Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential therapeutics for inflammatory bowel disease

Article abstract of DOI:10.1016/j.ejmech.2017.06.018

To develop effective therapeutics for inflammatory bowel disease (IBD), 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives, were designed and synthesized and their structure-activity relationships (SAR) were investigated. Compounds 7, 25, 26, 32, 39, 41, 52, 54, and 55 showed potent inhibitory effect (>70%) on the TNF-α-induced adhesion of monocytes to colon epithelial cells, which is one of the hallmark events leading to IBD. Such inhibitory activity of the compounds correlated with their suppressive activities against the TNF-α-induced production of ROS; ICAM-1 and MCP-1 expression, critical molecules involved in monocyte-epithelial adhesion; and NF-κB transcriptional activity. In addition, compounds 41 and 55 significantly suppressed the lipopolysaccharide (LPS)-induced expression of the TNF-α gene, with compound 55 showing better efficacy. This inhibition of TNF-α expression by compounds 41 and 55 corresponded to their additional inhibitory activity against AP-1 transcriptional activity, which is another transcription factor required for high level TNF-α expression. The strong inhibitory activity of compound 55 against an in vivo colitis model was confirmed by its dose-dependent inhibitory activity in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, demonstrating compound 55 as a new potential candidate for the development of therapeutics against IBD.

Full text of DOI:10.1016/j.ejmech.2017.06.018

Accepted Manuscript
Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-
inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential
therapeutics for inflammatory bowel disease
Tara Man Kadayat, Suhrid Banskota, Pallavi Gurung, Ganesh Bist, Til Bahadur Thapa
Magar, Aarajana Shrestha, Jung-Ae Kim, Eung-Seok Lee
PII:
S0223-5234(17)30464-6
10.1016/j.ejmech.2017.06.018
EJMECH 9513
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 March 2017
Revised Date: 17 May 2017
Accepted Date: 8 June 2017
Please cite this article as: T.M. Kadayat, S. Banskota, P. Gurung, G. Bist, T.B.T. Magar, A. Shrestha,
J.-A. Kim, E.-S. Lee, Discovery and structure-activity relationship studies of 2-benzylidene-2,3-
dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential therapeutics
for inflammatory bowel disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.06.018.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-
inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential
therapeutics for inflammatory bowel disease
#
#
Tara Man Kadayat , Suhrid Banskota , Pallavi Gurung, Ganesh Bist, Til Bahadur Thapa
Magar, Aarajana Shrestha, Jung-Ae Kim*, Eung-Seok Lee*
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
#
Authors are equal contributors.
*
Corresponding Authors:
email: jakim@yu.ac.kr (J.-A.K.)
email: eslee@yu.ac.kr (E.-S.L.)
1

ACCEPTED MANUSCRIPT
Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-
inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential
therapeutics for inflammatory bowel disease
#
#
Tara Man Kadayat , Suhrid Banskota , Pallavi Gurung, Ganesh Bist, Til Bahadur Thapa
Magar, Aarajana Shrestha, Jung-Ae Kim*, Eung-Seok Lee*
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
Abstract
To develop effective therapeutics for inflammatory bowel disease (IBD), 2-benzylidene-2,3-
dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives, were designed and
synthesized and their structure-activity relationships (SAR) were investigated. Compounds 7,
2
5, 26, 32, 39, 41, 52, 54, and 55 showed potent inhibitory effect (>70%) on the TNF-α-
induced adhesion of monocytes to colon epithelial cells, which is one of the hallmark events
leading to IBD. Such inhibitory activity of the compounds correlated with their suppressive
activities against the TNF-α-induced production of ROS; ICAM-1 and MCP-1 expression,
critical molecules involved in monocyte-epithelial adhesion; and NF-κB transcriptional
activity. In addition, compounds 41 and 55 significantly suppressed the lipopolysaccharide
(
LPS)-induced expression of the TNF-α gene, with compound 55 showing better efficacy.
This inhibition of TNF-α expression by compounds 41 and 55 corresponded to their
additional inhibitory activity against AP-1 transcriptional activity, which is another
transcription factor required for high level TNF-α expression. The strong inhibitory activity
of compound 55 against an in vivo colitis model was confirmed by its dose-dependent
inhibitory activity in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis,
demonstrating compound 55 as a new potential candidate for the development of therapeutics
against IBD.
Keywords: Inflammatory Bowel Disease; 2-Benzylidene-2,3-dihydro-1H-inden-1-one;
2

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Benzofuran-3(2H)-one; TNF-α; Structure-activity relationship
1
. Introduction
Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammatory
disease in the gastrointestinal tract [1]. The etiology of IBD is associated with a complex
interaction between genetic, environmental, and intestinal immune factors. Changes in the
innate immune response constitutes the pathogenic basis of the disease [2]. Among various
cytokines, tumor necrosis factor-α (TNF-α) is the major pro-inflammatory cytokine involved
in IBD. TNF-α perpetuates the inflammatory process by inducing the expression of other pro-
inflammatory cytokines and adhesion molecules, which is required for leukocyte attachment
and infiltration through the intestinal mucosa [3-6]. This recruitment of more inflammatory
cells into the damaged intestinal epithelium is one of the critical steps in the inflammation
and tissue injury encountered in IBD [7, 8]. TNF-α inhibitors have great benefit in the
treatment of chronic inflammatory conditions, including IBD by suppressing the expression
of pro-inflammatory cytokines and adhesion molecules, unlike cases of abscesses and
granulomatous infections, where the therapeutic blockade of TNF-α worsens the disease
prognosis. Until now, the most effective treatment for IBD patients is with an anti-TNF-α
antibody, such as infliximab [9-11]. Recently, another class of leukocyte migration inhibitors,
such as vedolizumab, which inhibits lymphocyte-endothelial cell adhesion, is launched in the
clinic [12]. On the other hand, the use of these expensive antibody therapeutics has some
adverse effects, such as serious infection, immunosuppression, and malignancies including
lymphoma [13-15]. To overcome these problems of biological agents, medicinal chemists are
in a continuous search for safer and more effective IBD therapeutics. In a recent study, an
orally active therapeutic candidate was derived by lanthionine synthetase C-like 2
(
LANCL2)-based screening; the candidate compound showed low bioavailability and no
3

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toxic effect, demonstrating its local action [16]. Regarding the complex etiology of IBD,
phenotype-based screening was attempted instead of a specific target-based approach to
develop an effective therapeutic inhibiting the pathological action of TNF-α [17-19].
Previously, the authors reported the anti-inflammatory and anti-angiogenic activity of a
propenone compound, 1-furan-2-yl-3-pyridin-2-ylpropenone (FPP-3), which is mediated by
the suppression of TNF-α induction and NF-κB activation [20-22]. Moreover, 1,3-
diphenylpropenone (DPhP) inhibited the TNF-α-induced adhesion of monocytes (U937 cells)
to colon epithelial cells (HT-29 cells) in vitro, and exhibited protective activity against the
trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model [23]. Based on these findings,
we aimed to optimize TNF-α-inhibitory activity of new compounds by modifying the A-ring
of DPhP with dihydro-1H-inden-1-one and benzofuran-3(2H)-one. Modifications were also
made to the B-ring with various substituted aryl or heterocyclic rings (Figure 1A).
Dihydro-1H-inden-1-one
A
R
1
B
A
O
Modification to A/B-ring
O
O
O
O
R
1
FPP-3
DPhP
O
Benzofuran-3(2H)-one
B
Modification to
Phenyl Ring
R
1
Hydroxylation at
Dihydro-1H-inden-1-one
R
1
HO
O
O
O
3
3-52
1
2-24
R1
Hydroxylation at
Benzofuran-3(2 )-one
2
-, 3-, or 4-Hydroxyphenyl
2-, 3-, or 4-Chlorophenyl
-, 3-, or 4-Fluorophenyl
-, 3-, or 4-Trifluoromethylphenyl
-, 3-, or 4-Trifluoromethoxylphenyl
2-, 3-, or 4-Pyridyl, Aryl
HO
R
1
O
O
O
R
1
H
2
2
2
O
5
3-58
2
5-32
Figure 1. Strategy for the design of the compounds.
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The 1-indanone moiety, which is found in a range of natural products is a valuable molecule
in pharmaceuticals. Several studies have shown that compounds possessing the indanone
moiety are of great pharmacological importance because they exhibit various biological
activities, such as anticancer, analgesic, anti-inflammatory, and antimicrobial properties [24-
2
7]. In addition, indanones are used as synthetic intermediates in the area of medicinal
chemistry research [28-33]. 2-Benzylidene indanone derivatives have cholinesterase
inhibitory activity for the treatment of Alzheimer’s disease [34]. Recently, 2-benzylidene
indanone analogues showing antitumor activity as tubulin polymerization inhibitors have
been described [35, 36]. Similarly, 2-benzylidene benzofuranones, naturally occurring
flavonoids, have been reported to possess chemopreventive, antifungal, antiviral, anticancer,
antibacterial, anti-tyrosinase, and anti-inflammatory activities [37-43]. Haudecoeur et al. [44]
described 2-benzylidene benzofuran-3(2H)-one as a hepatitis C Virus RNA-dependent RNA
polymerase inhibitor. A recent study has highlighted a series of polyhydroxylated benzofuran-
3
(2H)-one as a potential scaffold interfering with tau aggregation for the treatment of
Alzheimer’s disease [45].
As reported elsewhere [45-48], the introduction of hydroxyl, chloride, and fluoride
functionalities on the phenyl ring or substituted aryl group is important in drug discovery
research. These reported biological activities and the results of previous studies on propenone
derivatives, encouraged the development of a novel class of therapeutics for IBD. Herein, this
paper reports the design and synthesis of 2-benzylidene-2,3-dihydro-1H-inden-1-one and
benzofuran-3(2H)-one analogues containing pyridyl, hydroxyphenyl, chlorophenyl,
fluorophenyl, tri-fluoromethylphenyl, tri-fluoromethoxyphenyl, and various substituted aryl
moieties (Figure 1B). In addition, the TNF-α inhibitory effect, and their structure-activity
5

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relationships (SAR) are evaluated. Although, several studies revealed various biological
activities of compounds containing indanone and benzofuranone analogues, there are no
reports describing the synthesis and evaluation of the inhibitory effect of 2-benzylidene-2,3-
dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives against TNF-α nor has there
been any SAR study for the development of anti-IBD agents. To the best of the authors’
knowledge, this is the first study reporting 2-benzylidene indanone and benzofuranone
analogues as TNF-α inhibitor for discovering novel active small molecules as potential IBD
therapeutic agents. Compound 55, which showed strong inhibitory activities against TNF-α-
induced monocytic-colonic epithelial cell adhesion as well as LPS-induced TNF-α expression
was described as a potential candidate for IBD drug development by examining the
concentration-dependency of its inhibitory activity against colitis in vitro and in vivo animal
model of IBD.
2
. Chemistry
2
-Benzylidene-2,3-dihydro-1H-inden-1-one (compound 1) and its analogues for the SAR
exploration was synthesized using previously reported methods [28, 38, 48-51]. Initially,
compounds 1-24 were synthesized in yields ranging from 46.9-99.8% based on the aldol
condensation reaction of 1-indanone (I) and various aryl aldehydes, II (R=a-x) in the
presence of 5% aqueous NaOH in ethanol, as shown in Scheme 1. The introduction of ortho-,
meta-,
or
para-hydroxyphenyl/chlorophenyl/fluorophenyl/tri-fluoromethylphenyl/tri-
fluoromethoxyphenyl and substituted aryl groups in the R-position allowed to investigate
their effect in biological activity.
6

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R
O
Aq. NaOH
H
R
O
O
EtOH/H O
2
I (1-indanone)
II (R=a-x)
1 - 24
OH
OH
Cl
HO
N
R
=
N
N
a
b
c
d
e
f
g
h
Cl
F
Cl
F
CF3
F
Cl
Cl
O
S
i
j
k
l
m
n
o
p
CF3
OCF3
NO2
F3C
OCF3
F3CO
q
r
s
t
u
v
w
x
Scheme 1. Schematic representation of the synthesis of non-hydroxylated dihydro-1H-inden-
-ones 1-24 with ortho-, meta-, para-hydroxy/chloro/fluoro/tri-fluoromethyl/tri-
fluoromethoxy and substituted aryl groups. Reagents and conditions: aq. 5% NaOH, Ethanol,
-12 h, 0° or 25°C, 46.9-99.8% yield.
1
1
Analogues of 2-benzylidene benzofuranone (compounds 25-32) were synthesized, as shown
in Scheme 2. All target compounds were obtained in good yield (51.0-89.6%) by treating
benzofuranone (III) with aryl aldehydes, II (R=e-l) in the presence of aluminum oxide
(
Al O ) in dichloromethane (CH Cl ) or 5% aqueous KOH in methanol [38, 48]. Considering
2 3 2 2
the excellent inhibitory activity of compounds 5, 7, 25-27, and 30-32 (Table 1 and 2) and
importance of hydroxyphenyl and chlorophenyl groups in medicinal chemistry, a series of
hydroxylated 2-benzylidene indanone and benzofuranone analogues (Scheme 3A and 3B)
were prepared in 28.8 to 99.5% yields using the method reported elsewhere [50, 51]. Using
the thionylchloride (SOCl ) catalyzed aldol condensation reaction, various commercially
2
7

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available hydroxy-indanone (IVa-d) or 6-hydroxy-benzofuranone was treated with aryl
aldehydes, II (R =e-k) to afford compounds 34, 35, 37, 38, 40, 41, 43-52, and 54-58. On the
1
other hand, compounds 33, 36, 39, 42, and 53 bearing a hydroxyl group in the ortho-position
of the phenyl ring were difficult to synthesize or resulted very low yields; hence, they were
prepared using the 5M NaOH catalyzed aldol condensation reaction.
Scheme 2. Schematic diagram of the synthesis of non-hydroxylated benzofuran-3(2H)-ones
2
5-32. Reagents and conditions: (i) Al O , Dichloromethane, 25°C; (ii) aq. 5% KOH,
2 3
Methanol,1-3 h, 0° or 25°C, 51.0-89.6% yield.
8

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Scheme 3. Schematic representation for the synthesis of (A) hydroxylated dihydro-1H-inden-
1
-ones 33-52, and (B) hydroxylated benzofuran-3(2H)-ones 53-58. Reagents and conditions:
(
i) aq. 5M NaOH, Ethanol, 1-6 h, at 0° C; (ii) SOCl , Ethanol, 2-12 h, 25°C, 28.8 to 99.5%
2
yield.
All synthesized compounds were characterized by liquid chromatography mass spectrometry
1
13
(
LCMS), H nuclear magnetic resonance (NMR) spectroscopy, and C NMR spectroscopy
The purity of all the compounds tested was determined by analytical HPLC, and unless stated
otherwise, the purity was ≥95%. The stereochemistry of the exocyclic double bond in all
1
synthesized compounds may be E or Z. These configurational isomers have characteristic H
chemical shifts in the NMR spectrum. The chemical shift of the olefinic proton (=CH-) is
deshielded in the E isomer of 2-benzylidene indanone derivatives and appears downfield
(above 7 ppm) compared to the Z isomer (less than 7 ppm). In the case of the 2-benzylidene
9

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benzofuranone derivatives the exocyclic double bond is in Z. The chemical shift of the
olefinic proton (=CH-) is shielded in the Z isomer and appears upfield (less than 7 ppm).
Table S1 and S2 of Supplementary Data present the physicochemical characterization data,
such as yield (%), purity by HPLC (%), and melting point (°C).
3
3
. Results and Discussion
.1. Inhibitory activity of synthesized compounds on TNF-α-induced adhesion of
monocytes to colon epithelial cells
The recruitment of leukocytes in the mucosa by TNF-α, a major pro-inflammatory cytokine,
is an important step of intestinal inflammation in IBD. In the screening of effective
compounds against the intestinal inflammation in vitro, the inhibitory activities of all the
synthesized compounds (1-58) at a concentration of 10 µM against TNF-α-induced adhesion
of monocytes to HT-29 human colonic epithelial cells were measured (Table 1-4). 5-
Aminosalicylate (5-ASA), which is the active metabolite of the clinically used IBD drug,
sulfasalazine (SSZ), was used as a reference compound for the assay. Compared to 5-ASA,
which showed 3.7% inhibition at 10 µM and 52.8% inhibition at 20 mM against the TNF-α-
induced adhesion of monocytic cells to colonic epithelial cells, the majority of the
synthesized compounds exhibited almost five to twenty-fold better inhibitory activity.
Interestingly, 2-benzylidene-indanone and benzofuranone derivatives were more effective in
inhibiting the TNF-α-induced adhesion of monocytic cells to colonic epithelial cells than
previously reported propenone derivatives [23].
As shown in Table 1, in the series of non-hydroxylated indanone analogues 1-24, compound
1
synthesized as a parent compound was found to be more potent (55.3% inhibition) than 5-
ASA. Among compounds 2-24, most of the compounds showed better activity than 5-ASA,
1
0

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with compound 7 with a para-hydroxyphenyl ring showing the highest inhibition (72.6%) at
1
0 µM concentration. Similarly, compounds 4, 5, 6, 16, 17, 22, and 23 exhibited more than
ten-fold higher inhibition than 5-ASA at 10 µM. Similarly, in the series of non-hydroxylated
benzofuranone derivatives (25-32), all the compounds showed more than ten times higher
inhibitory activity than the 5-ASA against the TNF-α -induced adhesion of monocytes to
human colonic epithelial cells (40-85.6% inhibition). Among them, compounds 25, 26, and
3
2 with ortho- and meta-hydroxyphenyl ring and 2-chloro thiophene ring, respectively,
showed more than 70% inhibition (Table 2).
1
1

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Table 1. Structures and inhibitory activities on TNF-α-induced adhesion of monocytes to
colon epithelial cells for non-hydroxylated dihydro-1H-inden-1-ones 1-24.
a
1
0 µM concentration except for 5-ASA (20 mM).
b
The data represents the mean ± S.E.M. from three different experiments performed in
triplicate.
1
2

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Table 2. Structures and inhibitory activities on TNF-α-induced adhesion of monocytes to
colon epithelial cells for non-hydroxylated benzofuran-3(2H)-ones 25-32.
a
1
0 µM concentration except for 5-ASA (20 mM).
b
The data represents mean ± S.E.M. from three different experiments performed in triplicate.
A series of hydroxylated 2-benzylidene indanone derivatives (33-52) were prepared to
investigate the SAR of the hydroxyl substitution (at C-4, C-5, C-6, and C-7 position) on the
indanone ring. Among them, compounds 39, 41, and 52 exhibited 71.3%, 80.3%, and 78.4%
inhibition, respectively, against the TNF-α-induced adhesion of monocytes (Table 3).
Similarly, in the series of 6-hydroxy benzofuranone derivatives (53-58), all the compounds
showed more than twelve time better inhibitory activity than 5-ASA (Table 4). Compounds
5
4 and 55 displayed more than 70% inhibitory effect against TNF-α-induced adhesion.
1
3

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Table 3. Structures and inhibitory activities on the TNF-α-induced adhesion of monocytes to
colon epithelial cells for hydroxylated dihydro-1H-inden-1-ones 33-52.
a
1
0 µM concentration except for 5-ASA (20 mM).
b
The data represents mean ± S.E.M. from three different experiments performed in triplicate.
1
4

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Table 4. Structures and inhibitory activities on the TNF-α-induced adhesion of monocytes to
colon epithelial cells for 6-hydroxylated benzofuran-3(2H)-ones 53-58.
a
1
0 µM concentration except for 5-ASA (20 mM).
b
The data represents the mean ± S.E.M. from three different experiments performed in
triplicate.
3
.2. Structure-activity relationship (SAR) studies
One of the major focuses of this study was to demonstrate the SAR of the synthesized
compounds with their inhibitory effects on the TNF-α-induced adhesion of monocytes to
colon epithelial cells by introducing different substituents in the indanone and benzofuranone
derivatives. First, the SAR of the parent compound (1) and its analogues 2-24, which contain
pyridyl, hydroxyphenyl, chlorophenyl, fluorophenyl, tri-fluoromethylphenyl, tri-
fluoromethoxyphenyl, and various substituted aryl moieties, was investigated. The results
listed in Table 1 indicated that the introduction of phenyl, pyridyl, hydroxyphenyl,
fluorophenyl, tri-fluoromethylphenyl, and tri-fluoromethoxyphenyl groups at the R-position
1
5

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of 1-indanone analogues was favorable for inhibitory activities. The addition of a hydroxyl
substituent at the para-position (7) of the phenyl ring of the 2-benzylidene indanone (1) was
crucial for the potent inhibitory activity. Interestingly, the presence of chlorine functionality
(8-12) resulted in a loss of inhibitory activities of the compounds, which suggests that
chlorine substitution in 2-benzylidene indanone analogues is not important for its activity.
On the other hand, all the benzofuranone containing compounds, 25-32, exhibited strong
inhibitory activities. It is interesting to note that chloro-substituted benzofuranones (28-32)
showed relatively stronger inhibitory effects than those of the corresponding indanone
analogues (8-12), which showed very weak inhibitory effects. The results listed in Table 2
indicated that the presence of chlorine or hydroxyl functionalities in 2-benzylidene
benzofuranone analogues is important for the inhibitory activities against the TNF-α-induced
adhesion of monocytes to HT-29 human colonic epithelial cells.
The SAR study of the hydroxylated 2-benzylidene indanone derivatives was performed (33-
5
2). From the results listed in Table 3, the position of the hydroxyl (-OH) group on the
indanone ring is important for its inhibitory activity. Dihydroxylated compounds 33-35,
which contain a -OH group at the C-4 position and ortho-, meta-, or para-hydroxyl group at
the phenyl ring of 2-benzylidene indanone (1) showed relatively weaker inhibitory activity.
On the other hand, the presence of a –OH group at the C-5, C-6, or C-7 position (36-44)
resulted in strong inhibitory activity. Compound 41 bearing a -OH (R) group at the C-6
position and para-hydroxyphenyl group at the R position showed the highest inhibitory
1
activity (80.3%). Interestingly, compounds 39-44, 51, and 52 containing a -OH (R) group at
the C-6 and C-7 position of indanone, and a phenol or para-cholorphenyl group at the R1
position displayed better inhibitory activity (60.5-80.3% inhibition) than the parent
1
6

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compound 1. These results confirmed that the introduction of a –OH group at C-6 or C-7
position of indanone is crucial for its inhibitory activity. The further introduction of a
commercially available 3-hydroxy-4-methoxyphenyl functional group at the R position of 4-,
1
5
-, 6-, and 7-hydroxy-indanone (45-48) showed low inhibitory activity. In addition, to
illustrate the importance of the hydroxyl group in the benzofuranone moiety, compounds 53-
8 were prepared using commercially available 6-hydroxy benzofuranone. The results listed
5
in Table 4 show that the addition of –OH group at the C-6 position of the benzofuranone ring
is important for its activity. Compound 55 with para-hydroxyphenyl ring showed the highest
inhibition (80.3%) at 10 µM, and the inhibition was concentration-dependent (Figure 2).
Figure 3 summarizes the overall SARs of all the synthesized compounds.
Figure 2. Inhibition of TNF-α-induced monocyte-epithelial adhesion in concentration
dependent manner by compound 55. HT-29 cells and 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-
Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) fluorescence-labeled U937cells
were pretreated with different concentrations of compound 55 for 1 h prior to the treatment
with TNF-α. (A) Representative images of the adhesion of monocyte to epithelial cells by
fluorescence microscopy. (B) The logarithm plot shows the % inhibition of adhesion.
1
7

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Figure 3. Summary of the SAR of the prepared compounds.
3
.3. Selected compounds inhibit TNF-α-induced adhesion and chemokine molecules in
HT-29 colonic epithelial cells
Based upon the inhibitory activity of the prepared compounds on the TNF-α-induced
adhesion of monocytes to colon epithelial cells, nine compounds exhibiting more than 70%
inhibitory activity (Figure 4) were selected and further examined for their IC values. Table 5
50
lists the IC value of the selected compounds including compound 1. The IC value for the
5
0
50
most effective compound 55 was 0.50±0.05 µM, which was 40,000 times stronger than 5-
ASA (IC : 20.4±2.2 mM), and the least effective compound 1 was 5.75 ± 0.94 µM (Table 5).
5
0
To determine their mode of action, this study first investigated whether the inhibitory effects
of the nine compounds on the TNF-α-induced monocyte-epithelial adhesion were due to the
suppression of adhesion molecule expression in HT-29 cells. TNF-α-induced expression of
intercellular adhesion molecule (ICAM)-1 was suppressed significantly by the selected
compounds (Figure 5A). The order of the inhibitory activity of the nine compounds was 55 >
1
8

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4
1 > 25 > 52 > 32 > 26 > 54 > 7 > 39, all of which had stronger activity than parent
compound 1. Compound 55 showed the strongest and most concentration-dependent
inhibitory activities (Figure 5B).
Figure 4. Compounds exhibiting more than 70% inhibitory activities on the TNF-α-induced
adhesion of monocytes to colon epithelial cells at 10 µM.
Table 5. IC50 values of the selected compounds against TNF-α-induced adhesion of
monocytes to colon epithelial cells [µM concentration except for 5-ASA (mM)].
a
c
Compound
IC50 (µM)
1
7
5.75 ± 0.94
1.53 ± 0.23
0.95 ± 0.13
3.17 ± 0.21
3.72 ± 0.17
2.29 ± 0.19
0.83 ± 0.12
1.64 ± 0.29
1.68 ± 0.50
0.50 ± 0.05
20.4 ± 2.2 (mM)
25
26
32
39
41
52
54
55
b
5
-ASA
a
b
µM concentration for compounds; mM concentration of 5-ASA.
The data represents mean ±ꢀS.E.M. from three different experiments performed in
triplicate.
c
1
9

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Figure 5. Inhibitory effects of the selected compounds on TNF-α-induced ICAM-1
expression. (A) HT-29 cells were pretreated with each compound (10 µM) and 5-ASA (20
mM) for 1 h prior to the treatment with TNF-α (10 ng/ml) for another 3 h. The extracted
proteins were then analyzed for ICAM-1. (B) The cells were pretreated with different
concentrations of compound 55 prior to a treatment with TNF-α and the extracted proteins
were analyzed for the ICAM-1 expression. The bar diagram represents the quantitative
#
change in the expression of ICAM-1. *P<0.05 vs. the vehicle-treated controls. P<0.05 vs.
TNF-α-treated group.
2
0

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In addition, the TNF-α-induced expression of monocyte chemoattractant protein (MCP)-1,
one of the chemokines that regulate the movement of circulating leukocytes to the site of
inflammation [52], was suppressed significantly in a similar order as ICAM-1 by all the
compounds (Figure 6A) with compound 55 showing the best and concentration-dependent
activity (Figure 6B).
Figure 6. Inhibitory effects of the selected compounds on the TNF-α-induced expression of
monocyte chemoattractant protein (MCP-1). (A) The protein extracts obtained from the cells
pretreated with each compound (10 µM) and 5-ASA (20 mM) prior to the treatment with
TNF-α, were analyzed for MCP-1 expression. (B) Effect of compound 55 on TNF-α-induced
MCP-1 in a concentration–dependent manner. The graph represents the quantitative change in
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the expression of MCP-1. *P<0.05 vs. the vehicle-treated controls. P<0.05 vs. TNF-α-
treated group.
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.4. Inhibitory effects of the selected compounds on TNF-α-induced NF-κB
transcription activity in HT-29 cells
Upon binding to the TNFR receptors, TNF-α activates the signaling pathway. This leads to
the activation of nuclear factor-κB (NF-κB), which is the most well-known transcription
factor regulating many target genes, such as adhesion molecules, cytokines, and chemokines
[
53, 54]. This study examined whether the inhibitory effects of the compounds on the
expression of ICAM-1 and MCP-1 were associated with the inhibition of NF-κB
transcriptional activity. In a NF-κB dual-luciferase reporter assay, the TNF-α-induced
increase in NF-κB transcriptional activity was inhibited significantly by the nine selected
compounds (Figure 7). The inhibitory activities of these compounds were better than those of
the propenone derivatives reported in a previous study [23]. The order of the NF-κB
inhibitory activity of the compounds was 55 > 41 > 25 > 7 > 32 > 54, 52 > 26, 39 > 1. The
order of the NF-κB inhibitory activity of 55 and 41 corresponded to the inhibitory effects of
the compounds on ICAM-1 and MCP-1 expression.
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Figure 7. Selected compounds inhibiting the TNF-α-induced NF-ĸB transcriptional activity.
The HT-29 cells were transfected with the luciferase reporter plasmid containing the human
NF-ĸB responsive element. The reporter activity in the transfected cells was accessed after
the cells were pretreated with 5-ASA (20 mM) and the indicated compounds (10 µM) prior to
the treatment with TNF-α for 3 h. The bar graphs indicates the meanꢀ±ꢀS.E.M. from three
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independent experiments. *Pꢀ<ꢀ0.05 compared to the vehicle-treated control group. Pꢀ<ꢀ0.05
compared to the TNF-α -treated group.
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.5. Selected compounds inhibit TNF-α-induced ROS production in HT-29 cells
As NF-κB is a redox-sensitive transcription factor [55] and reactive oxygen species (ROS)
are crucial mediators for NF-κB activation in downstream signaling of TNF-α [56, 57], this
study also examined the inhibitory activities of the compounds on the TNF-α-induced
production of ROS. The TNF-α (10 ng/ml)-induced generation of ROS was inhibited
significantly by the compounds with the sequential order of 55 > 41 > 25 > 32 > 7 > 54, 52 >
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9 > 26. They had stronger activity than parent compound 1 (Figure 8A). The effects of
compound 55, which showed concentration-dependent activity (Figure 8B), were similar to
those of apocynin (100 µM), a reference antioxidant compound.
Figure 8. Inhibitory effects of the selected compounds on TNF-α-induced ROS production.
(
A) HT-29 cells were pretreated with apocynin (100 µM) or indicated compounds (10 µM)
for 1 h prior to treatment with TNF-α for 30 min. The intracellular ROS level was
determined by fluorescence microscopy using 2′,7′-dichlorofluorescin diacetate DCF-DA).
(
The bar graph represents the quantitation of the fluorescence intensity. *Pꢀ<ꢀ0.05 compared to
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the vehicle-treated control group. Pꢀ<ꢀ0.05 compared to the TNF-α-treated group. (B) HT-29
cells were pretreated with the indicated concentration of compound 55 prior to the treatment
with TNF-α and the intracellular ROS using DCF-DA (10 µM) determined by fluorescence
microscopy. The bar represents the quantitation of ROS production in terms of the
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fluorescence intensity. *Pꢀ<ꢀ0.05 compared to the vehicle-treated control group. Pꢀ<ꢀ0.05
compared to TNF-α -treated group.
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.6. Compounds 41 and 55 inhibit LPS-induced TNF-α expression
In addition to the inhibitory activities of the nine compounds, this study further examined the
effectiveness of the compounds on the suppression of TNF-α gene expression, which is also
regulated by NF-κB. To induce the maximum expression of TNF-α, the cells were stimulated
with lipopolysaccharide (LPS). Unlike the NF-κB inhibitory activity of the nine compounds,
LPS-induced TNF-α expression was inhibited by 41 and 55, 55 being stronger than 41
(Figure 9A). In the case of the reference compound, 5-ASA at the same concentration (10 µM)
as 41 and 55 did not have an inhibitory effect on LPS-induced TNF-α expression, but at high
concentrations (20 mM), it inhibited LPS-induced TNF-α expression significantly (Figure
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B). In contrast, compound 55 showed concentration-dependent suppression, and at a 10 µM
concentration, its inhibitory effect was strong enough to reduce TNF-α expression to the level
of the LPS-untreated controls (Figure 9C).
Figure 9. Inhibitory effects of compounds 41 and 55 on LPS-induced TNF-α expression. (A)
After pretreatment with compounds or 5-ASA (20 mM) for 1 h, the cells were treated with
LPS (1µg/ml) for another 3 h. The total proteins were extracted and analyzed for TNF-α
expression. (B) Effect of 5-ASA (20 mM) on LPS-induced TNF-α. (C) The bar diagram
represents the quantitative change in TNF-α expression. *P<0.05 compared to the vehicle
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treated control group. P<0.05 compared to the LPS alone treated group.
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.7. Compounds 41 and 55 inhibit AP-1 transcriptional activity
To differentiate actions of compounds 41 and 55 from the other selected compounds in the
suppression of TNF-α expression, this study further examined whether the two compounds
may have additional activity except for the NF-κB inhibitory activity. The inhibitory effects
of 41 and 55 on LPS-induced TNF-α expression suggest that these compounds may have AP-
1
inhibitory activity because LPS-induced maximal expression of TNF-α is regulated not
only by NF-κB, but also by other transcription factors, such as AP-1 [58, 59]. In an AP-1
luciferase reporter assay, the TNF-α-enhanced AP-1 transcriptional activity was blocked by
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1 and 55 (Figure 10). As IBD is a complex disease, compound 55, which showed strong
inhibitory activities against TNF-α-induced monocytic-colonic epithelial cell adhesion as
well as LPS-induced TNF-α expression, is as an excellent candidate for IBD drug
development.
Figure 10. Inhibitory effects of compounds 41 and 55 on TNF-α-induced AP-1
transcriptional activity. The reporter activity was accessed in HT-29 cells transfected with the
luciferase reporter plasmid containing the human AP-1 responsive element. The transfected
cells were pretreated with 10 µM of different compounds and 20 mM of 5-ASA for 1 h prior
to the treatment with TNF-α for 3 h. The result represents the meanꢀ±ꢀS.E.M. from three
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independent experiments. *Pꢀ<ꢀ0.05 compared to the vehicle-treated control group. Pꢀ<ꢀ0.05
compared to the TNF-α -treated group.
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.8. Ameliorating effect of compound 55 on TNBS-induced colitis in rats. Compound 55
was further assessed as a candidate for IBD drug development by examining its in vivo
efficacy. Corresponding to the concentration-dependent inhibitory activity of compound 55
on an in vitro inflammation model (Figure 2A, 5B, 6B, 8B, and 9C), the oral administration
of compound 55 suppressed significantly and dose-dependently TNBS-induced rat colitis.
Sulfasalazine (300 mg/kg) was used as the positive control drug. Compared to the sham-
operated control group, the rats with TNBS treatment-induced colitis showed signs of
inflammation, bloody diarrhea, and wasting conditions with sluggish and weak movement. In
the TNBS-treated rats, the body weight showed a significant reduction followed by a
stagnated increase (Figure 11A), and the colon tissue weight was increased significantly
(Figure 11B) due to the edematous congestion (Figure 11C). Oral administration of 55
suppressed TNBS-induced colitis in a dose-dependent manner, i.e., there was a significant
recovery in body weight decrease and colon tissue edematous inflammation. A higher dose
(30 mg/kg) of 55 showed a similar recovery effect to that of 300 mg/kg sulfasalazine. In the
colon tissues, TNBS induced a dramatic increase in the level of MPO, a biochemical marker
of inflammation, which was suppressed significantly by 55 in a dose-dependent manner
(Figure 11D).
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Figure 11. Ameliorating effect of compound 55 on TNBS-induced colitis in rats. TNBS was
administered rectally to the rat to induce colitis. The control group received 50% ethanol in
saline as a vehicle. (A) Record of body weight from day 0 to day 5. (B) The length and
weight of colon after the dissection was measured. The bar graph represents the colon weight
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(
g/cm). *Pꢀ<ꢀ0.05 compared to the vehicle-treated control group. Pꢀ<ꢀ0.05 compared to the
TNBS -treated group. (C) Macroscopic appearance of the removed tissue. (D) The amount of
MPO was measured in the colon tissue using a MPO assay kit. The result is expressed as
meanꢀ±ꢀS.E.M. from 3 different rats.
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