml of TNF
mPGES1, followed by addition of 50 M of compounds for 15 min and then AA (15
for another 15 min. The amount of PGE2 in the cell culture supernatant is estimated
a
to induce the production of COX-2 and
m
m
M)
Fig. 2. Pseudoperoxidase activity plot (shown as absorbance) for the consumption of
13(S)-HpODE by compounds 1c, 7f and Zileuton.
using EIA kit. The data is normalized with that of TNF
the treatment of compounds.
a and AA induced cells without

432
M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
these inflammatory enzymes. Licofelone, 1c and 7c inhibit the
mRNA expression of COX-2 and mPGES1 (Fig. 4a) while that of COX-
1 is not altered by any of them. However, the extent of inhibition of
mPGES1 by 1c is statistically significant when compared to that of
COX-2. Hence, it is presumed that these isocoumarins could be
acting both by inhibiting the expression of these enzymes as well as
by inhibiting the activity of these enzymes at varying levels.
Moderate inhibition of mRNA expression of COX-2 and no inter-
ference with COX-1 also indicates reasonable selectivity for
mPGES1 by these 3-aryl isocoumarins.
approximately 1 fold higher in 1c and 7c treated cells in compari-
son to the untreated control cells. NF b have contradicting roles of
being both apoptotic and anti-apoptotic in nature [43]. TGF family
of proteins, are mainly reported to induce the cells to undergo
apoptosis [44]. IL6 is predominantly a pro-inflammatory cytokine
[45]. From these results it appears that the anti-inflammatory ef-
k
fects of these two isocoumarins are routed mainly through IFN
g
and TGF pathways. Further studies to detect the apoptotic status
b
of the compounds treated cells could reveal the exact function of
these above said factors.
The results are normalized to that of the untreated cells and b-
Actin (****p < 0.0001). Statistical significance was calculated by 2-
way ANOVA method.
4. Conclusion
To fight against multifaceted diseases such as inflammation or
cancer, targeting more than one enzyme becomes a superior
strategy than designing an inhibitor selectively for one target. Such
dual or multi-targeted inhibitors with good pharmacokinetic
properties could be a valuable aid in the management of certain
cancers. Many natural products that are anti-inflammatory such as
curcumin, resveratrol, garcinol, epigallocatechingallate (EGCG) are
all identified to be acting on multiple targets [46]. Here in this
study, isocoumarins which are also natural product derivatives
have been synthesized in our lab and tested for multi-targeted
actions against the enzymes involved in the leukotriene and
prostaglandin synthesis.
Zileuton is the only marketed drug against 5-LOX and hence
there is a need for finding out more inhibitors against this enzyme
to treat anti-inflammatory disorders including asthma, allergy etc
[47]. Here in this present work, 1c and 7f are identified as potent
inhibitors of the 5-LOX enzyme. Also, compound 7f has been
identified as a promising inhibitor with non-redox mechanism
whereas 1c exhibits redox type of behaviour. In addition, com-
pound 1c is found to be superior with better antioxidant activity
(53%) which supported the above observation of redox mechanism
of action by it.
3.8. Inhibition of mRNA expression of inflammatory cytokines
To elucidate the mechanism further, real time PCR analysis was
carried out for various inflammatory cytokines and transcription
factors (TF). A significant increase is observed in the expression of
IFN
compared to the untreated cells (Fig. 4b). Though IFN
dominantly identified as pro-inflammatory cytokine, its anti-
inflammatory effects are also reported [40]. IFN has been shown
g
in both 1c (5.6 fold) and 7c (9.6 fold) treated HeLa cells when
g
is pre-
g
to protect arthritis induced by streptococcus in rats by acting as
anti-inflammatory molecule [41]. It functions mainly by inhibiting
the other pro-inflammatory cytokines and activate cytokine an-
tagonists [40]. IFN
[42]. The expressions of IL-6, TGF
g
also triggers apoptotic pathway in leukocytes
3 and NF b are found to be
b
k
The in vivo efficacy of many potential drug candidates is not as
appreciable as much as the in vitro activity. MK886, for instance, has
an IC50 of 1.6
mM in vitro against mPGES1 but could not demonstrate
similar potential when tested in whole blood (~20% inhibition for
100
mM) and gingival fibroblasts (no significant reduction at
2e4
mM) [48e50]. In contrast, in human cell lines such as Caco-2
and HT-29, PGE2 production is found to be increased when tested
at 10 M concentration [51]. Licofelone on the other hand is a
potent suppressor of PGE2 production in vivo (EC50 0.1 M) in
A549 cells. The problem with this drug is that it simultaneously
inhibits COX-1 (IC50 0.8 M). [52] Inhibiting the latter might shunt
m
m
m
the normal production of other prostaglandins such as PGI2
resulting in the disturbances of physiological processes.
From these observations it is heartening to note that, 1c and 7c,
with their potential to suppress the PGE2 production in the cancer
cell line, HeLa, appears to be very promising for further improve-
ment. Testing their efficacies in other in vivo models could help in
authenticating the proficiency of these compounds.
To summarise, a few 3-aryl isocoumarins synthesized and tested
here exhibit excellent inhibition of 5-LOX in vitro and PGE2 in vivo in
HeLa cells. Current study has identified a novel scaffold (1c) with
dihydroxyl groups at C3-aryl substitution as a dual inhibitor of both
5-LOX and PGE2 which could be further optimized for reducing its
IC50 value. The molecular mechanism of action could be due to the
activation of several anti-inflammatory and apoptotic cytokines
and transcription factors. Structural optimization and further
studies are warranted to decrease their IC50 to nanomolar range
before they could be considered for treatment against inflamma-
tion and cancer.
Fig. 4. a) Real time PCR analysis showing the fold decrease in the mRNA expression of
mPGES1, COX-2 and COX-1 treated with Licofelone (5 M), 1c and 7c (50 M). b) Real
Time PCR analysis showing mRNA expression of pro and anti-inflammatory cytokines
in HeLa cells treated with 1c and 7c at 50 M concentration.
m
m
m

M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
433
proliferation of HL-60 leukemia cells by suppressing the expression of mPGES-
1 and reducing prostaglandin E2 synthesis, Int. J. Hematol. 94 (5) (2011)
472e478.
Author contributions
SS carried out the 5-LOX experiments and antioxidant studies;
MR performed the PGE2 analysis and real time PCR experiments
and KS synthesized the 3-aryl isocoumarin derivatives. MD and ISA
designed the experiments, analyzed the data and corrected the
manuscript.
[9] A. Koeberle, U. Siemoneit, U. Bühring, H. Northoff, S. Laufer, W. Albrecht,
O. Werz, Licofelone suppresses prostaglandin E2 formation by interference
with the inducible microsomal prostaglandin E2 synthase-1, J. Pharmacol.
Exp. Ther. 326 (3) (2008) 975e982.
[10] T. Hanke, F. Dehm, S. Liening, S.-D. Popella, J. Maczewsky, M. Pillong, J. Kunze,
C. Weinigel, D. Barz, A. Kaiser, Aminothiazole-featured pirinixic acid de-
rivatives as dual 5-lipoxygenase and microsomal prostaglandin E2 synthase-1
inhibitors with improved potency and efficiency in vivo, J. Med. Chem. 56 (22)
(2013) 9031e9044.
Acknowledgments
[11] A.M. Schaible, H. Traber, V. Temml, S.M. Noha, R. Filosa, A. Peduto, C. Weinigel,
D. Barz, D. Schuster, O. Werz, Potent inhibition of human 5-lipoxygenase and
microsomal prostaglandin E 2 synthase-1 by the anti-carcinogenic and anti-
inflammatory agent embelin, Biochem. Pharmacol. 86 (4) (2013) 476e486.
[12] M. Hieke, C. Greiner, T.M. Thieme, M. Schubert-Zsilavecz, O. Werz, H. Zettl,
MR and SS acknowledge the WOS-A, DST (SR/WOS-A/LS-514/
2012; SR/WOS-A/CS-126/2013 respectively), India for the project
funding and
fellowship
and
KS acknowledges UGC
A novel class of dual mPGES-1/5-LO inhibitors based on the a-naphthylpir-
(2006201010225 (i) EU-IV), New Delhi for the SRF fellowship. SS
thanks Nisha S. Devi for 5-LOX assay development. The authors
acknowledge the gift of pT3 5-LOX plasmid from Prof. Olof Råd-
mark, Karolinska Institute, Stockholm, Sweden, Department of
Science and Technology (DST), New Delhi, for funding towards ESI-
MS facility, Department of Chemistry, IIT, Madras, under FIST and
BRNS for funding of project-2013.
inixic acid scaffold, Bioorg. Med. Chem. Lett. 21 (5) (2011) 1329e1333.
[13] M. Elkady, R. Niess, A.M. Schaible, J. Bauer, S. Luderer, G. Ambrosi, O. Werz,
S.A. Laufer, Modified acidic nonsteroidal anti-inflammatory drugs as dual in-
hibitors of mPGES-1 and 5-LOX, J. Med. Chem. 55 (20) (2012) 8958e8962.
[14] http://wikidiff.com/isocoumarin/coumarin.
[15] S. Pal, V. Chatare, M. Pal, Isocoumarin and its derivatives: an overview on their
synthesis and applications, Curr. Org. Chem. 15 (5) (2011) 782e800.
[16] W.-Z. Chen, L.-L. Fan, H.-T. Xiao, Y. Zhou, W. Xiao, J.-T. Wang, L. Tang, First
total synthesis of natural products cajanolactone A and cajanonic acid A, Chin.
Chem. Lett. 25 (5) (2014) 749e751.
[17] K. Sudarshan, M.K. Manna, I.S. Aidhen, Synthesis of 3-arylisocoumarins by
using acyl anion chemistry and synthesis of thunberginol A and cajanolact one
A, Eur. J. Org. Chem. 8 (2015) 1797e1803.
[18] K. Tianpanich, S. Prachya, S. Wiyakrutta, C. Mahidol, S. Ruchirawat,
P. Kittakoop, Radical scavenging and antioxidant activities of isocoumarins
and a phthalide from the endophytic fungus Colletotrichum sp. J. Nat. Prod. 74
(1) (2010) 79e81.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.066.
Abbreviations
[19] L.C. Di Stasi, D. Camuesco, A. Nieto, W. Vilegas, A. Zarzuelo, J. Galvez, Intestinal
anti-inflammatory activity of paepalantine, an isocoumarin isolated from the
capitula of Paepalanthus bromelioides, in the trinitrobenzenesulphonic acid
model of rat colitis, Plantamedica 70 (4) (2004) 315e320.
AA
COX-2 cyclooxygenase-2
EGCG epigallocatechingallate
EP receptor prostaglandin E2 receptor
arachidonic acid
~
[20] G. Qadeer, N.H. Rama, M. Garduno-Ramírez, Synthesis and anti-inflammatory
activity of fluorinated isocoumarins and 3, 4-dihydroisocoumarins, J. Fluor.
Chem. 128 (6) (2007) 641e646.
[21] H. Kumagai, T. Masuda, M. Ohsono, S. Hattori, H. Naganawa, T. Sawa,
M. Hamada, M. Ishizuka, T. Takeuchi, Cytogenin, a novel antitumor substance,
J. Antibiot. (Tokyo) 43 (11) (1990) 1505e1507.
FLAP
HIF1
5-LOX activating protein
hypoxia inducible factor 1 alpha
a
[22] T. Nakashima, S. Hirano, N. Agata, H. Kumagai, K. Isshiki, T. Yoshioka,
M. Ishizuka, K. Maeda, T. Takeuchi, Inhibition of angiogenesis by a new iso-
coumarin, NM-3, J. Antibiot. (Tokyo) 52 (4) (1999) 426e428.
[23] A. Obaid-ur-Rahman, K. Muhammad, T.H. Muhammad, H. Muhammad,
Q. Ghulam, H.R. Nasim, K. Alexander, M.K. Khalid, Synthesis and anti-cancer,
anti-metastatic evaluation of some new fluorinated isocoumarins and 3,4-
dihydroisocoumarins, J. Fluor. Chem. 135 (2012) 240e245.
[24] L. Du, Z. Zhang, X. Luo, K. Chen, X. Shen, H. Jiang, Binding investigation of
human 5-lipoxygenase with its inhibitors by SPR technology correlating with
molecular docking simulation, J. Biochem. 139 (4) (2006) 715e723.
[25] R. Wisastra, P.A. Kok, N. Eleftheriadis, M.P. Baumgartner, C.J. Camacho,
H.J. Haisma, F.J. Dekker, Discovery of a novel activator of 5-lipoxygenase from
an anacardic acid derived compound collection, Bioorg. Med. Chem. 21 (24)
(2013) 7763e7778.
5-HPETE 5(S) hydroperoxyeicosatetraenoic acid
13(S) HpODE 13(S)-hydroperoxyoctadecadienoic acid
IFN
IL6
g
interferon gamma
interleukin 6
5-LOX
LT
5-lipoxygenase
leukotriene
mPGES1 microsomal prostaglandin E2 synthase
NF-kB
PG
nuclear factor-
prostaglandin
k-light chain enhancer of activated B cells
TGF
b
transforming growth factor beta
[26] R. Briante, F. Febbraio, R. Nucci, Antioxidant properties of low molecular
weight phenols present in the Mediterranean diet, J. Agric. Food Chem. 51
(2003) 6975e6981.
References
[27] D. Villano, M.S. Fernandez-Pachon, M.A. Troncoso, M.C. Garcia-Parrilla, Com-
parison of antioxidant activity of wine phenolic compounds and metabolites
in vitro, Anal. Chim. Acta 538 (2005) 391e398.
[1] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454
(7203) (2008) 428e435.
[2] P.J. Jakobsson, S. Thoren, R. Morgenstern, B. Samuelsson, Identification of
ꢀ
[28] F. Natella, M. Nardini, M. di Felice, C. Scaccini, Benzoic and cinnamic acid
derivates as antioxidants: structure-activity relation, J. Agric. Food Chem. 47
(1999) 1453e1459.
human prostaglandin
inducible enzyme, constituting
Acad. Sci. U. S. A. 96 (1999) 7220e7225.
E
synthase:
a microsomal, glutathione-dependent,
a
potential novel drug target, Proc. Natl.
[29] M. Badeau, H. Adlercreutz, P. Kaihovaara, J.M. Tikkanen, Estrogen A-ring
structure and antioxidant effect on lipoproteins, J. Steroid Biochem. Mol. Biol.
96 (2005) 271e278.
[30] J. Jeong, S. Kang, I. Lee, J. Lee, H. Jung, C. Choi, Antioxidant and chemo-
sensitizing effects of flavonoids with hydroxyl and/or methoxy groups and
structure-activity relationship, J. Pharm. Pharm. Sci. 10 (4) (2007) 537e546.
www.cspsCanada.org.
[3] G.A. Green, Understanding NSAIDs: from aspirin to COX-2, Clin. Cornerstone 3
(5) (2001) 50e59.
[4] O. Rådmark, O. Werz, D. Steinhilber, B. Samuelsson, 5-lipoxygenase: regula-
tion of expression and enzyme activity, Trends Biochem. Sci. 32 (7) (2007)
332e341.
[5] M.A. Hull, S.C. Ko, G. Hawcroft, Prostaglandin EP receptors: targets for treat-
ment and prevention of colorectal cancer? Mol. Cancer Ther. 3 (8) (2004)
1031e1039.
[31] P.M. O'byrne, E. Israel, J.M. Drazen, Antileukotrienes in the treatment of
asthma, Ann. Intern. Med. 127 (6) (1997) 472e480.
~
[6] E. Terzuoli, S. Donnini, A. Giachetti, M.A. Iniguez, M. Fresno, G. Melillo,
[32] H.C. Steel, G.R. Tintinger, A.J. Theron, R. Anderson, Itraconazole-mediated in-
hibition of calcium entry into platelet-activating factor-stimulated human
neutrophils is due to interference with production of leukotriene B4, Clin. Exp.
Immunol. 150 (1) (2007) 144e150.
M. Ziche, Inhibition of hypoxia inducible factor-1alpha by dihydrox-
yphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E
synthase-1/vascular endothelial growth factor expression and reduces tu-
mor angiogenesis, Clin. Cancer Res. 16 (16) (2010) 4207e4216.
[33] S. Sinha, S. Thamini, S. Yuvaraj, M. Sreedharan, M. Doble, 2-Amino-4-aryl
thiazole : a promising scaffold identified as a potent 5-LOX inhibitor, RSC
Adv. 6 (2016) 19271e19279.
[7] X. Xue, Y.M. Shah, Hypoxia-inducible factor-2a is essential in activating the
COX2/mPGES-1/PGE2 signaling axis in colon cancer, Carcinogenesis 34 (1)
(2013) 163e169.
[8] Y. Li, S. Yin, D. Nie, S. Xie, L. Ma, X. Wang, Y. Wu, J. Xiao, MK886 inhibits the
[34] J.P. Falgueyret, J.H. Hutchinson, D. Riendeau, Criteria for the identification of

434
M. Ramanan et al. / European Journal of Medicinal Chemistry 124 (2016) 428e434
non-redox inhibitors of 5-lipoxygenase, Biochem. Pharmacol. 45 (4) (1993)
978e981.
[44] T. Motyl, K. Grzelkowska, W. Zimowska, J. Skierski, P. Ware,ski, T. Płoszaj,
L. Trzeciak, Expression of bcl-2 and bax in TGF- 1-induced apoptosis of L1210
b
[35] P. Sivakumar, P. Prabhakar, M. Doble, Synthesis, antioxidant evaluation, and
quantitative structureeactivity relationship studies of chalcones, Med. Chem.
Res. 20 (4) (2011) 482e492.
[36] W. Sha, C. Olesch, H. Hanaka, O. Rådmark, A. Weigert, B. Brüne, Necrosis in
DU145 prostate cancer spheroids induces COX-2/mPGES-1-derived PGE2 to
promote tumor growth and to inhibit T cell activation, Int. J. Cancer 133 (7)
(2013) 1578e1588.
[37] B.L. Fiebich, K. Lieb, N. Kammerer, M. Hull, Synergistic inhibitory effect of
ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary
rat microglia, J. Neurochem. 86 (1) (2003) 173e178.
[38] E. Wong, C. DeLuca, C. Boily, S. Charleson, W. Cromlish, D. Denis, S. Kargman,
B.P. Kennedy, M. Ouellet, K. Skorey, G.P. O'Neill, P.J. Vickers, D. Riendeau,
Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2)
in human osteosarcoma cells, Inflamm. Res. 46 (2) (1997) 51e59.
[39] Y. Li, S. Yin, D. Nie, S. Xie, L. Ma, X. Wang, Y. Wu, Effect of mPGES-1 inhibitor
MK886 on apoptosis and drug resistance of HL-60/A cells, J. Exp. Hematol.
Chin. Assoc. Pathophysiol. 20 (4) (2012) 829e834.
leukemic cells, Eur. J. Cell Biol. 75 (4) (1998) 367e374.
[45] J. Scheller, A. Chalaris, D. Schmidt-Arras, S. Rose-John, The pro-and anti-in-
flammatory properties of the cytokine interleukin-6, Biochim. Biophys. Acta
(BBA) Mol. Cell Res. 1813 (5) (2011) 878e888.
[46] A. Koeberle, O. Werz, Multi-target approach for natural products in inflam-
mation, Drug Discov. Today 19 (12) (2014) 1871e1882.
[47] G.W. Carter, P.R. Young, D.H. Albert, J. Bouska, R. Dyer, R.L. Bell, J.B. Summers,
D. Brooks, 5-lipoxygenase inhibitory activity of zileuton, J. Pharmacol. Exp.
Ther. 256 (3) (1991) 929e937.
[48] D. Riendeau, R. Aspiotis, D. Ethier, Y. Gareau, E.L. Grimm, J. Guay, S. Guiral,
ꢀ
H. Juteau, J.A. Mancini, N. Methot, Inhibitors of the inducible microsomal
prostaglandin E 2 synthase (mPGES-1) derived from MK-886, Bioorg. Med.
Chem. Lett. 15 (14) (2005) 3352e3355.
[49] M.G. Sciulli, F. Seta, S. Tacconelli, M.L. Capone, E. Ricciotti, G. Pistritto,
P. Patrignani, Effects of acetaminophen on constitutive and inducible pros-
tanoid biosynthesis in human blood cells, Br. J. Pharmacol. 138 (4) (2003)
634e641.
ꢀ
[40] H. Mühl, J. Pfeilschifter, Anti-inflammatory properties of pro-inflammatory
[50] T. Båge, T. Modeer, T. Kawakami, H.C. Quezada, T. Yucel-Lindberg, Regulation
interferon-
g
, Int. Immunopharmacol. 3 (9) (2003) 1247e1255.
of prostaglandin E synthases: effects of siRNA-mediated inhibition of micro-
somal prostaglandin E synthase-1, Biochim. Biophys. Acta (BBA) Mol. Cell Res.
1773 (10) (2007) 1589e1598.
[41] S. Wahl, J. Allen, K. Ohura, D. Chenoweth, A. Hand, IFN-gamma inhibits in-
flammatory cell recruitment and the evolution of bacterial cell wall-induced
arthritis, J. Immunol. 146 (1) (1991) 95e100.
[51] F. Cianchi, C. Cortesini, L. Magnelli, E. Fanti, L. Papucci, N. Schiavone,
L. Messerini, A. Vannacci, S. Capaccioli, F. Perna, Inhibition of 5-lipoxygenase
by MK886 augments the antitumor activity of celecoxib in human colon
cancer cells, Mol. Cancer Ther. 5 (11) (2006) 2716e2726.
[42] P. Bernabei, E.M. Coccia, L. Rigamonti, M. Bosticardo, G. Forni, S. Pestka,
C.D. Krause, A. Battistini, F. Novelli, Interferon-g receptor 2 expression as the
deciding factor in human T, B, and myeloid cell proliferation or death,
J. Leukoc. Biol. 70 (6) (2001) 950e960.
[43] V.R. Baichwal, P.A. Baeuerle, Apoptosis: activate NF-kB or die? Curr. Biol. 7 (2)
[52] S. Thoren, P.J. Jakobsson, Coordinate up-and down-regulation of glutathione-
dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells, Eur.
J. Biochem. 267 (21) (2000) 6428e6434.
(1997) R94eR96.