Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar

Article abstract of DOI:10.1021/jm8013822

The efflux pumps ABCB1 (p-gp, MDR1) and ABCG2 (BCRP) are expressed to a high extent by endothelial cells at the blood-brain barrier (BBB) and other barrier tissues and are involved in drug resistance of tumor (stem) cells. Whereas numerous ABCB1 inhibitors are known, only a few ABCG2 modulators with submicromolar activity have been published. Starting from tariquidar (4) analogues as ABCB1 modulators, minimal structural modifications resulted in a drastic shift in favor of ABCG2 inhibition. Highest potency was found when the 3,4-dimethoxy-2-(quinoline-3-carbonylamino)benzoyl moiety in 4 was replaced with a 4-methoxycarbonylbenzoyl moiety bearing a hetarylcarboxamido group in 3-position, e.g., quinoline-3-carboxamido (5,IC₅₀: 119 nM) or quinoline-2-carboxamido (6,IC₅₀: 60 nM, flow cytometric mitoxantrone efflux assay, topotecan-resistant MCF-7 breast cancer cells); the selectivity for ABCG2 over ABCB1 was about 100-500 fold and the compounds were inactive at ABCC2 (MRP2). Chemosensitivity assays against MCF-7/Topo cells revealed that the nontoxic inhibitor 6 completely reverted ABCG2-mediated topotecan resistance at concentrations > 100 nM, whereas 5 showed ABCG2 independent cytotoxicity. ABCG2 inhibitors might be useful for cancer treatment with respect to reversal of multidrug resistance, overcoming the BBB and targeting of tumor stem cells.

Full text of DOI:10.1021/jm8013822

1190
J. Med. Chem. 2009, 52, 1190–1197
Potent and Selective Inhibitors of Breast Cancer Resistance Protein (ABCG2) Derived from the
p-Glycoprotein (ABCB1) Modulator Tariquidar
Matthias Ku¨hnle,† Michael Egger,‡ Christine Mu¨ller,† Anne Mahringer,§ Gu¨nther Bernhardt,† Gert Fricker,§ Burkhard Ko¨nig,‡
and Armin Buschauer*,†
Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy, and Institute of Organic Chemistry, Faculty of Chemistry and
Pharmacy, UniVersity of Regensburg, UniVersita¨tsstrasse 31, D-93053 Regensburg, Germany, Institute of Pharmacy and Molecular
Biotechnology, UniVersity of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany
ReceiVed NoVember 1, 2008
The efflux pumps ABCB1 (p-gp, MDR1) and ABCG2 (BCRP) are expressed to a high extent by endothelial
cells at the blood-brain barrier (BBB) and other barrier tissues and are involved in drug resistance of
tumor (stem) cells. Whereas numerous ABCB1 inhibitors are known, only a few ABCG2 modulators with
submicromolar activity have been published. Starting from tariquidar (4) analogues as ABCB1 modulators,
minimal structural modifications resulted in a drastic shift in favor of ABCG2 inhibition. Highest potency
was found when the 3,4-dimethoxy-2-(quinoline-3-carbonylamino)benzoyl moiety in 4 was replaced with a
4-methoxycarbonylbenzoyl moiety bearing a hetarylcarboxamido group in 3-position, e.g., quinoline-3-
carboxamido (5, IC50: 119 nM) or quinoline-2-carboxamido (6, IC50: 60 nM, flow cytometric mitoxantrone
efflux assay, topotecan-resistant MCF-7 breast cancer cells); the selectivity for ABCG2 over ABCB1 was
about 100-500 fold and the compounds were inactive at ABCC2 (MRP2). Chemosensitivity assays against
MCF-7/Topo cells revealed that the nontoxic inhibitor 6 completely reverted ABCG2-mediated topotecan
resistance at concentrations >100 nM, whereas 5 showed ABCG2 independent cytotoxicity. ABCG2 inhibitors
might be useful for cancer treatment with respect to reversal of multidrug resistance, overcoming the BBB
and targeting of tumor stem cells.
Introduction
circulation. A high ABCG2 expression at the luminal surface
of the endothelium of microvessel also suggests a tutelary
function at the blood-brain barrier. In the therapy of CNS
diseases, the aforementioned physiological functions often lead
to low drug concentrations in the brain. Numerous anticancer
agents are ABCB1 and ABCG2 substrates, which are actively
pumped out by the transporters located at the blood-brain
barrier. This is one of the primary causes of the failure of
chemotherapy in the treatment of malignant brain tumors.7
Furthermore, ATP-binding cassette (ABC) transporters play a
more distinctive role in conjunction with multidrug resistance
(MDR). It is estimated that multidrug resistant tumors account
for up to half of all cancer-related deaths.8,9 MDR is caused by
the overexpression of efflux transporters such as ABCB1 and
ABCG2, located in the plasma membrane of cancer cells,
actively extruding a vast number of structurally unrelated
compounds, including many commonly used anticancer drugs.
Since its discovery in 1998,10,11 ABCG2 has been the subject
of many investigations concerning its role in MDR, and
overexpression of the transporter is associated with high-level
of resistance to a large number of cytostatics. In addition,
ABCG2 transporters have recently attracted interest with respect
to a new concept of tumor development and progression, the
so-called cancer stem cell hypothesis. The classical stochastic
model of tumor development and progression assumes that all
cancer cells are tumor initiating and participate in the tumor
growth. By contrast, the cancer stem cell concept is based on
the idea that only a small side population of cancer cells
proliferates, in analogy to the hematopoietic stem cells in the
bone marrow. The cells of this side population divide slowly,
are capable of long-term self-renewal, and express ABCG2.12-14
Such cells have been found in numerous established tumor cell
lines as well as in tumor biopsies15-17 and might be responsible
for the long-term failure of many cancer chemotherapies. As a
ABC transporters use the energy of ATP-hydrolysis to
transport a broad variety of substrates across the cell membrane.
These efflux transport proteins include ABCB1 (p-glycoprotein
170, p-gp), ABCC2 (multidrug resistance related protein 2),1,2
and ABCG2 (BCRP, ABCPa, MXR)3,4 and appear to play a
protective role in normal tissues.5 For example, in the placenta,
ABCG2 appears to reduce the passage of substrates from the
mother to the fetus and, as reasoned from mice studies,6
decreases the concentration of certain substrates in the fetal
* To whom correspondence should be addressed. Phone: +49-941
9434827. Fax: +49-941 9434820. E-mail: armin.buschauer@chemie.uni-
regensburg.de.
†
Department of Pharmaceutical/Medicinal Chemistry II, Institute of
Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg.
‡
Institute of Organic Chemistry, Faculty of Chemistry and Pharmacy,
University of Regensburg.
§
Institute of Pharmacy and Molecular Biotechnology, University of
Heidelberg.
a Abbreviations: ABCP, ATP-binding cassette transporter expressed in
placenta; aq, aqueous; ATP, adenosine triphosphate; BBB, blood-brain
barrier; BCRP, breast cancer resistance protein; Boc, tert-Butyloxycarbonyl,
CDI, 1,1′-carbonyldiimidazole; CNS, central nervous system; DAD, diode
array detector; DCM, dichloromethane; DIPEA, diisopropylethylamine;
DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMSO,
dimethylsulfoxide; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide;
EDTA, ethylenediaminetetraacetic acid; EI-MS, electron-impact ionization
mass spectrometry; ELSD, evaporative light scattering detection; ES-MS,
electrospray ionization mass spectrometry; FACS, fluorescence activated
cell sorter; HBTU, (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate); HOBt, hydroxybenzotriazole; HR-MS, high resolution
mass spectrometry; LSI-MS, liquid-secondary-ion mass spectrometry; MCF-
7/Topo cells: human breast cancer cells resistant to topotecan due to
overexpression of ABCG2; MDR, multidrug resistance; MRP, multidrug-
related protein; MXR, mitoxantrone resistance protein; NOESY, nuclear
Overhauser enhancement spectroscopy; p-gp, p-glycoprotein; SEM, standard
error of the mean; TFA, trifluoroacetic acid; Tf, triflyl, trifluoromethylsul-
fonyl; THF, tetrahydrofuran; TLC, thin layer chromatography; Tr, trityl,
triphenylmethyl.
10.1021/jm8013822 CCC: $40.75
2009 American Chemical Society
Published on Web 01/26/2009

ABCG2 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1191
Figure 1. Structures of the ABCG2 modulators 1 (fumitremorgin C) and 2 (Ko143), the dual ABCB1/ABCG2 inhibitor 3 (elacridar), the ABCB1
inhibitor 4 (tariquidar), and the initial ABCG2 selective lead structure 5 derived from 4.
result of their ABCG2-mediated drug resistance and slow
proliferation, they are inefficient targets for classical cytostatic
drugs.
metabolite of irinotecan ([1,4′-bipiperidine]-1′-carboxylic acid
(4S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-
1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl ester).28 Sev-
eral ABCB1 inhibitors have also been reported to modulate
ABCG2. It was demonstrated that 329 acts as an ABCG2
inhibitor, as does (although with lower potency) the ABCB1
inhibitor 430 and the analogue of 3 and 4, WK-X-34 (N-[2-
[[[4-[2-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)ethyl]-
phenyl]amino]carbonyl]phenyl]-3,4-dimethoxybenzamide).31,32
A very recent structure-activity relationship study on ana-
logues of 4 revealed preferential inhibition of ABCB1 compared
to ABCG2. In these compounds the anthranilic acid portion was
either acylated with different hetaroyl residues at the o-amino
group or was replaced with (hetero)aromatic carboxylic acids.33
During our work on tariquidar-like compounds as ABCB1
modulators,20,34,35 we discovered that, surprisingly, minimal
structural changes at the benzamide core of 4 (Figure 1) resulted
in a potent and selective ABCG2 inhibitor (5, Figure 1 and Chart
1). On the basis of this finding, we prepared a series of new
analogues that were characterized with respect to their activity
and selectivity and are presented in this work.
Thus, in many respects, the potent and selective inhibition
of ABCG2 might be a promising approach in cancer therapy.
The reversal of the multidrug resistance mediated by ABCG2
and the specific targeting of the stem cell like side population
are conceivable applications. Specific inhibition of ABCG2 in
combination with a cytostatic, which is a substrate of ABCG2,
might eradicate the tumor stem-cell population, provided that
ABCG2 expression is actually a characteristic of these cells.
Another interesting strategy might be the coadministration
of potent ABCG2 inhibitors with drugs that are substrates of
the efflux pump to increase drug levels in the brain.18 The latter
has already been proven by the combination of the cytostatic
paclitaxel (taxol A), an ABCB1 substrate, and the second
generation ABCB1 inhibitor valspodar (6-[(2S,4R,6E)-4-Methyl-
2-(methylamino)-3-oxo-6-octenoic acid]cyclosporin D),19 as well
as the third generation modulators, elacridar (GF120918) and
tariquidar (XR 9576).20 (3 and 4, Figure 1).
Because of the relatively recent discovery of the ABCG2
transporter, only a few ABCG2 inhibitors have been reported
so far.21-25 Fumitremorgin C (FTC, 1, Figure 1), a diketopip-
erazine, isolated from the fermentation broth of Aspergillus
fumigatus, was reported first.26 However, its neurotoxicity
precluded its use in in vivo experiments. The most potent
ABCG2 inhibitor known so far is the FTC analogue 2
(Ko143).27 Its low cytotoxicity made it promising for in vivo
studies, and the bioavailability of orally administered topotecan
((4S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-
pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-di-
one) could be increased by a combination with 2.27 Novobiocin
(N-[7-[[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-methyl-
ꢀ-L-lyxo-hexopyranosyl]oxy]-4-hydroxy-8-methyl-2-oxo-2H-1-
benzopyran-3-yl]-4-hydroxy-3-(3-methyl-2-butenyl)benza-
mide), a coumermycin derivative and inhibitor of the prokaryotic
enzyme gyrase, was also identified as an ABCG2 inhibitor. In
cytotoxicity and flow cytometric assays, micromolar concentra-
tions of novobiocin overcame ABCG2-mediated resistance to
mitoxantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)ami-
no]ethyl]amino]anthracene-9,10-dione), topotecan, and the active
Results and Discussion
Synthesis. The design of the compounds described in this
work was based on the serendipitous observation that compound
5 (Figure 1 and Chart 1) was only weakly active against ABCB1
but was found to be a potent ABCG2 inhibitor instead.
Compared to the third generation ABCB1 inhibitor and anthra-
nilic acid derivative 4, the substitution pattern of carboxylic acid
and amine functionalities at the central aromatic core of 5 was
changed and the two methoxy groups were replaced by an ester
group. Because this change apparently had a strong impact on
the selectivity for ABCG2 versus ABCB1 (Table 1) we decided
to prepare two small series of compounds in which either the
3-quinolinecarboxylic acid moiety was replaced by various
heteroaromatic systems or the ester group was exchanged by
methoxy and methyl substituents at different positions (Chart
1).
The general synthetic route for the title compounds (Scheme
1) comprises the formation of an amide bond between 3-ni-
trobenzoic acid analogues 16a-e and the aromatic amine 17,

1192 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Ku¨hnle et al.
Table 1. Inhibition of ABCG2, ABCB1, and ABCC2 by Reference
Compounds and the New Modulators 5-15 Determined in the
Mitoxantrone (ABCG2), the Calcein-AM (ABCB1) Efflux, and the
CMFDA Accumulation Assay (ABCC2)
Chart 1. Variation of the Lead Structure 5 (Subseries 5-11 and
12-15)
ABCG2
maximal
ABCG2 IC50 inhibitory ABCB1 IC50 ABCC2 IC50
a b
,
compound
[nM]a
effect [%]
[nM]c
[nM]
1 (fumitremorgin C) >11000
100
inactived
inactived
193 ( 18
223 ( 8
9450 ( 417
>29000f
>34000
inactived
>57000f
>20000f
>14000f
>6000f
>17000
>15000
>15000f
nd
inactivee
>50000
inactivee
inactivee
inactivee
>20000
nd
2 (Ko143)
225 ( 33
250 ( 45
916 ( 197
119 ( 22
60 ( 10
183 ( 50
179 ( 35
552 ( 125
632 ( 222
1015 ( 403
317 ( 131
858 ( 210
977 ( 244
1990 ( 355
nd
82 ( 5
46 ( 2
39 ( 3
41 ( 3
56 ( 6
55 ( 3
25 ( 2
44 ( 2
38 ( 3
47 ( 4
63 ( 7
36 ( 3
43 ( 6
52f
3 (elacridar)
4 (tariquidar)
5
6
7
8
9
10
11
12
13
14
nd
>20000
>50000
>20000
inactivee
inactivee
inactivee
>50000
>1000
<150
15
MK571g
h
LTC4
nd
nd
a Mean values ( SEM, calculated from two to three independent
experiments. b Maximal inhibitory effects [%] are expressed as inhibition
caused by the highest concentration of the compound tested (7 or 10 µM,
respectively) relative to the inhibitory effect caused by 10 µM 1 (100%
inhibition). c Mean values ( SEM, calculated from two to five independent
experiments. d No effect up to a concentration of 10 µM; 1: 0.8% inhibition
at 10 µM, 22% inhibition at 100 µM; 2: 1.6% inhibition at 10 µM. e No
effect up to a concentration of 50 µM. f N ) 1. g MK571: 3-(((3-(2-(7-
chloroquinoline-2-yl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)m-
ethyl)thio)propanoic acid. h LTC4: leukotriene C4. IC50 values were calcu-
lated using SIGMA PLOT 9.0, four parameter logistic curve fitting; nd )
not determined.
Scheme 1. General Synthetic Route for Compounds 5-15a
which was synthesized as described before.36 Subsequent
hydrogenation of the nitro group, followed by acylation of the
resulting aromatic amines 19a-e with different heteroaromatic
carbonyl chlorides, yielded the compounds 5-15.
Inhibition of the ABC Transporters ABCG2, ABCB1,
and ABCC2. The synthesized modulators 5-15 and the
reference compounds 1-4 were investigated for inhibition of
ABCG2 and ABCB1 by flow cytometry in a mitoxantrone efflux
assay and a calcein-AM efflux assay. In MCF-7/Topo cells, red
fluorescent mitoxantrone is not accumulated but extruded by
the ABCG2 transporter. Therefore, ABCG2 inhibitors can easily
be recognized by the flow cytometric determination of intra-
cellular mitoxantrone levels. Changes in the mitoxantrone efflux
caused by different concentrations of the ABCG2 modulators
can be measured by the relative fluorescence intensity of
the cells. Similarly, in ABCB1 overexpressing KBv1 cells, the
accumulation of calcein, a fluorescent substrate of ABCB1, can
be quantified by flow cytometric analysis. In the presence of
inhibitors of ABCB1, higher intracellular calcein levels lead to
increased relative fluorescence intensities of the cells. The
modulation of ABCC2 was investigated on ABCC2-overex-
pressing MDCK cells by incubation with chloromethylfluores-
cein-diacetate (CMFDA) in the absence and presence of
increasing concentrations of test compounds. After intracellular
formation of the ABCC2 substrate glutathione methylfluorescein
a Reagents and conditions: (i) (1) SOCl2; (2) 17, NEt3, CH2Cl2; (ii)
HBTU, HOBt, DIPEA, 17, CH2Cl2; (iii) H2, Pd/C, EtOAc/MeOH; (iv)
HetC(O)Cl · HCl, NEt3, CH2Cl2/DMF.
(GSMF), the extent of intracellular fluorescence was monitored
with a plate reader in a concentration-dependent manner.
To classify the new compounds with respect to their inhibitory
potency against the targets ABCG2, ABCB1, and ABCC2
known modulators were investigated as references (Table 1).

ABCG2 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1193
Whereas 1 shows only an IC50 value in the micromolar range
against ABCG2, its analogue 2 is a highly potent ABCG2
modulator with a maximal inhibitory effect of 82% referred to
the control. Both compounds were inactive against the ABCB1
transporter, suggesting that the diketopiperazine partial structure
confers some selectivity against ABCG2. However, the acridone
carboxamide derivative 329 strongly inhibits both transporters
without a preference to one of the two targets, whereas 4 was
about equipotent with 3 at ABCB1 but about four times less
potent at ABCG2. Compounds 3 and 4 have the same N-
substituent (dimethoxytetrahydroquinolinylethylphenyl) at ac-
ridonecarboxamide and benzamide, respectively. As shown very
recently, the ABCG2 preference of acridonecarboxamides can
be improved by replacing the amide substituent in 3 with
methoxyphenylethyl residues.22 Here we report that the prefer-
ence for one of the ABC transporters depends on the core amide
moiety in analogues of 4 with kept N-substituent.
Minimal structural changes at the benzamide core of 4
resulted in a change from ABCB1 to ABCG2 inhibition. The
shift of the quinoline-3-carboxamido substituent from position
2 (4) to position 3 of the benzamide moiety (13) proved to be
key to increase the selectivity for ABCG2 over ABCB1: whereas
the moderate inhibition of ABCG2 was maintained (13: IC50
858 nM, 4: IC50 916 nM), the inhibition of ABCB1 decreased
by a factor of >75 (IC50 >17000 vs 223 nM for 13 and 4,
respectively). An additional step toward potent and selective
ABCG2 inhibitors was the introduction of an ester instead of
the methoxy groups. Comparing the methyl ester 5 with 4 and
13, on one hand, the structural modification led to a strong
increase in the modulatory potency against ABCG2, and on the
other hand, the affinity against the original target ABCB1 was
dramatically reduced. Similar to the lead structure 5, the
substances 6-11 were potent inhibitors of ABCG2 with up to
500-fold lower activity at ABCB1. None of the novel derivatives
of 4 (up to a concentration of 50 µM) exerted any inhibitory
effect on ABCC2 (MRP2),
Figure 2. Concentration-response curves of ABCG2 inhibition by
selected analogues of 4 (5, 6, 9, and 11) and the reference compounds
2 (Ko143) and 4 (tariquidar).
solubility of the analogues of 4 presented in this work might
contribute to this phenomenon. Stability investigations as well
as the search for more stable bioisosteric replacements of the
ester group and compounds with higher water solubility are the
subjects of ongoing studies.
Cytotoxicity and Reversal of Drug Resistance. On the basis
of the results of the mitoxantrone efflux assay, the ability of
the two most potent modulators (5 and 6) to overcome multidrug
resistance mediated by ABCG2 was investigated using a kinetic
chemosensitivity assay. As a reference, in a further chemosen-
sitivity assay, the known ABCG2-inhibitor 2 was used. For this
purpose, ABCG2 positive topotecan-resistant MCF-7 breast
cancer cells (MCF-7/Topo cells) were incubated with the
inhibitors alone and in combination with topotecan at a nontoxic
concentration. The results are shown in Figure 3.
Incubation of MCF-7/Topo breast cancer cells with 2 alone
resulted in nontoxic effects up to a concentration of 500 nM.
The combination of 2 at a concentration of 100 nM with
topotecan at a nontoxic concentration of 100 nM yielded an
overcome of the efflux pump mediated resistance. MCF-7/Topo
breast cancer cells, which were incubated with the newly
synthesized tariquidar analogue 6 alone, showed only a weak
chemosensitivity up to concentrations of 500 nM (Figure 3C).
However, compound 6 at a concentration of 100 nM combined
with a nontoxic concentration of topotecan (100 nM) resulted
in a strong cytostatic effect. The effective inhibition of ABCG2
led to a total reversal of the ABCG2 mediated topotecan
resistance (Figure 3D). Surprisingly, at a concentration of 100
nM, compound 5 had a cytostatic effect against proliferating
MCF-7/Topo cells (Figure 3E), which was only slightly
enhanced by the combination with topotecan (Figure 3F).
Chemosensitivity assays with proliferating ABCG2 negative
U-373 MG glioblastoma cells indicate that the toxicity of 5 is
independent from ABCG2 modulation. In addition, quiescent
U-373 MG cells were not affected by 5, indicating that the
antiproliferative effect is cell cycle dependent (data not shown).
Specific modulators are desired as pharmacological tools
for the functional analysis and characterization of the ABCG2
transporter as the mechanism of action, and the selection and
binding of substrates is far from being understood. Some
anticancer drugs, e.g., topotecan, are substrates of different
ABC transporters. This might be one of the reasons why the
reversal of MDR by inhibition of one of these proteins often
fails in the clinics,37,38 suggesting a potential therapeutic
Obviously, the shift of the hetarylcarboxamido substituent
from the 2- to the 3-position at the benzamide core of 4
dramatically changes the selectivity of the compounds for
ABCG2 over ABCB1. All compounds with bicyclic hetaryl-
carboxamides in position 3 (5-8) were highly potent ABCG2
modulators (IC50 values within the range of the known ABCG2
inhibitor 2) superior to the substances with monocyclic het-
eroaromatic moieties (9-11). The most potent inhibitor (IC50:
60 ( 10 nM) was obtained with a quinoline-2-carboxamido
substituent (6). Although the methoxy-substituted compounds
12-14 showed some selectivity for ABCG2, these compounds
were less potent than 5. The decrease in activity was most
pronounced with 5-methoxy substitution (14). Presumably, the
carbonyl oxygen of the ester group in 5-11 contributes to the
interaction with ABCG2. As the esters 5-11 may not be
considered druglike due to susceptibility toward enzymatic
hydrolysis, as an example, compound 5 was converted to the
carboxylic acid. In the flow cytometric mitoxantrone efflux
assay, the cleavage product turned out to be about 80 times
less potent than the parent compound 5 as an inhibitor of
ABCG2 (data not shown), i.e., higher potency resides in the
esters.
As shown in Figure 2, the quinoline-3-carboxamide 5 and
the structural isomer 6 were about as potent as 2, the most potent
ABCG2 modulator described so far. However, the maximum
inhibition obtained by 2 was not reached. Although the lower
efficacy compared to 1 appears to be characteristic of the new
ABCG2 inhibitors related to 3,22 4, and 5, the low water

1194 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Ku¨hnle et al.
Figure 3. Effect of reference compound 2 alone (A) and in combination with 100 nM topotecan (B) on proliferating MCF-7/Topo cells (long-term
drug exposure); vehicle (g), 100 nM topotecan (b) and 2 at different concentrations: 10 nM (9), 50 nM (2), 100 nM (1), and 500 nM ([). Effect
of compound 6 alone (C) and in combination with 100 nM topotecan (D) on proliferating MCF-7/Topo cells (long-term drug exposure); vehicle
(g), 100 nM topotecan (b) and 6 at different concentrations: 10 nM (2), 100 nM ([), and 500 nM (1). Effect of compound 5 alone (E) and in
combination with topotecan (F) on proliferating MCF-7/Topo cells (permanent incubation with the test compounds); vehicle (g), 100 nM topotecan
(b) and 5 at different concentrations: 1 nM (9), 10 nM (2), 100 nM ([), and 500 nM (1). Open stars represent the proliferation (absorbance, right
ordinate in A-F) of the respective untreated cells (vehicle control). Thereby the A-F contain all the information necessary to reconstruct the
individual growth curves of the treated cell populations (closed symbols) according to the equations in the Experimental Section (chemosensitivity
assays).
value of dual inhibitors of ABC transporters.39 Selective
inhibition should be superior to dual ABCB1/ABCG2 inhibi-
tion, if the coadministered drug is preferentially transported
by one specific efflux pump. Additionally, combination
therapy could improve the pharmacokinetics of transported
drugs, thereby increasing oral bioavailability,40 plasma half-
lives, and brain penetration.18,41
among the most potent and selective ABCG2 inhibitors known
so far. Such compounds could be useful in combination with
cytostatics, which are ABCG2 substrates, to overcome drug
resistance of tumor cells, to modulate ABCG2 at the blood-brain
barrier, and thereby to improve the outcome of cancer chemo-
therapy of malignant brain tumors. Kinetic chemosensitivity
assays revealed two main effects of the new compounds. First,
weakly toxic modulators such as compound 6 were able to
reverse the ABCG2 mediated topotecan resistance in MCF-7/
Topo breast cancer cells. Second, compound 5 showed ad-
ditional cytotoxicity. The latter is an unexpected interesting
aspect because ABCG2 modulators with intrinsic antiprolifera-
Conclusions
Starting from the ABCB1 preferring dual ABCB1/ABCG2
modulator 4 (tariquidar), structural modifications at the benza-
mide core were performed. The synthesized compounds are

ABCG2 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1195
1H, H-Ar), 8.28-8.36 (m, 3H, H-Ar), 8.34 (bs, 1H, CONH),
9.45 (d, 4J ) 1.6 Hz, 1H, H-Ar), 12.31 (bs, 1H, CONH). 13C NMR
(75 MHz, CDCl3/MeOD): δ ) 27.8 (-), 32.9 (-), 50.7 (-), 52.8
(+), 55.0 (-), 55.9 (+), 56.0 (+), 59.4 (-), 109.4 (+), 111.3 (+),
117.9 (+), 118.7 (Cquat), 118.8 (+), 120.8 (+), 122.5 (+), 123.3
(Cquat), 124.8 (Cquat), 125.4 (Cquat), 127.7 (+), 128.5 (+), 129.3 (+),
129.4 (Cquat), 130.2 (+), 130.4 (+), 131.9 (+), 136.1 (Cquat), 137.8
(+), 140.1 (Cquat), 140.8 (Cquat), 146.6 (Cquat), 147.5 (Cquat), 147.8
(Cquat), 149.5 (Cquat), 163.9 (Cquat), 164.8 (Cquat), 167.4 (Cquat). IR
(KBr) [cm-1]: ν ) 3303, 2940, 2831, 1697, 1655, 1570, 1519. UV/
vis (CHCl3) λmax [nm] (lg ε): 309 (4.273), 289 (4.316), 245 (4.776).
HRMS calcd. for C38H37N4O6 [M•+]: 645.2713; found: 645.2709.
Drugs and Chemicals Used for Assays. Mitoxantrone stocks
were obtained by diluting Novantron (Wyeth Pharma, Muenster,
Germany) in 70% ethanol to a concentration of 2 mM. Test
compounds were dissolved in DMSO (Merck, Darmstadt, Germany)
at a concentration of 10 mM. Compound 1 (gift of Dr. Susan Bates,
NIH) was also dissolved in DMSO and diluted to a concentration
of 1 mM. A 10 mM stock solution of 2 in DMSO was kindly
provided by Dr. A.H. Schinkel (Netherlands Cancer Institute). All
stocks were stored at -20 °C. Topotecan stocks were prepared by
diluting Hycamtin (GlaxoSmithKline, Munich, Germany) in 70%
ethanol to a concentration of 0.1 mM and stored at 4 °C. Compound
4 (free base) was synthesized according to the literature with slight
modifications.36 Compound 3 was kindly provided by GlaxoSmith-
Kline (Research Triangle Park, NC). Calcein-AM, purchased from
Biotrend (Cologne, Germany), was dissolved in DMSO (Merck,
Darmstadt, Germany) to achieve a final concentration of 1 mM.
The aliquoted stock solutions were stored at -20 °C. The 1 mM
stock solution of vinblastine (vinblastine sulfate, Sigma, Munich,
Germany) was made in 70% ethanol.
Cell Lines and Culture Condition. MCF-7/Topo cells, an
ABCG2 overexpressing subclone of MCF-7 breast cancer adeno-
carcinoma cells (ATTC HTB-22) were obtained by passaging the
MCF-7 cells with increasing concentrations of topotecan in the
culture medium to a maximum concentration of 0.55 µM. Having
reached the final concentration of topotecan, the cells were passaged
after trypsinization using 0.05% trypsin/0.02% EDTA (PAA
Laboratories, Pasching, Austria) every 3-5 days. The treated cells
showed sufficient quantities of the ABCG2 transporter after three
passages in Eagle’s minimum essential medium (Sigma, Deisen-
hofen, Germany) containing L-glutamine, 2.2 g/L NaHCO3 (Merck,
Darmstadt, Germany), 0.11 g/L sodium pyruvate (Serva, Heidelberg,
Germany), 5% fetal calf serum (Biochrom, Berlin, Germany), and
topotecan at a concentration of 0.55 µM.
KBv1 cells, an ABCB1 overexpressing subclone of KB cells
(ATCC CCL-17), were maintained in Dulbecco’s modified Eagle’s
medium (Sigma, Deisenhofen, Germany) supplemented with 10%
FCS (Biochrom, Berlin, Germany) and 270 ng/mL vinblastine.
U-373 MG cells, an ABCB1 and ABCG2 nonexpressing human
glioblastoma cell line, were cultured in Eagle’s minimum essential
medium (Sigma, Deisenhofen, Germany) containing L-glutamine,
2.2 g/L NaHCO3 (Merck, Darmstadt, Germany), 0.11 g/L sodium
pyruvate (Serva, Heidelberg, Germany), and 5% fetal calf serum
(Biochrom, Berlin, Germany). All cells were cultured in a water-
saturated atmosphere (95% air/5% CO2) at 37 °C in 75 cm2 and
175 cm2 culture flasks (NUNC, Wiesbaden, Germany and Greiner,
Frickenhausen, Germany) respectively. Mycoplasma contamination
was routinely monitored by polymerase chain reaction (Venor GeM,
Minerva Biolabs GmbH, Berlin, Germany), and only mycoplasma
free cultures were used for testing.
ABCC2 overexpressing MDCKII-MRP2 cells transfected with
human ABCC2 were a kind gift from Prof. Dr. P. Borst (Nether-
lands Cancer Institute, Amsterdam). The cells were grown in
Dulbecco′s modified Eagle medium supplemented with 5% FCS
(Biochrom, Berlin, Germany).
Modulation of ABCG2 (BCRP, ABCP, MXR): Determina-
tion in the Flow Cytometric Mitoxantrone Efflux Assay. The
assay was essentially performed as described.35 Briefly, 3-5 days
after passaging, the ABCG2 overexpressing MCF-7/Topo cells were
trypsinized and resuspended in culture medium at 25 °C. After
tive activity may offer a new chemotherapeutic approach
addressing the tumor stem cell concept.42
Experimental Section
General. Commercial reagents and starting materials were
purchased from Aldrich, Fluka, or Acros and used without further
purification. Flash chromatography was performed on silica gel
(Merck silica gel Si 60 40-63 µm); products were detected by
TLC on alumina plates coated with silica gel (Merck silica gel 60
F254, thickness 0.2 mm) and visualized by UV light (λ ) 254 nm).
Melting points were determined with a Bu¨chi SMP 20 and are
uncorrected. NMR spectra were recorded with Bruker Avance 300
(1H: 300.1 MHz; 13C: 75.5 MHz; T ) 300 K), Bruker Avance 400
(1H: 400.1 MHz; 13C: 100.6 MHz; T ) 300 K), and Bruker Avance
600 (1H: 600.1 MHz; 13C: 150.1 MHz; T ) 300 K) instruments.
Chemical shifts are reported in δ/ppm relative to external standards
and coupling constants J are given in Hz. Abbreviations for the
characterization of the signals: s ) singlet, d ) doublet, t ) triplet,
m ) multiplet, bs ) broad singlet, dd ) double doublet. The relative
numbers of protons is determined by integration. Error of reported
values: chemical shift 0.01 ppm (1H NMR), 0.1 ppm (13C NMR),
coupling constant 0.1 Hz. The used solvent for each spectrum is
reported. Mass spectra were recorded with Finnigan MAT TSQ
7000 (ESI) and Finnigan MAT 90 (HRMS), IR spectra with a Bio-
Rad FT-IR-FTS 155 spectrometer and UV/vis spectra with a Cary
BIO 50 UV/vis/NIR spectrometer (Varian). For experimental details
and analytical data for the intermediates 16d, 18a-e, 19a-e and
1
for the target compounds 5, 7-15, H and 13C NMR spectra and
HPLC tracings of key target compounds cf. Supporting Information.
General Procedure A for the Preparation of Carbonyl
Chlorides. The corresponding (hetero-) aromatic carboxylic acid
was suspended in SOCl2 (10-15 mL) and heated to reflux for two
hours. Excess SOCl2 was removed under reduced pressure, and the
resulting white solid was dried under vacuum.
General Procedures B and C for the Preparation of the
Amide Bonds. (B) The aromatic amine (17, 19a-d) (1 equiv) and
NEt3 (3 equiv) were dissolved in CH2Cl2, and the aromatic carbonyl
chloride (1.5 equiv) derived from the corresponding acid via general
procedure A was added in small portions. The solution was stirred
at room temperature for 24 h, diluted with CH2Cl2, washed with
water and saturated aqueous solution of Na2CO3 (3×), dried over
MgSO4, and concentrated to give the crude product, which was
purified by flash chromatography on silica gel or recrystallization.
(C) The aromatic carboxylic acid (1.1 equiv), DIPEA (2 equiv),
HOBt (1.2 equiv), and HBTU (1.2 equiv) were dissolved in CH2Cl2
at 0 °C and stirred for 20 min. The amine (1 equiv) was added in
small portions, and the solution was allowed to warm to room
temperature and stirred for 24 h. The solution was diluted with
CH2Cl2, washed with water (2×) and saturated aqueous solution
of Na2CO3 (3×), dried over MgSO4, and concentrated to give the
crude product, which was purified by flash chromatography on silica
gel.
General Procedure D for the Reduction of the Nitro Group.
The corresponding nitro compound was dissolved in a mixture of
ethyl acetate and methanol, palladium on activated charcoal (10%
m/m) was added, and the solution was stirred under 5 bar H2
atmosphere overnight. The catalyst was filtered off, and the solvents
were removed to obtain the amines in quantitative yields.
Methyl 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-
2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2-carbonylami-
no)benzoate (6). The compound was synthesized following general
procedure C and purified by flash chromatography on silica gel
(5% MeOH/CHCl3, Rf ) 0.36) to obtain a pale-yellow solid (70
1
mg, 10%); mp ) 176 °C (decomposition). H NMR (300 MHz,
CD2Cl2): δ ) 2.85-3.02 (m, 8H, 4 CH2), 3.78 (s, 2H, NCH2), 3.84
(s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.07 (s, 3H, COOCH3), 6.54
(s, 1H, H-Ar), 6.60 (s, 1H, H-Ar), 7.24-7.27 (m, 2H, H-Ar,
AA′BB′), 7.64-7.67 (m, 2H, H-Ar, AA′BB′), 7.62-7.67 (m, 1H,
H-Ar), 7.69 (dd, 3J ) 8.2 Hz, 4J ) 1.6 Hz, 1H, H-Ar), 7.77-7.84
(m, 1H, H-Ar), 7.88-7.91 (m, 1H, H-Ar), 8.17 (d, 3J ) 8.2 Hz,

1196 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Ku¨hnle et al.
adjusting the cells to a number of 1 × 106 per mL with culture
medium, mitoxantrone was added to the cell suspension to achieve
a concentration of 20 µM. Different concentrations of the test
compound, solvent, and 1 (final concentration 10 µM) were added,
respectively. The cell suspensions were vortexed and incubated for
30 min at 37 °C/5% CO2 to allow maximal mitoxantrone uptake
into the cells. After one washing step with 0.8 mL of ice-cold PBS,
the cells of the sample containing 1 were resuspended in 0.5 mL
of PBS and placed on ice in the dark until the measurement to
avoid mitoxantrone efflux (determination of the 100% mitoxantrone
uptake). All other samples were resuspended in 1 mL of drug-free
culture medium and incubated for 1 h at 37 °C/5% CO2 in which
an equilibrium of mitoxantrone could be reached between the
cytoplasm and the surrounding medium. Subsequently, after the
medium was removed by centrifugation, the cell pellets were rinsed
once with 0.8 mL of ice-cold PBS and finally resuspended in 0.5
mL of PBS for the flow cytometry. A FACS calibur (Becton
Dickinson, Heidelberg, Germany) was used to analyze the fluo-
rescence intensity of the cells. Mitoxantrone accumulation in the
cells was monitored by using an excitation wavelength of 635 nm,
whereas emission was detected at a wavelength of 661 nm. A
minimum of 20000 events was collected per sample, and the events
were gated according to forward scatter and sideward scatter to
exclude clumps and debris. Analysis of the raw data was performed
with the WinMDI 2.8 software. The geometric means were
calculated from the fluorescence intensity histogram and related to
the controls. Afterward, the mean values of 3 independent measure-
ments were plotted against the concentration of the test compounds.
Addition of increasing concentrations of the modulator led to
sigmoidal concentration response curves. IC50 values were calcu-
lated using SIGMA PLOT 9.0, four parameter logistic curve fitting.
Errors were calculated as standard error of the mean.
(180 µL/well) while shaking the microplates for about 3 h.
Absorbance (a parameter proportional to cell mass) was measured
at 578 nm using a BIOTEK 309 Autoreader (TECNOMARA,
Fernwald, Germany).
Drug effects were expressed as corrected T/C-values for each
group according to
T - Co
C - C0
T ⁄ Ccorr
)
× 100 [%]
where T is the mean absorbance of the treated cells, C the mean
absorbance of the controls, and C0 the mean absorbance of the cells
at the time (t ) 0) when drug was added. When the absorbance of
treated cells T is less than that of the culture at t ) 0 (C0), the
extent of cell killing was calculated as
C0 - T
C0
cytocidal effect [%] )
× 100
For assays performed on quiescent U-373 MG cells, a high sowings
density was chosen in order to observe as much as possible the
effects of the compounds against resting cells. To detect maximum
cytocidal effect rotenone, an ubiquinon reductase inhibitor, blocking
ATP synthesis, served as positive control.
Acknowledgment. This work was supported by the Graduate
Training Program (Graduiertenkolleg) GRK 760, “Medicinal
Chemistry: Molecular RecognitionsLigand-Receptor Interac-
tions”, of the Deutsche Forschungsgemeinschaft and the Fonds
der Chemischen Industrie. M.E. thanks the Elitenetzwerk Bayern
for a graduate fellowship. We thank Carolin Fischer for the
synthesis of the carboxylic acid corresponding to ester 5, Dr.
A. H. Schinkel, Netherlands Cancer Institute (Amsterdam) for
kindly providing 2 (Ko143), Dr. Susan Bates from the NIH
(Bethesda, MD) for the reference compound 1 (fumitremorgin
C), and Dr. P. Borst (Netherland Cancer Institute, Amsterdam)
for providing the ABCC2 overexpressing MDCKII-MRP2 cells.
Modulation of ABCB1 (p-gp): Determination in the Flow
Cytometric Calcein-AM Assay. The assay was performed as
described.43
Modulation of ABCC2 (MRP2): Determination in the CM-
FDA Assay. Cells were kept in culture in 24-well plates. They
were washed twice with Krebs-Ringer buffer (KRB) at 37 °C prior
to experiments and then incubated with 1 µM CMFDA in the
absence or the presence of the test compounds at increasing
concentrations. Typically, the modulators were dissolved in DMSO.
Control experiments confirmed that the cell monolayers tolerated
up to 1% DMSO in the incubation medium without functional
impairment. All monolayers were incubated for 90 min at 37 °C
and under constant circular shaking at 50 rpm. Subsequently, the
culture plates were placed on ice, medium in the apical compartment
was removed, and the cells were washed twice with ice-cold KRB.
The cells were lysed by incubation with 200 µL of 1% Triton X-100
in KRB for 30 min under constant shaking. Finally, the culture
plates were subjected to fluorescence quantification in a plate reader
(Tecan Safire XFLUOR4, Tecan Safire, Crailsheim, Germany) with
filter settings of λex of 485 nm and λem of 516 nm. The extent of
transport inhibition in presence of ABCC2 modulators was calcu-
lated from fluorescence intensities using the software GraphPad
Prism (GraphPad Software, Inc., San Diego, CA).
Supporting Information Available: Experimental details and
analytical data for the intermediates 16d, 18a-e, 19a-e and for
the target compounds 5, 7-15, 1H- and 13C NMR spectra and HPLC
analysis including tracings of key target compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
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