Aromatic 2-(thio)ureidocarboxylic acids as a new family of modulators of multidrug resistance-associated protein 1: Synthesis, biological evaluation, and structure - Activity relationships

Article abstract of DOI:10.1021/jm900688v

Four series of aromatic carboxylic acids were prepared with a urea or thiourea moiety at the neighboring position to the carboxyl group and benzene or thiophene as aromatic scaffold. Using a calcein AM assay, these compounds were evaluated as inhibitors o

Full text of DOI:10.1021/jm900688v

4586 J. Med. Chem. 2009, 52, 4586–4595
DOI: 10.1021/jm900688v
Aromatic 2-(Thio)ureidocarboxylic Acids As a New Family of Modulators of Multidrug
Resistance-Associated Protein 1: Synthesis, Biological Evaluation, and Structure-Activity
Relationships
Hans-Georg Hacker,† Stefan Leyers,‡ Jeanette Wiendlocha,‡ Michael Gutschow,*,† and Michael Wiese*,‡
¨
¨
†Pharmaceutical Institute, Pharmaceutical Chemistry I and ‡Pharmaceutical Institute, Pharmaceutical Chemistry II, University of Bonn,
An der Immenburg 4, 53121 Bonn, Germany
Received January 22, 2009
Four series of aromatic carboxylic acids were prepared with a urea or thiourea moiety at the
neighboring position to the carboxyl group and benzene or thiophene as aromatic scaffold. Using a
calcein AM assay, these compounds were evaluated as inhibitors of multidrug resistance-associated
protein 1 (MRP1) and selected compounds were examined toward P-glycoprotein (P-gp) as well as
breast cancer resistance protein (BCRP) to assess selectivity for MRP1. Two 2-thioureidobenzo[b]-
thiophene-3-carboxylic acids (48, 49) were identified as particularly potent inhibitors of MRP1, with
IC50 values of around 1 μM. The structural features of this new family of nontoxic MRP1 inhibitors
include a (thio)urea disubstituted with preferentially two alkyl groups at the terminal nitrogen and an
additional fused aromatic ring.
Introduction
protein (BCRP), can confer multidrug resistance to cancer
cells.1-3,20
Failure to respond to chemotherapy is a serious impedi-
ment in the treatment of cancer. In this regard, the develop-
ment of multidrug resistance (MDRa) is frequently observed,
which leads to a reduced sensibility of cancer cells toward a
broad spectrum of cytostatics. The mostly involved mechan-
ism of MDR is associated with the overexpression of trans-
porters of the ATP binding cassette (ABC) family. These
efflux pumps are localized in the cell membrane and reduce
the intracellular concentration of multiple structurally and
functionally unrelated drugs by an active extrusion at the cost
of ATP hydrolysis.1-5
P-glycoprotein (P-gp), the first discovered member of ABC
efflux transporters, possesses their typical architecture with
two transmembrane domains usually consisting of six trans-
membrane helices and two cytoplasmic nucleotide-binding
sites.6 P-gp directed modulators belong to various chemical
classes, most of them being well-established drugs in different
pharmacological areas. Considerable effort has been devoted
to identify new P-gp modulators,7-14 and pharmacophore
patterns of P-gp ligands addressing different binding sites
have been derived.15-19
Subsequent to the discovery of P-gp, MRP1 was identified
in 1992 as the first member of the ABCC subfamily.21 This
190 kDa membrane-spanning protein is encoded by the
ABCC1 gene mapped to chromosome 16p13.1. MRP1 shares
the common structural motif of ABC proteins. Similar to P-
gp, MRP1 is composed of the large core segment containing
the nucleotide binding sites and two transmembrane domains
but additionally possesses a third transmembrane domain
with five helices and an extracellular N-terminus.22-24 Its
amino acid sequence resembles that of P-gp only to a modest
extent of ∼15%.25
The ubiquitous expression of MRP1 in human tissues, e.g.,
in endothelial cells of brain capillaries, epithelial cells of the
digestive, urogenital, and respiratory tracts, endocrine glands,
and the hematopoietic system implies an important role for
MRP1 in the protection against xenobiotics and endogenous
toxins.24,25 Since its discovery in the H69AR cancer cell line,
MRP1 has been found in multidrug-resistant cell lines derived
from different tissues and tumor types, for example, in lung,
colon, breast, bladder, and prostate cancer as well as leuke-
mia.21,26-28 MRP1 has unusually broad substrate specificity.
Like P-gp, it is capable of transporting a wide variety of
anticancer agents, e.g., anthracyclines, Vinca alkaloids, and
methotrexate. In contrast to P-gp, MRP1 is facilitating the
extrusion of negatively charged compounds such as numerous
glucuronate, sulfate, and glutathione conjugates, including
the eicosanoid leukotriene C4.22,24,29-31
Besides the well-known role of P-gp in multidrug resis-
tance, other ABC transporters, e.g., the multidrug resistance-
associated proteins (MRPs) and the breast cancer resistance
*To whom correspondence should be addressed. For M.G.: phone,
þ49 228 732317; fax, þ49 228 732567; E-mail, guetschow@uni-bonn.de.
For M.W.: phone, þ49 228 735213; fax, þ49 228 737929; E-mail,
mwiese@uni-bonn.de.
a Abbreviations: MRP, multidrug resistance-associated protein; cal-
cein AM, acetoxymethyl ester of calcein; P-gp, P-glycoprotein; MDR,
multidrug resistance; ABC, ATP binding cassette; BCRP, breast cancer
resistance protein; WT, wild-type; MTT, (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide.
Much effort has been focused on the finding of inhibitors
for MRP1, but the number of published compounds is con-
siderably lower than those available for P-gp.3,32,33 Whereas
most P-gp inhibitors failed to affect MRP1, cyclosporin A and
verapamil proved active against MRP1. Additionally, several
specific inhibitors have been described within the last 15 years.
r
pubs.acs.org/jmc
Published on Web 07/06/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4587
Table 1. 2-Ureidobenzoic Acids 1-16
prepared IC50 ( SD
compd
NR1R2
NHBn
N(Et)2
R3
R4
from
(μM)
1
2
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
B
C
A
A
A
A
C
C
A
A
A
A
A
C
B
nid
26.4 ( 10.1
ni
N(Me)CH2CO2Me
4a,b,c N(Me)cyclohexyl
12.3 ( 2.8
ni
5
N(Me)Ph
N(Me)Bn
6
42.6 ( 1.6
ni
7
N(Me)CH2CH2Ph
1-pyrrolidinyl
4-morpholinyl
N(Me)cyclohexyl
N(Me)Ph
8
43.1 ( 11.1
ni
9
10
Me
22.8 ( 6.2
ni
11
12
Me
Me
Me
Me
Me
N(Me)Bn
ni
13
14
N(Me)CH2CH2Ph
4-morpholinyl
4-morpholinyl
ni
ni
15
Me
43.4 ( 13.3
5.38 ( 0.68
16a,b,c N(Et)2
-CHdCH-CHdCH-
a Compounds showing no effect in the P-gp assay. b Compounds (at
31.6 μM)inhibited BCRPbetween 25% and 65% relative tothe inhibitory
effect of XR9577 (at 10 μM). c No cytotoxicity (IC50 > 100 μM). d ni, no
inhibition of the MRP1-mediated transport.
Figure 1. Structures of known MRP1 inhibitors.
The leukotriene D4 receptor antagonist MK 571 inhibited the
leukotriene C4 transport by membrane vesicles from MRP1-
transfected cells,34,35 and it was frequently used in recent
pharmacological studies (Figure 1).36-43 MK 571 exhibited
an Ki value of 0.6 μM in MRP1-mediated leukotriene C4
transport,34 IC50 values of 3.3 μM in ATP-dependent [3H]
para-aminohippurate transport,44 and 7.6 μM in the calcein
AM assay.45
Scheme 1a
Among various flavonoids,46 dehydrosilybin was a parti-
cularly active inhibitor of leukotriene C4 transport.47 The
selective estrogen receptor modulator raloxifene was used as
a lead structure to conceive inhibitors of MRP1-mediated
MDR. The analogue LY329146 with a bis(methylsulfonyl)
amino group in place of one hydroxyl group in raloxifene
showed an improved activity.48 The tricyclic isoxazole
LY402913 was identified as a potent and selective modulator
by reversing drug resistance to different MRP1 substrates in
HeLa-T5 and A2780 cells. Furthermore, LY402913 delayed
tumor progression in a xenograft mouse model.49,50 Another
seriesof selective andhighly efficient MRP1inhibitors derived
from a pyrrolo[3,2-d]pyrimidine template was reported by
Wang et al. Structural modifications led to pyrrolo- and
indolopyrimidines, exhibiting IC50 values of approximately
100 nM. Some derivatives such as XR12890 displayed good
pharmacokinetic profiles, e.g., high oral bioavailability and
limited interactions with cytochromes P450.51,52 Recently, we
identified a thieno[2,3-d][1,3]thiazin-4-one derivative I being
active at MRP1 and 2.45
a Reagents and conditions: (a) HNR1R2, H2O, room temperature, or
HNR1R2, EtOH, H2O, reflux. (b) (1) N,N0-carbonyldiimidazole,
CH2Cl2 or THF, room temperature; (2) HNR1R2, room temperature.
(c) (1) HNR1R2, acetone, reflux; (2) 0.25 M HCl, 5 °C.
carboxylic acids showed modulating activity on MRP1. On
the basis of these initial results, it was intended to provide four
series of aromatic carboxylic acids. All compounds bear either
a substituted urea or thiourea moiety at the neighboring
position to the carboxyl group. As an aromatic scaffold, either
a (substituted) benzene or thiophene ring was chosen. The
evaluation of these compounds as MRP1 inhibitors is pre-
sented herein.
The starting point for this study was the evaluation of an
in-house library of low molecular weight compounds for
modulating effects on MRP1 in a fluorometric calcein AM
assay.45,53 Initially, the selection was focused on carboxylic
acids with respect to the anionic nature of the MRP1 sub-
strates. More than 75 structurally diverse carboxylic acids
were inspected. Among them, some aromatic 2-(thio)ureido
Chemistry
The final 2-ureidobenzoic acids 1-16 are listed in Table 1,
their preparation is shown in Scheme 1. The majority of

4588 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 2. 2-Thioureidobenzoic Acids 17-24
Hacker et al.
¨
Table 3. 2-Ureidothiophene-3-carboxylic Acids 25-28
compd
NR1R2
N(Et)2
IC50 ( SD (μM)
compd
25a,b,c N(Et)2
NR1R2
R3
R4
prepared from IC50 ( SD (μM)
17
21.6 ( 5.3
16.2 ( 4.2
198 ( 29
29.1 ( 9.1
24.0 ( 6.9
18.3 ( 4.6
20.7 ( 2.5
nid
-(CH2)3-
-(CH2)4-
-(CH2)4-
E
F
E
E
11.6 ( 3.1
nid
18a,b,c
19
N(Me)cyclohexyl
N(Me)Ph
N(Me)Bn
26
NHCH(Me)2
27a,b,c N(Et)2
8.29 ( 4.22
ni
20
21
28
4-morpholinyl -(CH2)4-
N(Me)CH2CH2Ph
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
a Compounds showing no effect in the P-gp assay. b Compounds (at
31.6 μM) inhibited BCRP between 25% and 65% relative to the inhibitory
effect of XR9577 (at 10 μM). c No cytotoxicity (IC50 > 100 μM). d ni, no
inhibition of the MRP1-mediated transport.
22
23
24
a Compound showing no effect in the P-gp assay. b Compound (at 31.6
μM) inhibited BCRP less than 20% relative to the inhibitory effect of
XR9577 (at 10 μM). c No cytotoxicity (IC50 > 100 μM). d ni, no inhibition
of the MRP1-mediated transport.
Scheme 3a
Scheme 2a
a Reagents and conditions: (a) HNR1R2, CH2Cl2, room temperature.
(b) (1) 1 M NaOH, EtOH (1:1) reflux; (2) H2O; (3) 2 M HCl.
such compounds was obtained from the reaction of isatoic
anhydrides A with benzylamine or appropriate secon-
dary amines.54 To prepare 2-(3,3-diethylureido)benzoic acid
(2), anthranilic acid (B, R3 = R4 = H) was pretreated with
N,N0-carbonyldiimidazole followed by the addition of die-
thylamine. Similarly, the naphthalene derivative 16 was ob-
tained from 3-amino-2-naphthalenecarboxylic acid (B, R3R4 =
-CHdCH-CHdCH-). The preparation of further 2-ureido-
benzoic acids was accomplished when N-(mesyloxy)phthali-
mides C were reacted with secondary amines.55
The two-step synthetic route to the 2-thioureidobenzoic
acids 17-24 (Table 2) is depicted in Scheme 2. The methyl
benzoate precursors D were prepared from secondary amines
and methyl 2-isothiocyanatobenzoate56 in dichloromethane
and subsequently saponified with aqueous ethanolic sodium
hydroxide.
2-Ureidothiophene-3-carboxylic acids 25-28 (Table 3)
were synthesized by two alternative routes (Scheme 3). Thie-
no[2,3-d][1,3]oxazine-4-ones E underwent ring-opening upon
treatment with sodium hydroxide. Because a disubstituted
amino group prevented Dimroth rearrangement to pyrimidi-
nediones, the desired noncyclized ureas 25, 27, and 28 were
obtained. Trifluoroacetic acid promoted the cleavage of the
tert-butyl ester group in F to give the corresponding isopro-
pylurea 26. Both routes started with the Gewald thiophene
synthesis57,58 to alkyl 2-aminothiophene-3-carboxylates,
which were converted either with isopropyl isocyanate to F
or in five reaction steps to the heterocycles E.59
a Reagents and conditions: (a) (1) 1.5 M NaOH, acetone (2:1), reflux;
(2) 3 M HCl. (b) TFA, 0 °C to room temperature.
by saponification of the ethyl esters H or by sodium hydro-
xide-promoted cleavage of the thiazinone ring of compounds
I. With respect to R3 and R4, either non-, mono-, and
disubstituted thiophenes (30-39) or fused compounds (40-
50) have been synthesized. All compounds were accessible by
an initial Gewald reaction. To obtain the aromatic fused ring
system in 48-50, the corresponding isothiocyanatobenzo[b]-
thiophene G (R3R4 = -CHdCH-CHdCH-) afforded
three additional steps. The final compound 29 was achieved
following an analogous route, i.e., conversion of methyl 3-
aminothiophene-2-carboxylate to the isothiocyanate and the
diethylthioureido derivative, which was cyclized with concen-
trated sulfuric acid to the corresponding 1,3-thiazine-4-one
and reopened to 29.59
Biology
An accumulation assay using the MRP1 substrate calcein
AM was performed to determine the influence of the test
compounds on the transporter protein MRP1.33,45,53 Calcein
AM is a lipophilic, nonfluorescent acetoxymethyl ester of
calcein that easily penetrates cellular membranes. Intracellu-
lar calcein AM can either be transported out of the cell by
MRP1 or cleaved by unspecific cytosolic esterases to produce
the fluorescent calcein. The human ovarian cancer cell line
2008 stably expressing MRP1 was used. In 2008 MRP1 cells,
the efflux of calcein AM leads to a decreased intracellular
fluorescence, compared to the corresponding wild-type cell
The preparation of thiophenecarboxylic acids with a
thiourea moiety at the neighboring position is shown in
Scheme 4. Compounds 30-50 (Table 4) were either obtained

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4589
Table 4. 3-(Diethylthioureido)thiophene-2-carboxylic Acid (29, Left) and 2-(Thioureido)thiophene-3-carboxylic Acids 30-50 (Right)
compd
NR1R2
R3
R4
prepared from
IC50 ( SD (μM)
29a,b,c
30a,b,c
31a,c,d
32
4.18 ( 0.70
4.46 ( 0.58
4.51 ( 0.49
14.5 ( 5.5
nih
N(Et)2
N(Me)cyclohexyl
N(Et)2
H
H
H
I
H
H
I
H
CH(Me)2
Ph
33
4-morpholinyl
N(Et)2
H
H
H
H
I
34c,e
35
Ph
Ph
Me
Me
Me
H
14.3 ( 4.7
27.5 ( 3.3
20.4 ( 2.1
4.19 ( 1.51
18.3 ( 4.5
>100
4-morpholinyl
N(Et)2
H
36
Me
Me
Me
Me
37a,b,c
38
N(Me)cyclohexyl
4-morpholinyl
N(Et)2
I
I
39
CONH2
I
40a,b,c
41
N(Et)2
N(Me)2
N(Et)2
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
I
2.56 ( 1.11
ni
I
42a,b,c
43
I
2.71 ( 0.35
ndi
N(Me)cyclohexyl
4-morpholinyl
4-methyl-1-piperazinyl
N(Et)2
I
44
I
ni
45
I
ni
46a,c,f
47a,b,c
48a,c,g
49a,b,c
50
-(CH2)3CH(CH3)-
-(CH2)5-
-CHdCH-CHdCH-
-CHdCH-CHdCH-
-CHdCH-CHdCH-
I
4.06 ( 1.05
5.97 ( 3.83
0.932 ( 0.041
1.23 ( 0.29
ni
N(Et)2
N(Et)2
I
H
H
H
N(Me)cyclohexyl
4-morpholinyl
a Compounds showing no effect in the P-gp assay. b Compounds (at 31.6 μM) inhibited BCRP between 25% and 65% relative to the inhibitory effect
of XR9577 (at 10 μM). c No cytotoxicity (IC50 > 100 μM). d Partial inhibition of BCRP with IC50app = 10.2 ( 3.6 μM. e 20% inhibition at 31.6 μM, 40%
inhibition at 100 μM in the P-gp assay. f Partial inhibition of BCRP with IC50app = 9.10 ( 1.71 μM. g Partial inhibition of BCRP with IC50app = 6.16 (
1.36 μM. h ni, no inhibition of the MRP1-mediated transport. i nd, not detectable due to concentration-dependent decrease of fluorescence in both wild-
type and MRP1-transfected cells.
Scheme 4a
Figure 2. Fluorescence-time curves for different concentrations of
compound 48 determined with the calcein AM assay in 2008 MRP1
cells. Presented data are averages from a typical experiment with
two replicates belonging to a series of three independent experi-
ments.
to 2008 WT cells has no effect on fluorescence (Figure 3).
When performing the assay, XR9577,60 a selective blocker of
P-gp and BCRP, having no effect on MRP1,61 was added to
inhibit P-gp, which is present to a minor degree in both
MRP1-expressing and wild-type cells. The assay medium
contained cobalt ions to quench extracellular calcein fluores-
cence so that only the fluorescence of intracellular calcein is
measured.
a Reagents and conditions: (a) HNR1R2, CH2Cl2, room temperature.
(b) (1) Concd H2SO4, room temperature; (2) H2O; or (1) polyphosphoric
acid, 170 °C; (2) ice, EtOH; (c) (1) 1 M NaOH, EtOH (1:1) reflux; (2)
H2O; (3) 2 M HCl. (d) (1) 3 M NaOH, dioxane (1:2), (2) ice, H2O; (3) 3 M
HCl.
line, 2008 WT. An MRP1 inhibitor blocks calcein AM efflux,
leading to an enhancement of calcein concentration and
intracellular fluorescence (Figure 2). In contrast, as the
wild type does not exhibit MRP1, addition of an inhibitor
A subset of active compounds was defined and tested
for the ability to affect P-gp in a similar calcein AM assay

4590 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Hacker et al.
¨
IC50 values of 2-ureidothiophene-3-carboxylic acids 25-28
are in given in Table 3. A 2-(3-isopropylureido) or 2-[(4-
morpholinylcarbonyl)amino] residue gave inactive com-
pounds. The activities of the two diethylurea derivatives
25 and 27 (IC50 = 11.6 and 8.29 μM) were between those
of the two benzoic acid-derived diethylureas 2 and 16.
Both compounds, 25 and 27, did not affect P-gp-mediated
transport but were also weak inhibitors of BCRP. They did
not exhibit cytotoxicity. Four thiophene-3-carboxylic acids
with 2-acylamino or 2-alkoxycarbonylamino groups in place
of the 2-ureido function were also evaluated but had no effect
on MRP1-mediated transport (see Supporting Information,
Chart S3).
Thiophenecarboxylic acids 29-50 with an ortho-thioureido
substituent are outlined in Table 4. Again, the carboxyl group
was required for activity as the corresponding methyl ester of
29 and the corresponding carbonitrile of 42 did not affect
MRP1 (see Supporting Information, Chart S4). Within the
thioureido residue, a diethylamino or cyclohexylmethylamino
substitution pattern was advantageous over a more polar one,
i.e., morpholino or methylpiperazino. The two isomeric thio-
phenes 29 and 30 were equipotent.
Starting from 2-(3-diethylthioureido)thiophene-3-car-
boxylic acid (30), the influence of substituents in positions 4
and 5 was examined. The potency was somewhat diminished
by introducing two methyl groups (30 versus 36), or an
isopropyl group (32), but was lost by replacing the 5-methyl
by a polar carboxamide moiety (39). A fused cycloaliphatic
ring was tolerated in most cases and provided slightly im-
proved inhibitors, i.e., the tri- and tetramethylene compounds
(30 versus 40 and 42). Introduction of a methyl group into the
tetramethylene chain of 42 resulted in a slight decrease of
activity (46). As in the 2-ureidobenzoic acid series (compound
16) a bicyclic aromatic system was advantageous. An addi-
tional fused benzene ring in the benzothiophene 48 accounted
for the most potent MRP1 inhibitor of our study with an IC50
value of 0.932 μM. Increased potency of 48 compared to the
analogous cycloaliphatic derivative 42 could be due to pla-
narity of the fused aromatic system. Removing the steric
fixation of the annelated benzene ring in favor of a substituted
benzene ring considerably dropped activity (48 versus 34).
Although it was demonstrated by means of X-ray crystal-
lography that the biaryl structure in 34 is nearly in plane (data
not shown), the extended aromatic system might lead to steric
repulsion. An exchange of the diethylamino group in 48 for a
cyclohexylmethylamino substituent in the thioureido residue
led to the other potent benzothiophene 49 (IC50=1.23 μM).
Again, the exchange of the dialkyl moiety for a polar mor-
pholino substitutent in 50 resulted in a loss of activity.
When comparing the MRP1-inhibiting properties of ana-
logously substituted thiophenes (25, 27 versus 40, 42), thiour-
eas were superior to ureas. The bioisosteric benzene-
thiophene exchange improved the activity as can be seen from
the comparison of the parent diethylthioureidocarboxylic
acids (17 versus 29 and 30).
Figure 3. Concentration-effect curve of compound 48 in MRP1-
transfected cells (open circles) in comparison to the wild-type cells
(closed circles). Curves represent the average of at least five inde-
pendent experiments. For normalization of data, slopes from
fluorescence-time curves were transformed to relative units by
subtracting the lowest determined single value from all other data
and thus setting it to 0%. The highest measured single value was
defined as 100%.
using P-gp overexpressing A2780/ADR cells. Furthermore,
these compounds were investigated for modulation of BCRP
in MCF-7 MX cells in an accumulation assay with the
fluorescent substrate Hoechst 33342.62 Cytotoxicity of the
subset of compounds was determined in 2008 WT cells by
measuringviabilityviaMTTassay.63 TheMTTassaywas also
used to assess potentiating effects of two exemplary 2-urei-
dothiophene-3-carboxylic acids (31, 48) in combination with
the cytotoxic drugs vinblastine and daunorubicin.
Results
In search of MRP1 inhibitors, we identified active repre-
sentatives of 2-ureidobenzoic acids. All 2-ureidobenzoic acids
(1-16) investigated are outlined in Table 1. The presence of a
diethylureido and a cyclohexylmethylureido moiety provided
active compounds (2, 4, 10, 16). A replacement of the cyclo-
hexyl residue by a phenyl, benzyl, or phenethyl group mostly
led to a loss of potency (4 versus 5, 7; 10 versus 11-13). While
the introduction of a 5-methyl substituent (R4 = Me) did not
improve biological activity, an additional fused benzene ring
was advantageous (2 versus 16). The naphthalene derivative
16 (IC50 = 5.38 μM) was the only potent substance among the
2-ureidobenzoic acids. Compound 16 did not inhibit P-gp,
showed a weak inhibition of BCRP and was not cytotoxic.
Whereas seven of the 16 2-ureidobenzoic acids inhibited
MRP1, no active compound was identified from a series of
13 related anthranilic acid derivatives (see Supporting
Information, Chart S1), i.e., benzoic acids with a 2-alkylami-
no, 2-arylamino, 2-acylamino, 2-alkoxycarbonylamino, and
2-(3-aroylureido) substituent.
Compounds 17-24 are benzoic acids with a thiourea moiety
at position 2 (Table 2). As in the ureidobenzoic acid series, a
cyclohexylmethylureido moiety in 18 (IC50=16.2 μM) accounted
for activity. This substance did not inhibit P-gp but showed also
a weak inhibition of BCRP. No cytotoxicity was observed for
18. Toward MRP1, all compounds, except of 19 and 24, had
IC50 values below 30 μM. When comparing analogous ureas
with thioureas, a clear effect of the oxygen-sulfur exchange
could not be observed (2, 4-9 versus 17-22, 24). Five benzoic
acids with a 2-(3-aroylthioureido) residue and optional aromatic
substituents have also been investigated and proved to be
inactive. The replacement of the carboxyl group in 17 by a
cyano function abolished activity, indicating the importance of
carboxyl group (see Supporting Information, Chart S2).
Selected MRP1 inhibitors of this series (Table 4) were
evaluated as modulators of P-gp, but only 34 showed a
weak inhibitory activity. However, inhibition of BCRP was
observed in all these cases. Compounds31, 46, and 48behaved
as partial inhibitors of BCRP with a maximum inhibitory
effect of 60% relative to XR9577 and apparent IC50 values
between 6 and 10 μM. The selected MRP1 inhibitors of
this series were not cytotoxic. In the modified MTT assay,
MRP1-expressing cells were treated with 31 or 48 (in a fixed

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4591
inhibition of Hoechst 33342 efflux mediated by BCRP. Over-
lapping inhibitors of different ABC transporter are discussed
in the literature.3,23,70-72 Our finding that carboxylic acids are
capable of an interaction with BCRP is in agreement with a
recently published report revealing MK571 to be a BCRP
inhibitor.71 On the other hand, all MRP1 inhibitors with IC50
values less than 10 μM did not affect P-gp. As P-gp generally
does not transport negatively charged compounds, it might be
concluded that the presence of a carboxylic acid moiety
accounts for this selectivity.
Figure 4. Structural features for bioactive aromatic 2-(thio)ureido-
carboxylic acids.
concentrationof 31.6μM)and vinblastine ordaunorubicin (in
different concentrations). These combinations did not result
in an increased cytotoxicity of vinblastine and daunorubicin,
respectively (see Supporting Information, Figures S1-S4).
Experimental Section
General Methods and Materials. Thin-layer chromatography
was carried out on Merck aluminum sheets, silica gel 60 F254.
Preparative column chromatography was performed on Merck
Discussion
On the basis of the structure-activity relationships detailed
above, we combined the advantageous structural features
as can be deduced from the biological activities of the four
series of aromatic carboxylic acids (Figure 4). The best MRP1
inhibitors of the present series are more potent than verapa-
mil, indomethacin, MK 571, and cyclosporin A for which
IC50 values between 5 and 12 μM had been determined in the
calcein AM assay.45 They have the same order of activity
as dehydrosilybin (IC50 = 1.1 μM),32 LY329146 (IC50 = 0.8
μM),32 LY402913 (IC50 = 1.8 μM),64 and compound I
(IC50=1.2 μM)45 but are less potent than the recently re-
ported pyrrolo- and indolopyrimidines such as XR12890.51,52
The structural features derived for the aromatic carboxylic
acids of this study (Figure 4) reflects their differences to other
classes of MRP1 inhibitors (Figure 1). The essential compo-
nents are a carboxyl group at a (hetero)aromatic scaffold with
a (thio)urea function in ortho-position. The terminal nitrogen
of the (thio)urea should be disubstituted, preferentially with
two alkyl groups (e.g., diethyl or cyclohexylmethyl). The
(hetero)aromatic ring (A) consists of benzene or thiophene,
while the orientation of the sulfur seems to have minor
influence on activity. An additional fused benzene ring (B)
improves the inhibitory activity against MRP1. One the basis
of this study, further investigations are ongoing in our labora-
tories. Structural modifications of the title compounds will
include an exchange of rings A and/or B for other heteroaro-
matics, additional modifications at N-3 of the (thio)urea (e.g.,
cyclohexylethyl or di(iso)propyl), and a bioisosteric replace-
ment of the carboxyl group against a tetrazole moiety.65
We evaluated toxicity for 16 selected compounds and did
not observe cytotoxic properties. However, compounds 31
and 48 failed to reverse multidrug resistance of MRP1-
expressing cells against vinblastine and daunorubicin, respec-
tively. Although different transport mechanisms of substrates
are well characterized for MRP1, the mechanism of inhibition
is poorly understood.2,5,32,33,66 Perrotton et al. investigated
modulating effects of (R)- and (S)-verapamil on MRP1.67
Both enatiomers induced an increase in calcein AM accumu-
lation in MRP1-overexpressing cells, thus implying effective
inhibition of the efflux pump. In potentiation assays, only (R)-
verapamil reverted resistance of MRP1-BHK-21 cells to
vincristine. As 31 and 48 in our case, (S)-verapamil did not
show a cytotoxicity-potentiating effect. The different modula-
tion of MRP1 by both enatiomers was discussed as explana-
tion of controversial results of verapamil in reversion of
chemoresistance.40,67-69
silica gel 60 (70-230 mesh). Melting points were determined by a
Boetius melting point apparatus (PHMK, VEB Wagetechnik
¨
¨
Rapido, Radebeul, Germany) and are uncorrected. 1H NMR
(300 MHz) and 13C NMR spectra (75 MHz) were recorded on a
Varian Gemini 300. 1H NMR (500 MHz) and 13C NMR spectra
(125 MHz) were recorded on a Bruker Avance DRX 500. Chemi-
cal shifts δ are given in ppm referring to the signal center using the
solvent peaks for reference: CDCl3 7.26/77.0 ppm and DMSO-d6
2.49/39.7 ppm. Elemental analyses were performed with a Vario
EL apparatus. Solvents and reagents were obtained from Acros
(Geel, Belgium), Fluka (Taufkirchen, Germany), or Sigma-Al-
drich (Steinheim, Germany). Cobalt sulfate was purchased from
Merck (Darmstadt, Germany). XR957760 (see Supporting Infor-
mation, Chart S5) was prepared following the conditions reported
by Roe et al.73 Synthetic procedures and analytical data for
compounds 1, 5-16, 18-24, 26-30, precursor for 34, precursor
for 35, precursor for 49, precursor for 50, as well as compounds
32-47, 49, and 50 are listed in the Supporting Information. All
tested compounds possessed a purity of not less than 95%.
2-(3,3-Diethylureido)benzoic Acid (2). Anthranilic acid (B,
R3 = R4 = H) (688 mg, 5.0 mmol) was added in portions to a
stirring solution of N,N0-carbonyldiimidazole (811 mg, 5.0 mmol)
in CH2Cl2 (50 mL). After 30 min, diethylamine (731 mg, 10.0
mmol) was added dropwise and the mixture was stirred overnight.
The organic layer was washed with 1 M HCl (2 ꢀ 5 mL), filtered,
and evaporated to dryness to yield 2 (637 mg, 54%): mp 144-
148 °C (lit.55 151 °C). 1H NMR (500 MHz, CDCl3) δ 1.24 (t, J =
7.3 Hz, 6H), 3.43 (q, J = 7.3 Hz, 4H), 6.96 (ddd, J = 8.2, 7.2,
1.3 Hz, 1H), 7.52 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 8.05 (dd, J = 8.2,
1.6 Hz, 1H), 8.62 (dd, J=8.5, 1.0Hz, 1H), 10.50(s, 1H). 13CNMR
(125 MHz, CDCl3) δ 13.67, 41.73, 112.60, 119.56, 120.52, 131.58,
135.56, 144.40, 154.55, 172.83. Anal. (C12H16N2O3) C, H, N.
2-[3-(Methoxycarbonylmethyl)-3-methylureido]benzoic Acid (3).
A solution of N-(mesyloxy)phthalimide (C, R3 = R4 = H) (1.81 g,
7.5 mmol) in anhydrous acetone (25 mL) was stirred under argon
and heated to reflux. A solution of sarcosine methyl ester hydro-
chloride (3.14 g, 22.5 mmol) and triethylamine (2.28 g, 22.5 mmol)
in acetone (7 mL) was added dropwise over 10 min. The mixture
was refluxed for additional 15 min and evaporated to dryness. The
residue was stirred with 0.25 M HCl (100 mL) and filtrated. The
filtrate was kept a 5 °C for 14 days and the precipitate was collected
by filtration to obtain 3 (400 mg, 20%): mp 147-149 °C. 1H NMR
(500 MHz, DMSO-d6) δ3.07 (s, 3H), 3.66 (s, 3H), 4.16 (s, 2H), 7.02
(ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.52 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H),
7.96 (dd, J = 8.0, 1.4 Hz, 1H), 8.42 (dd, J = 8.5, 1.0 Hz, 1H), 10.67
(s, 1H), 13.56 (br s, 1H). 13C NMR (125 MHz, CDCl3) δ 35.30,
50.01, 51.89, 114.69, 118.65, 120.92, 131.15, 134.28, 154.89, 170.45,
170.49. Anal. (C12H14N2O5) C, H, N.
2-(3-Cyclohexyl-3-methylureido)benzoic Acid (4). General Pro-
cedure for 2-Ureidobenzoic Acids from Isatoic Anhydride A. A
mixture of isatoic anhydride (A, R3 = R4 = H) (3.26 g, 20 mmol)
and 50 mL of an ethanolic solution of N-methylcyclohexylamine
(4 M) was refluxed for 15 min, diluted with H2O (200 mL), kept at
The most potent representatives of the four series were
evaluated for modulating effects on BCRP- and P-gp-
mediated transport. All selected substances showed a weak

4592 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Hacker et al.
¨
8 °C overnight, and filtrated. The filtrate was acidified with 1 M
H2SO4, and the precipitate was filtered off. It was partitioned
between ethyl acetate (50 mL) and 0.5 M NaOH (50 mL). The
aqueous layer was washed with ethyl acetate (2 ꢀ 50 mL), cooled,
and acidified with 1 M H2SO4. The precipitate was collected by
-CHdCH-CHdCH-) (1.05 g, 65%): mp 136-138 °C (lit.59
137-138 °C). 1H NMR (500 MHz, DMSO-d6) δ 1.26 (t, J = 6.8
Hz, 6H), 1.42 (t, J = 7.1 Hz, 3H), 3.79-3.86 (m, 4H), 4.46 (q, J =
7.1 Hz, 2H), 7.29 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.41 (ddd, J = 8.2,
7.3, 1.3 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 8.20 (dt, J = 8.2, 1.0 Hz,
1H), 12.56 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 12.25, 14.28,
46.06, 61.27, 105.21, 122.00, 122.49, 123.80, 125.72, 132.70, 133.18,
156.96, 167.18, 174.77. Anal. (C16H20N2O2S2) C, H, N.
1
suction filtration to obtain 4 (3.92 g, 71%): mp 146-147 °C. H
NMR (500 MHz, DMSO-d6) δ 1.05-1.18 (m, 10H), 2.83 (s, 3H),
3.90-3.98 (m, 1H), 6.97 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.50 (ddd,
J = 8.5, 7.2, 1.6 Hz, 1H), 7.94 (dd, J = 8.2, 1.6 Hz, 1H), 8.47 (dd,
J = 8.5, 1.0 Hz, 1H), 10.83 (s, 1H), 13.51 (br s, 1H). 13C NMR (125
MHz, DMSO-d6) δ 25.09, 25.58, 28.12, 30.07, 53.79, 114.44,
188.80, 120.39, 131.11, 134.16, 143.67, 154.27, 170.56. Anal.
(C15H20N2O3) C, H, N.
2-(3-Cyclohexyl-3-methylthioureido)thiophene-3-carboxylic Acid
(31). General Procedure for 2-Thioureidothiophene-3-carboxylic
Acids from 2-Thioureidothiophene-3-carboxylates H. A mixture
of ethyl 2-(3-cyclohexyl-3-methylthioureido)thiophene-3-car-
boxylate (H, R1 = Me, R2 = cyclohexyl, R3 = R4 = H) (653
mg, 2.0 mmol), 1 M NaOH (10 mL), and EtOH (10 mL) was
refluxed for 1 h. The reaction was allowed to cool to room
temperature, and H2O (30 mL) was added. The mixture was
filtered, cooled to 0 °C, and acidified with 2 M HCl. The precipitate
was removed by suction filtration and washed with H2O(50 mL)to
obtain 31 (480 mg, 78%): mp 174-175 °C. 1H NMR (500 MHz,
DMSO-d6) δ 1.06-1.87 (m, 10H), 3.15 (s, 3H), 4.42-5.19 (m, 1H),
6.79 (d, J = 5.7 Hz, 1H), 7.15 (d, J = 6.0 Hz, 1H), 12.08 (s, 1H),
13.20 (br s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 24.91, 25.40,
29.32, 32.29, 59.18, 113.21, 115.40, 123.69, 152.73, 167.88, 175.20.
Anal. (C13H18N2O2S2) C, H, N.
2-(3,3-Diethylthioureido)benzoic Acid (17). General Procedure
for 2-Thioureidobenzoic Acids 17-24. A mixture of methyl 2-
(3,3-diethylthioureido)benzoate (D, R1 = R2 = Et)74 (533 mg,
2.0 mmol), 1 M NaOH (10 mL), and EtOH (10 mL) was refluxed
for 1 h. The reaction was allowed to cool to room temperature,
and H2O (30 mL) was added. The solution was filtered, cooled to
0 °C and acidified with 2 M HCl. The precipitate was removed by
suction filtration and washed with H2O (50 mL) to obtain 17 (394
mg, 78%): mp 114-116 °C. 1H NMR (500 MHz, DMSO-d6) δ
1.23 (t, J = 7.1 Hz, 6H), 3.76 (q, J = 7.0 Hz, 4H), 7.11 (ddd, J =
8.2, 7.1, 1.3 Hz, 1H), 7.50 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.90 (dd,
J = 7.9, 1.6 Hz, 1H), 8.49 (dd, J = 8.5, 1.0 Hz, 1H), 10.62 (s, 1H),
13.53 (br s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 12.50, 45.03,
119.10, 122.65, 123.93, 130.49, 132.40, 142.97, 169.83, 178.41.
Anal. (C12H16N2O2S) H, N. C: calcd, 57.12; found, 56.55.
2-(3,3-Diethylureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carboxylic Acid (25).75 General Procedurefor 2-Ureidothiophene-3-
carboxylic Acids from Thieno[2,3-d][1,3]oxazin-4-ones E. A mix-
ture prepared from 2-(diethylamino)-6,7-dihydro-4H,5H-cyclo-
penta[4,5]thieno[2,3-d][1,3]oxazin-4-one (E, R1 = R2 = Et, R3R4
= -(CH2)3-) (132 mg, 0.50 mmol), 1.5 M NaOH (14 mL), and
acetone (7 mL) was refluxed for 5 min. After being cooled, the
mixture was filtrated into 3 M HCl (14 mL), and the precipitate
was collected by filtration to yield 25 (110 mg, 78%): mp 171-173
°C. 1H NMR (300 MHz, DMSO-d6) δ 1.17 (t, J = 6.8 Hz, 6H),
2.23-2.37 (m, 2H), 2.69-2.89 (m, 4H), 3.36 (q, J = 6.8 Hz, 4H),
10.88 (s, 1H), 12.77 (s, 1H). 13C NMR (75 MHz, DMSO-d6) δ
14.28, 28.03, 29.07, 30.60, 41.86, 106.43, 129.56, 141.52, 152.75,
2-(3,3-Diethylthioureido)benzo[b]thiophene-3-carboxylic Acid
(48). General Procedure for 2-Thioureidobenzo[b]thiophene-3-
carboxylic Acids 48-50. A mixture of ethyl 2-(3,3-diethylthiour-
eido)benzo[b]thiophene-3-carboxylate (H, R1 =R2 = Et, R3R4 =
-CHdCH-CHdCH-) (672 mg, 2.0 mmol), 1 M NaOH (10
mL), and EtOH (10 mL) was refluxed for 1 h. The reaction was
allowed to cool to room temperature, and H2O (30 mL) was added.
The solution was cooled to 0 °C and acidified with 2 M HCl. The
precipitate was removed by suction filtration and washed with
H2O (35 mL). Recrystallization from EtOH yielded 48 (250 mg,
41%): mp 153-155 °C (lit.59 142-144 °C). 1H NMR (500 MHz,
DMSO-d6) δ 1.26 (t, J = 6.8 Hz, 6H), 3.70-3.90 (m, 4H), 7.27
(ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.38 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H),
7.86 (ddd, J = 7.9, 1.3, 0.6 Hz, 1H), 8.23 (dt, J = 8.2, 1.0 Hz, 1H),
13.02 (s, 1H), 13.78 (br s, 1H). 13C NMR (125 MHz, DMSO-d6) δ
12.28, 46.13, 105.85, 121.88, 122.54, 123.58, 125.53, 132.67, 133.84,
156.85, 169.09, 174.86. Anal. (C14H16N2O2S2) C, H, N.
155.77, 168.34. Anal. (C13H18N2O3S H2O) C, N. H: calcd, 6.71;
found, 6.27.
Cell Lines. The human ovarian cancer cell line 2008 WT76 and
the multidrug-resistant cell line 2008 MRP176 were kindly
provided by Piet Borst (The Netherlands Cancer Institute,
Amsterdam, The Netherlands). The ovarian carcinoma cell
line (A2780) and its P-gp-overexpressing subline (A2780/
ADR) were purchased from European Collection of Animal
Cell Cultures (ECACC, no. 93112519 [A2780], no. 93112520
[A2780adr], United Kingdom) and were used for P-gp determi-
nation. The breast cancer resistant cell line (MCF-7 MX) and
the parental cell line (MCF-7) were kindly provided by Dr. E.
Schneider, Wadsworth Center, Albany, NY.
The cell lines were cultured in RPMI 1640 medium, supple-
mented with 10% fetal calf serum (FCS) and 1% antibiotics
(100000 units/L penicillin and 100 mg/L streptomycin). The cells
were maintained at 37 °C in a humidified atmosphere containing
5% CO2. Expression of MRP1, P-gp, and BCRP was confirmed
and determined by Western blot (data not shown).
Calcein AM Accumulation assays.45,53,77 For estimation of
MRP1 activity, the cell line 2008 MRP1 and the corresponding
wild-type cell line were used. Cells were harvested by trypsina-
tion and centrifuged. After resuspension in medium, the cells
were counted with a Casy I cell counter model TT (Schaerfe
System, Reutlingen, Germany). The cell density was adjusted
to 300000 per mL. The cell suspension (70 μL) containing 21000
cells were transferred into a 96-well microplate. One column
of wells was without cells, only containing buffer, the next two
following columns contained the cell suspension, either wild-
type or transfected cells. Into each well, 10 μL of solutions of
the test compounds in Krebs-HEPES buffer at different
3
Ethyl 2-(3-Cyclohexyl-3-methylthioureido)thiophene-3-carboxy-
late (H, R1 = Me, R2 = Cyclohexyl, R3 = R4 = H). General Pro-
cedure for 2-Thioureidothiophene-3-carboxylates H from 2-(isothio-
cyanato)thiophene-3-carboxylates G. N-Methylcyclohexylamine
(736 mg, 6.5 mmol) was added dropwise to a stirring solution of
ethyl 2-(isothiocyanato)thiophene-3-carboxylate (G, R3 = R4 =
H)59 (1.07 g, 5.0 mmol) in CH2Cl2 (20 mL). The reaction mixture
was stirred at room temperature for 3 h. The organic layer was
washed with 0.5 M HCl (2 ꢀ 5mL),driedoverNa2SO4, filtered, and
evaporated to dryness. Recrystallization from EtOH yielded H (R1
=Me,R2 = cyclohexyl, R3 =R4 = H) (1.26 g, 77%): mp 143-145
°C. 1H NMR (500 MHz, DMSO-d6) δ 1.11-1.82 (m, 13H), 3.16 (s,
3H), 4.32 (q, J = 7.0 Hz, 2H), 4.71-5.09 (m, 1H), 6.83 (d, J = 5.7
Hz, 1H), 7.18 (d, J = 5.7 Hz, 1H), 11.70 (s, 1H). 13C NMR (125
MHz, DMSO-d6) δ 14.30, 24.92, 25.38, 29.22, 31.75, 59.32,
60.80, 112.46, 115.90, 123.12, 152.85, 165.91, 175.17. Anal.
(C15H22N2O2S2) C, H, N.
Ethyl 2-(3,3-Diethylthioureido)benzo[b]thiophene-3-carboxylate
(H, R1 = R2 = Et, R3R4 = -CHdCH-CHdCH-). General
Procedure for Ethyl 2-Thioureidobenzo[b]thiophene-3-carboxylates
H (R3R4 = -CHdCH-CHdCH-). Diethylamine (549 mg,
7.5 mmol) was added dropwise to a solution of ethyl 2-(isothiocya-
nato)benzo[b]thiophene-3-carboxylate (G, R3R4 = -CHdCH-
CHdCH-)59 (1.3 g, 5.0 mmol) in CH2Cl2 (5 mL) at 0 °C. The
mixture was stirred at room temperature for 2 h and acidified with
1 M HCl/EtOH. The precipitate was collected by suction filtration
and recrystallized from EtOH to yield (H, R1 = R2 = Et, R3R4 =

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4593
concentrations, 10 μL of a 10 μM cobalt sulfate solution in
Krebs-HEPES buffer, and 10 μL of a 100 μM solution of
XR9577 in Krebs-HEPES buffer were added. After a 30-min
preincubation, each well was supplied with 33 μL of a calcein
AM solution in Krebs-HEPES buffer to obtain a final con-
centration of 0.5 μM. Fluorescence was measured over 30 min.
Indomethacin (100 μM) was used as positive control. All
compounds showing more than 50% inhibition at a concentra-
tion of 100 μM were considered to be active, and their inhibitory
potency was characterized by determination of complete con-
centration-response curves. All other compounds were rated
inactive (ni, no inhibition). Different concentrations of indo-
methacin (0.316-178 μM) were generally included in the mea-
surements to obtain IC50 values for the test compounds.
For measuring P-gp activity, A2780/ADR cells and the
corresponding wild-type cells were used and the calcein AM
accumulation assay was performed as described above, except
that XR9577 and cobalt ions were omitted. Calcein is not
effluxed by P-gp, thus a quenching of extracellular fluorescence
by cobalt is not necessary.78,79 Instead, XR9577 (10 μM) served
as a positive control. A concentration-response curve for
XR9577 was generated with concentrations 0.010-10 μM. A
detailed description is given elswhere.63 Selected MRP1 inhibi-
tors were evaluated at two different concentrations (100 and
31.6 μM). Compounds showing less than 15% inhibition at a
concentration of 100 μM were classified as inactive.
Hoechst 33342 Assay.62 For the determination of BCRP
activity, MCF-7 and MCF-7 MX cells were cultivated in T175
flasks. After a confluence of 80-90% was reached, cells were
harvested by gentle trypsination (0.05% trypsin/0.02% EDTA)
and then carried over into a 50 mL tube. After this step, cells
were centrifugated (1200 rpm, 4 °C, 4 min). Subsequently, the
cell pellet was resuspended in fresh culture medium, and the cell
density was determined using a Casy I model TT cell counter
device (Schaerfe System, Reutlingen, Germany). Followed by
another centrifugation, cells were washed three times with
Krebs-HEPES buffer and seeded into black 96-well plates
(Greiner, Frickenhausen, Germany) at a density of approxi-
mately 20000 cells per well in a volume of 90 μL. The plate was
divided into two parts: the first, third, seventh, and tenth column
of the 12 columns of the 96-well plate (32 wells) only contained
90 μL of Krebs-HEPES buffer, the remaining eight columns
(64 wells) were filled with 90 μL cell suspension. The 32 wells
containing buffer were defined as “background” wells. Ten μL
of various test compounds in different concentrations was
added to a total volume of 100 μL so that each concentration
contained one “background” well and two wells including cell
suspension. The prepared 96-well plate was kept under 5% CO2
and 37 °C for 30 min. After this preincubation period, 20 μL of a
30 μM Hoechst 33342 solution (protected from light) was added
to each well. Fluorescence was measured immediately in con-
stant intervals (120 s) up to 2400 s at an excitation wavelength
of 355 nm and an emission wavelength of 460 nm applying a
37 °C tempered BMG POLARstar microplate reader. XR9577
(10 μM) served as positive control in all measurements. A
concentration-response curve for XR9577 was generated with
concentrations between 0.316 nM and 10 μM (see Supporting
Information, Figure S2).
incubation, 20 μL of MTT solution (5 mg/mL) was added to
each well. Incubation with MTT was terminated after 50-70
min (before cell-damaging formazan needles were formed) by
injecting 150 μL of a mixture of 2-propanol:concd HCl (50
mL:165 μL) to each well. Then, the 96-well plates were kept at
5 °C for 1-2 h. Finally, the absorption was measured at 595 nm
(test wavelength) and 690 nm (reference wavelength). Absorp-
tion at the reference wavelength was subtracted from the
absorption at the test wavelength.
In a second series of experiments, effects of combinations
of selected compounds (31, 48) with the cytostatic drugs vin-
blastine and daunorubicin were evaluated. A fixed concentra-
tion (31.6 μM) of 31 or 48 was combined with distinct
concentrations of vinblastine or daunorubicin (0.1 nM-100
μM) using 2008 MRP1 cells. Indomethacin (31.6 μM) served
as positive control in this setting. After 72 h, the MTT assay was
applied as described above. The two compounds 31 and 48 were
omitted in the control experiments, where 2008 MRP1 and 2008
WT cells were treated with different concentrations of the
cytostatic drugs.
Assay Data Analyses. The fluorescence measured in the
“background” wells was subtracted from the fluorescence mea-
sured in the corresponding wells which were supplemented with
cells. These data were applied for the following analysis: the
slope of each fluorescence-time curve was calculated by linear
regression and used as a dependent parameter. From these data,
concentration-response curves were generated by nonlinear
regression using the four-parameter logistic equation with vari-
able Hill slope (GraphPad Prism 5.0 software, San Diego, CA).
For normalization of data, slopes from absorption-time curves
or fluorescence-time curves were transformed to relative units
by subtracting the lowest determined single value from all other
data and thus setting it to 0%. The highest measured single value
was defined as 100%.
Acknowledgment. We are indebted to Friederike Lohr,
Florian Grundmann, Yasemin Atabas, and Dieter Baumert
for assistance, as well as Markus Pietsch for providing the
analytical data of compound 25. This work has been sup-
ported by the German Research Society (GRK 804).
Supporting Information Available: Synthetic procedures and
analytical data for the remaining compounds, elemental ana-
lyses, structures of inactive compounds and of XR9577, cyto-
toxicity data, as well as exemplary concentration-effect curves
from the BCRP assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
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