Novel symmetrical cage compounds as inhibitors of the symmetrical mrp4-efflux pump for anticancer therapy

Article abstract of DOI:10.3390/ijms22105098

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutation

Full text of DOI:10.3390/ijms22105098

International Journal of
Molecular Sciences
Communication
Novel Symmetrical Cage Compounds as Inhibitors of the
Symmetrical MRP4-Efflux Pump for Anticancer Therapy
David Kreutzer , Henry Döring , Peter Werner , Christoph A. Ritter and Andreas Hilgeroth 1,*
1
1
1
2
1
Research Group of Drug Development, Institute of Pharmacy, Martin Luther University Halle-Wittenberg,
06120 Halle, Germany; david.kreutzer94@web.de (D.K.); henry.doering@arcor.de (H.D.);
peter.werner@mail.de (P.W.)
Department of Clinical Pharmacy, Institute of Pharmacy, Ernst Moritz Arndt University Greifswald,
2
17489 Greifswald, Germany; ritter@uni-greifswald.de
*
Correspondence: andreas.hilgeroth@pharmazie.uni-halle.de; Tel.: +49-345-55-25168
Abstract: Within the last decades cancer treatment improved by the availability of more specifically
acting drugs that address molecular target structures in cancer cells. However, those target-sensitive
drugs suffer from ongoing resistances resulting from mutations and moreover they are affected
by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be
treated with the common drugs, so that there have been long efforts to develop drugs to combat
that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in
cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective
efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those
inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4
we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a
MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition
of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active
compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant
anticancer drug as proof-of-principle to encourage for further preclinical studies.
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ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Kreutzer, D.; Döring, H.;
Werner, P.; Ritter, C.A.; Hilgeroth, A.
Novel Symmetrical Cage Compounds
as Inhibitors of the Symmetrical
MRP4-Efflux Pump for Anticancer
Therapy. Int. J. Mol. Sci. 2021, 22,
Keywords: anticancer drug; drug resistance; structure activity; synthesis; inhibition
5
098. https://doi.org/10.3390/
ijms22105098
Academic Editor: Silvie Rimpelova
1
. Introduction
Cancer remains the second frequent cause of death worldwide, although cancer
therapies of the last decades improved by the development of novel drugs and the discovery
of novel target structures in cancer cells [ ]. The classical chemotherapeutic treatments
with antineoplastic agents affect all dividing cells and have strong side effects [ ]. Ongoing
resistances enforced the search for novel structures and resulting drugs [ ]. With the
understanding of the molecular pathways and regulators of cell growth and division the
number of potential target structures for drugs increased [ ]. Hence, deregulated protein
kinases in cancer cells could be identified and X-ray crystal structure analysis helped to
find the binding region for potential inhibitors [ ]. Novel small-molecule inhibitors were
Received: 29 March 2021
Accepted: 7 May 2021
Published: 12 May 2021
1–3
4
5
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
3
6
developed and also antibodies were synthesized using biotechnology procedures that
address the receptor regions of the respective protein kinases [7]. However, mostly single
mutations led to changes in the inhibitor binding protein region and thus cause resistances
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
to the respective drugs [8]. Moreover, the costs for such monoclonal antibody therapies
are high and a burden for the social health systems [
challenge to find novel drugs.
9]. Therefore, it will be an ongoing
As the main cause of anticancer drugs resistance is the occurrence of transmembrane
efflux pumps, those structures were early considered as promising target structures [10].
As they reduce the intracellular level of the anticancer drug by outwards transport, the
cancer cell becomes resistant [11]. The known cancer-relevant efflux pumps transport
4
.0/).
Int. J. Mol. Sci. 2021, 22, 5098. https://doi.org/10.3390/ijms22105098
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5098
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various drugs with different structures and thus establish a multidrug resistance (MDR)
that can hardly be defeated [11]. Also, the novel more specifically acting anticancer drugs
become affected by the MDR phenomenon.
Early inhibitors like verapamil, quinidine or cyclosporine A of efflux pump activity
have been evaluated in cancer cells that overexpressed the respective efflux pump to be
effective [12]. Partly, they could not be used in clinical trials due to own pharmacologi-
cal effects like verapamil as calcium antagonist, quinidine as antiarrhythmic agent and
cyclosporine A as immunosuppressant drug [12]. Candidates of the third generation with
dofequidar, zosuquidar, tariquidar, elacridar, and biricodar were developed as specific
efflux pump inhibitors but proved to inhibit also different efflux pumps. In clinical trials
they were disappointing because the benefit in tumor therapy was low [11,13,14]. The
reason for that was a missing knowledge of an efflux pump expression by the respective
tumor [11]. Therefore, recent studies show that in case of a detected expression of an efflux
pump the use of such inhibitors is successful [11,15,16].
A strategy to find novel inhibitors or candidates for a further profiling has been the
screening of drug libraries [11,12]. Compounds can be identified with their own pharmaco-
logical properties that have to be rebuilt to reduce those properties, but maintain the efflux
pump inhibition potential [12]. Our strategy was to synthesize novel inhibitors that may be
of favor for the inhibition of the symmetric efflux pump multidrug resistant protein (MRP)
4
for that inhibitors are absolutely rare [17]. Similar to the longer known efflux pump
P-glycoprotein, MRP4 owns a symmetric molecular structure with two transmembrane
domains subdivided in each six α-helical units [18,19].
We synthesized cage dimers of two 1,4-dihydropyridines arranged in a head to tail
fashion to provide the favorable C symmetry in the substituent arrangement at the sym-
2
metric molecular scaffold. Within the substituents we varied the positioning and number
of residues and discuss the results of determined MRP4 inhibition properties. These were
determined in a fluorescence uptake assay with a fluorescent MRP4 substrate in two pan-
creatic cancer cell lines one without expressing MRP4 and one with MRP4 by a fluorescence
ratio of the uptaken substrate in both cell lines. Substances with highest ratios and as
compared with a standard MRP4 inhibitor characterized best inhibitors. Furthermore, the
best inhibitor was evaluated to resensitize the MRP4-overexpressing cell line for a common
MRP4 anticancer drug as proof-of-concept.
2. Results and Discussion
2.1. Formation of the 1,4-Dihydropyridine Cage Dimer Formation
As a reduced pyridine the 1,4-dihydropyridine scaffold owns two neighbored double
bonds. A double bond may undergo a [2+2] cycloaddition reaction with another double
bond to form cyclobutane compounds after irradiation with light under double bond excita-
tion [20]. Pyrone compounds are known to form photodimers by the reaction of neighbored
double bonds under UV light irradiation as cage compounds with resulting neighborhood
cyclobutane rings that form the cage compound walls [21]. Such photodimers own a mirror
symmetry in case of a head to head conjunction or a C symmetry in case of a head to tail
2
conjunction [21,22].
Therefore, we irradiated solutions of synthesized 1,4-dihydropyridines 1 with neigh-
borhood double bonds similar to pyrone compounds to observe the formation of crystalliz-
ing cage compounds from the solutions that owned a head to tail arrangement of the former
1
,4-dihydropyridine subunits and thus own a C symmetry of the molecular framework
2
2
(Scheme 1). The used 1,4-dihydropyridines 1 have been synthesized from an aromatic
aldehyde, methyl propiolate and a benzylamine compound in acetic acid as a procedure
different from the known Hantzsch synthesis of 1,4 dihydropyridines that use a dicarbonyl
compound instead of the methyl propiolate [23].

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R1
R2
O
R4
MeO
OMe
O
R1
O
O
R3
MeO
OMe
R2
h ν
N
N
O
THF
N
R4
R3
O
R1
MeO
OMe
R4
R2
R3
1
2
R4
HO
N
OH
R1
R2
R3
LiAlH4
THF, -8 °C
N
HO
R1
OH
3a-k
Scheme 1. Cage dimer target formation via 1,4-dihydropyridines.
R4
R2
R3
Those Hantzsch 1,4-dihydropyridines with characteristic alkyl substituents at the
- and 6-positions of the molecular scaffold are not sensitive to light except of the 1,4-
2
dihydropyridine drug nifedepine as dimethyl 2,6-dimethyl-4-(2-nitrophenyl)- 1,4-dihydrop-
yridine-3,5-dicarboxylate. Nifedipine undergoes a intramolecular redox reaction of the
1
,4-dihydropyridine and the nitro function at the 4-phenyl residue to form a pyridine
compound [24]. Finally, the four methyl ester residues in compounds have been reduced
with lithium aluminium hydride to give alcoholic functions in target compounds 3a that
2
–
k
were of favor for the compound solubility and may serve as both hydrogen bond acceptors
and donators in interaction with amino acid residues of the protein target compound
MRP4.
2.2. MRP4 Efflux Pump Inhibition with the 1,4-Dihydropyridines
Recent studies prove the role of MRP4 in various kinds of solid cancer like neurob-
lastoma where it attributes to the poor clinical outcome, pancreatic and prostate cancer
and melanoma and myeloid leukemia with effects on cell proliferation and differentia-
tion [11
on MRP4 as promising target for anticancer therapy is relatively new compared to longer
known efflux pumps like P-glycoprotein [25 28 30]. Therefore, there is need for novel
,25–28]. The number of MRP4 inhibitors is strongly limited so far because the focus
,
–
MRP4 inhibitors. Our successful synthesis provides symmetric molecules that may serve
as inhibitors of the symmetric efflux pump MRP4. We introduced methoxy functions in
the aromatic residues that have been varied in positioning within the symmetric phenyl
residues and in the number. Methoxy functions are long known as favorable substituents
for efflux pump inhibition as they may serve as hydrogen bond acceptor functions to amino
acid residues of the potential MRP4 binding site [31].
In our assay system, for determination of the MRP4 inhibition we used two pancreatic
cancer cell lines one without the expression of MRP4 and the other one with expressing
MRP4 after vector plasmid transfection. We determined the uptake of the fluorescent MRP4

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substrate calcein in both cell lines each without an inhibitor and with an inhibitor. In case
of the inhibition of MRP4 the uptake of calcein is increased due to the lowered outwards
transport by the efflux pump. The fluorescence values of each inhibitor-treated cell line
are corrected by the fluorescence of the untreated control cells and the resulting values are
given as fluorescence activity ratios (FARs) of the MRP4-expressing and non-expressing
cells, so that the increasing values reflect the degree of MRP4 inhibition. The FAR values
are shown in Table 1.
Table 1. MRP4 inhibition data of target compounds 3a–k with varied substitution patterns expressed
as FAR values.
Cpd.
R1
R2
R3
R4
FAR Value a
3
a
H
H
OMe
OMe
H
H
H
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
OMe
OMe
H
OMe
OMe
OMe
H
1.15
1.15
1.34
1.56
1.52
1.70
1.55
1.36
1.25
1.41
1.74
0.82
3
b
OMe
OMe
H
3
c
3
3
d
e
H
OMe
OMe
OMe
H
OMe
OMe
OMe
H
3
f
OMe
OMe
OMe
H
OMe
OMe
3
g
3
h
3
3
i
j
3
k
OMe
MK571
a
Mean of three determinations.
We started with compound 3a without substitution of the aromatic residues. The FAR
value of 1.15 was much better than that of the used MRP4 inhibitor MK571 with a value
of 0.82 as best known standard inhibitor. The introduction of a para methoxy function
in the N-benzyl residues in compound 3b led to an unchanged activity. The additional
introduction of a para methoxy function in the phenyl residues increased the activity of
compound 3c to a FAR value of 1.34. Then we varied the positioning of the favorable four
methoxy functions within the aromatic residues.
If the para methoxy functions of the N-benzyl residues move to the meta positions
in compound 3d the FAR value further increased compared to compound 3c. If the para
methoxy phenyl functions of compound 3d also move to the meta position in compound 3e
the activity was almost unchanged. If the meta methoxy benzyl functions of compound 3e
now move to the para positions in compound 3f best activities were reached with a FAR
value of 1.70.
We then increased the number of methoxy functions to investigate a suggested increas-
ing effect on activity according to the results with the discussed first compounds. If another
methoxy function was introduced in the N-benzyl residues of the presently best compound
3f the activity of derivative 3g was similar to that of the both meta methoxy phenyl and
N-benzyl substituted compound 3e. If the meta methoxy phenyl function moved to the
para position of the phenyl residues in compound 3h the activity was lowered and similar
to that of the both para methoxy phenyl and N-benzyl substituted compound 3c. If both
methoxy functions were concentrated in the phenyl residues of derivative 3i with a meta
methoxy N-benzyl function the activity was lower than that of compound 3g with both
methoxy functions at the N-benzyl residues and the meta methoxy function at the phenyl
residues. If the N-benzyl meta methoxy moved to the para position in derivative 3j the
activity increased to slightly exceed that of compound 3h with both methoxy functions at
the N-benzyl residues and the para methoxy function at the phenyl residues. Therefore,
it can be stated that those six methoxy functions with a non-symmetrical distribution at
the aromantic residues is generally not of favor to increase the activity although increas-
ing numbers of methoxy functions were suggested to be of favor as suggested from the
previous compounds.

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Then we further increased the number of methoxy functions to eight in compound
3k
with an again symmetric distribution on the aromatic residues. The FAR value with
1
.74 of that compound was the best one of all tested compounds. Therefore, it can be
concluded from the allover results that an increasing number of methoxy functions is of
favor for the MRP4 inhibiting properties in case of a symmetric distribution within the
symmetrically arranged aromatic residues. The general suggestion that methoxy functions
are favorable for the efflux pump inhibiting properties can be confirmed with our study
results also for the so far not investigated MRP4. However, the result that the effect is
limited to those compounds with a symmetric distribution within the aromatic residues
supports the existence of a symmetric binding side or arrangement for inhibitors of that
symmetric efflux pump.
2.3. In Vitro MRP Resistance Studies of Drug Reversal
In various kinds of solid cancer the role of MRP4 in anticancer drug resistance has been
suggested based on mRNA analyses of the MRP4 encoding gene [11]. In order to confirm
such observations the expression of MRP4 should be proved. However, the number of
MRP4 inhibitors is strongly limited with MK571 as presently best known inhibitor that
is a cysteinyl leukotriene receptor antagonist with additional pharmacological properties
as phosphodiesterase inhibitor [25
dimers we found a tool to evaluate the structure-activity relationships of MRP4 inhibition
as discussed.
From that compound class we selected the best inhibitor 3k for further studies to
,32]. With our evaluated compound class of novel cage
3
prove that the MRP4 inhibition effect really contributes to a MRP4 reversal of an anticancer
drug resistance as suggested by early publications. We chose 6-mercaptopurin as a known
MRP4 substrate that is a commonly used antimetabolite in the chemotherapy of various
kinds of cancer. With our MRP4 expressing pancreatic cell line we had a cell model that
could be used for the study.
First we determined the toxicity of 6-mercaptopurin in the non-MRP4 expressing cell
line. For that purpose the MTT assay was used to determine the toxicity as degree of a
reduced formazan formation from the MTT reagent by the mitochondrial dehydrogenases.
In the presence of 50
3 ± 24.7% (mean ± SD, n = 3, Figure 1).
Next we determined the 6-mercatopurin toxicity in the MRP4-expressing pancreatic
µM 6-mercaptopurine cell viability of Colo357 cells was reduced to
7
cell line where the toxicity was expected to be much lower due to the MRP4 mediated
outwards transport of the drug. We found an only slight reduction of cell viability in
Colo357_MRP4 cells to 89.5
efflux pump activity as cause for the lowered 6-mercaptopurin toxicity. Then we used
M of MK571 as potential inhibitor and found a reduction in cell viability to 85.8 ± 18.3%
mean SD, n = 3, Figure 1) which means no effect on the MRP4 efflux inhibition. Next
we used our best inhibitor 3k with an almost double stronger effect on MRP4 as evaluated
FAR value. The use of 10 M of compound 3k resulted in a 6-mercaptopurin toxicity
corresponding to a reduction in cell viability to 63.0 16.9% (mean SD, n = 3, Figure 1).
Thus we observed a marked yet not significant (p = 0.125, unpaired t-test) effect of a reduced
-mercaptopurin toxicity for the MRP4-expressing cell line as result of an inhibition of
±
16.7% (mean
±
SD, n = 3, Figure 1) that confirmed the MRP4
10 µ
(
±
µ
±
±
6
MRP4 as proof-of-principle. An alternative use of naturally MRP4-overexpressing cell lines
like the breast cancer cell lines MDA-MB-436 and BT549 was not taken in consideration
because those cell lines overexpress also different efflux pumps like P-glycoprotein, BCRP
and MRP1 more than MRP4 in MDA-MB-436 and MRP1 and MRP2 in BT549 [29,33–35].

Int. J. Mol. Sci. 2021, 22, 5098
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Figure 1. Effect of the symmetric cage compound 3k on cell viability of MRP4 expressing Colo357
pancreatic cancer cells (Colo357_MRP4). Cells were incubated with 50
µM 6-mercaptopurine (6-MP)
in the absence or presence of 10
at 37 C and thereafter cell viability was assessed using an MTT assay as described in the Section 3.
µM compound 3k or the unselective MRP4 inhibitor MK571 for 48 h
◦
Values represent mean
±
SEM of three independent experiments. Due to an insolubility of 6-MP at
higher concentrations the effect was determined at the given one. Alternatively, the measurement
of a concentration-dependent effect and the depiction in complete sigmoid curves was not possible
with reliable values.
3. Material and Methods
3
.1. Chemical Reagents and Instruments
1
Commercial reagents were used without further purification. The H-NMR spectra
500 MHz) were measured using tetramethylsilane (Acros Organics, Fair Lawn, NJ, USA)
(
as internal standard. Thin layer chromatography (TLC) was performed on E. Merck 5554
silica gel plates (Merck KGaA, Darmstadt, Germany). The mass spectra were recorded on a
Finnigan LCQ Classic mass spectrometer (Thermo Electron, Langenselbold, Germany).
3.2. General Procedure for the Synthesis of Compounds 1
One equivalent of the aromatic aldehyde, two equivalents of the methyl propiolate
(
Acros Organics, Fair Lawn, NJ, USA) and one equivalent of the benzylamine were dis-
◦
solved in 2 mL of freshly distilled acetic acid and heated under reflux and stirring at 100 C
for two hours. After finishing the reaction mixture was cooled down to room temperature
and water was added. Then extraction followed with chloroform and was repeated for two
times. The unified organcic layer was dried over sodium sulfate and reduced in volume.
Then methanol was added under cooling to give the final products.
3.3. General Procedure for the Cage Dimer Formation of Compounds 2
One and a half equivalent of the respective monomeric 1,4-dihydropyridine
1 was
dissolved in dry THF (Sigma Aldrich, St. Louis, MO, USA) in a quartz flask. After
®
fumigating with argon the flask was closed and irradiated with Ultra Vitalux lamps
(
6
Osram, Munic, Germany) that produce light of wavelengths > 270 nm from a distance of
0 cm at room temperature. The reaction was controlled by the use of TLC. A precipitation
of the formed cage dimers was observed. After finishing of the irradiation that followed
after an observed disappearance of the fluorescent starting compound on the TLC sheets
the formed product was filtered off or separated from the solution under cooling and a
following filtration procedure. A final recrystallization was carried out from methanol.
2
2

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3
.4. General Procedure for the Ester Group Reduction in the Cage Dimers to Form the Target
Compounds 3a–k
Seventy micromole of the respective cage dimer
fumigating of the solution with argon it was stirred and cooled down to
.12 mmol of lithium aluminium hydride (Sigma Aldrich, St. Louis, MO, USA) were
2
were dissolved in dried THF. After
◦
−
8 C. Then
1
added dropwise as a one molare solution in THF under stirring for additional three hours
at the low temperature. The reaction was followed by the use TLC. After a complete
disappearance of the starting compound on the TLC sheet portions of a 20% solution of
◦
potassium hydroxide and of ice water were added at a temperature of 0 C. Then the
organic layer was extracted with chloroform for several times. The organic layer was
unified, dried over sodium sulfate and filtered. Then the solution volume was reduced
under low pressure and a mixture of chloroform, diethyl ether and petrol ether was added
dropwise. The precipitated target compounds 3a–k were recrystallized from methanol.
3
[
.4.1. 3,9-Dibenzyl-1,5,7,11-tetrakishydroxymethyl-6,12-diphenyl-3,9-diazhexacyclo-
6.4.0.02 .0 .0 ]dodecane 3a
.7
4.11 5.10
◦
1
Yield 92%; white solid; mp 226–231 C; H NMR (DMSO-d )
δ
7.79 (d, J = 8.0 Hz, 2H,
6
2
7
-H of phenyl), 7.34–7.29 (m, 10H, benzylic H), 7.27 (d, J = 8.0 Hz, 2H, 6-H of phenyl), 7.12,
.05, 7.03 (t, J = 8.0, 8,0, 8,0 Hz, 6H, 3-, 4-, 5-H of phenyl), 4.41 (part X of a ABX system, 4H,
CH OH), 4.13 (s, 4H, NCH ), 3.66 (s, 2H, 6-, 12-H), 3.16–3.06 (part AB of the ABX system,
2
2
+
8
2
H, CH OH), 2.95 (s, 4H, 2-, 4-, 8-, 10-H); MS (ESI), m/z = 615 [M + H ]; IR (KBr): 3441,
930, 1600, 1493 cm . Anal. (C H N O
C 77.14, H 6.79, N 4.46.
2
−
1
×
0.5 H O) Calc. C 77.02, H 6.95, N 4.49; Found
2
40
42
2
4
3
.4.2. 3,9-Bis(4-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-diphenyl-3,9-
2
.7 4.11 5.10
diazhexa-cyclo[6.4.0.0 .0 .0 ]dodecane 3b
◦
1
Yield 55%; white solid; mp 197–202 C; H NMR (DMSO-d ) δ 7.81 (d, J = 7.6 Hz, 2H,
6
6
6
-H of phenyl), 7.27–7.24 (m, 4H, 2-, 6-H of benzyl), 7.13–7.00 (m, 6H, 3-, 4-, 5-H of phenyl),
.87–6.85 (m, 4H, 3-, 5-H of benzyl), 4.40 (part X of a ABX system, 4H, CH OH), 4.05 (s, 4H,
2
NCH ), 3.74 (s, 6H, OCH ), 3.65 (s, 2H, 6-, 12-H), 3.11–3.04 (part AB of the ABX system, 8H,
2
3
+
CH OH), 2.93 (s, 4H, 2-, 4-, 8-, 10-H); MS (ESI), m/z = 675 [M + H ]; IR (KBr): 3458, 2913,
2
−
607, 1512 cm . Anal. (C H N O ) Calc. C 74.69, H 6.87, N 4.15; Found C 74.45, H 6.75,
42 46 2 6
1
1
N 3.95.
3
.4.3. 3,9-Bis(4-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(4-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3c
◦
1
Yield 45%; white solid; mp 238–241 C; H NMR (DMSO-d )
δ
7.70 (d, J = 8.7 Hz, 2H,
-H of phenyl), 7.24 (m, 4H, 2-, 6-H of benzyl), 7.17 (d, J = 8.4 Hz, 2H, 2-H of phenyl), 6.88
m, 4H, 3-, 5-H of benzyl), 6.67 (d, J = 8.4 Hz, 2H, 3-H of phenyl), 6.53 (d, J = 8.7 Hz, 2H, 5-H
of phenyl), 4.36 (part X of a ABX system, 4H, CH OH), 4.02 (s, 4H, NCH ), 3.74, 3.69 (2 × s,
6
6
(
2
2
1
2H, OCH ), 3.57 (s, 2H, 6-, 12-H), 3.16–3.05 (part AB of the ABX system, 8H, CH OH), 2.89
3
2
+
(
1
s, 4H, 2-, 4-, 8-, 10-H); MS (ESI), m/z = 757 [M + Na ]; IR (KBr): 3357, 2926, 2835, 1609, 1462,
−
246, 1077 cm . Anal. (C H N O ) Calc. C 71.90, H 6.86, N 3.81; Found C 71.55, H 6.95,
44 50 2 8
1
N 3.75.
3
.4.4. 3,9-Bis(3-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(4-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3d
◦
1
Yield 38%; white solid; mp 203–205 C; H NMR (DMSO-d )
δ
7.01 (d, J = 8.6 Hz,
H, 6-H of phenyl), 7.22–6.85 (m, 4H, 2-, 6-H of benzyl), 6.78 (d, J = 8.4 Hz, 2H, 2-H of
phenyl), 6.75–6.70 (m, 4H, 3-, 5-H of benzyl), 6.68 (d, J = 8.4 Hz, 2H, 3-H of phenyl), 6.52
d, J = 8.6 Hz, 2H, 5-H of phenyl), 4.39 (part X of a ABX system, 4H, CH OH), 4.08 (s, 4H,
6
2
(
2
NCH ), 3.69, 3.64 (2
×
s, 12H, OCH ), 3.58 (s, 2H, 6-, 12-H), 3.19–3.09 (part AB of the ABX
2
3
+
system, 8H, CH OH), 2.92 (s, 4H, 2-, 4-, 8-, 10-H); MS (ESI), m/z = 735 [M + H ]; IR (KBr):
2

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3
442, 2915, 2835, 1608, 1455, 1252, 1038 cm 1. Anal. (C H N O ) Calc. C 71.90, H 6.86,
−
44 50 2 8
N 3.81; Found C 71.87, H 6.90, N 3.58.
3
.4.5. 3,9-Bis(3-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3e
◦
1
Yield 32%; white solid; mp 204–206 C; H NMR (DMSO-d )
δ
6.92–6.83 (m 8H, arylic
6
H of benzyl), 7.48–6.62 (m, 8H, arylic H of phenyl), 4.44–4.39 (part X of a ABX system, 4H,
CH OH), 4.17-4,10 (m, 4H, NCH ), 3.69, 3.63 (2
×
s, 6H, OCH ), 3.58 (s, 2H, 6-, 12-H), 3.52,
3
2
2
3
.50 (2
×
s, 6H, OCH ), 3.18–3.09 (part AB of the ABX system, 8H, CH OH), 2.96 (s, 4H,
3
2
+
2
-, 4-, 8-, 10-H); MS (ESI), m/z = 735 [M + H ]; IR (KBr): 3428, 2954, 2869, 1597, 1455, 1378,
−
259, 1073 cm . Anal. (C H N O ) Calc. C 71.90, H 6.86, N 3.81; Found C 71.65, H 6.75,
44 50 2 8
1
1
N 3.82.
3
.4.6. 3,9-Bis(4-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3f
◦
1
Yield 38%; white solid; mp 206–208 C; H NMR (DMSO-d )
δ
7.25 (m, 4H, 2-, 6-H
of benzyl), 6.86 (m, 4H, 3-, 5-H of benzyl), 7.53–6.61 (m, 8H, arylic H of phenyl), 4.39–4.37
part X of a ABX system, 4H, CH OH), 3.93–4,13 (m, 4H, NCH ), 3.73, 3.71, 3.69 (3 s, 9H,
OCH ), 3.57 (s, 2H, 6-, 12-H), 3.49 (s, 3H, OCH ), 3.21–3.06 (part AB of the ABX system, 8H,
6
(
×
2
2
3
3
+
CH OH), 2.93 (s, 2H, 2-, 8-H), 2.90 (s, 2H, 4-, 10-H); MS (ESI), m/z = 733 [M
376, 2953, 2835, 1607, 1463, 1259, 1073 cm . Anal. (C H N O ) Calc. C 71.90, H 6.86,
−
H ]; IR (KBr):
2
−
1
3
44
50
2
8
N 3.81; Found C 71.63, H 6.72, N 3.90.
3
.4.7. 3,9-Bis(3,4-dimethoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3g
◦
1
Yield 41%; white solid; mp 200–203 C; H NMR (DMSO-d )
δ
6.87–6.79 (m, 6H,
6
benzylic H), 7.48–6.63 (m, 8H, phenylic H), 4.41–4.36 (part X of a ABX system, 4H, CH OH),
2
4
.04 (s, 4H, NCH ), 3.71, 3.69 (2
×
s, 6H, OCH ), 3.59 (s, 2H, 6-, 12-H), 3.58, 3.53, 3.49, 3.45
2
3
(
2
4 × s, 12H, OCH ), 3.21–3.09 (part AB of the ABX system, 8H, CH OH), 2.97-2.93 (m, 4H,
3
2
+
-, 4-, 8-, 10-H); MS (ESI), m/z = 817 [M + Na ]; IR (KBr): 3536, 2933, 2830, 1603, 1464, 1259,
−
027 cm . Anal. (C H N O ) Calc. C 69.50, H 6.85, N 3.52; Found C 69.10, H 6.67,
46 54 2 10
1
1
N 3.77.
3
.4.8. 3,9-Bis(3,4-dimethoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(4-
2
.7 4.11 5.10
methoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3h
◦
1
0
Yield 20%; white solid; mp 204–206 C; H NMR (DMSO-d ) δ 7.71 (m, 2H, AA -part
6
0
0
0
0
0
0
of the AA BB systems, 6-H of phenyl), 7.21 (m, 2H, AA BB -part of the AA BB system, 2-H
0
0
0
of phenyl), 6.86-6.80 (m, 6H, benzylic H), 6.67 (m, 2H, BB -part of the AA BB system, 3-H
0
0
of phenyl), 6.57 (m, 2H, BB-part of the AA BB system, 5-H of phenyl), 4.35 (part X of a ABX
system, 4H, CH OH), 4.05 (s, 4H, NCH ), 3.72, 3.69 (2 s, 6H, OCH ), 3.61 (s, 2H, 6-, 12-H),
×
2
2
3
3
4
1
.58, 3.49 (2
×
s, 12H, OCH ), 3.20–3.10 (part AB of the ABX system, 8H, CH OH), 2.91 (s,
3 2
+
H, 2-, 4-, 8-, 10-H); MS (ESI), m/z = 817 [M + Na ]; IR (KBr): 3536, 2954, 2837, 1607, 1463,
−
259, 1072 cm . Anal. (C H N O ) Calc. C 69.50, H 6.85, N 3.52; Found C 69.37, H 6.87,
46 54 2 10
1
N 3.53.
3
.4.9. 3,9-Bis(3-dimethoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3,4-
2
.7 4.11 5.10
dimethoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3i
◦
1
Yield 28%; white solid; mp 179–182 C; H NMR (DMSO-d )
δ
6.89–6.80 (m, 8H,
6
benzylic H), 7.40–6.68 (m, 6H, phenylic H), 4.40 (part X of a ABX system, 4H, CH OH), 4.08
2
(
3
s, 4H, NCH ), 3.70, 3.68, 3.64 (3
.24–3.19 (part AB of the ABX system, 8H, CH OH), 3.05 (m, 2H, 2-, 8-H), 2.91 (m, 2H, 4-,
×
s, 15H, OCH ), 3.53 (s, 2H, 6-, 12-H), 3.42 (s, 3H, OCH ),
2
3
3
2

Int. J. Mol. Sci. 2021, 22, 5098
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+
1
0-H); MS (ESI), m/z = 817 [M + Na ]; IR (KBr): 3478, 2930, 2869, 1599, 1463, 1258, 1073
−
1
cm . Anal. (C H N O ) Calc. C 69.50, H 6.85, N 3.52; Found C 69.21, H 6.69, N 3.32.
46
54
2
10
3
.4.10. 3,9-Bis(4-methoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3,4-
2
.7 4.11 5.10
dimethoxyphenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3j
◦
1
Yield 36%; white solid; mp 206–208 C; H NMR (DMSO-d )
δ
7.23 (m, 4H, 2-, 6-H of
benzyl), 6.86 (m, 4H, 3-, 5-H of benzyl), 7.44–6.53 (m, 6H, arylic H of phenyl), 4.36 (part
X of a ABX system, 4H, CH OH), 4.02 (m, 4H, NCH ), 3.73, 3.70 (2 s, 12H, OCH ), 3.53
6
×
2
2
3
(
3
s, 2H, 6-, 12-H), 3.49 (s, 6H, OCH ), 3.16–3.11 (part AB of the ABX system, 8H, CH OH),
3
2
+
.01 (s, 2H, 2-, 8-H), 2.91 (s, 2H, 4-, 10-H); MS (ESI), m/z = 817 [M + Na ]; IR (KBr): 3487,
−
929, 2868, 1609, 1463, 1249, 1073 cm . Anal. (C H N O ) Calc. C 69.50, H 6.85, N 3.52;
46 54 2 10
1
2
Found C 69.48, H 6.60, N 3.52.
3
.4.11. 3,9-Bis(3,4-dimethoxybenzyl)-1,5,7,11-tetrakishydroxymethyl-6,12-bis(3,4-
2
.7 4.11 5.10
dimethoxy-phenyl)-3,9-diazhexacyclo[6.4.0.0 .0 .0 ]dodecane 3k
◦
1
Yield 90%; white solid; mp 227–231 C; H NMR (DMSO-d )
δ
6.93–6.80 (m, 6H, arylic
6
H of benzyl), 7.43–6.57 (m, 6H, arylic H of phenyl), 4.36 (part X of a ABX system, 4H,
CH OH), 4.02, 4.05 (2
×
s, 4H, NCH ), 3.72, 3.71 (2
×
s, 12H, OCH ), 3.70, 3.68, 3.59 (3
×
s,
2
2
3
9
H, OCH ), 3.54 (s, 2H, 6-, 12-H), 3.49 (s, 3H, OCH ), 3.20–3.12 (part AB of the ABX system,
3
3
+
8
H, CH OH), 3.01 (s, 2H, 2-, 8-H), 2.91 (s, 2H, 4-, 10-H); MS (ESI), m/z = 877 [M + Na ]; IR
2
−
1
(KBr): 3323, 2926, 2835, 1594, 1463, 1255, 1072 cm . Anal. (C H N O ) Calc. C 67.43,
42
58
2
12
H 6.84, N 3.28; Found C 67.24, H 6.60, N 3.22.
3.5. MRP4 Inhibition Assay
The human pancreatic carcinoma cell lines colo357 and colo357_MRP4 were used.
Colo357_MRP4 cells were generated by transfection of the colo357 cells with a pcDNA3.1-
Hygro/MRP4 construct described in [36] using FuGENE transfection reagent (Roche,
Mannheim, Germany) according to the manufacturer’s protocol. After 48 h, the cells
were split, and stable transfectants were selected using medium containing hygromycin B.
MRP4 overexpression was confirmed by Western blotting after separating proteins on a
8% polyacrylamide gel, transfer to nitrocellulose membranes and detecting MRP4 protein
using the MRP4 rabbit polyclonal antiserum SNG [36] in a 1:1000 dilution followed by a
goat anti-rabbit-IgG HPR-conjugate at a 1:2000 dilution as shown in the Supplementary
Materials. Both cell lines were cultured in RPMI-1640 medium that was supplemented
◦
with fetal bovine serum (10%) and penicillin/streptomycin (1%) at 37 C and under carbon
dioxide atmosphere (5%).
In the assay, each 200.000 cells were given in an Eppendorf tube. The cells were
◦
centrifuged at 2000 rpm at 4 C. The supernatant was removed and the samples were
stored on ice. Then they were resuspended in RPMI-1640 medium and test compounds
and MK571 control were added from stock solutions of 4000
µ
M in DMSO to give a final
◦
concentration of 10
µM. The samples were cultured at 37 C for 20 min and 1200 rpm in a
thermomixer. Then the fluorescent calcein was added from a PBS solution to give a final
concentration of 0.005 M. The samples were centrifuged again and the supernatant was
µ
removed. Then PBS was added and the samples were centrifuged again. That washing
procedure was repeated. Finally, the fluorescence of the resuspended cells was measured by
flow cytometry using 10.000 cells and a MACSQuant Analyzer (Miltenyi Biotec, Bergisch
Gladbach, Germany). The measurement was conducted each for three times for inhibitor
treated and untreated cells of both cell lines.
The FAR values were calculated as ratio of the fluorescence of the treated colo357_MRP4
to the tretated colo357 cells with each values corrected by the fluorescence of the untretated
colo cells.

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3
.6. MTT-Based Viability Assay
0.000 cells of each cell line were cultured in wells of a 96-well plate at 37 C un-
der a carbon dioxide atmosphere (5%) for 24 h. Then 50 M of the MRP4 substrate
-mercaptopurin, the test compound and MK571, each in a 10 M final concentration
were added. The plate was incubated for 48 h under the original culture conditions. Then
the MTT reagent was added to each well (10 L of a stock solution of 5 mg/mL in PBS)
and incubation continued for 4 h. Then 100 L DMSO were added to each well to solve
◦
1
µ
6
µ
µ
µ
the formazan reduction product. The plate was shaken for 30 min on a plate shaker and
finally the formazan absorption was measured at 570 nm and 630 nm as reference wave
length on an Infinite 200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland).
For further analyses, reference values were subtracted from primary values at 570 nm and
resulting values were normalized to values of cells treated with the solvent DMSO, which
represent 100% cell viability. Measurements were repeated three times independently,
mean
±
SD of triplicate values were calculated and statistical analysis was performed
using GraphPadPrism version 5.02 (GraphPad Software, San Diego, CA, USA).
4. Conclusions
Due to ongoing resistances against anticancer drugs the challenge to combat cancer
is the development of novel anticancer drugs that affect novel target structures. The
expression of transmembrane efflux pumps mostly under therapy becomes a central
problem as they may also transport those novel drugs out of the cells and thus constitute
the multidrug resistance. The discovery to find effective inhibitors of such efflux pumps
would mean a great progress for anticancer therapy.
We discovered a novel class of symmetric cage compounds that effectively inhibits the
activity of the symmetric efflux pump MRP4. As only few inhibitors of MRP4 are known
an insight in structure–activity relationships of MRP4 inhibitors has not been possible so
far. Our compound class gives first information that methoxy functions are of favor for the
inhibitory properties. Increasing numbers of such methoxy functions enlarge the activity
in case of a symmetric distribution within the symmetrically arranged aromatic residues.
Those results suggest a symmetric binding of the inhibitors to a so far unknown binding
region. Additionally, one best compound was able to resensitise the MRP4-expressing
subline to the used anticancer drug. These results encourage for further preclinical studies
to combat the cause of the MDR in cancer.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22105098/s1. The following are available online, Western blot of MRP4 overexpression.
Author Contributions: Investigation, D.K., synthesis, H.D., data curation, P.W., validation and
formal analysis, C.A.R., conceptualization and writing, A.H. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Additional data are available from the authors on request.
Acknowledgments: The authors acknowledge the financial support of the Open Access Publication
Fund of the Martin Luther University Halle-Wittenberg.
Conflicts of Interest: The authors declare no conflict of interest.
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