Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein

Article abstract of DOI:10.1016/j.bmc.2011.10.074

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP binding cassette family of transport proteins. BCRP has been found to confer multidrug resistance in cancer cells. A strategy to overcome resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. The aim of the current study was to investigate different multi-substituted chalcones for their BCRP inhibition. We synthesized chalcones and benzochalcones with different substituents (viz. OH, OCH₃, Cl) on ring A and B of the chalcone structure. All synthesized compounds were tested by Hoechst 33342 accumulation assay to determine inhibitory activity in MCF-7 MX and MDCK cells expressing BCRP. The compounds were also screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity in the calcein AM accumulation assay and were found to be selective towards inhibition of BCRP. Substituents at position 2′ and 4′ on chalcone ring A were found to be essential for activity; additionally there was a great influence of substituents on ring B. Presence of 3,4-dimethoxy substitution on ring B was found to be optimal, while presence of 2- and 4-chloro substitution also showed a positive effect on BCRP inhibition.

Full text of DOI:10.1016/j.bmc.2011.10.074

Bioorganic & Medicinal Chemistry 20 (2012) 346–355
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigation of chalcones and benzochalcones as inhibitors of breast
cancer resistance protein
⇑
Kapil Juvale, Veronika F.S. Pape, 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:
Received 22 July 2011
Revised 24 October 2011
Accepted 27 October 2011
Available online 3 November 2011
Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP binding cassette family of transport
proteins. BCRP has been found to confer multidrug resistance in cancer cells. A strategy to overcome
resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. The
aim of the current study was to investigate different multi-substituted chalcones for their BCRP inhibi-
tion. We synthesized chalcones and benzochalcones with different substituents (viz. OH, OCH3, Cl) on
ring A and B of the chalcone structure. All synthesized compounds were tested by Hoechst 33342 accu-
mulation assay to determine inhibitory activity in MCF-7 MX and MDCK cells expressing BCRP. The com-
pounds were also screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1
(MRP1) inhibitory activity in the calcein AM accumulation assay and were found to be selective towards
inhibition of BCRP. Substituents at position 20 and 40 on chalcone ring A were found to be essential for
activity; additionally there was a great influence of substituents on ring B. Presence of 3,4-dimethoxy
substitution on ring B was found to be optimal, while presence of 2- and 4-chloro substitution also
showed a positive effect on BCRP inhibition.
Keywords:
Chalcones
Multidrug resistance
Breast cancer resistance protein
Inhibitors
Hoechst 33342 assay
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and its synthetic analog Ko143, broad spectrum modulators like
XR9576 (tariquidar),11 naturally occurring flavonoids and their
Chemotherapy is a major form of treatment for cancers. But
unfortunately the majority of cancers are either resistant to che-
motherapy or acquire resistance during treatment. A frequent
mechanism by which human cancers develop resistance is overex-
pression of certain ATP-binding cassette (ABC) transporters confer-
ring resistance to structurally unrelated anticancer agents, the so
called multidrug resistance (MDR). Among these transporters
especially P-glycoprotein (P-gp),1 Multidrug Resistance-associated
Protein 1 (MRP1)2 and the breast cancer resistance protein (BCRP)
are found to be involved in MDR.3
Transfection studies from various laboratories have confirmed
that overexpression of BCRP cDNA in different cell types confers
resistance to a variety of anticancer agents and decrease drug accu-
mulation in the cells.3,4 BCRP belongs to subfamily G of the ABC-
transporter superfamily and is designated as ABCG2. BCRP has
been found to transport large molecules with amphiphilic charac-
ter using energy from ATP hydrolysis.5,6 BCRP substrates include
the anticancer drugs mitoxantrone,7 topotecan,7 irinotecan8 and
its active metabolite SN-38, etoposide and flavopiridol.9 Several
inhibitors of BCRP have been developed during the last decade in
an attempt to overcome the resistance mediated by this trans-
porter. These involve specific inhibitors like fumitremorgin C10
derivatives.12
Chalcones, bioprecursors of flavonoids, have also been reported
to inhibit P-gp and BCRP.13 It was shown by Liu et al., that function-
alized basic chalcones are inhibitors of P-gp,14 while Han et al.
reported that non-basic chalcones showed no effect on P-gp while
inhibiting BCRP.15 Recently Zoldakova et al. reported the inhibition
of BCRP and P-gp by a combretastin A4 analogous chalcone and its
Pt-complex.16
In the current study we investigated the effect of different sub-
stitution patterns on the inhibitory activity of chalcones. For this
reason we synthesized several multi-substituted derivatives with
varying substituents on ring A and ring B of chalcones (Table 1).
Synthesis of selected chalcones was done by modified Claisen–
Schmidt condensation reaction. The compounds were investigated
for BCRP inhibition in MCF-7 MX and MDCK BCRP cell lines using
the Hoechst 33342 assay.17 To check selectivity the chalcones were
also tested for their P-gp and MRP1 inhibition.18 Reversal of resis-
tance due to BCRP inhibition was investigated using the two anti-
cancer agents mitoxantrone and SN-38 in presence of selected
compounds.
Substitution at position 20 and 40 in ring A of chalcones was
found to be essential for the activity. On ring B presence of 3,4-
dimethoxy substitution yielded maximum inhibitory effect on
BCRP, while presence of 2-chloro or 4-chloro substituents on ring
B also showed a positive effect. Compounds 8 and 25 were found
⇑
Corresponding author. Tel.: +49 228 735213; fax: +49 228 737929.
E-mail address: mwiese@uni-bonn.de (M. Wiese).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.074

K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
347
Table 1
Synthesized chalcones and their inhibitory potencies against MCF-7 MX and MDCK BCRP cells using Hoechst 33342 assay
O
6'
2
O
O
1'
3
4
5'
6'
A
3'
2
2
6'
1
6
1'
A
3
4
3
4
B
5'
4'
4'
1'
1
6
1
6
2'
5'
4'
A
B
B
3'
5
2'
2'
5
3'
5
1-30
31-37
38-44
Chalcone
Substituents on ring A
Substituents on ring B
MCF-7 MX IC50 SD [
lM]
MDCK BCRP IC50 SD [lM]
1
2
3
4
5
6
7
8
H
H
3,4-OCH3
2-Cl
H
4-OCH3
3,4-OCH3
H
4-OCH3
3,4-OCH3
3- OCH3
2-Cl
4-Cl
H
4-OCH3
3,4-OCH3
3-OCH3
2-Cl
n.a.
n.a.
n.a.
n.a.
5.07 0.27
n.a.
n.a.
n.a.
n.a.
n.a.
4.97 0.31
n.a.
20-OH
20-OH
20-OH
20,40-OH
20,40-OH
33.32 7.09
0.93 0.03
2.80 0.17
3.21 0.69
6.07 2.37
31.02 6.39
4.20 0.76
2.17 0.41
3.15 0.59
6.85 0.76
13.51 1.02
n.a.
2.34 0.62
2.20 0.40
3.13 0.57
3.09 0.44
n.a.
5.83 0.32
0.75 0.03
5.27 0.35
2.19 0.43
3.28 0.64
11.04 2.32
2.72 0.42
24.32 3.11
16.92 1.02
2.50 0.45
11.89 1.97
n.a.
12.05 1.69
0.85 0.13
1.94 0.17
3.28 0.50
6.37 0.70
30.95 1.34
2.20 0.75
1.06 0.17
1.47 0.31
3.32 0.57
10.72 2.82
n.a.
1.91 0.78
1.03 0.29
2.63 0.29
1.13 0.21
n.a.
3.00 0.50
0.53 0.22
2.82 0.26
1.16 0.34
3.80 0.13
7.80 2.01
1.73 0.13
9.23 1.00
9.83 0.72
1.95 0.39
5.68 0.50
n.a.
20,40-OH
9
20,40-OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Ko143
20,40-OH
20,40-OH
20-OH,40-OCH3
20-OH,40-OCH3
20-OH,40-OCH3
20-OH,40-OCH3
20-OH,40-OCH3
20-OH,40-OCH3
20-OH,40-OCH3
20,40-OCH3
20,40-OCH3
20,40-OCH3
20,40-OCH3
20,40-OCH3
20-OH,40,60-OCH3
20-OH,4’,60-OCH3
20-OH,40,60-OCH3
30,40-OCH3
30,40-OCH3
30,40-OCH3
30,40-OCH3
20-OH
4-Cl
3,4-Cl
4-OCH3
3,4-OCH3
2-Cl
4-Cl
3,4-Cl
4-OCH3
3,4-OCH3
2-Cl
4-OCH3
3,4-OCH3
2-Cl
4-Cl
H
4-OCH3
3,4-OCH3
3-OCH3
2-Cl
4-Cl
3,4-Cl
H
4-OCH3
3,4-OCH3
3-OCH3
2-Cl
20-OH
20-OH
20-OH
20-OH
20-OH
n.a.
n.a.
20-OH
26.12 3.77
2.77 0.41
3.81 0.62
1.84 0.87
1.93 0.15
5.21 0.75
n.a.
22.39 6.88
2.58 0.94
2.89 0.73
0.72 0.14
1.93 0.15
5.58 0.60
n.a.
20-OH
20-OH
20-OH
20-OH
20-OH
20-OH
4-Cl
3,4-Cl
20-OH
8.78 1.66
0.33 0.05
7.26 1.10
0.26 0.02
Data are expressed as mean SD (n = 3). n.a. = compounds showed no inhibitory effect. Ko143 was used for comparison.
to be the most active ones with IC50 values in the high nanomolar
range, being only about threefold less active than Ko143 the most
potent BCRP inhibitor.
acetophenones and benzaldehydes. Compounds 1–30 were syn-
thesized by the classical method in which aqueous alkaline base
(NaOH) was used as catalyst and reaction was carried out at room
temperature with stirring. Compounds 31–44 were synthesized
under ultrasound bath using LiOH as catalyst. Ultrasonication led
to much smaller reaction time and products obtained were almost
always pure as compared to classical method. All the synthesized
compounds are listed in Table 1.
2. Results and discussion
2.1. Chemistry
Based on the results obtained in earlier studies14,15 we synthe-
sized different chalcones with varying substituents on ring A and
ring B. These chalcones could be divided into three different
subtypes: chalcones (1–30), 30,40-benzochalcones (31–37) and
50,60-benzochalcones (38–44). All the compounds were synthesized
by modified Claisen–Schmidt condensation of different substituted
2.2. Hoechst 33342 assay: inhibition of BCRP by chalcones
All the synthesized chalcones were tested for BCRP inhibition by
the Hoechst 33342 assay using two different cell lines MCF-7 MX
and MDCK BCRP showing resistance due to presence of BCRP.

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K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
Hoechst 33342 is a substrate of BCRP3,19 and has been used as a
tool for investigation of BCRP inhibitors.17,20 To generate the con-
centration response curves averages of fluorescence values at pla-
teau of fluorescence–time curves of Hoechst 33342 were plotted
against the corresponding logarithmic concentrations. The activity
data of investigated compounds are presented in Table 1. A scatter
plot of the pIC50 values for the investigated chalcones obtained in
the Hoechst 33342 assay with MCF-7 MX and MDCK BCRP cells
gave a good correlation (r2 = 0.87) and is shown in Figure 1.
It was observed that compounds with no substituents on ring A
(1, 2) were inactive and presence of single substitution at position
20 (3–5) did not lead to improved inhibition. But compounds with
multiple substituents at positions 20, 30, 40 and 60 of ring A showed
increased BCRP inhibition. In case of chalcones with 3,4-dimethoxy
substituents on ring B (8, 14, 20, 25), it was observed that com-
pounds with hydroxyl group at position 20 had slightly increased
activity as compared to compounds with methoxy substituents.
But no substantial difference was noted for position 40 of ring A.
Presence of a methoxy group at position 60 had only very slight ef-
fect on activity. Compounds with 30,40-dimethoxy substituent (27–
30) showed comparable activity to that having 20,40-dimethoxy
substituents (19–23).
In the study by Han et al., effects of chloro substituents at differ-
ent positions of ring B were investigated.15 It was observed that
presence of 2-chloro and 4-chloro substituents led to increased
BCRP inhibition. This encouraged us to study chalcones with vary-
ing methoxy and chloro substituents on ring B, allowing to com-
pare the effect of methoxy and chloro substituents.
In case of all types of chalcones it was clearly observed that sub-
stitution was needed on ring B, as unsubstituted chalcones were
found to be inactive. Chalcones with 3,4-dimethoxy substitution
showed maximum efficacy of BCRP inhibition, with the chalcones
8, 14, 20, 25 being most potent. A 3-methoxy substituent in case
of compounds (9, 15) was found to be slightly better compared
to 4-methoxy substituent on ring B (7, 13), having the same sub-
stituents at ring A. Chalcones with 2-chloro and 4-chloro substitu-
ents were found to be active, while presence of a 3,4-dichloro
substituent (18, 23) led to inactivity.
2.3. Screening of chalcones for P-gp and MRP1 inhibition
To check the selectivity of the chalcones towards BCRP inhibi-
tion it was important to investigate their effect on the other ma-
jor ABC transporters P-gp and MRP1. For this purpose we tested
all chalcones at 10 lM final concentration using calcein AM
accumulation assay. Figure 3 illustrates the effects of chalcones
on accumulation of calcein in P-gp overexpressing A2780adr
cells and Figure 4 illustrates their effect on accumulation of cal-
cein in MRP1 overexpressing 2008 MRP1 cells. For comparison
purpose cyclosporine A (10 lM) was used as a standard. All
the chalcones showed only very weak effect on P-gp, while none
of the chalcones showed any inhibition of MRP1. This indicates
that the investigated chalcones are selective for BCRP inhibition.
2.4. MTT cytotoxicity assay
To investigate the influence of chalcones on the antiprolifera-
tive effect of cytotoxic agents in presence of the BCRP transporter,
cytotoxicity of mitoxantrone and SN-38 was evaluated in both
MDCK BCRP and MCF-7 MX cells. For this purpose, cell viability
was determined using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyltetrazoliumbromide) assay for the most potent chalcone
(25) and benzochalcone (40). EC50-values of the two anticancer
agents were determined in the absence and presence of three dif-
ferent concentrations of the selected compounds. Both compounds
were able to reverse the resistance of the BCRP expressing cell
lines, proving their functional efficacy. Figure 5 shows a represen-
tative dose–response curve of SN-38 in presence of compound 40.
The shift in the dose–response curve of MDCK BCRP towards lower
concentration indicates the dose dependent BCRP inhibition by
compound 40. Figure 6 illustrates the effect of compound 25 on
the EC50 of mitoxantrone in MCF-7 cells. Even the lowest concen-
tration of compound 25 (1 lM) led to a reversal of resistance and
the higher concentrations were able to reverse the resistance
completely.
3. Conclusion
We also investigated some benzochalcones to study the effect of
variation of ring A structure. In general 50,60-benzochalcones (38–
44) were found to be more potent as compared to 30,40-benzochal-
cones (31–37). Among all three subtypes of chalcones studied,
30,40-benzochalcones (31–37) were found to be least active.
The effect of different substituents on BCRP inhibitory potency
of chalcones is summarized in Figure 2.
Different types of chalcones have been previously shown to be
inhibitors of BCRP, P-gp and MRP1. In the current study we inves-
tigated chalcones, 30,40-benzochalcones and 50,60-benzochalcones
for their inhibitory effects and selectivity towards BCRP. The aim
of the current work was to study the effect of different substituents
on both rings A and B. Also we determined IC50 values of chalcones
against BCRP using two different cell lines MCF-7 MX and MDCK
BCRP, which allowed us to compare the inhibitory potential of
these compounds. It was clearly observed that among all chal-
cones, compounds with 3,4-dimethoxy substituents at ring B were
most active. The inhibitory effect of the most active compounds
was confirmed by MTT cytotoxicity assay. Compounds 8 and 25
were found to be most potent, being only 3 fold less active as
compared to the most active BCRP inhibitor Ko143. We also
investigated all chalcones for their inhibitory effect on P-gp and
6.5
6.0
5.5
5.0
4.5
+ 2-Cl
+ OCH3
R6'
O
R2
R5'
R4'
R3
R4
3,4-OCH3 > 3-OCH3 > 4-OCH3
substitution needed,
otherwise inactive
R2'
4.5
5.0
5.5
6.0
6.5
R3'
+ OCH3, OH
substitution needed
OH > OCH3, H
Figure 1. Scatterplot of the pIC50 values determined by the Hoechst 33342 assay
with MDCK BCRP and MCF-7 MX cells. Each point is an average of at least three
independent experiments. The squared correlation coefficient r2 = 0.87.
Figure 2. Effect of different substituents on BCRP inhibition by chalcones (1–30).

K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
349
1000
800
600
400
200
0
Figure 3. Effect of chalcones 1–44 on the accumulation of calcein AM in P-gp overexpresssing A2780adr cells. All the chalcones were investigated at 10
lM concentration
(n = 3). Cyclosporine A (Cs A), 10 lM, was used as a positive control leading to total inhibition. Data is expressed as response in percentage of control (vehicle).
600
400
200
0
Figure 4. Effect of chalcones 1–44 on the accumulation of calcein AM in MRP1 overexpresssing 2008 MRP1 cells. All the chalcones were investigated at 10
(n = 3). Cyclosporine A (Cs A), 10 M, was used as standard. Data is expressed as response in percentage of control (vehicle).
lM concentration
l
MRP1, showing selectivity of these chalcones towards BCRP
inhibition. The outcome of this study may be useful for the further
modifications and investigation of chalcones as BCRP inhibitors.
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, Bruker Advance 500 (126 MHz); chemical shifts are ex-
pressed 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 distortionless 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 were all within 0.4% of
the theoretical values except when indicated.
4. Experimental
4.1. Chemistry
The general synthetic scheme of target chalcones is depicted in
Scheme 1. All the chalcones were synthesized by base catalyzed
Claisen–Schmidt condensation, from selected benzaldehydes and
acetophenones. Syntheses and characterization of chalcones 1–36
and 38–43 have been described earlier.21–26
4.2. General procedures for synthesis of chalcones
All chemicals were purchased from Acros Organics, Alfa Aesar
or Sigma-Aldrich. During the synthesis reaction progress was mon-
itored using analytical thin layer chromatography (TLC) on silica
gel plates (Silica Gel 60 F254 from Merck). Melting points of newly
synthesized compounds 37 and 44 were determined by Stuart
open capillary melting point instrument and are uncorrected.
For synthesis of chalcones we have used 2 different methods.
Chalcones 1–30 were synthesized by method A and chalcones
31–44 were synthesized by method B. For Method B we used LiOH
instead of usual base NaOH for Claisen–Schmidt synthesis and the

350
K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
completion of the reaction as indicated by TLC, the mixture was
poured onto crushed ice and acidified with dilute HCl. Precipitated
product was filtered by suction and washed to neutral. The solid
was recrystallised from dilute ethanol to get crystalline chalcone.
Method B: To a mixture of 5 mmol of selected acetophenone and
5 mmol of selected benzaldehyde in 50 ml round bottom flask,
20 ml methanol and 35 mmol of LiOH was added. The reaction
mixture was kept in an ultrasonic bath for 1–5 h, until reaction
completion was indicated by TLC. The reaction was worked up as
described for method A.
4.2.1. (E)-3-(3,4-Dimethoxyphenyl)-1-phenylprop-2-en-1-one
(1)
Synthesized from acetophenone and 3,4-dimethoxybenzalde-
hyde, yield 61%. 1H NMR (CDCl3) d: 7.90 (d, J = 15.1 Hz, 1H), 7.80
(dd, J = 7.4, 1.4 Hz, 2H), 7.54 (dd, J = 13.6, 11.3 Hz, 2H), 7.46 (t,
J = 7.4 Hz, 2H), 7.21–7.16 (m, 2H), 6.99 (d, J = 7.4 Hz, 1H), 3.82 (d,
J = 21.9 Hz, 6H). 13C NMR (CDCl3) d: 191.02, 150.98, 150.02,
144.78, 139.01, 133.69, 130.20, 129.13, 128.52, 123.94, 123.01,
112.69, 111.32, 55.94, 56.15. Anal. Calcd for C17H16O3:⁄0.2 H2O:
C, 65.03; H, 9.30; O, 25.67. Found: C, 65.21; H, 9.16.
4.2.2. (E)-3-(2-Chlorophenyl)-1-phenylprop-2-en-1-one (2)
Synthesized from acetophenone and 2-chlorobenzaldehyde,
yield 64%. 1H NMR (CDCl3) d: 1H NMR (500 MHz, Chloroform) d
7.97 (d, J = 14.9 Hz, 1H), 7.69 (dd, J = 7.3, 1.5 Hz, 2H), 7.52–7.49
(m, 1H), 7.48–7.39 (m, 4H), 7.37 (d, J = 14.8 Hz, 1H), 7.34–7.25
(m, 2H). 13C NMR (CDCl3) d 191.56, 140.83, 138.01, 132.95,
132.04, 131.48, 130.91, 129.14, 128.18, 127.85, 128.15, 127.08,
125.72. Anal. Calcd for C15H11ClO: C, 74.23; H, 4.57; Cl, 14.61; O,
6.59. Found: C, 74.08; H, 4.62.
Figure 5. Representative dose–response curve of SN-38 in presence of compound
40. Arrows indicate dose dependent effect of chalcone on sensitisation of MDCK
BCRP cells towards SN-38. Closed squares: MDCK wild type cells, open square:
MDCK BCRP cells, triangle: MDCK BCRP cells + 1
M 40, diamond: MDCK BCRP cells + 5 M 40.
lM 40, circle: MDCK BCRP cells +
3
l
l
4.2.3. (E)-1-(2-Hydroxyphenyl)-3-phenylprop-2-en-1-one (3)
Synthesized from 2-hydroxyacetophenone and benzaldehyde,
yield 69%. 1H NMR (DMSO-d6) d: 12.45 (s, 1H), 8.24 (dd, 1H),
8.02 (d, J = 15.6 Hz, 1H), 7.92–7.88 (m, 2H), 7.84 (d, J = 15.6 Hz,
1H), 7.56 (ddd, J = 8.4, 7.2, 1.7 Hz, 1H), 7.48–7.45 (m, 3H), 7.03–
6.98 (m, 2H). 13C NMR (DMSO-d6) d: 193.75, 161.94, 144.87,
136.42, 134.58, 131.07, 131.00, 129.26, 129.08, 121.99, 120.93,
119.29, 117.85. Anal. Calcd for C15H12O2: C, 80.34; H, 5.39. Found:
C, 80.51; H, 5.28.
4.2.4. (E)-1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-
1-one (4)
Synthesized from 2-hydroxyacetophenone and 4-methoxy-
benzaldehyde, yield 78%. 1H NMR (DMSO-d6) d: 12.92 (s, 1H),
7.92–7.88 (m, 2H), 7.61 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 15.4 Hz,
1H), 7.47 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), 7.01 (dd, J = 8.4, 1.1 Hz,
1H), 6.94 (d, J = 8.8 Hz, 2H), 6.93–6.90 (m, 1H), 3.85 (s, 3H). 13C
NMR (CDCl3) d: 193.64, 163.52, 162.00, 145.32, 136.11, 130.52,
129.50, 127.33, 120.10, 118.72, 118.56, 117.58, 114.50, 55.43. Anal.
Calcd for C16H14O3: C, 75.57; H, 5.55. Found: C, 75.24; H, 5.72.
Figure 6. Effect of compound 25 on the EC50 of mitoxantrone in MCF-7 cells was
investigated at 1, 3, and 5 M concentrations. Untreated cells were used as control.
The figure shows average and standard deviation obtained from three independent
experiments.
l
reaction was carried out in an ultrasonic bath. The general reaction
scheme is depicted in Scheme 1.
Method A: 20% NaOH (5 ml) was added dropwise to a previously
cooled mixture of 5 mmol of selected acetophenone and 5 mmol of
selected benzaldehyde in 25 ml ethanol under vigorous stirring.
The mixture was stirred at room temperature for 24–72 h. After
4.2.5. (E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxyphenyl)prop-
2-en-1-one (5)
Synthesized from 2-hydroxyacetophenone and 3,4-dimethoxy-
benzaldehyde, yield 75%. 1H NMR (DMSO-d6) d: 12.89 (s, 1H),
O
O
a / b
R
R'
R
R'
O
OH
OH
substituted acetophenone
benzaldehyde
chalcone
Scheme 1. General scheme for synthesis of chalcones 1–44. (a) For compounds 1–30: reaction was carried out by stirring at rt for 24–72 h using ethanol as solvent and NaOH
as catalyst. (b) For compounds 31–44: reaction was carried out in an ultrasonic bath at rt for 1–5 h using methanol as solvent and LiOH as catalyst.

K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
351
7.91 (dd, J = 8.1, 1.6 Hz, 1H), 7.87 (d, J = 15.4 Hz, 1H), 7.50–7.46 (m,
4.2.11. (E)-3-(4-Chlorophenyl)-1-(2,4-dihydroxyphenyl)prop-2-
en-1-one (11)
1H), 7.27–7.23 (m, 1H), 7.15 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 1.1 Hz,
1H), 7.00 (d, J = 1.1 Hz, 1H), 6.95–6.88 (m, 2H), 3.94 (d,
J = 12.6 Hz, 6H). 13C NMR (CDCl3) d: 193.54, 163.51, 151.79,
149.29, 145.62, 136.13, 129.49, 127.57, 123.56, 120.06, 118.69,
118.58, 117.77, 111.14, 110.26, 56.00, 55.98. Anal. Calcd for
Synthesized from 2,4-dihydroxyacetophenone and 4-chloro-
benzaldehyde, yield 84%. 1H NMR (DMSO-d6) d: 12.58 (s, 1H),
10.58 (s, 1H), 7.95–7.90 (m, 2H), 7.73 (dd, J = 8.9, 5.2 Hz, 1H),
7.55 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.39–7.29 (m, 2H),
6.36 (dd, J = 8.8, 2.4 Hz, 1H), 6.23 (d, J = 2.3 Hz, 1H). 13C NMR
(DMSO-d6) d: 202.78, 165.00, 164.33, 142.26, 137.90, 133.80,
131.25, 129.81, 128.85, 122.28, 113.01, 108.23, 102.43. Anal. Calcd
for C15H11ClO3: C, 65.58; H, 4.0. Found: C, 65.52; H, 4.21.
C17H16O4: C, 71.82; H, 5.67. Found: C, 71.79; H, 5.48.
4.2.6. (E)-1-(2,4-Dihydroxyphenyl)-3-phenylprop-2-en-1-one
(6)
Synthesized from 2,4-dihydroxyacetophenone and benzalde-
hyde, yield 64%. 1H NMR (DMSO-d6) d: 13.35 (s, 1H), 8.19 (d,
J = 9.0 Hz, 1H), 7.96 (d, J = 15.4 Hz, 1H), 7.88 (dd, J = 6.7, 3.2 Hz,
2H), 7.79 (d, J = 15.5 Hz, 1H), 7.46 (dd, J = 5.0, 1.9 Hz, 3H), 6.42
(dd, J = 8.9, 2.4 Hz, 1H), 6.30 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-
d6) d: 191.61, 165.93, 165.41, 143.75, 134.75, 133.28, 130.78,
129.36, 129.11, 129.03, 128.65, 121.46, 113.18, 108.41, 102.73.
Anal. Calcd for C15H12O3: C, 74.99; H, 5.03. Found: C, 75.06; H, 5.16.
4.2.12. (E)-1-(2-Hydroxy-4-methoxyphenyl)-3-phenylprop-2-
en-1-one (12)
Synthesized from 2-hydroxy-4-methoxyacetophenone and
benzaldehyde, yield 62%. 1H NMR (DMSO-d6) d: 13.49 (s, 1H),
7.95 (d, J = 15.1 Hz, 1H), 7.63 (d, J = 1.7 Hz, 1H), 7.61 (d, J = 9.3 Hz,
1H), 7.56–7.53 (m, 2H), 7.43–7.35 (m, 3H), 6.65 (dd, J = 7.6,
1.4 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 3.87 (s, 3H). 13C NMR (DMSO-
d6) d: 192.78, 165.22, 164.48, 144.23, 135.03, 130.98, 129.17,
128.88, 129.87, 128.07, 128.07, 122.22, 114.91, 106.23, 101.47,
56.04. Anal. Calcd for C16H14O3: C, 75.57; H, 5.55. Found: C,
75.62; H, 5.39.
4.2.7. (E)-1-(2,4-Dihydroxyphenyl)-3-(4-methoxyphenyl)prop-
2-en-1-one (7)
Synthesized from 2,4-dihydroxyacetophenone and 4-methoxy-
benzaldehyde, yield 79%. 1H NMR (DMSO-d6) d: 13.51 (s,
J = 3.0 Hz, 1H), 10.64 (s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.85 (d,
J = 8.8 Hz, 2H), 7.79 (d, J = 14.4 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H),
7.02 (d, J = 8.8 Hz, 2H), 6.41 (dd, J = 8.9, 2.4 Hz, 1H), 6.28 (d,
J = 2.4 Hz, 1H), 3.82 (s, J = 1.8 Hz, 3H). 13C NMR (DMSO-d6) d:
191.66, 165.93, 165.17, 161.62, 143.86, 133.09, 131.97, 131.09,
127.43, 118.74, 114.66, 114.58, 113.17, 108.27, 102.73, 55.56. Anal.
Calcd for C16H14O4: C, 71.10; H, 5.22. Found: C, 71.50; H, 4.96.
4.2.13. (E)-1-(2-Hydroxy-4-methoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (13)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 4-
methoxybenzaldehyde, yield 71%. 1H NMR (DMSO-d6) d: 13.56 (s,
1H), 8.26 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.85 (s, 1H),
7.81 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.55 (dd, J = 9.0, 2.5 Hz, 1H),
6.50 (d, J = 2.5 Hz, 1H), 3.83 (d, J = 8.1 Hz, 6H). 13C NMR (DMSO)
d: 192.04, 165.99, 165.85, 161.72, 144.38, 132.67, 131.22, 127.36,
118.65, 114.59, 114.01, 107.43, 101.08, 55.87, 55.55. Anal. Calcd
for C17H16O4⁄0.2 H2O: C, 71.20; H, 5.91. Found: C, 71.14; H, 5.69.
4.2.8. (E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-
dimethoxyphenyl)prop-2-en-1-one (8)
Synthesized from 2,4-dihydroxyacetophenone and 3,4-dime-
thoxybenzaldehyde, yield 72%. 1H NMR (DMSO-d6) d: 13.55 (s,
1H), 8.20 (d, J = 9.0 Hz, 1H), 7.79 (dd, J = 40.4, 15.4 Hz, 2H), 7.54
(d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.4, 1.9 Hz, 1H), 7.02 (d, J = 8.4 Hz,
1H), 6.42 (dd, J = 8.9, 2.4 Hz, 1H), 6.28 (d, J = 2.4 Hz, 1H), 3.84 (d,
J = 22.4 Hz, 6H). 13C NMR (DMSO-d6) d: 191.68, 165.97, 165.18,
151.59, 149.22, 144.41, 133.12, 127.59, 124.40, 118.73, 113.15,
111.74, 111.02, 108.22, 102.74, 55.93, 55.77. Anal. Calcd for
4.2.14. (E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (14)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 3,4-
dimethoxybenzaldehyde, yield 89%. 1H NMR (DMSO-d6) d: 13.61 (s,
1H), 8.28 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 15.3 Hz, 1H), 7.79 (d,
J = 15.3 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 8.4, 1.9 Hz,
1H), 7.03 (d, J = 8.4 Hz, 1H), 6.56 (dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d,
J = 2.5 Hz, 1H), 3.87–3.81 (m, 9H). 13C NMR (DMSO-d6) d: 192.07,
166.00, 165.94, 151.72, 149.23, 144.93, 132.69, 127.54, 124.55,
118.66, 113.98, 111.75, 111.14, 107.46, 101.04, 55.95, 55.88,
55.78. Anal. Calcd for C18H18O5⁄0.25 H2O: C, 67.81; H, 5.81. Found:
C, 67.71; H, 5.77.
C17H16O5: C, 67.99; H, 5.37. Found: C, 67.73; H, 5.51.
4.2.9. (E)-1-(2,4-Dihydroxyphenyl)-3-(3-methoxyphenyl)prop-
2-en-1-one (9)
Synthesized from 2,4-dihydroxyacetophenone and 3-methoxy-
benzaldehyde, yield 67%. 1H NMR (DMSO-d6) d: 13.11 (s, 1H),
8.08 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.61 (d,
J = 15.3 Hz, 1H,), 7.54–6.58 (m, 6H), 3.89 (s, 3H). 13C NMR
(DMSO-d6) d: 191.66, 165.93, 165.17, 161.62, 143.86, 133.09,
131.97, 131.09, 127.43, 118.74, 114.66, 114.58, 113.17, 108.27,
102.73, 55.56. Anal. Calcd for C16H14O4: C, 71.10; H, 5.22. Found:
C, 70.89; H, 5.37.
4.2.15. (E)-1-(2-Hydroxy-4-methoxyphenyl)-3-(3-
methoxyphenyl)prop-2-en-1-one (15)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 3-
methoxybenzaldehyde, yield 68%. 1H NMR (DMSO-d6) d: 13.40 (s,
1H), 8.29 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 15.5 Hz, 1H), 7.79 (d,
J = 15.5 Hz, 1H), 7.51–7.48 (m, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.37
(t, J = 7.9 Hz, 1H), 7.03 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.56 (dd,
J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 3.84 (d, J = 8.2 Hz, 6H).
13C NMR (DMSO-d6) d: 192.04, 166.19, 165.88, 159.80, 144.24,
136.06, 132.90, 130.05, 122.06, 121.64, 117.01, 114.01, 113.67,
107.55, 101.06, 55.89, 55.45. Anal. Calcd for C17H16O4: C, 71.82;
H, 5.67. Found: C, 71.66; H, 5.486.
4.2.10. (E)-3-(2-Chlorophenyl)-1-(2,4-dihydroxyphenyl)prop-2-
en-1-one (10)
Synthesized from 2,4-dihydroxyacetophenone and 2-chloro-
benzaldehyde, yield 81%. 1H NMR (DMSO-d6) d: 13.17 (s, 1H),
10.79 (s, 1H), 8.21 (dd, J = 7.3, 2.2 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H),
8.09 (d, J = 15.5 Hz, 1H), 8.01 (d, J = 15.5 Hz, 1H), 7.56 (dd, J = 7.7,
1.6 Hz, 1H), 7.46 (ddd, J = 6.2, 4.4, 1.2 Hz, 2H), 6.43 (dd, J = 8.9,
2.4 Hz, 1H), 6.31 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6) d:
191.15, 165.99, 165.70, 138.16, 134.53, 133.49, 132.41, 132.18,
130.21, 128.88, 127.84, 124.40, 113.24, 108.61, 102.80. Anal. Calcd
for C15H11ClO3: C, 65.58; H, 4.04. Found: C, 65.39; H, 4.19.
4.2.16. (E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (16)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 2-
chlorobenzaldehyde, yield 74%. 1H NMR (DMSO-d6) d: 13.19 (s,
1H), 8.26 (d, J = 9.1 Hz, 1H), 8.22 (dd, J = 7.4, 2.1 Hz, 1H), 8.11 (d,
J = 15.5 Hz, 1H), 8.04 (d, J = 15.4 Hz, 1H), 7.57 (dd, J = 7.7, 1.6 Hz,

352
K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
1H), 7.47 (ddd, J = 14.5, 7.1, 1.8 Hz, 2H), 6.57 (dd, J = 9.0, 2.5 Hz,
1H), 6.52 (d, J = 2.5 Hz, 1H), 3.85 (s, J = 6.4 Hz, 3H). 13C NMR
(DMSO-d6) d: 191.55, 166.38, 165.83, 138.54, 134.58, 133.01,
132.30, 132.26, 130.18, 128.92, 127.82, 124.31, 114.08, 107.72,
101.13, 55.96. Anal. Calcd for C16H13ClO3⁄0.2 H2O: C, 65.74; H,
4.62. Found: C, 65.71; H, 4.388.
128.23, 127.93, 121.14, 106.31, 98.75, 56.13, 55.76. Anal. Calcd for
17H15ClO3: C, 67.44; H, 4.99. Found: C, 67.73; H, 5.183.
C
4.2.22. (E)-3-(4-Chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-
en-1-one (22)
Synthesized from 2,4-dimethoxyacetophenone and 4-chloro-
benzaldehyde, yield 72%. 1H NMR (DMSO-d6) d: 7.95 (dd, J = 7.1,
2.3 Hz, 1H), 7.79 (d, J = 15.6 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), (d,
J = 7.4 Hz, 2H), 7.43 (dd, J = 11.2, 7.7 Hz, 3H), 6.69 (d, J = 2.3 Hz,
1H), 6.65 (dd, J = 8.6, 2.3 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H). 13C
NMR (DMSO-d6) d: 189.08, 164.42, 161.22, 135.76, 133.99,
132.84, 132.26, 131.57, 130.78, 130.03, 128.74, 128.02, 121.12,
106.45, 98.64, 56.23, 55.68. Anal. Calcd for C17H15ClO3: C, 67.44;
H, 4.99. Found: C, 67.25; H, 5.02.
4.2.17. (E)-3-(4-Chlorophenyl)-1-(2-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (17)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 4-
chlorobenzaldehyde, yield 79%. 1H NMR (DMSO-d6) d: 13.34 (s,
1H), 8.27 (d, J = 9.1 Hz, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.94 (d,
J = 8.4 Hz, 2H), 7.79 (d, J = 15.5 Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H),
6.56 (dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 3.84 (s, 3H).
13C NMR (DMSO-d6) d: 191.87, 166.24, 165.85, 142.72, 135.40,
133.66, 132.90, 130.88, 129.07, 122.19, 114.01, 107.59, 101.08,
55.91. Anal. Calcd for C16H13ClO3: C, 66.56; H, 4.54. Found: C,
66.31; H, 4.54.
4.2.23. (E)-3-(3,4-Dichlorophenyl)-1-(2,4-
dimethoxyphenyl)prop-2-en-1-one (23)
Synthesized from 2,4-dimethoxyacetophenone and 3,4-dichlo-
robenzaldehyde, yield 67%. 1H NMR (DMSO-d6) d: 8.01 (d,
J = 2.0 Hz, 1H), 7.73 (dd, J = 8.4, 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz,
1H), 7.63–7.57 (m, 2H), 7.49 (d, J = 15.9 Hz, 1H), 6.68 (d,
J = 2.3 Hz, 1H), 6.64 (dd, J = 8.6, 2.3 Hz, 1H), 3.89 (s, J = 2.1 Hz,
3H), 3.85 (s, J = 1.9 Hz, 3H). 13C NMR (DMSO-d6) d: 189.23,
164.32, 160.56, 138.40, 136.02, 132.40, 132.24, 131.90, 131.17,
130.32, 129.27, 128.14, 121.33, 106.22, 98.80, 56.17, 55.78. Anal.
Calcd for C17H14Cl2O3: C, 60.55; H, 4.18. Found: C, 60.72; H, 4.23.
4.2.18. (E)-3-(3,4-Dichlorophenyl)-1-(2-hydroxy-4-
methoxyphenyl)prop-2-en-1-one (18)
Synthesized from 2-hydroxy-4-methoxyacetophenone and 3,4-
dichlorobenzaldehyde, yield 67%. 1H NMR (DMSO-d6) d: 13.29 (s,
1H), 8.31–8.28 (m, 2H), 8.09 (d, J = 15.5 Hz, 1H), 7.87 (dd, J = 8.4,
2.0 Hz, 1H), 7.76 (d, J = 15.5 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 6.57
(dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 3.85 (s, 3H). 13C
NMR (DMSO-d6) d: 191.75, 166.39, 165.96, 141.37, 135.58,
133.05, 133.02, 131.99, 131.13, 130.40, 129.48, 123.59, 114.03,
107.66, 101.08, 55.96. Anal. Calcd for C16H12Cl2O3: C, 59.46; H,
3.74. Found: C, 59.41; H, 3.79.
4.2.24. (E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (24)
Synthesized from 2-hydroxy-4,6-dimethoxyacetophenone and
4-methoxybenzaldehyde, yield 55%. 1H NMR (DMSO-d6) d: 13.75
(s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 16.1 Hz, 1H), 6.94 (d,
J = 8.8 Hz, 2H), 6.80 (d, J = 16.1 Hz, 1H), 6.31–6.27 (m, 2H), 3.78
(s, 3H), 3.70 (s, 6H). 13C NMR (DMSO-d6) d: 193.33, 161.93,
161.32, 158.12, 143.76, 130.40, 127.55, 127.06, 127.02, 125.04,
114.58, 111.45, 93.81, 91.26, 90.97, 55.93, 55.57, 55.46. Anal. Calcd
for C18H18O5⁄0.5 H2O: C, 67.64; H, 6.27. Found: C, 67.47; H, 6.00.
4.2.19. (E)-1-(2,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)prop-
2-en-1-one (19)
Synthesized from 2,4-dimethoxyacetophenone and 4-methoxy-
benzaldehyde, yield 85%. 1H NMR (DMSO-d6) d: 7.68–7.64 (m, 2H),
7.58 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 15.8 Hz, 1H), 7.39 (dd, J = 15.8,
0.7 Hz, 1H), 7.01–6.96 (m, 2H), 6.67 (d, J = 2.2 Hz, 1H), 6.65–6.61
(m, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.79 (d, J = 3.7 Hz, 3H). 13C
NMR (DMSO-d6) d: 189.51, 163.84, 161.15, 160.16, 141.45,
131.97, 130.23, 127.58, 124.92, 121.82, 114.59, 106.03, 98.80,
56.05, 55.69, 55.44. Anal. Calcd for C18H18O4: C, 72.47; H, 6.08.
Found: C, 72.58; H, 5.95.
4.2.25. (E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-4,6-
dimethoxy-phenyl)prop-2-en-1-one (25)
Synthesized from 2-hydroxy-4,6-dimethoxyacetophenone and
3,4-dimethoxybenzaldehyde, yield 71%. 1H NMR (DMSO-d6) d:
13.40 (s, 1H), 7.62 (d, J = 7.4 Hz, 2H), 7.29 (s, J = 6.1 Hz, 2H), 7.01
(d, J = 8.7 Hz, 1H), 6.14 (d, J = 2.3 Hz, 1H), 6.11 (d, J = 2.3 Hz, 1H),
3.88 (s, J = 2.9 Hz, 3H), 3.82 (s, J = 4.8 Hz, 3H), 3.81 (d, J = 2.1 Hz,
6H). 13C NMR (DMSO-d6) d: 192.41, 165.39, 165.36, 161.88,
151.28, 149.15, 143.13, 127.77, 125.38, 122.92, 111.97, 111.02,
106.63, 94.06, 91.18, 56.27, 56.17, 55.76, 55.67. Anal. Calcd for
4.2.20. (E)-1-(2,4-Dimethoxyphenyl)-3-(3,4-
dimethoxyphenyl)prop-2-en-1-one (20)
Synthesized from 2,4-dimethoxyacetophenone and 3,4-dime-
thoxybenzaldehyde, yield 76%. 1H NMR (DMSO-d6) d: 7.56 (d,
J = 8.5 Hz, 1H), 7.47 (d, J = 15.8 Hz, 1H), 7.39 (d, J = 15.7 Hz, 1H),
7.30 (d, J = 1.9 Hz, 1H), 7.26 (dd, J = 8.3, 2.0 Hz, 1H), 6.99 (d,
J = 8.3 Hz, 1H), 6.68 (d, J = 2.2 Hz, 1H), 6.63 (dd, J = 8.6, 2.2 Hz,
1H), 3.88 (s, 3H), 3.84 (s, J = 2.4 Hz, 3H), 3.80 (d, J = 7.4 Hz, 6H).
13C NMR (DMSO-d6) d: 189.79, 163.74, 160.08, 151.03, 149.12,
142.03, 131.85, 127.81, 125.21, 122.70, 121.93, 111.91, 110.95,
105.99, 98.85, 56.03, 55.73, 55.70,55.70. Anal. Calcd for C19H20O5:
C, 69.50; H, 6.14. Found: C, 69.29; H, 6.01.
C
19H20O6⁄0.33 H2O: C, 65.13; H, 5.95. Found: C, 65.00; H, 6.12.
4.2.26. (E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4,6-
dimethoxyphenyl)prop-2-en-1-one (26)
Synthesized from 2-hydroxy-4,6-dimethoxyacetophenone and
2-chlorobenzaldehyde, yield 68%. 1H NMR (DMSO-d6) d: 13.22 (s,
1H), 7.92–7.88 (m, 2H), 7.77 (d, J = 15.7 Hz, 1H), 7.57–7.54 (m,
1H), 7.46–7.42 (m, 2H), 6.15 (d, J = 2.3 Hz, 1H), 6.13 (d, J = 2.3 Hz,
1H), 3.88 (s, 3H), 3.82 (s, 3H). 13C NMR (DMSO-d6) d: 192.04,
165.94, 165.61, 162.08, 136.79, 134.10, 132.65, 131.79, 130.47,
130.20, 128.36, 128.01, 106.51, 94.06, 91.26, 56.38, 55.83. Anal.
Calcd for C17H15ClO4: C, 64.06; H, 4.74. Found: C, 63.69; H, 4.80.
4.2.21. (E)-3-(2-Chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-
en-1-one (21)
Synthesized from 2,4-dimethoxyacetophenone and 2-chloro-
benzaldehyde, yield 69%. 1H NMR (DMSO-d6) d: 7.92 (dd, J = 7.2,
2.3 Hz, 1H), 7.82 (d, J = 15.8 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.59
(d, J = 15.8 Hz, 1H), 7.56–7.53 (m, 1H), 7.43 (ddd, J = 6.7, 5.7,
1.8 Hz, 2H), 6.69 (d, J = 2.3 Hz, 1H), 6.65 (dd, J = 8.6, 2.3 Hz, 1H),
3.90 (s, 3H), 3.85 (s, 3H). 13C NMR (DMSO-d6) d: 189.06, 164.39,
160.57, 135.96, 134.07, 132.74, 132.30, 131.61, 130.13, 129.92,
4.2.27. (E)-1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)prop-
2-en-1-one (27)
Synthesized from 3,4-dimethoxyacetophenone and 4-methoxy-
benzaldehyde, yield 81%. 1H NMR (DMSO-d6) d: 7.87 (dd, J = 8.4,
1.9 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 15.5 Hz, 1H), 7.67

K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
353
(d, J = 15.5 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H),
7.01 (d, J = 8.7 Hz, 2H), 3.86 (d, J = 5.6 Hz, 6H), 3.81 (s, J = 3.5 Hz,
3H). 13C NMR (DMSO-d6) d: 187.41, 161.34, 153.22, 148.93,
143.15, 130.91, 130.78, 127.62, 123.27, 119.64, 114.51, 111.06,
110.97, 55.91, 55.76, 55.50. Anal. Calcd for C18H18O4: C, 72.47; H,
6.08. Found: C, 72.12; H, 6.20.
118.25, 114.63, 113.47, 55.57. Anal. Calcd for C20H16O3⁄0.25 H2O:
C, 77.95; H, 5.58. Found: C,77.83; H, 5.37.
4.2.33. (E)-3-(3,4-Dimethoxyphenyl)-1-(1-hydroxynaphthalen-
2-yl)prop-2-en-1-one (33)
Synthesized from 2-acetyl-1-hydroxynaphthalene and 3,4-
dimethoxybenzaldehyde, yield 79%. 1H NMR (DMSO-d6) d: 14.08
(s, 1H), 8.36 (d, J = 8.2 Hz, 1H), 8.25 (d, J = 9.0 Hz, 1H), 8.14 (d,
J = 15.4 Hz, 1H), 7.94 (d, J = 9.2 Hz, 3H), 7.92 (d, J = 3.9 Hz, 1H),
7.72 (m, 2H), 7.59 (d, J = 8.2, 1H), 7.47 (d, J = 8.9 Hz, 1H), 3.83 (d,
6H). 13C NMR (DMSO-d6) d: 193.60, 158.12, 150.2, 149.90,
144.56, 135.26, 130.93, 129.67, 127.68, 127.35, 126.52, 124.99,
123.25, 122.24, 121.93, 121.12, 120.83, 113.57, 110.67, 55.92,
55.57. Anal. Calcd for C21H18O4: C, 75.43; H, 5.43. Found: C,
75.37; H, 5.51.
4.2.28. (E)-1,3-Bis(3,4-dimethoxyphenyl)prop-2-en-1-one (28)
Synthesized from 3,4-dimethoxyacetophenone and 3,4-dime-
thoxybenzaldehyde, yield 81%. 1H NMR (DMSO-d6) d: 7.80 (d,
J = 15.5 Hz, 1H), 7.66–7.63 (m, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.41
(d, J = 2.0 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 2.3 Hz, 1H),
7.02 (d, J = 3.5 Hz, 1H), 6.76 (d, J = 1.0 Hz, 1H), 3.86–3.80 (m,
12H). 13C NMR (DMSO-d6) d: 197.32, 153.17, 153.09, 149.10,
148.61, 143.59, 130.88, 129.90, 123.59, 123.33, 122.74, 119.73,
111.72, 110.93, 110.43, 55.86, 55.81, 55.57, 55.54. Anal. Calcd for
C19H20O5: C, 69.50; H, 6.14. Found: C, 69.34; H, 6.23.
4.2.34. (E)-1-(1-Hydroxynaphthalen-2-yl)-3-(3-
methoxyphenyl)prop-2-en-1-one (34)
4.2.29. (E)-3-(2-Chlorophenyl)-1-(3,4-dimethoxyphenyl)prop-2-
en-1-one (29)
Synthesized from 2-acetyl-1-hydroxynaphthalene and 3-
methoxybenzaldehyde, yield 67%. 1H NMR (DMSO-d6) d: 14.05 (s,
1H), 8.33 (d, J = 8.7 Hz, 2H), 8.18 (d, J = 15.4 Hz, 1H), 7.93 (dd,
J = 15.4, 7.3 Hz, 3H), 7.86 (d, J = 8.8 Hz, 1H), 7.72 (dddd, J = 14.0,
8.2, 6.9, 1.3 Hz, 2H), 7.63–7.60 (m, 1H), 7.44–7.41 (m, 1H), 3.85
(s, 3H). 13C NMR (DMSO-d6) d: 219.48, 193.80, 163.41, 159.87,
147.20, 145.50, 137.26, 136.00, 130.36, 127.78, 126.34, 125.95,
124.60, 124.36, 123.81, 123.71, 121.45, 117.39, 113.88, 55.54. Anal.
Calcd for C20H16O3⁄0.33 H2O: C, 77.64; H, 5.68. Found: C, 77.59; H,
5.44.
Synthesized from 3,4-dimethoxyacetophenone and 2-chloro-
benzaldehyde, yield 73%. 1H NMR (DMSO-d6) d: 7.99 (d,
J = 4.4 Hz, 1H), 7.65 (dd, J = 8.5, 2.0 Hz, 1H), 7.52 (dd, J = 7.8,
1.7 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 7.9, 1.3 Hz, 1H),
7.25–7.22 (m, 1H), 7.18–7.14 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H),
3.83 (s, 3H), 3.79 (s, 3H). 13C NMR (DMSO-d6) d: 196.75, 153.22,
148.65, 141.75, 137.78, 133.08, 129.66, 129.41, 128.37, 127.75,
127.29, 124.94, 122.77, 110.98, 110.47, 55.85, 55.61. Anal. Calcd
for C17H15ClO3⁄0.25 H2O: C, 66.45; H, 5.08. Found: C, 66.67; H,
5.293.
4.2.35. (E)-3-(2-Chlorophenyl)-1-(1-hydroxynaphthalen-2-
yl)prop-2-en-1-one (35)
4.2.30. (E)-3-(4-Chlorophenyl)-1-(3,4-dimethoxyphenyl)prop-2-
en-1-one (30)
Synthesized from 2-acetyl-1-hydroxynaphthalene and 2-chlo-
robenzaldehyde, yield 72%. 1H NMR (DMSO-d6) d: 14.81 (s, 1H),
8.39–8.35 (m, 1H), 8.32 (dd, J = 7.5, 2.1 Hz, 1H), 8.29 (d, J = 9.0 Hz,
1H), 8.23 (d, J = 3.0 Hz, 2H), 7.93 (d, J = 8.1 Hz, 1H), 7.73 (ddd,
J = 8.2, 6.9, 1.3 Hz, 1H), 7.63–7.57 (m, 2H), 7.51–7.44 (m, 3H). 13C
NMR (DMSO-d6) d: 193.37, 163.51, 139.61, 137.33, 134.79,
132.53, 132.17, 130.78, 130.23, 129.12, 127.85, 127.78, 126.39,
125.17, 124.55, 123.97, 123.87, 118.52, 113.49. Anal. Calcd for
Synthesized from 3,4-dimethoxyacetophenone and 4-chloro-
benzaldehyde, yield 77%. 1H NMR (DMSO-d6) d: 7.95 (d,
J = 15.6 Hz, 1H), 7.92 (s, J = 1.9 Hz, 1H), 7.90 (dd, J = 8.5, 2.0 Hz,
2H), 7.68 (d, J = 15.6 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.51 (d,
J = 8.6 Hz, 2H), 7.10 (d, J = 8.5 Hz, 1H), 3.86 (d, J = 7.8 Hz, 6H). 13C
NMR (DMSO-d6) d: 187.38, 153.50, 148.99, 141.70, 134.99,
133.96, 130.63, 130.63, 130.55, 129.03, 129.03 123.62, 122.90,
111.08, 110.99, 55.95, 55.78. Anal. Calcd for C17H15ClO3: C, 67.44;
H, 4.99. Found: C, 67.19; H, 5.01.
C19H13ClO2: C, 73.91; H, 4.24. Found: C, 74.05; H, 4.19.
4.2.36. (E)-3-(4-Chlorophenyl)-1-(1-hydroxynaphthalen-2-
yl)prop-2-en-1-one (36)
4.2.31. (E)-1-(1-Hydroxynaphthalen-2-yl)-3-phenylprop-2-en-1-
one (31)
Synthesized from 2-acetyl-1-hydroxynaphthalene and 4-chlo-
robenzaldehyde, yield 76%. 1H NMR (DMSO-d6) d: 14.95 (s, 1H),
8.37 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 9.0 Hz, 1H), 8.20 (d, J = 15.5 Hz,
1H), 8.02 (d, J = 8.6 Hz, 2H), 7.95 (d, J = 5.2 Hz, 1H), 7.93 (d,
J = 1.6 Hz, 1H), 7.74–7.70 (m, 1H), 7.64–7.61 (m, 1H), 7.56 (d,
J = 8.5 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H). 13C NMR (DMSO-d6) d:
193.63, 163.40, 143.92, 137.25, 135.72, 133.57, 131.14, 130.67,
129.14, 127.76, 126.34, 125.17, 124.55, 123.80, 121.96, 118.43,
113.48. Anal. Calcd for C19H13ClO2: C, 73.91; H, 4.24. Found: C,
73.85; H, 4.33.
Synthesized from 2-acetyl-1-hydroxynaphthalene and benzal-
dehyde, yield 88%. 1H NMR (DMSO-d6) d: 15.00 (s, 1H), 8.37 (dd,
J = 8.3, 0.7 Hz, 1H), 8.30 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 15.5 Hz,
1H), 7.97 (dd, J = 6.2, 3.4 Hz, 2H), 7.93 (d, J = 8.3 Hz, 1H), 7.73
(ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.60 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H),
7.51–7.48 (m, 3H), 7.46 (d, J = 8.9 Hz, 1H). 13C NMR (DMSO-d6) d:
193.72, 163.37, 145.42, 137.22, 134.59, 131.19, 130.60, 129.45,
129.08, 127.74, 126.30, 125.17, 124.58, 123.78, 121.18, 118.38,
113.47. Anal. Calcd for C19H14O2: C, 83.19; H, 5.14. Found: C,
82.87; H, 5.26.
4.2.37. (E)-3-(3,4-Dichlorophenyl)-1-(1-hydroxynaphthalen-2-
yl)prop-2-en-1-one (37)
4.2.32. (E)-1-(1-Hydroxynaphthalen-2-yl)-3-(4-
methoxyphenyl)prop-2-en-1-one (32)
Synthesized from 2-acetyl-1-hydroxynaphthalene and 3,4-
dichlorobenzaldehyde, yield 64%, mp 161 °C. 1H NMR (DMSO-d6-
d6) d: 14.89 (s, J = 1.5 Hz, 1H), 8.37 (d, J = 1.9 Hz, 1H), 8.33 (d,
J = 9.1 Hz, 1H), 8.27 (d, J = 15.5 Hz, 1H), 7.94 (dd, J = 8.4, 2.3 Hz,
2H), 7.74 (d, J = 8.3 Hz, 2H), 7.71 (ddd, J = 8.2, 5.0, 1.3 Hz, 1H),
7.60 (m, J = 11.3, 8.2, 6.9, 1.2 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H). 13C
NMR (DMSO-d6) d: 193.57, 163.53, 142.58, 137.36, 135.49,
133.35, 132.09, 131.21, 130.81, 130.63, 129.78, 127.82, 126.43,
125.27, 124.56, 123.87, 123.38, 118.49, 113.54. Anal. Calcd for
Synthesized from 2-acetyl-1-hydroxynaphthalene and 4-
methoxybenzaldehyde, yield 84%. 1H NMR (DMSO-d6) d: 15.18 (s,
1H), 8.36 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 8.04 (d,
J = 15.4 Hz, 1H), 7.94 (d, J = 9.2 Hz, 3H), 7.92 (d, J = 3.9 Hz, 1H),
7.72 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H), 7.59 (ddd, J = 8.2, 5.0, 1.1 Hz,
1H), 7.45 (d, J = 8.9 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H).
13C NMR (DMSO-d6) d: 193.59, 163.26, 161.97, 145.58, 137.11,
131.53, 130.45, 127.71, 127.29, 126.22, 125.13, 123.73, 118.42,
C19H12Cl2O2: C, 66.49; H, 3.52. Found: C, 66.23; H, 3.61.

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K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
4.2.38. (E)-1-(2-Hydroxynaphthalen-1-yl)-3-phenylprop-2-en-1-
one (38)
4.2.43. (E)-3-(4-Chlorophenyl)-1-(2-hydroxynaphthalen-1-
yl)prop-2-en-1-one (43)
Synthesized from 1-acetyl-2-hydroxynaphthalene and benzal-
dehyde, yield 59%. 1H NMR (DMSO-d6) d: 8.23 (d, J = 8.5 Hz, 1H),
7.71 (d, J = 15.7 Hz, 1H), 7.58 (d, J = 7.3 Hz, 2H), 7.45–7.39 (m,
3H), 7.37 (t, J = 7.4 Hz, 2H), 7.33–7.28 (m, 1H), 7.16 (ddd, J = 8.4,
6.9, 1.5 Hz, 1H), 6.94–6.85 (m, 1H), 6.61 (d, J = 9.2 Hz, 1H). 13C
NMR (DMSO-d6) d: 190.38, 175.79, 136.69, 135.95, 134.84,
133.55, 132.31, 129.58, 128.85, 128.66, 127.87, 127.69, 126.22,
124.34, 123.14, 118.88, 116.93. Anal. Calcd for C19H14O2: C,
83.19; H, 5.14. Found: C, 83.04; H, 5.28.
Synthesized from 1-acetyl-2-hydroxynaphthalene and 4-chlo-
robenzaldehyde, yield 68%. 1H NMR (DMSO-d6) d: 10.00 (s, 1H),
8.16 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 7.67 (dd, J = 6.5,
5.1 Hz, 3H), 7.62 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.28
(d, J = 9.0 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 5.83 (dd, J = 13.2,
3.1 Hz, 1H). 13C NMR (DMSO-d6) d: 192.74, 163.20, 137.79,
134.31, 133.31, 130.95, 129.68, 129.04, 129.00, 128.83, 128.74,
128.65, 128.13, 125.12, 124.92, 119.11, 112.01. Anal. Calcd for
C19H13ClO2: C, 73.91; H, 4.24. Found: C, 73.68; H, 4.26.
4.2.44. 44(E)-3-(3,4-Dichlorophenyl)-1-(2-hydroxynaphthalen-
1-yl)prop-2-en-1-one (44)
4.2.39. (E)-1-(2-Hydroxynaphthalen-1-yl)-3-(4-
methoxyphenyl)prop-2-en-1-one (39)
Synthesized from 1-acetyl-2-hydroxynaphthalene and 4-chlo-
robenzaldehyde, yield 71%, mp 123 °C. 1H NMR (DMSO-d6) d:
8.42 (d, J = 8.5 Hz, 1H), 7.82 (dd, J = 12.2, 8.7 Hz, 2H), 7.59 (dd,
J = 11.2, 5.1 Hz, 2H), 7.41 (dd, J = 5.3, 4.1 Hz, 2H), 7.31 (d,
J = 15.7 Hz, 1H), 7.15 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 6.92–6.86 (m,
1H), 6.60 (d, J = 9.2 Hz, 1H). 13C NMR (DMSO-d6) d: 190.07,
176.41, 138.00, 136.07, 134.86, 133.78, 131.60, 131.40, 130.92,
130.44, 129.58, 129.13, 127.83, 127.54, 126.43, 124.40, 122.99,
119.00, 116.65. Anal. Calcd for C19H12Cl2O2: C, 66.49; H, 3.52.
Found: C, 66.60; H, 3.42.
Synthesized from 1-acetyl-2-hydroxynaphthalene and 4-
methoxybenzaldehyde, yield 62%. 1H NMR (DMSO-d6) d: 10.15 (s,
1H), 7.88 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.62 (d,
J = 8.8 Hz, 2H), 7.56 (dd, J = 8.4, 0.9 Hz, 1H), 7.41 (ddd, J = 8.4, 6.8,
1.4 Hz, 1H), 7.31 (dt, J = 8.0, 3.9 Hz, 1H), 7.28–7.22 (m, 2H), 7.10
(d, J = 16.1 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 3.78 (s, J = 1.5 Hz, 3H).
13C NMR (DMSO-d6) d: 196.35, 161.46, 152.86, 144.30, 131.66,
131.11, 130.57, 128.40, 128.30, 127.79, 127.19, 127.04, 126.70,
123.47, 123.19, 120.40, 118.53, 114.61, 114.08, 55.48. Anal. Calcd
for C20H16O3: C, 78.93; H, 5.30. Found: C, 78.76; H, 5.30.
4.3. Biological testing
4.2.40. (E)-3-(3,4-Dimethoxyphenyl)-1-(2-hydroxynaphthalen-
1-yl)prop-2-en-1-one (40)
4.3.1. Chemicals
Synthesized from 1-acetyl-2-hydroxynaphthalene and 3,4-
dimethoxybenzaldehyde, yield 68%. 1H NMR (DMSO-d6) d: 10.12
(s, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.54 (dd,
J = 8.4, 0.9 Hz, 1H), 7.41 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.33–7.30
(m, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 12.5, 9.5 Hz, 2H),
7.21–7.17 (m, 1H), 7.15 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 8.4 Hz,
1H), 3.77 (d, J = 2.8 Hz, 6H). 13C NMR (DMSO-d6) d: 196.59,
152.73, 151.32, 149.16, 144.94, 131.69, 130.95, 128.29, 127.77,
127.28, 127.15, 126.94, 123.45, 123.26, 123.16, 120.50, 118.55,
111.76, 110.92, 55.75, 55.72. Anal. Calcd for C21H18O4: C, 75.43;
H, 5.43. Found: C, 75.50; H, 5.35.
Ko143 was purchased from Tocris Bioscience (Bristol, United
Kingdom). Mitoxantrone hydrochloride was purchased from
Molekula (Gillingham, United Kingdom). All other chemicals were
purchased from Sigma–Aldrich Chemicals (Taufkirchen, Germany)
unless otherwise specified.
4.3.2. Cell culture
The breast cancer cell line MCF-7 MX was kindly provided by
Dr. E. Schneider (Wadsworth Center, Albany, NY, USA). The cells
were grown in RPMI-1640 medium supplemented with 20% fetal
bovine serum (FBS), 50
and 2 mM -glutamine.
lg/ml streptomycin, 50 U/ml penicillin G,
L
4.2.41. (E)-1-(2-Hydroxynaphthalen-1-yl)-3-(3-
methoxyphenyl)prop-2-en-1-one (41)
The cell line MDCK BCRP was a generous gift from Dr. A. Schin-
kel (The Netherlands Cancer Institute, Amsterdam, The Nether-
lands). MDCK BCRP cells were generated 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 fluo-
rescent protein. The cell line was grown in DMEM medium supple-
Synthesized from 1-acetyl-2-hydroxynaphthalene and 3-
methoxybenzaldehyde, yield 63%. 1H NMR (DMSO-d6) d: 8.29 (d,
J = 8.5 Hz, 1H), 7.72 (d, J = 15.7 Hz, 1H), 7.41 (d, J = 9.3 Hz, 2H),
7.36 (d, J = 15.7 Hz, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.16 (dd, J = 6.9,
1.4 Hz, 2H), 7.13–7.11 (m, 1H), 6.92–6.85 (m, 2H), 6.60 (d,
J = 9.2 Hz, 1H), 3.77 (s, 3H). 13C NMR (DMSO) d: 190.67, 159.70,
140.32, 138.24, 137.75, 136.00, 134.67, 133.48, 132.71, 129.84,
128.83, 127.84, 126.24, 124.34, 123.06, 120.13, 116.89, 114.46,
112.75, 55.23. Anal. Calcd for C20H16O3: C, 78.93; H, 5.30. Found:
C, 78.67; H, 5.46.
mented with 10% FBS, 50 lg/ml streptomycin, 50 U/ml penicillin G.
Human ovarian carcinoma cell lines A2780 and its corresponding
MDR1 overexpressing doxorubicin resistant A2780adr cell line
were purchased from European collection of animal cell cultures
(ECACC, Nos. 93112519 and 93112520, UK). The cell lines were
grown in RPMI-1640 medium supplemented with 10% FBS,
50 lg/ml streptomycin, 50 U/ml penicillin G, and 365 lg/ml L-glu-
4.2.42. (E)-3-(2-Chlorophenyl)-1-(2-hydroxynaphthalen-1-
yl)prop-2-en-1-one (42)
tamine. The human ovarian cancer cell line 2008 stably expressing
MRP1was used for MRP1 testing. The cell line 2008MRP1 was
grown in same medium as described for A2780 with addition of
Synthesized from 1-acetyl-2-hydroxynaphthalene and 2-chlo-
robenzaldehyde, yield 56%. 1H NMR (DMSO-d6) d: 8.53 (d,
J = 8.6 Hz, 1H), 7.82 (d, J = 15.6 Hz, 1H), 7.76 (dd, J = 7.3, 2.2 Hz,
1H), 7.65 (d, J = 15.6 Hz, 1H), 7.48 (dd, J = 7.0, 2.3 Hz, 1H), 7.40
(d, J = 9.1 Hz, 2H), 7.34–7.30 (m, 2H), 7.15 (ddd, J = 8.5, 6.8,
1.5 Hz, 1H), 6.87 (ddd, J = 7.9, 6.9, 1.2 Hz, 1H), 6.57 (d,
J = 9.2 Hz, 1H). 13C NMR (DMSO-d6) d: 190.19, 176.91, 136.23,
135.64, 134.82, 133.80, 133.20, 129.85, 129.77, 129.73, 128.76,
127.80, 127.67, 127.60, 126.46, 124.33, 122.92, 118.89, 116.50.
Anal. Calcd for C19H13ClO2: C, 73.91; H, 4.24. Found: C, 73.84;
H, 4.31.
400 lg/ml G-418 (Geneticin).
Cells were maintained in a 5% CO2 humidified atmosphere at
37 °C. After confluence of 80–90%, subculturing was performed
with 0.05% trypsin and 0.02% EDTA.
4.3.3. Accumulation assays
4.3.3.1. Hoechst 33342 assay.
performed as described earlier with small modification.17 The final
concentration of the Hoechst was kept at 1 M. Cell lines were cul-
tivated under standard conditions in T75- or T175-flasks. When
The Hoechst 33342 assay was
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K. Juvale et al. / Bioorg. Med. Chem. 20 (2012) 346–355
355
reaching a confluence of 80–90%, cells were harvested by gentle
trypsination (0.05% trypsin/0.02% EDTA) and then transferred to a
50 ml tube followed by centrifugation (266 g, 4 °C, 4 min). The cell
pellet obtained was resuspended in fresh culture medium and the
cell density was determined using a 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
cells were seeded into 96-well tissue culture plates (Sarstedt, New-
ton, USA) in the appropriate density evaluated for each cell line
(1250 cells per well for MDCK and 10,000 cells per well for MCF-
7 cells) in a volume of 100 ll and kept under 5% CO2 at 37 °C for
6 h. Attachment of cells was controlled by microscopy and the test
compound as well as the mitoxantrone or SN-38 were added to
achieve the required final concentration in a final volume of
200
was added (40
MTT was terminated after 1 h by removing the supernatants and
lysing the cells with 100 l DMSO per well. Viability of the cells
ll per well. After an incubation period of 72 h, the MTT reagent
ll of a 5 mg/mL solution per well). Incubation with
20,000 cells per well in a volume of 90 ll when using MCF-7 MX
or MDCK BCRP cells. The non-BCRP overexpressing counter parts
of these two cell lines MCF-7 and MDCK were used as controls to
identify unspecific interactions of the investigated substances
l
was measured spectrophotometrically by absorbance at 544 nm
and background corrected at 710 nm using a BMG POLARstar
microplate reader.
which could influence the detected fluorescence intensities. 10
of various test compounds in different concentrations were added
to a total volume of 100 l. The prepared 96 well plate was kept
under 5% CO2 and 37 °C for 30 min. After this preincubation period,
20 l of a 6 M Hoechst 33342 solution (protected from light) was
ll
l
Acknowledgements
l
l
We would like to thank Dr. A. H. Schinkel (The Netherlands Can-
cer Institute, Amsterdam, The Netherlands) for kindly providing
the MDCK BCRP cell line. KJ thanks DAAD (Germany) for financial
support.
added to each well.
Fluorescence was 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 applying a 37 °C tempered
BMG POLARstar microplate reader (BMG LABTECH, Offenburg,
Germany).
For the analyses of the data obtained from the assay, first fluores-
cence of KHB was subtracted from the fluorescence reading obtained
from MCF-7 or MDCK cells. Average of fluorescence values from
100 min to 109 min was calculated for each concentration and from
this data, concentration–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).
References and notes
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uptake assay.
Calcein-AM uptake assay was performed as de-
scribed earlier with small modifications.27 Cells (A2780adr for P-gp
and 2008 MRP1) were grown under standard conditions in T75-
flasks. After reaching confluence of approximately 80%, cells were
harvested by short trypsination (0.05% trypsin/0.02% EDTA). Pel-
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with a Casy I Modell TT cell counter (Schaerfe System GmbH,
Reutlingen, Germany). Cells were washed three times with
Krebs-HEPES buffer, and then seeded into colorless 96 well plates
(Greiner, Frickenhausen, Germany) at a density of approximately
30,000 cells in a volume of 90
compounds were added, resulting in a final volume of 100
well. The prepared 96 well plates were preincubated for 30 min.
After the preincubation period, 33 l of a 1.25 M Calcein AM solu-
tion which was protected from light were added to each well. The
fluorescence 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 micro-
plate reader tempered at 37 °C. For calculation of inhibitory effects
the first linear part of the fluorescence time curves was used.
ll per well. Then, 10
ll of the test
l
l per
l
l
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The influence of chalcones on
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MDCK and MCF-7 cells by MTT cytotoxicity assay. Assays were per-
formed as described earlier with minor modifications.28 Briefly,