Synthesis of B-ring substituted flavones and evaluation of their antitumor and antioxidant activities

Article abstract of DOI:10.1007/s00044-012-0423-1

A series of flavones, substituted at the ring B, were synthesized using either Claisen-Schimdt Condensation or Baker-Venkataraman rearrangement. The synthesized compounds were tested for in vitro cytotoxic activity by sulforhodamine B assay against three cell lines of different origin, viz. HepG2, MCF-7, and MOLT-4. The compounds were also tested for a possible antioxidant activity by determination of inhibition of lipid peroxidation. Quercetin was taken as a standard for antioxidant activity. Compound 1c showed the highest cytotoxic activity against MCF-7 (GI50 < 0.1 μM) and MOLT-4 (GI50 < 0.1 μM) cell lines and was comparable to adriamycin, the standard used. Compounds 1b, 1g, and 1h also showed promising activity against MCF-7 and MOLT-4 cell lines. In the absence of a hydroxyl group, one or more methoxy groups present on the B-ring (compounds 1c-1e) were major determinants of inhibition of lipid peroxidation.

Full text of DOI:10.1007/s00044-012-0423-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4293–4299
DOI 10.1007/s00044-012-0423-1
ORIGINAL RESEARCH
Synthesis of B-ring substituted flavones and evaluation of their
antitumor and antioxidant activities
•
Akshada J. Joshi Manoj K. Gadhwal
•
•
Urmila J. Joshi Priscilla D’Mello
•
•
Ragini Sinha Girjesh Govil
Received: 3 October 2012 / Accepted: 14 December 2012 / Published online: 6 January 2013
Ó Springer Science+Business Media New York 2013
Abstract Aseriesofflavones, substitutedattheringB,were
synthesized using either Claisen–Schimdt Condensation or
Baker–Venkataraman rearrangement. The synthesized com-
pounds were tested for in vitro cytotoxic activity by sulfo-
rhodamine B assay against three cell lines of different origin,
viz. HepG2, MCF-7, and MOLT-4. The compounds were also
tested for a possible antioxidant activity by determination of
inhibition of lipid peroxidation. Quercetin was taken as a
standard for antioxidant activity. Compound 1c showed the
highest cytotoxic activity against MCF-7 (GI50\ 0.1 lM)
andMOLT-4(GI50 \ 0.1 lM)celllinesandwascomparable
to adriamycin, the standard used. Compounds 1b, 1g, and 1h
also showed promising activity against MCF-7 and MOLT-4
cell lines. In the absence of a hydroxyl group, one or more
methoxy groups present on the B-ring (compounds 1c–1e)
were major determinants of inhibition of lipid peroxidation.
possess a number of pharmacological properties, which
include antioxidant, anti-neoplastic, and anti-inflammatory
activity (Kanadaswami et al., 2005). Mechanisms have
been proposed to explain the anticancer activity of flavo-
noids. These include, inhibition of CYP450 family of
enzymes (Androutsopoulos et al., 2010), which play a
major role in the activation of many suspected human
carcinogens, induction of apoptosis via p53 dependent
mechanism (Vidya Priyadarsini et al., 2010). Inhibition of
protein tyrosine kinase (Huang et al., 2009) and inhibition
of interaction of carcinogens with DNA (Kang et al., 1999)
also play a role in the anticancer activity of flavonoids.
Flavonoids are also reported to inhibit some enzymes
associated with the process of carcinogenesis including
cyclin-dependent kinases (Dong et al., 2010), aurora
kinases (Xie et al., 2012), aromatase (Chumsri et al.,
2011), and topoisomerases (Bandele and Osheroff, 2007).
Flavonoids serve as adjuvants in the cancer chemotherapy.
This is attributed to their ability to reduce the dose of
clinically used anticancer compounds and hence the asso-
ciated toxicity (Zhao et al., 2010). Flavonoids can signifi-
cantly reduce the development of resistance to standard
anticancer drugs by their ability to interact with p-glyco-
protein and ABC transporter (Alvarez et al., 2009). Oxi-
dative stress has also been listed as a major component of
diseases such as inflammatory diseases and cancer (Reuter
et al., 2010). The beneficial effect of flavonoids in these
diseases has been partially attributed to the ability of the
flavonoids to reduce oxidative stress.
Keywords B-ring substituted flavones ꢀ
Antitumor activity ꢀ Sulforhodamine B assay ꢀ
Antioxidant activity ꢀ Lipid peroxidation
Introduction
Flavonoids are a group of polyphenolic compounds, with a
characteristic diphenylpropane skeleton. The compounds
A. J. Joshi ꢀ M. K. Gadhwal ꢀ U. J. Joshi (&) ꢀ P. D’Mello
Prin. K. M. Kundnani College of Pharmacy, 23-Jote Joy
Building, R. S. Marg, Cuffe Parade, Colaba, Mumbai 400005,
India
Several naturally occurring flavonoids are present in the
form of glycosides. The glycosides are polar or large to
penetrate the intestinal membranes and are not easily
absorbed (Hu, 2007). The release of the aglycone by the
action of the microfloral enzymes is needed (Liu et al.,
2003; Liu and Hu, 2002) for the compound to become
e-mail: urmilajjoshi@hotmail.com
R. Sinha ꢀ G. Govil
National Facility for High Field NMR, Tata Institute of
Fundamental Research, Homi Bhabha Road, Navy Nagar,
Mumbai 400005, India
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Med Chem Res (2013) 22:4293–4299
absorbable. Although aglycone forms of flavonoids are
permeable, the bioavailability is low due to poor water
solubility and a greater extent of conjugation (Ng et al.,
2004; Zhang et al., 2005). This calls for structural modi-
fications of flavonoids to increase their bioavailability. The
extensive conjugation of flavonoids occurs due to the
presence of multiple hydroxyl groups (Hu, 2007). Thus, a
logical approach toward increasing the bioavailability of
flavonoids is to target at reduction in the number of
hydroxyl groups. Synthetic flavones devoid of any hydro-
xyl group have been previously reported (Lu et al., 1996).
Such compounds have been tested for anticancer activities
on various cell lines.
2 (0.15 mmol) were reacted to form a corresponding ben-
zoyl ester, 3. This was then dissolved in pyridine and
treated with a base (KOH), forming a 1,3-diketone inter-
mediate, 4. This diketone was refluxed for 1 h with acid
(AcOH/H2SO4) to give desired flavones (Cardenas et al.,
2006) (Scheme 1). The crude products were recrystallized
from methanol and purified by column chromatography
over silica gel yielding the desired products 1a–1d.
Synthesis of 1e–1h
Synthesis of compounds 1e–1h was carried out according
to the Claisen–Schmidt condensation reaction using lith-
ium hydroxide (LiOHꢀH2O) as a base catalyst. In this
reaction, 2-hydroxyacetophenone, 1 (1 mmol, 1 equiv.) in
EtOH (2.5 ml) was treated with LiOHꢀH2O (4 mg,
0.1 mmol) under magnetically stirred condition for 10 min
at room temperature. The corresponding benzaldehyde
derivative, 5 (1 mmol, 1 equiv.) was added dropwise to the
stirring mixture. The mixture was stirred until complete
consumption of the starting materials. Ethanol was
removed under reduced pressure. The residue was diluted
with water (5 ml), neutralized with 1 % aqueous HCl. The
products obtained were recrystallized from methanol to
give hydroxychalcones, 6. These chalcones, after refluxing
with iodine in dimethylsulfoxide (DMSO) for about 2 h,
were then cyclized giving corresponding flavones (Bhagat
et al., 2006; Cabrera et al., 2007) (Scheme 2). The products
were purified by column chromatography over silica gel
yielding the desired products 1e–1h.
In an attempt to study the effect of structural modifi-
cations in the B-ring of the flavones on their anticancer and
antioxidant activity, we have synthesized some flavones
with variations introduced in the B-ring and have studied
the effect of such modifications on the anticancer and
antioxidant activity of the synthesized compounds.
Experimental
Synthesis and characterization of B-ring substituted
flavone analogs
Synthesis of 1a–1d
The chemical synthesis of compounds 1a–1d was carried
out according to the Baker–Venkataraman transformation.
The benzoic acid derivatives were converted to the corre-
sponding benzoyl chloride derivatives using PCl5. Then,
following Baker–Venketaraman reaction, 2-hydroxyaceto-
phenone, 1 (0.1 mmol) and a substituted benzoyl chloride,
All the compounds were characterized on the basis of 1H
NMR, 13C NMR, and elemental analysis. NMR spectra
were recorded on a Bruker Avance 500 spectrophotometer.
Samples were dissolved (0.1 mmol/ml) in hexadeuterated
Scheme 1 General procedure for synthesis of flavones (1a–1d) Reagents and conditions. a Pyridine. b KOH/pyridine. c H2SO4/AcOH, reflux
Scheme 2 General procedure
for synthesis of flavones
(1e–1h) Reagents and
conditions. a LiOH, EtOH, rt.
b I2/DMSO, reflux
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dimethylsulfoxide (DMSO-d6), and spectra were recorded
at 323 K. Elemental analyses were made on a FLASH EA
1112 series (Thermo Finnigan, Italy) apparatus.
(d, 2H, H-30,50), 6.69 (s, 1H, H-3), 3.85 (s, 3H, OMe at 40
position); 13C NMR d ppm 177.151 (C-4), 163.103 (C-3),
162.373 (C-40), 156.198 (C-10), 134.358 (C-2, C-9),
128.641 (C-6, C-7), 125.782 (C-30, C-50), 125.096 (C-60),
123.838 (C-5), 118.693 (C-8), 115.148 (C-20), 106.0 (C-10),
56.03 (OMe at 40 position); Elem. Anal. Calcd. for
C16H12O3: C, 76.176; H, 4.795; found: C, 76.052; H, 4.517.
1
2-Phenylchromen-4-one (1a) 39.43 % yield; H NMR d
ppm 8.102 (d, 2H, H-20,60), 8.072 (d, 1H, H-5), 7.846
(t, 1H, H-7), 7.781 (d, 1H, H-8), 7.609 (m, 3H, H-30,40,50),
7.516 (t, 1H, H-6), 7.011 (s, 1H, H-3); 13C NMR d ppm
183.178 (C-4), 163.820 (C-2), 157.158 (C-9), 134.9 (C-10),
132.24 (C-10), 131.644 (C-40), 128.8 (C-30,50), 126.91
(C-20,60), 125.7 (C-7), 125.29 (C-6), 123.43 (C-5), 117.6
(C-8), 105.64 (C-3); Elem. Anal. Calcd. for C15H10O2: C,
81.064; H, 4.535; found: C, 80.791; H, 4.256.
2-(40-Chlorophenyl)-chromen-4-one (1f) 81.83 % yield;
1H NMR d ppm 8.16 (d, 2H, H-20,60), 8.06 (d, 1H, H-5),
7.88 (d, 1H, H-6), 7.82 (d, 1H, H-8), 7.66 (d, 2H, H-30,50),
7.52 (t, 1H, H-7), 7.10 (s, 1H, H-3); 13C NMR d ppm
177.389 (C-4), 161.912 (C-3), 156.198 (C-10), 137.149
(C-9), 134.768 (C-2), 130.565 (C-30, C-50), 128.530 (C-40),
128.339 (C-6, C-7), 125.720 (C-60), 125.182 (C-20),
123.577 (C-5), 118.815 (C-8), 107.862 (C-10); Elem. Anal.
Calcd. for C15H9O2Cl: C, 70.186; H, 3.534; found: C,
70.005; H, 3.254.
2-(40-Chloro-30-nitrophenyl)-chromen-4-one (1b) 41.3 %
1
yield; H NMR d ppm 8.771 (s, 1H, H-20), 8.406 (d, 1H,
H-50), 8.077 (d, 1H, H-60), 7.993 (d, 1H, H-5), 7.868 (t, 1H,
H-6), 7.837 (d, 1H, H-8), 7.533 (t, 1H, H-7), 7.208 (s, 1H,
H-3); 13C NMR d ppm 177.015 (C-4), 167.828 (C-2),
157.2 (C-9), 146.79 (C-30), 133.24 (C-10), 130.761 (C-10),
129.168 (C-50), 126.314 (C-7), 126.27 (C-6), 125.1 (C-40),
123.48 (C-5), 104.95 (C-3); Elem. Anal. Calcd. for
C15H8O4N1Cl1: C, 59.716; H, 2.673; N, 4.644; found: C,
57.99; H, 2.53; N, 4.61.
2-(40-Nitrophenyl)-chromen-4-one (1g) 82.39 % yield;
1H NMR d ppm 8.378 (d, 4H, H-20, 30, 50, 60), 8.087 (d, 1H,
H-5), 7.876 (d, 1H, H-6), 7.821 (d, 1H, H-8), 7.538 (t, 1H,
H-7), 7.202 (s, 1H, H-3); 13C NMR d ppm 177.525 (C-4),
160.772 (C-2), 156.230 (C-9), 149.670 (C-40), 137.660
(C-10), 135.137 (C-10), 128.274 (C-20,60), 126.256 (C-6),
125.348 (C-7), 124.540 (C-30,50), 123.834 (C-5), 119.090
(C-8), 109.704 (C-3); Elem. Anal. Calcd. for C15H9O4N1:
C, 67.413; H, 3.394; N, 5.242; found: C, 67.301; H, 3.442;
N, 5.104.
2-(30,50-Dimethoxyphenyl)-chromen-4-one (1c) 36.2 %
1
yield; H NMR d ppm 8.076 (d, 1H, H-5), 7.860 (d, 1H,
H-8), 7.820 (t, 1H, H-6), 7.513 (t, 1H, H-7), 7.234 (s, 1H,
H-60), 7.231 (s, 1H, H-20), 7.075 (s, 1H, H-3), 6.742 (s, 1H,
H-40), 3.872 (s, 6H, OMe at 30 and 50 positions); 13C NMR
d ppm 177.617 (C-4), 162.808 (C-2), 161.574 (C-30,50),
156.258 (C-9), 134.708 (C-10), 133.759 (C-10), 125.974
(C-6), 125.310 (C-7), 123.886 (C-5), 119.139 (C-8),
108.032 (C-40), 105.089 (C-20,60), 104.235 (C-3), 56.366
(OMe at 30 and 50 positions); Elem. Anal. Calcd. for
C17H14O4: C, 72.328; H, 4.999; found: C, 72.732; H, 4.688.
1
2-(30-Nitrophenyl)-chromen-4-one (1h) 73.4 % yield; H
NMR d ppm 8.840 (s, 1H, H-20), 8.550 (d, 1H, H-40), 7.885
(m, 4H, H-8, 50, 60), 7.876 (t, 1H, H-6), 7.539 (t, 1H, H-7),
7.215 (s, 1H, H-3); 13C NMR d ppm 177.35 (C-4), 164.398
(C-2), 156.651 (C-9), 148.36 (C-30), 135.684 (C-10),
134.739 (C-10), 133.65 (C-60), 129.03 (C-50), 125.993
(C-6), 124.86 (C-7), 124.51 (C-40), 123.672 (C-5), 122.834
(C-20), 117.212 (C-8), 107.724 (C-3); Elem. Anal. Calcd.
for C15H9O4N1: C, 67.413; H, 3.394; N, 5.242; found: C,
67.092; H, 3.286; N, 5.351.
2-(30,40-Dimethoxyphenyl)-chromen-4-one (1d) 36.2 %
1
yield; H NMR d ppm 8.04 (d, 1H, H-5), 7.674 (d, 1H,
H-8), 7.592 (t, 1H, H-6), 7.37 (t, 1H, H-7), 7.122 (d, 1H,
H-60), 7.015 (s, 1H, H-3), 6.874 (s, 1H, H-20), 6.792 (d, 1H,
H-50), 3.537 (s, 6H, OMe at 30 and 40 positions); 13C NMR
d ppm 177.06 (C-4), 163.651 (C-2), 157.52 (C-30), 156.82
(C-9), 156.481 (C-40), 133.763 (C-10), 130.25 (C-10),
125.751 (C-7), 123.986 (C-6), 123.294 (C-5), 117.592
(C-8), 108.793 (C-60), 108.52 (C-50), 105.724 (C-3), 56.32
(OMe at 30 and 40 positions); Elem. Anal. Calcd. for
C17H14O4: C, 72.328; H, 4.999; found: C, 72.044; H, 4.868.
Evaluation of cytotoxicity by sulforhodamine B assay
The cell lines were grown in RPMI 1640 medium con-
taining 10 % fetal bovine serum and 2 mM L-glutamine.
For the screening experiment, cells were inoculated into
96-well microtiter plates in 100 ll at plating densities as
shown in the study details above, depending on the dou-
bling time of individual cell lines. After cell inoculation,
the microtiter plates were incubated at 37 °C, 5 % CO2,
95 % air, and 100 % relative humidity for 24 h prior to
addition of experimental drugs.
2-(40-Methoxyphenyl)-chromen-4-one (1e) 80.52 % yield;
1H NMR d ppm 8.06 (d, 2H, H-20,60), 8.04 (d, 1H, H-5),
7.81 (d, 1H, H-8), 7.79 (t, 1H, H-6), 7.48 (t, 1H, H-7), 7.14
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Effect of synthesized compounds on different
human cancer cell lines
HepG2
MCF-7
MOLT-4
Fig. 1 Effect of synthesized flavones at a concentration of 10-4 lg/ml
on cell growth of different human cancer cell lines. All values are
expressed as mean SD of at least three independent experiments.
Compounds 1c and 1h were found to possess statistically significant
activity (*p \ 0.05) against MCF-7 cell lines whereas compounds 1b,
1c, and 1h were found to possess statistically significant activity
(*p \ 0.05) against MOLT-4 cell lines when compared to standard
adriamycin
After 24 h, one 96-well plate containing 5 9 103 cells/
well was fixed in situ with trichloroacetic acid (TCA) to
represent a measurement of the cell population at the time
of drug addition (Tz). Experimental drugs were initially
solubilized in DMSO at 100 mg/ml and diluted to 1 mg/ml
using water and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate (1 mg/ml)
was thawed and diluted to each of the following concen-
trations. 10-6, 10-5, 10-4, and 10-3 lg/ml with complete
medium containing test article. Aliquots of 10 ll of these
different drug dilutions were added to the appropriate
microtiter wells already containing 90 ll of medium,
relative to control wells, and expressed as the ratio of
average absorbance of the test well to the average absor-
bance of the control wells 9 100 (Vichai and Kirtikara,
2006).
Using the six absorbance measurements [time zero (Tz),
control growth (C), and test growth in the presence of drug
at the four concentration levels (Ti)], the percentage growth
was calculated at each of the drug concentration levels.
Percentage growth inhibition was calculated as:
½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢃ 100 for concentrations for
which Ti ꢄ Tz ðTi ꢁ TzÞ positive or zero
½ðTi ꢁ TzÞ=Tzꢂ ꢃ 100 for concentrations for which
Ti \Tz. ðTi ꢁ TzÞ negative
resulting in the required final drug concentrations of 10-7
,
10-6, 10-5, and 10-4 lg/ml.
Figure 1 shows the results of antitumor testing of the
synthesized compounds against three different cell lines
viz. HepG2, MCF-7, and MOLT-4. These results are
expressed in terms of percent growth in the presence of the
test compounds. The GI50 values are shown in Table 1.
Plates were incubated at standard conditions for 48 h and
assay was terminated by the addition of cold TCA. Cells
were fixed in situ by the gentle addition of 50 ll of cold
30 % (w/v) TCA (final concentration, 10 % TCA) and
incubated for 60 min at 4 °C. The supernatant was dis-
carded; plates were washed five times with tap water and air
dried. Sulforhodamine B (SRB) solution (50 ll) at 0.4 %
(w/v) in 1 % acetic acid was added to each of the wells, and
plates were incubated for 20 min at room temperature
(Voigt, 2005). After staining, unbound dye was recovered
and the residual dye was removed by washing five times
with 1 % acetic acid. The plates were air dried. Bound stain
was subsequently eluted with 10 mM trizma base, and the
absorbance was read on a plate reader at a wavelength of
540 nm with 690 nm reference wavelength. Percent growth
was calculated on a plate-by-plate basis for test wells
Statistical analysis
The statistical significance of the results obtained was
analyzed by one-way analysis of variance (ANOVA) at
p \ 0.05, using adriamycin (ADR) as the standard.
Lipid peroxidation inhibition by TBARS test (Okaha,
1979)
Liver homogenate was prepared from male albino wistar
rats. The liver was quickly excised, washed several times
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4297
as mean of at least three independent experiments. A curve
of concentration against percentage inhibition was plotted
for determination of half the maximal inhibitory concen-
tration (IC50 in lg/ml). The IC50 values for the lipid per-
oxidation inhibition by TBARS test are shown in Fig. 2.
Table 1 Tumor cell cytotoxicity of different natural and synthesized
flavonoids on human cancer cell lines
Compound
R
GI50 (lM)a
Human cell lines
HepG2 MCF-7 MOLT-4
1a
40-H
50.6
93.8
69.3
40.9
\0.1
–
–
50.7
\0.1
–
1b
40-Cl, 30-NO2
Discussion
1c
30,50-Dimethoxy 58.8
1d
30,40-Dimethoxy
40-OCH3
40-Cl
–
–
Eight synthetic flavones without hydroxyl substituents,
and having varying substitutions on the B-ring were syn-
thesized and subjected to determination of antitumor activity
on three cell lines, viz. HepG2, MCF-7, and MOLT-4. The
compounds were also tested to determine their ability to
inhibit lipid peroxidation. One of the synthesized com-
pounds, 1c (30,50-dimethoxyflavone), showed a high antitu-
mor activity against both MCF-7 and MOLT-4 cell lines.
Among the other compounds, 30-nitro substituted com-
pound, 40-nitro substituted compound, and 40-chloro-30-nitro
substituted compound i.e., 1h, 1g, and 1b, respectively,
showed appreciable activity against MCF-7 cell line. All
these four compounds showed activity better than chrysin.
The 30-nitro derivative (1h) also showed appreciable activ-
ity, higher than chrysin, against MOLT-4 cell lines. The
activity of most of the compounds against HepG2 cell lines
was very negligible. Inactivity against the HepG2 cell lines
was an unexpected result as the corresponding flavonol
previously synthesized in our laboratory showed good
activity against the HepG2 cell lines (Joshi et al., 2012).
Antioxidant activity testing produced some unexpected
results. It has been reported that the presence of phenolic
hydroxyl groups is the major structural feature associated with
antioxidant activity. None of the compounds in the present
study contained a phenolic hydroxyl group. The presence of a
2, 3-double bond and a carbonyl function at C-4 is reported to
be another structural feature of antioxidant activity (Rice-
Evans et al., 1996). This feature, however, is present in all the
1e
61.8
–
99.2
–
1f
–
1g
40-NO2
22.1
94.9
–
18.7
31
98.2
36
1h
30-NO2
Chrysin
69
58.6
\0.1
Adriamycin (ADR) standard
\0.1
\0.1
The symbol (–) indicates those compounds that inhibited cell growth less than
50 % at a 100 lM concentration
a
The molar drug concentrations required to cause 50 % growth inhibition
(GI50) were determined from dose–response curves. Results represent mean
values of at least three different experiments
with ice cold saline solution (0.15 M KCl), and homoge-
nized with tris buffer (pH 7.4) and centrifuged at 2000 rpm
for 10 min to get a clear supernatant. Then the supernatant
(0.5 ml) was mixed with 0.1 ml KCl (0.15 M), 0.4 ml Tris
buffer (pH 7.4), and test solution of different concentrations
(0–200 lg/ml in methanol). Lipid peroxidation was initiated
by adding 0.1 ml FeSO4 (15 mM) and 0.1 ml ascorbic acid
(6 mM). The homogenates were incubated at 37 °C for 1 h.
After incubation, 1 ml TCA (10 %) was added to the mix-
ture and the sample was centrifuged at 2000 rpm for 20 min.
Supernatant was removed and 1 ml thiobarbituric acid
(TBA) (0.8 %) was added to this fraction followed by
incubation at 90 °C for 20 min for development of colored
complex. After cooling, absorbance was measured at
532 nm and percent inhibition was calculated. Percent
inhibition value obtained at a concentration was expressed
Fig. 2 IC50 values for
inhibition of lipid peroxidation
by synthesized compounds
Compounds
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Androutsopoulos VP, Papakyriakou A, Vourloumis D, Tsatsakis AM,
Spandidos DA (2010) Dietary flavonoids in cancer therapy and
prevention: substrates and inhibitors of cytochrome P450 CYP1
enzymes. Pharmacol Therapeut 126:9–20
Bandele OJ, Osheroff N (2007) Bioflavonoids as poisons of human
topoisomerase IIa and IIb. Biochemistry 46:6097–6108
Bhagat S, Sharma R, Sawant DM, Sharma L, Chakraborti AK (2006)
LiOHꢀH2O as a novel dual activation catalyst for highly efficient
and easy synthesis of 1,3-diaryl-2-propenones by Claisen–
Schmidt condensation under mild conditions. J Mol Catal A
244:20–24
Cabrera M, Simoens M, Falchi G, Lavaggi ML, Piro OE, Castellano
EE, Vidal A, Azqueta A, Monge A, de Cerain AL (2007)
Synthetic chalcones, flavanones, and flavones as antitumoral
agents: biological evaluation and structure–activity relationships.
Bioorg Med Chem 15:3356–3367
Cardenas M, Marder M, Blank VC, Roguin LP (2006) Antitumor
activity of some natural flavonoids and synthetic derivatives on
various human and murine cancer cell lines. Bioorg Med Chem
14:2966–2971
Chumsri S, Howes T, Bao T, Sabnis G, Brodie A (2011) Aromatase,
aromatase inhibitors, and breast cancer. J Steroid Biochem
125:13–22
Dong H, Lin W, Wu J, Chen T (2010) Flavonoids activate pregnane X
receptor-mediated CYP3A4 gene expression by inhibiting
cyclin-dependent kinases in HepG2 liver carcinoma cells.
BMC Biochem 11:23
Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid antioxidants:
chemistry, metabolism and structure–activity relationships.
J Nutr Biochem 13:572–584
Hu M (2007) Commentary––bioavailability of flavonoids and poly-
phenols––call to arms. Mol Pharmaceut 4:803
HuangYT, HwangJJ, LeePP,KeFC, HuangJH,HuangCJ, Kandaswami
C, Middleton E, Lee MT (2009) Effects of luteolin and quercetin,
inhibitors of tyrosine kinase, on cell growth and metastasis-
associated properties in A431 cells overexpressing epidermal
growth factor receptor. Brit J Pharmacol 128:999–1010
Joshi UJ, Gadge AS, D’Mello P, Sinha R, Srivastava S, Govil G
(2012) Anti-inflammatory, antioxidant and anticancer activity of
quercetin and its analogues. Int J Pharm Biomed Res 2:
1756–1766
Kanadaswami C, Lee LTA, Lee PPH, Hwang JJ, Ke FC, Huang YT,
Lee MT (2005) The antitumor activities of flavonoids. In Vivo
19:895–909
Kang ZC, Tsai SJ, Lee H (1999) Quercetin inhibits benzo [a] pyrene-
induced DNA adducts in human Hep G2 cells by altering
cytochrome P-450 1A1 gene expression. Nutr Cancer 35:
175–179
synthesized compounds. Although the DPPH radical scav-
enging activity is the preliminary test done to evaluate the
antioxidant activity, this test requires a hydrogen donor in the
compounds under study. Since this feature is absent in the
synthesized compounds, we decided to evaluate the synthe-
sized compounds for their ability to inhibit lipid peroxidation.
The results revealed that compounds with one or more sub-
stituent/s on the B-ring cause inhibition of lipid peroxidation
comparable to chrysin. When the substituent/s was oneor more
methoxy groups, the inhibitory activity was shown to be higher
than chrysin. These results were unexpected since none of the
synthesized compounds contain an acidic H or a hydrogen
bond donor feature. The literature, however, reports lipid
peroxidation inhibitory activity of polymethoxylated flavo-
noids (Mora et al., 1990) and these have been reported as
inhibitors of non-enzymatic lipid peroxidation. It is also
reported (Heim et al., 2002) that the torsion angle between
B-ring and the rest of the molecule also affects the antioxidant
activity of the molecules. Planarity permits conjugation,
electron dislocation, and an increase in the radical scavenging
ability. This theory also explains why flavonols with a 3-OH
show a higher antioxidant ability than the corresponding
flavones. Takingthese reportsintoconsideration, the increased
antioxidant activity of the B-ring substituted flavones may be
explained as being due to both increased lipophilicity and thus
membrane partitioning ability, particularly of the methoxyf-
lavones. An additional hypothesis may be a favorable alter-
ation in the torsion angle of flavones upon substitution at the
B-ring, which results in an increase in the antioxidant activity.
Although most of the compounds showing some antitumor
activity possessed antioxidant activity equivalent or better
than chrysin, there was no correlation found between anti-
oxidant and antitumor activity of the synthesized compounds.
In conclusion, compound 1b, 1c, 1g, and 1h show
promising antitumor activity against MCF-7 and MOLT-4
cell lines, whereas methoxy substituent/s on the B-ring
were major determinants of the antioxidant activity in the
absence of the phenolic hydroxyl groups. Further work in
this area may be useful for designing molecules with better
antitumor activity and to understand the necessary struc-
tural features for the antioxidant activity of flavones.
Liu Y, Hu M (2002) Absorption and metabolism of flavonoids in the
caco-2 cell culture model and a perused rat intestinal model.
Drug Metab Dispos 30:370–377
Liu Y, Dai Y, Xun L, Hu M (2003) Enteric disposition and recycling
of flavonoids and ginkgo flavonoids. J Altern Complement Med
9:631–640
Lu YF, Santostefano M, Cunningham BDM, Threadgill MD, Safe S
(1996) Substituted flavones as aryl hydrocarbon (Ah) receptor
agonists and antagonists. Biochem Pharmacol 51:1077–1087
Mora A, Paya M, Rios JL, Alcaraz MJ (1990) Structure-activity
relationships of polymethoxyflavones and other flavonoids as
inhibitors of non-enzymic lipid peroxidation. Biochem Pharma-
col 40:793–797
Acknowledgments The authors thank the Indian National Science
Academy and University of Mumbai for financial support. The sup-
port by Dr. A. Juvekar of ACTREC and Ms. Mamta Joshi and
Mr. Devidas of National Facility for High Field NMR located at TIFR
is gratefully acknowledged.
References
Ng SP, Wong KY, Zhang L, Zuo Z, Lin G (2004) Evaluation of the
first-pass glucuronidation of selected flavones in gut by Caco-2
monolayer model. J Pharm Pharm Sci 8:1–9
Okaha H (1979) Assay for lipid peroxide in animal tissue by
thiobarbituric acid reaction. Anal Biochem 95:351–358
Alvarez AI, Real R, Perez M, Mendoza G, Prieto JG, Merino G
(2009) Modulation of the activity of ABC transporters
(P-glycoprotein, MRP2, BCRP) by flavonoids and drug response.
J Pharm Sci 99:598–617
123

Med Chem Res (2013) 22:4293–4299
4299
Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB (2010) Oxidative
stress, inflammation, and cancer: how are they linked? Free
Radic Bio Med 49:1603–1616
Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant
activity relationships of flavonoids and phenolic acids. Free
Radic Bio Med 20:933–956
Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay
for cytotoxicity screening. Nat Protoc 1:1112–1116
Vidya Priyadarsini R, Senthil Murugan R, Maitreyi S, Ramalingam K,
Karunagaran D, Nagini S (2010) The flavonoid quercetin
induces cell cycle arrest and mitochondria-mediated apoptosis
in human cervical cancer (HeLa) cells through p53 induction and
NF-kB inhibition. Eur J Pharmacol 649:84–91
Xie F, Lang Q, Zhou M, Zhang H, Zhang Z, Zhang Y, Wan B, Huang
Q, Yu L (2012) The dietary flavonoid luteolin inhibits aurora B
kinase activity and blocks proliferation of cancer cells. Eur J
Pharm Sci 46:388–396
Zhang L, Lin G, Chang Q, Zuo Z (2005) Role of intestinal first-pass
metabolism of baicalein in its absorption process. Pharmaceut
Res 22:1050–1058
Zhao Q, Wang J, Zou MJ, Hu R, Zhao L, Qiang L, Rong JJ, You QD,
Guo QL (2010) Wogonin potentiates the antitumor effects of low
dose 5-fluorouracil against gastric cancer through induction of
apoptosis by down-regulation of NF-kappaB and regulation of its
metabolism. Toxicol Lett 197:201–210
Voigt W (2005) Sulforhodamine B assay and chemosensitivity.
Methods Mol Med 110:39–48
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