Design and Discovery of Some Novel Chalcones as Antioxidant and Anti-Inflammatory Agents via Attenuating NF-κB

Article abstract of DOI:10.1002/ardp.201500349

Concerning the role of antioxidant and anti-inflammatory agents for hepatic fibrosis patients, the current study deals with the development of novel chalcone derivatives 5a-i via efficient synthetic methodology in a two-step reaction involving Claisen-Sch

Full text of DOI:10.1002/ardp.201500349

ARC HH PHARM
Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
Archiv der Pharmazie
Full Paper
Design and Discovery of Some Novel Chalcones as Antioxidant and
Anti-Inflammatory Agents via Attenuating NF-kB
Jun Chu and Chun-Liang Guo
Department of Hepatobiliary Surgery, Affiliated Hospital of Logistics University of PAPF, Tianjin,
P. R. China
Concerning the role of antioxidant and anti-inflammatory agents for hepatic fibrosis patients, the
current study deals with the development of novel chalcone derivatives 5a–i via efficient synthetic
methodology in a two-step reaction involving Claisen–Schmidt condensation. The obtained target
2 2
analogs were screened for in vitro antioxidant activity by various methods (H O , DPPH, ferrous
reducing power, and nitric oxide), where they exhibit considerable radical scavenging activity. These
compounds were also evaluated for inhibitory potency against NF-kB activation induced by LPS for
the determination of their anti-inflammatory activity. The inhibition values indicate that the entire
set of compounds efficiently inhibits the NF-kB activation provoked by LPS. Among the series,
compound 5i was identified as the most potent inhibitor of NF-kB, with a relative NF-kB activity of
1
.12 ꢀ 0.53. It also inhibits various inflammatory mediators, such as TNF-a, IL-1b, IL-6, and PGE
2
.
Keywords: Anti-inflammatory / Antioxidant / Chalcones / Hepatic fibrosis
Received: September 24, 2015; Revised: November 16, 2015; Accepted: November 20, 2015
DOI 10.1002/ardp.201500349
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
cell (HSC) activation, and proliferation whereas, the hepato-
cyte, Kupffer cell, and HSC communicate through oxygen
stress, which causes inhibition of electron transport, increas-
ing ROS production, and decreasing mitochondrial glutathi-
one [4]. Thus, the use of antioxidants diminishes fibrosis and
HSCs, by halting the associated cell communication [5].
On the other hand, the studies concluded that chronic
inflammation of the liver is the key component to initiate
fibrosis by the infiltration and activation of immune cells [6].
This will in turn initiate both inflammatory and wound
healing responses, including subsequent necrosis and apo-
ptosis of parenchymal cells and replacement by connective
tissue and extracellular matrix (ECM) proteins. According
to recent studies, it has been suggested that activation of
the proinflammatory nuclear factor-kB (NF-kB) pathway in
hepatocytes is a key factor responsible for inflammation of
fibrotic liver [7]. The family of NF-kB transcription factors
belongs to the key regulators of inflammatory processes.
Therefore, aberrant activation of NF-kB can lead to constitu-
tive overproduction of proinflammatory cytokines, which is
associated with a number of chronic inflammatory disorders
as seen in the case of patients with liver diseases such as
hepatitis B, hepatitis C, or hepatocellular carcinoma [8].
The liver is a vital organ of the body necessary for performing
critical functions to maintain homeostasis and health. It
executes tasks related to digestion, metabolism, immunity,
storage of nutrients within the body and detoxification and
degradation of foreign (e.g., drugs) and naturally occurring
chemicals [1]. Thus, improper functioning of liver due to these
four diseases, like cirrhosis, fatty liver, hepatitis, and liver
cancer can sternly have an effect on health and can be life
threatening. Out of them, hepatic fibrosis patients are prone
to serious complications by almost all systems, leading to
high morbidity rates and even death because of liver cirrhosis
and hepatic failure [2, 3]. At the molecular level, it includes
hepatocyte damage, Kupffer cell activation, hepatic stellate
Correspondence: Dr. Jun Chu, Department of Hepatobiliary
Surgery, Affiliated Hospital of Logistics University of PAPF, No.
2
20 Chenglin Road, Hedong District 300162, Tianjin, P. R. China.
E-mail: junchuchai@hotmail.com
Fax: þ86 022 60578778
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Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
J. Chu and C.-L. Guo
ARC HH PHARM
Archiv der Pharmazie
–
1
Concerning the above, dual targeting of drugs on inflamma-
tory cascade and production of ROS species could contribute
the beneficial and much more pronounced effect than
individual drug alone.
around 1612 cm which is attributable aromatic C –– C bonds.
The N–H bond was confirmed by the presence of absorption
ꢁ
1
bands around at 3498 cm . The absorptions at 786 and
ꢁ
1
868 cm
were characterized for the F and Cl groups. In
1
In this regard, chalcones, a b-ketone scaffold developed via
Claisen–Schmidt condensation [9] reaction between aromatic
aldehydes and ketone have proved to exhibit numerous
pharmacological activities ranging from anticancer [10],
antibacterial [11], antifungal [12], anti-HIV [13], antimalar-
ial [14], and other. More specifically, these chalcones are
also proved to exhibit antioxidant and anti-inflammatory
activities [15, 16]. Thus, in extension of this, we wish to report
the development of novel series of chalcones substituted
with numerous groups and tested for antioxidant and anti-
inflammatory properties.
H NMR spectra, the characteristic shift corresponds to N–H
and aliphatic C–H of side chain was observed around
at 9.14–9.17 and 8.12–8.15 ppm, respectively. The chemical
shift of aromatic hydrocarbon was seen at 8.11–7.42 ppm.
The singlet at 5.38 ppm for proton was attributable to
an aromatic hydroxyl group. The C NMR shift of all final
compounds was observed in the expected region. Finally, all
the structures of title compounds were confirmed by mass
and elemental analyses.
1
3
Biological activity
In vitro antioxidant activity
As seen in Table 1, the entire set of target analogs was
assessed for antioxidant activity in various in vitro methods.
All the synthesized compounds showed considerable activity
ranging from mild to moderate against hydrogen peroxide.
The results revealed compound 5a as most potent derivative
and exhibited 93.82% of inhibition. This was followed by
decrease in activity for its isomeric congeners. Thesame pattern
of percentage of inhibition was revealed for next analogs, i.e.,
5d, 5e, and 5f. However, a marked increase in activity was
reported by compound 5i, whereas rest of the compounds
Results and discussion
Chemistry
The synthesis of the target compounds was achieved in two-
step reaction as depicted in Scheme 1. Initially, the synthesis
of N-(3-acetylphenyl)-4-fluorobenzamide (3) was achieved
by reaction between 1-(3-amino-phenyl)ethanone (1) and
4
-fluorobenzoyl chloride (2) via nucleophillic reaction in the
presence of activating base NaOH. The furnished product (3)
was then allowed to react with substituted aromatic aldehyde
2 2
showed mild inhibition of H O scavenging activity.
(
4) to afford the corresponding chalcone derivatives (5a–l)
In the case of DPPH free radical scavenging activity,
compounds 5i, 5c, and 5a were identified as most potent
and showed excellent activity than standard ascorbic acid
whereas the rest of compounds exhibited mild to moderate
scavenging activity. For determination of ferrous reducing
ability of compounds, among the series, except compounds
5g, 5h, and 5i entire set of analogs showed excellent reducing
power than standard.
The nitric oxide radical scavenging activity result revealed
that all the synthesized derivatives showed excellent activity
than standard ascorbic acid. Among the series, in the
inhibitory assay, compound 5a was revealed as the most
potent analog and 5h as least potent.
via Claisen–Schmidt condensation. The target compounds
were obtained in excellent yields and their structures were
ascertained on the basis of various spectroscopic and
analytical techniques.
The melting points of the all synthesized compounds 5a–l
1
13
and their H NMR and
C NMR data along with the
elemental analysis (percentage C, H, and N analysis) and mass
analysis are given in the Experimental section. FT-IR spectra
of the synthesized compound clearly show C –– H stretching
absorption bands at 3066 cm due to aromatic ring and C –– O
stretching absorption bands at around 1718 cm , respec-
–
1
–
1
tively. Compound 5i has also showed strong absorption band
Scheme 1. Synthesis of compounds 5a–i. (a) 5% NaOH; (b) ethanol, aq. NaOH.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
Novel Chalcones as Antioxidant and Anti-Inflammatory Agents
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Table 1. Antioxidant activity of target analogs.
Ferrous reducing
Nitric oxide radical
scavenging activity
Entry
H
2
O
2
DPPH
power
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
93.82
84.56
83.44
66.43
63.28
60.69
32.14
28.94
63.35
86.36
–
45.76
51.34
54.56
40.87
40.94
35.18
33.56
28.13
51.35
–
56.96
55.16
50.71
43.54
48.36
41.59
38.42
35.82
39.04
–
76.33
71.67
73.17
61.15
56.42
55.87
45.61
39.19
44.84
–
BHT
Ascorbic acid
43.54
40.87
35.76
NF-kB inhibition assay
stimulated group (with the relative NF-kB activity (NF-kB/TK,
fold): 5.75 ꢀ 1.63). Structure–activity relationships (SAR) of
novel chalcone derivatives demonstrated that compounds
with para electron-withdrawing substituents (5g–i) showed
more potent activities than those with electron-donating
substituents (5a–f). Whereas, compounds having para, ortho,
and meta substituents on the aromatic ring demonstrated
that an electron-donating group (5d–f) slightly improved the
NF-kB inhibitory activity with the order of potency being
The target compounds 5a–i assayed their inhibitory potency
against NF-kB activation induced by LPS. The results of assay
are summarized in Table 2. The inhibition values indicate that
the entire set of compounds efficiently inhibits the NF-kB
activation provoked by LPS. Compounds 5a, 5b, and 5c,
containing an isomeric hydroxyl group showed mild inhibitory
activity, and out of which, meta-substituted derivative was
found most active. In the next instance, compounds contain-
ing the methoxy group at various positions on the phenyl
exhibit mild to moderate activity. Moreover, among the
synthesized chalcone derivatives, compound 5i was proved as
the most efficient inhibitor of NF-kB. The minor decline in
activity was reported by compounds 5g and 5h containing
halogen electron withdrawing group. It was revealed that the
inhibitory activity of the compounds was greatly influenced
by the nature of the substituent and their position. In the
study, the control group was calculated without stimulating
by LPS (with the relative NF-kB activity (NF-kB/TK, fold):
3 3 3
4-OCH > 2-OCH > 3-OCH . Moreover, in the case of com-
pounds (5g–i) with a halogen atom on the 4-position of
aromatic ring, the order of inhibitory activity is F > Br > Cl.
Cell viability
To assess the effect of compound 5i on the viability of cells, it
has been tested against the RAW264.7 cells via MTT assay. As
shown in Fig. 1, it was indicated that compound 5i did not
induce any loss of cell viability even at the higher tested
concentration of 50 mg/mL. Thus, it could be suggested that
compound 5i was found non-toxic to macrophages and its
1
.53 ꢀ 0.52) whereas, the LPS group was defined as the
Table 2. Effect of target compounds on NF-kB transcrip-
tional activity in LPS-stimulated RAW264.7 cells.
Relative NF-kB activity
Analogs
(NF-kB/TK, fold)
a)
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
3.85 ꢀ 1.62
a)
4.66 ꢀ 1.43
a)
4.01 ꢀ 1.45
a)
3.12 ꢀ 0.39
a)
4.76 ꢀ 1.25
a)
3.73 ꢀ 1.11
a)
3.76 ꢀ 0.32
a)
2.14 ꢀ 0.34
a)
1.12 ꢀ 0.53
Control
LPS
1.53 ꢀ 0.52
b)
5.75 ꢀ 1.63
N ¼ 3.
a)
p < 0.01 versus LPS.
Figure 1. Effect of compound 5i on the viability of RAW264.5
cells.
b)
p < 0.01 versus control.
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NF-kB inhibition is entirely dependent on the ability to exert
pharmacological effect.
Effect of compound 5i on pro-inflammatory mediators
To explain the inhibitory effect of the compound 5i on the
production of inflammatory mediators, various immuno-
2
assays were conducted for TNF-a, IL-1b, IL-6, and PGE . In
the current study, RAW264.7 cells were treated with different
concentrations of compound 5i for 1 h before the treatment
of cells with 1 mg/mL of lipopolysaccharide to stimulate
inflammatory process. After 1 day of incubation, inured
media were collected, and then the level of inflammatory
mediators was measured. As shown in Figs. 2–5, compound 5i
Figure 4. Effect of compound 5i on IL-16. The values presented
##
ꢂ
ꢂꢂ
are mean ꢀ SEM. p < 0.01 versus control, p < 0.05, p < 0.01
versus LPS.
exhibited a dose-dependent inhibitory effect on the produc-
tion of all inflammatory mediators. The 10 mg/mL of
compound 5i decreased TNF-a level as compared to blank
group (Fig. 2). Moreover, It has been found that no significant
difference in inhibitory potency was observed against TNF-a
between dexamethasone at 1 mM and treatment group of 5i
above 5 mg/mL. As shown in Fig. 3, the inhibitory effect of 5i
on the production of IL-1b was remarkably compared with
those of other cytokines whereas, it was surprising to note
that the level of IL-6 in the presence of 5i at 10 mM was
comparable to that of dexamethasone at 1 mM (Fig. 4). The
Figure 2. Effect of compound 5i on TNF-a. The values presented
##
ꢂ
ꢂꢂ
are mean ꢀ SEM. p < 0.01 versus control, p < 0.05, p < 0.01
inhibitory effect of 5i on production of PGE
pronounced than those on the production of cytokines
Fig. 5). The treatment with 10 mg/mL of 5i significantly
2
was more
versus LPS.
(
Figure 3. Effect of compound 5i on IL-b. The values presented
2
Figure 5. Effect of compound 5i on PGE . The values presented
##
##
ꢂ
ꢂꢂ
ꢂ
ꢂꢂ
are mean ꢀ SEM. p < 0.01 versus control, p < 0.05, p < 0.01
are mean ꢀ SEM. p < 0.01 versus control, p < 0.05, p < 0.01
versus LPS.
versus LPS.
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Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
Novel Chalcones as Antioxidant and Anti-Inflammatory Agents
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discovery initiatives for the treatment of the liver cirrhotic
patients.
Experimental
Chemistry
All commercially available solvents and reagents of analytical
grade were used without further purification. Melting points
were determined with digital thermometer and were
uncorrected. FT-IR were recorded on Perkin Elmer Spectro-
1
photometer. H NMR spectra were recorded on Bruker Avance
1
3
II 400 NMR and C NMR spectra on Bruker Avance II 100 NMR
spectrometer in DMSO using TMS as an internal standard.
Mass spectra were obtained on VG-AUTOSPEC spectrometer
equipped with electrospray ionization (ESI) sources. Elemen-
tal analysis was carried out on Vario EL-III CHNOS elementor
analyzer.
Figure 6. The orientation of compound 5i in the active site of
NF-kB.
General procedure for the preparation N-(3-acetylphenyl)-
4-fluorobenzamide (3)
In a 20 mL of 5% of sodium hydroxide solution taken in round
bottom flask, 1-(3-amino-phenyl)ethanone 1 (1 g, 7.40 mM)
was suspended. Afterwards, 2 mL 4-fluorobenzoyl chloride
2
dwindled the level of PGE , which was similar to that of aspirin
at 10 mM used as a positive control in this experiment.
(
2), 0.5 mL at a time was added to above mixture with
Docking study of compound 5i with NF-kB protein model
The pronounced activity of compound 5i has prompted us to
perform the molecular docking analysis to exemplify the key
structural parameters necessary for this activity. For this,
the NF-kB protein model has been taken from the protein
database (i.e., 1NFK.pdb) and co-crystallized ligand was
removed for further processing. After that, the protein was
optimized and the binding site was identified by the “binding
site tool” of Discovery Studio 2.5. The legend was then
allowed to dock with protein p50 NF-kB protein with the
help of CDOCKER. The procedure was followed as per the
manufacturer guidelines.
constant shaking and vigorous stirring for 20 min. The
reaction mixture was heated under reflux on water bath
for 30 min until the odor of the 4-fluorobenzoyl chloride
disappeared. Resultant reaction mixture was made alkaline,
the solid benzoyl derivative was filtered off, and recrystallized
it from petroleum ether and ethyl acetate to obtain
compound 3.
Yield: 82%; mp: 117–118°C; MW: 257.26; R
f
: 0.72; FT-IR
ꢁ
1
(nmax; cm KBr): 3478, 3294, 3073, 2961, 1715, 1602, 1503,
ꢁ
1 1
1483, 1285, 1163, 1058, 842 cm ; H NMR (400 MHz, DMSO,
TMS) d ppm: 8.38 (s, 1H, –NH), 8.14 (s, 1H, ArH), 8.04 (d,1H,
J ¼ 8.7 Hz, ArH), 7.63 (d, 1H, J ¼ 7.2 Hz, ArH), 7.38 (t, 1H,
Results showed that compound 5i was efficiently docked
into the active site of NF-kB via proficient CDOCKER energy of
J ¼ 7.8 Hz, ArH,), 7.94–7.83 (m, 4H, ArH), 2.58 (s, 3H, CH
3
);
1
3
C NMR (100 MHz, CDCl
135.7, 133.4, 129.7, 128.9, 126.5, 124.3, 118.9, 115.2, 26.6;
12FNO
3
) d ppm: 197.4, 166.2, 164.7, 136.9,
ꢁ
11.45. As shown in Fig. 6, the compound is able to interact
with Lys145, key catalytic residues via the H-bond. It will also
reveal the formation of two non-bonded p–cation interaction
with His141 and Lys145.
mass: 258.28 (Mþ1); elemental analysis for C15
H
2
:
Calculated: C, 70.03; H, 4.70; N, 5.44. Found: C, 70.10;
H, 4.69; N, 5.44.
General procedure for synthesis of substituted chalcone
derivatives 5a–i
Conclusion
N-(3-Acetylphenyl)-4-fluorobenzamide (3) (0.01 mol) and
substituted benzaldehyde (4) (0.01 mol) were dissolved in
50 mL ethanolic solution and stirred for 30 min. It was
followed by drop-wise addition of aqueous sodium hydroxide
(0.05 mol) with further stirring continued for 24 h. After
completion of the reaction, as monitored by TLC using
the mobile phase as n-butanol/acetic acid/water (4:3:1), a
crude product was obtained as substituted chalcone, 5(a–i).
The resultant solid was then filtered off, washed with water,
dried, and re-crystallized from ethanol.
We have developed a series of novel chalcones derivatives
via efficient synthetic methodology. The molecules were
obtained in excellent yields. In antioxidant assay, these
analogs showed an excellent ability to scavenge the genera-
tion of free radicals in different in vitro systems. Moreover, in
NF-kB assay, these molecules showed considerable inhibitory
activity. Together with excellent antioxidant activity, dock
score and anti-inflammatory activity via inhibition of NF-kB,
these molecules proved an excellent lead for the newer drug
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Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
J. Chu and C.-L. Guo
ARC HH PHARM
Archiv der Pharmazie
(
E)-4-Fluoro-N-(3-(3-(4-hydroxyphenyl)acryloyl)phenyl)-
115.6, 114.2, 55.7; mass: 376.42 (Mþ1); elemental analysis for
benzamide (5a)
23 3
C H18FNO : Calculated: C, 73.59; H, 4.83; N, 3.73. Found: C,
Yield: 73%; mp: 215–216°C; MW: 361.37; R
f
: 0.58; FT-IR (nmax
;
73.61; H, 4.84; N, 3.75.
ꢁ
1
cm KBr): 3478, 3291, 3073, 2968, 1713, 1608, 1513, 1489,
ꢁ
1 1
1
285, 1169, 1068, 846, 782 cm
;
H NMR (400 MHz, DMSO,
(E)-4-Fluoro-N-(3-(3-(3-methoxyphenyl)acryloyl)phenyl)-
TMS) d ppm: 9.15 (s, 1H, –NH), 8.14 (s, 1H, ArH), 8.11–7.42 (m,
benzamide (5e)
4
7
6
H, ArH), 8.06–8.02 (m, 2H, CH), 7.92 (d,1H, J ¼ 8.4 Hz, ArH),
.68 (d, 1H, J ¼ 7.8 Hz, ArH), 7.58 (t, 1H, J ¼ 7.1 Hz, ArH), 7.54–
Yield: 72%; mp: 238–239°C; MW: 375.39; R
f
: 0.71; FT-IR (nmax
;
ꢁ
1
cm KBr): 3490, 3287, 3072, 2978, 1717, 1609, 1518, 1487,
13
ꢁ1
1
.68 (m, 4H, ArH), 5.38 (s, 1H, Ar–OH); C NMR (100 MHz,
1282, 1178, 1072, 859, 778 cm ; H NMR (400 MHz, DMSO,
CDCl
3
) d ppm: 189.6, 166.3, 163.8, 157.4, 145.3, 138.8, 136.4,
TMS) d ppm: 9.15 (s, 1H, –NH), 8.12 (s, 1H, ArH), 8.05–8.03 (m,
2H, CH), 8.12–7.42 (m, 4H, ArH), 7.89 (d, 1H, J ¼ 8.4 Hz, ArH),
7.63 (d, 1H, J ¼ 7.3 Hz, ArH), 7.59–6.87 (m, 4H, ArH), 7.60 (t, 1H,
1
1
C
33.9, 130.6, 129.9, 128.9, 127.8, 127.3, 124.5, 121.9, 118.4,
14.9, 113.7; mass: 362.41 (Mþ1); elemental analysis for
1
3
22
H16FNO
3
: Calculated: C, 73.12; H, 4.46; N, 3.88. Found: C,
J ¼ 6.5 Hz, ArH), 3.87 (s, 3H, Ar–OCH
3
); C NMR (100 MHz,
7
3.14; H, 4.45; N, 3.91.
CDCl
35.1, 133.7, 129.7, 129.1, 128.5, 127.4, 127.1, 124.3, 121.3,
120.8, 119.3, 115.2, 113.5, 55.8; mass: 376.45 (Mþ1); elemental
analysis for C23 18FNO : Calculated: C, 73.59; H, 4.83; N, 3.73.
3
) d ppm: 189.5, 166.3, 164.8, 160.5, 145.2, 138.1, 136.4,
1
(
E)-4-Fluoro-N-(3-(3-(3-hydroxyphenyl)acryloyl)phenyl)-
benzamide (5b)
Yield: 78%; mp: 219-221 C; MW: 361.37; R
H
3
°
f
: 0.63; FT-IR (nmax
;
Found: C, 73.58; H, 4.86; N, 3.72.
ꢁ
1
cm KBr): 3482, 3295, 3073, 2971, 1712, 1608, 1509, 1482,
ꢁ
1 1
1
287, 1172, 1064, 848, 773 cm
;
H NMR (400 MHz, DMSO,
(E)-4-Fluoro-N-(3-(3-(2-methoxyphenyl)acryloyl)phenyl)-
TMS) d ppm: 9.14 (s, 1H, –NH), 8.13 (s, 1H, ArH), 8.12–7.48 (m,
benzamide (5f)
4
7
6
H, ArH), 8.06–8.01 (m, 2H, CH), 7.89 (d,1H, J ¼ 8.2 Hz, ArH),
.63 (d, 1H, J ¼ 7.6 Hz, ArH), 7.56 (t, 1H, J ¼ 6.8 Hz, ArH), 7.53–
Yield: 79%; mp: 241–242°C; MW: 375.39; R
f
: 0.63; FT-IR (nmax
;
ꢁ
1
cm KBr): 3495, 3282, 3078, 2985, 1709, 1612, 1509, 1487,
13
ꢁ1
1
.64 (m, 4H, ArH), 5.39 (s, 1H, Ar–OH); C NMR (100 MHz,
1287, 1171, 1076, 863, 787 cm ; H NMR (400 MHz, DMSO,
CDCl
3
) d ppm: 189.4, 166.2, 164.7, 158.3, 145.1, 138.2, 136.3,
TMS) d ppm: 9.14 (s, 1H, –NH), 8.13 (s, 1H, ArH), 8.33–7.43 (m,
2H, CH), 8.12–7.48 (m, 4H, ArH), 7.89 (d, 1H, J ¼ 8.4 Hz, ArH),
7.63 (d, 1H, J ¼ 7.3 Hz, ArH), 7.66–6.94 (m, 4H, ArH), 7.62 (t, 1H,
1
1
35.4, 133.7, 130.2, 129.8, 128.9, 127.4, 124.3, 121.3, 121.1,
19.5, 117.7, 115.7, 114.8; mass: 362.37 (Mþ1); elemental
1
3
analysis for C22
H16FNO
3
: Calculated: C, 73.12; H, 4.46; N, 3.88.
J ¼ 6.4 Hz, ArH), 3.84 (s, 3H, Ar–OCH
3
); C NMR (100 MHz,
Found: C, 73.13; H, 4.48; N, 3.87.
CDCl
35.2, 133.7, 129.8, 129.2, 128.9, 127.5, 125.7, 124.1, 121.5,
120.9, 119.5, 115.6, 114.2, 56.2; mass: 376.42 (Mþ1); elemental
analysis for C23 18FNO : Calculated: C, 73.59; H, 4.83; N, 3.73.
3
) d ppm: 189.3, 166.5, 164.7, 159.2, 141.5, 138.3, 137.8,
1
(
E)-4-Fluoro-N-(3-(3-(2-hydroxyphenyl)acryloyl)phenyl)-
benzamide (5c)
H
3
Yield: 83%; mp: 223–225°C; MW: 361.37; R
f
: 0.68; FT-IR (nmax
;
Found: C, 73.62; H, 4.84; N, 3.75.
ꢁ
1
cm KBr): 3484, 3298, 3072, 2968, 1718, 1612, 1509, 1482,
ꢁ
1 1
1
282, 1178, 1061, 854, 778 cm
;
H NMR (400 MHz, DMSO,
(E)-N-(3-(3-(4-Chlorophenyl)acryloyl)phenyl)-4-
TMS) d ppm: 9.17 (s, 1H, –NH), 8.15 (s, 1H, ArH), 8.30–7.42 (m,
fluorobenzamide (5g)
2
7
6
H, CH), 8.14–7.42 (m, 4H, ArH), 7.87 (d, 1H, J ¼ 8.4 Hz, ArH),
Yield: 62%; mp: 268–269°C; MW: 379.81; R
f
: 0.87; FT-IR (nmax
;
ꢁ
1
.63 (d, 1H, J ¼ 7.6 Hz, ArH), 7.58 (t, 1H, J ¼ 6.9 Hz, ArH), 7.60–
cm KBr): 3491, 3283, 3072, 2987, 1712, 1609, 1516, 1493,
13
ꢁ1
1
.48 (m, 4H, ArH), 5.42 (s, 1H, Ar–OH); C NMR (100 MHz,
1287, 1178, 1094, 868, 781 cm ; H NMR (400 MHz, DMSO,
CDCl
3
) d ppm: 189.1, 166.3, 164.8, 158.4, 145.3, 138.7, 136.4,
TMS) d ppm: 9.15 (s, 1H, –NH), 8.12 (s, 1H, ArH), 8.13–7.48 (m,
4H, ArH), 8.06–7.62 (m, 2H, CH), 7.88 (d, 1H, J ¼ 8.4 Hz, ArH),
7.64 (d, 1H, J ¼ 7.6 Hz, ArH), 7.61 (t, 1H, J ¼ 6.5 Hz, ArH), 7.68–
1
1
35.7, 133.7, 130.1, 129.9, 128.3, 127.4, 124.1, 121.5, 121.3,
19.8, 117.6, 115.8, 114.9; mass: 362.45 (Mþ1); elemental
1
3
analysis for C22
H16FNO
3
: Calculated: C, 73.12; H, 4.46; N, 3.88.
3
7.46 (m, 4H, ArH); C NMR (100 MHz, CDCl ) d ppm: 189.5,
Found: C, 73.15; H, 4.45; N, 3.91.
166.3, 164.7, 145.2, 138.3, 136.1, 133.5, 133.7, 133.1, 129.8,
29.1, 128.9, 128.5, 127.4, 124.1, 121.7, 119.8, 115.6; mass:
15ClFNO : Calcu-
lated: C, 69.57; H, 3.98; N, 3.69. Found: C, 68.58; H, 3.97;
N, 3.71.
1
(
E)-4-Fluoro-N-(3-(3-(4-methoxyphenyl)acryloyl)phenyl)-
380.85 (Mþ1); elemental analysis for C22
H
2
benzamide (5d)
f
Yield: 68%; mp: 233–234°C; MW: 375.39; R : 0.74; FT-IR (nmax;
ꢁ
1
cm KBr): 3492, 3289, 3078, 2972, 1713, 1618, 1513, 1482,
ꢁ
1 1
1
287, 1172, 1068, 851, 772 cm
;
H NMR (400 MHz, DMSO,
(E)-N-(3-(3-(4-Bromophenyl)acryloyl)phenyl)-4-
TMS) d ppm: 9.15 (s, 1H, –NH), 8.14 (s, 1H, ArH), 8.06–8.02 (m,
fluorobenzamide (5h)
2
7
6
H, CH), 8.14–7.48 (m, 4H, ArH), 7.89 (d,1H, J ¼ 8.4 Hz, ArH),
Yield: 65%; mp: 282–283°C; MW: 424.26; R
f
: 0.78; FT-IR (nmax
;
ꢁ
1
.64 (d, 1H, J ¼ 7.5 Hz, ArH), 7.62 (t, 1H, J ¼ 6.3 Hz, ArH), 7.62–
cm KBr): 3489, 3291, 3076, 2983, 1709, 1618, 1502, 1498,
13
ꢁ1
1
.94 (m, 4H, ArH), 3.82 (s, 3H, Ar–OCH
3
); C NMR (100 MHz,
1282, 1175, 1094, 875, 789 cm ; H NMR (400 MHz, DMSO,
CDCl
3
) d ppm: 189.7, 166.2, 164.7, 159.8, 145.1, 138.2, 136.4,
TMS) d ppm: 9.15 (s, 1H, –NH), 8.13 (s, 1H, ArH), 8.12–7.42 (m,
4H, ArH), 8.06–7.61 (m, 2H, CH), 7.89 (d, 1H, J ¼ 8.2 Hz, ArH),
1
33.5, 130.7, 129.8, 129.2, 127.5, 127.1, 124.3, 121.1, 119.5,
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Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
Novel Chalcones as Antioxidant and Anti-Inflammatory Agents
ARC HH PHARM
Archiv der Pharmazie
7
7
1
1
4
.64 (d, 1H, J ¼ 7.6 Hz, ArH), 7.61 (t, 1H, J ¼ 6.5 Hz, ArH), 7.61–
Ferrous reducing power
13
.58 (m, 4H, ArH); C NMR (100 MHz, CDCl
3
) d ppm: 189.4,
According to the previous procedure, 100 mM of the
synthesized compounds (1.0 mL) was mixed with 2.5 mL of
phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium
ferricyanide (1%). The resulting mixture was incubated at
50°C for 20 min with TCA (10%: 2.5 mL). Then the mixture was
centrifuged at 3000 rpm for 10 min. The resulting supernatant
66.3, 164.5, 145.1, 138.2, 136.3, 134.2, 133.8, 131.5, 129.7,
29.3, 128.6, 127.4, 124.1, 122.3, 121.3, 119.5, 115.8; mass:
2
24.28 (Mþ1); elemental analysis for C22H15BrFNO : Calcu-
lated: C, 62.28; H, 3.56; N, 3.30. Found: C, 62.31; H, 3.57;
N, 3.28.
(
2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of
(
E)-4-Fluoro-N-(3-(3-(4-fluorophenyl)acryloyl)phenyl)-
ferric chloride (1%) and the absorbance was measured at
700 nm. Higher absorbance of the reaction mixture indicated
greater reducing power. Thus, the reducing power of
compounds was compared with that of standard antioxidant.
benzamide (5i)
Yield: 76%; mp: 273–275°C; MW: 363.36; R : 0.71; FT-IR (nmax;
f
cm KBr): 3498, 3282, 3066, 2989, 1718, 1612, 1509, 1489,
1
ꢁ
1
ꢁ
1 1
287, 1175, 1098, 876, 792 cm
; H NMR (400 MHz, DMSO,
TMS) d ppm: 9.15 (s, 1H, –NH), 8.13 (s, 1H, ArH), 8.12–7.44 (m,
Nitric oxide radical scavenging activity
4
7
7
1
1
3
H, ArH), 8.06–7.58 (m, 2H, CH), 7.87 (d, 1H, J ¼ 8.4 Hz, ArH),
Nitric oxide radicals were generated from sodium nitroprus-
side solution. One milliliter of 10 mM sodium nitroprusside
was mixed with 1 mL of 100 mM target compounds in
phosphate buffer (0.2 M, pH 7.4). The mixture was incubated
at 25°C for 150 min. After incubation, the reaction mixture
was mixed with 1.0 mL of pre-prepared Griess reagent (1%
sulphanilamide, 0.1% napthyl ethylenediamine dichloride,
and 2% phosphoric acid). The absorbance was measured at
.64 (d, 1H, J ¼ 7.6 Hz, ArH), 7.61 (t, 1H, J ¼ 6.5 Hz, ArH), 7.72–
1
3
3
.19 (m, 4H, ArH); C NMR (100 MHz, CDCl ) d ppm: 189.5,
66.4, 164.7, 162.3, 145.3, 138.1, 136.4, 133.5, 130.8, 130.2,
29.8, 129.2, 127.4, 124.1, 121.5, 119.5, 115.6, 114.9; mass:
64.38 (Mþ1); elemental analysis for C22
15 2 2
H F NO : Calcu-
lated: C, 72.72; H, 4.16; N, 3.85. Found: C, 72.71; H, 4.18;
N, 3.82.
5
46 nm and percentage of inhibition was calculated using the
Antioxidant activity [17]
Hydrogen peroxide (H O ) scavenging activity
2 2
same formula as given below. The decreasing absorbance
indicates a high nitric oxide scavenging activity.
Phosphate buffer (pH 7.4) was taken for preparing a
solution of H (40 mM), which was used to conduct this
experiment. The 1 mM concentrations of various target
compounds were added to the above prepared H
solution (0.6 mL, 40 mM). After 10 min, the absorbance of
at 230 nm was determined against a blank solution
containing phosphate buffer without drug. The percentage
scavenging of hydrogen peroxide of synthetic compounds
and standard compounds was calculated using the follow-
ing formula:
ꢀ
À
Áꢁ
2
O
2
Acontrol ꢁ Asample
Percentage of inhibition ¼
ꢄ 100
A
control
2
O
2
where Acontrol is the absorbance of nitric oxide and Asample is
the absorbance of the reaction mixture (nitric oxide with
sample).
2 2
H O
NF-kB inhibition assay
RAW264.7 macrophages were purchased from American
Type Culture Collection (ATCC, USA). Cells were cultured in
DMEM with 10% FBS, 100 mg/mL streptomycin, and 100 U/mL
penicillin in humidified 5% CO at 37°C. The 80% confluency
of RAW264.7 cells was achieved in six-well plates and
transfected with 1 mg of the NF-kB reporter construct, along
with 0.5 mg of pSVGal plasmid using LipofectAMINE 2000
(Invitrogen) in Opti-MEM medium (Gibco). After 24 h of
transfection, cells were treated with LPS or target derivatives
(100 mM) for an additional 2 h, and then lysed using the
reporter lysis buffer (Promega). Luciferase assays were
performed using 20 mL of cell extract and 100 mL of luciferin
substrate (Promega), and the luciferase activity was measured
using a Dual-Luciferase Reporter Assay System (Promega)
according to the manufacturer’s instructions. Data are
normalized for transfection efficiency by dividing firefly
luciferase activity with that of Renilla luciferase.
ꢀ
ꢁ
ðA0 ꢁ A1Þ
A0
Percentage scavenged ½H2O2ꢃ ¼
where A
ꢄ 100
2
0
is the absorbance of the control and A
1
is the
absorbance in the presence of the sample of MO and
standards.
DPPH radical scavenging activity
In this method, briefly, 1 mL of synthesized compounds as
1
00 mM was mixed with 3.0 mL DPPH (0.5 mmol/L in metha-
nol); the resultant absorbance was recorded at 517 nm after
0 min of incubation at 37°C. Following formula was used to
3
assess the percentage of scavenging activity of the target
compounds:
ꢀ
À
Áꢁ
Acontrol ꢁ Asample
Percentage of inhibition ¼
ꢄ 100
A
Cell viability assay
control
The MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay was used to assess the cytotoxic level of
compound 5i on RAW264.7 cells. The cells were grown in
where Acontrol is the absorbance of DPPH and Asample is the
absorbance of the reaction mixture (DPPH with sample).
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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69

Arch. Pharm. Chem. Life Sci. 2016, 349, 63–70
J. Chu and C.-L. Guo
ARC HH PHARM
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3
9
6-well plates at a density of 5 ꢄ 10 cells/well. After 24 h, the
structure and all ionizable residues were set at their default
protonation state at a neutral pH. For ligand 6e, 10 ligand
binding poses were ranked according to their CDOCKER
energies, and the predicted binding interactions were
analyzed. In last, the best 10 ligand binding poses were
chosen and carried out in situ ligand minimization using
standard protocol.
cells in the culture wells were re-freshed with fresh medium
and were treated with different concentrations of 5i. After
8 h of incubation, cells were rewashed and 20 mL of MTT
5 mg/mL) was added and incubated for 4 h. Finally, DMSO
4
(
(
150 mL) was added to solubilize the formazan salt formed and
the amount of formazan salt was determined by measuring
1
the OD at 540 nm using an GENios microplate reader (Tecan
Austria GmbH, Groedig, Austria). Relative cell viability was
determined by the amount of MTT converted into formazan
salt. Viability of cells was quantified as a percentage
compared to the control (OD of treated cells – OD of blank/
OD of control – OD of blank ꢄ 100) and dose–response curves
were developed. The data were expressed as mean from at
least three independent experiments and p < 0.05 was
considered significant.
Statistical analysis
Statistical analyses were performed using ANOVA followed by
the Bonferroni t-test for multigroup comparisons; p < 0.05
was considered significant for all tests. Data are expressed as
mean ꢀ standard deviation. The statistical analysis was carried
out with the help of GraphPad Prism 5 Software.
The authors have declared no conflicts of interest.
Immunoassays
Enzyme immunoassay of TNF-a, IL-1b, and IL-6
Inflammatory mediators such as TNF-a, IL-1b, and IL-6 were
measured by solid phase sandwich enzyme-linked immuno-
sorbent assay (ELISA) according to the manufacturer’s
protocol (Code RPN 2751, RPN 2718, and RPN 2708, Amersham
Pharmacia Biosciences, Piscataway, NJ, USA) without prior
extraction or purification. Standard curves from 10.24–400,
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3
20 pg/well was used to construct the standard curve. PGE
2
concentrations in the samples were calculated by using
nonlinear regression of a four parameter logistic model.
Docking study
CDOCKER is a grid-based molecular docking method that
works on CHARMM force fields. During docking process, the
protein was firstly held rigid and the ligand was allowed to
freely rotate. Two hundred random ligand conformations
were then generated from the initial ligand through a series
of process including high temperature molecular dynamics,
followed by random rotations; refinement by grid-based
(
GRID 1) simulated annealing, and a final grid-based or full
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̊
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ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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