Design, synthesis, and structure-activity relationship study of halogen containing 2-benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.ejmech.2017.03.049

As a continuous effort to discover new potential anti-inflammatory agents, we systematically designed and synthesized sixty-one 2-benzylidene-1-indanone derivatives with structural modification of chalcone, and evaluated their inhibitory activity on LPS-stimulated ROS production in RAW 264.7 macrophages. Systematic structure-activity relationship study revealed that hydroxyl group in C-5, C-6, or C-7 position of indanone moiety, and ortho-, meta-, or para-fluorine, trifluoromethyl, trifluoromethoxy, and bromine functionalities in phenyl ring are important for inhibition of ROS production in LPS-stimulated RAW 264.7 macrophages. Among all the tested compounds, 6-hydroxy-2-(2-(trifluoromethoxy) benzylidene)-2,3-dihydro-1H-inden-1-one (compound 44) showed the strongest inhibitory activity of ROS production. Further studies on the mode of action revealed that compound 44 potently suppressed LPS-stimulated ROS production via modulation of NADPH oxidase. The findings of this work could be useful to design 2-benzylidene-indanone based lead compounds as novel anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2017.03.049

Accepted Manuscript
Design, synthesis, and structure-activity relationship study of halogen containing 2-
benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in
RAW 264.7 macrophages
Aarajana Shrestha, Hye Jin Oh, Mi Jin Kim, Nirmala Tilija Pun, Til Bahadur Thapa
Magar, Ganesh Bist, Hongseok Choi, Pil-Hoon Park, Eung-Seok Lee
PII:
S0223-5234(17)30216-7
10.1016/j.ejmech.2017.03.049
EJMECH 9309
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 February 2017
Revised Date: 21 March 2017
Accepted Date: 23 March 2017
Please cite this article as: A. Shrestha, H. Jin Oh, M.J. Kim, N.T. Pun, T.B.T. Magar, G. Bist, H. Choi,
P.-H. Park, E.-S. Lee, Design, synthesis, and structure-activity relationship study of halogen containing
2-benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in RAW 264.7
macrophages, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.049.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship study of halogen containing 2-
benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in
RAW 264.7 macrophages
#
#
Aarajana Shrestha , Hye Jin Oh , Mi Jin Kim, Nirmala Tilija Pun, Til Bahadur Thapa Magar,
*
*
Ganesh Bist, Hongseok Choi, Pil-Hoon Park , Eung-Seok Lee
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
#
Authors are equal contributors.
*
Corresponding Authors:
Tel: +82 53 810-2826, fax: +82 53 810-4654, email: parkp@yu.ac.kr (P.-H. Park)
Tel: +82 53 810-2827, fax: +82 53 810-4654, email: eslee@yu.ac.kr (E.-S.L.)
1

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship study of halogen containing 2-
benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in
RAW 264.7 macrophages
#
#
Aarajana Shrestha , Hye Jin Oh , Mi Jin Kim, Nirmala Tilija Pun, Til Bahadur Thapa Magar,
*
*
Ganesh Bist, Hongseok Choi, Pil-Hoon Park , Eung-Seok Lee
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
Abstract
As a continuous effort to discover new potential anti-inflammatory agents, we systematically
designed and synthesized sixty-one 2-benzylidene-1-indanone derivatives with structural
modification of chalcone, and evaluated their inhibitory activity on LPS-stimulated ROS
production in RAW 264.7 macrophages. Systematic structure-activity relationship study
revealed that hydroxyl group in C-5, C-6, or C-7 position of indanone moiety, and ortho-,
meta-, or para-fluorine, trifluoromethyl, trifluoromethoxy, and bromine functionalities in
phenyl ring are important for inhibition of ROS production in LPS-stimulated RAW 264.7
macrophages. Among all the tested compounds, 6-hydroxy-2-(2-(trifluoromethoxy)
benzylidene)-2,3-dihydro-1H-inden-1-one (compound 44) showed the strongest inhibitory
activity of ROS production. Further studies on the mode of action revealed that compound 44
potently suppressed LPS-stimulated ROS production via modulation of NADPH oxidase. The
findings of this work could be useful to design 2-benzylidene-indanone based lead
compounds as novel anti-inflammatory agents.
Keywords: Halogen containing 2-benzylidene-1-indanone; Anti-inflammatory agents;
Inhibition of ROS production; RAW 264.7 macrophages; Structure-activity relationship
2

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1
. Introduction
Reactive oxygen species (ROS) are constantly produced by aerobic respiration and various
other catabolic processes [1]. These molecules contain unstable oxygen and are highly
reactive with the diverse intracellular components [2]. While ROS in physiologically relevant
levels functions as important signaling molecules and are implicated in beneficial biological
responses, excessive production of ROS results in the damages to the various intracellular
components, such as protein, lipid, and DNA, collectively referred to as oxidative stress [3].
There is a growing appreciation that excessive ROS production plays key roles in the
development of a number of pathological conditions, including tumorigenesis,
neurodegeneration, and aging [4]. In particular, recent studies have clearly demonstrated that
ROS directly activate various signaling cascades in immune cells, which lead to the
activation of innate and acquired immune system, which consequently enhance ROS
production that exert an important role in the development and progression of inflammatory
disorders [5]. Therefore, suppression of undue ROS production is a promising strategy for the
prevention and treatment of chronic diseases, and optimal pharmacological agents inhibiting
ROS production could be applied for the management of the various diseases associated with
chronic inflammtion.
Cellular ROS level is tightly regulated by multiple anti-oxidant defense systems. It has been
reported that various non-enzymatic systems, including glutathione and thioredoxin, and
enzymatic systems, including cytochrome P450, xanthine oxidase and NADPH oxidase, are
implicated in the regulation of ROS [6]. Although a number of different mechanisms
participate in the regulation of ROS level, the major sources for determination of intracellular
ROS level is depending on experimental conditions, including cellular environments and cell
types. NADPH oxidase, a membrane-associated enzyme composed of multiple subunits,
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catalyzes the production of superoxide through electron transfer from NADPH. Recent
studies have revealed that NADPH oxidase system plays a principal role in the modulation of
cellular ROS level in macrophages [7, 8].
Chalcones, α,β-unsaturated aromatic ketones, are naturally occurring component found in
various edible natural plants and considered as precursor for synthesis of flavonoids and
isoflavonoids [9, 10]. Chalcones as leading skeletons have been intensively reported for
development of new therapeutic candidates. Compounds containing chalcone, as a central
core, have been studied for various biological activities such as anti-inflammatory [11],
anticancer [12], antibacterial [13], antimalarial and antileishmanial agents [14]. Interestingly,
chalcone derivatives are reported to inhibit cyclooxygenase 2 (COX-2), inducible NO
synthetase (iNOS) expression, and ROS and NO synthesis in lipopolysaccharide (LPS)
stimulated cells [12, 15, 16].
Quantitative Structure Activity Relationships (QSAR) revealed that substitutions of electron-
withdrawing groups like halogen atoms in ring B of chalcones (Figure 1a) enhance anti-
inflammatory activity [17]. The incorporation of halogen atoms into molecules is reported as
an important strategy in medicinal chemistry to optimize pharmacokinetic property of drug
like increment of lipophilicity which enhances membrane permeability and potency. The
presence of halogen atom also influences the conformation due to its bulkiness that aid to
occupy deeper pockets of target [18]. Similarly, the unique properties like high
electronegativity and small size of fluorine have dominated drug design considerations that
results in metabolic stability and selectivity, enhances binding interactions, and changes in
physical properties [19, 20]. Moreover, hydroxyl group has also been exploited extensively to
improve pharmacological profile of bioactive compounds. The presence of hydroxyl moiety
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in chalcones has been reported to exhibit important biological activities, such as anticancer,
anti-inflammatory [15, 21], antimalarial [14], antibacterial [22], antiviral [23], and antifungal
activity [24]. Previously, our research group reported that a series of fluorinated and
hydroxylated chalcones (Figure 1 b,c) showed importance of meta- or para-hydroxyl group,
and ortho-trifluoromethoxy group for inhibiting ROS production in LPS-stimulated RAW
2
64.7 macrophages [25, 26]. These findings encouraged us to continue efforts to the
discovery of useful anti-inflammatory agents by further structural modification of ring A to 2-
benzylidene-1-indanone derivatives (Figure 2). Indanone moiety is found in various natural
products and has a valuable biological relevance. Several studies have shown that compounds
possessing indanone moiety are of great pharmacological importance as they exhibit various
biological activities such as anti-inflammatory, anticancer, analgesic, and antimicrobial
properties [27-30].
Figure 1. Structures of chalcone and previously synthesized compounds [25, 26] a) chalcone,
b) hydroxychalcone derivatives, c) chalcone derivatives containing hydroxyl and fluorine
moiety.
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Here in this study, we designed sixty compounds as analogues of 2-benzylidene-1-indanone
(1) by substituting hydroxyl group at C-4, C-5, C-6, or C-7 position of indanone and
halogenated functionalities such as fluorine, trifluoromethyl, trifluoromethoxy, and bromine
at different (ortho-, meta- or para-) position of phenyl ring (Figure 2). Compounds were
synthesized in four different series (A, B, C, and D), and evaluated for their inhibitory
activity on LPS-stimulated ROS production in RAW 264.7 macrophages. A systematic
structure-activity (SAR) analysis was performed to determine relationships between
structures and their inhibitory effects. Further inhibitory effect of compound 44 against
NADPH oxidase activity in RAW 264.7 macrophages was also described.
Figure 2. Strategy of design of target compounds based on previously synthesized
compounds.
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2
2
. Results and Discussion
. 1. Synthetic chemistry
As depicted in Scheme 1 and 2, base catalyzed Claisen-Schmidt condensation reaction [26,
3
1
1] was used to synthesize designed 2-benzylidene-1-indanone derivatives (1-61). First of all,
1
-indanone was mixed with aryl aldehydes (R = a-m) in ethanol using 5 % aqueous NaOH to
prepare non-hydroxylated 2-benzylidene-1-indanones 1-13 (Scheme 1) in yields ranging from
0-90%. Similarly, as described in Scheme 2 condensation reaction was utilized to synthesize
3
hydroxylated 2-benzylidene-1-indanones 14-61 by treating hydroxyl-indanone with aryl
1
aldehydes (R = b-m) using 50% aqueous KOH in ethanol without protection of hydroxyl
group of indanone in 48-99% yields.
(E)-2-benzylidene-2,3-dihydro-1H-inden-1-one (1) and its sixty analogues, 2-61, were
synthesized in four different series each containing fifteen compounds to study structure-
activity relationship (SAR) as shown in Figure 3. Series A (compounds 2-4, 14-16, 26-28, 38-
4
0, 50-52) contains indanone derivatives with fluorine group at the ortho-, meta-, or para-
position of phenyl ring. Similarly, series B (compounds 5-7, 17-19, 29-31, 41-43, 53-55),
Series C (compounds 8-10, 20-22, 32-34, 44-46, 56-58), and Series D (compounds 11-13, 23-
2
5, 35-37, 47-49, 59-61) contain trifluoromethyl, trifluoromethoxy, and bromine, respectively
at the position of ortho-, meta-, or para- of phenyl ring, and with or without hydroxyl group
at C-4-, C-5, C-6, or C-7 position of indanone. Position of hydroxyl group at the indanone,
and substitution of fluorine, trifluoromethyl, trifluoromethoxy, and bromine at different
position of phenyl ring allowed us to investigate the differences in inhibitory activities of
halogen and hydroxyl containing indanone derivatives against LPS-stimulated ROS
production in RAW 264.7 macrophages. Purity (%) by HPLC, retention time, yield (%), and
7

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melting point of all the prepared compounds are listed in Table S1 and S2 of Supplementary
Data.
O
R1
R1
H
1
(
R = a-m)
O
O
1
-indanone
(i)
1-13
F
F
F
1
R =
a
b
e
c
d
g
CF3
F3C
F3CO
Br
CF3
f
OCF3
OCF3
h
k
i
j
Br
Br
l
m
Scheme 1. General synthetic method for non-hydroxylated 2-benzylidene-1-indanone
derivatives 1-13; Reagents and conditions: (i) 5% aq. NaOH, EtOH, 15 min - 3 h, 0 °C, 30-
9
0% yields.
8

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O
R1
HO
R1
1
HO
H
(
R = b-m)
O
O
(
i)
Hydroxylated indanone
14-61
F
CF3
F
F
F3C
CF3
1
R =
b
c
d
e
f
g
OCF3
Br
F3CO
OCF3 Br
Br
h
i
j
k
l
m
Scheme 2. General synthetic method for hydroxylated 2-benzylidene-1-indanone derivatives
1
4-61; Reagents and conditions: (i) 50% aq. KOH, EtOH, 15 min - 3 h, 25 °C, 48-99% yields.
9

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Figure 3. Structures of the prepared 2-benzylidene-indanone derivatives classified by series
A to D.
1
0

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2
.2. Inhibition of LPS-stimulated ROS production in RAW 264.7 macrophages
Malvidin, O-methylated anthocyanidin and one of the most abundant polyphenols found in
red wine, has been studied for antioxidant and anti-inflammatory effects in RAW 264.7
macrophages [32, 33]. In addition, malvidin has structural similarity to the synthesized
compounds. Therefore, in this study, we used malvidin as a positive control. It is reported that
malvidin inhibits 50% of LPS-stimulated ROS production at 9.0± 0.8 µM concentration [34].
In this study, all the sixty-one synthesized compounds were assayed to determine their
inhibitory activity (IC50 value) on LPS-stimulated ROS production in RAW 264.7
macrophages.
As shown in Table 1, treatment with compound 1 induced cytotoxicity (i.e., cell viability was
less than 90 % compared with control) and inhibitory effect on ROS production was weak
(
IC value was higher than 10 µM). In addition, Table 1 includes inhibitory activity of fifteen
50
compounds of series A which possess fluorine in ortho-, meta-, or para-position of phenyl
ring as a common skeleton in non-hydroxylated and hydroxylated 2-benzylidene-1-indanone
derivatives. Interestingly, all the 7-hydroxy-indanone containing compounds (50-52) showed
significant inhibitory activity (3.82 ± 0.63, 5.61 ± 1.07, and 5.06 ± 1.17 µM of IC50,
respectively). This result indicated that hydroxyl group at C-7 position of indanone in
combination with fluorophenyl ring is important for inhibitory activity against LPS-induced
ROS production in RAW 264.7 macrophages.
1
1

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Table 1. Inhibitory effects (IC ) of fluorine substituted 2-benzylidene-1-indanone derivatives
5
0
(Series A) on ROS production in LPS-stimulated RAW 264.7 macrophages.
Compound
R
R1
Inhibition of
Toxicity
ROS production
a
(
IC , µM)
50
Malvidin
9.0 ± 0.8
>10
b
1
2
3
4
H
H
Toxic
H
2-F
3-F
4-F
2-F
3-F
4-F
2-F
3-F
4-F
2-F
3-F
4-F
2-F
3-F
4-F
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
14
15
16
26
27
28
38
39
40
50
51
52
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
6-OH
6-OH
6-OH
7-OH
7-OH
7-OH
3.82 ± 0.63
5.61 ± 1.07
5.06 ± 1.17
a
N: Non-toxic; Each data represents mean ± SEM. from at least three different experiments;
b
Less than 90% cell viability at 10 µM; Malvidin was used as a positive control.
Similarly, in the series B compounds 31, 41, 42, 53, and 55 with trifluoromethyl group in
ortho-, meta-, or para-position of phenyl ring exhibited stronger inhibitory activities (IC =
5
0
1
2

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1
.94-5.55 µM) than positive control (Table 2). Compound 53 containing 7-hydroxy-indanone
along with ortho-trifluoromethyl group in phenyl ring showed the most significant inhibitory
activity (1.94 ± 0.19 µM of IC ) on LPS-stimulated ROS production in RAW 264.7
5
0
macrophages. Compounds 41 and 42 which contain trifluoromethyl in ortho- and meta-
position of phenyl ring, and hydroxyl group at C-6 position of indanone showed inhibition of
ROS production with IC values 2.61 ± 0.59 and 4.55 ± 0.17 µM, respectively. Likewise,
5
0
compounds 31 and 55 which possess para-trifluoromethyl in phenyl ring and C-5 and C-7
hydroxy-indanone moieties showed significant inhibitory activity (5.55 ± 1.94 and 3.89 ±
1
.07 µM of IC50, respectively) on LPS-stimulated ROS production in RAW 264.7
macrophages.
1
3

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Table 2. Inhibitory effects (IC ) of trifluoromethyl substituted 2-benzylidene-1-indanone
5
0
derivatives (Series B) on ROS production in LPS-stimulated RAW 264.7 macrophages.
Compound
R
R1
Inhibition of
Toxicity
ROS production
a
(
IC , µM)
50
5
6
7
H
2-CF
3-CF
4-CF
2-CF
3-CF
4-CF
2-CF
3-CF
4-CF
2-CF
3-CF
4-CF
2-CF
3-CF
4-CF
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
17
18
19
29
30
31
41
42
43
53
54
55
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
6-OH
6-OH
6-OH
7-OH
7-OH
7-OH
5.55 ± 1.94
2.61 ± 0.59
4.55 ± 0.17
> 10
1.94 ± 0.19
> 10
3.89 ± 1.07
a
N: Non-toxic; Each data represents mean ± SEM. from at least three different experiments.
As shown in Table 3, compounds in series C possess trifluoromethoxy in ortho-, meta-, or
para-position of phenyl ring as a common skeleton in 2-benzylidene-1-indanone derivatives.
Interestingly, all the 7-hydroxy-indanone containing compounds (56-58) showed significant
1
4

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inhibitory activity of LPS-stimulated ROS production in RAW 264.7 macrophages (3.25 ±
0
.54, 4.62 ± 1.32, and 3.15 ± 0.92 µM of IC respectively). Similarly, 5-hydroxy-indanone
50,
containing compounds 32 and 33 bearing ortho-, and meta-trifluoromethoxy group showed
strong to moderate inhibitory activity (3.38 ± 0.45 and 8.01 ± 0.97 µM, respectively).
Moreover, except 20, all the compounds (8, 32, 44, and 56) containing trifluoromethoxy in
ortho-position of phenyl ring exhibited significant inhibitory activity (1.33 ± 0.03 to 7.27 ±
1
.17) of LPS-stimulated ROS production in RAW 264.7 macrophages. Compound 44
exhibited the most significant inhibitory activity (IC = 1.33 ± 0.03 µM) against LPS-
5
0
stimulated ROS production in RAW 264.7 macrophages.
1
5

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Table 3. Inhibitory effects (IC ) of trifluoromethoxy substituted 2-benzylidene-1-indanone
5
0
derivatives (Series C) on ROS production in LPS-stimulated RAW 264.7 macrophages.
Compound
R
R1
Inhibition of
Toxicity
ROS production
a
(
IC , µM)
50
8
9
H
2-OCF
3-OCF
4-OCF
2-OCF
3-OCF
4-OCF
2-OCF
3-OCF
4-OCF
2-OCF
3-OCF
4-OCF
2-OCF
3-OCF
4-OCF
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
7.27 ± 1.17
> 10
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
10
20
21
22
32
33
34
44
45
46
56
57
58
H
> 10
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
6-OH
6-OH
6-OH
7-OH
7-OH
7-OH
> 10
> 10
> 10
3.38 ± 0.45
8.01 ± 0.97
> 10
1.33 ± 0.03
> 10
> 10
3.25 ± 0.54
4.62 ± 1.32
3.15 ± 0.92
a
N: Non-toxic; Each data represents mean ± SEM. from at least three different experiments.
Furthermore, as shown in table 4, all the compounds (35-37, 47-49) containing 5- and 6-
hydroxylated indanone moieties displayed significant inhibitory activities (8.18 ± 2.30, 4.05
±
0.81, 6.05 ± 0.70, 4.00 ± 1.78, 3.90 ± 1.67, and 4.27± 1.40 µM of IC50, respectively) on
LPS-stimulated ROS production in RAW 264.7 macrophages. However, among all the
1
6

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synthesized compounds, only compound 49 which contain bromine at para-position of
phenyl ring and hydroxyl group at C-6 position of indanone showed toxic effect.
Table 4. Inhibitory effects (IC ) of bromine substituted 2-benzylidene-1-indanone
5
0
derivatives (Series D) on ROS production in LPS-stimulated RAW 264.7 macrophages.
Compound
R
R1
Inhibition of
Toxicity
ROS production
a
(
IC , µM)
50
1
1
H
2-Br
3- Br
4- Br
2-Br
3- Br
4- Br
2-Br
3- Br
4- Br
2-Br
3- Br
4- Br
2-Br
3- Br
4- Br
> 10
> 10
> 10
> 10
> 10
> 10
N
N
N
N
N
N
N
N
N
N
N
12
13
23
24
25
35
36
37
47
48
49
59
60
61
H
H
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
6-OH
6-OH
6-OH
7-OH
7-OH
7-OH
8.18 ± 2.30
4.05 ± 0.81
6.05 ± 0.70
4.00 ± 1.78
3.90 ± 1.67
4.27 ± 1.40
> 10
b
Toxic
N
N
N
> 10
> 10
a
N: Non-toxic; Each data represents mean ± SEM. from at least three different experiments;
b
Less than 90% cell viability at 10 µM; Malvidin was used as a positive control.
1
7

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2
.3. Structure-activity relationship studies
Presence of different functionalities in the position of ortho-, meta-, or para- of phenyl ring,
and with or without hydroxyl group at C-4-, C-5, C-6, or C-7 position of indanone ring
allowed us for systematic structure-activity relationship (SAR) study. Except compound 8, all
the non-hydroxylated 2-benzylidene-indanones 1-13, did not show significant inhibitory
activities (IC > 10 µM), whereas, most of the hydroxylated 2-benzylidene-indanones 14-61,
5
0
exhibited better inhibitory activities than the positive control, malvidin. The results suggested
that the hydroxylated 2-benzylidene-indanone derivatives showed stronger inhibitory
activities than corresponding non-hydroxylated 2-benzylidene-indanones (Figure 4a).
Furthermore, results listed in the Tables 1-4 indicated that position of hydroxyl group on C-5,
C-6, or C-7 position of indanone moiety plays important role for inhibitory activity of
compounds against LPS-stimulated ROS production in RAW 264.7 macrophages. It is
interesting to notice that all the compounds containing hydroxyl group at C-4 position of
indanone (14-25) did not show significant inhibitory activities. However, introduction of
hydroxyl group from the C-4 of indanone to the C-5, C-6, or C-7 position of indanone with
fluoride functionality in phenyl ring generally enhanced the inhibitory activity in increasing
order, respectively (Figure 4b). Interestingly, except compound 54, all the 7-hydroxylated
indanone derivatives with fluorine, trifluoromethyl, and trifluoromethoxy at ortho-, meta-, or
para-position of phenyl ring showed significant inhibition. The results indicated that
hydroxyl group at C-7 position of indanone is crucial for inhibitory activity on LPS-
stimulated ROS production in RAW 264.7 macrophages. In contrast, bromine containing 7-
hydroxylated 2-benzylidene-indanones 59-61 did not show considerable inhibitory activity.
On the other hand, results of bromine containing C-5 and C-6 hydroxylated indanone
derivatives showing significant inhibitory activities indicated that bromine functionality at
phenyl ring was important in compounds with hydroxyl group at C-5 and C-6-position of
1
8

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indanone for inhibitory activity on LPS-stimulated ROS production in RAW 264.7
macrophages (Figure 4c). Interestingly, trifluoromethoxy group at ortho-position of phenyl
ring with hydroxyl group at C-6 position of indanone was found to be important in exhibiting
inhibitory activity on LPS-stimulated ROS production in RAW 264.7 macrophages (Figure
4
d). Among all the synthesized compounds, compound 44 which showed the most potent
inhibitory activity of ROS production was selected for further evaluation to investigate its
mechanistic action.
1
9

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R1
R1
a)
b)
c)
R = 5-OH, 6-OH, 7-OH
R
>
O
O
R = F, CF , OCF , Br
1
3
3
Hydroxylated
Non-hydroxylated
Indanone derivatives
Indanone derivatives
Strong ROS Inhibition
Weak ROS Inhibition
R1
R1
R1
R1
R1
OH
HO
>
>
>
>
HO
O
O
O
O
O
OH
R = F, CF , OCF
1
3
3
Br
Br
Br
O
O
HO
OH
O
O
Br
Br
HO
O
OH
HO
d)
OH
F3CO
F3CO
F3CO
F3CO
F3CO
>
>
>
>
HO
O
O
O
O
O
OH
44
56
32
8
20
IC50 = 1.33
M
IC50 = 3.25
M
IC50 = 3.38
M
IC50 = 7.27
M
IC50 = >10 M
Figure 4. Structure-activity relationship study based on a) hydroxylated and non-
hydroxylated 2-benzylidene-indanone derivatives, b) position of hydroxyl group in indanone
moiety with fluoride functionality in phenyl ring, c) bromine-containing 2-benzylidene-
indanone derivatives, and d) position of trifuloromethoxy at ortho-position of phenyl ring in
2
-benzylidene-indanone derivatives.
2
0

ACCEPTED MANUSCRIPT
2
.4. Mode of action for compound 44
In continuing experiments, we further elucidated the mechanism underlying regulation of
LPS-stimulated ROS production. For this, we examined the effect of compound 44 on
NADPH oxidase activity, because NADPH oxidase is one of the main enzyme systems
responsible for the modulation of ROS production in macrophages. In addition, in
preliminary experiments, we observed that DPI treatment significantly suppressed LPS-
stimulated ROS production (data not shown), indicating that NADPH oxidase plays a crucial
role in LPS-induced ROS production in macrophages
As indicated in figure 5, LPS-stimulated NADPH oxidase activation was returned to the
almost normal level by pretreatment with compound 44. This inhibitory effect was prominent
even at very low concentration (1 µM), suggesting that compound 44 suppressed LPS-
stimulated ROS production via modulation of NADPH oxidase. These results further raise a
possibility that NADPH oxidase would be a promising target molecule for the suppression of
ROS production by the compounds used in this study.
Figure 5. Effect of compound 44 on LPS-stimulated NADPH oxidase activation in RAW
2
64.7 macrophages. RAW 264.7 macrophages were pretreated with compound 44 (1 µM) for
2
1

ACCEPTED MANUSCRIPT
1
h followed by incubation with LPS (1 µg/ml) for additional 24 h. NADPH oxidase activity
was analyzed by lucigenin-based assay as described in the materials and methods. Values
represent fold increase in comparison to the cells treated with LPS and are expressed as mean
*
#
±
SEM (n = 3). P < 0.05 compared with control cells; P < 0.05 compared with cells treated
with LPS.
3
. Conclusion
On the basis of importance of halogen substituents on hydroxylated chalcones, we designed a
new series of 2-benzylidene-indanone derivatives by structural modification of chalcone.
Total sixty-one 2-benzylidene-indanone derivatives were synthesized and evaluated for their
inhibitory activity on LPS-stimulated ROS production in RAW 264.7 macrophages.
Compounds 8, 31-33, 35-37, 41, 42, 44, 47-53, and 55-58 showed better inhibitory activity,
with IC50 values in the range of 1.33-8.11 µM, than positive control, malvidin. Structure-
activity relationship study revealed that the modification of phenyl ring A of chalcone by
hydroxylated indanone containing fluorine, trifluoromethyl, trifluoromethoxy, bromine at
ortho-, meta-, or para-position of phenyl ring B of chalcone is crucial to enhance inhibitory
activity without cytotoxic effect except compound 49. Moreover, a great impact of
trifluoromethoxy substituent in ortho-position of phenyl ring was obtained supporting the
previously reported results of our research work. Compound 44, which showed the most
potent inhibitory effect on ROS production, was selected for further mechanistic study. In
continuing experiments for the elucidation of the underlying mechanisms, we demonstrated
that compound 44 potently suppressed NADPH oxidase activity. Results of this study
demonstrate the effectiveness of 2-benzylidene-indanone analogues as potential lead
compounds to develop new anti-inflammatory agents.
2
2

ACCEPTED MANUSCRIPT
4
. Experimental
Commercially available starting materials and reagents were purchased from Sigma-Aldrich,
TCI Chemicals, Alfa-Aesar, and Junsei, were used without further purification. HPLC grade
acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA. Column
chromatography was carried out using silica gel (Kieselgel 60, 230-400 mesh, Merck). Thin
layer chromatography (TLC) was performed on silica gel plates (Kieselgel 60 F254, Merck)
having layer thickness of 0.25 mm. Melting points were recorded using open capillary tube
1
on electrothermal 1A 9100 digital melting point apparatus and were uncorrected. H NMR
1
3
(
250 MHz) and C NMR (62.5 MHz) spectra were recorded in Bruker AMX 250 (250 MHz,
FT) spectrometer using CDCl and DMSO-d as solvent, and tetramethylsilane (TMS) as
3
6
internal standard. All the chemical shifts values (δ) were recorded in parts per million (ppm)
and coupling constants (J) in hertz (Hz).
HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled
HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode
array detector (SPD-M10A VP) utilizing Shimadzu LC Solution program. Sample volume of
®
1
0 µL was run in Waters X- Terra 5 µM reverse-phase C column (4.6 ⅹ 250 mm) with a
1
8
gradient elution of 85% to 100% of B in A for 10 min followed by 70% to 85% of B in A for
0 min at a flow rate of 0.7 mL/min at 254 nm UV detection, where mobile phase A was
2
doubly distilled water with 50 mM ammonium formate (AF) and B was 100% ACN. Purity of
compound is stated as percent (%) and retention time in minutes.
®
Mass spectra of the compound were recorded with Advion Expression CMS ESI-MS
spectrometry (Advion, Ithaca, NY, USA), using methanol: water: formic acid (80: 20: 0.1) as
a mobile phase.
2
3

ACCEPTED MANUSCRIPT
4
. 1. General method for preparation of 1-13
1
A mixture of non-hydroxylated indanone (1.0 eq.) and aryl aldehyde (R =a-m) (1.2 eq.) in
ethanol (3 mL) was stirred at 0ΩC and 5% aqueous NaOH solution (3 mL) was added
dropwise. The reaction was stirred for 15 min to 3 h. The progress of the reaction was
checked by using TLC. Upon completion, ice was added to the reaction mixture and then
neutralized by 6M aqueous HCl solution (pH adjusted to 2) to obtain precipitation. The
resulted precipitate was filtered, washed with excess water and dried. The solid residue was
further purified by recrystallization using ethyl acetate and n-hexane to afford compound 1-
1
3 in 30% to 90% yield. The exocyclic double bond in compound may be E (trans) or Z (cis).
1
These configurational isomers have characteristic H chemical shifts in the NMR spectra. The
chemical shift of the olefinic proton (=CH-) is deshielded in the E isomer of compound and
1
appears downfield (above 7 ppm) compared to the Z isomer (less than 7 ppm). H NMR data
of compounds 1-61 supported the assignment of E-stereochemistry.
4
.1.1. Synthesis of (E)-2-benzylidene-2,3-dihydro-1H-inden-1-one (1)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and benzaldehyde (0.12 mL, 1.20 mmol) for 15 min to yield 198 mg (0.90 mmol, 90%) as an
off-white solid. TLC (ethyl acetate / n-hexane = 1:15) R = 0.23; Mp 132.1-133.2 °C, HPLC:
f
+
Retention time: 8.32 min, purity: 100%; ESI LC/MS: m/z calcd for C H O [M+H] 221.09;
1
6
12
found 221.05.
1
H NMR (250 MHz, DMSO-d ) δ 7.90 (d, J = 7.6Hz, 1H, indeno H-7), 7.68-7.64 (m, 3H,
6
phenyl H-2, H-6, indeno H-5), 7.61-7.53 (m, 2H, indeno H-4, =CH-), 7.49-7.36 (m, 4H,
indeno H-6, phenyl H-3, H-4, H-5), 4.04 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, DMSO-d ) δ 194.23, 145.61, 138.04, 135.43, 134.76, 134.57, 133.88,
6
2
4

ACCEPTED MANUSCRIPT
1
30.68, 129.61, 128.90, 127.65, 126.14, 124.43, 32.42.
4
.1.2. Synthesis of (E)-2-(2-fluorobenzylidene)-2,3-dihydro-1H-inden-1-one (2)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 2-fluorobenzaldehyde (0.13 mL, 1.20 mmol) for 2 h to yield 174 mg (0.73 mmol, 73%)
o
as a white solid. TLC (ethyl acetate/n-hexane = 1:5 v/v) R = 0.28; Mp: 143.8-145.2 C;
f
+
HPLC: Retention time: 5.65 min, purity: 98.2%; ESI-MS: m/z calcd for C H FO [M+Na] :
1
6
11
+
2
61.1; found [M+Na] : 261.9.
1
H NMR (250 MHz, CDCl ) δ 7.91-7.88 (m, 2H, indeno H-7, =CH-), 7.69 (td, J = 7.65,
3
7
.53Hz, 1H, phenyl H-5), 7.63-7.50 (m, 2H, indeno H-5, H-4), 7.43-7.32 (m, 2H, indeno H-6,
phenyl H-6), 7.21 (t, J = 7.55Hz, 1H, phenyl H-4), 7.13 (t, J = 8.20Hz, 1H, phenyl H-3), 3.98
(s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 193.82, 161.78 (d, J = 252.11Hz), 149.54, 137.91, 136.52,
3
134.73, 131.25 (d, J = 8.64Hz), 129.99 (d, J = 2.31Hz), 127.70, 126.14, 125.57 (d, J =
5.54Hz), 124.53, 124.25 (d, J = 3.73Hz), 123.51 (d, J = 12.04Hz), 116.12 (d, J = 22.06Hz),
32.24 (d, J = 2.14Hz).
4
.1.3. Synthesis of (E)-2-(3-fluorobenzylidene)-2,3-dihydro-1H-inden-1-one (3)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 3-fluorobenzaldehyde (0.13 mL, 1.20 mmol) for 2 h to yield 114 mg (0.48 mmol, 48%)
o
as a white solid. TLC (ethyl acetate/n-hexane = 1:6 v/v) R = 0.25; Mp: 136.5-137.8 C;
f
+
HPLC: Retention time: 5.59 min, purity: 99.1%; ESI-MS: m/z calcd for C H FO [M+Na] :
1
6
11
+
2
61.1; found [M+Na] : 261.2.
1
H NMR (250 MHz, CDCl ) δ 7.89 (d, J = 7.60Hz, 1H, indeno H-7), 7.64-7.53 (m, 3H,
3
2
5

ACCEPTED MANUSCRIPT
indeno H-5, H-4, =CH-), 7.44-7.32 (m, 4H, phenyl H-4, H-5, H-6, indeno H-6), 7.12-7.04 (m,
1
H, phenyl H-2), 4.01 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 194.03, 162.93 (d, J = 245.13Hz), 149.46, 137.80, 137.53 (d,
3
J = 7.64Hz),135.86, 134.83, 132.41 (d, J = 6.38Hz), 130.40 (d, J = 8.20Hz), 127.79, 126.71
d, J = 2.79Hz), 126.19, 124.51, 116.75 (d, J = 9.66Hz), 116.40 (d, J = 9.18Hz), 32.29.
(
4
.1.4. Synthesis of (E)-2-(4-fluorobenzylidene)-2,3-dihydro-1H-inden-1-one (4)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 4-fluorobenzaldehyde (0.13 mL, 1.20 mmol) for 2 h to yield 207 mg (0.87 mmol, 87%)
o
as an off-white solid. TLC (ethyl acetate/n-hexane = 1:6 v/v) R = 0.25; Mp: 163.9-164.6 C;
f
+
HPLC: Retention time: 5.47 min, purity: 97.4%; ESI-MS: m/z calcd for C H FO [M+Na] :
1
6
11
+
2
61.1; found [M+Na] : 261.1.
1
H NMR (250 MHz, CDCl ) δ 7.88 (d, J = 7.45Hz, 1H, indeno H-7), 7.66-7.57 (m, 4H,
3
phenyl H-2, H-6, indeno H-4, =CH-), 7.54 (t, J = 7.13Hz, 1H, indeno H-5), 7.40 (t, J =
6
.88Hz, 1H, indeno H-6), 7.16-7.09 (m, 2H, phenyl H-3, H-5), 3.98 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 194.11, 163.31(d, J = 250.28Hz), 149.43, 137.92, 134.63,
3
1
3
34.23, 132.31 (2C), 132.49, 131.65 (d, J = 3.33Hz), 127.71, 126.13, 124.43, 116.26, 115.92,
2.27.
4
.1.5. Synthesis of (E)-2-(2-(trifluoromethyl)benzylidene)-2,3-dihydro-1H-inden-1-one (5)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 2-(trifluoromethyl) benzaldehyde (0.16 mL, 1.20 mmol) for 2 h to yield 216 mg (0.75
mmol, 75%) as a white solid. TLC (ethyl acetate/n-hexane = 1:4 v/v) R = 0.26; Mp: 144.0-
f
o
1
44.9 C; HPLC: Retention time: 5.87 min, purity: 98.4%; ESI-MS: m/z calcd for C H F O
1
7
11 3
2
6

ACCEPTED MANUSCRIPT
+
+
[
M+H] : 289.1; found [M+H] : 289.9.
1
H NMR (250 MHz, CDCl ) δ 7.96-7.94 (m, 1H, phenyl H-3), 7.90 (d, J = 7.63Hz, 1H,
3
indeno H-7), 7.75-7.68 (m, 2H, indeno H-4, =CH-), 7.64-7.56 (m, 2H, indeno H-5, phenyl H-
6
), 7.49-7.38 (m, 3H, phenyl H-4, H-5, indeno H-6), 3.88 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 193.41, 149.70, 138.21, 137.84, 134.87, 134.19, 131.73,
3
1
29.92, 129.64, 129.45, 128.70, 127.76, 126.32 (d, J = 5.53Hz), 126.15, 124.63, 123.86 (d, J
=
272.45Hz), 31.53.
4
.1.6. Synthesis of (E)-2-(3-(trifluoromethyl)benzylidene)-2,3-dihydro-1H-inden-1-one (6)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 3-(trifluoromethyl) benzaldehyde (0.16 mL, 1.20 mmol) for 2 h to yield 159 mg (0.55
mmol, 55%) as a white solid. TLC (ethyl acetate/n-hexane = 1:6 v/v) R = 0.22; Mp: 168.1-
f
o
1
69.6 C; HPLC: Retention time: 8.01 min, purity: 98.33%; ESI-MS: m/z calcd for
+
+
C H F O [M+H] : 289.1; found [M+H] : 289.7.
17
11 3
1
H NMR (250 MHz, CDCl ) δ 7.90-7.87 (m, 2H, indeno H-7, phenyl H-2), 7.80 (d, J =
3
7
.30Hz, 1H, phenyl H-4), 7.64-7.54 (m, 5H, =CH-, phenyl H-5, H-6, indeno H-4, H-5), 7.42
(t, J = 69.8Hz, 1H, indeno H-6), 4.03 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 193.94, 149.38, 137.63, 136.29, 136.07, 134.97, 133.63,
3
1
31.95, 131.61, 131.09, 129.45, 127.85, 126.74 (d, J = 3.99Hz), 126.23, 125.96 (d, J =
1
25.96Hz),124.54, 32.18.
4
.1.7. Synthesis of (E)-2-(4-(trifluoromethyl)benzylidene)-2,3-dihydro-1H-inden-1-one (7)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 4-(trifluoromethyl) benzaldehyde (0.16 mL, 1.20 mmol) for 3 h to yield 159 mg (0.55
2
7

ACCEPTED MANUSCRIPT
mmol, 55%) as a white solid. TLC (ethyl acetate/n-hexane = 1:6 v/v) R = 0.25; Mp: 189.5-
f
o
1
90.9 C; HPLC: Retention time: 7.24 min, purity: 98.2%; ESI-MS: m/z calcd for C H F O
1
7
11 3
+
+
[
M+H] : 289.1; found [M+H] : 289.8.
1
H NMR (250 MHz, CDCl ) δ 7.90 (d, J = 7.63Hz, 1H, indeno H-7), 7.76-7.64 (m, 5H,
3
phenyl H-2, H-3, H-5, H-6, =CH-), 7.61 (d, J = 6.95Hz, 1H, indeno H-4), 7.56 (t, J = 7.63Hz,
1
H, indeno H-5), 7.42 (t, J = 7.00Hz, 1H, indeno H-6), 4.04 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 193.88, 149.43, 138.79, 137.69, 136.94, 134.98, 131.88,
3
1
30.57 (4C), 127.89, 126.21, 125.85 (d, J = 3.64Hz), 125.73 (d, J = 3.87Hz), 124.60, 32.29.
4
.1.8. Synthesis of (E)-2-(2-(trifluoromethoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (8)
The procedure described in Section 4.1 was followed with 1-indanone (0.13 g, 1.00 mmol)
and 2-(trifluoromethoxy) benzaldehyde (0.17 mL, 1.20 mmol) for 3 h to yield 220 mg (0.72
mmol, 72%) as a white solid. TLC (ethyl acetate/n-hexane = 1:6 v/v) R = 0.20; Mp: 127.8-
f
o
1
29.3 C; HPLC: Retention time: 8.46 min, purity: 97.90%; ESI-MS: m/z calcd for
+
+
C H F O [M+H] : 305.1; found [M+H] : 305.4.
17
11
3
2
1
H NMR (250 MHz, CDCl ) δ 7.91-7.88 (m, 2H, =CH-, indeno H-7), 7.74 (dd, J = 7.10,
3
3.50Hz, 1H, phenyl H-6), 7.60 (td, J = 7.01, 1.18Hz, 1H, indeno H-5), 7.51 (d, J = 7.55Hz,
1H, indeno H-4), 7.46-7.30 (m, 4H, indeno H-6, phenyl H-3, H-4, H-5), 3.96 (d, J = 1.73Hz,
2H, indeno H-3).
1
3
C NMR (62.5 MHz, CDCl ) δ 193.66, 149.52, 148.40, 137.78, 137.28, 134.82, 130.59,
3
1
3
29.95, 128.83, 127.75, 126.87, 126.33, 126.13, 124.56, 121.45, 120.45 (d, J = 257.04Hz),
2.04.
4
.1.9. Synthesis of (E)-2-(3-(trifluoromethoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (9)
2
8