Synthesis, docking studies and antioxidant activity of some chalcone and aurone derivatives

Article abstract of DOI:10.1007/s00044-012-0413-3

Chalcones and aurones are found to possess high antioxidant activity. They are known to inhibit tyrosinase enzyme involved in synthesis of melanin. A series of substituted chalcones and aurones have been synthesized and tested for their antioxidant activi

Full text of DOI:10.1007/s00044-012-0413-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0413-3
ORIGINAL RESEARCH
Synthesis, docking studies and antioxidant activity of some
chalcone and aurone derivatives
•
Tamanna Narsinghani Mukesh C. Sharma
•
Sakshi Bhargav
Received: 14 September 2012 / Accepted: 8 December 2012
Ó Springer Science+Business Media New York 2012
Abstract Chalcones and aurones are found to possess
high antioxidant activity. They are known to inhibit tyros-
inase enzyme involved in synthesis of melanin. A series of
substituted chalcones and aurones have been synthesized
and tested for their antioxidant activity. Postulated struc-
tures of the newly synthesized compounds are in agreement
with their IR, 1H NMR and MS. The docking study of this
series of compounds was performed on crystal structure of
tyrosinase from Bacillus megaterium using VlifeMDS 3.0
software. Antioxidant activity data obtained from four
methods, i.e., DPPH free radical scavenging assay, iron
chelating assay, reducing power assay and hydrogen per-
oxide scavenging assay, indicate that the activity increased
with dimethylamino group on position 4/40 of ring B as
evident from the significant activities of SB7 and SB8 in
case of chalcones and aurones, respectively. The poor
activities of SB4 and SB5 in DPPH scavenging ability and
reducing power assays could be because of presence of
chloro group on B-ring. Furthermore, the activity is facili-
tated with the presence of hydroxyl group on A-ring
(preferably on position 5/50) in both chalcones and aurones.
counteract the harmful effects of free radicals and other
oxidants. Free radicals are responsible for causing a large
number of diseases including cancer (Kinnula and Crapo,
2004), cardiovascular diseases (Singh and Jialal, 2006),
neural disorders (Sas et al., 2007), Alzheimer’s disease
(Smith et al., 2000), mild cognitive impairment (Guidi
et al., 2006), Parkinson’s disease (Bolton et al., 2000),
alcohol-induced liver disease (Arteel, 2003), ulcerative
colitis (Ramakrishna et al., 1997), aging (Hyun et al.,
2006), and atherosclerosis (Upston et al., 2003). Antioxi-
dants may be of great benefit in improving the quality of
life by preventing or postponing the onset of degenerative
diseases. In addition, they have a potential for substantial
savings in the cost of health care delivery.
Therefore, antioxidants are intensively used, particu-
larly as treatment for stroke and neuro-degenerative dis-
eases. Antioxidants anti aging benefits are found to be
derived from the antioxidants’ ability to heal the cell and
prevent the free radicals from reproducing. Antioxidants
are also widely used as ingredients in dietary supplements
in the hope of maintaining health and preventing diseases
such as cancer and coronary heart disease. In addition to
these uses of natural antioxidants in medicine, these
compounds have many industrial uses, such as preserva-
tives in food and cosmetics and preventing the degrada-
tion of rubber and gasoline. As a result, compounds
possessing antioxidant activity are becoming increasingly
important in disease prevention and therapy (Hamid et al.,
2010).
Keywords Chalcones ꢀ Aurones ꢀ Antioxidant ꢀ
Docking ꢀ Tyrosinase
Introduction
The human body has a complex system of natural enzy-
matic and non-enzymatic antioxidant defenses which
The chalcone and aurone derivatives have displayed a
broad spectrum of pharmacological activities including
antioxidant activities. Changes in their structure have
offered a high degree of diversity that has proven useful for
the development of new antioxidants having improved
potency and lesser toxicity. Also the nucleus has been
T. Narsinghani (&) ꢀ M. C. Sharma ꢀ S. Bhargav
School of Pharmacy, Devi Ahilya Vishwavidyalaya,
Takshashila Campus, Khandwa Road, Indore 452017, MP, India
e-mail: kashishnarsinghani@rediffmail.com
123

Med Chem Res
found to be used as antioxidant in a wide range of for-
mulations targeting for the antiaging activity.
explored (Dominguez et al., 2003; Khan, 2012; Okombi
et al., 2006; Dubois et al., 2012).
Chalcones, 1,3-diaryl-2-propen-1-ones (Fig. 1) precur-
sors of open chain flavonoids and isoflavonoids present in
edible plants, have attracted increasing attention due to
numerous potential pharmacological applications. They
have displayed a broad spectrum of pharmacological
activities, among which antimalarial, anticancer (Lawrence
et al., 2006; Cabrera et al., 2007; Boumendjel et al., 2008),
antiprotozoal (antileishmanial and antitrypanosomal), anti-
inflammatory, antibacterial (Sugamoto et al., 2008; Fvila
et al., 2008), antifilarial, antifungal, antimicrobial, larvi-
cidal, anticonvulsant, antioxidant (Stevens et al., 2003;
Gacche et al., 2007; Vogel et al., 2008; Jung et al., 2008)
activities have been reported. Chalcone derivatives are
found to inhibit tyrosinase enzyme involved in melanin
production. In chalcones, antioxidant activity is attributable
to phenolic–OH group attached to the ring structure
(Okawa et al., 2001).
Materials and methods
Physical measurements
Melting point of all the compounds was determined by
open capillary melting point apparatus. Thin layer chro-
matography was performed by using prepared precoated
silica gel-G TLC plates. The IR spectra were recorded on
FT-IR, Shimadzu-8400S at School of Pharmacy, DAVV,
Indore (MP). Mass spectra of compounds were obtained on
Bruker microTOF-QII 10348 mass spectrometer using ESI
source at IIT, Indore (MP), Micromass Quattro II using ESI
source at Sophisticated Analytical Instrumentation Facility
(SAIF), CDRI, Lucknow and LC/MS/MS at RGPV,
1
Bhopal. H-NMR spectra of compounds were obtained on
Aurones, 2-benzylidenebenzofuran-3-(2H)-ones (Fig. 1)
occur rarely in nature and are less studied subclass of
flavonoids. Aurones contribute to the bright yellow color of
some flowering plants some such as snapdragon, cosmos
and dahlia. They are biosynthesized from chalcones in the
presence of enzyme aureusidin synthase (Ono et al., 2006).
These heterocyclic compounds are found to possess insect
antifeedant activity, anticancer (Lawrence et al., 2003;
Vogel et al., 2008), antileishmanial (Huang et al., 2007)
and antibacterial properties (Ferreira et al., 2004), inhibi-
tory activity against a variety of enzymes and proteins,
however only few study exist targeting their antioxidant
activity (Hadj-esfandiari et al., 2007; Venkateswarlu et al.,
2004, 2009).
Bruker Advance II 400 NMR spectrometer at Sophisticated
Analytical Instrumentation Facility (SAIF), Panjab Uni-
versity and Bruker DRX-300(300 MHz FT NMR) at
Sophisticated Analytical Instrumentation Facility (SAIF),
CDRI, Lucknow. For the in vitro tests, a Shimadzu
UV–Vis double beam spectrophotometer was used.
Synthesis of compounds
Synthesis of chalcones
To a stirred solution of the appropriate acetophenone
(1 equiv) and a substituted benzaldehyde (1 equiv) in 25 ml
ethanolic KOH (20 % w/v aqueous solution, 6 ml) was
added and the mixture was stirred at room temperature for
24–36 h. The reaction was monitored by performing TLC
using n-hexane: ethyl acetate (7:3) as mobile phase. The
reaction mixture was cooled to 0 °C (ice-water bath) and
acidified with HCl (10 % v/v aqueous solution). In most
cases, a yellow precipitate was formed, which was filtered
and washed with 10 % aqueous HCl solution. In the cases
where an orange oil was formed, the mixture was extracted
This work is thus aimed to synthesize and evaluate some
chalcone and aurone derivatives for their antioxidant
activity. The synthesized compounds were also docked on
the enzyme tyrosinase. Docking procedures basically aim
to identify the correct conformation of ligands in the
binding pocket of a protein and to predict the affinity
between the ligand and the protein. Docking is one method
in which the binding of an inhibitor to a receptor can be
O
O
Fig. 1 General structure of
chalcone and aurone
b
4
2
2'
3
2
9
10
3'
1
2'
B
5
6
3
4
a
1'
A
1'
A
3'
B
4'
6'
O
6
8
6'
4'
7
5
5'
5'
1,3-diaryl-2-propen-1-ones (Chalcones)
2-benzylidenebenzofuran-3-(2H)-ones
(Aurones)
123

Med Chem Res
with CH2Cl2, the extracts were dried (Na2SO4) and the
solvent was evaporated to give the chalcone (Scheme 1;
Table 1) as a solid (Detsi et al., 2009).
C-20), 2.50 (s, 1H, –OH group on C-50), 7.73 (d, 1H, Ha),
7.82 (d, 1H, Hb), 6.88 (d, 1H, H-5), 6.96 (d, 1H, H-6), 7.01
(d, 1H, H-30), 7.38 (d, 1H, H-40), 7.14 (s, 1H, H-2), 7.55 (s,
1H, H-60), 3.80 (s, 3H, –OCH3 group on C-3), 3.81 (s, 3H,
–OCH3 group on C-4); MS (LC/MS) m/z: 301.13 (M?H)?;
Mol. formula: C17H16O5; Mol. Weight: 301.31.
Synthesis of aurones
The substituted chalcone (0.002 mol) was dissolved in
dimethyl sulfoxide (DMSO) (10 ml) and after adding
mercuric acetate (0.003 mol); the contents were refluxed
for 6 h, cooled and poured into ice-cold water. The reaction
was monitored by performing TLC using n-hexane:ethyl
acetate (7:3) as mobile phase (Bhasker and Reddy 2011;
Barros et al., 2004). A solid mass separated out which was
filtered, washed well with water, dried, and crystallized
from ethanol to get aurones (Scheme 2; Table 2).
20,40-Dihydroxy-4-dimethylamino
chalcone
(SB2)
Prepared following the general procedure starting from 2,
4-dihydroxy-acetophenone (1.52 g, 0.01 mol) and
4-dimethylamino benzaldehyde (1.49 g, 0.01 mol). The
dark orange product was recrystallized from ethanol. Yield:
57 %; Rf = 0.62; m.p. 150–152 °C; IR (KBr) m (cm-1):
3105.18 (CH–Ar), 1593.09 (CH=CH), 1694 (C=O),
3328.91 [O–H (aromatic)], 2781.16 (N–CH3), 1232.43 [C–
O (aromatic)]. 1H-NMR (300 MHz, DMSO) d: 12.5 (s, 1H,
–OH group on C-20), 9.67 (s, 1H, –OH group on C-40), 7.70
(d, 1H, Ha), 7.93 (d, 1H, Hb), 6.24 (s, 1H, H-30), 6.35 (d,
1H, H-50), 6.38 (d, 2H, H-3, H-5), 6.77 (d, 2H, H-2, H-6),
7.67 (d, 1H, H-60), 3.04 (s, 6H, N(CH3)2 group on C-4); MS
(LC/MS) m/z: 284.15 [M?H]?; Mol. formula: C17H17O3;
Mol. Weight: 284.32.
20,50-Dihydroxy-3,4-dimethoxy chalcone (SB1) Prepared
following the general procedure starting from 2,5-dihydroxy-
acetophenone (1.52 g, 0.01 mol) and 3,4-dimethoxy benz-
aldehyde (1.66 g, 0.01 mol). The yellowish orange product
was recrystallized from ethanol. Yield: 42 %; Rf = 0.64;
m.p. 118–120 °C; IR (KBr) m (cm-1): 3060.82 (CH–Ar),
1604.66 (CH=CH), 1693.38 (C=O), 3274.9 [O–H (aro-
matic)], 2835.16 (O–CH3), 1257.5 [C–O (aromatic)];
1H-NMR (300 MHz, DMSO) d: 13.3(s, 1H, –OH group on
6-Hydroxy-40-dimethylamino aurone (SB3) Prepared
following the general procedure starting from 20,
CHO
OH
O
OH
O
R3
20% aqueous KOH,
EtOH, rt, 24–36h.
R3
R4
R4
R5
+
R1
R1
R5
R2
R2
Substituted
Benzaldehyde
Substituted Chalcone
Substituted
Acetophenone
Scheme 1 Synthesis of chalcones
Table 1 Substituted chalcones
S no.
Compounds
R1
R2
R3
R4
R5
SB1
SB2
SB5
SB7
SB10
20,50-Dihydroxy-3,4-dimethoxy chalcone
20,40-Dihydroxy-4-dimethylamino chalcone
20,40-Dihydroxy-4-chloro chalcone
20,50-Dihydroxy-4-dimethylamino chalcone
20,40-Dihydroxy-3,4-dimethoxy chalcone
H
OH
H
H
H
H
H
H
OCH3
H
OCH3
N(CH3)2
Cl
OH
OH
H
H
H
OH
H
H
N(CH3)2
OCH3
OH
OCH3
123

Med Chem Res
O
OH
O
R3
R2
Hg(OAc)2,
R4
R5
R3
DMSO, reflux 6hr.
R4
R1
O
R1
R5
R2
Substituted Chalcone
Substituted
Aurone
Scheme 2 Synthesis of aurones
Table 2 Substituted aurones
O
4
R3
3
R2
R1
9
10
2'
5
6
R4
2
1'
A
3'
4'
O
B
8
6'
7
R5
5'
S no.
Compounds
R1
R2
R3
R4
R5
SB3
SB4
SB6
SB8
SB9
6-Hydroxy-40-dimethylamino aurone
6-Hydroxy-40-chloro aurone
5-Hydroxy-30,40-dimethoxy aurone
5-Hydroxy-40-dimethylamino aurone
6-Hydroxy-30,40-dimethoxy aurone
OH
OH
H
H
H
H
H
H
H
H
N(CH3)2
Cl
H
H
OH
OH
H
OCH3
H
OCH3
N(CH3)2
OCH3
H
OH
OCH3
40-dihydroxy-4-dimethylamino chalcone (566 mg, 0.002
mol). The orange brown product was recrystallized from
ethanol. Yield: 43 %; Rf = 0.58; m.p. 98–100 °C; IR (KBr)
m (cm-1): 3060.82 (CH–Ar), 1581.52 (CH=CH), 1693.38
(C=O), 3487.06 [O–H (aromatic)], 2796.59 (N–CH3),
1245.93 [C–O (aromatic)]; 1H-NMR (300 MHz, DMSO) d:
10.59 (s, 1H, –OH group on C-6), 7.76 (s, 1H, H-10), 6.24
(s, 1H, H-7), 6.36 (d, 1H, H-5), 6.39 (d, 2H, H-30, H-50),
6.80 (d, 2H, H-20, H-60), 7.70 (d, 1H, H-4), 3.30
(s, 6H, N(CH3)2 group on C-40); MS (LC/MS) m/z:
282.13 [M?H]?; Mol. formula: C17H15O3N; Mol. Weight:
282.31.
275.06 [M?H?2]?; Mol. formula: C15H9O3Cl; Mol.
Weight: 275.68.
20,40-Dihydroxy-4-chloro
chalcone
(SB5) Prepared
following the general procedure starting from 2,4-dihy-
droxy-acetophenone (1.52 g, 0.01 mol) and 4-chloro
benzaldehyde (1.4 g, 0.01 mol). The yellow product was
recrystallized from ethanol. Yield: 76 %; Rf = 0.78; m.p.
155–160 °C; IR (KBr) m (cm-1): 3089.75 (CH–Ar),
1585.38 (CH=CH), 1691.46 (C=O), 3575.78 [O–H (aro-
matic)], 763.76 (C–Cl), 1215.07 [C–O (aromatic)]; 1H-
NMR (300 MHz, DMSO) d: 13.3 (s, 1H, –OH group on
C-20), 2.50 (s, 1H, –OH group on C-40), 7.74 (d, 1H, Ha),
8.18 (d, 1H, Hb), 6.80 (s, 1H, H-30), 6.95 (d, 1H, H-50), 7.44
(d, 2H, H-3, H-5), 7.64 (d, 2H, H-2, H-6), 7.59 (d, 1H,
H-60); MS (ESI) m/z: 277.05 [M?H?2]?; Mol. formula:
C15H11O3Cl; Mol. Weight: 277.7.
6-Hydroxy-40-chloro aurone (SB4) Prepared following
the general procedure starting from 20,40-dihydroxy-4-
chloro chalcone (549 mg, 0.002 mol). The yellow prod-
uct was recrystallized from ethanol. Yield: 54 %;
Rf = 0.74; m.p. 120–125 °C; IR (KBr)
m
(cm-1):
3049.25 (CH–Ar), 1593.09 (CH=CH), 1674.1 (C=O),
5-Hydroxy-30,40-dimethoxy aurone (SB6) Prepared fol-
lowing the general procedure starting from 20,50-dihydroxy-
30,40-dimethoxy chalcone (600 mg, 0.002 mol). The dark
orange product was recrystallized from ethanol. Yield:
38 %; Rf = 0.61; m.p. 110–112 °C; IR (KBr) v (cm-1):
3105.18 (CH–Ar), 1544.88 (CH=CH), 1685.67 (C=O),
3562.28 [O–H (aromatic)], 769.54 (C–Cl), 1261.36 [C–O
1
(aromatic)]; H-NMR (300 MHz, DMSO) d: 7.79 (s, 1H,
–OH group on C-6), 7.05 (s, 1H, H-10), 6.30 (s, 1H,
H-7), 5.59 (d, 1H, H-5), 6.51 (d, 2H, H-30, H-50), 7.64
(d, 2H, H-20, H-60), 7.74 (d, 1H, H-4); MS (LC/MS) m/z:
123

Med Chem Res
20,40-Dihydroxy-3,4-dimethoxy
chalcone
(SB10)
3458.13 [O–H (aromatic)], 2829.38 (s, O–CH3), 1249.79
[C–O (aromatic)]; 1H-NMR (300 MHz, DMSO) d: 9.70 (s,
1H, –OH group on C-5), 7.19 (s, 1H, H-10), 7.81 (d, 1H,
H-7), 7.40 (d, 1H, H-6), 7.47 (d, 1H, H-50), 7.56 (d, 1H,
H-60), 7.52 (s, 1H, H-4), 7.75 (s, 1H, H-20), 3.44 (s, 3H,
–OCH3 group on C-30), 3.48 (s, 3H, –OCH3 group on C-40);
MS (ESI) m/z: 300.9 [M?H?H]?; Mol. formula:
C17H14O5; Mol. Weight: 300.29.
Prepared following the general procedure starting from 2,
4-dihydroxy-acetophenone (1.52 g, 0.01 mol) and 3,4-
dimethoxy benzaldehyde (1.66 g, 0.01 mol). The yellowish
orange product was recrystallized from ethanol. Yield:
43 %; Rf = 0.52; m.p. 120–125 °C; IR (KBr) m (cm-1):
3078.18 (CH–Ar), 1485.09 (CH=CH), 1693.38 (C=O),
3558.42 [O–H (aromatic)], 2846.74 (O–CH3), 1213.14 [C–
O(aromatic)]; 1H-NMR (300 MHz, DMSO) d: 13.5 (s, 1H,
–OH group on C-20), 10.45 (s, 1H, –OH group on C-40), 7.31
(d, 1H, Ha), 7.46 (d, 1H, Hb), 6.28 (d, 1H, H-5), 7.29 (d, 1H,
H-6), 6.92 (s, 1H, H-30), 6.39 (d, 1H, H-50), 7.75 (s, 1H,
H-2), 6.42 (d, 1H, H-60), 3.82 (s, 3H, –OCH3 group on C-3),
3.84 (s, 3H, –OCH3 group on C-4); MS (ESI) m/z: 301.21
[M?H]?; Mol. formula: C17H16O5; Mol. Weight: 301.31.
20,50-Dihydroxy-4-dimethylamino
chalcone
(SB7)
Prepared following the general procedure starting from 2,
5-dihydroxy-acetophenone (1.52 g, 0.01 mol) and
4-dimethylamino benzaldehyde (1.49 g, 0.01 mol). The
brown product was recrystallized from ethanol. Yield:
62 %; Rf = 0.67; m.p. 98–100 °C; IR (KBr) m (cm-1):
3035.75 (CH–Ar), 1581.52 (CH=CH), 1706.88 (C=O),
3433.06 (O–H (aromatic)), 2796.59 (N–CH3), 1218.93
[C–O (aromatic)]; 1H-NMR (300 MHz, DMSO) d: 12.6 (s,
1H, –OH group on C-20), 9.67 (s, 1H, –OH group on C-50),
7.65 (d, 1H, Ha), 8.06 (d, 1H, Hb), 6.24 (d, 1H, H-30), 6.34
(d, 1H, H-40), 6.72 (d, 2H, H-3, H-5), 6.75 (d, 2H, H-2,
H-6), 6.36 (s, 1H, H-60), 3.07 (s, 6H, N(CH3)2 group on
C-4); MS (ESI) m/z: 283.9 [M]?; Mol. formula:
C17H17O3N; Mol. Weight: 283.32.
Docking study on tyrosinase using VlifeMDS 3.0
To explore the interactions of the synthesized compounds,
we carried out binding simulations by means of the Bio-
Predicta module of Vlife MDS 3.0 suite (Vlife MDS,
2006). The docking study (Kang et al., 2012) was per-
formed on crystal structure of tyrosinase from Bacillus
megaterium (PDB code: 3NM8). The computational work
was performed on a HP Compaq PC running on Intel
core2duo processor. The molecular structures of the com-
pounds in the data set were sketched using VLife MDS
(Molecular Design Suite) 3.0 software supplied by VLife
Sciences Technologies Pvt. Ltd., Pune, India. Energy
minimization was performed using the MMFF94 (Halgren,
1996) force field and Gasteiger–Marsili (Gasteiger and
Marsili, 1980) charges followed by AM-1 (Austin Model-
1) Hamiltonian method available in MOPAC module with
5-Hydroxy-40-dimethylamino aurone (SB8) Prepared fol-
lowing the general procedure starting from 20,50-dihydroxy-
4-dimethylamino chalcone (566 mg, 0.002 mol). The brown
product was recrystallized from ethanol. Yield: 49 %;
Rf = 0.64; m.p. 110–112 °C; IR (KBr) m (cm-1): 3153.4
(CH–Ar), 1589.23 (CH=CH), 1637.45 (C=O), 3336.62
[O–H (aromatic)], 2806.23 (N–CH3), 1220.86 [C–O (aro-
matic)]; 1H-NMR (300 MHz, DMSO) d: 12.45 (s, 1H, –OH
group on C-5), 7.54 (s, 1H, H-10), 7.30 (1H, H-6), 6.83 (d,
1H, H-7), 7.14 (d, 2H, H-30, H-50), 7.46 (d, 2H, H-20, H-60),
7.20 (s, 1H, H-4), 3.06 (s, 6H, N(CH3)2 group on C-40); MS
(ESI) m/z: 281 [M]?; Mol. formula: C17H15O3N; Mol.
Weight: 281.31.
˚
the convergence criterion 0.001 kcal/mol A.
The 3D structure of the tyrosinase was retrieved from
PDB by giving the PDB ID (PDB entry 3NM8;
www.rcsb.org) in the database. The water molecules were
removed and hydrogen atoms were added. The docking
simulations were done and potential hydrogen bonding,
pi-stacking, VDW, and hydrophobic interactions between the
protein and the synthesized compounds were recorded. The
water molecules were removed and hydrogens were added.
The energy of the protein was minimized using MMFF.
The ligands were prepared using Prepare Ligands and
subsequently docked using Grid Docking. All the docked
ligands were scored using the Dock Score function. The
best pose was identified and used for subsequent analyses.
6-Hydroxy-30,40-dimethoxy
aurone
(SB9) Prepared
following the general procedure starting from 20,60-dihy-
droxy-30,40-dimethoxy chalcone (600 mg, 0.002 mol). The
yellowish orange product was recrystallized from ethanol.
Yield: 42 %; Rf = 0.49; m.p. 118–120 °C; IR (KBr) m
(cm-1):3028.03 (CH–Ar), 1554.52 (CH=CH), 1695.31
(C=O), 3595.07 [O–H (aromatic)], 2866.02 (O–CH3),
1288.36 [C–O (aromatic)]; 1H-NMR (300 MHz, DMSO) d:
13.53 (s, 1H, -OH group on C-6), 6.97 (s, 1H, H-10), 6.91
(s, 1H, H-7), 6.29 (d, 1H, H-5), 7.08 (d, 1H, H-50), 7.44 (d,
1H, H-60), 7.73 (d, 1H, H-4), 7.78 (s, 1H, H-20), 3.92 (s, 3H,
–OCH3 group on C-30), 3.87 (s, 3H, –OCH3 group on C-40);
MS (ESI) m/z: 298.8 [M]?; Mol. formula: C17H14O5; Mol.
Weight: 298.29.
In vitro assays
Each in vitro experiment was performed at least in tripli-
cate. Ascorbic acid was used as the standard in all the four
methods.
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Med Chem Res
DPPH free radical scavenging activity
phosphate buffer saline of pH 7.4. Absorbance of hydrogen
peroxide at 230 nm was determined after 10 min against a
blank solution containing phosphate buffer without
hydrogen peroxide. For each concentration, a separate
blank sample was used for background subtraction. For
control sample, absorbance of hydrogen peroxide solution
was taken at 230 nm (Patel and Patel, 2010). The per-
centage inhibition activity (Table 3) was calculated from
the formula [(A0 - A1)/A0] 9 100, where A0 is the absor-
bance of the control, and A1 is the absorbance of test/
standard taken as ascorbic acid (50–300 lg/ml).
0.1 mM solution of DPPH in methanol was prepared and
1.0 ml of this solution was added to 3.0 ml of solution of
compound in methanol at different concentrations (50, 100,
150, 200, 250, 300 lg/ml). Thirty minutes later, the
absorbance was measured at 517 nm. A blank was pre-
pared without adding compound. Ascorbic acid at various
concentrations (50–300 lg/ml) was used as standard.
Lower absorbance value of the reaction mixture indicates
higher free radical scavenging activity. The capability to
scavenge the DPPH radical was calculated using the fol-
lowing equation:
Reducing power method
% Antiradical activity
2 ml of each sample and standard solutions were spiked
with 2.5 ml of 1 % potassium ferricyanide solution. This
mixture was kept at 50 °C in water bath for 20 min. After
cooling, 2.5 ml of 10 % trichloroacetic acid was added,
(when precipitate formed, centrifuged at 3,000 rpm for
10 min) 2.5 ml of supernatant was mixed with 2.5 ml of
distilled water and 1 ml of 0.1 % ferric chloride and kept
for 10 min. Control was prepared in similar manner
excluding samples (Collins, 2005). The absorbance of
resulting solution was measured at 700 nm (Table 3).
Control absorbance ꢁ Sample absorbance
¼
ꢂ 100
Control absorbance
The antioxidant activity of compounds (Table 3) were
expressed as IC50 and compared with standard (Nikhat and
Satyanarayana 2009).
Iron chelating activity
Iron chelating activity (Table 3) is a measure of antioxidant
activity. Different concentrations of compound (50–300
lg/ml) and ascorbic acid solution (50–300 lg/ml) each as
2 ml in methanol were incubated with methanolic o-phe-
nanthroline solution (1 ml, 0.05 % w/v) and ferric chloride
solution (2 ml, 200 lM) at ambient temperature for
10 min. After incubation, the absorbance of solutions was
measured at 510 nm (Benzie and Strain, 1996).
Results and discussion
Chemistry
Substituted chalcones (SB1, SB2, SB5, SB7, SB10) have
been synthesized via the Claisen–Schmidt condensation
reaction between appropriately substituted acetophenones
and benzaldehydes in basic conditions (20 % aqueous
KOH) (Scheme 1; Table 1). The synthesis of the desired
aurones (SB3, SB4, SB6, SB8, SB9) was accomplished via
the oxidative cyclization methodology using mercury(II)
Hydrogen peroxide scavenging assay
The solution of hydrogen peroxide (100 mM) was prepared
by the addition of various concentrations of compound
(50–300 lg/ml) to hydrogen peroxide solution (2 ml) in
Table 3 Antioxidant activity of substituted chalcones and aurones
Compound no.
DPPH scavenging
ability IC50 lg/ml SD
H2O2 scavenging
ability IC50 lg/ml SD
Iron chelating activity
assay IC50 lg/ml SD
Reducing power
IC50 lg/ml SD
SB1
132.22 0.7
65.8 0.7
89.59 1.8
135.88 0.53
193.91 0.36
178.13 0.36
129.02 0.85
206.57 0.19
153.85 2.1
174.07 1.9
248.16 0.76
70.1 0.92
159.22 1.02
140.16 1.08
179.33 0.31
179.18 0.74
159.97 0.29
185.05 1.2
90.81 0.86
149.98 0.51
213.83 0.68
166.73 2.5
126.12 0.5
111.79 0.69
53.6 1.1
SB2
SB3
109.92 1.02
266.12 0.49
264.95 1.6
222.19 0.7
24.32 0.87
243 0.13
70.1 2.2
SB4
188.31 0.31
155.87 0.68
151.43 0.25
47.79 1.3
SB5
SB6
SB7
SB8
64.16 1.2
SB9
173.29 0.94
35.2 1.5
168.23 0.83
134.45 0.34
53.24 0.72
SB10
Ascorbic acid
98.77 0.53
445.92 1.4
123

Med Chem Res
acetate in dimethyl sulfoxide (Scheme 2; Table 2) The
compounds were purified by recrystallization. In all cases,
the compounds were obtained in moderate to satisfactory
yields (38–76 %). Postulated structures of the newly syn-
Table 4 Dock score of substituted chalcone and aurone derivatives
Compound Compounds
no.
Dock score
(kcal/mol)
20,50-Dihydroxy-3,4-dimethoxy chalcone
-5.35
1
SB1
thesized compounds are in agreement with their IR, H
SB2
20,40-Dihydroxy-4-dimethylamino chalcone -25.62
NMR and MS.
SB3
6-Hydroxy-40-dimethylamino aurone
6-Hydroxy-40-chloro aurone
20,40-Dihydroxy-4-chloro chalcone
5-Hydroxy-30,40-dimethoxy aurone
-42.23
-40.89
-40.19
-34.43
SB4
SB5
Molecular docking
SB6
SB7
20,50-Dihydroxy-4-dimethylamino chalcone -31.95
Docking was performed on crystal structure of tyrosinase
from B. megaterium (PDB code: 3NM8) using VLife MDS
3.0 software. Docking studies showed that Ile39A,
Gly143A, Ile139A, Lys47A, Lys47B, Ala44B, Gly43B,
and Gln142A present in tyrosinase are highly conserved
and may play a major role in substrate binding or catalysis.
Standard nordihydroguaiaretic acid (NDGA) is also found
to bind to these amino acids (Fig. 2). Some other conserved
residues surrounding the binding site are His49B and
Glu141B.
SB8
5-Hydroxy-40-dimethylamino aurone
6-Hydroxy-30,40-dimethoxy aurone
20,40-Dihydroxy-3,4-dimethoxy chalcone
NDGA (Nordihydroguaiaretic acid)
-91.39
-31.82
-16.91
-41.88
SB9
SB10
Standard
20,40-Dihydroxy-4-chloro chalcone, SB5 (Table 4; Fig. 3)
has shown dock score (-40.19 kcal/mole) comparable to the
standard (-41.88 kcal/mol), while 5-hydroxy-40-dimethyl-
amino aurone, SB8 (Fig. 4) displayed better dock score
(-91.39 kcal/mol) compared to NDGA (-41.88 kcal/mol).
Antioxidant activity
All synthesized compounds were evaluated for invitro
antioxidant activity (Table 3) using four different antioxi-
dant assays. The radical scavenging ability of the com-
pounds was tested against the 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) stable free radical and hydrogen perox-
ide. Also the measurement of the reducing ability, the
Fe3?–Fe2? transformation was investigated along with iron
chelating ability.
Fig. 3 Docking view of 20,40-dihydroxy-4-chloro chalcone (SB5)
Fig. 2 Docking view of NDGA
Fig. 4 Docking view of 5-hydroxy-40-dimethylamino aurone (SB8)
123

Med Chem Res
DPPH free radical scavenging activity
scavenge hydrogen peroxide was determined according to
the method of Ruch (Nabavi et al., 2008). The compounds
were capable of scavenging hydrogen peroxide in a con-
centration-dependent manner. The scavenging ability of all
the compounds was found to be better compared to the
reference compound ascorbic acid (IC50 445.92 1.4 lg/
ml). Among the tested compounds, the best scavenging
activity was presented by SB10 (IC50 70.1 0.92 lg/ml)
and SB1 (IC50 89.59 1.8 lg/ml). The presence of
methoxy group on 3, 4 position of B-ring increases the
hydrogen peroxide scavenging activity (Table 3) compared
to electron-withdrawing substituents at para position.
The interaction of the synthesized compounds with the
stable free radical DPPH indicates their radical scavenging
ability in an iron-free system. The interaction of the tested
chalcones and aurones with DPPH was found to be con-
centration dependent. Among the tested compounds, the
best DPPH radical scavenging activity (Table 3) was pre-
sented by SB7 containing p-dimethylamino group (IC50
24.32 0.87 lg/ml). Also, SB10 (IC50 35.2 1.5 lg/ml)
and SB2 (IC50 65.8 0.7 lg/ml) exhibited higher activity
than the reference compound ascorbic acid (IC50
98.77 0.53 lg/ml). SB1 (IC50 132.22 0.7 lg/ml), and
SB3 (IC50 109.92 1.02 lg/ml) exhibited satisfactory
activity compared to reference compound. The activity
seems to increase with the presence of electron-donating
substituents on B-ring. Electron-withdrawing substituents
do not favor activity.
Reducing power method
Fe(III) reduction is often used as an indicator of electron-
donating activity, which is an important mechanism of
phenolic antioxidant action. In the reducing power assay,
the presence of antioxidants in the samples would result in
the reduction of Fe3? to Fe2? by donating an electron.
Amount of Fe2? complex was then monitored by measur-
ing the formation of Perl’s Prussian blue at 700 nm.
Increasing absorbance at 700 nm indicates an increase in
reducing ability (Nabavi et al., 2008). It was found that the
reducing powers of all the synthesized compounds also
increased with the increase in their concentrations. The
best reducing power was presented by SB7 containing
p-dimethylamino group (IC50 47.79 1.3 lg/ml). SB2
(IC50 53.60 1.1 lg/ml), SB8 (IC50 64.16 1.2 lg/ml)
and SB3 (IC50 70.10 2.2 lg/ml) exhibited satisfactory
activity compared to reference compound (Table 3). The
IC50 value of the reference compound was recorded as
53.24 0.72 lg/ml. Reducing power seems to depend on
the position of the hydroxyl group on ring A (compare
SB7 [ SB2, SB1 [ SB10, and SB8 [ SB3). On B-ring,
electron-donating substituents seem to increase the activity.
There is a correlation between the activity of chalcones and
the corresponding aurones in this assay: a highly active
chalcone gives a highly active aurone (e.g., SB7 cyclized to
SB8 and these compounds are highly active).
Iron chelating activity
Iron is essential for life because it is required for oxygen
transport, respiration, and activity of many enzymes.
However, iron is an extremely reactive metal and catalyzes
oxidative changes in lipids, proteins, and other cellular
components (Satheeshkumar et al., 2011). Iron binding
capacity of the synthesized compounds and the reference
compound ascorbic acid were examined (Table 3). Maxi-
mum chelating of metal ions was found to be shown by
SB7 (IC50 90.81 0.86 lg/ml) which was higher than the
reference compound. Compounds SB2 (IC50 140.16
1.08 lg/ml) and SB8 (IC50 149.98 0.51 lg/ml) exhib-
ited satisfactory activity compared to reference compound.
Iron chelating activity seems to depend on the position of
the hydroxyl group on ring A (compare SB7 [ SB2,
SB1 [ SB10, and SB8 [ SB3). On the other hand, the
activity seems to increase with the presence of electron-
donating substituents on ring B. There is a correlation
between the activity of chalcones and the corresponding
aurones in this assay: a highly active chalcone gives a
highly active aurone (e.g., SB7 cyclized to SB8 and these
compounds are highly active). The IC50 value of the ref-
erence compound ascorbic acid was recorded as
126.12 0.5 lg/ml.
Conclusion
Among the results obtained from the four methods for the
evaluation of antioxidant activity of chalcones (SB1, SB2,
SB5, SB7, and SB10), the compound SB7 (20,50-dihydroxy-
4-dimethylamino chalcone) with p-dimethylamino group on
B-ring and 20,50-dihydroxy group on A-ring of chalcone has
shown the most promising antioxidant activity in DPPH
free radical scavenging assay, iron chelating assay and
Hydrogen peroxide scavenging assay
Scavenging of hydrogen peroxide by synthesized com-
pounds may be attributed to the phenolics, which can
donate electrons to hydrogen peroxide thereby neutralizing
it to water. The ability of the compounds to effectively
123

Med Chem Res
reducing power assay. Compound SB2 (20,40-dihydroxy-4-
dimethylamino chalcone) and SB8 (5-hydroxy-40-dimeth-
ylamino aurone) have also shown good activities following
SB7 in iron chelating assay and reducing power assay.
Among the aurones (SB3, SB4, SB6, SB8, and SB9),
SB8 has shown best activity in iron chelating assay,
hydrogen peroxide scavenging assay and reducing power
assay. SB8 has also shown best dock score in case of
docking studies. On the other hand, SB4 (6-hydroxy-
40chloro aurone) is found to be least active in two of the
above methods, namely DPPH free radical scavenging
assay and reducing power assay.
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Antioxidant activity increased with dimethylamino
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Acknowledgments The authors are grateful to the Head, School of
Pharmacy, Devi Ahilya Vishwavidyalaya and Honorable Vice
Chancellor, Devi Ahilya Vishwavidyalaya Indore for providing nec-
essary facilities to carry out the research work. One of the authors
(SK) is thankful to AICTE for providing junior research fellowship.
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