Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-Spectrum ABCB1, ABCC1, and ABCG2 Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.0c00961

In the search for highly effective modulators addressing ABCG2-mediated MDR, 23 pyrimidines were synthesized and biologically assessed. Seven derivatives with (a) nitrogen- and/or halogen-containing residue(s) had extraordinary potencies against ABCG2 (IC50 < 150 nM). The compounds competitively inhibited ABCG2-mediated Hoechst 33342 transport but were not substrates of ABCG2. The most potent MDR reverser, compound 19, concentration-dependently increased SN-38-mediated cancer cell death at 11 nM (EC50), time-dependently doubled SN-38 toxicity in a period of 7 days at 10 nM, and half-maximally accelerated cell death combined with SN-38 at 17 nM. No induction of ABCG2 was observed. Furthermore, 11 pyrimidines were revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC50 values below 10 μM against each transporter, classifying them as some of the 50 most potent multitarget ABC transporter inhibitors. The most promising representative, compound 37, reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the three most potent ABC transporter inhibitors and reversers of ABC transporters-mediated MDR.

Full text of DOI:10.1021/acs.jmedchem.0c00961

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Article
Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors
and Broad-spectrum ABCB1, ABCC1, and ABCG2 Antagonists
Katja Silbermann, Jiyang Li, Vigneshwaran Namasivayam, Fabian
Baltes, Gerd Bendas, Sven Marcel Stefan, and Michael Wiese
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00961 • Publication Date (Web): 31 Jul 2020
Downloaded from pubs.acs.org on August 25, 2020
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Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-
spectrum ABCB1, ABCC1, and ABCG2 Antagonists.
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Katja Silbermann , Jiyang Li , Vigneshwaran Namasivayam, Fabian Baltes, Gerd
Bendas, Sven Marcel Stefan*, Michael Wiese*
Pharmaceutical and Cellbiological Chemistry, Pharmaceutical Institute, Rheinische
Friedrich-Wilhelms-University Bonn, An der Immenburg 4, 53121 Bonn, Germany
Key Words. ABC transporter; ABCG2 (BCRP) inhibition; ABCB1 (P-gp); ABCC1
(MRP1); Multidrug Resistance (MDR); triple (multi-target) inhibitor.
#
These two authors contributed equally to this work
*
Corresponding authors:
Michael Wiese (mwiese@uni-bonn.de)
Sven Marcel Stefan (s.m.stefan@medisin.uio.no)
Abstract
In search for highly effective modulators addressing ABCG2-mediated MDR, 23
pyrimidines were synthesized and biologically assessed. Seven derivatives with (a)
nitrogen- and/or halogen-containing residue(s) had extraordinary potencies against
ABCG2 (IC < 150 nM). The compounds competitively inhibited ABCG2-mediated
50
Hoechst 33342 transport, but were not substrates of ABCG2. The most potent MDR
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reverser compound 19 concentration-dependently increased SN-38-mediated cancer
cell death at 11 nM (EC ), time-dependently doubled SN-38 toxicity in a period of
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seven days at 10 nM, and half-maximally accelerated cell death combined with SN-38
at 17 nM. No induction of ABCG2 was observed. Furthermore, eleven pyrimidines were
revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC values below
0
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10 µM against each transporter, classifying them as some of the 50 most potent multi-
target ABC transporter inhibitors. The most promising representative compound 37
reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the 3 most
potent ABC transporter inhibitors and reversers of ABC-transporters-mediated MDR.
Introduction
Multidrug resistance (MDR) in cancer chemotherapy remains until today a major
1
2,3
as well as chemo-treated patients,4,5
obstacle in oncology. In both chemo-naive
the overexpression of ATP-binding cassette (ABC) transport proteins is associated
with a negative prognosis and poor outcome of first-line chemotherapeutic intervention.
Recurrence of certain cancers, such as leukemia,4,6,7 as well as lung, pancreatic,
2
8
ovarian,3,9 or breast cancer10,11 was already connected to the overexpression of ABC
3, , , ,
,
transporters. 6 7 8 10 11 These efflux pumps are not only negative prognostic markers
for the development of metastasis,12 but collectively lead to decreased progression-
3, , , , ,
,
and disease-free survival as well as increased mortality. 6 7 8 9 10 11 One of the most
important representatives in this regard is ABCG2 (breast cancer resistance protein;
BCRP).6,7,8 ABCG2 confers resistance to a broad spectrum of chemically unrelated
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anticancer drugs, such as camptothecins, epipodophyllotoxins, mitoxantrone, and
tyrosine kinase inhibitors (TKIs).13,14 Overexpression of this ABC transporter was
found in several cancer cell lines derived from leukemia, as well as cancers from lung,
ovary, colon, prostate gland, or breast.13,15 One strategy to treat certain advanced
and/or relapsed cancers is the use of camptothecin (1; Figure 1) analogs [e.g.,
topotecan (2; Figure 1),16,17 or irinotecan (3; Figure 1) ] as second-line treatment.
However, cell lines derived from certain malignancies acquired ABCG2 expression19,20
after exposure to 121 and its analogs 220,22,23 and 324 as well as its active metabolite
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SN-38 (4; Figure 1),24,25 leading to cross-resistance.20 22 23 25 Other tumor cell
lines even showed inherent ABCG2 expression and resistance toward
camptothecins.26 One concept to overcome resistance against 1 in certain cancers
,21,
,
,24,
was the design of compound 1 analogs that were less recognized by ABCG2, which
led to promising candidates, such as diflomotecan,27 gimatecan,28 or lurtotecan.29
Nonetheless, ABCG2-overexpressing cells always possessed
a
basic
(cross-)resistance against these agents that could not be abolished by structural
2
4,
,30,31
variations. 29
In addition, certain representatives were shown to induce ABCG2
on mRNA and protein levels.30,31
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HO
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3 (irinotecan)
4 (SN-38)
Figure 1: Compound 1 and its derivatives commonly used in second-line anticancer
regimens.
Another approach was the design of inhibitors of ABCG2 which blocked the efflux of
compound 1 analogs and increased their persistence inside of cancer cells. Several
tyrosine kinase inhibitors were proven to reverse ABCG2-mediated resistance against
camptothecins.14 This was firstly described for canertinib (5; Figure 2),32 which
reversed ABCG2-mediated resistance against 4. Until today, only very few TKIs did
likewise at low triple-digit to high double-digit nanomolar concentrations,33,34,35,36,37
such as ceritinib (6; Figure 2).38 Most compounds exerted their reversing potential in
the micromolar concentration range, and clinical evaluations of TKIs in combination
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with either 239,40,41 or 342,43,44,45,46 had only limited success. This was attributed to no
40,
,
benefit of the combination compared to single-drug treatment 43 46 and/or severe
,41,
,
,
,
adverse side effects. 39 42 44 45 46
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In need of the development of more potent and less adverse side effects-associated
adjuvant modulators of ABCG2-related cancers, a bulk of compounds were generated
on the basis of the main scaffolds of TKIs. These were mostly either
quinazoline,32,33,36,47 quinoline,35,48,49 pyrimidine38,50,51 or pyridine37,52 derivatives.
32
Subsequent optimization of the backbone derived from compound 5 resulted in either
highly potent quinazoline-containing (e.g., 7; Figure 2),53,54 or broad-spectrum
quinoline-containing ABCG2 inhibitors (e.g., 8; Figure 2).55 Further improvement
resulted in the most potent ABCG2-mediated MDR-reverser known until today (9; EC50
=
12.7 nM; Figure 2).56 The most recent and final improvement was the downsizing of
the structures to a pyrimidine scaffold, yielding highly potent ABCG2 inhibitors and
reversers of ABCG2-mediated MDR (e.g., 10; Figure 2).57
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O S O
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(ceritinib)
N
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(canertinib)
OH
HN
N
10 (compound 47, HN
Krapf et al. 2017)
NO2
N
N
N
7
(compound 12/20,
Juvale et al. 2012/2013)
NO2
HN
S
HN
N
N
O
O
N
8
(compound 29,
Krapf et al. 2016)
9 (compound 54,
Krapf et al. 2017)
Figure 2: Depiction of inhibitor- and MDR reverser-development regarding ABCG2-
mediated drug transport; compound 5: very first TKI associated with inhibition of
32
ABCG2 and reversal of ABCG2-mediated compound 1 analog resistance; compound
6
: most active TKI regarding MDR reversal against topotecan in ABCG2-
38
overexpressing cells (EC = ~65 nM); compound 7: one of the first reported HTS-
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derived quinazolines as ABCG2 inhibitors;53,54 compound 8: exceptional
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representative of a triple ABCB1/ABCC1/ABCG2 inhibitor; compound 9: one of the
56,
most potent ABCG2-mediated MDR reversers known until today (EC = 12.7 nM); 57
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compound 10: one of only 7 synthetic pyrimidine derivatives as ABCG2 inhibitors.57
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As only 7 pyrimidine derivatives have been described as ABCG2 inhibitors in
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literature, the aim of the present study was further exploration of the pyrimidine class
as ABCG2 inhibitors. The substitution pattern has carefully been selected according to
53,
,55,56,57,58,59,60,61
our experience from previous studies 54
targeting ABCG2.32,33,36,39,40,47,48,52 This included introduction of flexible nitrogen-
cyano or nitro)- and oxygen-(hydroxy or methoxy)-containing substituents with or
as well as reports of TKIs
(
without combination of halogens (fluorine, chlorine, bromine), but also bulky, rigid
nitrogen- and oxygen containing heterocycles. Compounds with high inhibitory power,
low cytotoxicity, highly effective MDR reversal capability, and ABCG2-selectivity were
in focus, neither being substrates of this transporter, nor interfering with its expression.
As our past investigations frequently led to the discovery of multi-target ABCB1,
ABCC1, and ABCG2 inhibitors,54,55,61,62,63,64,65,66 a second task of the present work was
the screening of the 23 synthesized compounds against ABCB1 and ABCC1 as well
as evaluating their capability to reverse ABCB1-, ABCC1-, and ABCG2-mediated MDR.
As certain cancers simultaneously overexpress specific ABC transporters,67,68 and
selective inhibition and/or downregulation of one transporter (e.g. ABCG2) might lead
to compensatory up-regulation of another (e.g., ABCB1 and/or ABCC1),69,70,71 broad-
spectrum ABC transporter inhibitors are considered to be useful tools in a novel
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strategy to approach multidrug-resistant cancers.72,73 However, until today only ~1000
compounds have been evaluated regarding inhibition of ABCB1, ABCC1, and ABCG2.
This group of compounds comprised of less than 120 triple ABCB1/ABCC1/ABCG2
inhibitors, less than 50 of these exerting their effect at 10 µM or
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38,
,54,
,
,
,
,
,
,
,74,75,76,77,78,79,80,81,82,83,84
lower, 49 55 61 62 63 64 65 66
and less than 20 compounds
at 5 µM or lower against all three transporters.55,63,74,77,78,79,80,82,83 While ~75
compounds were reported in the literature as reversers of ABCB1-, ABCC1-, and
ABCG2-mediated MDR,47,48,50,50,52,65,66,85,86,87,88,89 only 2 compounds have been
proven to exert their MDR reversing effect due to inhibition of these ABC transporters,
9
-deazapurine 55 (11; Figure 3) and thienopyrimidine 14 (12, Figure 3).65,66
NH
NH2
O
N
N
N
N
N
N
N
S
N
N
N
11 (compound 55,
12 (compound 14,
Stefan et al. 2017)
Silbermann et al. 2019)
Figure 3: Reported triple reversers of ABCB1-, ABCC1-, and ABCG2-mediated MDR
that proved to exert their effect due to inhibition of ABCB1, ABCC1, and ABCG2: 9-
65
66
deazapurine 55 (11) and thienopyrimidine 14 (12).
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Results and Discussion
Chemistry
Synthesis of 4-anilino-6-methyl-2-phenylpyrimidine derivatives was achieved in three
steps as depicted in Scheme 1. First, a Pinner pyrimidine synthesis was applied by
condensing a benzamidine with ethyl acetoacetate in a freshly prepared sodium
ethoxide solution to form the pyrimidine ring (13). Exposure to excess phosphoryl
chloride yielded 4-chloro-6-methyl-2-phenylpyrimidine (14). Finally, the chlorinated
pyrimidine was reacted with differently substituted anilines to obtain the desired
products (18-40).
1
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HN
NH2
OH
N
O
O
a
N
+
O
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b
R2
R1
HN
N
Cl
c
N
N
N
NH2
R
18-40
14
1
2
R = 18, 29: cyano
R = 18, 19, 30, 31: cyano
R =37: pyridine
3
3
4
8: pyrazine
9: 1,4-dioxane
0: phthalimide
1
2
2
2
9: methyl
20: cyanomethyl
22, 33: nitro
32: fluoro
1, 32: nitro
3: hydroxy
4: hydroxymethyl
34: bromo
25, 34: methoxy
6, 35: fluoro
27, 33: chloro
35, 36: methoxy
21, 23-28: H
2
2
3
2
8, 30: bromo
1, 36: trifluoromethyl
0, 22: H
Scheme 1: General synthesis of 4-anilino-6-methyl-2-phenylpyrimidine derivatives.
Reagents and conditions: (a) NaOEt/EtOH, reflux, overnight; (b) POCl reflux, 8-9 h;
3
(
c) i-PrOH, microwave, 110°C, 30 min.
Biological Investigation
Inhibitory Activity against ABCG2. The synthesized compounds 18-40 were
1
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investigated for their inhibitory activity against ABCG2 using ABCG2-overexpressing
MDCK II BCRP cells in Hoechst 33342 and pheophorbide A assays. In short, blockage
in both cases results in enhanced intracellular accumulation of these fluorescent
ABCG2 substrates. The degree of inhibition of the evaluated compounds correlates
with the measured amount of fluorescence. Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-
octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-
b]indole-3-propanoic acid 1,1-dimethylethyl ester; 41] was chosen as reference
compound. The results of the biological investigations are summarized in Table 1. The
evaluated compounds were compared to certain pyrimidine analogs reported by Krapf
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
57
et al. as these marked the starting scaffold of the present investigation. The within
this work designed and synthesized compounds can be divided into six sub-groups: (i)
nitrogen-containing derivatives (18-22); (ii) oxygen-containing analogs (23-25); (iii)
halogen-containing pyrimidines (26-28); (iv) nitrogen- and halogen-containing
derivatives (29-33); (v) oxygen- and halogen-containing analogs (34-36); as well as
(vi) pyrimidines with bulky rigid heterocyclic residues (37-40). Compound 15
represents the unsubstituted pyrimidine derivative which had an IC value of 0.648 µM
50
in the Hoechst 33342 assay.57 In earlier studies cyano or nitro substituents had a
61
positive impact on ABCG2 inhibition which could also be shown for the 3-cyano (16;
IC50 (Hoechst 33342) = 0.122 µM) and 4-cyano (17; IC50 (Hoechst 33342) = 0.128 µM) analogs.57
In sub-group (i), the 3,4-dicyano analog 18 possessed slightly inferior inhibitory activity
(IC50 (Hoechst 33342) = 0.188 µM; IC50 (Pheophorbide A) = 0.516 µM). The 3-methyl 4-cyano
derivative 19 showed excellent inhibitory power in the Hoechst 33342 assay (IC =
50
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0.0935) with a maximal inhibition level (Imax) value similar to 41. On the other hand,
introduction of a carbon between the main scaffold and the 4-cyano (20) led to a major
loss of inhibitory power.
0
1
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9
0
1
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0
1
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4
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8
9
0
The most potent pyrimidine-based ABCG2 inhibitor known until today with its 3-nitro 4-
57
hydroxy substitution pattern had an IC value of 0.0988 µM (10). Investigation of the
50
3-nitro (21) and 4-nitro (22) analogs resulted also in very potent inhibitors, the first
being as potent as the cyano derivatives 16-17, the latter exceeded the inhibitory
potency of compound 10 by a factor of ~2 in the Hoechst 33342 assay (Figure 4 A).
Interestingly, in the pheophorbide A assay, both compounds 21 and 22 were
equipotent (IC ≈ 0.150 µM), but with a depressed maximum inhibition level (Imax) of <
50
70%.
Compound set (ii) comprised of pyrimidines with solely oxygen-containing substituents.
Here, the 3-hydroxy analog 23 was 2-fold less potent than the cyano derivatives 16-17
in the Hoechst 33342. In the pheophorbide A assay, compound 23 was equipotent to
the 3,4-dicyano derivative 18. As already shown for compound 20, the insertion of a
methylene linker at position 3 resulted in a nearly 20-fold loss of inhibitory activity of
compound 24 in the Hoechst 33342 assay. On the other hand, the 3-methoxy
derivative 25 was equally potent as the 3-hydroxy compound 23, but with a higher Imax
value similar to the standard inhibitor 41.
In group (iii), representatives with solely halogen-containing substituents were
synthesized [(3-fluoro (26), 3-chloro (27), or 3-bromo (28)]. These variations resulted
in compounds with equal inhibitory activity of around ~1 µM in the Hoechst 33342
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assay. Strikingly, compound 28 was ~2-fold more potent compared to compounds 26-
2
7 in the pheophorbide A assay (IC = 0.386 µM).
50
In the next step, combinations of the above stated substituents were evaluated. Within
compound set (iv), compounds 29-30 (3-cyano-4-fluoro and 3-bromo-4-cyano,
respectively) were found to be the most potent representatives as determined in the
pheophorbide A assay, with IC50 values in the high double-digit nanomolar
concentration range. These data were confirmed in the Hoechst 33342 assay, although
the determined IC values were ~2-3.5-fold higher. However, it must be taken into
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
account that both compounds had depressed Imax values in the pheophorbide A assay
59 and 40%, respectively) as shown in Figure 4 B, which artificially decreases the IC50
value. This so-called ‘partial inhibition’ frequently occurs in the
(
literature.58,61,65,90,91,92,93,94,95 Potential explanations could be reduced solubility or
increased intrinsic toxicity of the compounds, but also an actual partial antagonisms
against the transporter. The other way around, the 3-trifluoromethyl 4-cyano analog 31
had superior power against ABCG2 in the Hoechst 33342 assay, exceeding the most
potent pyrimidine 10 up to now (IC = 0.0693 µM) and being around 10-fold more
50
potent than the unsubstituted representative 15. Conversely, inhibition of ABCG2-
mediated pheophorbide A transport was over 5-fold weaker. Discrepancies like these
occur some times and depend on the manner of fluorescence dye and are influenced
by, for example, its polarity, lipophilicity, membrane distribution, velocity of diffusion,
affinity to transport protein.96,97
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1
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1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
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6
A
100
75
50
25
0
0
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9
0
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9
0
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0
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8
9
0
-

-8
-7
-6
-5
log [M]
B
100
7
5
0
5
25
0
-

-8
-7
-6
log [M]
Figure 4: (A) Concentration-effect curves of compound 22 (■, IC = 0.0522 µM) in
50
comparison to reference inhibitor 41 (●, IC = 0.220 µM) in the Hoechst 33342 assay.
50
(
B) Concentration-effect curves of compound 30 (■, IC = 0.0594 µM) in comparison
50
to reference inhibitor 41 (●, IC = 0.274 µM) in the pheophorbide A assay. Shown is
50
mean ± standard error of the mean (SEM) of at least three independent experiments.
Unexpectedly, the 3-nitro 4-fluoro derivative 32 did not result in superior inhibitory
power as described for its 3-cyano analog 29. It was by factors of ~3 and ~9 less active
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in the Hoechst 33342 and pheophorbide A assays, respectively. The 3-chloro 4-nitro
derivative 33 was the second most potent inhibitor in the Hoechst 33342 assay (IC =
50
0.0589 µM), with a notable Imax value of above 75%. This could not be confirmed by
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the pheophorbide A data, as the determined IC value was ~4-fold higher.
50
Given the positive impact of methoxy groups in some scaffolds on ABCG2
inhibition,58,90 certain representatives were synthesized containing either 3- or 4-
methoxy and a neighbored halogen in group (v). The 3-methoxy 4-bromo derivative 34
had in both assay an inhibitory power against ABCG2 of ~300 nM but a decreased Imax
value in the pheophorbide A assay of about 50%. The 4-methoxy derivatives 35 and
3
6 were less preferred (IC values of ~1 µM and ~0.5 µM, respectively). Collectively,
50
the methoxy derivatives did not meet the expected results.
In group (vi), heteroaromatic and heterocyclic substituents at positions 3 and 4 of the
pyrimidine scaffold were introduced to evaluate rigid nitrogen and oxygen substituents.
The pyridine derivative 37 was by a factor of ~2 superior compared to the quinoxaline
analog 38 in the pheophorbide A assay. Unfortunately, these compounds could not be
evaluated in the Hoechst 33342 assay due to auto-fluorescence. The non-aromatic
oxygen analog to compound 38, the 1,4-dioxane 39, was inferior to its aromatic
nitrogen counterpart, which might be due to loss of planarity and/or aromaticity. The
phthalimide derivative 40 was inferior to its closed-ring pyrimidine analogs 37-39.
Collectively, the results concerning these compounds showed that a certain flexibility
of the substituents is needed for good inhibitory activity.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1: Inhibitory potencies of synthesized pyrimidine derivatives as obtained in the
Hoechst 33342 and pheophorbide A assays, respectively, using ABCG2-
overexpressing MDCK II BCRP cells. Data are expressed as mean ± SEM (n ≥ 3).
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
R2
R1
4
3
6
1
HN
4
2
N
2
N
18-40
Hoechst 33342
IC ± SEM
No. R1
R2
pheophorbide A
Imax
± IC ± SEM
Imax
±
50
50
[μM]
SEM
[μM]
SEM
[%]
[
%]
0.0988 ± 0.0174 n.t.
1
1
1
0b NO2
5b
6b CN
OH
H
n.t.
n.t.
n.t.
n.t.
n.t.
H
0.648 ± 0.085
0.122 ± 0.014
0.128 ± 0.018
0.188 ± 0.010
n.t.
n.t.
n.t.
n.t.
H
n.t.
17b
H
CN
CN
CN
n.t.
1
8
9
CN
CH3
H
117 ± 10 0.548 ± 0.043
100a
76 ± 12
100a
68 ± 6
67 ± 10
59 ± 3
1
0.0935 ± 0.0183 106 ± 11 0.175 ± 0.011
20
CH CN 2.50 ± 0.19
100a
3.62 ± 0.27
2
2
1
2
NO2
H
H
0.130 ± 0.011
80 ± 5
0.146 ± 0.009
0.156 ± 0.016
0.516 ± 0.033
2
NO2
H
0.0522 ± 0.0037 79 ± 5
0.253 ± 0.027 74 ± 8
23 OH
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CH OH
H
4.22 ± 0.36
100a
2.52 ± 0.12
100a
2
OCH3
F
H
0.271 ± 0.061
1.11 ± 0.15
96 ± 6
0.503 ± 0.046
0.776 ± 0.151
1.00 ± 0.18
100a
100a
100 a
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27 Cl
H
0.811 ± 0.081
0.892 ± 0.091
0.307 ± 0.007
0.119 ± 0.006
100a
100a
100a
90 ± 6
100 a
2
8
9
Br
H
0.386 ± 0.046
0.0861 ± 0.0043
0.0594 ± 0.0036
0.385 ± 0.047
0.771 ± 0.086
0.228 ± 0.025
0.328 ± 0.031
1.31 ± 0.10
83 ± 3
59 ± 6
40 ± 5
2
CN
F
30 Br
CN
CN
F
3
1
2
CF3
NO2
0.0693 ± 0.0009 114 ± 7
92 ± 4
100a
100a
3
1.01 ± 0.12
33 Cl
NO2
Br
0.0589 ± 0.0015 76 ± 9
86 ± 9
34
35
36
37
OCH3
F
0.280 ± 0.042
0.861 ± 0.053
0.598 ± 0.075
n.t.c
100a
53 ± 3
100a
100a
OCH3
OCH3
CF3
100a
n.t.c
0.544 ± 0.035
0.151 ± 0.014
83 ± 5
79 ± 16
N
N
38
49
40
n.t.c
n.t.c
0.312 ± 0.045
1.63 ± 0.17
100a
N
O
100a
100a
0.808 ± 0.050
0.253 ± 0.007
O
O
105 ± 7
100
0.416 ± 0.043
101 ± 15
100
NH
O
41
0.220 ± 0.006
0.274 ± 0.014
n.t. = not tested
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1
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2
2
2
2
2
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3
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6
a
b
c
Constrained to 41.
Data taken from reference [57].
Data could not be analyzed due to auto-fluorescence of compound at wavelength
0
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0
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4
5
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8
9
0
355 nm.
Screening against ABCB1 and ABCC1. All compounds were examined for their
inhibitory power against ABCB1 and ABCC1 in order to investigate their selectivity over
ABCG2 and to potentially discover multi-target ABCB1/ABCC1/ABCG2 inhibitors. The
screening was performed at a concentration of 10 µM using a calcein AM assay with
either ABCB1-overexpressing A2780/ADR or ABCC1-overexpressing MDCKII MRP1
cells. In short, inhibition of the transporter(s) results in enhanced persistence of calcein
AM inside the cell, which becomes cleaved by unspecific esterases to fluorescent
calcein. As already stated above for the Hoechst 33342 and pheophorbide A assays,
the amount of measured fluorescence correlates to the degree of inhibition exerted by
the compounds. Cyclosporine A (10 µM) was used as a positive control. Figure 5 A
gives the results of the screening against ABCB1, while Figure 5 B depicts the results
regarding ABCC1.
Several pyrimidines, especially representatives with methoxy substituents (25),
halogen residues (26-28), or a combination of both substituents (34-36) had an
inhibitory effect on ABCB1 and ABCC1. On the other hand, derivatives with cyano (18,
19, 29-31) and nitro (21-22,32-33) groups inhibited neither ABCB1 nor ABCC1 besides
few exceptions (21-22). Compounds with an inhibition level of at least 25% were further
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evaluated for their IC value and the results are presented in Table 2. Generally, these
50
compounds had similar inhibitory potencies against both transporters. The most potent
ABCG2 inhibitors (19, 29, 31, 33) were ABCG2-selective without noticeable affinity to
the other two ABC transporters. Exceptions were compounds 21 and 22. The former
also slightly inhibited ABCC1, which made it (a rather ABCG2-seletive) dual
ABCC1/ABCG2 inhibitor. The latter inhibited both ABCB1 and ABCG2, but also with
great preference for ABCG2. Nevertheless, compound 22 can be considered as rare
finding of a triple ABCB1/ABCC1/ABCG2 inhibitor. In addition, compounds 25, 27-28,
34-38, and 40 were also found to be multi-target ABCB1/ABCC1/ABCG2 inhibitors,
which is extraordinary considering the amount of less than 120 compounds of this kind
given in literature. Five of the herein presented compounds (27-28, 34, 36-37) had
potencies ranging within the 50 most potent triple ABCB1/AABCC1/ABCG2 inhibitors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
known until today. 49 54 55 61 62 63 64 65 66 74 75 76 77 78 79 80 81 82 83 84
A
1
50
125
00
1
7
5
0
5
25
0
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
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3
4
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4
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5
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5
5
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5
5
6
B
150
1
25
00
75
0
1
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0
1
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5
6
7
8
9
0
1
5
0
5
0
2
18
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Figure 5: Inhibitory effect of screened compounds at 10 µM against ABCB1 using the
ABCB1-overexpressing cell line A2780/ADR (A) and ABCC1 using the ABCC1-
overexpressing cell line MDCK II MRP1 (B) applying a calcein AM assay. Data were
normalized by defining the effect of 10 µM cyclosporine A as positive control (100%)
and buffer medium as negative control (0%). Shown is mean ± SEM of at least three
independent experiments with duplicate measurements.
Table 2: Inhibitory potencies against ABCB1 and ABCC1 of compounds that had an
inhibition level of at least 25% in the initial screening. Data were obtained in a calcein
AM assay using A2780/ADR and MDCK II MRP1. Maximum inhibition was defined by
1
0 µM of cyclosporine A. Shown is mean ± SEM of at least three independent
experiments.
2
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9
Compound
IC [µM] ± SEM
IC [µM] ± SEM
50
50
A2780/ADR (ABCB1)
16.0 ± 1.6
n. t.
MDCK II MRP1 (ABCC1)
20
n. t.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
11.2 ± 2.8
9.26 ± 0.75
8.87 ± 1.00
7.01 ± 1.01
3.51 ± 0.05
3.53 ± 0.06
6.13 ± 0.37
19.5 ± 4.7
4.76 ± 0.72
7.23 ± 0.69
28.4 ± 5.0
30.4 ± 11.0
18.3 ± 1.2
6.63 ± 0.67
2
2
5
11.2 ± 2.7
13.4 ± 0.6
n. t.
2
26
2
7
8
5.92 ± 0.14
7.14 ± 0.33
5.03 ± 0.59
8.99 ± 0.55
7.30 ± 0.23
5.13 ± 0.20
13.4 ± 2.7
13.1 ± 1.5
17.7 ± 3.6
2
34
35
36
37
38
39
40
Cyclosporine A 1.20 ± 0.03
n. t. = not tested due to lack of activity in initial screening assay.
Determination of the Concentration-dependent Reversal of ABCG2-mediated MDR.
The ability of the most potent compounds to reverse ABCG2-mediated MDR was
verified by a MTT-based approach, using a dilution series of the ABCG2 substrate 4.
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In addition, certain concentrations of the tested compounds were added to evaluate
whether toxicity of compound 4 was enhanced by presence of the tested compounds
(Figure 6 A). This would be indicated by the shift of the concentration-effect curve to
0
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0
the left (closer to the curve of the parental cell line without ABCG2 expression), which
means that less compound 4 is necessary for half-maximal growth inhibition due to the
presence of the tested compound. This effect can be quantified by plotting the
resistance factors of the different experiments as well as the parental cell line against
the concentration of the tested compound. This half-maximal reversal concentration
(EC ) is the concentration at which the cancer cells were half-maximally sensitized
50
with respect to compound 4 and is comparable to IC values in the inhibition assays
50
(Figure 6 B).
All chosen compounds were able to achieve full MDR reversal. Their EC values were
50
in the low double-digit nanomolar concentration range (Table 3), which gave proof that
pyrimidines are very effective reversers of ABCG2-mediated MDR. The most efficient
MDR reverser was compound 19, which was able to sensitize MDCK II BCRP cells
with an EC value of 11.0 nM (Figure 6 A and B). This exceeds the efficacy of its
50
56
parent compound 9 and makes it one of the most potent MDR reversers reported
56,
,89
until today. 57 In addition, this EC value is of a 6- and 12- fold higher potency
50
compared to the results of the Hoechst 33342 or pheophorbide A assays, respectively.
This phenomenon can be explained by different affinities of the ABCG2 substrates for
the transporter and transport behavior of the different substrates.96,97 It might also be
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correlated to the different time frames of the assays (short-term vs. long-term), and has
already been observed in literature before.65,90
1
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A
150
1
25
00
75
1
5
2
0
5
0
-
-8
-7
-6
-5
compound 4 log [M]
B
40
30
20
10
0
I
I
I
I
-10
-9
-8
-7
-6
K
C
K
C
C
B
D
P
compound 19 log [M]
D
M
R
M
Figure 6: (A) MDR reversal assay obtained in a MTT-based cell viability assay for
compound 19. The curve shows sensitization of ABCG2-overexpressing MDCK II
BCRP cells with regard to compound 4. Compound 19 was used at concentrations of
0
µM (●), 0.001 µM (■), 0.010 µM (▲), 0.100 µM (▼), and 1.000 µM (♦) and was
compared to the wild type cell line without compound addition (○). Shown is mean ±
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SEM of at least three independent experiments with duplicate measurements. (B)
Calculated resistance factors derived from GI50 values of graph (A) were plotted
against the used concentrations of compound 19. A nonlinear regression of the
obtained curve yielded a half-maximal sensitization concentration (EC ) of 11 nM.
0
1
2
3
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5
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7
8
9
0
1
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3
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9
0
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9
0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
Shown is mean ± SEM of at least three independent experiments.
Table 3: Results of the MDR reversal assay obtained in a MTT-based viability assay.
Resistance factors were plotted against the corresponding concentration of the
investigated compound. The nonlinear regression of the obtained curve gave the half-
maximal reversal concentration (EC ). Shown is mean ± SEM.
50
Compound
EC [µM] ± SEM
50
19
0.0110 ± 0.0015
0.0159 ± 0.0009
0.0209 ± 0.0005
0.0150 ± 0.0020
0.0152 ± 0.0016
2
2
9
2
30
31
Determination of the Time-dependent Reversal of ABCG2-mediated MDR. In order to
investigate the MDR reversing property of the most potent representative – compound
19 – in more detail, a time-dependent MTT-based MDR reversal assay was conducted.
In short, cells were exposed to a concentration of 0.5 µM of compound 4 alone and in
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combination with 10 nM of compound 19 over a time period of seven days determining
the cell viability every day. These concentrations were chosen as 0.5 µM of compound
4
showed in preliminary experiments no toxic effect on the resistant cells over 3 days,
1
1
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0
and 10 nM of compound 19 nearly equals its EC value. Figures 7 A [normalized on
50
control (pure cell culture medium)] and B (normalized on compound 4) show the
obtained results. As one can see, 10 nM of compound 19 is sufficient to double the
toxic effect of compound 4 over a period of 7 days. A statistically significant effect was
seen after 5 days, which equals the timeline of a commonly used application in
antineoplastic chemotherapy (so-called 5-day-course; 5 days topotecan i.v. application
every 3 weeks).98 To verify the concentration-dependency of this effect over time, this
7-days-experiment was conducted using different concentrations of compound 19 (0,
10, 25, and 50 nM). Figure 7 C presents the obtained results, showing that increased
concentrations of compound 19 led to decreased cell viability over time. Plotting the
inflection points of the curves of Figure 7 C against the used concentrations of
compound 19 results in a time-concentration graphic as indicated by Figure 7 D.
Several measures can be deduced from this plot: (i) tmax, which represents the time
needed for the antineoplastic agent alone at a fixed concentration (in this case
compound 4 at 0.5 µM) to half-maximally kill the cancer cells (~7.25 days); (ii) tmin,
which represents the minimum time needed to achieve half-maximal cell death by the
antineoplastic agent at a fixed concentration in combination with a sensitizer (in this
case compound 19; ~4.25 days); (iii) ctmin (maximal cell death acceleration
concentration), which is the concentration of the modulator needed to achieve tmin (in
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this case compound 19 at ~50 nM); and (iv) c½tmax (half-maximal cell death acceleration
concentration), which gives the concentration of the modulator needed to achieve half-
maximal cell death by the used antineoplastic agent at ½ of tmax (in this case compound
19 at 17.26 nM in ~5.75 days in combination with compound 4 at 0.5 µM; Figure 8 D).
c½tmax is a compound-, cell line-, antineoplastic agent-, and assay-specific value, which
is described for the first time within this work. It is a critical measure for judging the
effectiveness of a modulator and its superiority to the treatment with an antineoplastic
agent alone, especially regarding its relation over time. Besides IC (relation to target)
0
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9
0
50
and EC (capability to reverse MDR), c should be the third standard measure in
½tmax
50
future compound evaluation.
In summary, it can be stated that compound 19 (i) half-maximally sensitizes ABCG2-
overexpressing cells at only 11 nM in combination with varying concentrations of
compound 4; (ii) sensitizes ABCG2-overexpressing cells in combination with 0.5 µM
compound 4 two times as effective as compound 4 does alone; and (iii) accelerates
cell death half-maximally at ~17.25 nM considering treatment with compound 4 alone
and the maximal possible acceleration of cell death represented by use of ~50 nM of
compound 19. In essence, this makes compound 19 most potent sensitizer of ABCG2-
mediated MDR in detail.
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A
*
***
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*
***
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****
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ay
ay
ay
ay
ay
ay
ay
d
d
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00
****
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0
*
***
****
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ay
ay
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ay
ay
ay
ay
d
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150
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time [days]
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10
20
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50
60
compound 19 [nM]
Figure 7: Time-dependent MDR reversal assay using a MTT-based approach. (A-B)
Compound 19 was used at a concentration of 10 nM and was combined with 0.5 µM
compound 4 over a period of 7 days. Shown is mean ± SEM of at least three
independent experiments with triplicate measurements. (A) Normalization was
conducted on the effect-value of cells in pure cell culture medium without addition of
compound 4 and/or compound 19. (B) Normalization was conducted on the effect-
value of compound 4 alone at the corresponding day. Significance was calculated
applying multiple t-test and is given as *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, **** p ≤
0
.0001. (C) Cell viability-versus-time assay using 0.5 µM of compound 4 and 0 nM (●),
10 nM (■), 25 nM (▲), 50 nM (▼) of compound 19. Shown is mean ± SEM of at least
three independent experiments with triplicate measurements. (D) The inflection points
derived from the curves in graphic (C) have been plotted against the used
concentration of compound 19. Nonlinear regression revealed that using 0.5 µM
compound 4 in combination with compound 19 the latter is able to accelerate half-
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maximal cell death by a factor of 1.7 (~4.25 days vs. ~7.25 days). The half-maximal
cell death acceleration concentration of compound 19 was ~17.25 nM.
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0
Evaluation of Multi-target ABCB1, ABCC1, and ABCG2 Inhibitors for Reversal of
ABCB1-, ABCC1-, and ABCG2-mediated MDR. The five most potent triple
ABCB1/ABCC1/ABCG2 inhibitors as indicated by Tables 1 and 2 – compounds 27, 28,
34, 36, and 37 – were investigated for their ability to reverse ABCB1-, ABCC1-, and
ABCG2-mediated MDR. While the first 4 compounds failed to sensitize ABCB1- and
ABCC1-overexpressing cells regarding doxorubicin (data not shown), compound 37
revealed to be a potent, broad-spectrum reverser of ABCB1-, ABCC1-, and ABCG2-
mediated MDR. It half-maximally sensitized A2780/ADR (ABCB1), MDCK II MRP1
(ABCC1), and MCDK II BCRP (ABCG2) cells at 8.41 ± 0.48 µM, 5.15 ± 0.68 µM, and
0.0164 ± 0.0023 µM, respectively (Figures 8 A–F). These results make compound 37
a rare example of a triple ABCB1/ABCC1/ABCG2 inhibitor that reversed ABCB1-,
ABCC1-, and ABCG2-mediated MDR, which has only been shown for compounds 11
and 12 in the literature before (Figure 3).65,66
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