Tariquidar-Related Chalcones and Ketones as ABCG2 Modulators

Article abstract of DOI:10.1021/acsmedchemlett.8b00289

ABC transporters, including ABCG2, play a vital role in defending the human body against the vast range of xenobiotics. Even though this is beneficial for human health, these protein transporters have been implicated in the emerging resistance of cancer c

Full text of DOI:10.1021/acsmedchemlett.8b00289

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Letter
Tariquidar-related chalcones and ketones as ABCG2 modulators
Diana Pena Solorzano, Matthias Scholler, Günther Bernhardt,
Armin Buschauer, Burkhard König, and Cristian Ochoa Puentes
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00289 • Publication Date (Web): 25 Jul 2018
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Tariquidar-related chalcones and ketones as ABCG2 modulators
Diana PeñaꢀSolórzano,1,2 Matthias Scholler,3 Günther Bernhardt,3* Armin Buschauer,3† Burkhard Köꢀ
nig2* and Cristian OchoaꢀPuentes1*
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1Laboratorio de Síntesis Orgánica Sostenible, Departamento de Química, Universidad Nacional de Colombia–Sede Bogotá,
2
5997 Bogotá, Colombia. Institute of Organic Chemistry, University of Regensburg, Dꢀ93040 Regensburg, Germany.
3Institute of Pharmacy, University of Regensburg, Dꢀ93040 Regensburg, Germany
†Died July 18, 2017
KEYWORDS: tariquidar, Chalcone, ketone, ABCG2, modulator.
ABSTRACT: ABC transporters, including ABCG2, play a vital role in defending the human body against the vast range of xenoꢀ
biotics. Even though this is beneficial for human health, these protein transporters have been implicated in the emerging resistance
of cancer cells to a variety of structurally and functionally diverse anticancer drugs. In order to investigate their role in resistance,
potent and selective ABCG2 modulators have been described in literature. A leading class of modulators are the tariquidar anaꢀ
logues, however, their susceptibility to hydrolysis limits their applicable use. To overcome this, we synthesized a novel series of
chalconeꢀ and ketoneꢀbased compounds inspired by reported tariquidar analogues. Compounds were characterized and evaluated
for their ABCG2 modulatory activity and ABC transporter selectivity. When compared to transporters ABCB1 and ABCC1, the
chalconeꢀbased compounds exhibited selectivity for ABCG2, while the ketoneꢀbased compounds showed only a slight preference
for ABCG2. From the former series, chalcone 16d (URꢀDP48) displayed similar activity to the reference fumitremorgin C, both
producing comparable maximal effects. The compound exhibited marked antiproliferative activity, while cytotoxicity was less
pronounced for the most active compound 17f from the ketone series. Chalconeꢀcontaining tariquidar analogues are promising
modulators to aid in functional investigations of ABCG2 transporters.
Cytotoxic drugs are well established in the treatment of many
cancer types. In order to improve the effectiveness of treatment,
multiple cytotoxic drugs with different mechanisms of action are
administered simultaneously. However, emerging resistance of
cells, known as multidrug resistance (MDR), has become an
(see Figure 1; general chalcone structure and compound 2) are
crucial for activity.12ꢀ13 Recent structureꢀactivity relationship
studies14 revealed that the 2′ꢀOHꢀ4′,6′ꢀdimethoxyphenyl group (Aꢀ
ring) could be replaced either by a 2′ꢀnaphthyl group or a 3′,4′ꢀ
methylenedioxyphenyl moiety. It was found that at least two
methoxy groups on the Bꢀring are necessary for optimal inhibiꢀ
tion. Additionally, substitution at positions 3, 4, and 5 (Bꢀring)
improved cytotoxicity, while the presence of a large Oꢀbenzyl
substituent at position 4 and a 2′ꢀnaphthyl substituent (Aꢀring)
decreased cytotoxicity.14 Recently published chalconeꢀbased
ABCG2 modulators are shown in Figure 1.15ꢀ16
increasing issue for effective cancer treatment.1
A wellꢀ
established mechanism of resistance is associated with an overexꢀ
pression of ATPꢀbinding cassette (ABC) transporters, including
Pꢀglycoprotein (Pꢀgp/ABCB1), multidrug resistance protein 1
(MRP1/ABCC1) and breast cancer resistance protein
(BCRP/ABCG2). Several anticancer drugs are substrates of these
proteins and are exported from the tumor cells, which prevents the
drugs from exerting their intracellular cytotoxic effects.2ꢀ3 Strateꢀ
gies to increase the efficacy of cancer chemotherapy involve the
inhibition or modulation of such cellular efflux pumps. Even
though, the role of these pumps in drug resistance has been invesꢀ
tigated for decades,4ꢀ6 ABCG2 transporters have only recently
received increasing attention with a desire to further understand
their structure,7ꢀ8 function,9ꢀ10 and molecular pharmacology.11
A new class of potent and selective ABCG2 inhibitors are the
tariquidar analogues.17ꢀ19 These compounds were developed to
investigate the functional role of ABCG2, however, their suscepꢀ
tibility to hydrolysis limits their use in biochemical and biological
studies. In search for more stable analogues, we propose a bioisoꢀ
steric approach for developing new tariquidarꢀlike compounds.
Two series of compounds were explored in this study – chalcones
and ketones. The design of the former series involved incorporaꢀ
tion of tariquidar’s key structural features necessary for ABCG2
inhibition. The latter series was designed to have a reduced moꢀ
lecular weight and more drugꢀlike properties by replacing the 1,4ꢀ
ethoxystyryl moiety by a methylene group. We hypothesized that
this may reduce the compound toxicity. All compounds were
evaluated for ABCG2, ABCB1 and ABCC1 modulatory activity
by fluorescence based assays (an increase in cellꢀassociated fluoꢀ
rescence is indicative of a decrease in transporterꢀmediated efflux)
as well as for cytotoxicity.
A class of compounds that are able to interact with ABC transꢀ
porters are chalcones. These chemical scaffolds are present in
various biomolecules and are synthetically accessible.12 Chalconeꢀ
based compounds exert a variety of biological effects including
antiꢀcancer, antiꢀinflammatory, chemoꢀpreventive, antiꢀoxidant
and antiꢀmicrobial effects.13 Compounds, containing this scaffold,
have been developed to inhibit the transport activity of ABCꢀ
cassette proteins, such as Pꢀgp. Further developments have resultꢀ
ed in compounds that selectively engage with ABCG2.14
Structural investigations of chalcones have shown that the
methoxy and hydroxy substituents at positions 2´ and 4´ on ring A
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Figure 1. General chalcone structure and chalcones, recently reported as ABCG2modulators.15ꢀ19
As shown in Scheme 1, amines 12aꢀb were prepared from
commercially available acetophenone via an electrophilic aroꢀ
matic substitution, followed by a Bechamp reduction.20 The amꢀ
ides 9aꢀb were obtained by reaction with quinaldic acid, pꢀ
toluenesulfonyl chloride (TsꢀCl) and the amines (8aꢀb). Comꢀ
pound 10 was prepared according to known procedures.21
while compounds 12aꢀb were obtained by a bromination with Nꢀ
bromosuccinimide. Tetrahydroisoquinoline 14 was obtained
commercially as a hydrochloride salt. The Bocꢀprotected tetrahyꢀ
droisoquinoline 13 was synthesized as previously described.18
This compound was reacted with 2ꢀmethoxyethyl 4ꢀmethylꢀ
benzenesulfonate or 2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethyl 4ꢀ
methylbenzenesulfonate, and subsequently deprotected to produce
the desired compounds as trifluoroacetic salts (15aꢀb). Finally, a
nucleophilic substitution between the tosylated compounds (11aꢀ
b) or brominated compounds (12aꢀb) and the respective tetrahyꢀ
droisoquinolines (14, 15aꢀb) led to the target compounds (16aꢀf
and 17aꢀf, Scheme 1) in moderate to good yields (54ꢀ92%,
Scheme 1). All synthesized compounds were characterized by
Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR),
High Resolution Mass Spectrometry (HRꢀMS), and the purity was
confirmed by analytical High Performance Liquid Chromatogꢀ
raphy (HPLC).
The synthesized compounds 16aꢀf, 17aꢀf, the reference comꢀ
pounds fumitremorgin C (FTC) and tariquidar were investigated
in a Hoechst 33342 microplate assay22 using ABCG2ꢀoverꢀ
expressing MCFꢀ7/Topo cells. Relative IC50 values23 i. e. the
concentration corresponding to the response midway between the
lower and the upper plateau of a full concentrationꢀresponse
curve, relative to the maximal response achieved by each indiꢀ
vidual compound, were calculated according to a four parameter
curve fit (GraphPad Prism 7) Relative IC50 values are summaꢀ
rized in Table 1. Concentration response curves of selected comꢀ
pounds obtained in the Hoechst 33342 assay are shown in Figure
2.
All chalcones (16aꢀf) exhibited maximal inhibitory activity beꢀ
tween 72ꢀ111% (Table 1), indicating an ABCG2 related decrease
in Hoechst 33342 efflux. Compounds with a methyl substituent at
the amino chalcone core (16dꢀf) were superior in potency and
maximal inhibitory effect than compounds bearing a hydrogen
atom (16aꢀc). This suggests an importance in the substitution
pattern at this particular position. Compound 16d (URꢀDP48)
showed similar potency to the reference compound FTC (Figure
2). Moreover, the maximal inhibitory effects of these compounds
(16dꢀf, 86%, 97% and 111% respectively) were higher than the
effects of tariquidar and even surpassed that of FTC. The insertion
of ethylene and triethylene glycol chains (R2 in the structure,
Scheme 1) at the tetrahydroisoquinoline moiety in compounds
16aꢀc had no effect on the IC50 or Imax values (Table 1). In conꢀ
trast, the maximal inhibitory effect achieved by the compounds
16dꢀf increased from 86 to 111% (Table 1).
Scheme 1. Synthesis of compounds 16ꢀ17aꢀf. Reagents and
conditions: (i) Quinaldic acid, TsꢀCl, TEA, 50 ºC, overnight; (ii)
NaOH 30%, rt, 24h; (iii) NBS, methanol, reflux, 2h (iv) a) 2ꢀ
methoxyethyl 4ꢀmethylbenzenesulfonate or 2ꢀ(2ꢀ(2ꢀmethoxyꢀ
ethoxy)ethoxy)ethyl 4ꢀmethylbenzenesulfonate, KOH, THF, N2,
6h; b) DCM, trifluoroacetic acid, rt, overnight; (v) 15aꢀb or 16aꢀ
b, K2CO3, acetonitrile, 90 °C, overnight.
A ClaisenꢀSchmidt condensation of the ketones 9aꢀb with the
aldehyde 10 resulted in the desired protected chalcones 11aꢀb,
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The partition coefficient (log P) is a measure of the differential
By contrast, all ketones (17a-f) exhibited lower modulatory acꢀ
tivities (47ꢀ86% maximal inhibition of Hoechst 33342 efflux) in
comparison to the chalcone series (16a-f). The synthesized keꢀ
tones were found to have lower partition coefficients compared to
the chalcones (3.40 to 4.91 vs. 5.46 to 6.97), which could explain
the reduction in ABCG2 modulation (Table 1). As ABCG2 is
located in the cell membrane, compound lipophilicity may be
essential for modulation. In addition, recently published cryoꢀEM
structural studies suggest that compounds can access one binding
pocket of ABCG2 via a hydrophobic membrane entrance from the
lipid bilayer.8 This reflects the importance of lipophilicity, which
should be considered when designing ABCG2 modulators. In
accordance to this hypothesis, the two most hydrophilic comꢀ
pounds in the ketone series (17b and 17c) showed the lowest
maximal inhibitory effect. Interestingly, compound 17f, which
also had a low calculated log P value, showed the highest maxiꢀ
mal inhibition in the Hoechst 33342 accumulation assay. This
may emphasize the importance of a methyl group at the R1 posiꢀ
tion in the central phenyl core.
solubility of a compound in a hydrophobic solvent (octanol) and a
hydrophilic solvent (water). The logarithm of these two values
enables compounds to be ranked in terms of hydrophilicity (or
hydrophobicity).24 As shown in Table 1, compounds bearing a
methyl substituent at the amino chalcone core (16dꢀf) exhibited a
higher calculated partition coefficient, and so, were more lipoꢀ
philic, in comparison to analogs with hydrogen as a substituent at
the same position (16aꢀc). These compounds also had the highest
values for the maximal inhibition of Hoechst 33342 efflux. Comꢀ
paring compounds 16a and 16d, the maximal inhibition for the
most lipophilic compound 16d (86%) was higher and the IC50
value was almost 5 times lower (0.88 µM, Table 1). Chalcones
16e and 16f showed a considerable increase in maximal inhibitory
effect (97% and 111%) in comparison to compound 16b and 16c
(72% and 81%). With the highest partition coefficient (log P)
amongst all synthesized compounds, chalcone 16d (URꢀDP48)
was the most potent inhibitor in the series, with similar activity to
the reference compound (FTC).
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In summary, the results of the Hoechst assay illustrated that the
synthesized chalcones are more potent and efficacious in ABCG2
modulation than the ketones. Additionally, similar potencies were
observed for chalcone 16d (IC50 0.880 µM) and the previously
reported ABCG2 modulators 2 (IC50 0.75 µM), 4 (IC50 1.4 µM), 5
(IC50 0.94 µM), and 6 (IC50 0.501 µM) described in Figure 1.
Table 1. ABCG2 inhibitory activity in comparison to reference
compounds, chalcones 16aꢀf and ketones 17aꢀf.
ABCG2a
compd
FTC
clogPd
1.23
IC50 (µM)b
0.731
0.092
0.526
0.085
Imax (%)c
100
Compound modulatory activities for ABCB1 and ABCC1 were
screened in a calcein accumulation assay, using tariquidar (10
µM) and reversan (30 µM) as reference compounds. Tariquidar
has been reported as a potent and specific inhibitor of ABCB1 (Pꢀ
gp), therefore it is commonly used as a reference compound for
this transporter inhibition assay.25ꢀ26 Reversan is a potent ABCC1
modulator.27ꢀ29 Screening was conducted at a fixed concentration
of 1 ꢁM and 10 ꢁM using KbꢀV1 (ABCB1) and MDCKII
(ABCC1) cell lines (Figure 3). Concentrationꢀresponse curves
were not constructed, as none of the compounds showed more
than 25% inhibition of calceinꢀAM efflux (ABCB1 and ABCC1)
at 10 ꢁM. The results from the calcein accumulation assay sugꢀ
gested that the chalcones (16aꢀf) and ketones (17aꢀf) had a low
inhibitory activity against ABCB1 and ABCC1 (values below
25%, Figure 3A, red and green bar respectively).
±
Tariquidar
±
69 ± 5
6.38
16a
16b
16c
16d
16e
16f
17a
17b
4.08 ± 0.29
2.13 ± 0.47
2.83 ± 0.30
0.88 ± 0.02
1.55 ± 0.36
2.72 ± 0.54
6.73 ± 1.02
14.39
79 ± 4
72 ± 8
81 ± 5
86 ± 10
97 ± 3
111 ± 4
77 ± 1
55 ± 2
6.51
6.18
5.46
6.97
6.64
5.92
4.45
4.12
±11.8
17c
17d
17e
1.13 ± 0.27
8.76 ± 1.59
47 ± 1
77 ± 1
79 ± 1
3.40
4.91
4.58
23.24
4.36
6.65 ± 1.27
±
17f
86 ± 2
3.86
aHoechst 33342 microplate assay using ABCG2ꢀ
B
overexpressing MCFꢀ7/Topo cells. Relative IC50 value23 calcu-
lated using GraphPad Prism 7 four parameter curve fitting and
presented as mean values ± SEM from three independent experꢀ
c
iments performed in triplicate. Maximal inhibitory effects (%)
are expressed as inhibition caused by the highest concentration
(100 µM) of the compound, relative to the inhibitory effect
caused by 10 ꢁM FTC (100% inhibition). dCalculated values
using ACD/Labs IꢀLab 2.0 ilab.acdlabs, Algorithm Version:
v5.0.0.184
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Figure 2. Concentration depended inhibition of ABCG2 mediated Hoechst 33342 efflux by chalcones 16aꢀf (A) and ketones 17aꢀf (B)
using MCFꢀ7/Topo cells; inhibition relative to 10 µM FTC (100%).
Figure 3. Comparison of ABC transporter mediated efflux inhibition by 10 µM of 16aꢀf (A) and 17aꢀf (B) on MCFꢀ7/Topo cells (ABCG2;
blue), KbV1 cells (ABCB1; red) and MDCKII/MRP1 cell (ABCC1; green); the accumulation of Hoechst 33342 (ABCG2) and calcein
(ABCB1 and ABCC1) was measured relative to 10 µM FTC (ABCG2), 10 µM tariquidar (ABCB1) and 30 µM reversan, respectively
(100%).
To evaluate the chemical stability, all synthesized chalcones
and ketones were incubated in assay medium (DMEM suppleꢀ
mented with 10% fetal calf serum) at 37°C. Aliquots were then
analyzed by HPLC over a period of 24 hours. Products of cleavꢀ
age and degradation of compounds were not detected (See SI,
page 62ꢀ63), indicating that all compounds were not affected by
the components present in the assay medium.
Figure 4. Concentration dependent effect of 16d (URꢀDP48)
(A) and 17f (B) on proliferating MCFꢀ7/Topo cells upon longꢀ
term exposure; 100 nM (purple circles); 1 µM (orange squares); 3
µM (green triangles); 10 µM (red inverted triangles); positive
control: 100 nM vinblastine (blue diamonds); growth curve of
vehicle (DMSO) treated cells (negative control) (black stars).
Compound 16d showed a concentration dependent cytostatic
effect on proliferating MCFꢀ7/Topo cells, which may be due to
the reactive αꢀβꢀunsaturated ketone core.31 In accordance to the
working hypothesis, cytotoxicity was reduced in the case of keꢀ
tone 17f, which is lacking the Michael acceptor system.
Finally, the effect of the modulators 16d and 17f on the prolifꢀ
eration of ABCG2ꢀoverexpressing MCFꢀ7/Topo cells was invesꢀ
tigated by means of a kinetic chemosensitivity assay (Figure 4).30
The cytostatic drug vinblastine was used as a positive control.
In summary, new types of tariquidarꢀrelated chalcone and keꢀ
tone derivatives were synthesized and investigated for their inꢀ
hibitory effect and selectivity toward ABCG2. All chalcones were
selective for ABCG2 inhibition. Compounds bearing a methyl
substituent at the A ring (16dꢀf) were most active and potent
(86%, 97%, and 111% respectively). Compound 16d showed
similar potency to the reference compound (FTC) with an IC50
value of 0.88 ꢁM in the Hoechst assay and a maximal inhibitory
effect of 86%. The compounds of the ketone series showed none
or only very small preference for ABCG2 over ABCB1 and
ABCC1. Compounds 17dꢀf surpassed the maximal inhibitory
effect of tariquidar although requiring higher concentrations. The
difference in ABCG2 modulation and selectivity when comparing
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(6) Sandor, V.; Fojo, T.; Bates, S. E., Future perspectives for the
the two series of compounds indicated that the lipophilicity plays
a critical role. The chemosensitivity assay demonstrated antiproꢀ
liferative activity of the synthesized chalcones, whereas cytotoxiꢀ
city was less pronounced in the ketone series. With further optiꢀ
mization, chalcone 16d displaying pronounced ABCG2 modulatꢀ
ing potential with a subꢀmicromolar IC50 value, is a promising
lead to aid in future investigations of ABCG2 to further underꢀ
stand it´s role in cancer drug resistance.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website.
Chemical synthesis, characterization of target compounds; protoꢀ
cols of biological assays (PDF).
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: Guenther.Bernhardt@chemie.uniꢀregensburg.de (G.B.);
burkhard.koenig@chemie.uniꢀregensburg.de
apu@unal.edu.co (C.OꢀP).
(B.K.);
cochoꢀ
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
Notes
The authors declare no competing financial interest.
(14) Rangel, L. P.; Winter, E.; Gauthier, C.; Terreux, R.; Chiaradiaꢀ
Delatorre, L. D.; Mascarello, A.; Nunes, R. J.; Yunes, R. A.;
CreczynskiꢀPasa, T. B.; Macalou, S., New structure–activity reꢀ
lationships of chalcone inhibitors of breast cancer resistance
protein: polyspecificity toward inhibition and critical substituꢀ
tions against cytotoxicity. Drug Des., Dev. Ther. 2013, 7, 1043.
(15) Winter, E.; Gozzi, G. J.; ChiaradiaꢀDelatorre, L. D.; Daflonꢀ
Yunes, N.; Terreux, R.; Gauthier, C.; Mascarello, A.; Leal, P.
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G.; König, B.; Buschauer, A., Quinoline CarboxamideꢀType
ABCG2 Modulators: Indole and Quinoline Moieties as Anilide
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ACKNOWLEDGMENT
D. P.ꢀS thanks COLCIENCIAS for a graduate fellowship and
C.O.ꢀP thanks COLCIENCIAS, the German Academic Exchange
Service (DAAD) and Universidad Nacional de Colombia for
providing financial support. We thank Ms. Ranit Lahmy for proof
reading the manuscript.
ABBREVIATIONS
ABCB1, ATPꢀbinding cassette transporter, subfamily B, member
1; ABCC1, ATPꢀbinding cassette transporter, subfamily C, memꢀ
ber 1; ABCG2, ATPꢀbinding cassette transporter, subfamily G,
member 2; BCRP, breast cancer resistance protein (= ABCG2);
IC50, concentration of inhibitor required to give 50% inhibition of
activity; MDR, multidrug resistance; MRP1, multidrug resistance
associated protein 1 (= ABCC1); pꢀgp, pglycoprotein (= ABCB1);
SEM, standard error of the mean.
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