Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Article abstract of DOI:10.1016/j.bmcl.2017.01.061

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.

Full text of DOI:10.1016/j.bmcl.2017.01.061

Accepted Manuscript
Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated
chalcones in RAW 264.7 macrophages with Structure-Activity Relationship
study
Ganesh Bist, Nirmala Tilija Pun, Til Bahadur Thapa Magar, Aarajana Shrestha,
Hye jin Oh, Amrita Khakurel, Pil-Hoon Park, Eung-Seok Lee
PII:
S0960-894X(17)30073-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.061
BMCL 24637
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 December 2016
11 January 2017
19 January 2017
Please cite this article as: Bist, G., Pun, N.T., Magar, T.B.T., Shrestha, A., Oh, H.j., Khakurel, A., Park, P-H., Lee,
E-S., Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7
macrophages with Structure-Activity Relationship study, Bioorganic & Medicinal Chemistry Letters (2017), doi:
http://dx.doi.org/10.1016/j.bmcl.2017.01.061
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Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated
chalcones in RAW 264.7 macrophages with Structure-Activity Relationship study
Ganesh Bist#, Nirmala Tilija Pun#, Til Bahadur Thapa Magar, Aarajana Shrestha, Hye jin Oh,
Amrita Khakurel, 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. Lee)

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated
chalcones in RAW 264.7 macrophages with Structure-Activity Relationship study
Ganesh Bist#, Nirmala Tilija Pun#, Til Bahadur Thapa Magar, Aarajana Shrestha, Hye jin Oh,
Amrita Khakurel, Pil-Hoon Park*, Eung-Seok Lee*
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
Abstract
Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies,
thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro,
trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified
Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized
compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were
evaluated. Structure-activity relationship (SAR) study revealed that the compounds
possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl
phenyl group, and meta/para-hydroxyphenyl group combined with ortho-
trifluoromethoxyphenyl group have an essential role in inhibiting the LPS- stimulated ROS
production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-
stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that
possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.
Keywords: fluorinated and hydroxylated chalcones, inhibition of LPS-stimulated ROS
production, SAR study

Partially reduced oxygen species such as hydroxyl radical, peroxyl radical, superoxide radical,
and hydrogen peroxide, formed during normal cellular metabolism are known as reactive
oxygen species (ROS) and are responsible for both beneficial and harmful activities.
Beneficial effects of ROS occur at low or moderate concentrations and include cellular
signaling, defense against infectious agents, and the induction of a mitogenic response. On
the other hand, overproduction of ROS, known as oxidative stress, can cause significant
damage to biological systems which can further contribute to the development of various
degenerative conditions such as DNA mutation, cancer, aging, neurodegenerative disorders,
cell death, and inflammation [1, 2]. Macrophages are considered to have important roles in
host immune defense systems during infection and disease development. Activation of
macrophages by stimuli such as bacterial lipopolysaccharide (LPS) [3, 4], induces the
production of ROS which is the key to the progression of many inflammatory diseases.
Reactive nitrogen species (RNS) such as peroxynitrite are formed by the rapid combination
of nitric oxide (NO) with superoxide radical. The RNS, in turn, induces nitrosative stress that
further contributes to the inflammatory burden of ROS [4]. Recently, malvidin, which is
included in the polyphenols of red wine, has been shown to inhibit ROS production triggered
by LPS in RAW 264.7 macrophages and the concentration that inhibits 50% of LPS-induced
ROS production has been found to be 9.0± 0.8 µM [5]. In addition, the in-vitro anti-oxidant
activity [6] along with anti-inflammatory [7], antibacterial [8], and neuroprotective [9]
activities of malvidin have been reported. Although diverse chemical compounds have been
reported to combat the oxidative as well as inflammatory conditions, potent compounds with
negligible unwanted effects are still lacking. Therefore, additional pharmacological agents
that aim to control the excessive production of ROS and possess minimal side effects could
be considered as one of the promising treatment options for several pathological conditions.
Chalcone (1,3-diphenylpropenone) is one of the major constituents obtained from natural
products and is an important precursor for synthetic manipulations. Chalcones, along with
their synthetic analogues, demonstrate diverse biological activities such as antibacterial,
antifungal, antimalarial [10, 11], antituberculosis [11], antifilarial [12], antileishmanial [13],
antioxidant, antidyslipidemia [14], neuroprotective [15], anticancer [16], modulation of P-
glycoprotein-mediated multidrug resistance [17], and also have anti-inflammatory effects
[18]. Previously, our research group has synthesized various chalcone derivatives (Fig. 1) by
replacing two phenyl rings of a chalcone with heteroaromatic rings, and reported those to

have anti-inflammatory properties through considerable dual COX/5-LOX inhibitory activity
[19] as well as anti-angiogenic and anti-tumor activity [20-22]. In addition, thienyl/furanyl-
hydroxyphenylpropenones, previously synthesized by our research group, as chalcone
derivatives, displayed significant inhibition of ROS production stimulated by LPS in RAW
264.7 macrophages [23].
From various studies, it has been found that most of the phenolic and polyphenolic
derivatives show antioxidant [24], anti-inflammatory [25], anti-angiogenic, and
antiproliferative properties [26, 27]. Further, hydroxylated chalcones found in nature as well
as their synthetic analogues are reported to have several pharmacological activities such as
antiprotozoal, anti-plasmodium activity [28], antifungal activity [29], xanthine oxidase
inhibitors, radical scavengers activity [30, 31], antibacterial [32, 33], anti-diabetic and anti-
obesity activity [34] including analgesic and anti-inflammatory activity [35]. Similarly, a
large bulky group such as halogen atoms tend to occupy most of the deeper pockets as well as
the reacting regions of the molecular targets, thereby inducing either antagonistic or agonist
activity. Recently, in drug discovery optimization, many studies are performed by introducing
fluorine atoms (-F, -CF3, or -OCF3) in the structure because of their capacity to enhance
potency, bioavailability, cell permeability, and also chemical and metabolic stability by
increasing molecules’ lipophilicity and acting as hydrogen bond acceptors [36]. Likewise,
several studies about the fluorinated chalcones have found in them significant
antiproliferative [37], anti-invasive activity [38], antiperoxidation, 5-lipoxygenase inhibitory
activity [39], MAO-B inhibitory activity, analgesic, antioxidant, and anti-inflammatory
activity [40]. From these various reports exhibiting the importance of the phenolic group
together with the introduction of the fluorine atom has inspired us to integrate the following
functional groups in the chalcones that we have synthesized as shown in Fig. 2.

Fig. 1. Structures of a) chalcone, b) chalcone derivatives, c) 1,3-diarylpropenones, and d)
thienyl/furanyl bearing phenyl/hydroxyphenyl propenones
Fig. 2. Design of phenyl/hydroxyphenyl bearing fluoro/trifluoromethyl/trifluoromethoxy
phenyl propenones as fluorinated and hydroxylated chalcones
In this following study, we have consistently designed and prepared thirty-six phenyl or
hydroxyphenyl bearing fluoro, trifluoromethyl, or trifluoromethoxy phenyl propenones as
fluorinated or both fluorinated and hydroxylated chalcones. Fluorinated functionalities on 3-
phenyl moiety and ortho, meta, or para-phenols of chalcone were prepared in order to
investigate the inhibitory effects on ROS production stimulated by LPS in RAW 264.7
macrophages. A Structure-activity relationship (SAR) study was conducted with respect to
ortho, meta, and para substitution along with fluorinated phenyl groups on chalcones.

Firstly, we designed and synthesized phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl,
or trifluoromethoxy phenyl propenones (3-38) via the single step modified Claisen-Schmidt
condensation reaction [41, 42] using appropriate aryl methyl ketone 1(R= a-d) and aryl
aldehyde 2 (R1= e-m) as summarized in Scheme 1. Compounds 3-38 were obtained through
base catalyzed reactions without any protection of hydroxyl group that involves the addition
of 50% aqueous KOH (1.0 mL) to a solution of aryl methyl ketone 1 (1.0 mmol) and aryl
aldehyde 2 (1.2 mmol) in ethanol (3 mL). The yields were 32.1 to 82.7%.
Total thirty-six compounds (3-38) were synthesized as shown in Fig. 3. Among them,
compounds 3-14 contained phenyl, ortho-, meta-, or para-hydroxyphenyl moiety along with
ortho-, meta-, or para-fluorophenyl moiety. Compounds 15-26 contained phenyl, ortho-,
meta-, or para-hydroxyphenyl moiety along with ortho-, meta-, or para-
trifluoromethylphenyl moiety. Similarly, compounds 27-38 contained phenyl, ortho-, meta-,
or para-hydroxyphenyl moiety along with ortho-, meta-, or para-trifluoromethoxyphenyl
moiety. The SAR was determined according to the position of hydroxyl, fluoro,
trifluoromethyl, or trifluoromethoxy group, as well as the presence or absence of hydroxyl
group in the phenyl ring of chalcone.

Scheme 1. Schematic representation for the synthesis of phenyl/hydroxylphenyl bearing
fluoro/trifluoromethyl/trifluoromethoxy phenyl propenones 3-38 as fluorinated and
hydroxylated chalcones. Reagents and conditions: (i) 50% aq. KOH (9.0 equiv., 1mL), EtOH
(3 mL), 1-3 h, 25°C, 32.1 to 82.7% yield.

Fig. 3. Synthesized phenyl/hydroxyphenyl bearing fluoro/trifluoromethyl/trifluoromethoxy
phenyl propenones 3-38

The prepared compounds were evaluated for inhibitory effect on ROS production stimulated
by LPS in RAW 264.7 macrophage as shown in Table 1.
Table 1. Inhibitory activities of compounds 3-38 on ROS production stimulated by LPS in
RAW 264.7 macrophages
ROS
ROS
Compounds
Inhibition
(IC50, µM)
Toxicity
Compounds
Inhibition
(IC50, µM)
Toxicity
>10
>10
N
N
1.44 ± 0.56
>10
N
N
3
22
4
23
>10
N
>10
N
5
24
>10
N
>10
N
6
25
>10
N
>10
N
7
26
>10
N
>10
N
8
27
>10
N
ND
Toxic*
9
28
9.15 ± 1.77
>10
N
1.61 ± 1.06
1.34 ± 0.44
>10
N
10
11
12
13
14
15
16
17
18
19
20
21
29
N
N
30
>10
N
N
Toxic*
31
>10
N
ND
32
>10
N
>10
N
33
>10
N
Toxic*
>10
N
34
ND
>10
N
35
>10
N
>10
N
36
>10
N
>10
N
37
38
>10
N
Toxic*
Toxic*
>10
N
ND
Malvidin [5]
9.0 ± 0.8
ND
*
N: Non-toxic, ND: Not Determined, Less than 90% cell viability at 1 µM, **Each data
represents mean ± S.D. from three different experiments performed in triplicate

Amongst the first twelve synthesized compounds 3-14, that had the basic skeleton of
hydroxyphenyl-fluorophenylpropenone either with a fluorine substituent at the ortho, meta,
or para position on the 3-phenyl ring of chalcone (3, 7, 11), or with both hydroxyl and
fluorine substituents at the ortho, meta, or para position on the 1, and 3-phenyl ring of
chalcone (4-6, 8-10, 12-14), compound 10 significantly inhibited LPS-stimulated ROS
production in RAW 264.7 macrophages (9.15 µM of IC50). This demonstrated that a hydroxyl
group at the para position along with a fluorine atom at the meta position is essential for
inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. Among the next
twelve synthesized compounds 15-26, that had the basic skeleton of hydroxyphenyl-
trifluoromethylphenyl propenone either with a trifluoromethyl substituent at the ortho, meta,
or para position on the 3-phenyl ring of chalcone (15, 19, 23), or with both hydroxyl and
trifluoromethyl substituents at the ortho, meta, or para position on the 1, and 3-phenyl ring of
chalcone (16-18, 20-22, 24-26), compound 22 significantly inhibited ROS production
stimulated by LPS in RAW 264.7 macrophages (1.44 µM of IC50). This also showed that a
hydroxyl group at the para position along with a trifluoromethyl group at the meta position is
essential for inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages.
Among the last twelve synthesized compounds 27-38, having the basic skeleton of
hydroxyphenyl-trifluoromethoxyphenyl propenone either with a trifluoromethoxy substituent
at the ortho, meta, or para position on the 3-phenyl ring of chalcone (27, 31, 35), or with both
hydroxyl and trifluoromethoxy substituents at the ortho, meta, or para position on the 1, and
3-phenyl ring of chalcone (28-30, 32-34, 36-38), compound 29 and 30 significantly inhibited
LPS-stimulated ROS production in RAW 264.7 macrophages (1.61 and 1.34 µM of IC50,
respectively). This demonstrated that a hydroxyl group at the meta or para position along
with a trifluoromethoxy group at the ortho position is essential for inhibiting the LPS-
stimulated ROS production in RAW 264.7 macrophages. Among the thirty-six synthesized
compounds, five compounds 16, 20, 21, 28, and 32 showed toxicity, which possessed ortho-
hydroxyphenyl moiety along with trifluoromethyl or trifluoromethoxy phenyl moiety. It was
observed that 3-phenyl bearing fluoro, trifluoromethyl, trifluoromethoxy phenyl propenones
without a hydroxyl substituent on 1-phenyl moiety had no significant inhibition of LPS-
stimulated ROS production in RAW 264.7 macrophages. Analyzing the structure-activity
relationship revealed that the compounds possessing para-hydroxyphenyl moiety along with
meta-fluoro or meta-trifluoromethyl phenyl group, and meta- or para-hydroxyphenyl group
along with ortho-trifluoromethoxyphenyl group showed significant inhibition of LPS-

stimulated ROS production in RAW 264.7 macrophages. It was also observed that most of
the ortho-hydroxyphenyl moiety along with trifluoromethyl, or trifluoromethoxyphenyl
moiety possessed toxic properties. Therefore, it can be concluded that a hydroxyl group at the
para position of the 1-phenyl ring along with a fluoro or trifluoromethyl group at the meta
position, or a trifluoromethoxy group at the ortho position of the 3-phenyl ring of chalcone
has an essential role in inhibiting LPS-stimulated ROS production in RAW 264.7
macrophages, which is explained in Fig. 4.
Fig. 4. Favorable substitution order on chalcone for the inhibition of ROS production
stimulated by LPS in RAW 264.7 macrophages from SAR study
In summary, systematical design and synthesis of the thirty-six phenyl or hydroxyphenyl
bearing fluoro, trifluoromethyl, or trifluoromethoxy phenyl propenones were performed using
Claisen-Schmidt condensation reaction. Inhibitory effects of these compounds on LPS-
stimulated ROS production in RAW 264.7 macrophages were examined. A concrete
structure-activity relationship was observed according to the positions of substitutions of
hydroxyl and fluorinated groups. The most significant inhibitory effect on LPS-stimulated
ROS production in RAW 264.7 macrophages was exhibited by compound 30 that possessed
para-hydroxyphenyl moiety as well as ortho-trifluoromethoxyphenyl moiety.

Acknowledgements This research was supported by the Yeungnam University research
grants in 2014.
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Figure captions
Fig. 1. Structures of a) chalcone, b) chalcone derivatives, c) 1,3-diarylpropenones, and d)
thienyl/furanyl bearing phenyl/hydroxyphenyl propenones
Fig. 2. Design of phenyl/hydroxyphenyl bearing fluoro/trifluoromethyl/trifluoromethoxy
phenyl propenones as fluorinated and hydroxylated chalcones
Fig. 3. Synthesized phenyl/hydroxyphenyl bearing fluoro/trifluoromethyl/trifluoromethoxy
phenyl propenones 3-38
Fig. 4. Favorable substitution order on chalcone for inhibition of ROS production stimulated
by LPS in RAW 264.7 macrophages for SAR study

Scheme caption
Scheme 1. Schematic representation for the synthesis of phenyl/hydroxylphenyl bearing
fluoro/trifluoromethyl/trifluoromethoxy phenyl propenones 3-38 as fluorinated and
hydroxylated chalcones. Reagents and conditions: (i) 50% aq. KOH (9.0 equiv., 1mL), EtOH
(3 mL), 1-3 h, 25°C, 32.1 to 82.7% yield.

Table caption
Table 1. Inhibitory activities of compounds 3-38 on ROS production stimulated by LPS in
RAW 264.7 macrophages

Graphical abstract
Graphical Abstract

Highlights
 Designed and synthesized new fluorinated and hydroxylated chalcones.
 Inhibitory effects on LPS-stimulated ROS production in RAW 264.7 macrophage were
evaluated.
 SAR showed p-OH along with m-F or CF3, and m- or p-OH along with o-OCF3 has an
essential role for inhibition.
 Compounds 30 showed the most potent inhibitory effect.