4-Substituted-2-phenylquinazolines as inhibitors of BCRP

Article abstract of DOI:10.1016/j.bmcl.2012.08.024

We investigated several 2-phenylquinazolines with different substitutions at position 4 for their BCRP inhibition. Compounds with phenyl ring attached via an amine-containing linker at position 4 were found to be potent inhibitors of BCRP. In general compounds with meta substitution of phenyl ring at position 4 were found to have higher inhibitory effect, compound 12 being the most potent and selective towards BCRP.

Full text of DOI:10.1016/j.bmcl.2012.08.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6766–6769
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Substituted-2-phenylquinazolines as inhibitors of BCRP
⇑
Kapil Juvale, Michael Wiese
Pharmaceutical Institute, University of Bonn, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated several 2-phenylquinazolines with different substitutions at position 4 for their BCRP
inhibition. Compounds with phenyl ring attached via an amine-containing linker at position 4 were found
to be potent inhibitors of BCRP. In general compounds with meta substitution of phenyl ring at position 4
were found to have higher inhibitory effect, compound 12 being the most potent and selective towards
BCRP.
Received 27 June 2012
Revised 31 July 2012
Accepted 3 August 2012
Available online 15 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
BCRP
Inhibitors
Multidrug resistance
Quinazolines
Hoechst 33342
Multidrug resistance (MDR) has been found to be a major
cause for failure of chemotherapy for different cancers. Over-
expression of ATP binding cassette (ABC) transporters is the most
common mechanism involved in MDR. ABC transporters like
P-glycoprotein (P-gp), multidrug resistance-associated protein 1
(MRP1) and breast cancer resistance protein (BCRP/ABCG2) are
the principal ABC transporters responsible for MDR.1,2
BCRP belongs to subfamily G of ABC transporters and consists of
6 transmembrane helices and a N-terminal intracellular nucleotide
binding domain. The functional unit of BCRP has been reported to
be a homodimer linked by a disulfide bridge.3
BCRP is involved in the transport of large molecules with
amphiphilic character such as the anticancer drugs mitoxantrone,4
etoposide, flavopiridol,6 topotecan,4 and irinotecan5 and its active
metabolite SN-38. Several attempts have been made to overcome
the MDR caused by over-expression of BCRP. One of the strategies
is to investigate inhibitors of BCRP. Several types of inhibitors of
BCRP that have been identified, such as naturally occurring flavo-
noids,7 fumitremorgin C8 and its nontoxic synthetic analog
Ko143 and broad spectrum modulators like XR9576 (tariquidar)9
and GF120918 (elacridar) are of widely-varying structure. Ko143
is the most potent and selective BCRP inhibitor.
recently we were able to show the effect of TKIs gefitinib and
PD158780 on BCRP expression.12 Both compounds were found to
reduce the total and surface BCRP expression in EGFR-positive
MDCK BCRP cells by interacting with the PI3K/Akt signaling path-
way. During this study, EGFR inhibitors containing a quinazoline
moiety were found to be particularly potent inhibitors of BCRP.
This work encouraged us to investigate quinazolines for their BCRP
inhibition potential. As the quinazoline TKIs investigated until now
for BCRP inhibition possess substituents only at positions 4, 6 and 7
of the quinazoline moiety, we investigated compounds with differ-
ent substitution, starting with 2-phenyl substituted derivatives for
their activity against BCRP.
In the current study, we have synthesized several 4-substituted
analogs of 2-phenylquinazoline. Quinazolines 1–17 contain a sim-
ple amine linker at position 4 to which a phenyl ring bearing differ-
ent substituents is attached (Scheme 1). Quinazolines 18–24 carry
a piperazine linker at position 4 of quinazoline (Scheme 2). The fi-
nal compounds were synthesized by reacting different substituted
F
The quinazoline compound prazosin (an a1 adrenergic blocker)
O
has been found to be a substrate of BCRP.10 Also the BCRP inhibi-
tory potential of quinazoline based TKIs (tyrosine kinase inhibi-
tors) such as gefitinib and PD153035 has been reported
(Fig. 1).11,12 Yanase et al. have suggested an interaction of gefitinib
with BCRP and a possible effect on BCRP expression.13 Very
HN
N
Cl
HN
N
Br
N
O
O
O
N
N
O
gefitinib
PD153035
⇑
Corresponding author. Tel.: +49 228 735213; fax: +49 228 737929.
E-mail address: mwiese@uni-bonn.de (M. Wiese).
Figure 1. Recently reported TKIs with quianzoline moiety. Both compounds
showed strong inhibition of BCRP and reduction of BCRP expression.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.024

K. Juvale, M. Wiese / Bioorg. Med. Chem. Lett. 22 (2012) 6766–6769
6767
O
Cl
O
O
H
NH
N
i
ii
NH2
NH2
N
N
S2
S1
NH2
iii
R
HN
NO2
N
R
HN
N
N
N
12
1-17
Scheme 1. Structure of the lead compound 12 and general synthesis of 4-substituted-2-phenylquinazolines. Reagents and conditions: (i) DMF, I2, K2CO3, heat for 4–5 h, 73%;
(ii) POCl3, reflux, 8 h, 86%; (iii) isopropanol, reflux, 3–4 h, 55–80%.
H
N
Cl
N
NH2
HCl
H
N
i
N
N
R
Cl
Cl
R
S2
S3
ii
R
N
N
N
N
18-24
Scheme 2. General synthesis of 2-phenyl-4-(4-phenylpiperazin-1-yl)quinazolines. Reagents and conditions: (i) diethyleneglycol monomethyl ether, reflux, 6–12 h, 65%; (ii)
K2CO3, isopropanol, reflux, 10–12 h, 45–64%.
anilines (1–17) or N-phenylpiperazines (18–24) with 4-chloro-2-
phenyl quinazoline, which was synthesized as reported earlier.14
All synthesized compounds were investigated for their BCRP
inhibition using the Hoechst 33342 assay15,16 with MDCK BCRP
cells, while the non BCRP expressing MDCK cells17,18 were used
as control. Table 1 shows the structural features of synthesized
compounds and their half-maximal inhibitory concentration ob-
tained in the Hoechst 33342 assay. Ko143 was used as a standard.
From Table 1 it is apparent that, of the anilinoquinazolines
1–17, those bearing a meta substituent on the aniline were found
to be more potent than ortho- or para-substituted analogs (see
compounds 3, 12, 15 and 16). Compound 12 was the most potent
Additionally compounds 18–24 bearing a piperazine ring in
place of amine linker at position 4 of quinazoline were investigated
for BCRP inhibition. However all the compounds with N-phenyl-
piperazinyl substitution at position 4 showed reduced inhibitory
activity. This reduction in activity might result from a change in
the length and type of the linker.
The anilinoquinazolines with inhibitory activity in the high
nanomolar range against BCRP (3, 10, 12, 13, 15 and 16), were
additionally screened for P-gp and MRP1 inhibition. Screening of
these compounds was done at a concentration of 10
cein AM assay16 using A2780 adr (for P-gp) and 2008 MRP1 (for
MRP1) cell lines.19,20 Cyclosporine A (10
M) was used as a stan-
lM, in the cal-
l
in the set, having an IC50 (0.13
l
M) even lower than that of the
dard for both P-gp and MRP1 inhibition. Amongst all screened
compounds, compound 3 and 10 showed substantial P-gp and
MRP1 inhibition, while compounds 12, 13 and 16 showed no to
very slight effect. Figure 3 shows the results obtained from the
screening of compounds for P-gp and MRP1 inhibition. Concentra-
tion response curves of compounds 3 and 10 against cyclosporine
A in calcein AM assay are shown in Figure 4. Although compound
10 was found to be very active against BCRP, it also showed
standard Ko143 (0.25 M). A comparison of the concentration re-
l
sponse curves for these two compounds is shown in Figure 2. How-
ever, IC50-values only are not suitable to estimate the real affinity
of inhibitors as additionally the distribution of the compounds into
the biomembrane has to be taken into account. Based on calculated
lipophilicities Ko143 is less lipophilic than our compounds and
thus should possess a higher intrinsic affinity towards BCRP.

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K. Juvale, M. Wiese / Bioorg. Med. Chem. Lett. 22 (2012) 6766–6769
Table 1
Quinazolines 1–24 and their IC50 values in the Hoechst 33342 assay
R
HN
R
N
N
N
N
N
N
1-17
18-24
Quinazoline
R
IC50 SDa
(lM)
1
2
3
4
5
6
7
8
H
2.57 0.28
3.75 0.53
0.57 0.04
6.83 0.97
1.93 0.28
1.66 0.11
2.25 0.07
1.32 0.10
1.93 0.11
0.19 0.01
5.93 0.10
0.13 0.03
0.97 0.24
2.46 0.14
0.14 0.04
0.25 0.10
1.53 0.13
8.92 1.82
5.12 0.57
5.91 1.73
8.22 1.36
8.90 2.35
7.95 0.35
9.35 2.79
0.25 0.02
20-Br
30-Br
40-Br
30-Cl
30-Cl,40-F
20-OCH3
30-OCH3
40-OCH3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Ko143
30,40-OCH3
20-NO2
30-NO2
40-NO2
30-NO2,40-F
30-CN
30-CF3
40-CF3
H
30-Br
30-Cl
30-Cl,40-F
30-OCH3
30-NO2
40-NO2
—
Figure 3. Influence of quinazolines at 10
calcein AM in P-gp (A) and MRP1 (B) overexpressing cells. Cyclosporine A (CsA,
10 M) was used as a standard. Response in absence of any compound was used as
lM concentration on accumulation of
l
negative control. Data are expressed as response in percentage of control (accu-
mulation of calcein AM in non P-gp or MRP1 expressing sensitive cells) and
presented as the mean SD of three independent experiments.
a
All values are means of at least 3 individual experiments.
Figure 2. Concentration response curves of compound 12 (closed circles,
IC50 = 0.13 0.03 M) and Ko143 (closed triangles, IC50 = 0.25 0.02 M) obtained
l
l
in the Hoechst 33342 assay. Each data point shown represents average of
3
independent experiments with error bars indicating standard deviation.
inhibition of P-gp (IC50 = 5.85 0.79
lM) as well as MRP1
(IC50 = 11.18 0.58 M), losing its selectivity for BCRP.
l
The results obtained clearly reveal the potential of quinazolines
as BCRP inhibitors. Also we were able to show that replacing the
linker at position 4 of the quinazoline moiety with a piperazine
linker leads to a strong decrease in activity.
As quinazolines, except a few TKIs, have not been explored for
their BCRP inhibition ability, these findings can be the basis for
further development of quinazolines as potent BCRP inhibitors.
Figure 4. Concentration response curves of compound 3 (closed circles) and 10
(closed squares) against cyclosporine A (closed triangles) obtained in the calcein
assay for P-gp inhibition (A) and MRP1 inhibition (B). Each data point shown
represents average of
standard deviation.
3 independent experiments with error bars indicating

K. Juvale, M. Wiese / Bioorg. Med. Chem. Lett. 22 (2012) 6766–6769
6769
18. Hoechst accumulation assay: when reaching a confluence of 80–90%, cells were
harvested by gentle trypsination (0.05% trypsin/0.02% EDTA) and then
transferred to a 50 ml tube followed by centrifugation (266 ꢀ g, 4 °C, 4 min).
The cell pellet obtained was resuspended in fresh culture medium and the cell
density was determined using a Casy I Modell TT cell counter device (Schaerfe
System GmbH, Reutlingen, Germany). Followed by another centrifugation cells
were washed three times with Krebs-Hepes buffer (KHB) and seeded into black
Acknowledgements
The authors thank to Dr. A.H. Schinkel (The Netherlands Cancer
Institute, Amsterdam, The Netherlands) for kindly providing the
MDCK BCRP cell line. K.J. thanks DAAD (Germany) for financial
support.
96 well plates (Greiner, Frickenhausen, Germany) at
approximately 20,000 cells per well in a volume of 90 l when using MDCK
BCRP cells. 10 l of various test compounds in different concentrations were
added to a total volume of 100 l. The prepared 96 well plate was kept under
5% CO2 and 37 °C for 30 min. After this preincubation period, 20 l of a 6 lM
a
density of
l
l
References and notes
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17. MDCK BCRP cells were generated by transfection of the canine kidney
epithelial cell line MDCKII with the human wild-type cDNA C-terminally
linked to the cDNA of the green fluorescent protein. Cells were maintained in a
5% CO2 humidified atmosphere at 37 °C. After confluence of 80–90%,
subculturing was performed with 0.05% trypsin and 0.02% EDTA.
Hoechst 33342 solution (protected from light) was added to each well.
Fluorescence was measured immediately in constant intervals (60 s) up to
120 min at an excitation wavelength of 355 nm and an emission wavelength of
460 nm applying a 37 °C tempered BMG POLARstar microplate reader (BMG
Labtech, Offenburg, Germany). For the analysis of the data obtained from the
assay, first fluorescence of KHB was subtracted from the fluorescence reading
obtained from MDCK cells. Average of fluorescence values in the steady state
(from 100 to 109 min) was calculated for each concentration and from these
data, concentration response curves were generated by nonlinear regression
using the four-parameter logistic equation with variable Hill slope (GraphPad
Prism v. 5.0, San Diego, CA, USA).
19. Human ovarian carcinoma cell lines A2780 and the corresponding MDR1
overexpressing doxorubicin resistant A2780adr cell line were purchased from
ECACC (Nos. 93112519 and 93112520). The cell lines were grown in RPMI-
1640 medium supplemented with 10% FBS, 50
ll/ml streptomycin, 50 U/ml
penicillin G, and 365 l/ml -glutamine. The human ovarian cancer cell line
l
L
2008 stably expressing MRP1was used for MRP1 testing. The cell line 2008
MRP1 was grown in the same medium as described for A2780 with addition of
400 lg/ml G-418 (Geneticin). Cells were maintained in a 5% CO2 humidified
atmosphere at 37 °C. After confluence of 80–90%, subculturing was performed
with 0.05% trypsin and 0.02% EDTA.
20. Cells were prepared as described above and washed three times with Krebs-
HEPES buffer and then seeded into colourless 96 well plates (Greiner,
Frickenhausen, Germany) at a density of approximately 30,000 cells in a
volume of 90
resulting in a final volume of 100
preincubated for 30 min. After the preincubation period, 33
l
l per well. Then, 10
l per well. The prepared 96 well plates were
l of a 1.25 lM
ll of the test compounds were added,
l
l
calcein AM solution, which was protected from light, were added to each well.
The fluorescence was measured immediately in constant time intervals (60 s)
up to 90 min using an excitation wavelength of 485 nm and an emission
wavelength of 520 nm with a BMG POLARstar microplate reader tempered at
37 °C. For calculation of inhibitory effects the first linear part of the
fluorescence time curves was used.