Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2

Article abstract of DOI:10.1016/j.ejmech.2013.06.035

Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting molecules with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a solution to overcome MDR.We synthesized a series of flavones, 7,8-benzofl avones and 5,6-benzo flavones with varying substituents at positions 3, 3′ and 4′ of the (benzo)fl avone structure. All synthesized compounds were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compounds were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addition most active compounds were investigated for their cytotoxicity. It was observed that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH₃ substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3′,4′-OCH₃ on phenyl ring lead to increase in activity as compared to other substituents. Compound 24, a 7,8-benzoflavone derivative was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concentrations.

Full text of DOI:10.1016/j.ejmech.2013.06.035

European Journal of Medicinal Chemistry 67 (2013) 115e126
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of flavones and benzoflavones as
inhibitors of BCRP/ABCG2
*
Kapil Juvale, Katja Stefan, Michael Wiese
Pharmaceutical Institute, University of Bonn, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance
Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to
confer MDR in cancer cells by transporting molecules with amphiphilic character out of the cells using
energy from ATP hydrolysis. Inhibiting BCRP can be a solution to overcome MDR. We synthesized a series
of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 30 and 40 of
the (benzo)flavone structure. All synthesized compounds were tested for BCRP inhibition in Hoechst
33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compounds
were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1
(MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In
addition most active compounds were investigated for their cytotoxicity. It was observed that in most
cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found
that presence of a 3-OCH3 substituent leads to increase in activity in comparison to presence of OH or no
substitution at position 3. Also, it was found that presence of 30,40-OCH3 on phenyl ring lead to increase in
activity as compared to other substituents. Compound 24, a 7,8-benzoflavone derivative was found to be
most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher
concentrations.
Received 31 October 2012
Received in revised form
12 June 2013
Accepted 13 June 2013
Available online 25 June 2013
Keywords:
Flavonoids
Benzoflavones
Breast cancer resistance protein (BCRP)
P-gp
MRP1
Multidrug resistance (MDR)
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
[3e5]. Until now 48 ABC transporters have been identified in
humans.
Cancer is the main cause of millions of deaths worldwide. Since
decades chemotherapy has been a major form of the treatment for
different types of cancers. Unfortunately, the majority of cancers
are either resistant to chemotherapy or acquire multidrug resis-
tance (MDR) during treatment. MDR is an acquired drug resistance
by tumour cells that consists of simultaneous emergence of cellular
resistance to toxic action of chemotherapeutic drugs originally used
and to other chemicals having different chemical structure and
mechanism of action. As a result of MDR, chemotherapeutic agents
fail to target the tumour cells and cancer becomes untreatable by
chemotherapy [1,2].
One of the mechanisms by which tumours develop multidrug
resistance is over expression of efflux transport proteins, especially
certain ATP-binding cassette (ABC) transporters in the plasma
membrane of cancer cells. ABC transporters utilize energy obtained
from hydrolysis of ATP to efflux chemically unrelated compounds
P-glycoprotein (P-gp) and the multidrug resistance associated
protein 1 (MRP1) belonging to subfamily ABCC have been shown to
confer resistance to a broad spectrum of chemotherapeutic agents.
More recently BCRP (breast cancer resistance protein) which is also
known as ABCG2 has been discovered and proved to cause resis-
tance in tumour cells too.
Although BCRP was first cloned in doxorubicin resistant MCF-7
breast cancer cells [6], later it has been also found to confer resis-
tance to mitoxantrone (hence named MXR) [7] and subsequently it
was found in the placenta (hence named ABCP) [8]. BCRP is an ABC
transporter protein consisting of 655 amino acids and having a
molecular weight of 72 kDa. It is a half transporter containing a
single NH2-terminal nucleotide binding domain (NBD) followed by
six transmembrane domains (TMD). It has been proposed that, to
achieve its functionality, BCRP needs to be homo- or hetero-
dimerized [6,7]. BCRP has been found to be present in many normal
tissues other than in tumour cells, including apical membrane of
placental syncytiotrophoblasts, endocrine cells of the pancreas,
epithelial cells in the small intestine and colon, liver caniculi,
bloodebrain and bloodetestis barrier, gallbladder epithelium and
* Corresponding author. Tel.: þ49 228 735213; fax: þ49 228 737929.
E-mail address: mwiese@uni-bonn.de (M. Wiese).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.035

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K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
Scheme 1. General scheme for the synthesis of target compounds. Reagents and conditions: a) EtOH, NaOH or LiOH, rt, 5e72 h; b) DMSO, I2, reflux, 7e8 h; c) EtOH, 25% H2O2, NaOH,
rt, 24e48 h; d) acetone, CH3I, K2CO3, reflux, 24 h.
stem cells [8e10]. In normal tissues BCRP protects the body from
various endogenous and exogenous toxins.
accumulation assays. The compounds were also investigated for
their effect on P-gp and MRP1. Compound 24, a 7,8-benzoflavone
bearing three methoxy substituents at positions 3, 30,40 was
found to be most potent having an inhibitory concentration only
about 2 fold less than Ko143 which is the most potent specific BCRP
inhibitor [26], while being almost 3 fold more potent than the
unsubstituted 7,8-benzoflavone.
Several substrates of BCRP have been identified, which include
anticancer drugs such as mitoxantrone [11], podophyllotoxins
etoposid and teniposid, flavopiridol [12], topotecan, irinotecan [13]
and its active metabolite SN-38. To overcome MDR acquired by over
expression of BCRP several efforts have been made by investigating
inhibitors of BCRP. These structurally unrelated compounds include
naturally occurring flavonoids [14e17], broad spectrum inhibitors
like tariquidar [18] and elacridar, chromons [19] and methoxy-
stilbenes [20]. Several research groups have investigated chal-
cones (precursor of flavonoids) for their inhibitory effect [21e24].
Fumitremorgin C was found to be a very potent inhibitor of BCRP
[13], but its use was limited due to neurotoxicity. Its non-toxic
analogue Ko143 is the most potent and selective inhibitor of
BCRP known today.
Recently a flavonoid, the 7,8-benzoflavone was identified as very
potent inhibitor of BCRP [15,25]. To further investigate benzo-
flavones for their potential to inhibit BCRP, we synthesized several
7,8- and 5,6-benzoflavones bearing varying substitutions at posi-
tions 3, 30 and 40. Also several substituted synthetic flavones were
investigated to allow comparison of their inhibitory potential with
that of the benzoflavones. These compounds were checked for their
inhibitory effect on BCRP in Hoechst 33342 and pheophorbide A
2. Result and discussion
2.1. Chemistry
In the current study we synthesized and investigated several
flavones (1e13), 7,8-benzoflavones (14e25) and 5,6-benzoflavones
(26e34). Flavones (1e9) and both type of benzoflavones were
synthesized from their precursor chalcones and benzochalcones.
The precursors were synthesized by ClaiseneSchmidt condensa-
tion using substituted acetophenones and benzaldehydes in pres-
ence of NaOH or LiOH as base in ethanol as reported earlier [23].
The synthesis of the studied compounds is depicted in Scheme 1
and Scheme 2. Synthesis of benzoflavones occurred via one of
two routes. The first route involved cyclization of a precursor
benzochalcone to the corresponding benzoflavone in presence of
iodine in DMSO to give benzoflavones with no substituent at
Scheme 2. General scheme for the synthesis of compounds 10e13. Reagents and conditions: a) acetone, CH3I, K2CO3, reflux, 24 h; b) acetone/water (2:1), dimethyl sulphate, KOH,
reflux, 3 h.

K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
117
Table 1
Synthesized flavonoids and their inhibitory potencies against MDCK BCRP cells in Hoechst 33342 assay and pheophorbide A assay. Data are expressed as mean ꢀ SD (n ¼ 3).
Flavons (1-13)
0
0
0
Compound
R3
R5
R7
R2
R3
R4
Hoechst 33342 IC50 ꢀ SD (
m
M)
Pheophorbide A IC50 ꢀ SD (
mM)
1
2
3
4
5
6
7
8
OH
OH
OH
OH
H
H
H
H
OCH3
H
H
H
H
OCH3
OCH3
OH
OH
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
1.01 ꢀ 0.10
12.4 ꢀ 0.47
1.21 ꢀ 0.12
7.74 ꢀ 0.35
1.18 ꢀ 0.29
1.18 ꢀ 0.18
0.822 ꢀ 0.169
5.98 ꢀ 0.45
0.540 ꢀ 0.079
3.27 ꢀ 0.15
1.77 ꢀ 0.14
8.95 ꢀ 0.65
1.28 ꢀ 0.17
6.50 ꢀ 0.58
1.34 ꢀ 0.19
2.14 ꢀ 0.31
1.88 ꢀ 0.24
5.94 ꢀ 0.74
0.570 ꢀ 0.093
3.89 ꢀ 0.16
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
H
OCH3
H
9
OCH3
OCH3
OCH3
OCH3
OCH3
10
11
12
13
OCH3
H
OCH3
*n.e. ¼ no inhibitory effect up to 10
mM.
position 3. In second route chalcones or benzochalcones were
converted to 3-OH (benzo)flavones using the AlgareFlynneOya-
mada reaction [27], by treatment with H2O2 and NaOH for 24 h. 3-
OH flavones or benzoflavones were further converted to 3-OCH3
flavones or benzoflavones using methyl iodide and potassium
carbonate in acetone. Compounds 12 and 13, the tetramethoxy
derivatives of quercetin and morin were synthesized by a method
described earlier and purified by column chromatography [27]. All
the synthesized compounds are listed in Tables 1 and 2.
for compound 24 compared to the standard Ko143. The inhibitory
concentration of all the compounds is given in Tables 1 and 2.
In case of flavones, two types of compounds were investigated
1) those having hydroxy substituents at position 3 of the flavone
Table 2
Synthesized 7,8- and 5,6-benzoflavones and their inhibitory potencies against
MDCK BCRP cells in Hoechst 33342 assay and Pheophorbide A assay. Data are
expressed as mean ꢀ SD (n ¼ 3).
All synthesized compounds were characterized by 1H NMR, 13
C
NMR and elemental analysis. Characterization and purity data of
precursor benzochalcones have been reported previously [23].
2.2. Biological investigation
The synthesized compounds were investigated for their BCRP
inhibition ability by measuring accumulation of Hoechst 33342 and
pheophorbide A in MDCK BCRP cells. All compounds were addi-
tionally checked for their P-gp and MRP1 inhibition in calcein AM
assay. Cytotoxicity of representative most active compounds was
assessed by MTT-cytotoxicity assay.
7,8-Benzoflavones (14-25)
5,6-benzoflavones (26-34)
0
0
Compound R3
R3
R4
Hoechst 33342
IC50 ꢀ SD ( M)
Pheophorbide A
IC50 ꢀ SD ( M)
m
m
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Ko143
H
H
H
H
OH
OH
H
H
H
1.31 ꢀ 0.12
1.40 ꢀ 0.18
1.29 ꢀ 0.09
1.13 ꢀ 0.19
0.457 ꢀ 0.124
1.59 ꢀ 0.14
4.97 ꢀ 0.88
1.52 ꢀ 0.08
17.9 ꢀ 0.29
2.31 ꢀ 0.04
3.07 ꢀ 0.38
0.468 ꢀ 0.034
4.01 ꢀ 0.77
6.05 ꢀ 1.62
1.69 ꢀ 0.07
0.458 ꢀ 0.053
13.6 ꢀ 2.40
6.12 ꢀ 1.54
4.84 ꢀ 0.99
12.5 ꢀ 0.65
8.02 ꢀ 1.39
5.25 ꢀ 0.67
0.354 ꢀ 0.042
OCH3 1.28 ꢀ 0.07
OCH3 OCH3 1.23 ꢀ 0.08
2.2.1. Hoechst 33342 and pheophorbide A assay to determine BCRP
inhibition
OCH3
H
H
F
H
0.522 ꢀ 0.083
2.89 ꢀ 0.73
Hoechst 33342 and pheophorbide A are substrates of BCRP and
have been used to determine the effect of BCRP inhibitors on their
accumulation in BCRP overexpressing cells. All the compounds
were investigated in BCRP overexpressing MDCK BCRP cells. The
accumulation of Hoechst 33342 and pheophorbide A in non-BCRP
expressing MDCK cells was also determined and was considered
as maximum accumulation, as a result of absence of BCRP
expression.
In the current study two types of flavonoids were investigated: A)
flavones and B) 7,8- and 5,6-benzoflavones. For the calculation of IC50
values of these compounds, concentration response curves were
generated from plots of averages of fluorescence values obtained
in Hoechst 33342 and pheophorbide A assays against the logarithmic
concentrations of the inhibitor. Fig. 1 depicts representative con-
centrationeresponse curves obtained in the Hoechst 33342 assay
OCH3 6.93 ꢀ 1.58
OH OCH3 OCH3 0.724 ꢀ 0.049
OH OCH3
F
H
22.5 ꢀ 4.90
2.71 ꢀ 0.41
OCH3
OCH3
H
H
OCH3 1.06 ꢀ 0.26
OCH3 OCH3 OCH3 0.426 ꢀ 0.019
OCH3 OCH3
F
H
2.44 ꢀ 0.25
8.32 ꢀ 0.61
H
H
H
OH
OH
H
H
OCH3 2.46 ꢀ 0.15
OCH3 OCH3 0.590 ꢀ 0.064
H
H
H
11.0 ꢀ 2.98
OCH3 8.93 ꢀ 2.37
OH OCH3 OCH3 6.12 ꢀ 0.35
OCH3
OCH3
H
H
H
11.1 ꢀ 1.37
OCH3 3.07 ꢀ 0.04
OCH3 OCH3 OCH3 4.27 ꢀ 0.64
e
0.215 ꢀ 0.017

118
K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
125
100
75
50
25
0
O
O
H
N
6.5
6.0
O
Ko 143
NH
O
N
H
O
Ko143
5.5
O
O
O
O
5.0
O
Compound 24
4.5
4.5
5.0
5.5
6.0
6.5
-
∞
-7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5
Concentration (log M)
pIC50 in Hoechst 33342 assay
Fig. 2. Scatterplot of pIC50 values for ABCG2 inhibition obtained in the Hoechst 33342
assay and the pheophorbide A assay. Each point indicates mean of IC50 values obtained
in three independent experiments and error bars indicate standard deviation. The
squared correlation coefficient is r2 ¼ 0.89.
Fig. 1. Representative concentrationeresponse curves of compound 24 (open circles,
M) obtained in
IC50 ¼ 0.43 ꢀ 0.02
m
M) and Ko143 (closed squares, IC50 ¼ 0.22 ꢀ 0.02
m
the Hoechst 33342 assay. Each data point shown represents the average of 3 inde-
pendent experiments and error bars indicate standard deviation.
assays as shown in Fig. 2. Also the Hill slopes of the doseeresponse
curves were similar and not significantly different from one, except
for a few experiments. This indicates that the inhibitory activity of
these compounds is not substrate specific. From the activity data of
all compounds, it was found that 7,8-benzoflavones are more active
as compared to flavones, while 5,6-benzoflavones are the least
active class with exception of compound 28, which was found to be
a potent inhibitor of BCRP. The effect of different substituents on
BCRP activity is summarized in Fig. 3.
To determine the type of interaction of flavones and benzo-
flavones with BCRP, enzyme kinetic studies were performed with
pheophorbide A as substrate and compounds 6 and 24 as inhibitors.
Fig. 4 presents the LineweavereBurk plots for both compounds,
which shows that both studied compounds are non-competitive
inhibitors of BCRP in presence of pheophorbide A as substrate.
nucleus (1e5) and 2) those having methoxy substituents at the
same position (6e13). Generally compounds possessing a 3-OH
substituent were very less active or even inactive when compared
to compounds bearing a 3-OCH3 substituent as can be seen from
Table 1. But this difference in activity was much smaller for com-
pounds bearing an unsubstituted phenyl ring at position 2 of
benzoflavones, as is shown by the negligible differences of IC50
values for compounds 18 and 22 or 29 and 32. The results obtained
from our functional assays are in accordance with the structure
activity relationship proposed earlier by several investigators
[15,16,28]. It was also observed that presence of methoxy groups on
side ring of flavones lead to an increase in activity. Presence of 30,40-
OCH3 substitution was found to be optimum for BCRP inhibition.
The comparison of IC50 values for compounds 10e13 shows the
importance of the presence of a 5-OH substituent (in compounds
12 and 13) for BCRP inhibition. Replacement of 5-OH with 5-OCH3
leads to a decrease in activity (compare compounds 10 and 11).
Compound 12, bearing 3-OCH3 and 5-OH substituents on the
flavone scaffold was found to be most potent in the flavone series
2.2.2. Calcein AM assay to determine P-gp and MRP1 inhibition
All the compounds were screened for their P-gp and MRP1 in-
hibition to investigate selectivity towards BCRP. Screening was
performed at 10 mM final inhibitor concentration using the calcein
AM assay. Cyclosporine A, a P-gp and MRP1 inhibitor, was used as
standard. Most compounds showed no or very weak inhibition of P-
gp and MRP1. Fig. 5 illustrates the effect of the compounds on calcein
accumulation in P-gp overexpressing A2780 adr cells. Fig. 6 shows
the effect of the compounds on accumulation of calcein in MRP1
overexpressing 2008 MRP1 cells. In case of flavones only compound
with an IC50-value of 0.54 mM.
The second series includes 7,8-benzoflavones and 5,6-
benzoflavones. Compound 14, the unsubstituted benzoflavone has
been shown to be a potent inhibitor of BCRP by Zhang et al. [15] and
was included in the current study for comparison purpose. As can
be seen from Table 2 also in case of 7,8- and 5,6-benzoflavones the
presence of a 3-OH substituent (in compounds 18e20 and 29e31)
led to a decrease in activity. While, particularly in case of 7,8-
benzoflavones the presence of a 3-OCH3 group (compounds 22e
24) led to a strong increase in BCRP inhibition. As for the flavones
30,40-OCH3 substituted benzoflavones were more potent BCRP in-
hibitors as compared to unsubstituted compounds and those
bearing a 40-OCH3 substituent only. Compound 17 having a 30-
OCH3, 4-F substitution was found to be most active
(IC50 ¼ 0.52 ꢀ 0.08
mM in Hoechst 33342 assay) amongst the
benzoflavones having no substitution at position 3. However, other
derivatives of this compound (21 and 25) showed less activity. From
Table 2, it can be seen that 5,6-benzoflavones are less potent as
compared to 7,8-benzoflavones with exception of compound 28
which showed strong BCRP inhibition (IC50 ¼ 0.59 ꢀ 0.06
mM in
Hoechst 33342 assay).
The BCRP inhibition analysis done by two different assays gave
Fig. 3. Effect of different substituents on activity of flavonoids. Dotted lines indicate
7,8- or 5,6-benzo substitution for benzoflavones.
comparable results with good correlation (r2 ¼ 0.89) between both

K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
119
Fig. 4. LineweavereBurk plot for BCRP inhibitors 6 (panel A) and 24 (panel B) at various concentrations with the BCRP substrate pheophorbide A. Inhibitor concentrations used
were, 0 M (open circles), 0.031 M (closed triangles), 0.1 M (closed squares), 0.18 M (open triangles), 0.31 M (closed circles) and 0.56 M (open squares).
m
m
m
m
m
m
10 was found to show substantial effect on both P-gp and MRP1.
While in case of benzoflavones, only 7,8-benzoflavones showed
considerable effect. Compounds showing more than 25% of
response when compared to control (non P-gp or MRP1 expressing
sensitive cells) were further investigated with a range of different
concentrations to calculate their IC50 values. P-gp and MRP1 inhi-
bition data of selected compounds are given in Table 3. Compound
16, a 7,8-benzoflavone bearing 30,40-OCH3 substituents was found to
be most potent and active for both P-gp and MRP1 inhibition. It was
even more potent than cyclosporine A in MRP1 inhibition. (Fig. 6)
Compound 24, the most active flavonoid in the current study
showed very weak P-gp and MRP1 inhibition with almost 50 times
less activity compared to BCRP inhibition.
of SN-38, the active metabolite of irinotecan, and Hoechst 33342
was evaluated in MDCK BCRP cells. The cytotoxic effect of both drugs
was determined in the absence and presence of 5 mM and 10 mM
concentrations of the selected compounds. Both compounds were
found to be able to reverse the resistance of the BCRP expressing cell
lines, proving their functional efficacy. Fig. 8 shows representative
doseeresponse curves of SN-38 in presence of compound 12 and of
Hoechst 33342 in presence of compound 24. The shift in the dosee
response curve of MDCK BCRP in presence of these compounds to-
wards lower concentrations indicates sensitization of MDCK BCRP
cells to cytotoxicity of SN-38 and Hoechst 33342.
3. Conclusion
2.2.3. MTT cytotoxicity assay
Naturally occurring as well as synthetic flavonoids have been
shown to inhibit BCRP and P-gp in several studies. In the current
study we synthesized and investigated several 7,8-benzoflavones,
5,6-benzoflavones along with comparison to multisubstituted fla-
vones for their BCRP inhibition potential. All the compounds were
evaluated using two different substrates of BCRP. From the activity
data obtained in the Hoechst 33342 and pheophorbide A assays we
were able to propose the effect of substituents as well as the effect
of attachment of a benzo-ring at position 7,8 or 5,6 of the flavone
moiety. 7,8-benzoflavones were found to be more potent than fla-
vones and 5,6-benzoflavones. The inhibitory effect of these com-
pounds was further confirmed by evaluating the influence of
selected compounds on the cytotoxicity of SN-38 and Hoechst
33342 in BCRP overexpressing cells. We also investigated all
To investigate the cytotoxicity of flavones and benzoflavones,
the most potent compounds (4, 12, 24 and 28) from each class were
selected. These compounds were studied for their cytotoxic effect
in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) cytotoxicity assay. For this purpose both MDCK BCRP
and sensitive MDCK cells were used. Very little toxicity could be
observed at 100
icity at lower concentrations. For most of the compounds GI50 could
be estimated to be more than 100 M and only for compound 12
GI50 was estimated to be around 85 M (Fig. 7).
mM and none of the compound showed cytotox-
m
m
The MTT assay was also used to investigate the effect of selected
flavones (12) and benzoflavones (24) on the antiproliferative effect
of cytotoxic agents in presence of the BCRP transporter. Cytotoxicity
Fig. 5. Effect of flavones and benzoflavones on accumulation of calcein in P-gp overexpressing A2780 adr cells. The compounds were investigated at 10
Cyclosporine A (CsA, 10 M) was used as a standard. Response in absence of any compound was used as negative control. Data are expressed as response in percentage of control
(accumulation of calcein in non P-gp expressing sensitive A2780 cells) and presented as the mean ꢀ SD of three independent experiments.
mM final concentration.
m

120
K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
Fig. 6. Effect of flavones and benzoflavones on accumulation of calcein in MRP1 overexpressing 2008 MRP1 cells. The compounds were investigated at 10
Cyclosporine A (CsA, 10 M) was used as a standard. Response in absence of any compound was used as negative control. Data are expressed as response in percentage of control
(accumulation of calcein in non MRP1 expressing sensitive 2008 cells) and presented as the mean ꢀ SD of three independent experiments.
mM final concentration.
m
compounds for their P-gp and MRP1 inhibition. Most of the com-
pounds were found to be selective towards BCRP. Cytotoxicity of
most active compounds was determined in MTT cytotoxicity assay.
Compound 24 was found to be a non-toxic potent BCRP inhibitor
described earlier [23]. Synthesis of final flavones and benzoflavones
was carried out as reported earlier with small modifications [27,29].
All chemicals were purchased from Acros Organics, Alfa Aesar or
SigmaeAldrich. During the synthesis reaction progress was moni-
tored using analytical thin layer chromatography (TLC) on silica gel
plates (Silica Gel 60 F254 from Merck). Purity of all compounds was
confirmed by NMR and elemental analysis. NMR spectra were
recorded in DMSO-d6 or CDCl3. 1H NMR spectra were obtained on
Bruker Advance 500 (500 MHz); 13C NMR on Bruker Advance 500
having 50 times selectivity towards BCRP. Compound 16,
a
substituted 7,8-benzoflavone could be a good broad spectrum MDR
modulator as it produced low IC50 values for all BCRP, P-gp and
MRP1.
4. Experimental section
(126 MHz); chemical shifts are expressed in d values (ppm) using
the solvent peak as an internal standard; multiplicity of resonance
peaks is indicated as singlet (s), doublet (d), triplet (t), quartet (q)
and multiplet (m). The 13C signals were assigned with the aid of
distortion less enhancement by polarization transfer (DEPT) and
attached proton test (APT), the J values are in Hertz. Elemental
analyses were performed on a Vario EL of Elementar. Found values
4.1. Chemistry
The general synthetic route to target flavones and benzoflavones
is depicted in Scheme 1 and Scheme 2. Syntheses and character-
ization of precursor chalcones and benzochalcones have been
Fig. 7. Toxicity of selected compounds was determined in the MTT assay using MDCK BCRP and sensitive MDCK cells. Compounds 4, 12, 24 and 28 were investigated up to 100 mM
concentration, for 72 h. Open circles indicate sensitive MDCK cells and closed squares indicate MDCK BCRP cells. Data are expressed as mean ꢀ SD obtained from least three
independent experiments.

K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
121
Table 3
DMSO)
d 176.92, 162.00, 152.86, 135.55, 131.82, 131.34, 129.63,
Inhibitory potencies of selected flavonoids using A2780 adr and 2008 MRP1 cells in
calcein AM assay. Data are expressed as mean ꢀ SD (n ¼ 3). Cyclosporine A was used
as standard.
129.35, 128.34, 127.78, 126.46, 125.47, 123.60, 122.36, 120.08, 119.74,
108.25. Anal. Calcd for C19H12O2: C, 83.81; H, 4.44; O, 11.75. Found:
C, 83.72; H, 4.52. PHPLC (MeOH/H2O, 35/65) 99.9%, k0 ¼ 3.85.
Compound
A2780 adr IC50 ꢀ SD (
m
M)
2008 MRP1 IC50 ꢀ SD (
mM)
10
11
14
15
16
17
20
22
23.4 ꢀ 2.78
27.1 ꢀ 3.53
24.6 ꢀ 3.98
19.7 ꢀ 2.82
4.00 ꢀ 0.22
n.a.
26.35 ꢀ 2.65
4.2.2. 2-(4-Methoxyphenyl)-4H-benzo[h]chromen-4-one (15)
Synthesized from (E)-1-(1-hydroxynaphthalen-2-yl)-3-(4-
methoxyphenyl)prop-2-en-1-one, yellow solid, 72% yield. 1H NMR
n.a.a
11.3 ꢀ 2.49
9.86 ꢀ 1.05
1.76 ꢀ 0.04
15.1 ꢀ 3.83
n.a.
27.5 ꢀ 4.44
19.0 ꢀ 2.48
21.8 ꢀ 2.74
2.81 ꢀ 0.62
(500 MHz, DMSO)
d 8.70e8.66 (m, 1H), 8.22e8.18 (m, 2H), 8.13e
8.09 (m, 1H), 8.00 (d, J ¼ 8.7 Hz, 1H), 7.93 (d, J ¼ 8.6 Hz, 1H), 7.84e
7.80 (m, 2H), 7.18e7.15 (m, 2H), 7.09 (s, 1H), 3.88 (s, 3H). 13C NMR
4.27 ꢀ 0.59
n.a
(126 MHz, DMSO)
d 176.83, 162.22, 152.78, 135.54, 129.60, 128.38,
23
24
n.a
128.35, 127.80, 125.37, 123.63, 123.52, 122.37, 120.17, 119.68, 114.83,
106.80, 55.65. Anal. Calcd for C20H14O3: C, 79.46; H, 4.67; O, 15.88.
Found: C, 79.33; H, 4.73. PHPLC (MeOH/H2O, 35/65) 99.56%, k0 ¼ 4.48.
21.9 ꢀ 2.33
Cyclosporine A
0.99 ꢀ 0.08
a
n.a. ¼ not active, showing a response of less than 25% of control in screening at
mM concentration.
10
4.2.3. 2-(3,4-Dimethoxyphenyl)-4H-benzo[h]chromen-4-one (16)
Synthesized from (E)-3-(3,4-dimethoxyphenyl)-1-(1-hydroxy-
naphthalen-2-yl)prop-2-en-1-one, yellowish brown solid, 64%
were all within ꢀ0.4% of the theoretical values except when indi-
cated. All the compounds were analyzed for purity by HPLC using
reversed phase column Eurospher II 100-5 C18H (50 ꢁ 4 mm)
connected with a pre-column Vertex-Plus Eurospher II 100-5 C18H
(5 ꢁ 4 mm) obtained from Knauer, Germany. Eluent flow-rate was
maintained at 1.5 ml/min. The capacity factor (k0) is given for each
compound.
yield. 1H NMR (500 MHz, DMSO)
d 8.66e8.62 (m,1H), 8.12e8.08 (m,
1H), 7.99 (d, J ¼ 8.6 Hz, 1H), 7.92 (d, J ¼ 8.6 Hz, 1H), 7.85 (dd, J ¼ 8.5,
2.2 Hz, 1H), 7.83e7.79 (m, 2H), 7.66 (d, J ¼ 2.2 Hz, 1H), 7.18 (s, 2H),
3.93 (s, 3H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO)
d 176.89,
162.22, 152.79,152.06,149.27, 135.55,129.58, 128.39,127.84, 125.35,
123.63, 122.31, 120.16, 119.98, 119.67, 112.01, 109.58, 107.16, 56.00,
55.86. Anal. Calcd for C21H16O4: C, 75.89; H, 4.85; O, 19.26. Found: C,
75.56; H, 5.02. PHPLC (MeOH/H2O, 35/65) 97.97%, k0 ¼ 4.06.
4.2. General procedure for synthesis of 7,8- and 5,6-benzoflavones
4.2.4. 2-(4-Fluoro-3-methoxyphenyl)-4H-benzo[h]chromen-4-
one (17)
The appropriate benzochalcone (2 mmol), I2 (0.14 g, 0.55 mmol)
was dissolved in DMSO (10 ml). The solution was refluxed for 7e
8 h. After completion of the reaction as indicated by TLC, the so-
lution was cooled to room temperature and poured onto crushed
ice. 1 N HCl solution (50 ml) was added and stirred for 15 min. This
solution was extracted with ethyl acetate and washed 3 times
with brine. The organic phase was evaporated under reduced
pressure to get crude product. It was further purified by column
chromatography using silica gel as solid phase and dichlor-
omethane:methanol (9.5:0.5) as eluent to get compounds 14e17
and 26e28.
Synthesized from (E)-3-(4-fluoro-3-methoxyphenyl)-1-(1-
hydroxynaphthalen-2-yl)prop-2-en-1-one, pale yellow solid, 76%
yield. 1H NMR (500 MHz, DMSO)
d 8.70e8.64 (m,1H), 8.12e8.04 (m,
3H), 7.98 (d, J ¼ 8.7 Hz, 1H), 7.94e7.89 (m, 1H), 7.83e7.78 (m, 2H),
7.40e7.33 (m, 1H), 7.15 (s, 1H), 3.95 (s, 3H). 13C NMR (126 MHz,
DMSO)
d 176.83, 160.98, 152.80, 152.71, 150.76, 135.57, 129.67,
128.36, 127.84, 125.51, 123.99, 123.74, 123.57, 122.53, 120.10, 119.69,
114.33, 113.98, 107.63, 56.47. Anal. Calcd for C20H13FO3: C, 74.99; H,
4.09; O, 14.98. Found: C, 74.68; H, 4.25. PHPLC (MeOH/H2O, 35/65)
96.18%, k0 ¼ 4.02.
4.2.1. 2-Phenyl-4H-benzo[h]chromen-4-one (14)
4.2.5. 3-Phenyl-1H-benzo[f]chromen-1-one (26)
Synthesized
phenylprop-2-en-1-one, beige solid, 68% yield. 1H NMR
(500 MHz, DMSO) 8.63e8.57 (m, 1H), 8.21e8.15 (m, 2H), 8.07e
from
(E)-1-(1-hydroxynaphthalen-2-yl)-3-
Synthesized from (E)-1-(2-hydroxynaphthalen-1-yl)-3-phenyl-
prop-2-en-1-one, yellowish solid, 59% yield. 1H NMR (500 MHz,
d
DMSO)
d
9.98e9.90 (m, 1H), 8.35 (d, J ¼ 9.0 Hz, 1H), 8.16e8.12 (m,
8.02 (m, 1H), 7.95 (d, J ¼ 8.7 Hz, 1H), 7.87 (d, J ¼ 8.6 Hz, 1H), 7.78e
7.74 (m, 2H), 7.63e7.58 (m, 3H), 7.13 (s, 1H). 13C NMR (126 MHz,
2H), 8.11e8.07 (m, 1H), 7.87 (d, J ¼ 9.0 Hz, 1H), 7.77 (ddd, J ¼ 8.5, 6.9,
B
A
100
100
50
0
50
0
-7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
-∞
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-∞
SN-38 Concentration (log M)
Hoechst 33342 Concentration (log M)
Fig. 8. Representative shift in doseeresponse curves of SN-38 and Hoechst 33342 cytotoxicity. Panel A depicts the effect of compound 12 on SN-38 cytotoxicity in BCRP cells and
panel B shows the effect to compound 24 on cytotoxicity of Hoechst 33342. Both compounds were investigated at 5 mM (open circles) and 10 mM (closed circles) concentrations.
BCRP cells without inhibitor (open triangles) are more resistant than sensitive cells (closed squares).

122
K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
1.5 Hz, 1H), 7.67 (ddd, J ¼ 8.1, 6.9, 1.2 Hz, 1H), 7.63e7.58 (m, 3H), 7.18
(s, 1H). 13C NMR (126 MHz, DMSO)
179.32, 160.38, 157.15, 135.90,
4.3.3. 3-Hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-
4-one (3)
d
131.78, 130.88, 130.51, 129.81, 129.29, 129.17, 128.71, 126.72, 126.31,
126.21, 118.28, 116.37, 109.90. Anal. Calcd for C19H12O2: C, 83.81; H,
4.44; O, 11.75. Found: C, 83.72; H, 4.52. PHPLC (MeOH/H2O, 35/65)
97.06%, k0 ¼ 3.72.
Synthesized from (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one, pale yellow solid, 66% yield. 1H
NMR (500 MHz, DMSO)
J ¼ 8.7 Hz, 1H), 7.02e6.99 (m, 2H), 6.48 (dt, J ¼ 5.9, 2.4 Hz, 2H), 3.81
(s, 3H), 3.78 (s, 3H). 13C NMR (126 MHz, DMSO)
171.97, 167.12,
d 12.58 (s, 1H), 7.90e7.86 (m, 2H), 7.69 (d,
d
4.2.6. 3-(4-Methoxyphenyl)-1H-benzo[f]chromen-1-one (27)
Synthesized from (E)-1-(2-hydroxynaphthalen-1-yl)-3-(4-
methoxyphenyl)prop-2-en-1-one, yellowish brown solid, 65%
165.28, 163.56, 162.98, 131.76, 131.47, 123.13, 113.95, 107.17, 105.78,
100.86, 55.69, 55.57. Anal. Calcd for C17H14O5: C, 68.45; H, 4.73; O,
26.82. Found: C, 68.19; H, 4.91. PHPLC (MeOH/H2O, 35/65) 99.56%,
k0 ¼ 4.48.
yield. 1H NMR (500 MHz, DMSO)
d 9.99e9.95 (m, 1H), 8.35 (d,
J ¼ 9.0 Hz, 1H), 8.12 (d, J ¼ 9.0 Hz, 2H), 8.11e8.08 (m, 1H), 7.88 (d,
J ¼ 9.0 Hz, 1H), 7.77 (ddd, J ¼ 8.5, 6.9, 1.5 Hz, 1H), 7.67 (ddd, J ¼ 8.1,
4.3.4. 2-(3,4-Dimethoxyphenyl)-3-hydroxy-7-methoxy-4H-
chromen-4-one (4)
6.9, 1.2 Hz, 1H), 7.14 (d, J ¼ 9.0 Hz, 2H), 7.09 (s, 1H), 3.87 (s, 3H). 13
C
NMR (126 MHz, DMSO)
d
179.24, 162.17, 160.52, 157.01, 135.67,
Synthesized from (E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-
130.49, 129.87, 129.07, 128.67, 128.15, 126.62, 126.23, 123.01, 118.27,
116.24, 114.76, 108.45, 55.67. Anal. Calcd for C20H14O3: C, 79.46; H,
4.67; O, 15.88. Found: C, 79.51; H, 4.59. PHPLC (MeOH/H2O, 35/65)
96.88%, k0 ¼ 5.60.
4-methoxyphenyl)prop-2-en-1-one, yellow solid, 62% yield. 1H
NMR (500 MHz, DMSO)
d
9.27 (s, 1H), 7.98 (d, J ¼ 8.9, 1H), 7.85 (dd,
J ¼ 2.1, 8.6 Hz, 1H), 7.79 (d, J ¼ 2.1 Hz, 1H), 7.28 (d, J ¼ 2.3 Hz, 1H),
7.13 (d, J ¼ 8.7, 1H), 7.03 (dd, J ¼ 2.4, 8.9 Hz, 1H), 3.92 (s, 3H), 3.84 (d,
J ¼ 3.1 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 172.18, 163.66, 156.47,
4.2.7. 3-(3,4-Dimethoxyphenyl)-1H-benzo[f]chromen-1-one (28)
Synthesized from (E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-
naphthalen-1-yl)prop-2-en-1-one,yellow solid, 77% yield. 1H NMR
150.32, 148.59, 144.94, 138.06, 126.20, 123.94, 121.41, 115.29, 114.64,
111.70, 111.10, 100.49, 56.22, 55.90, 55.76. Anal. Calcd for
C
18H16O6*0.5H2O: C, 64.09; H, 5.08; O, 30.83. Found: C, 63.94; H,
(500 MHz, DMSO)
d
9.98e9.94 (m, 1H), 8.33 (d, J ¼ 9.0 Hz, 1H), 8.07
5.17. PHPLC (MeOH/H2O, 35/65) 99.56%, k0 ¼ 4.48.
(dd, J ¼ 8.3, 1.0 Hz, 1H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.78e7.72 (m, 2H),
7.69e7.62 (m, 2H), 7.16 (s, 1H), 7.13 (d, J ¼ 8.6 Hz, 1H), 3.91 (s, 3H),
4.3.5. 3-Hydroxy-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-
chromen-4-one (5)
3.85 (s, 3H). 13C NMR (126 MHz, DMSO)
d 179.30, 160.51, 157.01,
151.97, 149.26,135.60, 130.48,129.89, 129.04,128.66,126.60,126.26,
123.10, 119.78, 118.33, 116.23, 111.94, 109.46, 108.72, 56.03, 55.86.
Anal. Calcd for C21H16O4: C, 75.89; H, 4.85; O,19.26. Found: C, 76.03;
H, 4.67. PHPLC (MeOH/H2O, 35/65) 99.9%, k0 ¼ 2.85.
Synthesized from (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-
(4-methoxyphenyl)prop-2-en-1-one, yellow solid, 70% yield. 1H
NMR (500 MHz, DMSO)
2H), 6.90 (d, J ¼ 8.9 Hz, 2H), 6.35 (s, 2H), 3.75 (s, 6 H), 3.69 (s, 3H).
13C NMR (126 MHz, DMSO)
191.14, 162.18, 159.23, 158.24, 148.88,
d
9.12 (d, J ¼ 1.7 Hz, 1H), 7.66 (d, J ¼ 8.9 Hz,
d
4.3. General procedure for synthesis of 3-hydroxy flavones and
benzoflavones
131.44, 127.52, 116.02, 114.13, 108.89, 91.27, 55.99, 55.62, 55.29.
Anal. Calcd for C18H16O6: C, 65.85; H, 4.91; O, 29.24. Found: C,
65.67; H, 5.11. PHPLC (MeOH/H2O, 35/65) 96.17%, k0 ¼ 3.32.
The appropriate chalcone or benzochalcone (5 mmol) was dis-
solved in EtOH (25 ml). NaOH 25% (10 ml) and H2O2 25% (10 ml)
were added in small portions. The solution was stirred overnight at
room temperature. After completion of the reaction as indicated by
TLC, the reaction mixture was poured onto crushed ice and was
acidified with dilute HCl. Yellowebrown precipitate was filtered
under suction, washed with water. The crude product was recrys-
tallized from ethanol to give compounds 1e5, 18e21 and 29e31.
4.3.6. 3-Hydroxy-2-phenyl-4H-benzo[h]chromen-4-One (18)
Synthesized from (E)-1-(1-hydroxynaphthalen-2-yl)-3-phenyl-
prop-2-en-1-one, white solid, 57% yield. 1H NMR (500 MHz,
DMSO)
d
9.78 (s, 1H), 8.66 (dd, J ¼ 6.1, 3.4 Hz, 1H), 8.34 (dd, J ¼ 8.5,
1.2 Hz, 2H), 8.10 (dd, J ¼ 6.0, 3.3 Hz, 1H), 8.05 (d, J ¼ 8.7 Hz, 1H), 7.89
(d, J ¼ 8.7 Hz, 1H), 7.84e7.79 (m, 2H), 7.74 (d, J ¼ 8.7 Hz, 1H), 7.64e
7.60 (m, 2H). 13C NMR (126 MHz, DMSO)
d 172.80, 151.96, 144.74,
140.52, 135.24, 131.61, 129.92, 129.73, 128.87, 128.48, 127.90, 127.68,
125.04, 124.89, 123.76, 123.21, 122.46, 120.28, 117.66. Anal. Calcd for
4.3.1. 3-Hydroxy-2-phenyl-4H-chromen-4-one (1)
Synthesized from (E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
C19H12O3: C, 79.16; H, 4.20; O, 16.65. Found: C, 79.28; H, 4.15. PHPLC
1-one, white solid, 59% yield. 1H NMR (500 MHz, CDCl3)
d
11.86 (s,
(MeOH/H2O, 35/65) 98.85%, k0 ¼ 1.13.
0H), 8.30e8.21 (m, 2H), 7.69 (ddd, J ¼ 1.6, 7.1, 8.6, 1H), 7.58 (d,
J ¼ 8.5, 1H), 7.53 (dd, J ¼ 4.7, 10.3, 2H), 7.49e7.43 (m, 1H), 7.40 (t,
J ¼ 7.5, 1H), 7.07 (dd, J ¼ 2.8, 5.9, 1H). 13C NMR (126 MHz, CDCl3)
4.3.7. 3-Hydroxy-2-(4-methoxyphenyl)-4H-benzo[h]chromen-4-
one (19)
d
173.49, 155.44, 144.98, 138.46, 133.64, 131.07, 130.20, 128.60,
Synthesized from (E)-1-(1-hydroxynaphthalen-2-yl)-3-(4-
128.31, 127.77, 126.30, 125.47, 124.53, 120.66, 118.28. Anal. Calcd for
methoxyphenyl)prop-2-en-1-one, pale yellow solid, 59% yield. 1H
C
15H10O3*1.0H2O: C, 70.31; H, 4.72; O, 24.97. Found: C, 70.22; H,
NMR (500 MHz, DMSO) d 9.60 (s, 1H), 8.68e8.62 (m, 1H), 8.32 (d,
4.83. PHPLC (MeOH/H2O, 50/50) 95.72%, k0 ¼ 5.92.
J ¼ 8.9 Hz, 2H), 8.14e8.07 (m, 1H), 8.04 (d, J ¼ 8.7 Hz, 1H), 7.89 (d,
J ¼ 8.7 Hz, 1H), 7.81 (dd, J ¼ 6.0, 3.3 Hz, 2H), 7.18 (d, J ¼ 9.0 Hz, 2H),
4.3.2. 3-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (2)
Synthesized from (E)-1-(2-hydroxyphenyl)-3-(4-methoxyphe-
nyl)prop-2-en-1-one, pale yellow solid, 61% yield. 1H NMR
3.87 (s, 3H). 13C NMR (126 MHz, DMSO)
d 172.46, 160.52, 151.70,
145.18, 139.53, 135.14, 129.58, 129.39, 128.46, 127.83, 124.75, 123.91,
123.73, 122.37, 120.30, 117.64, 114.38, 55.50. Anal. Calcd for
(500 MHz, DMSO)
d
9.39 (s, 1H), 8.20 (d, J ¼ 9.2 Hz, 2H), 7.91e7.86
C20H14O4: C, 75.46; H, 4.43; O, 20.10. Found: 75.37; H, 4.54. PHPLC
(m,1H), 7.82e7.69 (m, 2H), 7.45 (ddd, J ¼ 8.0, 6.8,1.3 Hz,1H), 7.12 (d,
(MeOH/H2O, 35/65) 97.51%, k0 ¼ 2.46.
J ¼ 9.2 Hz, 2H), 3.84 (s, 3H). 13C NMR (126 MHz, DMSO)
d 172.77,
160.60, 154.59, 145.74, 138.28, 133.61, 131.45, 129.54, 124.87, 123.73,
121.50, 118.44, 114.19, 55.50. Anal. Calcd for C16H12O4: C, 71.64; H,
4.51; O, 23.86. Found: C, 71.53; H, 4.64. PHPLC (MeOH/H2O, 50/50)
95.83%, k0 ¼ 4.71.
4.3.8. 2-(3,4-Dimethoxyphenyl)-3-hydroxy-4H-benzo[h]chromen-
4-one (20)
Synthesized from (E)-3-(3,4-dimethoxyphenyl)-1-(1-hydroxy-
naphthalen-2-yl)prop-2-en-1-one, pale yellow solid, 61% yield.

K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
123
1H NMR (500 MHz, DMSO)
d
9.61 (s, 1H), 8.61 (s, 1H), 8.10 (d, J ¼ 9.1,
(3 mmol) were added. The resulting mixture was refluxed for 24 h.
After completion of the reaction, acetone and methyl iodide were
removed under reduced pressure. To the resulting solid water was
added, the precipitated product was filtered and washed with
water to remove traces of K2CO3. The product was dried at room
temperature and recrystallized from ethanol to yield compounds
6e9, 22e25 and 32e34.
1H), 8.04 (d, J ¼ 8.7,1H), 7.95 (d, J ¼ 8.5,1H), 7.91 (d, J ¼ 2.0,1H), 7.89
(d, J ¼ 8.8, 1H), 7.83e7.79 (m, 2H), 7.20 (d, J ¼ 8.6, 1H), 3.88 (d,
J ¼ 12.2, 6H). 13C NMR (126 MHz, DMSO)
d 172.44, 151.70, 150.44,
148.68, 145.14, 139.66, 135.17, 129.60, 128.49, 127.92, 124.00, 123.72,
117.63, 111.96, 111.19, 55.83, 55.80. Anal. Calcd for C21H16O5*0.5H2O:
C, 72.41; H, 4.63; O, 22.96. Found: C, 72.35; H, 4.71. PHPLC (MeOH/
H2O, 35/65) 95.75%, k0 ¼ 2.85.
4.4.1. 3-Methoxy-2-phenyl-4H-chromen-4-one (6)
4.3.9. 2-(4-Fluoro-3-methoxyphenyl)-3-hydroxy-4H-benzo[h]
chromen-4-one (21)
Synthesized from 3-hydroxy-2-phenyl-4H-chromen-4-one, pale
yellow solid, 74% yield. 1H NMR (500 MHz, CDCl3)
d
8.26 (dd, J ¼ 8.0,
Synthesized from (E)-3-(4-fluoro-3-methoxyphenyl)-1-(1-
hydroxy-naphthalen-2-yl)prop-2-en-1-one, white solid, 54%
1.7 Hz, 1H), 8.12e8.06 (m, 2H), 7.66 (ddd, J ¼ 8.5, 7.1, 1.7 Hz, 1H),
7.55e7.48 (m, 4H), 7.39 (ddd, J ¼ 8.0, 7.1, 1.0 Hz, 1H), 3.89 (s, 3H). 13
C
yield. 1H NMR (500 MHz, DMSO)
d
9.85 (s, 1H), 8.72e8.63 (m, 1H),
NMR (126 MHz, CDCl3) d 175.14, 155.60, 155.28, 141.53, 133.45,
8.22e8.08 (m, 3H), 8.04 (d, J ¼ 8.7 Hz, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H),
130.72, 128.53, 128.50, 125.83, 124.66, 117.98, 60.13. Anal. Calcd for
7.86e7.77 (m, 2H), 7.41 (t, J ¼ 8.9 Hz, 1H), 3.95 (s, 3H). 13C NMR
C
16H12O3: C, 76.18; H, 4.79; O, 19.03. Found: C, 75.92; H, 4.96. PHPLC
(126 MHz, DMSO)
d
172.56, 151.77, 150.32, 148.47, 144.01, 143.78,
(MeOH/H2O, 30/70) 98.39%, k0 ¼ 1.17.
140.07,135.22,129.70,128.46,127.90,124.90,124.64,124.29,123.68,
122.52, 120.26, 117.67, 115.10, 114.13, 56.36. Anal. Calcd for
4.4.2. 3-Methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (7)
Synthesized from 3-hydroxy-2-(4-methoxyphenyl)-4H-chro-
men-4-one, pale yellow solid, 64% yield. 1H NMR (500 MHz, DMSO)
C
20H13FO4: C, 71.43; H, 3.90; O, 19.03. Found: C, 71.54; H, 3.78. PHPLC
(MeOH/H2O, 35/65) 97.42%, k0 ¼ 3.48.
d
8.09 (d, J ¼ 1.3 Hz, 1H), 8.08e8.06 (m, 1H), 8.06e8.04 (m, 1H), 7.80
4.3.10. 2-Hydroxy-3-phenyl-1H-benzo[f]chromen-1-one (29)
Synthesized from (E)-1-(2-hydroxynaphthalen-1-yl)-3-phenyl-
prop-2-en-1-one, white solid, 63% yield. 1H NMR (500 MHz,
(ddd, J ¼ 8.6, 7.0, 1.7 Hz, 1H), 7.75e7.71 (m, 1H), 7.47 (ddd, J ¼ 8.0,
7.0, 1.1 Hz, 1H), 7.16e7.11 (m, 2H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR
(126 MHz, DMSO)
d 173.80, 161.38, 155.10, 154.76, 140.22, 134.01,
DMSO)
d
8.49 (d, J ¼ 8.5 Hz,1H), 7.96 (d, J ¼ 9.0 Hz,1H), 7.86e7.82 (m,
130.20, 125.08, 125.01, 123.68, 122.75, 118.47, 114.35, 59.61, 55.58.
Anal. Calcd for C17H14O4: C, 72.33; H, 5.00; O, 22.67. Found: C, 72.47;
H, 5.38. PHPLC (MeOH/H2O, 50/50) 99.74%, k0 ¼ 6.36.
2H), 7.78 (d, J ¼ 9.0 Hz, 1H), 7.71 (td, J ¼ 7.5, 1.4 Hz, 1H), 7.65 (d,
J ¼ 9.0 Hz, 1H), 7.61 (dt, J ¼ 13.4, 4.6 Hz, 1H), 7.55e7.48 (m, 2H), 7.35
(ddd, J ¼ 8.0, 6.8, 1.1 Hz, 1H), 7.16 (d, J ¼ 8.9 Hz, 1H). 13C NMR
(126 MHz, DMSO)
d
172.28, 165.73, 159.67, 156.12, 142.48, 135.82,
4.4.3. 2-(3,4-Dimethoxyphenyl)-3-methoxy-4H-chromen-4-one (8)
Synthesized from 2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-
chromen-4-one, yellow solid, 73% yield. 1H NMR (500 MHz, DMSO)
134.73, 134.56, 129.88, 128.77, 127.98, 127.94, 124.59, 123.33, 119.04,
113.08. Anal. Calcd for C19H12O3*1.0H2O: C, 74.50; H, 4.61; O, 20.89.
Found: C, 74.57; H, 4.88. PHPLC (MeOH/H2O, 35/65) 98.15%, k0 ¼ 3.85.
d
8.08 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.81 (ddd, J ¼ 8.5, 6.9, 1.6 Hz, 1H),
7.78e7.75 (m, 1H), 7.72 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.68 (d, J ¼ 2.1 Hz,
1H), 7.48 (ddd, J ¼ 8.0, 6.9,1.2 Hz,1H), 7.16 (d, J ¼ 8.6 Hz,1H), 3.86 (d,
4.3.11. 2-Hydroxy-3-(4-methoxyphenyl)-1H-benzo[f]chromen-1-
One (30)
J ¼ 1.8 Hz, 6H), 3.82 (s, 3H). 13C NMR (126 MHz, DMSO)
d 173.81,
Synthesized from (E)-1-(2-hydroxynaphthalen-1-yl)-3-(4-meth
155.07, 154.75, 151.26, 148.62, 140.35, 134.00, 125.10, 125.00, 123.65,
122.76, 122.14, 118.57, 111.78, 111.68, 59.64, 55.82. Anal. Calcd for
oxyphenyl)prop-2-en-1-one, pale yellow solid, 57% yield. 1H NMR
(500 MHz, DMSO)
d
9.33 (s, 1H), 8.67 (dd, J ¼ 7.2, 1.7 Hz, 1H), 7.79e
C18H16O5: C, 69.22; H, 5.16; O, 25.61. Found: C, 69.28; H, 5.13. PHPLC
7.71 (m, 2H), 7.54 (d, J ¼ 7.5 Hz, 1H), 7.49 (d, J ¼ 7.5 Hz, 2H), 7.42
(MeOH/H2O, 50/50) 99.33%, k0 ¼ 3.95.
(dtd, J ¼ 19.9, 7.4, 1.6 Hz, 2H), 6.97 (d, J ¼ 7.5 Hz, 2H), 3.81 (s, 3H). 13
C
NMR (126 MHz, DMSO)
d
172.28, 167.89, 161.06, 159.95, 143.82,
4.4.4. 2-(3,4-Dimethoxyphenyl)-3,7-dimethoxy-4H-chromen-4-
134.65, 131.57, 131.44, 130.02, 128.79, 128.03, 127.00, 126.96, 124.54,
123.41, 118.94, 116.64, 114.47, 55.42. Anal. Calcd for C20H14O4: C,
75.46; H, 4.43; O, 20.10. Found: C, 75.27; H, 4.67. PHPLC (MeOH/H2O,
35/65) 95.28%, k0 ¼ 4.03.
one (9)
Synthesized
methoxy-4H-chromen-4-one, yellow solid, 71% yield. 1H NMR
(500 MHz, DMSO)
from
2-(3,4-dimethoxyphenyl)-3-hydroxy-7-
d
7.96 (d, J ¼ 8.9 Hz, 1H), 7.71 (dd, J ¼ 8.5, 2.1 Hz,
1H), 7.66 (d, J ¼ 2.1 Hz,1H), 7.27 (d, J ¼ 2.3 Hz,1H), 7.15 (d, J ¼ 8.6 Hz,
4.3.12. 3-(3,4-Dimethoxyphenyl)-2-hydroxy-1H-benzo[f]chromen-
1-one (31)
1H), 7.05 (dd, J ¼ 8.9, 2.4 Hz, 1H), 3.91 (s, 3H), 3.86 (s, 6H), 3.81 (s,
3H). 13C NMR (126 MHz, DMSO)
d 173.21, 163.86, 156.57, 154.50,
Synthesized from (E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-
naphthalen-1-yl)prop-2-en-1-one,yellowish brown solid, 66%
151.11, 148.60, 140.10, 126.36, 122.83, 121.93, 117.44, 114.71, 111.73,
111.56, 100.73, 59.62, 56.26, 55.84, 55.80. Anal. Calcd for C19H18O6:
C, 66.66; H, 5.30; O, 28.04. Found: C, 66.38; H, 5.64. PHPLC (MeOH/
H2O, 50/50) 97.28%, k0 ¼ 3.51.
yield. 1H NMR (500 MHz, DMSO)
d
9.83 (s, 1H), 7.55 (dd, J ¼ 8.4,
2.0 Hz, 2H), 7.43 (d, J ¼ 2.0 Hz, 2H), 7.29 (d, J ¼ 2.0 Hz, 1H), 7.18 (ddd,
J ¼ 14.2, 7.5, 4.5 Hz, 1H), 7.03 (d, J ¼ 8.5 Hz, 2H), 6.42 (d, J ¼ 15.9 Hz,
1H), 3.81 (s, 3H), 3.79 (s, 3H). 13C NMR (126 MHz, DMSO)
d
167.98,
4.4.5. 3-Methoxy-2-phenyl-4H-benzo[h]chromen-4-one (22)
Synthesized from 3-hydroxy-2-phenyl-4H-benzo[h]chromen-
4-one, yellowish brown solid, 62% yield. 1H NMR (500 MHz, DMSO)
167.22, 152.78, 150.95, 149.15, 148.47, 127.22, 123.32, 123.12, 122.74,
116.86, 112.11, 111.74, 111.47, 111.17, 110.55, 55.80, 55.62. Anal. Calcd
for C21H16O5: C, 72.41; H, 4.63; O, 22.96. Found: C, 72.18; H, 4.92.
PHPLC (MeOH/H2O, 35/65) 96.01%, k0 ¼ 4.22.
d
8.59 (dd, J ¼ 6.1, 3.4 Hz, 1H), 8.21e8.18 (m, 2H), 8.14e8.10 (m, 1H),
8.04 (d, J ¼ 8.7 Hz, 1H), 7.94 (d, J ¼ 8.7 Hz, 1H), 7.85e7.79 (m, 2H),
7.68e7.61 (m, 3H), 3.90 (s, 3H). 13C NMR (126 MHz, DMSO)
4.4. General procedure for synthesis of 3-methoxy flavones and
benzoflavones
d 173.83, 154.36, 152.10, 142.14, 135.39, 130.99, 130.70, 129.82,
129.02, 128.44, 128.40, 127.97, 125.34, 123.56, 122.40, 120.22,
120.05, 59.82. Anal. Calcd for C20H14O3: C, 79.46; H, 4.67; O, 15.88.
Found: C, 79.29; H, 4.81. PHPLC (MeOH/H2O, 35/65) 99.31%,
k0 ¼ 9.87.
To a solution of 3-hydroxyflavone or 3-hydroxybenzoflavone
(2 mmol) in acetone (25 ml), K2CO3 (2 mmol) and methyl iodide

124
K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
4.4.6. 3-Methoxy-2-(4-methoxyphenyl)-4H-benzo[h]chromen-4-
one (23)
4.4.11. 3-(3,4-Dimethoxyphenyl)-2-methoxy-1H-benzo[f]chromen-
1-one (34)
Synthesized from 3-hydroxy-2-(4-methoxyphenyl)-4H-benzo
Synthesized from 3-(3,4-dimethoxyphenyl)-2-hydroxy-1H-
[h]chromen-4-one, yellowish brown solid, 59% yield. 1H NMR
benzo[f]chromen-1-one, yellowish brown solid, 68% yield. 1H NMR
(500 MHz, DMSO)
d
8.59e8.54 (m, 1H), 8.20e8.16 (m, 2H), 8.11e
(500 MHz, DMSO)
d
8.79 (dd, J ¼ 7.4, 1.6 Hz, 1H), 7.85e7.78 (m, 2H),
8.07 (m, 1H), 8.01 (d, J ¼ 8.7 Hz, 1H), 7.90 (d, J ¼ 9.0 Hz, 1H), 7.80
7.68e7.62 (m, 1H), 7.45 (dtd, J ¼ 25.1, 7.4, 1.5 Hz, 2H), 7.08e6.91 (m,
(ddd, J ¼ 4.7, 2.2, 0.8 Hz, 2H), 7.19 (d, J ¼ 9.1 Hz, 2H), 3.88 (s, 6H). 13
C
2H), 6.79 (d, J ¼ 7.8 Hz, 1H), 3.59 (s, 6H), 3.51 (s, 3H). 13C NMR
NMR (126 MHz, DMSO)
d
173.58, 161.33, 154.37, 151.85, 141.34,
(125 MHz, DMSO) d 176.04, 155.52, 153.59, 152.27, 149.41, 137.83,
135.31, 130.13, 129.67, 128.41, 127.87, 125.16, 123.53, 122.87, 122.31,
120.23, 119.96, 114.52, 59.58, 55.56. Anal. Calcd for C21H16O4: C,
75.89; H, 4.85; O, 19.26. Found: C, 75.58; H, 5.04. PHPLC (MeOH/H2O,
35/65) 98.20%, k0 ¼ 6.54.
133.83, 130.92, 130.85, 128.01, 127.98, 126.47, 125.09, 123.58, 122.40,
119.04, 114.85, 112.34, 111.89, 61.45, 56.78. Anal. Calcd for C22H18O5:
C, 72.92; H, 5.01; O, 22.08. Found: C, 73.05; H, 5.12. PHPLC (MeOH/
H2O, 35/65) 96.83%, k0 ¼ 4.92.
4.4.7. 2-(3,4-Dimethoxyphenyl)-3-methoxy-4H-benzo[h]chromen-
4-one (24)
4.5. General procedure for the synthesis of pentamethyl ethers of
quercetin and morin
Synthesized from 2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-
benzo[h]chromen-4-one, yellowish brown solid, 69% yield. 1H NMR
To a solution of quercetin hydrate or morin (10 mmol) in acetone
(50 ml), K2CO3 (30 mmol) and methyl iodide (70 mmol, excess)
were added. The resulting mixture was refluxed for 24 h. After
completion of the reaction, acetone and methyl iodide were
removed under reduced pressure. To the resulting solid water was
added, the precipitated product was filtered and washed with
water to remove traces of K2CO3. The product was dried at room
temperature and recrystallized from ethanol to yield compounds
10 and 11.
(500 MHz, DMSO)
d
8.58 (dd, J ¼ 3.5,6.2 Hz, 1H), 8.12 (dd,
J ¼ 3.1,6.2 Hz, 1H), 8.03 (d, J ¼ 8.7 Hz, 1H), 7.92 (d, J ¼ 8.6 Hz, 1H),
7.84 (d, J ¼ 2.1 Hz, 1H), 7.83e7.82 (m, 1H), 7.81 (d, J ¼ 3.3 Hz, 1H),
7.77 (d, J ¼ 2.1 Hz, 1H), 7.22 (d, J ¼ 8.6 Hz, 1H), 3.92 (d, J ¼ 11.4 Hz,
6H), 3.76 (s, 3H). 13C NMR (126 MHz, DMSO)
d 173.61, 154.45, 151.90,
151.22,148.72, 135.37, 129.72, 128.47, 128.00, 125.24, 123.57, 122.89,
122.33, 121.99, 120.27, 119.99, 111.98, 111.62, 59.67, 55.85, 55.81
Anal. Calcd for C22H18O5: C, 72.92; H, 5.01; O, 22.08. Found: C,
72.68; H, 5.25. PHPLC (MeOH/H2O, 35/65) 98.74%, k0 ¼ 5.82.
4.5.1. 2-(3,4-Dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-
one (10)
4.4.8. 2-(4-Fluoro-3-methoxyphenyl)-3-methoxy-4H-benzo[h]
chromen-4-one (25)
Synthesized from quercetin, yellow solid, 82% yield. 1H NMR
Synthesized from 2-(4-fluoro-3-methoxyphenyl)-3-hydroxy-
(500 MHz, CDCl3)
J ¼ 8.3 Hz, 1H), 6.48 (d, J ¼ 2.3 Hz, 1H), 6.33 (d, J ¼ 2.3 Hz, 1H), 3.94
(s, 9H), 3.88 (s, 3H), 3.86 (s, 3H). 13C NMR (126 MHz, CDCl3)
174.00,
163.87, 161.02, 158.78, 152.52, 150.79, 148.65, 141.18, 123.41, 121.60,
111.26,110.76,109.49, 95.72, 92.42, 59.93, 56.39, 56.05, 55.95, 55.75.
Anal. Calcd for C20H20O7: C, 64.51; H, 5.41; O, 30.08. Found: C,
64.34; H, 5.62. PHPLC (MeOH/H2O, 50/50) 97.46%, k0 ¼ 2.69.
d
7.69 (s, 1H), 7.67 (d, J ¼ 2.1 Hz, 1H), 6.96 (d,
4H-benzo[h]chromen-4-one, yellow solid, 74% yield. 1H NMR
(500 MHz, DMSO)
d
8.41 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.75 (dt, J ¼ 8.1,
d
1.4 Hz, 2H), 7.61 (d, J ¼ 7.1 Hz,1H), 7.44 (dtd, J ¼ 25.6, 7.2,1.5 Hz, 2H),
6.88e6.81 (m, 3H), 3.61 (s, 3H), 3.55 (s, 3H). 13C NMR (125 MHz,
DMSO)
d 173.68, 158.40, 155.41, 154.67, 151.95, 151.02, 150.80,
136.86, 134.25, 129.19, 129.16, 128.22, 128.27, 127.72, 125.19, 124.54,
124.15, 123.62, 123.57, 123.22, 121.35, 115.26, 115.04, 114.78, 114.72,
61.45, 59.80, 57.76. Anal. Calcd for C21H15FO4: C, 71.99; H, 4.32; O,
18.27. Found: C, 72.18; H, 4.24. PHPLC (MeOH/H2O, 35/65) 97.83%,
k0 ¼ 4.49.
4.5.2. 2-(2,4-Dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-
one (11)
Synthesized from morin, yellowish brown solid, 76% yield. 1H
NMR (500 MHz, DMSO)
d
7.34 (d, J ¼ 8.4 Hz, 1H), 6.69 (d, J ¼ 2.3 Hz,
4.4.9. 2-Methoxy-3-phenyl-1H-benzo[f]chromen-1-one (32)
Synthesized from 2-hydroxy-3-phenyl-1H-benzo[f]chromen-1-
1H), 6.64 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.61 (d, J ¼ 2.3 Hz, 1H), 6.47 (d,
J ¼ 2.3 Hz, 1H), 3.83 (d, J ¼ 2.1 Hz, 9H), 3.79 (s, 3H), 3.60 (s, 3H). 13
C
one, yellow solid, 54% yield. 1H NMR (500 MHz, DMSO)
d
8.78
NMR (126 MHz, DMSO) d 172.12, 163.71, 162.44, 160.64, 158.85,
(dd, J ¼ 7.5, 1.6 Hz, 1H), 7.82e7.76 (m, 2H), 7.58e7.54 (m, 1H), 7.49
158.38, 152.86, 141.48, 131.46, 114.37, 112.26, 109.11, 105.34, 98.78,
96.01, 93.05, 59.57, 56.30, 56.10, 55.91, 55.63. Anal. Calcd for
(td, J ¼ 7.5, 1.5 Hz, 1H), 7.42 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.32 (dd, J ¼ 7.5,
1.3 Hz, 2H), 7.24 (t, J ¼ 7.3 Hz, 2H), 7.21e7.15 (m, 1H), 3.49 (s, 3H). 13
C
C20H20O7: C, 64.51; H, 5.41; O, 30.08. Found: C, 64.67; H, 5.34. PHPLC
NMR (125 MHz, DMSO)
d
172.01, 156.52, 152.44, 136.05, 133.83,
(MeOH/H2O, 50/50) 95.69%, k0 ¼ 4.64.
131.19, 130.92, 130.85, 130.59, 128.69, 128.47, 128.01, 127.98, 126.47,
125.09, 119.04, 116.89, 59.51. Anal. Calcd for C20H14O3: C, 79.46; H,
4.67; O, 15.88. Found: C, 79.14; H, 4.88. PHPLC (MeOH/H2O, 35/65)
96.51%, k0 ¼ 4.18.
4.6. General procedure for synthesis of tetramethyl ethers of
quercetin and morin
Quercetin hydrate or morin (10 mmol) was added to a mixture
of acetone (150 ml) and water (75 ml). To the resulting mixture, 30%
aqueous KOH (6 ml) was added and refluxed for 10 min. Dimethyl
sulphate (2.4 ml) was added and refluxed for 20 min. More amount
of KOH (3 ml) was added producing dark brown solution. To this
solution an additional amount of dimethyl sulphate (2.4 ml) was
added and refluxed for 20 min. Finally, again KOH (3.0 ml) and
dimethyl sulphate (3.5 ml) were added, refluxed further for 1.5 h
and allowed to cool to room temperature. Evaporation of acetone
gave a dark yellow to brown residue. This residue was purified
by column chromatography using hexan:ethylacetate (1:3) as
mobile phase. The first two fractions were combined to give pure 12
or 13.
4.4.10. 2-Methoxy-3-(4-methoxyphenyl)-1H-benzo[f]chromen-1-
one (33)
Synthesized from 2-hydroxy-3-(4-methoxyphenyl)-1H-benzo
[f]chromen-1-one, pale yellow solid, 63% yield. 1H NMR (500 MHz,
DMSO)
d
8.75 (dd, J ¼ 7.5, 1.7 Hz, 1H), 7.81e7.75 (m, 2H), 7.66e7.58
(m, 1H), 7.51 (dtd, J ¼ 25.1, 7.3, 1.7 Hz, 2H), 7.32 (d, J ¼ 7.5 Hz, 2H),
6.89 (d, J ¼ 7.5 Hz, 2H), 3.77 (s, 3H), 3.59 (s, 3H). 13C NMR (125 MHz,
DMSO)
d 173.14, 160.42, 155.62, 152.44, 138.05, 133.73, 130.92,
130.85, 129.87, 128.01, 127.98, 126.47, 125.09, 123.08, 119.04, 114.85,
113.56, 59.97, 58.03. Anal. Calcd for C21H16O4: C, 75.89; H, 4.85; O,
19.26. Found: C, 75.68; H, 5.03. PHPLC (MeOH/H2O, 35/65) 98.17%,
k0 ¼ 4.87.

K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
125
4.6.1. 2-(3,4-Dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-
chromen-4-one (12)
Casy I Modell TT cell counter device (Schaerfe System GmbH,
Reutlingen, Germany). Followed by another centrifugation cells
were washed three times with Krebs-HEPES buffer (KHB) and
seeded into black 96 well plates (Greiner, Frickenhausen, Germany)
at a density of approximately 20,000 cells per well in a volume of
Synthesized from quercetin, yellow solid, 51% yield. 1H NMR
(500 MHz, DMSO)
d
12.61 (s, 1H), 7.71 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.65
(d, J ¼ 2.1 Hz,1H), 7.15 (d, J ¼ 8.7 Hz,1H), 6.77 (d, J ¼ 2.1 Hz,1H), 6.37
(d, J ¼ 2.2 Hz, 1H), 3.87e3.84 (m, 9H), 3.82 (s, 3H). 13C NMR
90
were added to a total volume of 100
was kept under 5% CO2 and 37 ꢂC for 30 min. After this pre-
incubation period, 20 l of a 6 M Hoechst 33342 solution (pro-
tected from light) was added to each well yielding a final
concentration of Hoechst 33342 of 1 M. Fluorescence was
ml. 10
ml of various test compounds in different concentrations
(126 MHz, DMSO)
d
178.20, 165.34, 160.81, 156.51, 155.59, 151.52,
ml. The prepared 96 well plate
148.67, 138.51, 122.25, 122.22, 111.79, 111.52, 105.39, 97.95, 92.70,
59.94, 56.27, 55.85. Anal. Calcd for C19H18O7*0.5H2O: C, 62.12; H,
5.21; O, 32.67. Found: C, 62.27; H, 5.32. PHPLC (MeOH/H2O, 50/50)
96.32%, k0 ¼ 3.82.
m
m
m
measured immediately in constant intervals (60 s) up to 120 min at
an excitation wavelength of 355 nm and an emission wavelength of
460 nm using a 37 ꢂC tempered BMG POLARstar microplate reader
(BMG Labtech, Offenburg, Germany). For the analysis of the data
obtained from the assay, first fluorescence of KHB was subtracted
from the fluorescence reading obtained from MDCK cells. Average
of fluorescence values in the steady state (from 100 min to 109 min)
were calculated for each concentration and from these data, con-
centration response curves were generated by nonlinear regression
using the four-parameter logistic equation with variable Hill slope
(GraphPad Prism v. 5.0, San Diego, CA, USA).
4.6.2. 2-(2,4-Dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-
chromen-4-one (13)
Synthesized from morin, yellowish brown solid, 48% yield. 1H
NMR (500 MHz, DMSO)
d
12.62 (s, 1H), 7.39 (d, J ¼ 8.5 Hz, 1H), 6.72
(d, J ¼ 2.3 Hz, 1H), 6.66 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.61 (d, J ¼ 2.2 Hz,
1H), 6.38 (d, J ¼ 2.2 Hz, 1H), 3.84 (d, J ¼ 7.2 Hz, 6H), 3.81 (s, 3H), 3.69
(s, 3H). 13C NMR (126 MHz, DMSO)
d 178.24, 165.31, 162.85, 161.27,
158.56, 157.10, 156.82, 139.54, 131.58, 111.72, 105.77, 105.53, 98.80,
97.98, 92.48, 60.12, 56.24, 55.99, 55.67. Anal. Calcd for
C
19H18O7*0.5H2O: C, 62.12; H, 5.21; O, 32.67. Found: C, 61.99; H,
5.36. PHPLC (MeOH/H2O, 50/50) 97.25%, k0 ¼ 4.04.
4.7.3.2. Pheophorbide A assay. The synthesized compounds were
additionally investigated for BCRP inhibition in pheophorbide A
assay as described earlier using MDCK wt and MDCK BCRP cells
[32,33]. To perform the pheophorbide A assay, cells were prepared
as described for the Hoechst 33342 assay. Approximately 45,000
cells per well were seeded into U-shaped clear 96 well plates
4.7. Biology
4.7.1. Materials for biological studies
Chemicals: Ko143 was purchased from Tocris Bioscience (Bristol,
United Kingdom). All other chemicals were purchased from Sigmae
Aldrich Chemicals (Taufkirchen, Germany) unless otherwise spec-
ified. Stock solutions (10 mM) of investigated compounds were
prepared in DMSO.
(Greiner, Frickenhausen, Germany) in a volume of 160
20 l of various test compounds in different concentrations were
added. The prepared 96 well plate was kept under 5% CO2 and 37 ꢂC
for 30 min. After this preincubation period, 20 l of a 5 M pheo-
ml. To this
m
m
m
4.7.2. Cell culture
phorbide A solution (protected from light) was added to each well
and plates were further incubated for 120 min. Fluorescence was
measured by flow cytometry (FACSCalibur, Becton Dickinson Bio-
sciences, Heidelberg, Germany). For the measurement, pheo-
phorbide A was excited at an excitation wavelength of 488 nm and
emission was detected in the FL3 channel (670 nm). Concentration
response curves were generated by nonlinear regression using the
four-parameter logistic equation with variable Hill slope.
The cell line MDCK wt and MDCK BCRP were received as a
generous gift from Dr. A. Schinkel (The Netherlands Cancer Insti-
tute, Amsterdam, The Netherlands). MDCK BCRP cells were gener-
ated by transfection of the canine kidney epithelial cell line MDCKII
with the human wild-type cDNA C-terminally linked to the cDNA of
the green fluorescent protein (GFP) [30]. These cells were cultured
in Dulbecco’s modified eagle medium (DMEM) with 10% foetal calf
serum (FCS), streptomycin (50
mg/mL) and penicillin (50 U/mL).
Human ovarian carcinoma cell lines A2780 and the corresponding
MDR1 overexpressing doxorubicin resistant A2780 adr cell line
were purchased from ECACC (Nos. 93112519 and 93112520). The
cell lines were grown in RPMI-1640 medium supplemented with
4.7.3.3. Calcein-AM assay. For checking the selectivity of synthe-
sized compounds towards BCRP, these were further tested for their
P-gp and MRP1 inhibition in the calcein-AM assay [23,32]. For this
purpose A2780 adr cells for P-gp and 2008 MRP1 cells for MRP1
were used. After confluence of 80e90%, subculturing was per-
formed with 0.05% trypsin and 0.02% EDTA. Cells were prepared as
described above and washed three times with Krebs-HEPES buffer
and then seeded into colourless 96 well plates (Greiner, Frick-
enhausen, Germany) at a density of approximately 30,000 cells in a
10% FBS, 50
ml/mL streptomycin, 50 U/mL penicillin G, and 300 mg/L
L-glutamine. The human ovarian cancer cell line 2008 MRP1 stably
expressing MRP1 was a gift from Prof. Dr. Piet Borst (NCI, Amster-
dam, The Netherlands). The cell line 2008 MRP1 was grown in the
same medium as described for A2780 cells with addition of 400
mL G-418 (Geneticin) for maintaining ABCC1 over expression.
mg/
volume of 90
added, resulting in a final volume of 100
96 well plates were preincubated for 30 min. After the pre-
incubation period, 33 l of a 1.25 M calcein AM solution, which
ml per well. Then, 10
ml of the test compounds were
All the cells were maintained in a 5% CO2 humidified atmo-
sphere at 37 ꢂC. After confluence of 80e90%, subculturing was
performed with 0.05% trypsin and 0.02% EDTA.
ml per well. The prepared
m
m
was protected from light, were added to each well. The fluores-
cence was measured immediately in constant time intervals (60 s)
up to 90 min using an excitation wavelength of 485 nm and an
emission wavelength of 520 nm with a BMG POLARstar microplate
reader tempered at 37 ꢂC. For calculation of inhibitory effects the
first linear part of the fluorescence time curves was used.
4.7.3. Drug accumulation assays
4.7.3.1. Hoechst 33342 assay. To investigate the inhibitory effect of
synthesized compounds on BCRP, a Hoechst 33342 accumulation
assay was performed as described earlier with small modifications
[16,23,31]. In brief, after reaching a confluence of 80e90%, cells
were harvested by gentle trypsination (0.05% trypsin/0.02% EDTA)
and then transferred to a 50 ml tube followed by centrifugation
(266ꢁg, 4 ꢂC, 4 min). The cell pellet obtained was re-suspended in
fresh culture medium and the cell density was determined using a
4.7.4. MTT cytotoxicity assay
Cytotoxicity of the selected active flavones (4, 12) and benzo-
flavones (24, 28) was studied in MDCK BCRP and sensitive MDCK

126
K. Juvale et al. / European Journal of Medicinal Chemistry 67 (2013) 115e126
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USA) at a density of 2500 cells per well in a volume of 180 ml and
kept under 5% CO2 at 37 ꢂC for 6 h. Attachment of cells was
controlled under microscope and the test compounds were added
to the cells to achieve the required final concentration in a final
volume of 200
dium containing 0.1% (v/v) of DMSO. After an incubation period of
72 h, the MTT reagent was added (20 l of a 5 mg/ml solution) to
ml. Control experiments were performed with me-
m
each well. Plates were further incubated for 1 h and after that MTT
was terminated by removing supernatants and lysing the cells with
100
ml DMSO per well. Viability of cells was measured spectro-
photometrically by measuring absorbance at 544 nm and back-
ground corrected at 710 nm using BMG POLARstar microplate
reader.
Acknowledgements
We would like to thank Dr. A. H. Schinkel (The Netherlands
Cancer Institute, Amsterdam, The Netherlands) for kindly providing
the MDCK BCRP cell line. K.J. thanks to DAAD (Germany) for
doctoral fellowship.
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as inhibitors of breast cancer resistance protein, Bioorg. Med. Chem. 20 (2012)
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