Synthesis, anticancer and antioxidant activities of 2,4,5-trimethoxy chalcones and analogues from asaronaldehyde: Structure-activity relationship

Article abstract of DOI:10.1016/j.ejmech.2013.01.018

2,4,5-Trimethoxy chalcones and analogues were synthesized from asaronaldehyde derived from β-asarone. These novel compounds when tested against three human tumour cell lines (MCF-7, SW-982 and HeLa) using MTT assay, revealed that chalcones possessing electron donor groups in para position to carbonyl moiety of phenyl ring A, showed better inhibitory activity (2, 3, 4, 6, 7, 10, 17). When evaluated for antioxidant activities, compound 15 exhibited better free radical scavenging property in DPPH assay while compounds 2, 3, 5, 7, 9, 10, 11, 16, and 18 showed significant NO scavenging activity. All compounds exhibited very good phenyl hydrazine induced haemolysis of erythrocytes in phenylhydrazine assay. Structure-activity relationship (SAR) study using in-silico analysis matched well with in-vitro tumour cell inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2013.01.018

European Journal of Medicinal Chemistry 62 (2013) 435e442
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anticancer and antioxidant activities of
2,4,5-trimethoxy chalcones and analogues from
asaronaldehyde: Structureeactivity relationship
Suvarna Shenvi, Krishna Kumar, Kaushik S. Hatti, K. Rijesh, Latha Diwakar,
*
G. Chandrasekara Reddy
Vittal Mallya Scientific Research Foundation, # 94/3 & 94/5, 23rd Cross, 29th Main, BTM II Stage, Bangalore 560 076, India
a r t i c l e i n f o
a b s t r a c t
Article history:
2,4,5-Trimethoxy chalcones and analogues were synthesized from asaronaldehyde derived from
b-
Received 8 November 2012
Received in revised form
10 January 2013
Accepted 13 January 2013
Available online 23 January 2013
asarone. These novel compounds when tested against three human tumour cell lines (MCF-7, SW-982
and HeLa) using MTT assay, revealed that chalcones possessing electron donor groups in para position
to carbonyl moiety of phenyl ring A, showed better inhibitory activity (2, 3, 4, 6, 7, 10, 17). When eval-
uated for antioxidant activities, compound 15 exhibited better free radical scavenging property in DPPH
assay while compounds 2, 3, 5, 7, 9, 10, 11, 16, and 18 showed significant NO scavenging activity. All
compounds exhibited very good phenyl hydrazine induced haemolysis of erythrocytes in phenyl-
hydrazine assay. Structureeactivity relationship (SAR) study using in-silico analysis matched well with
in-vitro tumour cell inhibitory activity.
Keywords:
Chalcones
Flavones
Antioxidant
Anticancer
Flavonols
In-silico
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
inhibitory activities [30,31]. Fluorine substituted chalcones with
methoxy groups showed inhibition of nitric oxide production [32].
Chalcones (1,3-diaryl-2-propen-1-ones) which are precursors of
flavonoids constitute an important group of natural products pos-
sessing awidevarietyofbiological activities [1e17]. Flavonoids which
are naturally occurring polyphenols also possess diverse biological
applications [18]. The anticancer potential of flavonoids and their
biogenetic precursors have been investigated in great length [19,20].
Structureeactivity relationship has been reported [21,22] where
in the activity of chalcones was found to be dependent on the
presence and the position of hydroxyl and methoxy groups in both
A and B rings [23,24]. Presence of 20,40,50-trimethoxy groups on the
ring A of chalcones favours the antimalarial activity [25,26], while
30,40,50-trimethoxy substitution on ring A inhibits the transport
activity of glycoprotein and showed reversal of multidrug resist-
ance (MDR) activity [27,28]. 20,40,60-Trimethoxy and 30,40,50-trime-
thoxy chalcones showed nitric oxide scavenging and
antiproliferation activity [11,29]. Substitution at 20 and 40 positions
of ring A was essential for breast cancer resistance protein
Trimethoxy substituted chalcone and stilbene hybrids have been
found to show antiplasmodial activity [33].
Antioxidants are molecules capable of inhibiting the oxidation of
other molecules and thereby preventing the cell death that occurs
due to the release of free radicals. Free radicals such as superoxide
radical ðOO2ꢀÞ, hydroxyl radical (ꢁOH), hydrogen peroxide and lipid
peroxide radicals have been implicated in a variety of diseases such
as asthma, cancer, cardiovascular, diabetes, gastrointestinal
inflammation, periodontal disease and other inflammatory pro-
cesses [34,35]. Antioxidant activity of natural compounds like
chalcones and flavonoids is related to a number of different mech-
anisms such as free radical scavenging, hydrogen donation, singlet
oxygen quenching, and metal ion chelation. Antioxidant properties
of chalcones are influenced to a great extent by two aryl groups and
their substitution patterns. Hydroxyl is one of the key groups to
enhance the antioxidant activity due to its easy conversion to phe-
noxy radical through hydrogen transfer mechanism [36].
In continuation of our work [37], we have synthesized chalcones
containing 2,4,5-trimethoxy groups in ring B starting from asar-
onaldehyde derived from an abundantly available natural source
* Corresponding author. Tel.: þ91 80 26687216; fax: þ91 80 26687170.
E-mail address: gcreddy@vmsrf.org (G.C. Reddy).
i.e. b-asarone. Here, we report the synthesis and biological activities
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.018

436
S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
of these trimethoxy chalcones and some of their analogues as
anticancer and antioxidant agents. Antioxidant activities were
determined using assays viz., DPPH (2,2-diphenyl-1-picrylhydrazyl
free radical), NO (nitric oxide scavenging), PhNHNH2 (phenyl hy-
drazine induced haemolysis of erythrocytes). These compounds
were tested for their inhibitory activity against three human
tumour cell lines (MCF-7, SW-982 and HeLa) using MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
The structureeactivity relationship (SAR) and biological properties
of these newly synthesized molecules were compared to estimate
its drug-likeliness and undesired properties. Finally toxicity of
these 2,4,5-trimethoxy asarone derivatives were experimentally
and theoretically evaluated as safe drug leads.
poured into ice water and acidified with dil HCl to get the desired
2,4,5-trimethoxy chalcones (Scheme 1, 1e20) in good yields rang-
ing from 60 to 96% (Table 1). Chalcones were recrystallized from
ethanol/methanol and structures were established with the help of
NMR (nuclear magnetic resonance) and mass spectral studies.
Coupling constants (J values) of vinylic protons in 1H NMR indicated
that compounds 1e20 were trans (E) isomers (Jtrans
]
¼ 14e
c
c
16 Hz). Chalcones with 20-OH in ring A (6, 13 and 15) were cyclised
to get the corresponding flavonoids viz., flavones (21e23), flavonols
(24, 25), and flavanone (26). All these compounds were subjected to
biological testing and compared their potency with their
precursors.
2.2. Biological evaluation
2. Results and discussion
All 26 synthesized compounds i.e. 2,4,5-trimethoxy chalcones
and analogues were evaluated for their in-vitro anticancer activity
against the three human cancer cell lines viz., HeLa (human cervical
cancer), SW-982 (human synovial sarcoma), and MCF-7 (human
2.1. Chemistry
Acorus calamus (sweet flag) is a widespread, semi-aquatic plant
grown in temperate to sub-temperate regions.
2,4,5-trimethoxy-1-propenylbenzene) is a major active principle
(70e80%) found in A. calamus oil. -Asarone was oxidized with
KMnO4/NaHCO3 to get 2,4,5-trimethoxy benzaldehyde (i.e. asar-
onaldehyde, Scheme 1). The asaronaldehyde was reacted with
various commercially available acetophenones in ethanol/aq. KOH
at room temperature. After completion, the reaction mass was
b
-Asarone ((Z)-
breast cancer) in comparison with b-asarone and using pristimerin
(triterpene quinone methide) as a positive control (Table 2). Chal-
cones were found to have better activity than the cyclised flavones,
flavanone and flavonols. Compounds 2, 7 and 17 having Cl, F, Br
groups at 40 position are relatively more potent and it may be due to
electromeric effect contributed by halogen groups being situated at
para position to carbonyl group of the phenyl ring. This point is
b
Scheme 1. General procedure for synthesis of chalcones, flavones, flavonols and flavanone. (i) KMnO4/NaHCO3, r.t.; (ii) aq. KOH (40%), C2H5OH, r.t., with corresponding aceto-
phenone; (iii) DMSO/I2, 1e2 h, reflux; (iv) H2O2 (30%), NaOH (8 M) in EtOH, 2 h, r.t.; (v) glacial AcOH, 45 h, reflux.

S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
437
Table 1
2,4,5-Trimethoxy chalcones and analogues 1e26.
O
1
R
5'
O
O
O
6'
1'
4'
3'
2
R
2'
2
1'
6'
3'
1
R
1
8
5
3
O
2
7
6
2O'
4'
4 O
3
6
5
R
R
3
2
5'
5
O
R
4
O
4
R
21 - 23
1 - 20
O
O
O
O
O
1
R
1
R
O
O
O
2
O
2
R
OH
R
O
26
24, 25
Compounds
R1
R2
R3
R4
R5
% Yielda
Ref.
1
2
3
4
5
6
7
8
H
H
H
H
H
OH
H
H
H
H
H
H
OH
H
OH
OCH3
H
H
H
NO2
H
H
H
H
H
H
H
H
NO2
H
CF3
H
OCH3
H
H
H
Cl
OCH3
CH3
H
OH
F
Morphilino
Imidazolyl
Cl
NO2
H
H
OH
H
OCH3
Br
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
e
e
e
e
e
e
82
89
85
92
95
60
90
85
68
75
85
89
79
86
65
96
93
88
83
95
52
49
54
53
35
46
N
[26]
[33]
N
N
[31]
N
N
N
N
[26]
N
N
N
N
N
[26]
N
N
N
N
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
OH
H
H
CH2C6H5
H
H
CH2C6H5
H
H
H
OH
Cl
Cl
H
OAc
OH
e
OAc
e
e
e
e
N
N
N
N
e
e
e
e
e
e
OH
H
e
e
OH
e
e
N
N e new compound.
a
Percentage yield of isolated compound.
corroborated by the fact that when NO2 group (electron with-
drawing group) at para position as in 11 did not show significant
activity on these three cell lines. Expectedly, compound 30-nitro, 40-
chloro chalcone (10) was most prominent among all, showing
better activity as could be explained. Morphilino and imidazolyl
substitution at para position as in 8 and 9 did not exhibit any sig-
nificant shift in activity. 20,40-Dihydoxy compound (hydroxyl
groups being ortho, para to carbonyl) (6) showed higher activity
than 20,50-dihydoxy chalcone (15). Likewise compounds with
methoxy and methyl group at 40 position (3, 4) showed higher
activity.
Antioxidant activities of all compounds were evaluated by
means of DPPH (2,2-diphenyl-1-picrylhydrazyl free radical), NO
(nitric oxide scavenging) and PhNHNH2 (for phenyl hydrazine
induced haemolysis of erythrocytes) assays (Table 3). Compound 15
showed superior activity over other molecules in DPPH assay

438
S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
Table 2
Table 3
Anticancer activity of 2,4,5-trimethoxy chalcones and analogues (1e26).
Antioxidant activities of all 26 compounds.
IC50 valuesa
DPPH
(mg/ml)
a
Compounds
IC50
(
mM)
Compounds
HeLab
SW-982c
MCF-7d
NO
PhNHNH2
1
2
3
4
5
6
7
8
129.9 ꢂ 4.64
36.54 ꢂ 4.82
37.64 ꢂ 3.92
42.16 ꢂ 3.26
>150
37.64 ꢂ 3.75
28.6 ꢂ 3.05
>150
101.40 ꢂ 7.39
58.54 ꢂ 2.58
60.46 ꢂ 0.48
81.08 ꢂ 2.89
>150
32.42 ꢂ 1.32
32.49 ꢂ 2.53
>150
>150
1
2
3
4
5
6
7
8
4.309
4.593
4.394
4.325
4.506
4.368
4.710
4.269
4.757
4.472
4.754
5.153
5.073
4.310
2.653
5.023
4.318
5.002
4.293
4.650
4.808
4.487
4.973
4.680
4.496
4.566
3.039b
2.348
2.014
2.070
2.181
2.012
2.763
1.963
2.078
1.972
1.990
1.982
2.085
2.320
2.462
2.444
1.977
2.224
2.092
2.502
2.370
2.273
2.142
2.145
2.106
2.330
2.434
2.632b
2.209
2.298
2.178
2.236
2.225
2.373
2.216
2.121
2.481
2.151
2.337
2.302
2.233
2.163
2.103
2.134
2.300
2.167
2.285
2.327
2.147
2.141
2.125
2.144
2.154
2.140
3.658c
29.48 ꢂ 4.01
33.87 ꢂ 3.91
33.04 ꢂ 6.62
>150
33.76 ꢂ 8.44
29.96 ꢂ 5.67
>150
9
>150
>150
>150
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
A
16.48 ꢂ 1.54
20.57 ꢂ 0.52
16.35 ꢂ 5.99
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Std
>150
>150
>150
>150
>150
>150
>150
108.08 ꢂ 3.75
31.67 ꢂ 3.54
26.89 ꢂ 3.55
e
>150
>150
>150
>150
96.47 ꢂ 3.82
38.17 ꢂ 0.91
69.94 ꢂ 8.83
154.9 ꢂ 0.34
78.52 ꢂ 9.03
>150
>150
>150
>150
>150
>150
>150
1.28 ꢂ 0.21
>150
>150
95.53 ꢂ 16.3
>150
51.36 ꢂ 6.05
18.26 ꢂ 2.99
23.29 ꢂ 5.28
182.31 ꢂ 4.37
63.67 ꢂ 4.9
>150
>150
>150
>150
>150
57.90 ꢂ 3.33
>150
>150
>150
>150
>150
>150
>150
126.11 ꢂ 6.82
129.35 ꢂ 8.12
B
3.42 ꢂ 0.53
0.87 ꢂ 0.04
a
Values are the mean of triplicate of three independent experiments.
Standard ascorbic acid.
b
c
A:
b-asarone.
B: pristimerin (positive control).
Standard a-tocopherol.
a
IC50: each data represent mean ꢂ S.D. from three different test results in trip-
licate and expressed as the concentration of test compound which inhibits the cell
growth by 50%.
b
HeLa: human cervical cancer.
SW-982: human synovial sarcoma.
MCF-7: human breast cancer.
c
d
Table 4
Drug likeliness properties of all 26 compounds according to Osiris property Explorer
tool [38].
indicating better free radical scavenging capacity. All compounds
exhibited nitric oxide scavenging activity compared to ascorbic
acid. Also all the compounds inhibited phenyl hydrazine induced
haemolysis of erythrocytes revealing their ability to scavenge most
of the free radicals generated. Here again these compounds showed
Compounds Mol. wt C log P Drug-likeness Drug-score Toxicity risksa
Mb Tc Id
Re
1
2
3
4
5
6
7
8
343.30 3.06
332.70 3.81
328.30 3.09
312.30 3.51
298.30 3.19
330.30 2.59
316.30 3.25
383.40 2.58
364.40 2.53
377.70 3.68
343.30 3.06
366.30 3.95
348.30 3.51
344.30 2.79
330.30 2.59
388.40 2.88
377.20 3.89
510.50 5.72
314.30 2.89
ꢀ4.49
4.34
2.51
1.59
1.97
2.01
2.81
3.89
3.52
ꢀ5.56
ꢀ9.54
ꢀ8.35
2.68
2.51
2.12
2.53
ꢀ0.6
ꢀ2.99
2.91
2.48
2.49
0.61
2.99
2.75
0.33
2.12
0.22
0.39
0.45
0.4
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(þ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
better activity in comparison with standard molecule,
tocopherol.
a-
0.44
0.78
0.43
0.44
0.33
0.14
0.22
0.19
0.68
0.46
0.79
0.71
0.25
0.09
0.47
0.64
0.78
0.66
0.64
0.67
0.39
0.78
3. Structureeactivity relationship using in silico analysis
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
(ꢀ) (þ) (þ/ꢀ) (þ)
Structureeactivity relationship is an important factor in deter-
mining compounds with undesired effects or low bioactivity. Osiris
Property Explorer [38] is one such knowledge based activity pre-
diction tool which predicts drug likeliness, drug score and unde-
sired properties such as mutagenic, tumorigenic, irritant and
reproductive effect of novel compounds based on chemical frag-
ment data of available drugs and non-drugs as reported. Based on
these calculations (Table 4), chalcones 6, 13, 15 and 16 have been
predicted to show better activity. These observations correlated
well with in vitro results for compounds 6 and 16, thus matching
the prediction levels to the extent of 50%.
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(þ) (þ) (þ)
(ꢀ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(ꢀ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ) (þ) (þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
(þ)
414.1
3.05
328.30 2.98
328.30 2.98
346.70 3.98
362.70 3.3
344.0.3 1.21
330.30 2.79
4. Conclusion
a
Ranking as (þ) no bad effect, (þ/ꢀ) medium bad effect, (ꢀ) bad effect.
2,4,5-Trimethoxy chalcones and analogues were synthesized
from corresponding acetophenones and asaronaldehyde derived
b
c
M (mutagenic effect).
T (tumorigenic effect).
I (irritant effect).
R (reproductive effect).
d
e
from naturally occurring
b-asarone. A few flavones, flavanone and
flavonols from corresponding chalcones were also prepared. When

S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
439
these compounds were subjected to in-vitro testing against three
human tumour cell lines (MCF-7, SW-982 and HeLa) using MTT
assay, chalcones that are having electron donor groups in para
position to carbonyl moiety of phenyl ring A, showed better
inhibitory activity (2, 3, 4, 6, 7, 10, 17). When tested for antioxidant
activities, compound 15 showed very good free radical scavenging
activity in DPPH assay. All compounds exhibited significant NO
scavenging activity and showed good phenyl hydrazine induced
haemolysis of erythrocytes. Structureeactivity relationship (SAR)
study has been made and also compared in-vitro and in-silico re-
sults which matched well with each other.
115.99, 111.9, 97.34, 56.95 (OCH3), 56.69 (OCH3), 56.40 (OCH3). GCe
MS (m/z) ¼ 312 [M]þ (12), 281 (100), 265 (18), 237 (14), 119 (32).
5.1.2.3. (E)-1-Phenyl-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
(5). Yellow solid, C18H18O4, mp 107e109 ꢃC.
IR (KBr): 2931, 2839,1647,1630,1558,1512,1458,1400,1261,1211,
1137, 1028, 979, 810 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.90 (3H, s,
OCH3), 3.94 (3H, s, OCH3), 3.97 (3H, s, OCH3), 6.52 (1H, s, H-3), 7.13
(1H, s, H-6), 7.46e7.56 (3H, m), 7.45 (1H, d, J ¼ 15.8 Hz, eCH]CHe
CO), 7.98e8.04 (2H, m), 8.09 (1H, d, J ¼ 15.8 Hz, eCH]CHeCO).
13C NMR (CDCl3, 50 MHz):
d 191.37 (C]O), 155.06, 152.94, 143.63,
140.48, 139.18, 132.68, 128.84, 128.77, 120.50, 115.80, 111.77, 97.21,
56.92 (OCH3), 56.68 (OCH3), 56.41 (OCH3). GCeMS (m/z) ¼ 298 [M]þ
(16), 267 (100), 251 (8), 223 (8), 105 (26).
5. Experimental
5.1. Chemistry
5.1.2.4. (E)-1-(4-Flurophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-
1-one (7). Yellow solid, C18H17FO4, mp 224e227 ꢃC.
Melting points were recorded on an Acro melting point appa-
ratus using a calibrated thermometer. Thin layer chromatography
(TLC) and column chromatography (CC) were performed with [TLC
silica gel 60 F254, Merck] and silica gel (Kieselgel 60, 230e400 mesh,
Merck) respectively. Chromatograms were developed using
hexaneeEtOAc (8:2, v/v) and chloroformeMeOH (9:1, v/v). IR
spectra were recorded on Thermo-Nicolet instrument in KBr discs.
Mass spectra were recorded using GCMS-QP2010S (direct probe)
and on a QTOF microÔ AMPS MAX 10/6A system. PMR spectra and
13C NMR spectra were recorded in CDCl3/DMSO-d6 with TMS (tetra
methylsilane) as an internal standard on a Bruker AG spectrometer
IR (KBr): 2835, 1654, 1573, 1504, 1469, 1407, 1296, 1153, 1022,
986, 829 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.90 (3H, s, OCH3), 3.91
(3H, s, OCH3), 3.94 (3H, s, OCH3), 6.52 (1H, s, H-3), 7.15 (1H, s, H-6),
7.15e7.2 (2H, m), 7.42 (1H, d, J ¼ 15.7 Hz, eCH]CHeCO), 8.00e8.07
(2H, m), 8.08 (1H, d, J ¼ 15.7 Hz, eCH]CHeCO). 13C NMR (CDCl3, 50
MHZ): d 189.68 (C]O), 155.11, 153.02, 143.64, 140.69, 135.50,135.44,
131.38, 131.20, 120.02, 116.07, 115.69, 115.64, 111.85, 97.17, 56.91
(OCH3), 56.65 (OCH3), 56.40 (OCH3). GCeMS (m/z) ¼ 316 [M]þ (14),
285 (100), 269 (8), 241 (10), 170 (8), 123 (68).
and chemical shifts were recorded in
d
units.
5.1.2.5. (E)-1-(4-Morphilinophenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (8). Yellow solid, C22H25NO5, mp 206e209 ꢃC.
IR (KBr): 3421, 2839, 1639, 1604, 1573, 1508, 1446, 1407, 1299,
5.1.1. Synthesis of 2,4,5-trimethoxy benzaldehyde from
b-asarone
b-Asarone ((E)-2,4,5-trimethoxy-1-propenylbenzene) was con-
1211, 1126, 1026, 929, 821 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.32
verted into 2,4,5-trimethoxy benzaldehyde by using KMnO4/
NaHCO3 [39].
(4H, m), 3.84e3.87 (4H, m), 3.89 (3H, s, OCH3), 3.90 (3H, s, OCH3),
3.94 (3H, s, OCH3), 6.52 (1H, s, H-3), 6.89e6.94 (2H, m), 7.13 (1H, s,
H-6), 7.48 (1H, d, J ¼ 15.7 Hz, eCH]CHeCO), 7.98e8.0 (2H, m), 8.07
(1H, d, J ¼ 15.7 Hz, eCH]CHeCO). GCeMS (m/z) ¼ 383 [M]þ (8),
352 (100), 336 (10), 294 (10), 190 (8), 149 (10).
5.1.2. General procedure for synthesis of chalcones via Claisene
Schmidt condensation (1e20)
To a solution of 2,4,5-trimethoxy benzaldehyde (4 mmol) and
appropriate acetophenone (4 mmol) in C2H5OH (25 ml), 40% of
aqueous KOH (2 mmol) was added. The reaction mixture was stir-
red at r.t. till completion of reaction (monitored by TLC). Then the
reaction mass was poured into ice water and neutralized with
aqueous 10% HCl solution. The precipitate was filtered, washed
with excess of water, dried and recrystallized from methanol to
obtain pure chalcones. All structures were confirmed by mass and
NMR spectra as discussed below.
5.1.2.6. (E)-1-(4-Imidazolylphenyl)-3-(2,4,5-trimethoxyphenyl)prop-
2-en-1-one (9). Orangish yellow solid, C21H20N2O4, mp 189e192 ꢃC.
IR (KBr): 3541, 2835, 1639, 1600, 1558, 1508, 1434, 1411, 1288,
1207, 1126, 1026, 983, 829 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.91
(3H, s, OCH3), 3.92 (3H, s, OCH3), 3.95 (3H, s, OCH3), 6.53 (1H, s, H-
3), 7.13 (1H, s, H-6), 7.46 (1H, d, J ¼ 15.7 Hz, eCH]CHeCO), 7.36e
7.54 (4H, m), 8.08e8.16 (3H, m), 8.08 (1H, d, J ¼ 15.7 Hz, eCH]CHe
CO). 13C NMR (CDCl3, 50 MHZ):
d 190.04 (C]O), 160.24, 155.03,
152.83, 143.74, 140.05, 132.02, 131.42, 120.55, 116.16, 115.74, 112.11,
97.47, 57.04 (OCH3), 56.80 (OCH3), 56.46 (OCH3). GCeMS (m/
z) ¼ 364 [M]þ (18), 333 (100), 317 (10), 289 (8), 171 (10), 143 (10).
5.1.2.1. (E)-1-(3-Nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-
1-one (1). Brown solid, C18H17NO6, mp 185e188 ꢃC.
IR (KBr): 2932, 2839, 1650, 1566, 1523, 1473, 1400, 1350, 1261,
1215, 1145, 1026, 979, 840 cmꢀ1; 1HNMR (CDCl3, 200 MHz):
d
3.92
5.1.2.7. (E)-1-(3-Nitro,4-chlorophenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (10). Dark orange solid, C18H16ClNO6, mp 156e
159 ꢃC.
(3H, s, OCH3), 3.93 (3H, s, OCH3), 3.96 (3H, s, OCH3), 6.53 (1H, s, H-
3), 7.13 (1H, s, H-6), 7.45 (1H, d, J ¼ 15.6 Hz, eCH]CHeCO), 7.69
(1H, dd, J ¼ 7.9 Hz), 8.16 (1H, d, J ¼ 15.6 Hz, eCH]CHeCO), 8.32
(1H, d, J ¼ 7.8 Hz), 8.41 (1H, d, J ¼ 7.8 Hz), 8.81 (1H, br s). GCeMS (m/
z) ¼ 343 [M]þ (28), 312 (100), 282 (10), 266 (25), 150 (20).
IR (KBr): 2835, 1658, 1577, 1515, 1465, 1407, 1292, 1207, 1126,
1026, 991, 837 cmꢀ1 1H NMR (CDCl3, 200 MHz):
; d 3.81 (3H, s,
OCH3), 3.93 (3H, s, OCH3), 3.96 (3H, s, OCH3), 6.52 (1H, s, H-3), 7.10
(1H, s, H-6), 7.38 (1H, d, J ¼ 15.6 Hz, eCH]CHeCO), 7.68 (1H, d,
J ¼ 8.4 Hz), 8.11e8.18 (1H, dd, J ¼ 2 & J ¼ 8.4 Hz), 8.14 (1H, d,
J ¼ 15.6 Hz, eCH]CHeCO), 8.46 (1H, d, J ¼ 2 Hz). 13C NMR (CDCl3,
5.1.2.2. (E)-1-(4-Methylphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-
en-1-one (4). Yellow solid, C19H20O4, mp 130e132 ꢃC.
IR(KBr):3433, 2931, 2835,1651,1608,1585,1505,1465,1434,1407,
50 MHZ): d 187.68 (C]O), 155.68, 153.79, 148.31, 143.76, 142.86,
1299, 1207, 1176, 1033, 821 cmꢀ1; 1HNMR (CDCl3, 200 MHz):
d
2.43
138.67, 132.80, 132.55, 131.02, 125.67, 118.55, 115.18, 112.12, 97.0,
57.0 (OCH3), 56.67 (OCH3), 55.48 (OCH3). GCeMS (m/z) ¼ 377 [M]þ
(10), 346 (100), 316 (10), 300 (26), 288 (8), 184 (14), 138 (16).
(3H, s, CH3), 3.87 (3H, s, OCH3), 3.90 (3H, s, OCH3), 3.94 (3H, s, OCH3),
6.52 (1H, s, H-3), 7.13 (1H, s, H-6), 7.28 (2H, d, J ¼ 8.2 Hz), 7.45 (1H, d,
J ¼ 15.8 Hz, eCH]CHeCO), 7.92 (2H, d, J ¼ 8.2 Hz), 8.08 (1H, d,
J ¼ 15.8 Hz, eCH]CHeCO). 13C NMR (CDCl3, 50 MHz):
d
190.82 (C]
5.1.2.8. (E)-1-(3-Triflurophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-
en-1-one (12). Yellow solid, C19H17F3O4, mp 134e137 ꢃC.
O),154.99,152.85,143.67,143.37,140.0,136.58,129.53,128.90,120.59,

440
S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
IR (KBr): 2930, 2835, 1658, 1577, 1515, 1467, 1438, 1292, 1203, 1157,
114, 1026, 938, 844 cmꢀ1 1H NMR (CDCl3, 200 MHz):
3.91 (3H, s,
IR (KBr): 3448, 2835, 1647, 1604, 1562, 1515, 1465, 1407, 1296,
1207, 1126, 1029, 825 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
3.87 (3H,
;
d
d
OCH3), 3.95 (3H, s, OCH3), 3.98(3H, s, OCH3), 6.53 (1H, s, H-3), 7.12(1H, s,
H-6), 7.42 (1H, d, J¼ 15.7 Hz, eCH]CHeCO), 7.63 (1H, m), 7.81 (1H, brd,
J¼ 7.7Hz),8.12(1H,d,J¼ 15.7 Hz, eCH]CHeCO), 8.21 (2H, m). GCeMS
(m/z) ¼ 366 [M]þ (20), 335 (100), 291 (8), 277 (10), 173 (20), 145 (28).
s, OCH3), 3.90 (3H, s, OCH3), 3.94 (3H, s, OCH3), 6.13 (1H, s), 6.52 (1H,
s, H-3), 6.93 (2H, d, J ¼ 8.6 Hz), 7.12 (1H, s, H-6), 7.46 (1H, d,
J ¼ 15.8 Hz, eCH]CHeCO), 7.98 (2H, d, J ¼ 8.6 Hz), 8.09 (1H, d,
J ¼ 15.8 Hz, eCH]CHeCO). GCeMS (m/z) ¼ 314 [M]þ (12), 283
(100), 267 (8), 239 (8), 121 (23).
5.1.2.9. (E)-1-(2-Hydroxy,5-chlorophenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (13). Orange solid, C18H17ClO5, mp 160e163 ꢃC.
IR (KBr): 3422, 2930, 2902, 2833, 1640, 1563, 1496, 1417, 1266,
5.1.2.15. (E)-1-(2,5-Dicetoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-
2-en-1-one (20). Dark yellow solid, C22H22O8, mp 148e150 ꢃC.
IR (KBr): 2985,1759,1651,1562,1515,1415,1292,1180,1149,1029,
1187, 1081, 1011 cmꢀ1 1H NMR (CDCl3, 200 MHz):
; d 3.94 (3H, s,
OCH3), 3.95 (3H, s, OCH3), 3.96 (3H, s, OCH3), 6.53 (1H, s, H-3), 6.97
(1H, d, J ¼ 9.0 Hz), 7.13 (1H, s, H-6), 7.41 (1H, dd, J ¼ 2.5, 9.0 Hz), 7.50
(1H, d, J ¼ 15.4 Hz, eCH]CHeCO), 7.87 (1H, d, J ¼ 2.5 Hz), 8.25 (1H,
d, J ¼ 15.4 Hz, eCH]CHeCO),13.0 (1H, s, OH). GCeMS (m/z) ¼ 348
[M]þ (45), 317 (100), 194 (24), 179 (22), 155 (26), 127 (15).
1002, 855 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 2.2 (3H, s), 2.31 (3H, s),
3.87 (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.94 (3H, s, OCH3), 6.50 (1H, s,
H-3), 7.04 (1H, s), 7.10 (1H, d, J ¼ 16.0 Hz, eCH]CHeCO), 7.18 (1H, s,
H-6), 7.23 (1H, d, J ¼ 2.4 Hz), 7.41 (1H, d, J ¼ 2.4 Hz), 7.88 (1H, d,
J ¼ 16.0 Hz, eCH]CHeCO). GCeMS (m/z) ¼ 414 [M]þ (10), 383 (13),
341 (12), 299 (15), 194 (10), 137 (8), 43 (100).
5.1.2.10. (E)-1-(3-Methoxy,4-hydroxyphenyl)-3-(2,4,5-trimethoxyphe
nyl)prop-2-en-1-one (14). Light orange solid, C19H20O6, mp 181e
183 ꢃC.
5.1.3. General procedure for synthesis of flavones (21e23)
To a solution of 20-hydroxy-2,4,5-trimethoxy chalcones ((6, 15,
13) (1 equiv)) in DMSO (10.0 ml), I2 (catalytical amounts) was
added. The reaction mixture was heated to reflux for 1e2 h, cooled
and poured into water and extracted into EtOAc (3 ꢄ 25.0 ml). The
organic layer was washed with brine and dried over MgSO4. The
solvent was evaporated to get the product.
IR (KBr): 3050, 2950, 2835,1648,1593,1515,1465,1442,1380,1357,
1299, 1253, 1215, 1126, 1026, 972, 840 cmꢀ1 1H NMR (CDCl3,
;
200 MHz); d 3.90 (3H, s, OCH3), 3.92 (3H, s, OCH3), 3.94 (3H, s, OCH3),
3.98 (3H, s, OCH3), 6.10 (1H, s), 6.53 (1H, s, H-3), 6.99 (1H, d, J ¼ 4.4 Hz),
7.18 (1H, s, H-6), 7.48 (1H, d, J¼ 15.7Hz,eCH]CHeCO), 7.70e7.63 (2H,
m), 8.07 (1H, d, J ¼ 15.7 Hz, eCH]CHeCO). GCeMS (m/z) ¼ 344 [M]þ
(16), 313 (100), 298 (15), 207 (42), 194 (24), 167 (67), 152 (22).
5.1.3.1. 2-(2,4,5-Trimethoxyphenyl)-7-hydroxy chromen-4-one (21).
Light orange solid, C18H16O6, mp 228e231 ꢃC.
5.1.2.11. (E)-1-(2,5-Dihydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (15). Brick red solid, C18H20O6, mp 218e221 ꢃC.
IR (KBr): 3421, 2935, 1635, 1558, 1508, 1469, 1407, 1280, 1215,
IR (KBr): 3450, 3089, 2989, 2842, 1630, 1612, 1558, 1519, 1438,
1392, 1285, 1215, 1134, 1022, 975, 848, 817 cmꢀ1; 1H NMR (DMSO-
d6, 200 MHz):
d 3.82 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.98 (3H, s,
1176, 1122, 1022, 897, 840 cmꢀ1
.
13C NMR (DMSO-d6, 50 MHz):
OCH3), 6.80e7.02 (4H, m), 7.80e7.87 (2H, m). GCeMS (m/z) ¼ 328
d
193.75 (C]O),155.37,155.18,153.82,149.70,143.75,139.89,124.54,
[M]þ (100), 313 (21), 297 (14), 285 (15), 192 (20), 177 (18), 137 (45).
121.08, 118.58, 115.23, 114.93, 112.62, 98.38, 56.95 (2 ꢄ OCH3), 56.33
(OCH3) (identified as diacetoxy compound, 20). GCeMS (m/z) ¼ 330
[M]þ (54), 299 (100), 194 (70), 179 (56), 151 (40), 136 (30).
5.1.3.2. 2-(2,4,5-Trimethoxyphenyl)-6-hydroxy chromen-4-one (22).
Light brown solid, C18H16O6, mp 254e256 ꢃC.
IR (KBr): 3136, 2927, 1616, 1566, 1523, 1469, 1365, 1272, 1226,
5.1.2.12. (E)-1-(2,4,6-Trimethoxyphenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (16). Light yellow solid, C21H24O7, mp 132e134 ꢃC.
IR (KBr): 2842, 1650, 1604, 1515, 1465, 1407, 1257, 1207, 1153,
1149, 1018, 852, 833 cmꢀ1 1H NMR (DMSO-d6, 200 MHz):
; d 3.81
(3H, s, OCH3), 3.90 (3H, s, OCH3), 3.94 ( 3H, s, OCH3), 6.85 (1H, s),
7.20e7.65 (5H, m), 9.95 (1H, s, OH). GCeMS (m/z) ¼ 328 [M]þ (100),
313 (20), 297 (12), 285 (16), 192 (18), 177 (17), 137 (50).
1122, 1026, 945, 813 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.81 (3H, s,
OCH3), 3.76 (6H, s, 2 ꢄ OCH3), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3),
3.98 (3H, s, OCH3), 6.16 (2H, s), 6.47 (1H, s, H-3), 6.89 (1H, d,
J ¼ 16.0 Hz, eCH]CHeCO), 7.04 (1H, s, H-6), 7.65 (1H, d, J ¼ 16.0 Hz,
5.1.3.3. 2-(2,4,5-Trimethoxyphenyl)-6-chloro chromen-4-one (23).
Light orange solid, C18H15ClO5, mp 181e83 ꢃC.
eCH]CHeCO). 13C NMR (CDCl3, 50 MHz):
d
195.10 (C]O), 162.50,
IR (KBr): 3421, 1639, 1612, 1558, 1438, 1269, 1207, 1153, 1022,
159.09, 154.51, 152.64, 143.75, 139.97, 127.64, 116.09, 112.78, 111.45,
97.54, 91.28, 56.87 (OCH3), 56.43 (2 ꢄ OCH3), 56.32 (2 ꢄ OCH3),
55.80 (OCH3). GCeMS (m/z) ¼ 388 [M]þ (10), 357 (100), 341 (8), 195
(13), 180 (14), 151 (12), 137 (10).
852, 817 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.87 (3H, s, OCH3), 3.93
(3H, s, OCH3), 3.97 (3H, s, OCH3), 6.59 (1H, s, H-30), 7.17 (1H, s, H-60),
7.42 (1H, s), 7.47 (1H, d, J ¼ 8.8 Hz), 7.60 (1H, dd, J ¼ 2.5 and 8.8 Hz),
8.17 (1H, d, J ¼ 2.5 Hz). GCeMS (m/z) ¼ 346 [M]þ (100), 331 (26),
303 (24), 192 (18), 177 (17), 155 (44), 149 (22).
5.1.2.13. (E)-1-(3,5-Dibenzyloxyphenyl)-3-(2,4,5-trimethoxyphenyl)
prop-2-en-1-one (18). Yellow solid, C32H30O6, mp 154e156 ꢃC.
IR (KBr): 3436, 1658, 1585, 1512, 1438, 1292, 1215, 1161, 1126,
5.1.4. General procedure for synthesis of flavonol (24, 25)
A suspension of a mixture of powdered 20-hydroxychalcone
(0.10 g, 0.45 mmol) in EtOH, an aq. NaOH solution (8 M, 1.0 ml) and
a 30% hydrogen peroxide solution (0.25 g) was stirred at r.t. for 2 h.
The crude product was filtered off, washed with water to give fla-
vonol. Recrystallization of the crude product from MeOH gave pure
compound.
1029, 964, 833 cmꢀ1 1H NMR (CDCl3, 200 MHz):
; d 3.89 (3H, s,
OCH3), 3.90 (3H, s, OCH3), 3.94 (3H, s, OCH3), 5.10 (4H, s, 2 ꢄ CH2),
6.51 (1H, s, H-3), 7.10 (1H, s, H-6), 7.25ee7.28 (3H, m), 7.35e7.46
(10H, m), 7.32 (1H, d, J ¼ 15.8 Hz, eCH]CHeCO), 8.11 (1H, d,
J ¼ 15.8 Hz, eCH]CHeCO). 13C NMR (CDCl3, 50 MHz):
d 190.71 (C]
O), 160.38, 155.12, 153.06, 143.73, 141.30, 140.61, 136.95, 129.05,
128.55, 128.06, 120.34, 115.9, 111.69, 107.97, 106.97, 106.48, 97.26,
5.1.4.1. 2-(2,4,5-Trimethoxyphenyl)-6-chloro,3-hydroxy chromen-4-
one (24). Light orange solid, C18H15ClO6, mp 188e190 ꢃC.
IR (KBr): 3298,1608,1566,1519,1469,1407,1269,1211,1029, 871,
70.78 (2 ꢄ
feCH2), 57.03 (OCH3), 56.76 (OCH3), 56.46 (OCH3). GCe
MS (m/z) ¼ 510 [M]þ (16), 479 (50), 419 (4), 269 (6), 91 (100).
779 cmꢀ1; 1H NMR (CDCl3, 200 MHz):
d 3.89 (3H, s, OCH3), 3.97 (3H,
5.1.2.14. (E)-1-(4-Hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-
2-en-1-one (19). Orangish yellow solid, C18H18O5, mp 186e189 ꢃC.
s, OCH3), 4.0 (3H, s, OCH3), 6.65 (1H, s, H-30), 7.11 (1H, s, H-60), 7.47
(1H, d, J ¼ 9.0 Hz), 7.60 (1H, dd, J ¼ 2.2 and 9.0 Hz), 8.24 (1H, d,

S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
441
J ¼ 2.2 Hz). GCeMS (m/z) ¼ 362 [M]þ(100), 345 (82), 331 (76), 315
decrease from the DPPH radical to the DPPHeH form. The degree
of discolouration indicates the scavenging potential of the anti-
oxidant compounds or extracts in terms of hydrogen donating
ability.
(24), 287 (22), 275 (24), 207 (62), 179 (67), 155 (32).
5.1.4.2. 2-(2,4,5-Trimethoxyphenyl)-3,6-dihydroxy chromen-4-one
(25). Light orange solid, C18H16O7, mp 226e229 ꢃC.
1 mg/ml solution of DPPH in methanol was prepared as stock.
IR (KBr): 3286, 1610, 1567, 1521, 1463, 1407, 1265, 1209, 1027,
Different concentrations of the sample were prepared and 100 ml
885, 813 cmꢀ1; 1H NMR (DMSO-d6, 200 MHz):
d
3.72 (3H, s, OCH3),
of stock DPPH was added to each concentration and made up to
3 ml with methanol. Absorbance @517 nm was measured after
incubating for 15 min and the readings were taken against a blank
without the sample. Ascorbic acid at various concentrations was
used as standard. Lower the absorbance of the reaction mixture
indicates higher free radical scavenging activity. The capability to
scavenge the DPPH radical was calculated using the following
equation.
3.80 (1H, s, OCH3), 3.88 (3H, s, OCH3), 6.84 (1H, s, H-30), 7.1 (1H, s, H-
60), 7.74 (2H, m), 8.05 (1H, br s), 9.0 (1H, s, OH). GCeMS (m/z) ¼ 344
[M]þ (3), 313 (6), 238 (23), 163 (10), 78 (77), 63 (100).
5.1.5. General procedure for synthesis of flavanone (26)
20-Hydroxychalcone (1 g) in glacial AcOH (10 ml) was heated to
reflux for 45 h. Then the mixture was poured into cold water and
extracted with EtOAc (2 ꢄ 25 ml). EtOAc layer was washed with
brine and the organic layer dried over MgSO4. Solvent evaporated
to dryness. Required product was purified by column chromatog-
raphy (hexaneeEtOAc with increasing polarity).
Inhibitionð%Þ ¼ ð1 ꢀ A1=A0Þ*100
where A0 is the absorbance of control and A1 is the absorbance of
test substance.
5.1.5.1. 2-(2,4,5-Trimethoxyphenyl)-6-hydroxy chroman-4-one (26).
Pale yellow solid, C18H18O6, mp 175e177 ꢃC.
5.2.2.2. Nitric oxide scavenging activity [42]. The procedure of nitric
oxide scavenging activity is based on the principle that sodium
nitroprusside in aqueous solution at physiological pH sponta-
neously generates nitric oxide which interacts with oxygen to
produce nitrite ions that can be estimated using Griess reagent.
Scavengers of nitric oxide compete with oxygen, leading to reduced
production of nitrite ions.
10 mM of sodium nitroprusside was prepared in phosphate
saline buffer (pH 7.4) and 1 ml was added to different concen-
trations of sample. After incubation at room temperature for
2.5 h, 0.5 ml of Griess reagent was added and absorbance at
546 nm was recorded against a blank. Ascorbic acid was used as
the standard. The percentage of inhibition was calculated using
the formula.
IR (KBr): 3450, 3417, 2939, 2839, 1693, 1616, 1523, 1454, 1407,
1207, 1122, 1029, 991, 860 cmꢀ1; 1HNMR (CDCl3, 200 MHz):
d 2.79e
3.0 (2H, m), 3.89 (3H, s, OCH3), 3.90 (3H, s, OCH3), 3.91 (3H, s, OCH3),
5.76 (1H, dd, J ¼ 4.2 and 12.0 Hz), 6.07 (1H, br s), 6.55 (1H, s, H-30),
6.97 (1H, d, J ¼ 8.8 Hz), 7.10 (1H, dd, J ¼ 3.0 and 8.8 Hz), 7.15 (1H, s,
H-60), 7.44 (1H, d, J ¼ 3.0 Hz). 13C NMR (CDCl3, 50 MHZ):
d 190.34
(C]O), 156.86, 151.08, 150.92, 150.18, 143.68, 125.46, 121.35, 119.74,
119.21, 111.60, 111.16, 97.87, 74.86 (C-2), 57.14 (OCH3), 56.67 (OCH3),
56.60 (OCH3), 44.24 (C-3). GCeMS (m/z) ¼ 330 [M]þ (60), 299 (100),
194 (72), 179 (58), 151 (40), 136 (26).
5.2. Biology
5.2.1. 5.7.1Anticancer activity
Inhibitionð%Þ ¼ ð1 ꢀ A1=A0Þ*100
The cancerous cell lines were maintained in Dulbecco’s Modified
Eagle’s Medium (SigmaeAldrich Inc., USA) supplemented with 10%
foetal bovine serum (Gibco BRL., USA) in a CO2 incubator. The
cytotoxicity of the compounds was measured by MTT assay [40].
Threedifferent kinds ofcancerous celllines, viz., HeLa(cervical), MCF-
7 (breast) and SW-982 (synovial) were plated in a 96-well plate at the
density of 10,000 cells per well. After 24 h, the cells were treated with
various concentrations of chalcones from 250
pristimerin (triterpene quinine methide) (50
standard. The cells were further incubated for 72 h. The cytotoxicity
was measured by adding 5 mg/ml of MTT (SigmaeAldrich Inc., USA)
to each well and incubated for another 3 h. The purple formazan
where A0 is the absorbance of control and A1 is the absorbance of
test substance.
5.2.2.3. Assay for phenyl hydrazine induced haemolysis of erythro-
cytes [43]. The erythrocyte suspension (20% PCV) was prepared
from rat blood. To the different sample concentrations 1 ml of
0.5 mM phenyl hydrazine, 0.1 ml of 20% erythrocyte suspension
were added and made up the volume to 3 ml with phosphate buffer
saline. This was incubated at 37 ꢃC for 1 h and centrifuged at 1000 g
for 10 min. The supernatant was transferred to fresh test tubes and
m
M to 200 nM and
mMe50 nM) as
crystals were dissolved by adding 100 ml of DMSO to each well. The
absorbance was read at 570 nm in a spectrophotometer [Spectra
Max 340]. The cell death was calculated as follows:
the absorbance was recorded at 540 nm.
the positive control.
a-Tocopherol was used as
Cell death ¼ 100ꢀ½ðtest absorbance=control absorbanceÞꢄ100ꢅ
Acknowledgements
The test result is expressed as the concentration of a test com-
pound which inhibits the cell growth by 50% (IC50).
We express our sincere gratitude to Dr. Anil Kush, CEO, Vittal
Mallya Scientific Research Foundation, for his keen interest and
encouragement, Dr. Puja Ravikumar for helpful discussions and also
wish to thank Mr. AC Karunakara and Ms. Aparna Bhat for analytical
help.
5.2.2. Antioxidant activity
5.2.2.1. In-vitro antioxidant activity (DPPH method) [41]. The scav-
enging reaction between (DPPHꢁ) and an antioxidant (HeA) can be
written as:
Appendix A. Supplementary data
ðDPPHÞ þðHeAÞ/ DPPHeH þðAÞ
ðYellowÞ
ðPurpleÞ
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.01.
018.
Antioxidants react with DPPH, which is a stable free radical and
is reduced to the DPPHH and as consequence the absorbance’s

442
S. Shenvi et al. / European Journal of Medicinal Chemistry 62 (2013) 435e442
[22] M. Cabrera, M. Simoens, G. Falchi, M.L. Lavaggi, O.E. Piro, E.E. Castellano,
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