Synthesis and Biological Evaluation of 4-Anilino-quinazolines and -quinolines as Inhibitors of Breast Cancer Resistance Protein (ABCG2)

Article abstract of DOI:10.1021/acs.jmedchem.6b00330

Chemotherapeutic treatment of cancer often fails due to overexpression of the ATP-binding cassette (ABC) transport proteins, like ABCG2, triggering active efflux of various structurally unrelated drugs. This so-called multidrug resistance (MDR) may be rev

Full text of DOI:10.1021/acs.jmedchem.6b00330

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Article
Synthesis and biological evaluation of 4-Anilino-quinazolines and -
quinolines as inhibitors of Breast Cancer Resistance Protein (ABCG2)
Michael K Krapf, and Michael Wiese
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00330 • Publication Date (Web): 05 May 2016
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Synthesis and biological evaluation of 4ꢀAnilinoꢀquinazolines and ꢀquinolines as
inhibitors of Breast Cancer Resistance Protein (ABCG2)
*
Michael K. Krapf, Michael Wiese
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Pharmaceutical Institute
University of Bonn
An der Immenburg 4
53121 Bonn, Germany
Keywords
ABC transporter; inhibitor; ABCG2; quinazolines
ABSTRACT
Chemotherapeutic treatment of cancer often fails due to overexpression of ATPꢀbinding
cassette (ABC) transport proteins, like ABCG2, triggering active efflux of various structurally
unrelated drugs. This soꢀcalled multidrug resistance (MDR) may be reversed by selective,
potent and nonꢀtoxic inhibitors of ABCG2. As only a few potent inhibitors are known, new
compounds based on a 4ꢀsubstitutedꢀ2ꢀphenylquinazoline scaffold were investigated.
Substitution with hydroxy, cyano, nitro, acetamido and fluoro led to high inhibitory activities
toward ABCG2. The ability to reverse MDR of the most active compounds was confirmed in
a MTT efficacy assay. Moreover, a negligibly low intrinsic cytotoxicity was found resulting
in a high therapeutic ratio. Investigations of the inhibitory activity toward ABCB1 and
ABCC1 yielded a high selectivity toward ABCG2 for the quinazoline compounds. Quinolineꢀ
based analogues showed lower inhibitory activity and selectivity. The study yielded a variety
of promising compounds, some with superior properties compared to the standard inhibitor
Ko143.
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INTRODUCTION
Failure in cancer chemotherapy is most often caused by the development of MDR where
cancer cells become simultaneously resistant to structurally unrelated chemotherapeutic
agents. ABC transport proteins, especially Pꢀglycoprotein (Pꢀgp, ABCB1), the multidrug
resistance associated protein 1 (MRP1, ABCC1) and the breast cancer resistance protein
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(BCRP, ABCG2) play a key role in the development of MDR. These members of the ABC
family transport a wide variety of structurally unrelated substrates out of cells against a
concentration gradient by using the energy of ATP hydrolysis. The decreased intracellular
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concentrations may lead to failure of the cancer chemotherapy. To date, 48 human ABC
transport proteins have been discovered and were classified in seven subfamilies ABC A to
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ABC G based on phylogenetic similarity. In the context of MDR, the most important ABC
transport proteins in chronological order of their discovery are ABCB1, ABCC1 and ABCG2.
The ATP dependent efflux pump ABCB1 was discovered when the “reduced drug
permeation” of colchicine in Chinese hamster ovary cells was attributed to a cell surface
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glycoprotein. Later, the ABC transporter ABCC1 was found in the lung cancer cell line
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H69AR, which showed resistance toward doxorubicin and other chemotherapeutics.
Progressive investigations indicated the existence of another member of the ABC transporter
family. The study of human MCFꢀ7 breast cancer cells in the presence of the
chemotherapeutic agent mitoxantrone revealed resistance of some cells toward the cytostatic
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without overexpression of ABCB1 or ABCC1. In 1998, Doyle et al. eventually discovered
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ABCG2 in MCFꢀ7/AdrVp human breast carcinoma cells.
ABCG2 is a 72 kDa integral membrane protein consisting of six transmembrane helices and
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one amino terminal nucleotide binding domain (NBD). It is assumed that the formation of
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dimers or tetramers is essential for the function of the “half transporter” ABCG2. Indeed,
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the mechanisms and processes involved in the transport are still not fully elucidated.
In
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recent years, the pharmacotherapeutic significance of ABCG2 in cancer treatment emerged
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more and more.
Already in the early 1980s, clinical trials of first generation ABCB1 modulators like
verapamil, cyclosporine A and progesterone were carried out with the objective to reverse the
ABCB1 mediated MDR. Due to toxicity at high doses and low inhibitory potencies, early
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expectations were not met.
Later, more specific and potent inhibitors of ABCB1 were
developed. However, none of them met the expectations based on their high in vitro potency
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in clinical trials.
In case of ABCG2, only a few selective and potent inhibitors, active in submicromolar
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concentration range have been reported. The well known and selective first generation
inhibitor of ABCG2 fumitremorgin C (FTC) for instance, revealed neurotoxic effects which
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prevented any clinical use.
Shortly afterwards the less toxic and very potent second generation inhibitor Ko143 was
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developed. Very recently Ko143 was coꢀadministered with phototoxic porphyrine based
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agents in photodynamic therapy with promising results. As the intracellular uptake of the
applied photosensitizers is regulated by ABCG2, inhibition of the transport protein was found
to lead to an increased accumulation in ABCG2 overexpressing colon cancer cells HT29.
Irradiation of the cells resulted in significantly higher apoptosis rates by intracellular
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production of singlet oxygen than without inhibitor.
Thus, inhibitors of ABC transporters have been shown to possess some potential in cancer
treatment. As the number of potent ABCG2 inhibitors is rather limited, there is a need for
developing more potent and selective inhibitors of this protein. Quinazoline based tyrosine
kinase inhibitors, like for example gefitinib, have been reported to be potent inhibitors of
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ABCG2. We have reported the synthesis and biological evaluation of various substituted 4ꢀ
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anilino quinazolines that proved to be even more potent inhibitors of ABCG2.
Among the
synthesized derivatives 4ꢀanilinoꢀ2ꢀphenylquinazolines showed highest inhibitory activities
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when substituted with nitro, hydroxy, cyano or trifluoromethyl substituents in meta position
of the anilino moiety. It was found that para substitutions resulted in slightly increased IC50
values, while ortho substituted compounds yielded less potent compounds with highly
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increased IC50 values.
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In this study a variety of new substituents have been introduced aiming to give new insights in
the structure activity relationship. Variation was carried out only at the anilino moiety in meta
and para position to clearly illustrate the interrelations in this moderately complex system.
RESULTS AND DISCUSSION
Chemistry. Synthesis of all 4ꢀanilinoꢀ2ꢀphenylquinazoline and 4ꢀanilinoꢀ2ꢀphenylquinoline
derivatives was carried out by nucleophilic aromatic substitution of the corresponding 4ꢀ
chloro species with meta and para substituted anilines (Scheme 1). Substitutions in ortho
position of the anilino residue have been proven to result in a decreased inhibitory activity
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against BCRP and were not investigated in this study.
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The identity of all compounds was confirmed by H and C NMR spectra and the purity by
elemental analysis. Additionally, the melting point of each quinazoline derivative was
determined.
Biological evaluation.
Hoechst 33342 Accumulation Assay. Investigation of the inhibitory activity of the test
compounds against ABCG2 was performed using the Hoechst 33342 accumulation assay with
MDCK II BCRP cell line. Hoechst 33342 is a fluorescent dye and substrate of ABCG2, which
shows a significant increase in fluorescence when bound to DNA or embedded in lipophilic
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environment like the cell membrane. Hence, an increase of fluorescence by intracellular
accumulation of Hoechst 33342 can be correlated to the inhibitory activity of the compound.
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The structures and inhibitory activity data of all compounds obtained in the Hoechst 33342
accumulation assay are depicted in Table 1.
First, a 4ꢀcyano residue was introduced at the aniline moiety. Surprisingly, this modification
increased the inhibitory activity considerably from 140 nM of the 3ꢀcyano derivative 6 to 70
nM of the 4ꢀcyano derivative 1. In comparison to Ko143, which is currently the most active
inhibitor of ABCG2 found in the literature (IC : 221 nM in the Hoechst 33342 accumulation
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assay), compound 1 possesses a 3ꢀfold higher activity against ABCG2. The 4ꢀhydroxy and 4ꢀ
fluorsulfon substituted compounds 2 and 3 exhibit also high inhibitory potencies, resulting in
IC50 values of 204 nM and 556 nM, respectively. Interestingly, the hydrophilic nature of the
4ꢀhydroxy group is no drawback for the activity of compound 2. Furthermore, a disubstitution
with 3ꢀnitro and 4ꢀhydroxy substituents at the anilino moiety was investigated (compound 4).
This combination significantly increased the inhibitory activity to 80 nM compared to the 4ꢀ
hydroxy monosubstituted compound 2 as well as the 3ꢀnitro monusubstituted derivative 5.
Concentrationꢀresponse curves of the most potent compounds 1 and 4 in the Hoechst 33342
accumulation assay together with Ko143 as standard are shown in Figure 1.
To further increase the hydrophilicity of the compounds, an acetamido group was introduced
in meta position (7). With an IC of 278 nM compound 7 was similarly active as Ko143. For
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the methoxy substituted compounds the known pattern of an increased activity from para
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(comp. 10: 1930 nM) to meta substitution (comp. 9: 1320 nM) applies. While the 3,4ꢀ
dimethoxy substituted compound 11 yields an significantly enhanced IC50 value of 152 nM.
By the exchange of oxygen with sulphur at the 4ꢀmethoxy group of compound 9, a minor
improvement of the inhibitory activity could be achieved. Hence, an IC of 1190 nM was
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determined for the 4ꢀmethyl thioether 8. The substitutions with single halogen atoms
exhibited a distinct trend for the inhibitory activities of the compounds. For meta substitutions,
increasing atomic numbers of the halogen atom from 3ꢀfluoro (15) to 3ꢀiodo (18) resulted in a
twoꢀfold decrease of activity from 355 nM to 791 nM, respectively. In contrast, increasing
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atomic numbers of the halogen atom in para position, starting from 4ꢀfluoro (16) to 4ꢀchloro
(17) and 4ꢀiodo (19), resulted in an about twoꢀfold increase in activity from 1060 nM to 830
nM and 612 nM, respectively. Furthermore, the combination of 3ꢀmethoxy and 4ꢀbromo
yielded an excellent activity of 289 nM for compound 13. A major contribution to this
considerable activity is likely due to the paraꢀsubstitution of the halogen with a high atomic
number. The disubstitution of 3ꢀfluoro and 4ꢀmethoxy at the aniline moiety results in an IC50
of 872 nM for compound 12, which nearly exactly equals the mean pIC50 values of the
corresponding monosubstituted compounds 10 and 15. Moreover, a methyl ester and tertꢀ
butyl ester were investigated in para position to determine the impact of the used alkyl groups
on the activity. The methyl ester, compound 20, possesses a moderate activity of 944 nM,
while the more bulky and lipophilic tertꢀbutyl ester residue of compound 21 yields only an
IC50 value of 5650 nM. Additionally, a cleavage of the ester group of compound 21 may
occur yielding compound 22 bearing a carboxylate group. This compound with a substitution
of 4ꢀcarboxyl group, shows a slightly lower activity (IC50: 6380 nM) than 21. This is due to a
highly increased hydrophilicity, caused by deprotonation under physiological conditions.
Furthermore, the impact of alkyl residues with different steric requirements and lipophilic
properties were investigated. Hence, substitutions at the aniline moiety were carried out each
in meta and para for methyl (24, 25) and tertꢀbutyl (26, 27), respectively. Only minor
differences in the IC50 values were observed for the substitutions of 3ꢀmethyl (1150 nM) and
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4ꢀmethyl (1480 nM). Also the tertꢀbutyl substitutions in meta and para exhibited very similar
IC values of 1070 nM and 1060 nM, respectively. In comparison to the methyl moiety, the
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tendency of a slightly increased activity for the tertꢀbutyl residues was noticeable. In addition,
an ethinyl residue was introduced in meta position of compound 23. Although its logP value
was calculated in the range of the 3ꢀmethyl substituted compound 24 (data not shown), it
exhibits a considerably lower IC50 value of 672 nM.
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Furthermore, five different quinoline derivatives were synthesized for comparison with their
quinazoline analogues. The aim was to investigate the importance of the nitrogen atom in
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position 3 of the scaffold (Scheme 1: R ) for the inhibitory activity. The anilino moiety of the
quinoline scaffold was substituted in given order of compounds 28ꢀ32 with 3ꢀmethoxy, 3ꢀ
thiomethyl, 3ꢀacetamido, 4ꢀcyano and 3ꢀfluoro, respectively. With exception of compound 28,
all quinoline derivatives showed at least a twoꢀfold lower inhibitory activity against ABCG2
than their quinazoline counterparts. Only the 3ꢀmethoxy derivative retained an activity
comparable to the quinazoline analogue.
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From the results, some factors, related to the activity of a substance, can be derived: The
electronic effect (electron withdrawing and electron donating) of the substituents at the aniline
moiety as well as the steric requirements play a minor role. Substitution in meta and para
position yielded both high activities. However, a distinct trend for the halogen substitutions
was found. Apart from the carboxylate group, no correlation of logP and IC50 could be
determined. It was found that the quinazoline scaffold is preferable over quinoline for the
activity against ABCG2.
Pheophorbide A Accumulation Assay. Additionally, the IC values of selected compounds
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were determined in a pheophorbide A accumulation assay (Table 1). The chlorophyll
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catabolite pheophorbide A is a specific substrate of ABCG2 and used as fluorescent dye.
Like in the Hoechst 33342 accumulation assay, the MDCK II cell line was used. The
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scatterplot of the pIC values of both assays reveal a good correlation with r = 0.91 (Figure
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2). Doseꢀresponse curves of the most active compounds 1 and 4 in the pheophorbide A assay
is shown in Figure 3.
Screening for ABCB1 and ABCC1 Inhibition. Selectivity of all compounds toward ABCG2
was investigated by a calcein AM accumulation assay. For this purpose, the ABCC1
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overexpressing cell line H69AR and the ABCB1 overexpressing cell line A2780adr were used
as described in the experimental section. The inhibitory effect of the substances toward the
multispecific transporters ABCC1 and ABCB1 was screened at a concentration of 10 µM. For
comparison, Cyclosporine A (CsA) was used as positive control, leading to total inhibition of
both transporters, respectively. Compounds with an inhibitory potency of more than 25%
relative to the standard were additionally tested with more dilutions to generate full doseꢀ
response curves, and obtaining IC50 values. A bar chart of the screening results for the
inhibitory activity toward ABCB1 and ABCC1 is depicted in Figure 4 (a) and (b),
respectively. A summary of the IC50 values of the compounds giving more than 25% of
inhibition in the screening is given in Table 2.
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In the calcein AM accumulation assay with ABCB1 overexpressing cells, compounds 11, 24
and 28ꢀ31 exhibited a relative inhibitory activity of more than 25% of the positive control
cyclosporine A (IC50: 1.17 µM). The presence of methoxy groups clearly tends to increase the
inhibitory activity toward ABCB1 significantly. For compound 11, with a 3,4ꢀdimethoxy
substitution at the anilino moiety, an IC50 value of 1.86 µM was determined. Moreover,
compound 24 with a 3ꢀmethoxy substitution gave an IC50 of 3.00 µM. Among the other
quinazoline derivatives, no significant inhibitory activity toward ABCB1 was detected. On the
other hand, the quinoline derivatives 28ꢀ31 were found to be inhibitors of ABCB1. These
compounds contain a 3ꢀmethoxy, 3ꢀthiomethyl, 3ꢀacetamido and 4ꢀcyano residue,
respectively. The IC values for 28ꢀ31 were determined in given order as 2.78 µM, 1.42 µM,
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0.66 µM and 4.74 µM, respectively. Merely compound 32 with a 3ꢀfluoro substitution
exhibited only moderate inhibitory activity. In comparison with their quinazoline analogues it
becomes evident, that the quinoline scaffold clearly contributes to the inhibitory activity
toward ABCB1.
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A calcein AM accumulation assay with ABCC1 overexpressing cells exhibited, that only five
substances in the data set exceeded the 25% mark of inhibitory activity at a concentration of
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0 µM relative to the positive control CsA (IC50: 3.77 µM). A bar chart of the screening
results is presented in Figure 4 (b). The substitution with 3ꢀthiomethyl, 4ꢀcarboxymethyl and
ꢀcarboxylate for the quinazoline derivatives 8, 20 and 22, resulted in IC50 values of 3.73 µM,
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8.13 µM and 8.77 µM, respectively. All investigated quinolines showed an increase in activity
toward ABCC1. Compound 29 with a 3ꢀthiomethyl and compound 30 with a 3ꢀacetamido
group yielded IC50 values of 2.98 µM and 5.80 µM, respectively. Here, the 3ꢀthiomethyl
substitution on the aniline moiety contributes to the inhibitory activity against ABCC1, both
for the quinoline as well as the quinazoline scaffold. As in case of ABCB1 inhibition, most of
the quinazoline derivatives exhibit a lower inhibitory activity than the corresponding
quinolines. This is most likely due to the more basic character of the nitrogen atom in the
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quinoline structure with a calculated pK of 8.4 vs. 5.6 for the 4ꢀanilinoꢀ2ꢀphenylquinoline
and quianzoline, respectively. Meaning that the quinoline derivatives would be mostly
protonated while the quinazolines were neutral under physiological pH. The negatively
charged compound 22 with a carboxylate residue could be expected to interact with ABCC1
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as it is well known, that ABCC1 transports anionic substances with sufficient lipophilicity.
Again the methyl ester 20 has a very similar IC50 value as the corresponding carboxylate
derivative 21, pointing to the possibility that the ester gets cleaved by intracellular esterases to
the carboxylate.
Determination of the Intrinsic Cytotoxicity via MTT Assay. The intrinsic cytotoxicity of
selected compounds was determined in the MTT assay. For this purpose, MDCK II cells were
incubated with different dilutions of selected test compounds for 72 h. After this time period,
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) was added to the wells.
As living cells reduce the MTT dye to the insoluble formazan species with a purple colour,
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the cell viability was obtained by measuring the absorption of each well after solubilisation of
the formed formazan. A more detailed description of the MTT assay is given in the
experimental section.
The influence of selected compounds on the intrinsic cell toxicity is illustrated in Table 3. The
GI50 values correspond to the modulator concentration which reduces the cell survival to 50%
of the control. Due to the fact that small amounts of methanol (≤ 1.8%) and DMSO (≤ 1%)
are used for diluting the compounds, the toxic effect of MeOH and DMSO without modulator
was determined additionally. For the MeOH/DMSO dilution alone, a GI50 of 71.5 µM was
obtained. Furthermore, the GI50 value of Ko143 was determined in analogy to the other test
compounds as 11 µM. The mono substitution of 4ꢀhydroxy in comparison to a 4ꢀhydroxy and
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3ꢀnitro disubstitution shows high differences of the intrinsic cytotoxicity of the compounds 2
and 4, respectively. Hence, the disubstituted compound 4 is rather nonꢀtoxic with a GI value
5
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of 52.4 µM while the mono substituted 4ꢀhydroxy compound 2 exhibits a considerably higher
toxicity with a GI50 of 12.2 µM. Among all tested substances, compound 4 possesses the
lowest toxicity. Compound 1 on the other hand, possesses the second highest cytotoxicity of
the tested derivatives with a GI50 of 7.96 µM. This is interesting, since compound 1 and 4
yield the highest activity in the Hoechst 33342 and pheophorbide A accumulation assays.
Moreover, the 3,4ꢀdimethoxy substituted compound 11 showed a rather low toxicity, which
relates to a high therapeutic ratio. Among the halogen substituted derivatives 15ꢀ19 the 3ꢀ and
4
ꢀfluoro derivatives exhibited the highest GI50 values with 17.2 µM and 29.0 µM. In contrast,
the 3ꢀchloro, 3ꢀ and 4ꢀiodo substituted derivatives yielded lower GI50 values of 6.75 µM,
4.75 µM and 9.25 µM, respectively. To illustrate a possible benefit for in vivo applications,
1
the therapeutic ratio of the tested compounds was calculated and is included in Table 3
Compound 4 in particular, exhibits an outstanding ratio of high inhibitory activity and low
cytotoxicity, scoring GI50/IC50 = 655. This was determined to be more than 13 times higher
than the score of Ko143 (Figure 5).
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Determination of the Ability to Reverse MDR via MTT Assay. A MTT efficacy assay was
performed for the most active compounds 1 and 4, in order to confirm the reversal of the
MDR caused by ABCG2. Here, the MDCK II cell line was used with the ABCG2 substrates
and cytostatics SNꢀ38, Hoechst 33342 and mitoxantrone (MX), respectively. First, a dilution
of SNꢀ38 and Hoechst33342 was tested with MDCK II BCRP overexpressing cells in the
presence of different modulator concentrations. Due to the inhibitory effect of the compounds
toward ABCG2, the intracellular accumulation of the cytostatic is increased. This leads to a
decrease of the cell viability, which can be measured analogously to the MTT cytotoxicity
assay described above. Parental MDCK II cells were used as positive control, representing a
total inhibition of ABCG2.
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Both modulators 1 and 4 were able to fully reverse the MDR using the cytostatics SNꢀ38 and
Hoechst 33342 (Figure 6): With increasing modulator concentration, a shift of the ABCG2
overexpressing cells toward the parental cells is noticeable (dashed arrow). At the highest
concentration of 5 µM, a total sensitization of the ABCG2 overexpressing cells toward the
corresponding cytostatic was achieved with both compounds. This is indicated by identical
IC values of the ABCG2 overexpressing and the parental cells. Furthermore, the efficacy of
5
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MDR reversal was determined by plotting the pIC50 values obtained from each doseꢀeffect
curve in the efficacy assay against the logarithm of the compound concentration. This gives
concentrationꢀefficacy curves that can be fitted with the logistic equation yielding pEC50
characterizing the power of MDR reversal by a compound (Figure 7). Here “no inhibition” is
defined with ABCG2 overexpressing cells without modulator, which show maximal
resistance toward the cytostatic, and “total inhibition” of ABCG2 is defined by the pIC50
ꢀ
value of the parental cells. Thereby, the EC50 values for compound 1 and 4 were calculated
for SNꢀ38 and Hoechst 33342, respectively: For compound 1, an EC50 value of 0.041 µM
(SNꢀ38) and 0.053 µM (Hoechst 33342) was determined. This result is in good agreement
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with the IC ꢀvalue from the Hoechst 33342 accumulation assay (0.072 µM). Applying the
50
same method to compound 4, EC values of 0.12 µM (SNꢀ38) and 0.13 µM (Hoechst 33342)
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were obtained. Again, the EC50ꢀvalue for Hoechsst 33342 is in good agreement with the
accumulation assay (IC50: 0.080 µM).
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Additionally, the cytostatic MX was used to demonstrate the reversal of resistance of the
BCRP overexpressing MDCK II cells with compound 1 and 4. For this purpose, the cell
viability was measured as described above after 72 h incubation with different modulator
concentrations in the presence of 5 µM cytostatic. Decrease of cell viability caused by the
cytotoxicity of the compound was taken into account by performing an analogue dilution of
the substance without MX. Wells with only cells and medium were used as negative control
whereas wells with cells, medium and 10% DMSO were used as positive control, both in
presence and absence of MX. For both compounds, the reversal of the resistance was
demonstrated as shown in Figure 8. The halfꢀmaximal growth inhibition was achieved for
compound 1 at 4.41 nM and for 4 at 68.4 nM, respectively. In particular, an excellent efficacy
was revealed in context of coꢀadministration with cytostatics for compound 1. In all efflux
based MTT assays with cytostatics it significantly exceeds the IC50 values obtained in the
Hoechst 33342 accumulation assay. Compound 4 on the other hand was able to reproduce the
results from the Hoechst 33342 accumulation assay yielding a similar GI50 value in the MTT
efficacy assays with cytostatics.
CONCLUSIONS
This study investigates the inhibitory effect toward ABCG2 of 4ꢀanilinoꢀ2ꢀphenylquinazolines
with different meta and para substitutions at the aniline moiety. It turned out that cyano, nitro
and hydroxy substituents at both positions lead to derivatives with high inhibitory potencies.
Moreover, it was found that some disubstitutions, such as 3ꢀnitro, 4ꢀhydroxy, positively
contribute to the potency of the quinazoline derivative in comparison to the corresponding
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mono substitutions. However, electronic effects of the substituents at the aniline moiety as
well as their steric requirements were found to play a minor role for the potency of a
compound. Concerning the substitutions with halogens, a distinct trend of the atomic number
and the position at the anilino residue was found. Compounds 2 (4ꢀcyano) and 4 (3ꢀnitro, 4ꢀ
hydroxy) exhibit a 3ꢀfold higher potency than Ko143, the most potent ABCG2 inhibitor
known in literature so far. The most potent derivatives conatin a cyano or nitro group that are
not preferred substituents of drugs. However, the compounds are valuable pharmacological
tools. Additionally, disubstitution has proven to be beneficial as can be seen from comparison
of compounds 4 and 5, and the three compounds 10 vs. 11 and 13. This will be a starting point
for future optimization, trying to replace the nitro or cyano groups by substituents with better
toxicological profile.
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Furthermore, some quinoline analogues were investigated. The results showed, that the
nitrogen atom at position 3 is crucial for activity toward ABCG2. Further differences between
the quinazoline and quinoline scaffold became evident in the context of selectivity toward
ABCG2. Among the quinazoline derivatives only compounds 8, 20 and 22 exhibit some
inhibitory activity toward ABCC1 and 11 and 24 toward ABCB1, respectively. For the
quinoline derivatives on the other hand, a substantially lower selectivity was observed. The
ability to reverse MDR against SNꢀ38 and Hoechst 33342 was confirmed for the most potent
compounds 1 and 4 in a MTT efficacy assay. Moreover, their inhibitory potency as
determined in the Hoechst 33342 accumulation assay agreed with the degree of sensitization
of the ABCG2 overexpressing cells in the MTT efficacy assay. Investigation of the intrinsic
cytotoxicity revealed a remarkably high GI50 value for compound 4, resulting in a
considerably high therapeutic ratio, which exceeds that of Ko143 about 13ꢀfold.
Based upon these findings, new highly potent, selective and nonꢀtoxic compounds, based
upon a quinazoline scaffold, have been discovered and may be utilized for specific
applications.
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EXPERIMENTAL SECTION
Chemistry.Materials. Chemicals were purchased from Acros Organics (Geel, Belgium), Alfa
Aesar (Karlsruhe, Germany) or Sigma Aldrich (Steinheim, Germany) or Merck (Darmstadt,
Germany) and used without further purification. Microwave reactions were performed in 50
ml vials with a CEM Discover SP (CEM GmbH, KampꢀLintfort, Germany). The reaction
progress was monitored by thin layer chromatography (TLC) using an aluminium plate coated
with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). As eluent a mixture of
dichloromethane and methanol (9:1) was used. TLCs were analyzed in a UV cabinet with an
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excitation wavelength of 254 nm. The identity of the compounds was confirmed by NMR. Hꢀ
1
3
and C spectra were either obtained on a Bruker Advance 500 MHz (500/126 MHz) or
Bruker Advance 600 MHz (600/151 MHz). The compounds were dissolved in DMSOꢀd6,
which was also used as internal standard and the chemical shifts (δ) are expressed in ppm. For
1
3
the assignment of the C signals distortionless enhancement by polarization transfer (DEPT)
and attached proton test (APT) were used. Multiplicity of the signals is indicated as singulet
(
s), doublet (d), doublet of doublets (dd), triplet of doublets (td), triplet (t), doublet of triplets
dt), quartet (q) and multiplet (m). Coupling constants J are given in Hz. The purity of all
(
biologically evaluated compounds was determined to be > 95% by elemental analysis
recorded on a Vario EL V24 CHN Elemental Analyzer (Elementar Analysensysteme GmbH,
Hanau, Germany). Found values were in the range of ±0.4% of the theoretical values, unless
indicated.
General procedure for the preparation of 4ꢀanilinoꢀ2ꢀphenylquinazoline and 4ꢀanilinoꢀ2ꢀ
phenylquinoline derivatives
4ꢀchloroꢀ2ꢀphenylquinazoline (1 mmol) and a substituted aniline (1 mmol) were added to a 50
mL microwave tube and suspended in 25 mL isopropanol. The tube was sealed and the
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reaction mixture stirred under 100 watt microwave irradiation at 110 °C for 30 min.
Completion of the reaction was monitored by TLC. After cooling, a precipitate was formed
and filtered off by suction. If no precipitate was formed, the solvent was removed by rotary
evaporation and the obtained solids recrystallized from 75%% EtOH.
10
11
12
13
14
15
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17
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19
20
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General Procedure for the preparation of 4ꢀanilinoꢀ2ꢀphenylquinoline derivatives
4ꢀchloroꢀ2ꢀphenylquinoline (1 mmol) and a substituted aniline (1 mmol) were added to a 50
mL microwave tube and suspended in 25 mL isopropanol. The tube was sealed and the
reaction mixture stirred under 100 watt microwave irradiation at 110 °C for 15 min. Reaction
control was performed by TLC. Workup of the compounds was analogous to 4ꢀanilinoꢀ
quinazoline compounds. For some quinoline derivatives it was necessary to recrystallize from
acetone.
4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)benzonitrile (1). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀcyanoaniline (118 mg, 1 mmol)
1
to yield 2 as pale yellow solid (235 mg, 73%), mp 299ꢀ302 °C (decomposition). H NMR
(
500 MHz, DMSOꢀd ) δ 11.57 (s, 1H), 8.95 (d, J = 8.3 Hz, 1H), 8.45 – 8.36 (m, 2H), 8.30 (d,
6
J = 8.4 Hz, 1H), 8.21 – 8.12 (m, 2H), 8.12 – 8.03 (m, 1H), 8.02 – 7.92 (m, 2H), 7.86 – 7.76
1
3
(m, 1H), 7.72 – 7.65 (m, 1H), 7.65 – 7.57 (m, 2H). C NMR (126 MHz, DMSO) δ 159.08,
157.80, 143.23, 142.14, 135.84, 133.07, 132.94, 129.21, 129.12, 128.06, 124.66, 124.12,
1
22.56, 118.98, 113.37, 107.45. Anal. Calcd for C H N : C, 78.24; H, 4.38; N, 17.38. Found:
21
14
4
C, 78.03; H, 4.61; N, 17.56.
4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)phenol (2). The title compound was synthesized from 4ꢀ
chloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀaminophenol (109 mg, 1 mmol) to yield
1
1
as bright yellow solid (255 mg, 81%), mp 335ꢀ336 °C (decomposition). H NMR (500 MHz,
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
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4
4
4
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DMSOꢀd ) δ 11.53 (s, 1H), 9.77 (s, 1H), 8.87 (d, J = 8.3 Hz, 1H), 8.42 – 8.28 (m, 3H), 8.10 –
6
8
2
1
.00 (m, 1H), 7.83 – 7.75 (m, 1H), 7.74 – 7.66 (m, 1H), 7.66 – 7.53 (m, 4H), 6.96 – 6.88 (m,
13
H). C NMR (126 MHz, DMSO) δ 158.74, 157.21, 156.19, 135.81, 133.41, 131.69, 129.30,
15 3
29.10, 128.13, 128.09, 126.17, 124.57, 120.45, 115.35, 112.73. Anal. Calcd for C20H N O:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
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8
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3
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C, 76.66; H, 4.83; N, 13.41; Found: C, 76.72; H, 4.97; N, 13.34.
4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)benzenesulfonyl fluoride (3). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, mmol) and 4ꢀ
1
aminobenzenesulfonyl fluoride (175 mg, 1 mmol) to yield 3 as white solid (245 mg, 76%),
1
mp 278ꢀ279 °C (decomposition). H NMR (500 MHz, DMSOꢀd
6
) δ 11.68 (s, 1H), 9.02 (t, J =
2.0 Hz, 1H), 8.95 (d, J = 8.3 Hz, 1H), 8.49 – 8.38 (m, 3H), 8.28 (d, J = 8.4 Hz, 1H), 8.14 –
8.04 (m, 1H), 8.05 – 7.98 (m, 1H), 7.92 (t, J = 8.1 Hz, 1H), 7.87 – 7.76 (m, 1H), 7.72 – 7.62
13
(
m, 1H), 7.63 – 7.51 (m, 2H). C NMR (126 MHz, DMSO) δ 159.00, 157.86, 139.62, 135.69,
132.80, 131.97, 131.78, 131.09, 130.78, 129.07, 128.98, 127.96, 124.59, 124.43, 123.10,
122.71, 113.34. Anal. Calcd for C20
H, 3.86; N, 10.99.
3 2
H14FN O S: C, 63.31; H, 3.72; N, 11.08. Found: C, 63.68;
2ꢀnitroꢀ4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)phenol (4) The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀaminoꢀ2ꢀnitrophenol (154 mg, 1
1
mmol) to yield 4 as yellow solid (229 mg, 60%), mp 283ꢀ284 °C (decomposition). H NMR
(500 MHz, DMSOꢀd
6
) δ 11.63 (s, 1H), 11.30 (s, 1H), 8.89 (d, J = 8.3 Hz, 1H), 8.62 (d, J = 2.6
Hz, 1H), 8.42 (dt, J = 8.2, 1.1 Hz, 2H), 8.31 (d, J = 8.4 Hz, 1H), 8.13 – 7.96 (m, 2H), 7.86 –
1
3
7
.76 (m, 1H), 7.76 – 7.66 (m, 1H), 7.67 – 7.56 (m, 2H), 7.34 (dd, J = 9.0, 0.9 Hz, 1H). C
NMR (126 MHz, DMSO) δ 158.89, 157.56, 150.36, 141.61, 136.02, 135.84, 133.23, 132.33,
31.32, 129.25, 129.08, 128.71, 128.11, 124.45, 121.52, 120.67, 119.37, 112.95. Anal. Calcd
1
for C20
H
14
N
4
O
3
: C, 67.03; H, 3.94; N, 15.63. Found: C, 66.85; H, 4.30; N, 15.52.
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Nꢀ(3ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)phenyl)acetamide (7). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg,
1
mmol) and Nꢀ(4ꢀ
aminophenyl)acetamide (150 mg, 1 mmol) to yield 7 as white solid (135 mg, 38%), mp 291ꢀ
1
2
93 °C. H NMR (500 MHz, DMSOꢀd
6
) δ 11.46 (s, 1H), 10.22 (s, 1H), 8.88 (d, J = 8.3 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
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1
H), 8.42 (dt, J = 8.3, 1.2 Hz, 2H), 8.32 (d, J = 8.3 Hz, 1H), 8.28 (t, J = 2.0 Hz, 1H), 8.13 –
8.02 (m, 1H), 7.87 – 7.76 (m, 1H), 7.75 – 7.65 (m, 1H), 7.61 (dd, J = 8.4, 7.0 Hz, 2H), 7.53
13
(dt, J = 7.5, 1.9 Hz, 1H), 7.48 – 7.36 (m, 2H), 2.09 (s, 3H). C NMR (126 MHz, DMSO) δ
1
68.64, 159.13, 157.46, 140.83, 139.88, 137.30, 135.85, 133.25, 131.89, 129.46, 129.01,
28.82, 128.10, 124.60, 121.01, 119.37, 117.08, 115.22, 112.87, 24.15. Anal. Calcd. for
1
22 18 4
C H N O: C, 74.56; H, 5.12; N, 15.81. Found: C, 74.31; H, 5.26; N, 15.48.
Nꢀ(3ꢀ(methylthio)phenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (8). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀ(methylthio)aniline
1
(139 mg, 1 mmol) to yield 8 as pale yellow solid (243 mg, 71%), mp 138ꢀ139 °C. H NMR
(600 MHz, DMSOꢀd
6
) δ 10.56 (s, 1H), 8.71 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 7.2 Hz, 2H), 8.04
d, J = 8.1 Hz, 1H), 7.96 (d, J = 11.5 Hz, 2H), 7.70 (s, 2H), 7.56 (q, J = 6.9, 5.3 Hz, 3H), 7.42
t, J = 7.9 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 2.54 (s, 3H). Anal. Calcd. for C H N S: C,
(
(
21
17
3
73.44; H, 4.99; N, 12.24. Found: C, 73.56; H, 4.84; N, 11.91.
Nꢀ(3,4ꢀdimethoxyphenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (11) The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3,4ꢀdimethoxyaniline
1
(153 mg, 1 mmol) to yield 11 as bright yellow solid (171 mg, 48%), mp 135ꢀ137 °C. H NMR
6
(500 MHz, DMSOꢀd ) δ 11.46 (s, 1H), 8.90 (d, J = 8.3 Hz, 1H), 8.48 – 8.38 (m, 2H), 8.34 (d,
J = 8.4 Hz, 1H), 8.16 – 7.94 (m, 1H), 7.80 (s, 1H), 7.75 – 7.67 (m, 1H), 7.67 – 7.54 (m, 3H),
1
3
7
.39 (dd, J = 8.7, 2.5 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 3.81 (d, J = 10.7 Hz, 6H). C NMR
(126 MHz, DMSO) δ 158.67, 158.14, 148.49, 146.02, 134.00, 132.05, 131.27, 128.66, 128.33,
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126.59, 123.46, 114.91, 113.75, 111.89, 108.00, 55.92, 55.67. Anal. Calcd. for C H N O : C,
22
19
3
2
73.93; H, 5.36; N, 11.76. Found: C, 73.63; H, 5.52; N, 11.77.
Nꢀ(3ꢀfluoroꢀ4ꢀmethoxyphenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (12). The title compound was
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synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀfluoroꢀ4ꢀ
methoxyaniline (141 mg, 1 mmol) to yield 12 as light yellow solid (192 mg, 56%), mp 262ꢀ
1
2
1
1
63 °C (decomposition). H NMR (500 MHz, DMSOꢀd ) δ 11.65 (s, 1H), 8.95 (d, J = 8.3 Hz,
6
H), 8.43 – 8.29 (m, 3H), 8.11 – 8.02 (m, 1H), 7.83 (dd, J = 13.1, 2.5 Hz, 1H), 7.82 – 7.77 (m,
13
H), 7.73 – 7.67 (m, 1H), 7.67 – 7.60 (m, 3H), 7.31 (t, J = 9.3 Hz, 1H), 3.90 (s, 3H). C
NMR (126 MHz, DMSO) δ 158.93, 157.59, 151.75, 149.81, 145.45 (d, J = 10.4 Hz), 141.44,
1
1
7
35.86, 133.23, 132.40, 130.17 (d, J = 9.5 Hz), 129.15, 128.13, 124.52, 121.38, 120.65,
13.77 – 113.76 (d), 112.91, 112.75 (d, J = 21.9 Hz), 56.41. Anal. Calcd. for C H FN O: C,
21
16
3
3.03; H, 4.67; N, 12.17. Found: C, 72.70; H, 4.84; N, 12.38.
Nꢀ(4ꢀbromoꢀ3ꢀmethoxyphenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (13). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀbromoꢀ3ꢀ
methoxyaniline (202 mg, 1 mmol) to yield 13 as yellow solid (280 mg, 69%), mp 276ꢀ278 °C
1
(
decomposition). H NMR (500 MHz, DMSOꢀd
6
) δ 11.20 (s, 1H), 8.85 (d, J = 8.3 Hz, 1H),
8.52 – 8.31 (m, 2H), 8.22 (d, J = 8.4 Hz, 1H), 8.06 (t, J = 7.8 Hz, 1H), 7.89 – 7.75 (m, 2H),
1
3
7
.74 – 7.54 (m, 4H), 3.90 (s, 3H). C NMR (151 MHz, DMSO) δ 158.90, 157.69, 155.42,
38.56, 135.72, 132.78, 129.16, 129.09, 128.06, 124.47, 117.43, 114.66, 113.16, 108.65,
6.44. Anal. Calcd. for C H BrN O: C, 62.08; H, 3.97; N, 10.34. Found: C, 61.81; H, 4.23;
1
5
21
16
3
N, 10.29.
Nꢀ(4ꢀiodoꢀ3ꢀmethylphenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (14). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀiodoꢀ3ꢀmethylaniline
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(233 mg, 1 mmol) to yield 14 as beige solid (224 mg, 51%), mp 274ꢀ276 °C (decomposition).
1
H NMR (500 MHz, DMSOꢀd ) δ 11.48 (s, 1H), 8.91 (d, J = 8.4 Hz, 1H), 8.43 – 8.36 (m, 2H),
6
8.33 (d, J = 8.5 Hz, 1H), 8.12 – 8.01 (m, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 2.5 Hz,
1H), 7.83 – 7.75 (m, 1H), 7.74 – 7.66 (m, 1H), 7.66 – 7.58 (m, 2H), 7.52 (dd, J = 8.5, 2.6 Hz,
1H), 2.44 (s, 3H). 13C NMR (126 MHz, DMSO) δ 158.85, 157.54, 141.24, 138.74, 137.69,
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135.78, 133.12, 129.19, 129.11, 128.06, 125.38, 124.49, 123.57, 121.67, 113.07, 97.31, 27.76.
Anal. Calcd. for C H IN : C, 57.68; H, 3.69; N, 9.61. Found: C, 57.96; H, 3.82; N, 9.51.
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1
16
3
Nꢀ(3ꢀfluorophenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (15) The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀfluoroaniline (111 mg, 1 mmol)
1
to yield 15 as pale yellow solid (269 mg, 85%), mp 298ꢀ299 °C (decomposition). H NMR
(
500 MHz, DMSOꢀd ) δ 11.59 (s, 1H), 8.96 (d, J = 8.6 Hz, 1H), 8.39 (dq, J = 7.1, 1.5 Hz, 2H),
6
8
.35 (d, J = 8.9 Hz, 1H), 8.12 – 8.01 (m, 1H), 7.90 – 7.77 (m, 2H), 7.77 – 7.72 (m, 1H), 7.72
7.66 (m, 1H), 7.63 (t, J = 7.5 Hz, 2H), 7.56 (td, J = 8.2, 6.7 Hz, 1H), 7.16 (td, J = 8.5, 2.6
–
13
Hz, 1H). C NMR (126 MHz, DMSO) δ 162.92, 160.99, 159.15, 157.59, 139.08 (d, J = 6.3
Hz), 135.87, 133.14, 130.38 (d, J = 9.2 Hz), 129.19, 129.08, 128.10, 124.65, 120.17, 113.03,
112.74 (d, J = 16.3 Hz), 111.29 (d, J = 24.3 Hz).Anal. Calcd. for C H FN : C, 76.18; H,
20 14 3
4.48; N, 13.33. Found: C, 76.54; H, 4.60; N, 13.08.
Nꢀ(4ꢀfluorophenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (16) The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀfluoroaniline (111 mg, 1 mmol)
1
to yield 16 as beige solid (213 mg, 68%), mp 271ꢀ272 °C (decomposition). H NMR (500
MHz, DMSOꢀd
6
) δ 11.52 (s, 1H), 8.89 (d, J = 8.3 Hz, 1H), 8.43 – 8.34 (m, 2H), 8.30 (d, J =
8.7 Hz, 1H), 8.13 – 7.99 (m, 1H), 7.97 – 7.84 (m, 2H), 7.85 – 7.75 (m, 1H), 7.74 – 7.65 (m,
13
1
H), 7.64 – 7.54 (m, 2H), 7.44 – 7.32 (m, 2H). C NMR (126 MHz, DMSO) δ 161.01 –
159.08 (J = 233 Hz) , 159.15, 157.51, 141.64, 135.80, 133.53, 133.16, 132.26, 129.24 –
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129.06 (J = 21,7 Hz), 128.04, 126.68 – 126.62 (J = 7,9 Hz), 124.62, 121.35, 115.66 – 115.48
(
22,6 Hz), 112.90. Anal. Calcd. for C H FN : C, 76.18; H, 4.48; N, 13.33. Found: C, 76.32;
20 14 3
H, 4.87; N, 13.12.
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Nꢀ(4ꢀchlorophenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (17) The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀchloroaniline (128 mg, 1 mmol)
1
to yield 17 as pale yellow solid (227 mg, 69%), mp 288ꢀ289 °C (decomposition). H NMR
(500 MHz, DMSOꢀd
6
) δ 11.54 (s, 1H), 8.91 (d, J = 8.4 Hz, 1H), 8.45 – 8.34 (m, 2H), 8.31 (d,
J = 8.5 Hz, 1H), 8.15 – 8.01 (m, 1H), 7.99 – 7.86 (m, 2H), 7.87 – 7.75 (m, 1H), 7.77 – 7.66
13
(m, 1H), 7.66 – 7.47 (m, 4H). C NMR (126 MHz, DMSO) δ 159.08, 157.93, 150.63, 138.44,
138.32, 133.47, 130.45, 128.59, 128.52, 128.29, 128.04, 127.32, 126.17, 123.84, 123.14,
1
14.10. Anal. Calcd. for C H ClN : C, 72.40; H, 4.25; N, 12.66. Found: C, 72.17; H, 4.39;
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14
3
N, 12.58.
Nꢀ(3ꢀiodophenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (18). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀiodoaniline (219 mg, 1 mmol) to
1
yield 18 as pale yellow solid (145 mg, 34%), mp 226ꢀ227 °C. H NMR (500 MHz, DMSOꢀd )
6
δ 11.51 (s, 1H), 8.91 (d, J = 8.3 Hz, 1H), 8.49 – 8.38 (m, 3H), 8.32 (d, J = 8.4 Hz, 1H), 8.12 –
8.02 (m, 1H), 7.93 – 7.84 (m, 1H), 7.84 – 7.76 (m, 1H), 7.73 – 7.65 (m, 2H), 7.65 – 7.57 (m,
1
3
2
1
1
3
H), 7.32 (t, J = 8.0 Hz, 1H). C NMR (126 MHz, DMSO) δ 158.96, 157.42, 142.00, 138.74,
35.81, 134.41, 133.16, 132.70, 130.71, 129.20, 129.03, 128.06, 124.55, 123.36, 121.70,
13.02, 94.03. Anal. Calcd. for C H IN : C, 56.76; H, 3.33; N, 9.93. Found: C, 56.38; H,
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3
.68; N, 10.04.
Nꢀ(4ꢀiodophenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (19). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀiodoaniline (219 mg, 1 mmol) to
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yield 19 as pale yellow solid (166 mg, 39%), mp 271ꢀ272 °C (decomposition). H NMR (500
MHz, DMSOꢀd ) δ 11.50 (s, 1H), 8.91 (d, J = 8.4 Hz, 1H), 8.47 – 8.35 (m, 2H), 8.31 (d, J =
6
8
3
1
.4 Hz, 1H), 8.11 – 8.00 (m, 1H), 7.94 – 7.83 (m, 2H), 7.83 – 7.75 (m, 1H), 7.76 – 7.66 (m,
13
H), 7.66 – 7.56 (m, 2H). C NMR (126 MHz, DMSO) δ 159.17, 158.03, 142.34, 137.83,
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37.43, 136.14, 133.33, 132.90, 129.43, 129.29, 128.41, 126.57, 124.50, 122.06, 113.26,
91.09. Anal. Calcd. for C H IN : C, 56.76; H, 3.33; N, 9.93. Found: C, 56.65; H, 3.62; N,
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14
3
9.91.
methyl 4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)benzoate (20). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and methyl 3ꢀ
aminobenzoate (193 mg, 1 mmol) to yield 20 as white solid (195 mg, 55%), mp 273ꢀ275 °C
1
(
decomposition). H NMR (500 MHz, DMSOꢀd ) δ 10.11 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H),
6
8
.47 (d, J = 7.3 Hz, 2H), 8.20 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 4.1 Hz,
13
2
H), 7.65 (t, J = 5.8 Hz, 1H), 7.54 (q, J = 8.9, 7.9 Hz, 3H), 3.86 (s, 3H). C NMR (126 MHz,
DMSO) δ 166.06, 159.04, 157.86, 150.78, 144.26, 138.21, 133.63, 130.53, 130.08, 128.66,
28.35, 128.08, 126.34, 123.95, 123.27, 121.12, 114.29, 52.01, 40.20. Anal. Calcd. for
C H N O : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.29; H, 5.13 N, 11.43.
1
22
17
3
2
tertꢀbutyl 4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)benzoate (21). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and tertꢀbutyl 3ꢀ
aminobenzoate (193 mg, 1 mmol) to yield 21 as pale yellow solid (302 mg,76%%), mp 328ꢀ
1
3
30 °C (decomposition). H NMR (600 MHz, DMSOꢀd ) δ 11.44 (s, 1H), 8.91 (t, J = 7.8 Hz,
6
1
H), 8.47 – 8.35 (m, 2H), 8.28 (d, J = 7.9 Hz, 1H), 8.12 – 7.98 (m, 5H), 7.81 (dd, J = 9.2, 6.2
1
3
Hz, 1H), 7.68 (t, J = 7.2 Hz, 1H), 7.64 (dd, J = 8.2, 6.5 Hz, 2H), 1.57 (s, 9H). C NMR (126
MHz, DMSO) δ 166.91, 164.61, 158.99, 157.78, 141.68, 135.74, 132.92, 130.08, 129.81,
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129.18, 129.10, 128.13, 127.99, 124.52, 123.43, 122.20, 113.25, 80.86, 27.98. Anal. Calcd.
for C H N O : C, 75.55; H, 5.83; N, 10.57. Found: C, 75.43; H, 5.89 N, 10.87.
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4ꢀ((2ꢀphenylquinazolinꢀ4ꢀyl)amino)benzoic acid (22). The title compound was synthesized
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from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀaminobenzoic acid (137 mg, 1
1
mmol) to yield 22 as light yellow solid (254 mg, 74%), mp 336ꢀ 337 °C (decomposition). H
NMR (600 MHz, DMSOꢀd ) δ 11.98 (s, 1H), 11.79 (s, 1H), 9.03 (d, J = 8.3 Hz, 1H), 8.43 (d,
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J = 7.6 Hz, 3H), 8.25 – 7.93 (m, 5H), 7.81 (t, J = 7.7 Hz, 1H), 7.66 (dt, J = 36.7, 7.5 Hz, 3H).
1
3
C NMR (151 MHz, DMSO) δ 166.92, 159.17, 157.47, 141.35, 136.03, 133.33, 130.05,
129.45, 129.14, 128.22, 128.04, 124.89, 123.95, 113.11. Anal. Calcd. for C21 : C,
3.89; H, 4.43; N, 12.31. Found: C, 74.26; H, 4.64 N, 12.10.
15 3 2
H N O
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Nꢀ(3ꢀethynylphenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (23). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀethynylaniline (117
1
mg, 1 mmol) to yield 23 as pale yellow solid (213 mg, 66%), mp 222ꢀ224 °C. H NMR (500
MHz, DMSOꢀd ) δ 11.77 (s, 1H), 9.01 (d, J = 8.3 Hz, 1H), 8.49 – 8.31 (m, 3H), 8.14 – 8.00
6
(m, 2H), 7.98 – 7.87 (m, 1H), 7.86 – 7.75 (m, 1H), 7.74 – 7.65 (m, 1H), 7.61 (t, J = 7.6 Hz,
13
2
H), 7.54 (t, J = 7.9 Hz, 1H), 7.43 (dt, J = 7.6, 1.2 Hz, 1H), 4.27 (s, 1H). C NMR (126 MHz,
DMSO) δ 157.23, 141.16, 137.44, 135.91, 133.31, 131.87, 129.33, 129.27, 129.18, 128.99,
28.12, 127.57, 125.09, 124.78, 122.07, 121.00, 112.88, 82.95, 81.23. Anal. Calcd. for
C H N : C, 82.22; H, 4.70; N, 13.08. Found: C, 82.14; H, 5.05 N, 12.83.
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2ꢀphenylꢀNꢀ(mꢀtolyl)quinazolinꢀ4ꢀamine (24). The title compound was synthesized from 4ꢀ
chloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀmethylaniline (107 mg, 1 mmol) to yield
1
2
4 as pale yellow solid (247 mg, 79%), mp >350 °C. H NMR (500 MHz, DMSOꢀd
6
) δ 11.58
(s, 1H), 8.95 (d, J = 8.3 Hz, 1H), 8.51 – 8.28 (m, 3H), 8.15 – 7.97 (m, 1H), 7.87 – 7.76 (m,
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1H), 7.75 – 7.55 (m, 5H), 7.41 (t, J = 7.8 Hz, 1H), 7.22 – 7.10 (m, 1H), 2.40 (s, 3H).
C
NMR (126 MHz, DMSO) δ 159.01, 157.35, 141.16, 138.12, 137.07, 135.82, 133.26, 132.18,
29.25, 129.07, 128.63, 128.09, 127.05, 124.96, 124.59, 121.62, 121.19, 112.91, 21.20. Anal.
Calcd. for C21 : C, 81.00; H, 5.50; N, 13.49. Found: C, 80.84; H, 5.79; N, 13.55.
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17 3
H N
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2ꢀphenylꢀNꢀ(pꢀtolyl)quinazolinꢀ4ꢀamine. (25) The title compound was synthesized from 4ꢀ
chloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀmethylaniline (107 mg, 1 mmol) to yield
1
2
5 as pale yellow solid (256 mg, 82%), mp 254ꢀ256 °C (decomposition). H NMR (500 MHz,
DMSOꢀd
6
) δ 11.56 (s, 1H), 8.92 (d, J = 8.3 Hz, 1H), 8.45 – 8.28 (m, 3H), 8.14 – 8.00 (m, 1H),
7.87 – 7.76 (m, 1H), 7.76 – 7.66 (m, 3H), 7.65 – 7.55 (m, 2H), 7.39 – 7.26 (m, 2H), 2.37 (s,
1
3
3
H). C NMR (126 MHz, DMSO) δ 158.98, 157.39, 140.84, 135.90, 134.49, 133.36, 131.93,
129.31, 129.29, 129.12, 128.17, 124.65, 124.46, 120.85, 112.87, 20.86. Anal. Calcd. for
21 17 3
C H N : C, 81.00; H, 5.50; N, 13.49. Found: C, 81.37; H, 5.58; N, 13.23.
Nꢀ(3ꢀ(tertꢀbutyl)phenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (26). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 3ꢀ(tertꢀbutyl)aniline
1
(
149 mg, 1 mmol) to yield 26 as pale yellow solid (263 mg, 74%), mp 223ꢀ 224 °C. H NMR
6
(500 MHz, DMSOꢀd ) δ 11.63 (s, 1H), 8.98 (d, J = 8.3 Hz, 1H), 8.52 – 8.33 (m, 3H), 8.13 –
8.02 (m, 1H), 7.94 (t, J = 2.0 Hz, 1H), 7.81 (td, J = 7.7, 7.2, 1.2 Hz, 1H), 7.69 (td, J = 7.3, 1.2
Hz, 1H), 7.66 – 7.62 (m, 1H), 7.62 – 7.55 (m, 2H), 7.45 (t, J = 7.9 Hz, 1H), 7.40 – 7.35 (m,
13
1
1
1
H), 1.34 (d, J = 0.9 Hz, 9H). C NMR (126 MHz, DMSO) δ 158.99, 157.24, 151.37, 140.98,
36.81, 135.83, 133.34, 131.86, 129.31, 128.91, 128.45, 128.08, 124.65, 123.37, 121.69,
23 3
21.62, 120.89, 112.89, 34.72, 31.22. Anal. Calcd. for C24H N : C, 81.55; H, 6.56; N, 11.89.
Found: C, 81.47; H, 6.89; N, 11.70.
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Nꢀ(4ꢀ(tertꢀbutyl)phenyl)ꢀ2ꢀphenylquinazolinꢀ4ꢀamine (27). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinazoline (241 mg, 1 mmol) and 4ꢀ(tertꢀbutyl)aniline
(149 mg, 1 mmol) to yield 27 as pale yellow solid (233 mg, 66%), mp 287ꢀ 289 °C
1
(
decomposition). H NMR (500 MHz, DMSOꢀd
6
) δ 11.66 (s, 1H), 9.02 (d, J = 8.3 Hz, 1H),
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0
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2
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4
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8
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8.53 – 8.29 (m, 3H), 8.11 – 7.99 (m, 1H), 7.89 – 7.73 (m, 3H), 7.70 (td, J = 7.1, 6.7, 1.4 Hz,
13
1H), 7.62 (td, J = 7.2, 6.6, 1.3 Hz, 2H), 7.57 – 7.45 (m, 2H), 1.33 (d, J = 1.1 Hz, 9H). C
NMR (126 MHz, DMSO) δ 158.82, 157.46, 148.93, 140.82, 135.83, 134.56, 133.25, 131.92,
129.32, 129.07, 128.08, 125.45, 124.70, 123.87, 120.84, 112.86, 34.48, 31.27. Anal. Calcd.
23 3
for C24H N
: C, 81.55; H, 6.56; N, 11.89. Found: C, 81.68; H, 6.62; N, 11.89.
Nꢀ(3ꢀmethoxyphenyl)ꢀ2ꢀphenylquinolinꢀ4ꢀamine (28). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinoline (240 mg, 1 mmol) and 3ꢀmethoxyaniline (123 mg, 1 mmol)
1
to yield 28 as yellow solid (76 mg, 23%), mp 279 °C (decomposition). H NMR (500 MHz,
DMSOꢀd
6
) δ 14.41 (s, 1H), 11.17 (s, 1H), 8.93 (dd, J = 8.6, 1.2 Hz, 1H), 8.44 (dd, J = 8.6, 1.2
Hz, 1H), 8.07 – 7.97 (m, 1H), 7.95 – 7.83 (m, 2H), 7.82 – 7.71 (m, 1H), 7.69 – 7.52 (m, 3H),
1
3
7
.51 – 7.38 (m, 1H), 7.22 – 7.07 (m, 2H), 7.05 – 6.90 (m, 2H), 3.80 (d, J = 0.9 Hz, 3H). C
NMR (126 MHz, DMSO) δ 160.51, 154.74, 152.96, 139.40, 138.61, 134.02, 132.37, 131.94,
30.84, 129.35, 128.63, 127.00, 123.83, 120.90, 117.30, 116.74, 113.15, 111.14, 99.23, 55.58.
Anal. Calcd. for C22 O: C, 80.71; H, 5.85; N, 8.56. Found: C, 80.74; H, 5.81; N, 8.86.
1
19 2
H N
Nꢀ(3ꢀ(methylthio)phenyl)ꢀ2ꢀphenylquinolinꢀ4ꢀamine (29).
The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinoline (240 mg, 1 mmol) and 3ꢀ(methylthio)aniline
1
(
139 mg, 1 mmol) to yield 29 as pale yellow solid (243 mg, 71%), mp 238ꢀ240 °C. H NMR
(600 MHz, DMSOꢀd
6
) δ 14.42 (s, 1H), 11.22 (s, 1H), 8.93 (dd, J = 8.5, 3.1 Hz, 1H), 8.43 (dd,
J = 8.6, 3.1 Hz, 1H), 8.03 (td, J = 7.9, 3.1 Hz, 1H), 7.90 (dd, J = 7.8, 3.0 Hz, 2H), 7.78 (td, J =
7.9, 3.1 Hz, 1H), 7.72 – 7.54 (m, 3H), 7.54 – 7.42 (m, 2H), 7.42 – 7.32 (m, 1H), 7.27 (t, J =
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5.4 Hz, 1H), 6.98 (d, J = 3.2 Hz, 1H). C NMR (151 MHz, DMSO) δ 154.68, 152.99, 140.47,
1
39.37, 138.15, 134.10, 132.35, 132.01, 130.36, 129.38, 128.68, 127.08, 124.58, 123.89,
22.29, 121.45, 120.89, 116.80, 99.27, 14.71. Anal. Calcd. for C22 S: C, 76.93; H, 5.58;
1
19 2
H N
N, 8.16. Found: C, 77.31; H, 5.48; N, 7.99.
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Nꢀ(3ꢀ((2ꢀphenylquinolinꢀ4ꢀyl)amino)phenyl)acetamide (30). The title compound was
synthesized from 4ꢀchloroꢀ2ꢀphenylquinoline (240 mg, mmol) and Nꢀ(3ꢀ
1
aminophenyl)acetamide (150 mg, 1 mmol) to yield 30 as pale yellow solid (136 mg, 38%),
1
mp 281 °C (decomposition). H NMR (500 MHz, DMSOꢀd
6
) δ 14.25 (s, 1H), 11.05 (s, 1H),
10.38 (s, 1H), 8.85 (dd, J = 8.6, 1.3 Hz, 1H), 8.35 (dd, J = 8.6, 1.1 Hz, 1H), 8.08 – 8.01 (m,
1H), 7.98 (d, J = 2.1 Hz, 1H), 7.96 – 7.91 (m, 2H), 7.82 – 7.76 (m, 1H), 7.68 – 7.58 (m, 3H),
13
7
.52 – 7.43 (m, 2H), 7.23 (dt, J = 7.2, 1.9 Hz, 1H), 7.05 (s, 1H), 2.08 (s, 3H). C NMR (126
MHz, DMSO) δ 168.89, 154.61, 153.04, 140.80, 139.35, 137.52, 134.10, 132.39, 132.00,
30.23, 129.38, 128.67, 127.09, 123.66, 120.89, 119.61, 117.85, 116.73, 115.58, 99.01, 24.17.
Anal. Calcd. for C23 O: C, 77.94; H, 5.69; N, 11.86. Found: C, 78.20; H, 5.65; N, 11.97.
1
20 3
H N
4ꢀ((2ꢀphenylquinolinꢀ4ꢀyl)amino)benzonitrile (31). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinoline (240 mg, 1 mmol) and 4ꢀaminobenzonitrile (118 mg, 1
1
mmol) to yield 31 as pale yellow solid (170 mg, 53%), mp 290 °C (decomposition). H NMR
(500 MHz, DMSOꢀd
6
) δ 14.53 (s, 1H), 11.21 (s, 1H), 8.87 (d, J = 8.6 Hz, 1H), 8.38 (d, J = 8.5
Hz, 1H), 8.06 (t, J = 7.7 Hz, 1H), 8.02 – 7.93 (m, 4H), 7.82 (t, J = 9.0 Hz, 3H), 7.74 – 7.58 (m,
13
3
H), 7.33 (s, 1H). C NMR (126 MHz, DMSO) δ 156.48, 148.43, 142.42, 137.61, 131.24,
13
1
30.26, 129.83, 129.02, 128.05, 127.50, 124.69, 123.66, 118.99. C NMR (126 MHz, DMSO)
δ 153.65, 142.62, 134.14, 132.05, 129.33, 128.85, 127.34, 124.58, 123.89, 118.78, 117.58,
1
00.74. Anal. Calcd. for C22
2.82.
16 3
H N : C, 81.96; H, 5.00; N, 13.03. Found: C, 81.73; H, 5.39; N,
1
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Nꢀ(3ꢀfluorophenyl)ꢀ2ꢀphenylquinolinꢀ4ꢀamine (32). The title compound was synthesized
from 4ꢀchloroꢀ2ꢀphenylquinoline (240 mg, 1mmol) and 3ꢀfluoroaniline (111 mg, 1 mmol) to
1
yield 32 as yellow solid (188 mg, 60%), mp 283 °C (decomposition). H NMR (500 MHz,
0
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9
0
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9
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DMSOꢀd
6
) δ 14.44 (s, 1H), 11.22 (s, 1H), 8.92 (dd, J = 8.5, 1.4 Hz, 1H), 8.41 (dd, J = 8.5, 1.1
Hz, 1H), 8.13 – 8.00 (m, 1H), 8.00 – 7.89 (m, 2H), 7.85 – 7.75 (m, 1H), 7.72 – 7.55 (m, 4H),
1
3
7
.56 – 7.43 (m, 2H), 7.31 – 7.19 (m, 1H), 7.08 (s, 1H). C NMR (126 MHz, DMSO) δ 163.72,
161.78, 154.50, 153.24, 139.34 (d, J = 10.4 Hz), 134.15, 132.32, 132.02, 131.68 (d, J = 9.3
Hz), 129.35, 128.75, 127.18, 123.85, 121.25, 120.96, 116.89, 114.10 (d, J = 20.7 Hz), 112.48
(d, J = 23.7 Hz), 99.52. Anal. Calcd. for C22 O: C, 79.98; H, 5.11; N, 8.88. Found: C,
0.24; H, 4.81; N, 8.91.
16 2
H N
8
Biological investigations. Materials for biological investigation. Ko143 ((3S,6S,12aS)ꢀ
,2,3,4,6,7,12,12aꢀOctahydroꢀ9ꢀmethoxyꢀ6ꢀ(2ꢀmethylpropyl)ꢀ1,4ꢀ
1
dioxopyrazino[1′,2′:1,6]pyrido[3,4ꢀb]indoleꢀ3ꢀpropanoicacid1,1ꢀdimethylethyl ester) was
purchased from Tocris Bioscience (Bristol, United Kingdom). Compound XR9577 (Nꢀ[2ꢀ[[4ꢀ
[
2ꢀ(3,4ꢀdihydroꢀ1Hꢀisoquinolinꢀ2ꢀyl)ethyl]phenyl]carbamoyl]phenyl]quinolineꢀ3ꢀ
2
8 , 29
carboxamide) was synthesized as described in literature.
purchased from SigmaꢀAldrich (Taufkirchen, Germany).
All other chemicals were
For the cell based assays 10 mM stock solutions were prepared by dissolving the test
compound in DMSO. KrebsꢀHEPES buffer (KHB) was prepared from a solution of 118.6 mM
NaCl, 4.7 mM KCl, 1.2 mM MgSO , 2.5 mM CaCl , 11.7 mM Dꢀglucose monohydrate, and
4
2
10.0 mM HEPES in doubly distilled water. The solution was adjusted with 4 N NaOH (aq) at
37 °C to pH 7.0 and sterilized, using a 0.2 µM membrane filter.
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Cell culture. MDCK II BCRP cells were generated by transfection of the canine kidney
epithelial cell line MDCK II with human wildꢀtype cDNA Cꢀterminally linked to the cDNA
of the green fluorescent protein (GFP) and were a kind gift of Dr. A. Schinkel (The
Netherlands Cancer Institute, Amsterdam, The Netherlands). Cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) with 10%% fetal calf serum (FCS), 50
µg/mL streptomycin, 50 U/mL penicillin G and 2 mM Lꢀglutamine. Human ovarian
carcinoma cell line A2780adr, which overexpresses ABCB1 and shows resistance against
doxorubicin, was purchased from European Collection of Animal Cell Culture (ECACC, No
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93112520). Culture of these cells was carried out with RPMIꢀ1640 medium supplemented
with 20% FCS, 50 µg/mL streptomycin, 50 U/mL penicillin G and 2 mM Lꢀglutamine.
Overexpression of ABCB1 was ensured by treating the cells with doxorubicin for one passage
every 10 passages for less than 40 passages. The ABCC1 overexpressing small cell lung
cancer cell line H69AR was purchased from American Type Culture Collection (ATCC,
CRLꢀ11351). This cell line was grown in RPMI 1640 medium with 20% FCS, 50 mg/mL
streptomycin, 50 U/mL penicillin G and 2 mM Lꢀglutamine. All cells were kept in a 5% CO
2
humidified atmosphere at 37 °C. Subculturing of cells was performed after reaching a
confluence of 80ꢀ90% with 0.05% Trypsin and 0.02% EDTA. To remove excess medium the
cells were washed with PBS before trypsination. After detachment of the cells, they were
transferred to a 50 mL falcon and centrifuged (266 x g, 4 °C, 4 min). The supernatant was
removed and the obtained cell pellet resuspended in fresh medium. Determination of the cell
count for all cells was carried out with a CASY1 model TT cell counter equipped with a 150
µm capillary (Schaerfe System GmbH, Reutlingen, Germany). Cells were washed with KHB
three times before using them in cell based assays.
Hoechst 33342 accumulation assay. To investigate the inhibitory effect on ABCG2, the
Hoechst 33342 accumulation assay was performed as described earlier with small
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3,24,25,30,31,32,33,34,35,36,37,38,39,40,41,42
modifications.
Stockꢀsolutions of the compounds were
prepared in DMSO at a final concentration of 10 mM. Sterile filtered KHB, a small amount of
methanol and the stock solution were employed to prepare different dilutions of the
corresponding compound. The quantity of methanol and DMSO in the final concentration was
chosen not to exceed 5% and 0.1%, respectively.
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Cells were harvested after a confluence of 80ꢀ90% by gentle trypsination (0.05%
trypsin/0.02% EDTA) and centrifuged (266 x g, 4 °C, 4 min) to yield a cell pellet. After
resuspension of the pellet in fresh medium, the cell density was determined with a CASY1
model TT cell counter device. Residual medium was removed by threefold centrifugation of
the cell suspension in KHB.
For the assay, 90 µL of the washed cell suspension was added to 10 µL of different compound
dilutions to obtain a cell density of approximately 30,000 cells in each well (96 well plate,
Greiner, Frickenhausen, Germany). After a preincubation period of 30 min at 37 °C and 5%
2
CO , 20 µL of a 6 µM Hoechst 33342 solution (protected from light) was quickly added to
each well. Fluorescence was measured immediately in constant time intervals (60 s) up to 120
min with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a
BMG POLARstar microplate reader (BMG Labtech, Offenburg, Germany). For the analysis
of the data, first fluorescence of the KHB was subtracted from the fluorescence reading
obtained from the MDCK II cells. Average of the fluorescence measured between 100 and
1
09 min (steady state) were calculated for each concentration and plotted against the
logarithm of the compound concentration. Doseꢀresponse curves were fitted by nonlinear
regression using the fourꢀparameter logistic equation with variable Hill slope (GraphPad
Prism, version 5.0, San Diego, CA, USA).
Pheophorbide A assay. Selected compounds were also tested in the pheophorbide A assay
23,25,31,33,37,38,39,41
as described earlier using parental MDCK II and MDCK II BCRP cells.
The
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cells were prepared analogously to the Hoechst 33342 accumulation assay, using a Uꢀshaped
clear 96 well plate (Greiner). In each well 160 µL of a cell suspension, containing
approximately 45,000 cells, was added to 20 µL of the corresponding compound dilution and
2
preincubated at 5% CO and 37 °C for 30 min. Then 20 µL of a 5 µM pheophorbide A
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solution (protected from light) was added to each well, followed by a 120 min period of
incubation to reach steady state. After incubation the cells were resuspended in the wells and
the fluorescence measured by flow cytometry (FACS Calibur, Becton Dickinson Biosciences,
Heidelberg, Germany). Pheophorbide A was excited at a wavelength of 488 nm and emission
was detected in the FL3 channel (≥ 670 nm). Concentration response curves were obtained by
nonlinear regression using the fourꢀparameter logistic equation with variable Hillꢀslope.
Calcein AM assay. Selectivity of the synthesized compounds toward ABCG2 was
determined by investigating the inhibitory activity against ABCB1 and ABCC1 in the calcein
23,24,25,31,32,33,34,35,36,38,39,40,42,43,44,45
AM assay described earlier.
For this purpose the ABCB1
overexpressing cell line A2780adr and the ABCC1 overexpressing cell line H69AR were used.
At a confluence of 80ꢀ90% the cells were harvested and prepared as described above for the
MDCK II cells. After washing the cells three times with KHB, 90 µL suspension, containing
approximately 30,000 cells, of the corresponding cell line was seeded in each well of a
colourless flat bottom 96 well plate (Greiner), containing 10 µL of different compound
2
dilutions. The prepared plates were kept at 5% CO and 37 °C for 30 min. After preincubation,
33 µL of a 1.25 µM calcein AM solution (protected from light) was quickly added to each
well and measured immediately in a 37 °C tempered BMG POLARstar microplate reader.
Over a period of 60 min fluorescence was measured in constant time intervals (60 s) using an
excitation wavelength of 485 nm and an emission wavelength of 520 nm. The initial linear
part of the obtained fluorescence time curve was used to calculate the slope for each
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