Symmetric bis-chalcones as a new type of breast cancer resistance protein inhibitors with a mechanism different from that of chromones

Article abstract of DOI:10.1021/jm401879z

Potent ABCG2 inhibitors were recently identified as asymmetric chromones with different types of substituents. We here synthesized symmetric bis-chalcones that were differently substituted and screened for their ability to inhibit mitoxantrone efflux from

Full text of DOI:10.1021/jm401879z

Article
pubs.acs.org/jmc
Symmetric Bis-chalcones as a New Type of Breast Cancer Resistance
Protein Inhibitors with a Mechanism Different from That of
Chromones
Evelyn Winter,†,‡ Patrícia Devantier Neuenfeldt,§ Louise Domeneghini Chiaradia-Delatorre,§
Charlotte Gauthier,† Rosendo Augusto Yunes,§ Ricardo Jose Nunes,§ Tania Beatriz Creczynski-Pasa,‡
́
̂
and Attilio Di Pietro*,†
†Equipe Labellisee
́ ́
Ligue 2014, BMSSI UMR5086 CNRS/Universite Lyon 1, IBCP, 69367 Lyon, France
‡Department of Pharmaceutical Sciences, PGFAR, Federal University of Santa Catarina, 88040-900 Florianopolis, Santa Catarina,
Brazil
§Department of Chemistry, Federal University of Santa Catarina, 88040-900 Florianopolis, Santa Catarina, Brazil
ABSTRACT: Potent ABCG2 inhibitors were recently identi-
fied as asymmetric chromones with different types of
substituents. We here synthesized symmetric bis-chalcones
that were differently substituted and screened for their ability
to inhibit mitoxantrone efflux from ABCG2-transfected
HEK293 cells. Potent bis-chalcone inhibitors were identified,
the efficiency depending on both position of the central ketone
groups and the number and positions of lateral methoxy
substituents. The best derivative, namely, 1p, was selective for
ABCG2 over P-glycoprotein and MRP1, appeared not to be
transported by ABCG2, and was at least as active on various
drug-selected cancer cells overexpressing ABCG2. Compound
1p stimulated the ABCG2 basal ATPase activity by contrast to a chromone lead that inhibited it, suggesting different mechanisms
of interaction. Combination of both types of inhibitors produced synergistic effects, leading to complete inhibition at very low
concentrations.
nucleotide-binding domain and one transmembrane domain
with six α-helical spans. This transporter is present in various
membrane barriers protecting sensitive organs, as well as in
many types of cancer concerning breast, lung, pancreas, colon,
and leukemias.12,13 The first specific ABCG2 inhibitor of
natural origin was fumitremorgin C (FTC), which displayed a
serious neurotoxicity.14 Synthetic derivatives were developed
resulting in highly potent Ko143, which still retained some
residual toxicity.15,16 New-generation inhibitors of other types
have been developed, but very few have been tested with in
vivo animal models.17
Screening of different classes of flavonoids identified
interesting inhibitors such as hydrophobic flavones18,19 and
asymmetric chalcones with a variety of substituents20−22 for
establishing structure−activity relationships. The best com-
pound recently identified to be nearly as potent as Ko143 was
chromone 6g,23 which in this work was renamed as chromone
1 (Figure 1) for the comparative study with a number of
derivatives to characterize the inhibition mechanism toward
both drug efflux and ATPase activity.24 In the present work, to
better understand the structure−activity relationships and their
INTRODUCTION
■
One major obstacle for treating tumors by chemotherapy is
cancer cell multidrug resistance, which may be caused by
several factors including the overexpression of ATP-binding
cassette (ABC) transporters. These transporters are trans-
membrane proteins working as efflux pumps to reduce the
intracellular concentration of drugs.1,2 A total of 48 genes
encode ABC transporters in humans, of which only three are
recognized to be associated with low prognostic in cancer
patients: ABCB1/P-glycoprotein, ABCC1/MRP1 (multidrug
resistance protein 1), and ABCG2/BCRP (breast cancer
resistance protein).3 P-glycoprotein (P-gp) was the first
multidrug ABC transporter to be discovered and extensively
studied.4 MRP1 was later also associated with multidrug
resistance,5 while BCRP was more recently identified.6−8
One of the strategies to eliminate resistant tumors is to use
inhibitors of the multidrug ABC transporters. Combination of
inhibitors with anticancer drugs should increase drug
accumulation inside the cell. In vitro, a number of P-gp
inhibitors have been optimized up to third- or fourth-
generation compounds, but their intrinsic toxicity and low in
vivo activity prevented finalizing of clinical trials.9−11
ABCG2 is a protein composed of 655 aminoacids,
constituting a “half-transporter” containing only one cytosolic
Received: December 6, 2013
Published: March 10, 2014
© 2014 American Chemical Society
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are due to the resonance of the double-bond hydrogens
adjacent to the carbonyl group (Hα and Hβ). The 13C NMR
spectra of the compounds showed signals between 100.0 and
160.0 ppm due to the resonance of aryl and unsaturated
carbons. The signals at the region between 180 and 190 ppm
attributed to the carbon resonance of the CO group are
consistent with the expected structures.
BIOLOGICAL EVALUATION
■
Structure−Activity Relationships toward Inhibition of
ABCG2-Mediated Drug Efflux. The 33 synthesized sym-
metric bis-chalcones, constituting the series 1 and 2 that differ
by the central carbonyls position, were analyzed for their ability
to inhibit mitoxantrone efflux and then to increase its
accumulation in ABCG2-transfected HEK293 cells (Table 1).
The proximity of the carbonyl with the central phenyl ring in
the series 1 seems to be more favorable toward inhibition when
comparing 1i (96% inhibition at 5 μM, EC50 = 0.3 μM) to 2m
(41% inhibition), 1k (92% inhibition, EC50 = 0.4 μM) to 2o
(66% inhibition, EC50 = 1.8 μM), and 1e (96% inhibition, EC50
= 0.7 μM) to 2g (74% inhibition, EC50 = 1.3 μM). However, an
opposite effect was observed in 1b (43% inhibition) versus 2b
(73% inhibition) and in 1c (63% inhibition, EC50 = 2.1 μM)
versus 2d (97% inhibition, EC50 = 0.8 μM), suggesting that the
dependence on carbonyl position was modulated by the
substituents present on the lateral A and B phenyl rings.
In both series, the inhibition efficiency was highly dependent
on methoxy substituents, since all of the 14 most potent
compounds, for which the EC50 values were determined as
illustrated in Figure 2, contained one (2c and 2l), two (1c, 1e,
1p, 2d, 2e, 2g, and 2i), or three (1i, 1k, 1q, 2b, and 2o)
methoxy groups. This contrasted with the low efficiency
observed in their absence (in compounds 1d, 1f, 1g, 1h, 1j, 1m,
1n, 1o, 2f, 2h, 2j, 2k, and 2n).
The position of methoxy substituents was critical for activity,
with the following preference: position 2′ (2l, 95% inhibition,
EC50 = 1.1 μM) > 3′ (2c, 73% inhibition, EC50 = 2.3 μM) > 4′
(2a, 27% inhibition). It is notable that compounds with only
one methoxy group at position 4′ (2a and 1a) did not show a
significant activity. In addition, the substitution of the methoxy
group by a hydrophilic hydroxyl substituent at position 4′
clearly resulted in a negative effect on activity when comparing
2p (only 28% inhibition) and 1l (no inhibition) with 2d (97%
inhibition) and 1c (63% inhibition). Methoxy groups at
positions 6′ and 5′ were also favorable, since the inhibitor
leads of each series were 1p (2′,6′-dimethoxy, EC50 = 0.2 μM)
and 2i (3′,5′-dimethoxy, EC50 = 0.5 μM). Within the series 2,
except for 2l, the methoxy substituents at meta positions (3′
and 5′) appeared to bring the best contribution to inhibition
potency (in 2b, 2c, 2d, 2g, and 2i), in contrast to the series 1
where the ortho positions (2′ and 6′) were better (in 1e, 1i, 1k,
1p, and 1q). This might indicate the role of electron density of
the rings or the contribution of steric effects.
Figure 1. Structures of the previously published chromone 123 and the
two series of novel bis-chalcones.
influence on the interaction with ABCG2 protein, we
synthesized two series of symmetric bis-chalcones (series 1
and series 2), by varying the position of central carbonyls and
the number and nature (essentially methoxy) of substituents on
the lateral phenyl rings (Figure 1). The best compounds were
found to be potent and selective and, interestingly, to bind to a
different site than chromone 1 allowing combinations.
CHEMISTRY
■
The access to series 2 is shown in Table 1. Bis-chalcones 2a−p
were synthesized according to a previously reported method of
chalcones synthesis.25 These compounds were obtained by the
reaction between terephthalaldehyde with substituted aceto-
phenones, using 50% KOH in methanol, to provide the final
compounds with yields in the range 62−92%. All reagentes
were commercially available except 2,4,5-trimethoxyacetophe-
none (synthesized as previously described with 81% yield)26
and 2,4,6-trimethoxyacetophenone (synthesized as previously
described with 85% yield).27
The preparation scheme of derivatives belonging to series 1
is also shown in Table 1. The reactions conditions were the
same as those used for the series 2 of bis-chalcones. The 1,4-
diacetylbenzene reacts with substituted benzaldehydes in the
presence of 50% KOH and methanol, providing analogues 1a−
q with yields in the range 59−93%.
The bis-chalcones 1a, 1c, 1f, 1h, 1k, 1l, 1n, 2a, 2d, 2e, 2h, 2j,
2k, 2o, and 2p have already been reported in the literature,28
and 1b, 1d, 1e, 1g, 1i, 1j, 1m, 1o, 1p, 1q, 2b, 2c, 2f, 2g, 2i, 2l,
2m, and 2n are novel compounds.
All synthesized compounds were fully characterized by
Inhibition of Cancer Cells Overexpressing ABCG2,
Selectivity toward ABCG2, and Chemosensitization of
Cell Growth. The action mechanism of symmetric bis-
chalcones was further investigated with the lead compound of
each series (1p and 2i). The inhibitory effects were first
evaluated in mitoxantrone-selected cancer cell lines, which were
demonstrated to overexpress ABCG2, namely, lung cancer
H46029 and H2330 cell lines and pancreatic PANC-1 cell line.31
Table 2 shows that 1p displayed the same efficiency on H23
and PANC-1 (EC50 = 0.2 μM) and an even 3-fold higher
1
melting points, H NMR, and 13C NMR and confirmed by
mass spectrometry (see the Experimental Section). High
resolution mass spectra are in agreement with the exact mass,
1
confirming ≥95% purity. In H NMR spectra of compounds,
the aromatic hydrogens appear at 6.5−8.5 ppm depending on
the substituent on the aromatic (Ar) group. The singlet signals
at the region between 7.5 and 8.5 ppm are attributed to the
resonance of the 1,4-phenylene ring hydrogens. The doublet
signals with J ≈ 16 Hz at the region between 7.0 and 8.0 ppm
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Table 1. Inhibition of ABCG2-Mediated Mitoxantrone Efflux by Differently Substituted Bis-chalcones
a
(i) Methanol, 50% KOH, rt, 16 h. The asterisk (∗) indicates novel compounds. Mitoxantrone efflux was determined by measuring its cellular
accumulation by flow cytometry, relative to ABCG2-negative control cells. For high-affinity compounds, the maximal inhibition was observed at 5
b
μM. EC50 values were calculated by GraphPad Prism5 as the bis-chalcone concentrations producing half-maximal inhibition after incubating the
cells with increasing concentrations up to 10 μM. Data are the mean SD of at least three independent experiments.
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Figure 2. Concentration dependence of mitoxantrone-efflux inhibition by symmetric bis-chalcones in HEK293-tranfected cells. The curves are fitted
with GraphPad Prism5 and illustrate the EC50 values of the best compounds identified in Table 1. Data are the mean SD of at least three
independent experiments.
(Figure 3). This indicated that these bis-chalcones behave as
ABCG2-selective inhibitors.
Table 2. Ability of the Bis-chalcone Leads to Inhibition of
Different Cancer Cell Lines Overexpressing ABCG2
a
The inhibition by 1p and 2i on ABCG2-mediated
mitoxantrone efflux was confirmed by their ability to sensitize
to mitoxantrone toxicity the cell growth of ABCG2-transfected
cells, as monitored by MTT assays (Figure 4). The IC50
(cytotoxic concentration for 50% cell survival) of mitoxantrone
in ABCG2-transfected cells (26.6 nM) was decreased to 7.2 or
7.6 nM, respectively, in the presence of 1p or 2i at low
concentrations, namely, 0.1 and 0.5 μM, which were not
cytotoxic. These results showed that symmetric bis-chalcones
can fully chemosensitize the ABCG2-overexpressing cells, since
mitoxantrone presented an IC50 of 10 nM in control, sensitive
HEK293 cells. At increasing concentrations, the 1p and 2i
cytotoxicity on cell growth was not lower in ABCG2-transfected
cells than in control cells, indicating no cross-resistance, which
suggested the lack of any apparent transport by ABCG2 (data
not shown).
EC50 (μM)
ABCG2-overexpressing cancer cells
1p
2i
mitoxantrone-selected H460
mitoxantrone-selected H23
mitoxantrone-selected PANC-1
0.08 0.01
0.22 0.11
0.21 0.18
1.63 0.08
1.17 0.71
0.21 0.18
a
The EC50 values of inhibition, on ABCG2-mediated mitoxantrone
efflux, by both bis-chalcone leads were determined on various drug-
selected cancer cell lines, as described in Table 1 for transfected cells.
potency in H460 cells, with a very low EC50 value of 80 nM. As
for ABCG2-transfected cells, 2i seemed to be less potent than
1p, but the EC50 values remained in the micromolar range
(0.2−1.6 μM).
The two bis-chalcone leads (1p and 2i) were not able, at up
to 10 μM, to inhibit the drug-efflux activity of either P-
glycoprotein toward mitoxantrone or MRP1 toward calcein
Effects on Basal and Drug-Stimulated ATPase
Activity. The effects of the bis-chalcone leads 1p and 2i
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Figure 3. Effects of the bis-chalcone leads on drug efflux mediated by either P-glycoprotein or MRP1. Each bis-chalcone was assayed up to 10 μM for
its ability to alter the P-glycoprotein-mediated mitoxantrone efflux in ABCB1-transfected NIH-3T3 (A) or the MRP1-mediated calcein efflux in
ABCC1-transfected HEK293 cells (B), as determined by flow cytometry. The inhibition was calculated relative to control cells, not expressing the
transporter, displaying by a maximal drug accumulation.
Figure 4. Sensitization of cell growth to mitoxantrone. The viability of
HEK293-ABCG2 cells was evaluated by MTT assays after a 72 h
incubation with mitoxantrone (at 0−25 nM) in the absence of bis-
chalcone (filled circles) or with 0.1 μM 1p (filled triangles) or 0.5 μM
2i (empty circles). Parallel experiments were performed without bis-
chalcone and in HEK293-pcDNA3.1 (empty squares).
were studied on the ATPase activity of Sf9 insect cell
membranes overexpressing human ABCG2,32 which were
supplemented with cholesterol to generate a maximal
vanadate-sensitive activity.33 The “basal” ATPase activity,
measured in the absence of substrate drug, was strongly
inhibited by Ko143 and chromone 1 (Figure 5A). Interestingly,
it was not inhibited but, on the contrary, stimulated by 1p and
2i 1.6-fold and 1.8-fold respectively, similar to the transported
substrate quercetin, which produced a 3.6-fold stimulation.
The “coupled”, or “drug-stimulated”, ATPase activity
observed in the presence of 2 μM quercetin was highly
dependent on 1p concentration, a complete inhibition being
produced with an IC50 value (concentration producing 50%
inhibition of activity) of around 0.19 μM (Figure 5B). For 2i, a
slightly lower-affinity inhibition was observed (IC50 = 0.27
μM), and the maximal inhibition was limited to 72%.
Figure 5. Effects of 1p and 2i on ABCG2 basal and substrate-
stimulated ATPase activity. The effects of the different compounds on
vanadate-sensitive ATPase activity of ABCG2 were tested using 10 μg/
sample of ABCG2-containing membrane vesicles (prepared from Sf9
cells overexpressing human ABCG2 and loaded with cholesterol). The
experiments were performed in the absence of transport substrate (A)
or with 2 μM quercetin, where the coupled ATPase activity was the
difference between total and basal activities (B). The specific basal and
coupled ATPase activities, taken as 100%, were respectively 7.0 0.4
and 18.3 0.5 nmol of ATP hydrolyzed/min × mg of proteins.
Combination of 1p with Inhibitors of Different
Mechanism. The stimulation produced by 1p on the basal
ATPase activity suggested a distinct binding site compared with
two other potent inhibitors, Ko143 and chromone 1, that
inhibit such activity. The inhibition affinity of 1p was therefore
investigated in combination with low concentrations of either
chromone 1 or Ko143 (0.1 or 0.02 μM, respectively, producing
around 50% inhibition). Interestingly, synergistic effects were
The less potent inhibition of drug efflux produced by bis-
chalcones, especially 2i, by comparison to chromone 1 and
Ko143 might be related, at least partly, to their stimulation, in
contrast to inhibition, of basal ATPase activity.
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obtained in all cases: the highest effect was observed with
chromone 1, which increased by 15-fold the inhibition affinity
of 1p (Table 3), while a lower effect was induced by Ko143
different interaction mechanism than the asymmetric inhibitors
chromone 1 and Ko143.
Synergistic Combinations of Asymmetric and Sym-
metric Inhibitors at Low Concentrations. The synergistic
inhibitions observed for all combinations of inhibitors at low
concentrations, as also observed for some P-glycoprotein
inhibitors,42 suggest that both types of inhibitors may bind to
distinct sites within ABCG2, which however requires to be
further proven by direct structural data. The highest synergism,
observed with a low concentration of chromone 1 increasing to
15-fold the inhibition affinity toward 1p, might be due to a
proximity of the two inhibitory sites. This agrees with the
recent observation that chromone 1 binding could be shifted
upon a slight substituent modification leading to stimulation of
basal ATPase activity.24 Such a high synergism presents the
great advantage to reach a complete inhibition of drug efflux by
using very low concentrations of both inhibitors, within the
nanomolar range. This should be actually useful for future in
vivo assays in animal models, by limiting cytotoxicity and
related side effects. This should further improve the already
promising in vivo activity in mice of chromone 1 to sensitize
xenografted tumor growth to chemotherapy.
Table 3. Synergistic Combinations of 1p with Other
a
Inhibitors
studied
inhibitor
increased affinity
additional inhibitor
EC50 (μM)
(fold)
1p alone
0.227 0.043
+chromone 1
(0.1 μM)
0.015 0.010**
15
+Ko143
(0.02 μM)
0.126 0.025*
1.8
chromone 1
alone
0.094 0.001
+1p (0.1 μM)
+1p (0.1 μM)
0.031 0.015**
0.029 0.008
0.012 0.007
3.0
2.4
Ko143 alone
a
The EC50 value of inhibition, on ABCG2-mediated mitoxantrone
efflux from transfected HEK293 cells, was determined first for each
inhibitor used alone and then in the presence of the indicated
additional inhibitor producing around 50% inhibition. The increased
affinity of inhibition was the ratio of EC50 values without inhibitor to
EC50 with additional inhibitor. Data are the mean SD of at least
three independent experiments. The t-test compared all combinations
including additional inhibitors with the studied inhibitor alone: (∗) p <
0.05 and (∗∗) p < 0.01.
EXPERIMENTAL SECTION
■
Chemistry. Preparation of Compounds. Reagents used were
obtained commercially (Sigma-Aldrich), except 2,4,5-trimethoxyace-
tophenone and 2,4,6-trimethoxyacetophenone, synthesized as pre-
viously described.26,27
(1.8-fold increase). Similarly, a low concentration of 1p (0.1
μM) was able to increase the inhibition affinity of chromone 1
(by 3-fold) and of Ko143 (by 2.4-fold).
General Procedure for the Preparation of the Series 1 of Bis-
chalcones. Bis-chalcones 1a−q were prepared by aldolic condensa-
tion between 1,4-diacetylbenzene (1.0 mmol) and corresponding
benzaldehydes (2.0 mmol) in methanol (25 mL) and KOH (50% w/
v) at room temperature and with magnetic stirring for 24 h. Distilled
water and 10% hydrochloric acid solution were added for total
precipitation of the compounds, which were obtained by vacuum
filtration and then recrystallized in ethanol. The purity of the
synthesized bis-chalcones 1a−q was analyzed by melting points and by
thin−layer chromatography (TLC) using Merck silica precoated
aluminum plates of 200 μm thickness, with several solvent systems of
different polarities. Compounds were visualized with ultraviolet light
(λ = 254 and 360 nm) and using sulfuric vanillin solution followed by
heat application as developing agent.
DISCUSSION
■
The present analysis of 33 bis-chalcones has shown for the first
time the interest of symmetric, methoxy-substituted, inhibitors
of ABCG2 through their inhibition potency, even on cancer cell
lines, their selectivity and apparent absence of transport, and
finally their different binding site and/or inhibition mechanism
versus other known inhibitors allowing synergistic combina-
tions at low concentrations.
Originality of Symmetric ABCG2 Inhibitors. Our finding
that the best symmetric bis-chalcones behave as potent ABCG2
inhibitors, with submicromolar EC50 values, is consistent with
the high-affinity inhibition produced by dimeric inhibitors on P-
glycoprotein.34,35 Of special interest were flavonoid dimers
shown to be active on both P-glycoprotein36,37 and MRP1,38
bringing attention to their potential application to ABCG2. The
positive role of methoxy substituents toward ABCG2 inhibition
agrees with previous observations with methoxy trans-
stilbenes,39 asymmetric chalcones,20,21 and chromones.23
However, as shown for P-glycoprotein, other structural
parameters in addition to methoxy substituents might be
critical for inhibition.40−42 Bis-chalcones behave as selective
inhibitors of ABCG2 over P-glycoprotein and MRP1, as for
other flavonoidic compounds, and appear not to be transported
on the basis of the absence of cross-resistance, in contrast to
transport substrates.
General Procedure for the Preparation of the Series 2 of Bis-
chalcones. Bis-chalcones 2a−p were prepared by aldolic condensa-
tion between terephthaldeyde (1.0 mmol) and corresponding
acetophenones (2.0 mmol) in methanol (25 mL) and KOH (50%
w/v) at room temperature and with magnetic stirring for 24 h.
Distilled water and 10% hydrochloric acid solution were added for
total precipitation of the compounds, which were obtained by vacuum
filtration and then recrystallized in ethanol. The purity of the
synthesized bis-chalcones 2a−p was analyzed in the same way as for
the series 1.
Physicochemical Data of the Synthesized Compounds. All
1
compounds were characterized by melting points (mp) and H and
13C nuclear magnetic resonance spectroscopy (NMR) and confirmed
by mass spectrometry. Melting points were determined with a
1
́
Microquimica MGAPF-301 apparatus and are uncorrected. NMR ( H
and 13C) spectra were recorded on a Bruker Ac-200F (200 MHz) or
on a Varian Oxford AS-400 (400 MHz) instrument, using
tetramethylsilane as internal standard.
High-resolution mass spectra were recorded on a micrOTOF-QII
(Bruker Daltonics) mass spectrometer equipped with an automatic
syringe pump (KD Scientific) for sample injection (constant flow of 3
μL/min), by positive mode of electron spray ionization (ESI-MS)
technique (4.5 kV and 180 °C). An acetonitrile/methanol mixture was
used as solvent. The instrument was calibrated in the range m/z 50−
3000 using an internal calibration standard (low concentration tuning
The main originality of bis-chalcones 1p and 2i versus other
known potent inhibitors, such as Ko143 and chromone 1 which
inhibit the basal ATPase activity,24 is that they stimulate such
an activity, similar to transport substrates.24 Therefore, the bis-
chalcone leads appear to mimic substrates without being
transported. This indicates that symmetric bis-chalcones obey a
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mix solution), which is supplied from Agilent Technologies. Data were
processed via Bruker DataAnalysis software (version 4.0). When the
calculated and experimental masses were compared, the error was as
expected (<2 ppm), confirming ≥95% of purity for all compounds.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(4-methoxyphenyl)prop-
121.9 (Cα, Cα″). HRMS (APPI+) m/z calculated for C32H22O2 [M+],
439.1693; found, 439.1689.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(naphthalen-1-yl)prop-2-
1
en-1-one) (1g). Yield 81%, yellow solid, mp 230−231 °C. H NMR
(200 MHz, CDCl3) δ/ppm 8.72 (d, 2H, Hβ, Hβ″, J = 15.4 Hz), 8.21
(s, 4H, H2′, H3′, H5′, H6′), 7.65 (d, 2H, Hα, Hα″, J = 15.5 Hz),
8.29−7.55 (m, 14H, naphthyl ring). 13C NMR (50, MHz, CDCl3) δ/
ppm 189.8 (CO), 142.6 (Cβ, Cβ″), 141.3 (C1′, C4′), 133.7 (C1,
C1″), 132.0 (C4a, C4a″), 131.7 (C8a, C8a″), 128.7 (C2′, C3′, C4′,
C6′), 127.0 (C4, C4″), 126.3 (C7, C7″), 125.4 (C3, C3″), 125.2 (C8,
C8″), 124.4 (Cα, Cα″), 123.3 (C2, C2″). HRMS (APPI+) m/z
calculated for C32H22O2 [M+], 439.1693; found, 439.1699.
1
2-en-1-one) (1a). Yield 79%, yellow solid, mp 210−210.5 °C. H
NMR (200 MHz, CDCl3) δ/ppm 8.10 (s, 4H, H2′, H3′, H5′, H6′),
7.88 (d, 2H, Hβ, Hβ″, J = 15.5 Hz), 7.63 (d, 4H, H2, H2″, H6, H6″, J
= 8.8 Hz), 7.41 (d, 2H, Hα, Hα″, J = 15.6 Hz), 6.96 (d, 4H, H3, H3″,
H5, H5″, J = 8.8 Hz), 3.87 (s, 6H, p-OCH3). 13C NMR (50 MHz,
CDCl3) δ/ppm 190.1 (CO), 161.9 (C4, C4″), 145.5 (Cβ, Cβ″),
141.4 (C1′, C4′), 130.7 (C2, C2″, C6, C6″), 128.5 (C2′, C3′, C5′,
C6′), 127.4 (C1, C1″), 119.6 (Cα, Cα″), 111.4 (C3, C3″, C5, C5″),
55.4 (p-OCH3). HRMS (APPI+) m/z calculated for C26H22O4 [M+],
399.1591; found, 399.1594.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one) (1b). Yield 82%, yellow solid, mp 223−224 °C. 1H
NMR (400 MHz, CDCl3) δ/ppm 8.12 (s, 4H, H2′, H3′, H5′, H6′),
7.75 (d, 2H, Hβ, Hβ″, J = 15.6 Hz), 7.40 (d, 2H, Hα, Hα″, J = 15.6
Hz), 6.89 (s, 4H, H2, H2″, H6, H6″), 3.94 (s, 12H, m-OCH3), 3.92 (s,
6H, p-OCH3). 13C NMR (100 MHz, CDCl3) δ/ppm 190.1 (CO),
153.5 (C3, C3″, C5, C5″), 146.0 (Cβ, Cβ″), 141.3 (C1′, C4′), 130.0
(C4, C4″), 128.6 (C2′, C3′, C5′, C6′), 121.2 (Cα, Cα″), 105.8 (C2,
C2″, C6, C6″), 104.9 (C1, C1″), 61.0 (p-OCH3), 58.2 (m-OCH3).
HRMS (APPI+) m/z calculated for C30H30O8 [M+], 519.2013; found,
519.2019.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-phenylprop-2-en-1-one)
1
(1h). Yield 92%, light yellow solid, mp 200−201 °C. H NMR (200
MHz, CDCl3) δ/ppm 8.12 (s, 4H, H2′, H3′, H5′, H6′), 7.85 (d, 2H,
Hβ, Hβ″, J = 15.6 Hz), 7.67 m, 4H, H2, H2″, H6, H6″), 7.53 (d, 2H,
Hα, Hα″, J = 15.6 Hz), 7.46 (m, 6H, H3, H3″, H4, H4″, H5, H5″).
13C NMR (50 MHz, CDCl3) δ/ppm 190.0 (CO), 145.8 (Cβ, Cβ″),
141.3 (C1′, C4′), 134.6 (C1, C1″), 130.8 (C4, C4″), 129.0 (C2, C2″,
C6, C6″), 128.6 (C2′, C3′, C5′, C6′), 128.5 (C3, C3″, C5, C5″),
121.9 (Cα, Cα″). HRMS (APPI+) m/z calculated for C24H18O2 [M+],
339.1380; found, 339.1376.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(2,4,5-trimethoxyphenyl)-
prop-2-en-1-one) (1i). Yield 90%, yellow solid, mp 224.5−225 °C.
1H NMR (200 MHz, CDCl3) δ/ppm 8.09 (s, 4H, H2′, H3′, H5′,
H6′), 8.12 (d, 2H, Hβ, Hβ″, J = 15.6 Hz), 7,46 (d, 2H, Hα, Hα″, J =
15.6 Hz), 7.14 (s, 2H, H6, H6″), 6.53 (s, 2H, H3, H3″), 3.96 (s, 6H, p-
OCH3), 3.92 (s, 12H, o-CH3, m-OCH3). 13C NMR (100 MHz,
CDCl3) δ/ppm 190.8 (CO), 154.8 (C2, C2″), 152.8 (C4, C4″),
143.2 (C5, C5″), 141.6 (C1, C1″), 141.0 (C1′, C4′), 128.5 (C2′, C3′,
C5′, C6′), 120.1 (Cα, Cα″), 111.3 (C6, C6″), 96.6 (C3, C3″), 56.5
(p-OCH3), 56.0 (o-OCH3, m-OCH3). HRMS (APPI+) m/z calculated
for C30H30O8 [M+], 519.2013; found, 519.2011.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(3,4-dimethoxyphenyl)-
prop-2-en-1-one) (1c). Yield 84%, yellow solid, mp 176.5−177 °C.
1H NMR (400 MHz, CDCl3) δ/ppm 8.11 (s, 4H, H2′, H3′, H5′,
H6′), 7.79 (d, 2H, Hβ, Hβ″, J = 15.6 Hz), 7.39 (d, 2H, Hα, Hα″, J =
15.6 Hz), 7.27 (dd, 2H, H6, H6″, J = 8.2 Hz, J = 1.9 Hz), 7.18 (d, 2H,
H2, H2″, J = 1.9 Hz), 6.92 (d, 2H, H5, H5″, J = 8.2 Hz), 3.97 (s, 6H,
m-OCH3), 3.95 (s, 6H, p-OCH3). 13C NMR (50 MHz, CDCl3) δ/
ppm 190.1 (CO), 151.7 (C3, C3″), 149.3 (C4, C4″), 145.9 (Cβ,
Cβ″), 141.4 (C1′, C4′), 128.5 (C2′, C3′, C5′, C6′), 127.6 (C1, C1″),
123.4 (Cα, Cα″), 119.8 (C6, C6″), 111.1 (C2, C2″), 110.2 (C5, C5″),
55.9 (m-OCH3, p-OCH3). HRMS (APPI+) m/z calculated for
C28H26O6 [M+], 459.1802; found, 459.1808.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(benzo[d][1,3]dioxol-5-yl)-
prop-2-en-1-one) (1d). Yield 76%, yellow solid, mp 268−269 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 8.29 (s, 4H, H2′, H3′, H5′, H6′),
7.85 (m, 4H, Hβ, Hβ″, Hα, Hα″), 7.70 (s, 2H, H2, H2″), 7.37 (d, 2H,
H6, H6″, J = 8.0 Hz), 7.02 (d, 2H, H5, H5″, J = 7.9 Hz), 6.13 (s, 4H,
CH2, CH2″). 13C NMR (50 MHz, CDCl3) δ/ppm 189.1 (CO),
150.2 (C3, C3″), 148.5 (C4, C4″), 145.3 (Cβ, Cβ″), 141.2 (C1′, C4′),
129.5 (C1, C1″), 129.1 (C2′, C3′, C5′, C6′), 126.7 (Cα, Cα″), 120.4
(C6, C6″), 109.0 (C5, C5″), 107.5 (C2, C2″), 102.1 (CH2, CH2″).
HRMS (APPI+) m/z calculated for C26H18O6 [M+], 427.117 61;
found, 427.117 58.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(4-carboxyphenyl)prop-2-
1
en-1-one) (1j). Yield 61%, light yellow solid, mp >350 °C. H NMR
(200 MHz, DMSO-d6) δ/ppm 8.33 (s, 2H, COOH), 8.29 (d, 4H, H2,
H2″, H6, H6″, J = 8.4 Hz), 8.12 (d, 4H, H3, H3″, H5, H5″, J = 8.5
Hz), 8.05 (m, 2H, Hβ, Hβ″), 8.03 (s, 4H, H2′, H3′, H5′, H6′), 7.84
(d, 2H, Hα, Hα″, J = 15.6 Hz). 13C NMR (100 MHz, DMSO-d6) δ/
ppm 189.4 (CO), 167.2 (COOH), 143.7 (Cβ, Cβ″), 141.0 (C1′,
C4′), 139.0 (C1, C1″), 132.7 (C4, C4″), 130.1 (C2′, C3′, C5′, C6′),
129.4 (C3, C3″, C5, C5″), 128.9 (C2, C2″, C6, C6″), 124.6 (Cα,
Cα″). HRMS (APPI+) m/z calculated for C26H18O6 [M+], 425.1020;
found, 425.1023.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(2,4,6-trimethoxyphenyl)-
prop-2-en-1-one) (1k). Yield 83%, yellow solid, mp 278−279 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 8.28 (d, 2H, Hβ, Hβ″, J = 15.9 Hz),
8.07 (s, 4H, H2′, H3′, H5′, H6′), 7.87 (d, 2H, Hα, Hα″, J = 15.8 Hz),
6.15 (s, 4H, H3, H3″, H5, H5″), 3.92 (s, 12H, o-OCH3), 3.87 (s, 6H,
p-OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 191.8 (CO), 161.8
(C2, C2″, C6, C6″), 158.4 (C4, C4″), 142.1 (Cβ, Cβ″), 136.8 (C1′,
C4′), 128.4 (C2′, C3′, C5′, C6′), 125.9 (Cα, Cα″), 106.6 (C1, C1″),
90.5 (C3, C3″, C5, C5″), 55.7 (o-OCH3), 55.2 (o-OCH3). HRMS
(APPI+) m/z calculated for C30H30O8 [M+], 519.2013; found,
519.2009.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(2,5-dimethoxyphenyl)-
prop-2-en-1-one) (1e). Yield 82%, yellow solid, mp 170−170.5 °C.
1H NMR (400 MHz, CDCl3) δ/ppm 8.10 (d, 2H, Hβ, Hβ″, J = 16.0
Hz), 8.10 (s, 4H, H2′, H3′, H5′, H6′), 7.59 (d, 2H, Hα, Hα″, J = 15.6
Hz), 7.18 (d, 2H, H2, H2″, J = 3.0 Hz), 6.97 (dd, 2H, H4, H4″, J = 8.9
Hz, J = 3.0 Hz), 6.89 (d, 2H, H5, H5″, J = 8.9 Hz), 3.88 (s, 6H, o-
OCH3), 3.83 (s, 6H, m-OCH3). 13C NMR (100 MHz, CDCl3) δ/ppm
190.7 (CO), 153.4 (C6, C6″), 141.3 (Cβ, Cβ″), 141.1 (C1′, C4′),
128.6 (C2′, C3′, C5′, C6′), 124.1 (Cα, Cα″), 122.9 (C1, C1″), 117.6
(C5, C5″), 113.8 (C4, C4″), 112.4 (C2, C2″), 56.1 (o-OCH3), 55.8
(m-OCH3). HRMS (APPI+) m/z calculated for C28H26O6 [M+],
459.180 22; found, 459.180 16.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one) (1l). Yield 60%, brown solid,
1
mp 140−141.5 °C. H NMR (200 MHz, CDCl3) δ/ppm 8.07 (s, 4H,
H2′, H3′, H5′, H6′), 7.77 (d, 2H, Hβ, Hβ″, J = 15.6 Hz), 7.35 (d, 2H,
Hα, Hα″, J = 15.6 Hz), 7.24 (dd, 2H, H6, H6″, J = 8.2 Hz, J = 1.9 Hz),
7.15 (d, 2H, H2, H2″, J = 1.8 Hz), 6.97 (d, 2H, H5, H5″, J = 8.2 Hz),
3.98 (s, 6H, m-OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 190.1
(CO), 148.6 (C3, C3″), 146.8 (C4, C4″), 146.2 (Cβ, Cβ″), 142.0
(C1′, C4′), 128.5 (C2′, C3′, C5′, C6′), 127.1 (C1, C1″), 123.6 (Cα,
Cα″), 119.5 (C6, C6″), 114.9 (C5, C5″), 110.1 (C2, C2″), 56.0 (m-
OCH3). HRMS (APPI+) m/z calculated for C26H22O6 [M+],
431.1489; found, 431.1483.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(naphthalen-2-yl)prop-2-
1
en-1-one) (1f). Yield 80%, light yellow solid, mp 248−249 °C. H
NMR (400 MHz, CDCl3) δ/ppm 8.11 (s, 4H, H2′, H3′, H5′, H6′),
8.00 (d, 2H, Hβ, Hβ″, J = 15.9 Hz), 7.62 (d, 2H, Hα, Hα″, J = 15.7
Hz), 7.90−7.52 (m, 14H, naphthyl ring). 13C NMR (100 MHz,
CDCl3) δ/ppm 189.9 (CO), 145.9 (Cβ, Cβ″), 141.7 (C1′, C4′),
139.8 (C2, C2″), 134.5 (C4, C4″), 133.3 (C4a, C4a″), 132.0 (C8a,
C8a″), 130.9 (C8, C8″), 128.8 (C5, C5″), 128.6 (C2′, C3′, C5′, C6′),
127.8 (C7, C7″), 127.5 (C6, C6″), 126.8 (C1, C1″), 123.5 (C3, C3″),
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(quinoxalin-6-yl)prop-2-
1
en-1-one) (1m). Yield 89%, light yellow solid, mp > 350 °C. H
NMR (200 MHz, CDCl3) δ/ppm 8.90 (m, 4H, H4, H4″, H5, H5″),
2936
dx.doi.org/10.1021/jm401879z | J. Med. Chem. 2014, 57, 2930−2941

Journal of Medicinal Chemistry
Article
8.37 (d, 2H, H2, H2″, J = 1.5 Hz), 8.22 (s, 4H, H2′, H3′, H5′, H6′),
8.14 (m, 4H, H7, H7″, H8, H8″), 8.07 (d, 2H, Hβ, Hβ″, J = 15.7 Hz),
7.76 (d, 2H, Hα, Hα″, J = 15.6 Hz). 13C NMR (50 MHz, CDCl3) δ/
ppm 189.2 (CO), 146.0 (Cβ, Cβ″), 145.6 (C4, C4″), 144.0 (C5,
C5″), 143.7 (C3, C3″), 143.0 (C6, C6″), 141.1 (C1′, C4′), 136.3 (C1,
C1″), 130.8 (C7, C7″), 130.2 (C2, C2″), 128.9 (C2′, C3′, C5′, C6′),
128.4 (C8, C8″), 124.0 (Cα, Cα″). HRMS (APPI+) m/z calculated
for C28H18N4O2 [M+], 443.1503; found, 443.1501.
(s, 6H, p-OCH3). 13C NMR (100 MHz, CDCl3) δ/ppm 190.5 (C
O), 188.8 (CO′), 153.1 (C3′, C5′, C3″, C5″), 143.4 (C4′, C4″),
142.8 (Cβ′, Cβ″), 136.8 (C1, C4), 133.2 (C1′, C1″), 128.9 (C2, C3,
C5, C6), 122.7 (Cα′, Cα″), 106.1 (C2′, C6′, C2″, C6″), 61.0 (p-
OCH3), 56.4 (m-OCH3). HRMS (APPI+) m/z calculated for
C30H30O8 [M+], 519.2013; found, 519.2017.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(3-methoxyphenyl)prop-
2-en-1-one) (2c). Yield 87%, yellow solid, mp 176−177 °C. 1H NMR
(400 MHz, CDCl3) δ/ppm 7.81 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz), 7.69
(s, 4H, H2, H3, H5, H6), 7.62 (d, 2H, H6′, H6″, J = 7.8 Hz), 7.56 (d,
2H, Hα′, Hα″, J = 15.6 Hz), 7.55 (d, 2H, H2′, H2″, J = 1.5 Hz), 7.43
(dd, 2H, H5′, H5″, J = 8.2 Hz, J = 7.8 Hz), 7.15 (dd, 2H, H4′, H4″, J =
8.2 Hz, J = 2.7 Hz), 3.89 (s, 6H, m-OCH3). 13C NMR (100 MHz,
CDCl3) δ/ppm 189.9 (CO), 159.9 (C3′, C3″), 143.5 (Cβ′, Cβ″),
139.4 (C1′, C1″), 136.8 (C1, C4), 129.6 (C5′, C5″), 128.9 (C2, C3,
C5, C6), 123.0 (Cα′, Cα″), 121.0 (C6′, C6″), 119.4 (C4′, C4″), 112.8
(C2′, C2″), 55.5 (m-OCH3). HRMS (APPI+) m/z calculated for
C26H22O4 [M+], 399.1591; found, 399.1585.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(3,4-dimethoxyphenyl)-
prop-2-en-1-one) (2d). Yield 91%, yellow solid, mp 198−199 °C. 1H
NMR (400 MHz, CDCl3) δ/ppm 7.81 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz),
7.71 (m, 2H, H6′, H6″), 7.70 (s, 4H, H2, H3, H5, H6), 7.64 (m, 2H,
H2′, H2″), 7.61 (d, 2H, Hα′, Hα″, J = 15.6 Hz), 6.95 (d, 2H, H5, H5″,
J = 8,20 Hz), 3.98 (s, 12H, m-OCH3, p-OCH3). 13C NMR (100 MHz,
CDCl3) δ/ppm 183.5 (CO), 149.9 (C4′, C4″), 149.3 (C3′, C3″),
142.7 (Cβ′, Cβ″), 136.8 (C1, C4), 131.5 (C6′, C6″), 130.1 (C1′,
C1″), 128.8 (C2, C3, C5, C6), 122.5 (Cα′, Cα″), 110.7 (C5′, C5″),
109.9 (C2′, C2″), 56.0 (m-OCH3, p-OCH3). HRMS (APPI+) m/z
calculated for C28H26O6 [M+], 459.1802; found, 459.1804.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(4-(dimethylamino)-
phenyl)prop-2-en-1-one) (1n). Yield 62%, red solid, mp 204−205
1
°C. H NMR (400 MHz, CDCl3) δ/ppm 8.07 (s, 4H, H2′, H3′, H5′,
H6′), 7.81 (d, 2H, Hβ, Hβ″, J = 15.2 Hz), 7.55 (d, 4H, H2, H2″, H6,
H6″, J = 8.6 Hz), 7.33 (d, 2H, Hα, Hα″, J = 15.6 Hz), 6.69 (d, 4H, H3,
H3″, H5, H5″, J = 8.9 Hz), 3.04 (s, 12H, N(CH3)2). 13C NMR (100
MHz, CDCl3) δ/ppm 190.1 (CO), 152.1 (C4, C4″), 146.6 (Cβ,
Cβ″), 141.7 (C1′, C4′), 130.6 (C6, C6″), 128.3 (C2′, C3′, C5′, C6′),
122.3 (Cα, Cα″), 116.6 (C1, C1″), 111.8 (C5, C5″), 40.1 (N(CH3)2).
HRMS (APPI+) m/z calculated for C28H28N2O2 [M+], 425.2224;
found, 425.2218.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(4-(dimethylamino)-
naphthalen-1-yl)prop-2-en-1-one) (1o). Yield 59%, red solid, mp
1
159−160 °C. H NMR (200 MHz, CDCl3) δ/ppm 8.66 (d, 2H, Hβ,
Hβ″, J = 15.4 Hz), 8.07 (s, 4H, H2′, H3′, H5′, H6′), 7.52 (d, 2H, Hα,
Hα″, J = 15.4 Hz), 8.27−7.05 (m, 12H, naphthyl ring), 2.98 (s, 12H,
N-(CH3)2). 13C NMR (50 MHz, CDCl3) δ/ppm 198.3 (CO),
154.0 (C4, C4″), 142.8 (Cβ, Cβ″), 139.5 (C1′, C4′), 133.2 (C8a,
C8a″), 128.5 (C4a, C4a″), 128.3 (C2′, C3′, C5′, C6′), 126.9 (C1,
C1″), 126.0 (C7, C7″), 125.2 (C6, C6″), 125.1 (C5, C5″), 124.9 (C8,
C8″), 123.6 (Cα, Cα″), 121.7 (C2, C2″), 113.2 (C3, C3″), 44.8 (N-
(CH3)2). HRMS (APPI+) m/z calculated for C36H32N2O2 [M+],
525.2537; found, 525.2534.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(2,4-dimethoxyphenyl)-
prop-2-en-1-one) (2e). Yield 83%, yellow solid, mp 182.5−183 °C.
1H NMR (400 MHz, CDCl3) δ/ppm 7.78 (d, 2H, H3′, H3″, J = 8.5
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(2,6-dimethoxyphenyl)-
prop-2-en-1-one) (1p). Yield 91%, yellow solid, mp 253−254 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 8.30 (d, 2H, Hβ, Hβ″, J = 16.0 Hz),
8.10 (s, 4H, H2′, H3′, H5′, H6′), 8.00 (d, 2H, Hα, Hα″, J = 15.9 Hz),
7.33 (dd, 2H, H4, H4″, J = 8.4 Hz, J = 8.3 Hz), 6.61 (d, 4H, H3, H3″,
H5, H5″, J = 8.4 Hz), 3.93 (s, 12H, o-OCH3). 13C NMR (50 MHz,
CDCl3) δ/ppm 191.8 (CO), 160.4 (C2, C2″, C6, C6″), 141.6 (Cβ,
Cβ″), 136.5 (C1′, C4′), 131.8 (C4, C4″), 128.5 (C2′, C3′, C5′, C6′),
124.7 (Cα, Cα″), 112.6 (C1, C1″), 103.7 (C3, C3″, C5, C5″), 55.8 (o-
OCH3). HRMS (APPI+) m/z calculated for C28H26O6 [M+],
459.1802; found, 459.1808.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(2,3,4-trimethoxyphenyl)-
prop-2-en-1-one) (1q). Yield 93%, yellow solid, mp 178−179 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 8.11 (s, 4H, H2′, H3′, H5′, H6′),
8.03 (d, 2H, Hβ, Hβ″, J = 15.9 Hz), 7.56 (d, 2H, Hα, Hα″, J = 15.7
Hz), 7.42 (d, 2H, H6, H6″, J = 8.8 Hz), 6.74 (d, 2H, H5, H5″, J = 8.8
Hz), 3.97 (s, 6H, o-OCH3), 3.93 (s, 6H, p-OCH3), 3.90 (s, 3H, m-
OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 190.4 (CO), 156.0
(C4, C4″), 153.9 (C2, C2″), 142.5 (Cβ, Cβ″), 141.5 (C3, C3″), 141.0
(C1′, C4′), 128.5 (C2′, C3′, C5′, C6′), 124.0 (C1, C1″), 121.7 (Cα,
Cα″), 121.2 (C6, C6″), 107.6 (C5, C5″), 61.3 (p-OCH3), 60.8 (m-
OCH3), 56.0 (o-OCH3). HRMS (APPI+) m/z calculated for
C30H30O8 [M+], 519.2013; found, 519.2019.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(4-methoxyphenyl)prop-
2-en-1-one) (2a). Yield 83%, yellow solid, mp 248−249 °C. 1H NMR
(200 MHz, CDCl3) δ/ppm 8.06 (d, 4H, H2′, H2″, H6′, H6″, J = 8.9
Hz), 7.81 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz), 7.69 (s, 4H, H2, H3, H5,
H6), 7.59 (d, 2H, Hα′, Hα″, J = 15.6 Hz), 7.00 (d, 4H, H3′, H3″, H5′,
H5″, J = 8.9 Hz), 3.91 (s, 6H, p-OCH3). 13C NMR (50 MHz, CDCl3)
δ/ppm 188.4 (CO), 163.5 (C4′, C4″), 142.7 (Cβ′, Cβ″), 136.9
(C1, C4), 130.8 (C2′, C2″, C6′, C6″), 130.6 (C1″, C1″), 128.8 (C2,
C3, C5, C6), 122.8 (Cα′, Cα″), 113.9 (C3′, C3″, C5′, C5″), 55.4 (p-
OCH3). HRMS (APPI+) m/z calculated for C26H22O4 [M+],
399.1591; found, 399.1596.
Hz), 7.68 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz), 7.61 (s, 4H, H2, H3, H5,
H6), 7.58 (d, 2H, Hα′, Hα″, J = 15.6 Hz), 6.58 (dd, 2H, H4′, H4″, J =
8.5 Hz, J = 2.3 Hz), 6.51 (d, 2H, H6′, H6″, J = 2.3 Hz), 3.92 (s, 3H, p-
OCH3), 3.92 (s, 3H, o-OCH3′). 13C NMR (100 MHz, CDCl3) δ/ppm
190.1 (CO), 164.3 (C2′, C2″), 160.4 (C5′, C5″), 140.8 (Cβ′, Cβ″),
137.0 (C1, C4), 130.1 (C1′, C1″), 128.7 (C2, C3, C5, C6), 127.8
(C4′, C4″), 122.0 (Cα′, Cα″), 105.2 (C3′, C3″), 98.6 (C6′, C6″),
55.7 (o-OCH3, p-OCH3). HRMS (APPI+) m/z calculated for
C28H26O6 [M+], 459.1802; found, 459.1807.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(benzo[d][1,3]dioxol-5-yl)-
prop-2-en-1-one) (2f). Yield 78%, light yellow solid, mp 274−275
1
°C. H NMR (200 MHz, CDCl3) δ/ppm 7.97 (s, 4H, H2, H3, H5,
H6), 7.96 (m, 6H, Hβ′, Hβ″, H2′, H2″, H6′, H6″), 7.72 (d, 2H, Hα′,
Hα″, J = 15.5 Hz), 7.10 (d, 2H, H5′, H5″, J = 8.2 Hz), 6.17 (s, 4H,
CH2′, CH2″). 13C NMR (50 MHz, DMSO-d6) δ/ppm 190.0 (CO),
152.0 (C4′, C4″), 148.4 (C3′, C3″), 142.9 (Cβ′, Cβ″), 137.1 (C1,
C4), 129.7 (C2, C3, C5, C6), 125.6 (Cα′, Cα″), 125.1 (C1′, C1″),
118.8 (C6′, C6″), 108.4 (C5′, C5″), 104.1 (C2′, C2″), 102.5 (CH2′,
CH2″). HRMS (APPI+) m/z calculated for C26H18O6 [M+], 427.1176;
found, 427.1177.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(2,5-dimethoxyphenyl)-
prop-2-en-1-one) (2g). Yield 84%, yellow solid, mp 160−161 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 7.67 (d, 2H, Hβ′, Hβ″, J = 15.7 Hz),
7.62 (s, 4H, H2, H3, H5, H6), 7.46 (d, 2H, Hα′, Hα″, J = 15.7 Hz),
7.21 (d, 2H, H6′, H6″, J = 3.0 Hz), 7.05 (dd, 2H, H4′, H4″, J = 9.0 Hz,
J = 3.0 Hz), 6.95 (d, 2H, H3′, H3″, J = 9.0 Hz), 3.88 (s, 6H, o-OCH3,
m-OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 190.5 (CO), 153.6
(C5′, C5″), 152.6 (C2′, C2″), 141.8 (Cβ′, Cβ″), 136.9 (C1, C4),
129.4 (C1′, C1″), 128.8 (C2, C3, C5, C6), 127.6 (Cα′, Cα″), 119.4
(C4′, C4″), 114.4 (C6′, C6″), 113.3 (C3′, C3″), 56.4 (m-OCH3), 55.8
(o-OCH3). HRMS (APPI+) m/z calculated for C28H26O6 [M+],
459.1802; found, 459.1798.
(2E,2′E)-1,1′-(1,4-Phenylene)bis(3-(naphthalen-2-yl)prop-2-
1
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one) (2b). Yield 80%, yellow solid, mp 190−190.5 °C.
1H NMR (400 MHz, CDCl3) δ/ppm 7.82 (d, 2H, Hβ′, Hβ″, J = 15.6
Hz), 7.71 (s, 4H, H2, H3, H5, H6), 7.54 (d, 2H, Hα′, Hα″, J = 15.6
Hz), 7.29 (s, 4H, H2′, H2″, H6′, H6″), 3.96 (s, 12H, m-OCH3), 3.95
en-1-one) (2h). Yield 89%, light yellow solid, mp 229−230 °C. H
NMR (200 MHz, CDCl3) δ/ppm 7.91 (d, 2H, Hβ′, Hβ″, J = 15.5 Hz),
7.77 (s, 4H, H2, H3, H5, H6), 7.76 (d, 2H, Hα′, Hα″, J = 15.5 Hz),
8.56−7.55 (m, 14H, naphthyl ring). 13C NMR (50 MHz, DMSO-d6)
δ/ppm 189.3 (CO), 148.7 (Cβ′, Cβ″), 144.2 (C2′, C2″), 135.4
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(C1, C4), 134.7 (C4a′, C4a″), 132.7 (C8a′, C8a″), 130.8 (C1′, C1″),
130.0 (C8′, C8″), 129.2 (C2, C3, C5, C6), 129.1 (C6′, C6″), 128.9
(C4′, C4″), 128.0 (C5′, C5″), 127.0 (C7′, C7″), 124.5 (C3′, C3″),
122.0 (Cα′, Cα″). HRMS (APPI+) m/z calculated for C32H22O2
[M+], 439.1693; found, 439.1692.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(3,5-dimethoxyphenyl)-
prop-2-en-1-one) (2i). Yield 92%, yellow solid, mp 178−179 °C. 1H
NMR (400 MHz, CDCl3) δ/ppm 7.81 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz),
7.69 (s, 4H, H2, H3, H5, H6), 7.52 (d, 2H, Hα′, Hα″, J = 15.6 Hz),
7.16 (d, 4H, H2′, H6′, H2″, H6″, J = 2.3 Hz), 6.69 (dd, 2H, H4′, H4″,
J = 2.3 Hz, J = 2.3 Hz), 3.87 (s, 12H, m-OCH3). 13C NMR (100 MHz,
CDCl3) δ/ppm 189.7 (CO), 160.9 (C3′, C3″, C5′, C5″), 143.5
(Cβ′, Cβ″), 139.9 (C1′, C1″), 136.8 (C1, C4), 128.9 (C2, C3, C5,
C6), 122.9 (Cα′, Cα″), 106.3 (C2′, C2″, C6′, C6″), 105.0 (C4′, C4″),
55.6 (m-OCH3). HRMS (APPI+) m/z calculated for C28H26O6 [M+],
459.1802; found, 459.1806.
(C2′, C2″, C6′, C6″), 123.6 (Cα′, Cα″). HRMS (APPI+) m/z
calculated for C26H18O6 [M+], 425.1020; found, 425.1014.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(2,4,6-trimethoxyphenyl)-
prop-2-en-1-one) (2o). Yield 88%, yellow solid, mp 205−206 °C. 1H
NMR (200 MHz, CDCl3) δ/ppm 7.52 (s, 4H, H2, H3, H5, H6), 7.36
(d, 2H, Hβ′, Hβ″, J = 16.0 Hz), 6.98 (d, 2H, Hα′, Hα″, J = 16.0 Hz),
6.16 (s, 4H, H3′, H3″, H5′, H5″), 3.86 (s, 6H, p-OCH3), 3.77 (s, 12H,
o-OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 193.8 (CO), 162.5
(C4′, C4″), 158.9 (C2′, C2″, C6′, C6″), 142.6 (Cβ′, Cβ″), 136.7 (C1,
C4), 129.7 (Cα′, Cα″), 128.7 (C2, C3, C5, C6), 111.7 (C1′, C1″),
90.7 (C3′, C3″, C5′, C5″), 55.9 (o-OCH3), 55.4 (p-OCH3). HRMS
(APPI+) m/z calculated for C30H30O8 [M+], 519.20134; found,
519.20135.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one) (2p). Yield 62%, yellow solid,
1
mp 220−220.5 °C. H NMR (200 MHz, DMSO-d6) δ/ppm 9.67 (s,
2H, OH), 7.92 (d, 2H, H2′, H2″, J = 2.5 Hz), 7.77 (s, 4H, H2, H3,
H5, H6), 7.71 (m, 6H, H6′, H6″, Hα′, Hα″, Hβ′, Hβ″), 6.96 (d, 2H,
H5′, H5″, J = 8.2 Hz), 3.95 (s, 3H, m-OCH3). 13C NMR (50 MHz,
DMSO-d6) δ/ppm 187.1 (CO), 151. 7 (C3′, C3″), 147.5 (C4′,
C4″), 141.6 (Cβ′, Cβ″), 136.3 (C1, C4), 129.6 (C1′, C1″), 128.5 (C2,
C3, C5, C6), 123.2 (C6′, C6″), 122.4 (Cα′, Cα″), 114.6 (C5′, C5″),
111.1 (C2′, C2″), 55.5 (m-OCH3). HRMS (APPI+) m/z calculated
for C26H22O6 [M+], 431.1489; found, 431.1491.
Biology and Biochemistry. Compounds. Mitoxantrone, quer-
cetin, Ko143, ATP, and sodium orthovanadate were purchased from
Sigma Aldrich (France). Chromone 1 was obtained as previously
described.24 All other reagents were commercial products of the
highest available purity grade.
Cell Cultures. The human fibroblast HEK293 cell line transfected
with ABCG2 (HEK293-ABCG2) or the empty vector (HEK293-
pcDNA3.1), as well as the human lung cancer H460 and H23 cell lines,
and pancreas cancer PANC-1 cell line were kindly provided by Drs. R.
W. Robey and S. E. Bates (NCI, Bethesda, MD). The HEK293 cell
line transfected with ABCC1 (HEK293-ABCC1) or the empty vector
(HEK293-pcDNA5) was obtained as described,22 as well as for the
NIH-3T3 transfected with ABCB1 (NIH-3T3-ABCB1).43 The
HEK293 cell lines were maintained in Dulbecco’s modified Eagle
medium (DMEM high glucose), and the cancer cells were maintained
in RPMI-1640, supplemented with 10% fetal bovine serum (FBS), 1%
penicilin/streptomycin, and in some cases 0.75 mg/mL G418 (for
HEK293-pcDNA3.1 and HEK293-ABCG2), 200 μg/mL hygromycin B
(for HEK293-ABCC1 and HEK293-pcDNA5), 60 ng/mL colchicine
(for NIH-3T3-ABCB1), or mitoxantrone at variable concentrations in
selected cells: 20 nM in H460, 100 nM in PANC-1, and 10 nM in H23
cell lines.
Inhibition of ABCG2-Mediated Drug Efflux. Cells were seeded
at a density of 1.5 × 105 cells/well into 24-well culture plates. After a
48 h incubation, the cells were exposed to 5 μM mitoxantrone for 30
min at 37 °C in the presence or absence of compounds at various
concentrations, then washed with phosphate buffer saline (PBS) and
trypsinized. The intracellular fluorescence was monitored with a
FACSCalibur cytometer (Becton Dickinson), using the FL4 channel,
and at least 10 000 events were collected. The percentage of inhibition
was calculated by using the following equation:
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(naphthalen-1-yl)prop-2-
1
en-1-one) (2j). Yield 69%, light yellow solid, mp 162−163 °C. H
NMR (200 MHz, CDCl3) δ/ppm 8.35 (m, 2H, H8′, H8″), 8.00 (d,
2H, H5′, H5″, J = 8.2 Hz), 7.91 (m, 2H, H4′, H4″), 7.78 (d, 2H, H2′,
H2″, J = 7.0 Hz), 7.58 β (s, 4H, H2, H3, H5, H6), 7.56 (m, 8H, Hβ′,
Hβ″, H3′, H3″, H6′, H6″, H7′, H7″), 7.34 (d, 2H, Hα′, Hα″, J = 16.0
Hz). 13C NMR (50 MHz, CDCl3) δ/ppm 195.1 (CO), 144.3 (Cβ′,
Cβ″), 136.8 (C1, C4), 136.7 (C1′, C1″), 133.8 (C4a′, C4a″), 131.8
(C7′, C7″), 130.4 (C8a′, C8a″), 128.9 (C2, C3, C5, C6), 128.4 (C4′,
C4″), 127.9 (C5′, C5″), 127.5 (C2′, C2″), 127.2 (C3′, C3″), 126.5
(C6′, C6″), 125.5 (C8′, C8″), 124.4 (Cα′, Cα″). HRMS (APPI+) m/z
calculated for C32H22O2 [M+], 439.1693; found, 439.1695.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-phenylprop-2-en-1-one)
1
(2k). Yield 90%, light yellow solid, mp 190−190.5 °C. H NMR (200
MHz, CDCl3) δ/ppm 8.15 (d, 4H, H2′, H2″, H6′, H6″, J = 7.8 Hz),
8.01 (d, 2H, Hβ′, Hβ″, J = 15.6 Hz), 7.96 (s, 4H, H2, H3, H5, H6),
7.75 (d, 2H, Hα′, Hα″, J = 15.6 Hz), 7.65 (dd, 2H, H4′, H4″, J = 7.0
Hz, J = 7.0 Hz), 7.55 (dd, 4H, H3′, H3″, H5′, H5″, J = 7.8 Hz, J = 7.4
Hz).). 13C NMR (50 MHz, CDCl3) δ/ppm 189.5 (CO), 143.5
(Cβ′, Cβ″), 137.9 (C1′, C1″), 137.1 (C1, C4), 133.7 (C4′, C4″),
129.8 (C2′, C2″, C6′, C6″), 129.2 (C3′, C3″, C5′, C5″), 129.0 (C2,
C3, C5, C6), 123.4 (Cα′, Cα″). HRMS (APPI+) m/z calculated for
C24H18O2 [M+], 339.1380; found, 339.1379.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(2-methoxyphenyl)prop-
1
2-en-1-one) (2l). Yield 75%, gold yellow solid, mp 146−147 °C. H
NMR (200 MHz, CDCl3) δ/ppm 7.65 (dd, 2H, H6′, H6″, J = 7.4 Hz,
J = 1.5 Hz), 7.63 (d, 2H, Hβ′, Hβ″, J = 16.0 Hz), 7.61 (s, 2H, H2, H3,
H5, H6), 7.50 (ddd, 2H, H5′, H5″, J = 7.4 Hz, J = 7.4 Hz, J = 1.5 Hz),
7.43 (d, 2H, Hα′, Hα″, J = 16.0 Hz), 7.05 (dd, 2H, H4′, H4″, J = 7.4
Hz, J = 7.4 Hz), 7.01 (d, 2H, H3′, H3″, J = 8.20 Hz), 3.92 (s, 6H, o-
OCH3). 13C NMR (50 MHz, CDCl3) δ/ppm 182.6 (CO), 158.1
(C2′, C2″), 141.8 (Cβ′, Cβ″), 136.9 (C1, C4), 133.1 (C4′, C4″),
130.4 (C5′, C5″), 128.8 (C2, C3, C5, C6), 127.8 (C1′, C1″), 120.8
(Cα′, Cα″), 111.6 (C3′, C3″), 55.7 (o-OCH3). HRMS (APPI+) m/z
calculated for C26H22O4 [M+], 399.1591; found, 399.1593.
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(2,4,5-trimethoxyphenyl)-
prop-2-en-1-one) (2m). Yield 81%, yellow solid, mp 222.5−223 °C.
1H NMR (200 MHz, CDCl3) δ/ppm 7.67 (m, 8H, H2, H3, H5, H6,
Hβ′, Hβ″), 7.41 (s, 2H, H6′, H6″), 6.55 (s, 2H, H3′, H3″), 3.98 (s,
3H, m-OCH3), 3.95 (s, 3H, o-OCH3), 3.91 (s, 3H, p-OCH3). 13C
NMR (50 MHz, CDCl3) δ/ppm 189.5 (CO), 154.9 (C4′, C4″),
153.8 (C2′, C2″), 143.4 (C5′, C5″), 140.7 (Cβ′, Cβ″), 137.0 (C1,
C4), 128.7 (C2, C3, C5, C6), 127.9 (C1′, C1″), 120.3 (Cα′, Cα″),
113.1 (C6′, C6″), 97.0 (C3′, C3″), 56.8 (m-OCH3), 56.3 (p-OCH3),
56.1 (o-OCH3). HRMS (APPI+) m/z calculated for C30H30O8 [M+],
519.2013; found, 519.2017.
C − M
Cev − M
% inhibition =
× 100
where C is the intracellular fluorescence of resistant cells (transfected
HEK293-ABCG2 or drug-selected cancer cells) in the presence of
compounds and mitoxantrone, M is the intracellular fluorescence of
resistant cells in the presence of only mitoxantrone, and Cev is the
intracellular fluorescence of control cells (HEK293-pcDNA3.1 or
parental cancer cells) in the presence of compounds and mitoxantrone.
Effects on P-Glycoprotein- and MRP1-Mediated Drug Efflux.
NIH-3T3 cells transfected with ABCB1 were seeded at a density of 1.5
× 105 cells/well into 24-well culture plates and incubated for 48 h at
37 °C in 5% CO2, whereas HEK293 cells transfected with ABCC1
were seeded at 2 × 105 cells/well. The cells were respectively exposed
to 5 μM mitoxantrone or 0.2 μM calcein-AM for 30 min at 37 °C in
(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-(4-carboxyphenyl)prop-2-
en-1-one) (2n). Yield 69%, yellow solid, mp >350 °C. 1H NMR (200
MHz, CDCl3) δ/ppm 10.07 (s, 2H, COOH), 8.28 (d, 4H, H2′, H2″,
H6′, H6″, J = 8.3 Hz), 8.06 (m, 6H, Hβ′, Hβ″, H3′, H3″, H5′, H5″),
8.02 (s, 4H, H2, H3, H5, H6), 7.82 (d, 2H, Hα′, Hα″, J = 15.6 Hz).
13C NMR (50 MHz, CDCl3) δ/ppm 189.4 (CO), 167.0 (COOH),
144.1 (Cβ′, Cβ″), 141.4 (C1′, C1″), 137.1 (C1, C4), 134.9 (C4′,
C4″), 130.0 (C2, C3, C5, C6), 129.2 (C3′, C3″, C5′, C5″), 129.1
2938
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the presence or absence of bis-chalcones at 1 or 10 μM, then washed
with phosphate buffer saline (PBS) and trypsinized. The intracellular
fluorescence was monitored with a FACSCalibur cytometer (Becton
Dickinson), using the FL4 channel to mitoxantrone and FL1 to calcein
detection. At least 10 000 events were collected. The percentage of
inhibition was calculated relative to NIH-3T3 cells for P-glycoprotein
or to HEK293/pcDNA5 cells for MRP1, where the maximal
accumulation was equivalent to a complete inhibition.
ATPase Activity Assay. Vanadate-sensitive ATPase activity was
measured colorimetrically by determining the liberation of inorganic
phosphate from ATP.44 The Sf9 membranes were prepared as
described previously32 and loaded with cholesterol.33 The incubation
was performed in 96-well plates. Sf9 membranes (0.5 mg/mL) were
incubated in a 50 mM Tris-HCl, 50 mM NaCl buffer (pH 8.0)
containing the tested compounds with or without sodium
orthovanadate (0.33 mM). The reaction was started by the addition
of ATP-Mg (3.9 mM), and the plates were incubated for 30 min at 37
°C. The reaction was stopped with sodium dodecyl sulfate (10%) and
revealed with a mixture of ammonium molybdate reagent and 10%
ascorbic acid (1:4). The absorbance was measured at 880 nm, after a
30 min incubation, using a reader plate.
ABBREVIATIONS USED
■
ABC, ATP-binding cassette; BCRP, breast cancer resistance
protein (ABCG2); FTC, fumitremorgin C; MRP1, multidrug
resistance protein 1 (ABCC1); P-gp, P-glycoprotein (ABCB1)
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AUTHOR INFORMATION
■
(10) Cripe, L. D.; Uno, H.; Paietta, E. M.; Litzow, M. R.; Ketterling,
R. P.; Bennett, J. M.; Rowe, J. M.; Lazarus, H. M.; Luger, S.; Tallman,
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improve the outcome of older patients with newly diagnosed acute
myeloid leukemia: a randomized, placebo-controlled trial of the
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4077−4085.
Corresponding Author
*E-mail: a.dipietro@ibcp.fr. Phone: (+33) 47272 2629. Fax:
(+33) 47272 2604.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Drs. R. W. Robey and S. E. Bates, NCI Bethesda, MD, are
acknowledged for providing the mitoxantrone-selected cancer
cell lines, and Drs. C. Ozvegy-Laczka and B. Sarkadi are
acknowledged for the Sf9 membranes overexpressing human
ABCG2. E.W. was a recipient of a mobility fellowship from the
Brazilian CAPES (Process No. 8792127). P.D.N. and C.G.
were recipients of doctoral fellowships from Conselho Nacional
́
́
de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil,
and the Ligue Nationale Contre le Cancer, France, respectively.
Financial support was provided by the CNRS and Universite
Lyon 1 (UMR 5086), the Ligue Nationale Contre le Cancer
́
́
(Equipe Labellisee Ligue 2013), an international grant from
French ANR and Hungarian NIH (2010-INT-1101-01) and
grants from CNPq, Brazil. We thank Department of Chemistry
and Structural Molecular Biology Center from Federal
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