Flavonoid derivatives as selective ABCC1 modulators: Synthesis and functional characterization

Article abstract of DOI:10.1016/j.ejmech.2015.12.010

A series of chromones, bearing substituted amino groups or N-substituted carboxamide moieties in position 2, was synthesized and characterized in cellular assays for modulation of the ABC transporters ABCC1 (MDCKII-MRP1 cells), ABCB1 (Kb-V1 cells) and ABCG2 (MCF-7/Topo cells). The most potent ABCC1 modulators identified among these flavonoid-type compounds were comparable to the reference compound reversan regarding potency, but superior in terms of selectivity concerning ABCB1 and ABCG2 (2-[4-(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl]-5,7-dimethoxy-4H-chromen-4-one (51): ABCC1, IC₅₀ 11.3-1/4M; inactive at ABCB1 and ABCG2). Compound 51 was as effective as reversan in reverting ABCC1-mediated resistance to cytostatics in MDCKII-MRP1 cells and proved to be stable in mouse plasma and cell culture medium. Modulators, such as compound 51, are of potential value as pharmacological tools for the investigation of the (patho)physiological role of ABCC1.

Full text of DOI:10.1016/j.ejmech.2015.12.010

Accepted Manuscript
Flavonoid derivatives as selective ABCC1 modulators: Synthesis and functional
characterization
José Esteban Obreque-Balboa, Qiu Sun, Günther Bernhardt, Burkhard König, Armin
Buschauer
PII:
S0223-5234(15)30396-2
10.1016/j.ejmech.2015.12.010
EJMECH 8250
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2015
Revised Date: 23 November 2015
Accepted Date: 9 December 2015
Please cite this article as: J.E. Obreque-Balboa, Q. Sun, G. Bernhardt, B. König, A. Buschauer,
Flavonoid derivatives as selective ABCC1 modulators: Synthesis and functional characterization,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Flavonoid derivatives as selective ABCC1
modulators: Synthesis and functional
characterization
a
b,*
José Esteban Obreque-Balboa a,1, Qiu Sun b,1, Günther Bernhardt , Burkhard König
,
a,*
Armin Buschauer
a Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
b Institute of Organic Chemistry, University of Regensburg, D-93040 Regensburg, Germany.
* Corresponding Authors
E-mail addresses: Burkhard.Koenig@chemie.uni-regensburg.de (B. König),
Armin.Buschauer@chemie.uni-regensburg.de (A. Buschauer)
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. 1These authors contributed equally to this
work. Syntheses were performed by Q.S., analytical and biological investigations were
carried out by J.O.-B.

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ABSTRACT
A series of chromones, bearing substituted amino groups or N-substituted carboxamide
moieties in position 2, was synthesized and characterized in cellular assays for modulation of
the ABC transporters-ABCC1 (MDCKII-MRP1 cells), ABCB1 (Kb-V1 cells) and ABCG2
(MCF-7/Topo cells). The most potent ABCC1 modulators identified among these flavonoid-
type compounds were comparable to the reference compound reversan regarding potency, but
superior in terms of selectivity concerning ABCB1 and ABCG2 (2-[4-
(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl]-5,7-dimethoxy-4H-chromen-4-one
(51): ABCC1, IC50 11.3 µM; inactive at ABCB1 and ABCG2). Compound 51 was as
effective as reversan in reverting ABCC1-mediated resistance to cytostatics in MDCKII-
MRP1 cells and proved to be stable in mouse plasma and cell culture medium. Modulators,
such as compound 51, are of potential value as pharmacological tools for the investigation of
the (patho)physiological role of ABCC1.
Keywords:
ABC transporter, multidrug resistance, chromone, reversan, ABCC1, MRP1.

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Abbreviations
ABCB1, ATP-binding cassette transporter, subfamily B, member 1, also termed p-gp or P-
glycoprotein; ABCC1, ATP-binding cassette transporter, subfamily C, member 1, also termed
multidrug resistance associated protein 1 (MRP1); ABCG2, ATP-binding cassette transporter,
subfamily G, member 2, also termed breast cancer resistance protein (BCRP) or mitoxantrone
resistance protein (MXR); BSA, bovine serum albumin; Calcein-AM, calcein acetoxymethyl
ester; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; EMEM,
Eagle minimum essential medium; FCS, fetal calf serum; FTC, fumitremorgin C; IC50,
concentration of inhibition required to give 50% inhibition of activity; MDR, multidrug
resistance; SEM, standard error of the mean; TLC, thin layer chromatography.

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1. Introduction
The superfamily of human ATP-binding cassette (ABC) proteins comprises 49 members
divided into 7 subfamilies (ABCA - ABCG) [1-3]. The proteins ABCB1 (p-glycoprotein, p-
gp), ABCG2 (BCRP, MXR) and ABCC1 (MRP1) are amply expressed efflux pumps in
various tissues related with absorption, metabolism and excretion, e. g. lung, gut, kidney and
liver. In addition, these transporters are important components of physiological barriers, such
as the blood-brain barrier and the blood-cerebrospinal fluid barrier, mediating the transport of
endogenous compounds and playing a protective role against a wide range of structurally
diverse xenobiotics [4-8]. ABC transporters are known to lower the bioavailability of orally
administered drugs and to limit the access of pharmacotherapeutics to the central nervous
system (CNS). Moreover, ABCB1, ABCG2 and ABCC1 are the most prominent efflux
transporters expressed in cancer cells and related to resistance against numerous antitumor
agents such as anthracyclines, vinca alcaloids or epipodophyllotoxins, resulting in poor
outcome of chemotherapy [3-7, 9-11]. This phenomenon is known as multidrug resistance
(MDR) [11, 12]. Since its discovery in 1992 [13], ABCC1 has been the subject of many
investigations on its role in physiological processes and MDR [14-20]. Besides non-small-
lung cancer cells [13], ABCC1 overexpression was detected in acute myeloblastic leukemia,
acute lymphoblastic leukemia, prostate cancer, some types of breast cancer and especially
neuroblastoma [21]. Based on this evidence, the co-administration of an ABCC1 modulator
and a cytostatic, which is substrate of ABCC1, might improve the outcome in cancer therapy.
Several substances were described as modulators of ABCC1 (Fig. 1), including MK-571,
flavonoids,
imidazothiazole
derivatives,
dihydropyridines,
pyrrolopyrimidines,
indolopyrimidines, anellated isoxazoles such as LY465803 and LY455776, and reversan [21-
28]. Nevertheless, most of them are non-selective inhibitors or substrates of the ABCC1
transporter.

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Fig. 1. Structures of several ABCC1 (MRP1) modulators.
Selective modulators are required as pharmacological tools to investigate the role of ABC
transporters in health and disease, to improve access to the brain or to overcome
chemoresistance. Previously, potent and selective ABCB1 and ABCG2 modulators were
developed in our laboratory [29-33]. Surprisingly, we identified compounds with inhibitory
activity at ABCC1 among substances which were initially synthesized as putative ABCG2
modulators. This fortuitous observation, supported by different studies reporting flavonoids
[22, 23, 28] as substrates or inhibitors of ABC transporters, inspired the synthesis and
characterization of the title compounds with respect to ABCC1 selectivity.
2. Results and discussion
2.1 Chemistry
2.1.1 Synthesis of compounds 31-53
Our synthetic strategy for compounds 31-53 (Scheme 1) followed a published procedure
[34]. Commercially available hydroxylated acetophenones (1, 2) were methylated with
Me2SO4 in good yields. Ring closure of the obtained compounds (3, 4) was accomplished by

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treatment with CS2 in the presence of H2SO4 as a catalyst to give the chromene-2-thiones 5
and 6. Subsequent S-alkylation with ethyl iodide under basic conditions followed by the
oxidation of the ethylsulfanyl-4-chromones (7, 8) with m-CPBA led to the corresponding
sulfoxides (9, 10). Finally, the reaction of 9-10 with appropriate N-substituted piperazines 11-
28 or primary amines 29, 30 at ambient conditions afforded 31-46, 49-53 in 20-45% yield.
The aromatic amine 47 was obtained in 80% yield by reduction of 32 using Fe/HCl/H2O
according to a known protocol [35, 36] and chromatographic purification. Subsequent
amidation with acetyl chloride gave 48 as a colorless solid in 46% yield.
Scheme 1. Synthesis of compounds 31-53. Reagents and conditions: (a) Me2SO4, acetone,
65 °C. (b) (1) tBuOK, CS2, PhCH3, rt; (2) 10% H2SO4, r.t. (c) EtI, K2CO3, acetone, reflux. (d)
mCPBA, DCM, reflux. (e) DIPEA, N-substituted piperazines, EtOH, 80 °C. (f) Iron powder,
conc. HCl, EtOH, reflux. (g) Acetyl chloride, Et3N, DCM, r.t.

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2.1.2 Synthesis of compounds 62-65
Compounds 62-65 and the reference compounds 66 and 67 [37] were synthesized as shown
in Scheme 2. In the first step, by analogy with a described procedure [38] , Claisen
condensation was conducted yielding intermediates 55, 56, followed by acid-catalyzed
cyclization reaction, which gave esters 14, 15. After hydrolysis of the ester, products 62-67
were prepared under standard amide coupling conditions with HBTU [39].
Scheme 2. Synthesis of chromone-2-carboxamides 62-67. Reagents and conditions: (a)
NaOEt, diethyl oxalate. (b) conc. HCl, EtOH, reflux. (c) K2CO3, EtOH, THF, r.t. (d) HBr,
HOAc, reflux. (e) DIPEA, HBTU, DCM, r.t.
The amines required as building blocks for the synthesis of the title compounds were
prepared according to standard procedures as outlined in Supplementary Material.
2.2 Inhibition of ABCC1, ABCB1 and ABCG2 transporters
The synthesized compounds 31-53, 62-67 and reference substances were investigated for
modulation of ABCC1, ABCB1 and ABCG2 using calcein-AM and Hoechst 33342 efflux
assays in the microtiter plate format [32]. The data are summarized in Table 1 and Table 2.

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Table 1. Inhibition of ABCC1, ABCB1 and ABCG2 transporters by flavonoid-type modulators 31-53 and reference compounds
ABCC1a
ABCB1b
ABCG2c
Compound
R2
d,e
d,f
d,g
n
1
1
2
1
1
2
1
0
1
1
1
IC50 µMd
7.3 ± 2
Imax
%
IC50 µMd
inactive
inactive
Imax
%
IC50 µMd
inactive
Imax %
4-NO2
4-NO2
4-NO2
H
38.8 ± 3.5
130.1 ± 7.7
118.5 ± 6.4
-
-
-
31
32
33
34
35
36
37
38
39
40
41
13.2 ± 2.5
10.9 ± 1.8
>100
-
87.8 ± 6.7
36.9 ± 6.7
inactive
56.9 ± 2.5
33.0 ± 4.6
inactive
58.5 ± 6
inactive
inactive
inactive
32.8 ± 3.9
>100
54.8 ± 3.6
31.9 ± 2.7
-
-
3,4-F2
H
38.0 ± 16
6.8 ± 4.2
>100
140.6 ± 20
44.5 ± 7.6
-
14.3 ± 1
66.6 ± 11.3
>100
50.4 ± 5
-
-
4-OMe
4-NO2
3-NO2
4-COOMe
4-CN
-
>100
-
12.1 ± 2.2
18.8 ± 7.6
58.3 ± 10.3
26.7 ± 3.5
64.1 ± 3.5
-
12.2 ± 2.6
38.2 ± 6.4
>100
22.6 ± 2.0
44.0 ± 3.8
-
193.3 ± 24.8
118.5 ± 11.8
114.9 ± 5.2
29.2 ± 1.9
-
-
>100
99.7 ± 8.6
59.1 ± 4.0

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1
1
1
1
1
1
1
1
1
2-NO2
4-SO2Me
8.3 ± 1.1
>100
68.8 ± 4.0
>100
inactive
31.3 ± 3.8
36.6 ± 3.2
>100
-
37.6 ± 4.7
inactive
>100
72.6 ± 4.5
42
43
44
45
46
47
48
49
50
-
-
-
-
-
-
-
-
-
-
4-CF3
19.8 ± 2.6
20.8 ± 11.2
22.7 ± 5.8
>100
158.6 ± 7.3
21.8 ± 1.5
4-SCF3
136.5 ± 24.6
29.4 ± 1
>100
4-SO2CF3
98.8 ± 8.6
-
-
-
-
-
>100
4-NH2
-
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
4-NHAc
>100
-
Me
inactive
30.1± 7.2
-
4-NO2-benzoyl
benzo[c][1,2,5]oxadiaz
ol-5-ylmethyl
3,4-dimethoxyphenyl
morpholin-4-yl
19.7 ± 1.5
11.3 ± 1.8
119.6 ± 6.4
inactive
-
inactive
-
-
51
2
3
>100
53.3 ± 17.8
4.3 ± 0.2
>30
-
inactive
inactive
6.8 ± 0.3f
inactive
n.d.
-
>100
inactive
n.d.
52
53
14 ± 2.7
-
Reversan
MK571
100
143.3 ± 3.3
-
-
-
-
-
-
inactive
0.73 ± 0.09h
0.52± 0.09i
Fumitremorgin C
Tariquidar
inactive
inactive
100
69± 5
-
0.22 ± 0.01j
100
a Calcein-AM microplate assay using ABCC1-overexpressing MDCK-MRP1 or b ABCB1-overexpressing Kb-V1 cells. c Hoechst 33342 assay using ABCG2-
d
e
overexpressing MCF-7/Topo cells. Mean values ± SEM from two to four independent experiments performed in triplicate or sextuplicate. Inhibitory effect
f
(Imax) relative to the maximum response to reversan at a concentration of 30 µM (100%). Imax expressed as percent inhibition relative to tariquidar at a
g
h
i
concentration of 1 µM (100%). Imax relative to FTC at a concentration of 10 µM (100%). Ref. [40]. Ref. [41]. IC50 values were calculated using SigmaPlot
11.0, four parameter logistic curve fitting; inactive: no transporter inhibition up to a concentration as high as 100 µM; n.d. not determined; IC50 value > 100 µM
or >30 µM means that the inhibition achieved at the respective concentration did not allow a determination of the IC50 value.

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Table 2. Inhibition of ABCC1-, ABCB1-, and ABCG2-transporters by the new flavonoid-type modulators 62–65 and reference compounds 66, 67
ABCC1a
ABCB1b
IC50 µMd
ABCG2c
Compound
n
2
R1
R2
R3
IC50 µMd
18.5 ± 3.4
Imax
%
Imax
%
IC50 µMd
>100
Imax %
d,e
d,f
d,g
OMe
OMe
4-NO2
139.4 ± 7.6
83.9 ± 11.7
37.7 ± 3.4
-
62
1
1
1
1
-
OMe
OMe
OMe
H
OMe
OH
4-NO2
2-NO2
4-NO2
H
5.5 ± 0.6
8.3±1.1
1.8 ± 0.3
>200
73.1 ± 2.6
58.2 ± 3.3
inactive
33.4 ± 2.0
>100
-
63
64
65
66
67
68.8 ± 4.0
-
34.9 ± 2.5
12 ± 1.2
90.5 ± 2.7
23.1 ± 0.9
84.1 ± 9.9
108 ± 4.6
OH
70.5 ± 4.8
inactive
-
OCH2Ph
-
-
-
15.8 ± 1.4
inactive
34.3 ± 1.7
-
16.3 ± 4.7h
1.8 ± 0.3h
-
-
inactive
a Calcein-AM microplate assay using ABCC1-overexpressing MDCK-MRP1 or b ABCB1-overexpressing Kb-V1 cells. c Hoechst 33342 assay using ABCG2-
d
e
overexpressing MCF-7/Topo cells. Mean values ± SEM from two to four independent experiments performed in triplicate or sextuplicate. Inhibitory effect
f
(Imax) relative to the maximum response to reversan at a concentration of 30 µM (100%). Imax expressed as percent inhibition relative to tariquidar at a
concentration of 1 µM (100%). g Imax relative to FTC at 10 µM (100%). h Ref.[37]: ABCG2 inhibition reported for 66: 9.8 ± 2.5 at a concentration of 5 µM, and
IC50 reported for 67: 0.17 µM, determined on mitoxantrone flow cytometric assay using ABCG2-transfected HEK293 cells [37]. IC50 values were calculated
using SigmaPlot 11.0, four parameter logistic curve fitting; inactive: no transporter inhibition up to a concentration as high as 100 µM; IC50 value > 100 µM or
>200 µM means that the inhibition achieved at the respective concentration did not allow a determination of the IC50 value.

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In accordance with a previous publication [25], reversan was more potent than MK571 as an
MRP1 modulator in our assay (Table 1). Therefore, reversan was selected as the reference
compound. The most potent ABCC1 inhibitors identified among the compounds 31-53 and 62-
67 were comparable with reversan in terms of IC50 values (31, 32, 36, 38, 51, 63-65).
Interestingly, these compounds were different from the reference substance regarding maximal
response (Imax) and, especially, selectivity. Chromone derivative 33 exhibited modulator activity
in the two-digit micromolar range and showed a slight preference for ABCC1 compared to
ABCB1 and ABCG2. ABCC1 selectivity was improved with compound 32, the lower
homologue of 33, which was inactive at ABCB1 and much less potent at ABCG2. Solubility
studies performed with 32 and 33 as examples revealed that the dissolution of 33 in loading
buffer was slower than that of 32, but there was no difference in the amount of dissolved
compound. Interestingly, compared to buffer, the solubility was increased under assay-like
conditions, i. e. in the presence of protein and pluronic F127 (loading suspension devoid of
calcein-AM), from 53 % to 100 %, when a nominal concentration of 100 µM of 32 or 33 was
analyzed (cf. Supplementary Material). Insertion of a carbonyl group between the
phenylchromone moiety and N-substituted piperazines led to a slight decrease in the inhibitory
effect (62, 63), whereas the replacement of the methylene linker in 32 by a carbonyl group (50)
dramatically decreased the maximal response from 130% to approximately 20%. In addition, 64
and 65 were prepared to study the contribution of an H-bond donor in position 5 of the
phenylchromone core. Compared with the compounds bearing two methoxy groups, the IC50
value at ABCC1 decreased from 5.5 µM (63) to 1.8 µM (65). However, a moderate inhibitory
activity at ABCB1 or ABCG2 was observed. Changing the position of the nitro group on the
phenyl ring from para (32) to meta, reduced the ABCC1 inhibitory activity (39). The shift of the
11

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nitro group to the ortho position in the phenyl ring decreased the inhibition of ABCC1, while the
activity at ABCG2 increased (42). Keeping in mind that nitro groups can be reduced in vivo,
resulting in potentially toxic reactive metabolites [42], we replaced the nitro group by different
substituents. In general, electron-withdrawing groups on the phenyl ring markedly contributed to
inhibition of ABCC1. Compounds with strong electron withdrawing group in para position (32,
33, 35, 41, 44, 46, and 51) showed higher potencies and maximum effects. In contrast, lack of
substituents or introduction of electron-donating groups (34, 36, 37, and 47) resulted in a strong
decrease in inhibitory activity. Examples of concentration-response curves are shown in Fig. 2.
Fig. 2. Concentration dependent inhibition of ABCC1 transporter in MDCKII-MRP1 cells
(calcein-AM assay) by reversan, 32, 33, 51, 63, and 65. The inhibition is expressed relative to the
maximal effect in the presence of 30 µM of reversan set to 100%.
The flavonoids 66 and 67 were described as selective inhibitors of ABCG2 [37]. As the
scaffold of 66 and 67 is similar to that of 31-53 and 62-65 we synthesized and investigated these
substances for comparison. Compound 66 showed negligible inhibition and 67 was inactive at
ABCC1 (Table 2). In our assay systems, compound 66 inhibited ABCB1 with an IC50 value of
12

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15.8 µM (Imax 34 %;). In case of 67, the potency determined with MCF-7/Topo cells was 10-fold
lower than that reported for HEK293-ABCG2 cells [37], whereas the selectivity of 67 for
ABCG2 was confirmed (no IC50 values for inhibition of ABCB1 and ABCC1 by 66 and 67
provided in ref. [37]).
Taken together, the maximum inhibitory effect was strongly dependent, both on the
substituents (R1, R2) at the chromone and the presence of an electron-withdrawing substituent at
the phenyl moiety, in particular NO2 or CF3. Whereas reversan was equipotent at ABCB1 and
ABCC1, several of the new modulators revealed preference for ABCC1 with highest selectivity
residing in compound 51.
2.3 Chemosensitivity and reversal of drug resistance of ABCC1 expressing MDCKII-MRP1 cells
On the basis of the results obtained from the functional efflux assays, the effect of the most
potent and selective ABCC1 modulator (51) on the proliferation as well as the ability to
overcome drug resistance of MDCKII-MRP1 cells was investigated in a kinetic crystal violet
chemosensitivity assay, using the modulator or the reference compound reversan alone and in
combination with the cytostatic etoposide, an ABCC1 substrate. The results are shown in Fig. 3..
Additionally, compounds 32, 33, 36, 51, 62, 63, and 65 were investigated in kinetic assays at
spontaneously immortalized human keratinocytes (HaCaT cells) [43] with respect to
cytotoxicity. The compounds proved to be non-toxic against HaCaT cells, which are considered
a model of “normal” cells (cf. Supplementary Material).
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Fig. 3. Reversal of ABCC1 mediated drug resistance against etoposide and doxorubicin on
proliferating MDCKII-MRP1 cells. Effect of reversan alone (A), compound 51 alone (B),
etoposide (1.5 µM) alone (open inverted triangles) and in combination (colored symbols) with 1
and 5 µM of either reversan (C) or compound 51 (D), doxorubicin (50 nM) alone (filled inverted
14

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triangles) and in combination (colored symbols) with 1 and 5 µM of either reversan (E) or
compound 51 (F).
Incubation of MDCKII-MRP1 cells with reversan alone had no effect on cell proliferation up
to a concentration of 10 µM (Fig. 3A). By analogy with reversan, the proliferation of MDCKII-
MRP1 cells incubated with the ABCC1 modulator 51 alone was not affected (Fig. 3B).
Etoposide alone at a concentration of 1.5 µM produced only a transient antiproliferative effect
(Fig. 3C, D). By contrast the combination with reversan (1.0 µM) led to a complete reversal of
the ABCC1-mediated resistance, which was not further enhanced by increasing the concentration
of the modulator (Fig. 3C). The cytostatic effect of etoposide at 1.5 µM in combination with
reversan was the same as after the administration of etoposide alone at a concentration of 10 µM
(data not shown). The antiproliferative activity of etoposide (1.5 µM) was enhanced to the
maximal response when combined with 51, even at a concentration as low as 1.0 µM (Fig. 3D).
In the same way, the ABCC1 overexpressing MDCKII-MRP1 cells were incubated with a
different ABCC1 substrate, doxorubicin, at a per se nontoxic concentration of 50 nM in
combination with reversan (Fig. 3E) or 51 (Fig. 3F), leading to complete reversal of ABCC1-
mediated chemoresistance.
2.4 Stability of compounds in mouse plasma and culture medium
With respect to in vivo studies, the most potent modulator (51) was investigated for stability in
mouse plasma. Compound 51 was incubated at a temperature of 37 °C, and aliquots were
analyzed by HPLC over a period of 24 hours. As obvious from Fig. 4, no products of cleavage or
degradation of compound 51 were detected. Additionally, the compounds 32, 33, 36, 51, 62, and
15

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65 proved to be stable in culture medium (DMEM supplemented with 10% FCS) at a
temperature of 37 °C oer a period of 48 hours (cf. Supplementary Material).
Fig. 4. Chromatograms of compound 51 incubated in mouse plasma over a period of 24 h. HPLC
analysis, UV detection at 220 nm.
3. Conclusion
Among the synthesized flavonoids, several ABCC1 modulators were identified, which were
comparable to reversan in terms of potency, but superior regarding selectivity over ABCB1 and
ABCG2. The new compounds were capable of reverting chemoresistance of ABCC1
overexpressing cells and are expected to be of potential value as pharmacological tools for the
investigation of the (patho)physiological role of the ABCC1 transporter.
4. Experimental section
4.1 General chemistry
Starting materials and reagents were from Sigma Aldrich (Munich, Germany) and Alfa Aesar
(Karlsruhe, Germany) unless otherwise indicated and used without further purification.
Deuterated solvents for NMR spectroscopy were from Deutero (Kastellaun, Germany).
Automated flash chromatography was performed with a Biotage Isolera Spektra One system
16

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1
(Uppsala, Sweden) using silica gel 60M (40-63 µm, Merck). H, 13C and 2D NMR spectra were
measured at 298 K on an AVANCE 300 spectrometer (Bruker, Karlsruhe, Germany) (operating
at 300.13 MHz for 1H and 75.47 MHz for 13C), a Bruker AVANCE 400 spectrometer (operating
1
at 400.13 MHz for H and 100.62 MHz for 13C) and a Bruker AVANCE 600 spectrometer
1
(operating at 600.25 MHz for H and 150.93 MHz for 13C). Spectra were measured in
chloroform-d (99.8%) or methanol-d4 (99.8%) or DMSO-d6 (99.9%) and referenced against non-
deuterated (1H) /deuterated (13C) solvent. Shift values (δH and δC) were always given in ppm, J
values in Hz. Melting points were measured on a Stanford Research Systems OptiMelt MPA 100
(Gilching, Germany). Compounds 31-53 and 62-67 were confirmed to be > 95% pure using
analytical RP-HPLC analysis performed with a Eurosphere-100 C18 column (250 x 4 mm, 5 µm,
Knauer, Berlin, Germany) or a Kinetex-100 5µ XB-C18 column (250 x 4.0 mm; Phenomenex,
Aschaffenburg, Germany) at 30 °C on a Merck Hitachi system (Merck, Darmstadt, Germany)
consisting of an AS-2000A , a L-6200-A pump, a L-400A UV-VIS detector. UV-detection was
performed at 220 nm. Mixtures of MeCN (A) and 0.05 % aq. TFA (B) were used as mobile
phase, using a linear gradient (A/B: 15/85 to 80/20 or 85/15) over 30 min. High-resolution mass
spectra were measured on a Finnigan MAT SSQ 710 A spectrometer (Bremen, Germany) at 70
eV (HREIMS, positive and negative mode) or recorded on an Agilent 6540 UHD (Frankfurt,
Germany) (HRESIMS, positive and negative mode). Analytical TLC was performed on silica gel
coated alumina plates (TLC sheets ALUGRAM Xtra SIL G/UV254; Macherey-Nagel, Düren,
Germany). Analytes were detected with UV-light (254 or 366 nm).
Building blocks 5-30. For the synthesis of intermediates 5-10 (cf. Supplementary Material).
The amine building blocks 17, 26 and 29 were commercially available. Compounds 11[44], 12
[45], 13 [46], 14 [47], 15 [48], 16 [49], 18 [49], 19 [50], 20 [51], 21 [52], 22 [53], 23 [53], 27
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[54], 28 [55], and 30 [56] were obtained according to the literature [57], and 24 and 25 were
synthesized by analogy with the published protocols for mono-alkylation of piperazine (cf.
Supplementary Material).
4.1.1 General procedure for the synthesis of compounds 31-53
To a solution of 9 or 10 (1 equiv.) in ethanol, the pertinent N-substituted piperazine derivative
(1.0 equiv.) and DIPEA (5 equiv.) were added at 0 °C. The mixture was refluxed for 24 h. After
purification by flash column, the products 31-53 were obtained in moderate yields. Preparative
and analytical data of 31, 36 and 51 are given as examples below. For additional data cf. Supp.
Inf.
4.1.1.1 5-Methoxy-2-[4-(4-nitrobenzyl)piperazin-1-yl]-4H-chromen-4-one 31. Pale yellow
solid. Yield: 119 mg. 54 %. Purity 98% (HPLC detection at 220nm). Mp. 167−168 °C. 1H NMR
(400 MHz, CDCl3) δ 8.24 – 8.17 (m, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.43 (t, J = 8.4 Hz, 1H), 6.87
(dd, J = 8.4, 0.8 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 5.44 (s, 1H), 3.96 (s, 3H), 3.66 (s, 2H), 3.55 –
13
3.45 (m, 4H), 2.69 – 2.50 (m, 4H). C NMR (101 MHz, CDCl3) δ 177.8, 161.1, 159.6, 155.9,
147.4, 145.4, 132.2, 129.5, 123.7, 113.1, 108.9, 106.8, 88.9, 61.9, 56.5, 52.2, 44.5. HRMS (EI-
MS) calculated for C21H21N3O5 [M+H]+ 396.1554, found 396.1555.
4.1.1.2 5,7-Dimethoxy-2-(4-phenethylpiperazin-1-yl)-4H-chromen-4-one 36. Yellow solid.
1
Yield: 36 mg, 27%. Purity 95% (HPLC detection at 220 nm). Mp. 145−147 °C. H NMR (300
MHz, CD3OD) δ 7.35 – 7.10 (m, 5H), 6.57 (d, J = 2.4Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 5.36 (s,
13
1H), 3.87 (d, J = 4.2 Hz, 6H), 3.63 – 3.53 (m, 4H), 2.88-2.83 (m, 2H), 2.74 – 2.64 (m, 6H). C
NMR (75 MHz, CD3OD) δ 180.1, 165.4, 163.1, 161.7, 158.8, 141.0, 129.8, 129.6, 127.3, 107.2,
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97.1, 93.9, 87.7, 61.2, 56.5, 53.3, 45.3, 34.0. HRMS (EI-MS) calculated for C23H26N2O4 [M+H]+
395.1965, found 395.1958.
4.1.1.3 2-[4-(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl]-5,7-dimethoxy-4H-chromen-
4-one 51. White solid. Yield: 35 mg, 25%. Purity 95% (HPLC detection at 220nm). Mp.
204−206 °C. 1H NMR (300 MHz, CDCl3) δ 7.80 (d, J = 9.3 Hz, 1H), 7.72 (s, 1H), 7.50 (dd, J =
9.3, 1.1 Hz, 1H), 6.51 – 6.14 (m, 2H), 5.36 (s, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.63 (s, 2H), 3.47
13
(t, J = 3.0 Hz, 4H), 2.60 (t, J = 6.0 Hz, 4H). C NMR (75 MHz, CDCl3) δ 177.7, 163.0, 161.1,
160.6, 157.4, 149.3, 149.0, 142.2, 133.2, 116.5, 114.5, 107.4, 95.8, 92.4, 88.5, 62.4, 56.4, 55.6,
52.2, 44.6. HRMS (EI-MS) calculated for C22H22N4O5 [M+H]+ 423.1663, found 423.1667.
4.1.2 Synthesis of compounds 62-67
The intermediates 57 [58], 58 [59], 59 [60], 60 [37] and 61 [61], as well as the reference
compounds 66 [37] and 67 [37] were synthesized according to published protocols.
A 50-mL Schlenk flask was charged with 59-61 (1 equiv.), DIPEA (10 equiv.) and HBTU (3.0
equiv.) under nitrogen atmosphere. After dissolution in 10 mL of anhydrous DCM, the pertinent
N-substituted piperazine (11-13, 21 or 29, 1.0 equiv.) was slowly added at 0 °C. The mixture was
stirred at room temperature for 24 h. The organic phase was washed with water and brine and
dried over anhydrous Na2SO4. The products 62-67 were purified by flash chromatography
(EtOAc: MeOH = 30:1). Preparative and analytical data of 65 is given as an example. For
additional data cf. Supplementary Material.
4.1.2.1 5-Hydroxy-7-methoxy-2-[4-(4-nitrobenzyl)piperazine-1-carbonyl]-4H-chromen-4-one
65. White solid. Yield: 149 mg, 60%. Purity 98% (HPLC detection at 220nm). Mp. 184−186 °C.
1H NMR (400 MHz, CDCl3) δ 12.33 (s, 1H), 8.18 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H),
6.39 (d, J = 2.2 Hz, 1H), 6.37 (s, 1H), 6.36 (d, J = 2.2 Hz, 1H), 3.85 (s, 3H), 3.67 (d, J = 84 Hz,
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4H), 3.65 (s, 2H), 2.53 (d, J = 19.6 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 181.6, 166.1, 162.3,
160.5, 158.1, 157.2, 147.4, 145.3, 129.5, 123.7, 110.5, 106.1, 98.7, 93.1, 61.8, 55.9, 53.2, 52.4,
47.0, 42.4. HRMS (EI-MS) calculated for C22H21N3O7 [M+H]+ 440.1452, found 440.1450.
4.2 Cellular assays
4.2.1 Chemicals
Calcein-AM and Hoechst 33342 were from Biotium (Hayward, CA, USA). Calcein-AM was
dissolved in DMSO (Merck, Darmstadt, Germany) at a concentration of 1 mM, whereas a
solution of Hoechst 33342 (0.8 mM) was prepared in Millipore water. Pluronic F127 was
obtained from Sigma (Munich, Germany) and dissolved to achieve a final concentration of 20%
(m/m) in DMSO. Reversan and fumitremorgin C (FTC) were from Merck (Darmstadt,
Germany), dissolved in DMSO and diluted to achieve concentrations of 3 mM and 1 mM,
respectively. MK571 was purchased from Tocris Bioscience (Bristol, UK) and dissolved in
DMSO. Vinblastine, topotecan and etoposide were from Sigma (Munich, Germany) and
dissolved in 70% ethanol. Tariquidar was synthesized according to literature [62, 63] with minor
modifications [64]. Solutions of vinblastine and topotecan at a concentration of 0.1 mM were
stored at 4 °C, whereas all stock solutions were stored at -20 °C.
4.2.2 Cell culture conditions
Human Kb-V1 cells, a subclone of human Kb cells (ATCC CCL-17), overexpressing ABCB1
were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma, Munich, Germany)
supplemented with 3.7 g/L NaHCO3 (Merck, Darmstadt, Germany) and 110 mg/L sodium
pyruvate (Serva, Heidelberg, Germany) containing 10% fetal calf serum (FCS) (Biochrom,
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Berlin, Germany) and vinblastine at a final concentration of 330 nM to maintain ABCB1
expression [64].
The human breast adenocarcinoma cells MCF-7/Topo (ABCG2 overexpressing subclone of
MCF-7 cells, ATCC HTB-22) were incubated in Eagle’s Minimum Essential Medium containing
2.2 g/L NaHCO3 (Merck, Darmstadt, Germany) and 110 mg/L sodium pyruvate (Serva,
Heidelberg, Germany) and 10% FCS (Biochrom) and 550 nM topotecan to maintain the ABCG2
transport overexpression as described previously [31].
The MDCKII-MRP1 cells (Madin-Darby Canine Kidney, strain II; an epithelial cell line;
ATCC CRL-2936, transfected with the human ABCC1 transporter gene [65]) was a kind gift
from Prof. Dr. P. Borst from Netherland Cancer Institute (Amsterdam, NL). These cells were
maintained in DMEM supplemented with 10% FCS (Biochrom) [40].
HaCaT human skin keratinocyte cells (cryovial, 300493) were from Cell Line Service (CLS,
Eppelheim, Germany). The cells were cultured in RPMI-1640, containing 110 mg/L sodium
pyruvate, 10 mM HEPES, 2.0 g/L NaHCO3 and additional 2.5 g/L D-glucose.
All cells were cultured in a water-saturated atmosphere (5% CO2) at 37 °C in cell culture
flasks purchased from Sarstedt (Nümbrecht, Germany). Subculturing of cells was performed
every 3-7 days after trypsinization (0.05% trypsin/0.02% EDTA or 0.1% trypsin/0.04% EDTA,
GE Healthcare (PAA Laboratories, Pasching, Austria). HaCaT cells were pre-incubated with
0.05% EDTA for 10 min at 37 °C before trypsination. Additionally, the cells were routinely
monitored for mycoplasma contamination by PCR (Venor GeM, Minerva Biolabs, Berlin,
Germany) and only mycoplasma free cultures were used.
4.2.3 Modulation of ABCB1 and ABCG2
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The modulation of ABCB1 (calcein-AM assay) [29] and ABCG2 (Hoechst 33342 assay) was
determined in the microplate format as described [29].
4.2.4 Modulation of ABCC1
ABCC1 modulation was determined with the calcein-AM microplate assay by analogy with the
procedure established for ABCB1 [33]. Briefly, 3-5 days after passaging, the ABCC1
overexpressing MDCKII-MRP1 cells were trypsinized and resuspended in culture medium at 25
°C. The cells were seeded into flat-bottomed 96-well plates (Cellstar Greiner Bio-One,
Frickenhausen, Germany) at a density of 20,000 – 25,000 cells per well. The next day, cells were
washed with loading buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2 · 6 H2O,
1.5 mM CaCl2·2H2O, 10 mM glucose, pH 7.4) in order to remove unspecific serum esterases.
Afterwards, cells were incubated with loading suspension (loading buffer, 5 mg/mL BSA, 1.25
ꢀL/mL pluronic F127 (20 % in DMSO) containing 0.5 ꢀM of calcein-AM) and vehicle or the test
compound at increasing concentrations (10 nM – 100 µM) for 60 min (37 °C / 5% CO2).
Reversan at a final concentration of 30 µM was used as reference compound; the response
obtained under these conditions was defined as 100% inhibition of calcein-AM efflux. In
general, two to four independent experiments were performed in triplicate, in case of controls in
sextuplicate. Subsequently, the loading suspension was discarded and cells were fixed under
light protection using 100 µL of a solution of paraformaldehyde (4 % in PBS) for 20 min. After
three washing circles with 150 µL of loading buffer, fixed cells were overlaid with 100 µL of
loading buffer and relative fluorescence intensities were determined at a GENios Pro microplate
reader (Tecan Deutschland GmbH, Crailsheim, Germany). Measurement mode: fluorescence top;
excitation filter: 485/20 nm; emission filter: 535/25 nm; number of reads: 10; integration time:
40 µs; lag time: 0 µs; mirror selection: dichroic 3; plate definition file: GRE96ft.pdf; multiple
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reads per well (circle): 3x3; time between move and flash: 100 ms. On each plate, the optimal
gain was calculated by determination of the fluorescence in absence and presence of the
reference compound reversan (30 µM). All values were corrected by subtracting the fluorescence
intensity in the absence of ABCC1 modulator (DMSO control value), and the maximal response
was referred to the signal obtained in the presence of 30 µM reversan (100%). IC50 values were
calculated using SigmaPlot 11.0, “four parameter logistic curve” fitting. Errors are expressed as
standard error of the mean (SEM).
4.2.5 Kinetic cytotoxicity assay
The assay was performed according to an established protocol [66] with minor modifications
[33].
4.2.6 Determination of the stability of compound 51 in mouse plasma
Compound stability in mouse plasma was determined according to a previously established
procedure [33]. Blood from NMRI (nu/nu) mice was collected by heart puncture in deep
anesthesia using heparin-coated syringes. Samples were immediately centrifuged for 7 min at
4,500 g (Centrifuge 5415R, Eppendorf, Hamburg, Germany). The test compound was dissolved
in DMSO at a concentration of 7 mM. A 1:50 dilution of substance with mouse plasma was
prepared in 1.5 mL polypropylene reaction vessels (Eppendorf, Hamburg, Germany). The
samples were shortly vortexed and promptly incubated at 37 °C. Aliquots were obtained at
different incubation periods. The samples were deproteinized by mixing with two parts of ice-
cold acetonitrile (MeCN). For quantitative precipitation, the samples were vortexed and stored at
4 °C for 30 min. Afterwards the samples were centrifuged for 5 min at 14,000 g (Eppendorf
centrifuge 5415 R) and the supernatants were transferred into new reaction vessels. The samples
were diluted with the same volume of MeCN and stored at -80 °C until the HPLC analysis.
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RP-HPLC analysis was performed with a Eurosphere-100 C18 column (250 x 4 mm, 5 µm,
Knauer, Berlin, Germany), maintained at 30 °C, on a Merck Hitachi system (Merck, Darmstadt,
Germany) consisting of an AS-2000A , a L-6200-A pump, a L-400A UV-VIS detector. UV-
detection was performed at 220 nm. The samples were thawed at room temperature and 100 µL
were injected. Mixtures of MeCN (A) and 0.05 % aq. TFA (B) were used as mobile phase, using
a linear gradient (A/B: 10/90 to 80/20) over 30 min.
Acknowledgments
The authors thank Olga Schitkow and Maria Beer-Krön for excellent technical assistance, Dr.
Piet Borst, Netherland Cancer Institute (Amsterdam, NL), for kindly providing the MDCKII-
MRP1 cells, and to Dr. Joachim Wegener for collaboration with respect to the determination of
the chemosensitivity of HaCaT cells. Graduate fellowships granted to J. O.-B. by the German
Academic Exchange Service (DAAD) and to Q. S. by the Chinese Scholarship Council are
gratefully acknowledged.
Appendix A. Supplementary data
Preparation of building blocks and intermediates 3-25, 28. Synthesis of 27 and 30. Preparative
1
13
and analytical data of compounds 32-35, 37-50, 52, 53, 62-64, 66 and 67. H and C NMR
spectra of compounds 31-53 and 62-67. Purity and chromatograms of compounds 31-53 and 62-
67. Solubility of 32 and 33 in loading buffer and suspension. Investigation of the chemical
stability of compounds 32, 33, 36, 51, 62, 65. Investigation of the chemosensitivity of HaCat
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cells against selected modulators. Supplementary data related to this article can be found at
http://dx.doi.org/10
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