Search for ABCB1 modulators among 2-amine-5-arylideneimidazolones as a new perspective to overcome cancer multidrug resistance

Article abstract of DOI:10.3390/molecules25092258

Multidrug resistance (MDR) is a severe problem in the treatment of cancer with overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis, involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight into the mechanisms of action using either the luminescent Pgp-Glo Assay in vitro or docking studies to human Pgp was performed. The safety profile in vitro was examined. Structure-activity relationship (SAR) analysis was discussed. The most active compounds, representing both 2-substituted- (11) and Dimroth-rearranged 3-substituted (18) imidazolone topologies, displayed 1.38-1.46 fold stronger efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition via competitive action on the Pgp verapamil binding site was predicted in silico.

Full text of DOI:10.3390/molecules25092258

molecules
Article
Search for ABCB1 Modulators Among
2-Amine-5-Arylideneimidazolones as a New
Perspective to Overcome Cancer Multidrug Resistance
Aneta Kaczor 1 , Márta Nové 2, Annamária Kincses 2, gabriella Spengler 2
,
Ewa Szyman´ska 1 , gniewomir Latacz 1 and Jadwiga Handzlik 1,
*
1
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University
Medical College, 9 Medyczna Street, 30-688 Kraków, Poland; aneta.kaczor@doctoral.uj.edu.pl (A.K.);
ewa.szymanska@uj.edu.pl (E.S.); glatacz@cm-uj.krakow.pl (G.L.)
Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged,
Dóm tér 10, H-6720 Szeged, Hungary; nove.marta@med.u-szeged.hu (M.N.);
kincses.annamaria@med.u-szeged.hu (A.K.); spengler.gabriella@med.u-szeged.hu (G.S.)
Correspondence: j.handzlik@uj.edu.pl
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 March 2020; Accepted: 3 May 2020; Published: 11 May 2020
Abstract: Multidrug resistance (MDR) is a severe problem in the treatment of cancer with
overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series
of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two
different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis,
involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the
ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma
cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were
investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight
into the mechanisms of action using either the luminescent Pgp-Glo
™ Assay in vitro or docking studies
to human Pgp was performed. The safety profile in vitro was examined. Structure–activity relationship
(SAR) analysis was discussed. The most active compounds, representing both 2-substituted- (11) and
Dimroth-rearranged 3-substituted (18) imidazolone topologies, displayed 1.38–1.46 fold stronger
efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin
in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that
active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition
via competitive action on the Pgp verapamil binding site was predicted in silico.
Keywords: ABCB1 modulator; 5-arylideneimidazolone; Dimroth rearrangement; cancer MDR; Pgp;
T-lymphoma cancer cell; rhodamine 123 accumulation assay; Pgp docking
1. Introduction
Multidrug resistance (MDR) is nowadays one of the most important problems in the treatment
of various diseases including cancer. The lack of efficacy seen after usage of chemotherapeutic
drugs, effective previously, is connected to various mechanisms. One of them involves an increased
drug efflux through membrane transporters, e.g., P-glycoprotein (Pgp, ABCB1) [
ATP-binding cassette protein, encoded by the MDR1 (ABCB1) gene, which is present in both normal
and cancer cells [ ]. The structure includes two hydrophobic transmembrane domains, which contain
drug binding domain (DBD). In the cytoplasmic region, there are two nucleotide binding domains
(NBDs) in which coupling ATP hydrolysis takes place [ ]. ABCB1 obtains energy from the ATP
hydrolysis process and uses it to translocate to the cell exterior structurally unrelated compounds, e.g.,
1–4]. Pgp is an
5,6
5,7–9
Molecules 2020, 25, 2258; doi:10.3390/molecules25092258
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2258
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chemotherapeutics, xenobiotics [10
,11]. Hence, ABCB1 overexpression became the reason for the lack
of response in the applied therapy, due to lowered drug concentration, often below the therapeutic
point [ 12]. This mechanism seems to play an important role in cancer MDR, mostly because many
1–4,
anticancer drugs are ABCB1 substrates, e.g., doxorubicin, paclitaxel, vincristine [13]. Additionally, cells
with overexpression of this pump may inhibit the process of apoptosis [14]. This may lead to resistance
even against chemotherapeutics, which are not Pgp substrates [6]. In this context, searching for ABCB1
inhibitors seems to be a prominent way to overcome cancer MDR [15]. Earlier studies provided four
main groups of Pgp inhibitors, with respect to their potency, selectivity, and potential of drug–drug
interaction [16]. However, the toxicity of previously found ABCB1 inhibitors is a significant problem
that limits their therapeutic application [12]. For example, verapamil, which belongs to the first group,
demonstrates cardiotoxicity due to the higher affinity for the calcium channel than for ABCB1 [17].
Taking into account the important role of MDR in cancer, searching for new compounds that are able
to increase the concentration of chemotherapeutic drugs is currently a crucial task of cancer medicinal
chemistry [12].
On the other hand, compounds with the hydantoin (imidazole-2,4-dione) scaffold are a big
structural family in the search for new drugs with various therapeutic directions, to give active agents
with antiarrhythmic, anti-inflammatory, or other physiological properties [18–20]. Representatives
from this group displayed potential activity as antibiotic adjuvants able to block mechanism(s) of
bacterial MDR, e.g., efflux pumps (compounds 1, 2, Figure 1) [21,22].
Figure 1. 5-Arylideneimidazolones able to block mechanism of bacterial multidrug resistance (MDR)
(1,2) [21,22].
Furthermore, some (thio)hydantoin derivatives (3–5; Figure 2) demonstrated the ability to
modulate the ABCB1 efflux pump in cancer cells at the same or stronger level than verapamil (6;
Figure 2) [23,24].
Figure 2. Previously found (thio)hydantoin derivatives able to modulate ABCB1 in cancer cells (
and the reference modulator, verapamil (6) [23,24].
3–5)

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SAR analysis performed within the previous studies indicated that the arylidene moiety at position
5 of imidazolone was crucial for the modulatory action, while the presence of hydroxyl groups was
unprofitable [24].
Taking this into account, a series of new 5-arylideneimidazolones (7–20) were designed,
synthesized, and tested as ABCB1 modulators in mouse T-lymphoma cells within this study. The
designed compounds possess amine fragments, containing a morpholine moiety, at position 2 (group A,
Table 1) or at positions 2 and 3 (group B, Table 1) of the imidazolone ring. In order to determine MDR
efflux pump modulatory properties for the new compounds, a dye-substrate accumulation assay was
performed in two T-lymphoma cell lines, i.e., parental (PAR) and multidrug resistant one (MDR) with
overexpression of ABCB1. The cytotoxicity and antiproliferative effect of the obtained compounds
were examined in these cell lines. In search for probable molecular mechanisms of modulatory action
of active compounds, both in silico and in vitro studies were carried out. The safety of the most active
compounds was tested in vitro in the human HEK-293 cell line.
Table 1. Structures of tested 5-arylideneimidazolones (7–20).
Cpd
Group
R
n
Amine
7
8
A
A
—
—
9
A
—
10
11
12
A
A
A
—
—
—
13
A
—
14
15
B
B
2
3
—
—
16
17
18
B
B
B
3
3
3
—
—
—
19
20
B
B
3
3
—
—

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2. Results
2.1. Synthesis
The compounds (
7
–
20) were synthesized within 3–4-step synthesis pathways, according to
Scheme 1. The synthesis of compounds 16 and 19 is described elsewhere [22]. In the first step,
Knoevenagel condensation of thiohydantoin with suitable aromatic aldehydes was applied to give
5-arylideneimidazolone intermediates (21
in sodium ethanolate solution. Treatment of the resulting S-methylated derivatives (27
commercial secondary- (Group A, Table 1) or primary- (Group B, Table 1) amines allowed the target
2-amine-5-arylideneimidazolones ( 20) to be yielded. In the case of group B (14 20), the Dimroth
–
26) that were subsequently reacted with iodomethane
–
32) with
7–
–
rearrangement was observed to proceed via a similar mechanism to that described previously [22]. Thus,
the final products of group B contain the primary amine group at position 2 and the morpholine-alkyl
moiety at position 3 of the imidazolone core.
Scheme 1. Synthesis route for compounds 7–20; (i), CH3COONa, CH3COOH, reflux, 5–7 h; (ii)
CH3I, C2H5ONa, rt, 24 h; (iii) proper amine, oil bath 120–130 ◦C, 15 min; C2H5OH, reflux 5–7 h (iv)
Dimroth rearrangement.
2.2. Biological Screening
2.2.1. The Rhodamine 123 Accumulation Assay
Compounds 7–20 were investigated for their ABCB1 efflux pump inhibiting activity using the
rhodamine 123 accumulation assay. The study was carried out on mouse T-lymphoma cell lines,
including the parental sensitive line (PAR) and its multidrug-resistant subline (MDR) transfected with
the human MDR1 (ABCB1) gene. Results were acquired by flow cytometry measuring the intracellular
accumulation of the fluorescent ABCB1 substrate rhodamine 123. The fluorescence activity ratio (FAR),
as a measure of the efflux pump modulating properties, was calculated based on the fluorescence
data obtained [23–25]. Verapamil was used as a reference ABCB1-modulator with respect to two
assays performed for appropriate compounds (superscript 1 or 2, Table 2). Therefore, results were also
expressed as % of FAR for verapamil in the appropriate assay (quotient, Table 2) in order to enable
a good comparison of results within the whole series [26].

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Table 2. Effects of 5-arylideneimidazolones (7–20) on rhodamine 123 accumulation by multidrug
-resistant (MDR) mouse T-lymphoma cells.
Effects on Rhodamine 123 Accumulation
Concentration 2 µM
Concentration 20 µM
FAR FAR Quotient (%)
Cpd
FAR
FAR Quotient (%)
7 1
8 2
0.955
1.359
0.766
1.108
1.203
1.161
0.968
0.814
0.887
0.927
0.929
1.038
1.272
0.896
-
7.31
8.49
5.86
6.92
7.51
8.88
6.04
6.22
6.78
7.09
5.80
7.94
7.94
0.927
2.409
1.645
1.992
23.419
6.045
2.073
0.847
2.221
2.426
7.09
15.04
12.58
12.44
146.22
46.23
12.94
6.48
16.98
18.55
14.37
138.37
10.08
7.72
9 1
10 2
11 2
12 1
13 2
14 1
15 1
16 1
17 2
18 1
19 2
20 2
VER
DMSO
2.301
18.095
1.615
5.59
1.237
13.077 1/16.016 2
-
-
100.00
-
0.672 1/0.664 2 (V/V%)
5.141/4.15 2
VER, verapamil; FAR, fluorescence activity ratio; FAR quotient: FAR of a compound related to FAR of VER tested at
20 µM; 1,2 Compounds tested in two groups with respect to different FAR values for VER and DMSO. The highest
quotients underlined.
Results indicate that the arylideneimidazolones
moderate (12) ABCB1 modulatory properties (FAR = 2.07–6.05). Two compounds (11
were highly effective, almost 1.5 fold more active than verapamil tested at the same concentration
(20 M). The fluorescence activity ratio (FAR) for compound 11 was 23.419, with a quotient of 146.22%,
8,
12
,
13, and 15
–
17 demonstrated slight to
,
18), however,
µ
while compound 18 displayed 138.37% of the activity determined for verapamil. At 10 fold lower
concentration (2 µM), the whole series (7–20) was not active.
2.2.2. Cancer Cytotoxicity Assays
The cytotoxicity of compounds 7–20 was examined in both aforementioned PAR and MDR mouse
T-lymphoma cell lines, using the MTT assay to estimate IC50 values. The selectivity index for cytotoxic
action on MDR with respect to PAR cells was calculated (SI, Table 3). The morpholine-imidazolones
(7
–
20) showed weak (IC50 > 20
µM) to moderate (10 µM < IC50 < 20 µM) cytotoxic effects, and all
derivatives were, furthermore, more potent in PAR cells, as shown by the selectivity index (SI < 1).
This range of SI may indicate ABCB1-efflux pump substrate properties for the whole series (
especially distinct in the case of 19 (SI ≤ 0.29), and slightly weaker for 8, 12, 16, and 18.
7–20),

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Table 3. Cytotoxic effects of 2-amine-5-arylideneimidazolones (7–20) on cancer cells.
Cpd
Mouse T-Lymphoma Cells
MDR
PAR
IC50 (µM)
SD ±
IC50 (µM)
SD ±
SI
7
8
9
53.52
23.97
12.82
53.96
39.06
15.82
82.22
36.85
17.48
16.00
21.49
15.96
28.59
19.60
0.7
2.37
2.09
1.35
3.80
2.36
1.15
3.08
1.12
0.53
0.98
0.45
0.68
0.60
2.35
0.56
-
54.17
46.42
18.11
63.76
45.50
30.80
>100
57.37
20.76
31.29
23.41
27.75
>100
24.88
2.14
1.42
3.37
2.53
0.30
0.60
1.56
-
5.74
0.91
2.11
1.21
1.29
-
0.99
0.52
0.71
0.85
0.86
0.51
≤0.82
0.64
0.84
0.51
0.92
0.58
≤0.29
0.79
0.33
-
10
11
12
13
14
15
16
17
18
19
20
DOX
0.10
0.76
-
DMSO
>1%
>1%
PAR, parental T-lymphoma cells; MDR, multidrug-resistant T-lymphoma cells overproducing efflux pump Pgp;
DOX, doxorubicin; DMSO, dimethyl sulfoxide; SD, standard deviation; SI, selectivity index; starting concentration
of tested compounds: 100 µM.
The relatively strongest T-lymphoma cytotoxic action could be observed in the case of compound
9
with IC50 values of 12.82
µM (PAR) and 18.11
µM (MDR). The rest of the compounds displayed IC50
> 20 M in the MDR cells, and 15.80
µ
µM < IC50 < 20
µ
M in the case of the most active ones (12 15 16,
,
,
18, and 20) in parental T-lymphoma cells.
2.2.3. Influence on Cancer Cell Proliferation
Compounds (
mouse T-lymphoma cancer cells. The compounds (
effects (Table 4). Compared to the cytotoxicity results, the effects were even stronger, with IC50 values
in the range of 7.78 M ( ) to 26.42 M (10) in PAR cells, 10.82 M (18) to 48.58 M (13) in the case of
MDR. Compound 18 was the most active agent, with an IC50 of 10.82 M in the multidrug-resistant
subline of mouse T-lymphoma cells. Half of the tested compounds exhibited an IC50 lower than 20
7–
20) were also tested for their ability to inhibit proliferation of both PAR and MDR
7–
20) displayed moderate to weak antiproliferative
µ
9
µ
µ
µ
µ
µM
in MDR cells. The antiproliferative effects were 28–94 fold weaker than those of the reference anticancer
drug (doxorubicin) in PAR, and 6–28 fold in the resistant cells. Interestingly, the relatively most active
agent (18) showed a higher antiproliferative potency in MDR cells compared to the parental ones.
A similar trend was also observed for 10, whereas the antiproliferative action was comparable in both
PAR and MDR cells for compounds 11 and 16.

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Table 4. Antiproliferative effects of 2-amine-5-arylideneimidazolones (7–20) on cancer cells.
Cpd
Mouse T-Lymphoma Cells
MDR
PAR
IC50 (µM)
SD ±
IC50 (µM)
SD ±
SI
7
8
9
10
11
12
13
14
15
16
17
18
19
17.60
14.77
7.78
0.87
1.41
0.31
1.43
0.89
0.08
0.17
0.77
0.32
0.38
1.07
0.20
0.90
0.41
0.06
-
26.33
25.98
16.53
22.45
25.60
16.20
48.58
27.86
13.64
12.16
14.25
10.82
43.82
17.31
1.75
0.32
1.17
1.10
0.94
0.26
0.30
0.21
0.95
0.22
0.04
0.58
0.61
4.29
0.70
0.38
-
0.67
0.57
0.47
1.18
0.97
0.78
0.36
0.89
0.75
0.94
0.85
1.24
0.33
0.85
0.16
-
26.42
24.77
12.70
17.37
24.93
10.26
11.39
12.17
13.38
14.39
14.78
0.28
20
DOX
DMSO
>1%
>1%
PAR, parental T-lymphoma cells; MDR, multidrug-resistant T-lymphoma cells overproducing efflux pump Pgp; SD,
standard deviation; SI, selectivity index; starting concentration of tested compounds: 100 µM.
2.3. Insight into Molecular Mechanisms of the Efflux Pump Modulation
Three compounds with the best efflux pump modulatory properties, i.e., two strong agents 11
and 18 and the moderate one (12), were selected for a deeper insight into mechanisms of action at the
molecular level, using both in vitro and in silico assays. The ABCB1-efflux pump (Pgp) as an example
of a transport protein represents a complex mechanism. Thus, two main mechanisms for the action
of modulators can be involved, including: (i) a consumption of energy coming from ATP hydrolysis,
which involves ATPase and is necessary to activate the Pgp pump to perform substrate efflux, and
(ii) a competitive modulator-substrate action to obtain the pump binding site, which protects the
substrate to be expelled, although the pump is active. In order to estimate the first mechanism, the
arylideneimidazolones were tested in the intrinsic activity Pgp ATPase assay in vitro, whereas their
action on the Pgp-binding site was examined on the basis of molecular modeling.
2.3.1. Intrinsic Activity toward ABCB1 In Vitro
Compounds 11
the Pgp pump in the luminescence Pgp-Glo
and protocols [27 29]. In this assay, luminescence was measured. The Pgp-dependent decreases
,
12, and 18 were tested at 100
µM on their influence on the ATPase activity of
™
Assay, according to previously described methods
–
in luminescence reflect the ATP consumption by Pgp. The ABCB1 basal ATP consumption (basal
activity) is considered a difference between the luminescent signal of samples treated with Na3VO4, the
selective and strong Pgp inhibitor (100% inhibition observed), and the luminescence of untreated Pgp
samples. Thus, inhibitors give results <100% of the basal activity, while stimulators/substrates cause
a statistically significant increase in the basal activity. Verapamil at 200 µM was used as a reference
modulator with the substrate/ATPase stimulatory mode of action, and caffeine was used to give a
negative control as a reference inactive compound toward the ABCB1 transporter.
Compounds 11 and 18 caused a statistically significant (p < 0.001 and p < 0.0001, respectively)
increase in basal activity in the range corresponding to verapamil (11) or higher (18). Compound 12
also increased the basal activity with the statistical significance (p < 0.05); however, the action was
much weaker than those observed for 18, verapamil, and 11, and only slightly stronger than that of the
negative control caffeine (Figure 3).

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Figure 3. The effect of the ABCB1 substrate verapamil (VER) (200
caffeine (CFN), and compounds 11 12, and 18 (100 M) on ABCB1 basal activity. The compounds are
recognized as ABCB1 substrates if they stimulate its basal activity (>100%). Data are presented as
the mean SD. Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s
µM), ABCB1-negative compound
,
µ
±
comparison test (* p < 0.05, *** p < 0.001, **** p < 0.0001 compared to the basal activity).
This trend of action is in some accordance with the modulatory potency of the compounds found in
the rhodamine 123 accumulation assays (11
Pgp inhibitory action of the imidazolones (11
–
18 >> 12, Table 2), but also excludes an ATPase-involving
12, and 18), indicating their Pgp-substrate properties.
,
These results suggest that the most probable mechanism of the inhibition of rhodamine 123 efflux,
observed for the compounds in the accumulation assay, is based on the modulator-substrate competitive
action to the binding site of ABCB1.
2.3.2. Molecular Modeling
In order to support the mechanism hypothesis postulated on the basis of results for both
the Pgp ATPase and the rhodamine 123 accumulation assays, possible binding modes of the
arylideneimidazolone compounds at the human Pgp protein were investigated using the molecular
docking method.
Homology Model of Human Pgp
The homology model of human Pgp was constructed by multiple comparative modeling, and
three reported X-ray structures of murine Pgp in the inward facing conformation: 4Q9H, 4XWK,
and 4M1M, were selected as templates due to the relatively high diffraction resolution. To improve
the quality of the model, the secondary structure of the linker region (amino acids 630–698) was
additionally calculated.
Docking Results Analysis
Structures of the three selected imidazolone modulators 11, 12, and 18, as well as verapamil, were
subjected to induced fit docking to the predicted homology model. Obtained top-ranked poses were
carefully analyzed in order to assess the possibility of competition between verapamil and imidazolone
derivatives for occupation of the same binding site or overlapping binding sites in Pgp.
The highly hydrophobic transmembrane interior of Pgp is believed to be the putative site for
substrate recognition [30–32]. According to the literature data, the residues inside this cavity are
neutral, devoid of a positive or negative charge, and the substrates bind to the protein mainly by means
of hydrophobic interactions and van der Waals and hydrogen bonds [31]. Most of the published data

Molecules 2020, 25, 2258
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report docking of ligands at their neutral form; however, some authors also describe the docking of
positively charged species [33 35]. For these reasons, we decided to perform docking to the homology
model for both neutral and positively charged forms of the ligands in separate runs. The protonation
states at pH = 7 2 were assigned during ligand preparation. In the case of predicted ionized forms of
–
±
the imidazolone derivatives (11, 12, and 18), the positive charge was located on the nitrogen atom of
the morpholine moiety.
The binding space for verapamil was generated as a box of 25 Å size, defined by a centroid of
amino acids found in the cysteine-scanning mutagenesis studies to be a part of the verapamil binding
site [36–38]. Docking results for the neutral and charged forms of this drug showed similar poses
for both species with comparable values of the binding energy expressed by the IFD score (Table S1
in Supplementary). As a reference for further investigations, one of the top-ranked poses (with a
docking score
experimental data. The binding mode of verapamil in this pose follows the pattern of protein–ligand
interactions predicted by other authors [32 34 39 41]. The molecule of the ligand is located in the
−
7.471 for the neutral form of verapamil) was chosen for its very good agreement with
,
, –
surroundings, including mostly hydrophobic residues: Met 69, Phe 72, Ile 306, Phe 335, Phe 336, Leu 339,
Leu 724, Phe 728, Phe 732, Leu 762, Phe 957, Leu 975, Phe 978, Phe 983, and Met 986 (Figure 4). Benzene
rings of the drug were found to interact with Phe 983 and Phe 336 by π–π stacking. Furthermore,
three hydrogen bonds were observed in this binding mode: two H-bonds formed by methoxy groups
interacting with Tyr 307 and Tyr 953, and a H-bond between Tyr 310 and the amine group of verapamil.
Figure 4. On the left: Position of the top-ranked docking pose of verapamil in human Pgp homology
model; On the right: Binding mode and molecular interactions observed for the top-ranked docking
pose of verapamil.
For the investigated 5-arylideneimidazolone derivatives 11, 12, and 18, the receptor grid box was
set to include the whole binding cavity formed by the transmembrane region of the human Pgp, as
defined for the P08183 sequence in the UniProt database [42]. Analysis of IFD docking results showed
that almost all of the obtained top-ranked poses for the neutral forms of compounds were located at
the top of the cavity (close to the outer leaflet of the membrane), interacting with residues of TM1, TM2,
TM5, TM6, TM7, TM8, TM9, TM11, and TM12. In contrast to the case of verapamil, docking scores,

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XP glide scores, and IFD scores demonstrated by the docked protonated forms of imidazolones were
significantly lower than those seen for the neutral forms; therefore, we did not investigate them further.
Out of docking poses obtained for the neutral forms of compounds, top-ranked poses detected
for at least two out of three docked imidazolones were collected in three clusters (Table S2 in the
Supplementary). The molecular interactions for each pose are shown for individual compounds with
the best docking score (Figures 5–7).
Figure 5.
(a) Binding mode of compounds 18 and 12 adopting docking pose 1; (b) molecular interactions
for the best docking pose 1 represented by compound 18.
Figure 6.
(a) Binding mode of compounds 11, 18, and 12 adopting docking pose 2; (b) molecular
interactions for the best docking pose 2 represented by compound 11.

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Figure 7.
(a) Binding mode of compounds 11 and 18 adopting docking pose 3; (b) molecular interactions
for the best docking pose 3 represented by compound 11.
The ligand–protein interaction pattern for pose 1 adopted by compound 18 (with the highest
docking score and the highest binding energy among all obtained poses, Table S2 in the Supplementary),
and by compound 12, corresponded to the binding mode of verapamil. In this pose 18, it was found
to form H-bonds with Tyr 307 and Tyr 310 in a similar way to verapamil, and additional H-bonds
between the amino group of the ligand and Ser 733, Ser 979, and the backbone chain of Ala 729 were
observed (Figure 5). The aromatic fluorene moiety of 18 interacted by π–π stacking with Phe 72 and Tyr
953. The whole ligand molecule was involved in numerous hydrophobic contacts with surrounding
residues, most of which is known to be a part of the verapamil binding site (Table S2) [31]. Furthermore,
according to literature reports, some of the amino acids listed in Table S2 for pose 1 are also a part of
the putative binding site for rhodamine dyes - R site (Met 68, Met 69, Phe 72, Phe 336, Phe 732, Tyr 953,
Phe 957, Leu 975, Phe 978) [31,33].
Pose 2, found in the case of all three docked imidazolones 11, 12, and 18, was, in fact, very similar
to pose 1, with the difference in location of the morpholine tail attached to the imidazolone ring at
the 2- or 3-position (Figure 6). This part of the molecule in pose 2 was oriented toward TM5 and
TM6 (instead of TM7, like in the case of pose 1) and formed hydrophobic contacts with Ile 306, Tyr
307, Tyr 310, and Leu 339. The aromatic moiety of ligands was involved in π–π stacking interactions
with surrounding phenylalanines and tyrosines (Phe 978 and Tyr 953 in the case of 11). A hydrogen
bond with Ser 733 was detected for compounds 11 and 18, while in the case of 12, this interaction was
replaced by a H-bond with Ser 979.
An interesting pose 3 with high docking scores and high binding energy values (only slightly
below those calculated for pose 1 and 2, Table S2, Supplementary) was observed for compounds 11 and
18. In this case, imidazolone derivatives adopted a more extended conformation, located among TM5,
TM7, TM8, and TM12 in almost perfectly parallel orientation to
directed to the outer leaflet of the lipid bilayer. It was found that compound 11 in this position formed
hydrogen bonds with surrounding polar residues: Asn 721, gln 838, and Tyr 307 (Figure 7), and π–π
α-helices, with an aromatic “head”
stacking interactions with Phe 732, Phe 759, and Tyr 310. Additionally, the ligand was involved in
hydrophobic contacts with the amino acids that belong mostly to TM5 (Phe 303, Ala 311, Phe 314, Trp
315, Thr 318), TM7 (Leu 724, gln 725, Phe 728, Ile 735, Ile 736), TM8 (Ser 756, Ser 766, Phe 770), and
TM12 (Phe 983, Met 986) (Table S2). The binding mode observed for pose 3 resembled to some extent

Molecules 2020, 25, 2258
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that proposed by Pajeva for Hoechst 33342, according to which Tyr 307, Asn 721, and Phe 770 were
identified as important residues forming a part of the H-site [33].
Summing up, results of the docking studies confirm that imidazolones (11, 12, 18) are highly
probable to interact with the transmembrane interior of Pgp with a mechanism similar to verapamil.
Analysis of the docking results showed that the binding pocket for imidazolones most likely involves
the residues responsible for binding verapamil and, partially, residues of the putative binding site for
rhodamine dyes. These results are in good accordance with those coming from the Pgp ATPase assay.
Thus, they support our hypothesis of imidazolone modulator–rhodamine 123 substrate competitive
action to the same Pgp-binding site as the most probable mechanism of the dye-efflux inhibition caused
by the imidazolones.
2.4. Safety In Vitro
In order to determine the safety of the most active ABCB1 modulators (11, 18), cell-based toxicity
assays in vitro were performed using the standard colorimetric MTS procedure in the human embryonic
kidney HEK-293 cell line. Results are shown in Figure 8. Both compounds significantly decreased
HEK-293 cell viability at the highest used concentrations (50 and 100
more toxic than 11 as, at the same concentration (50 M), 18 decreased the cell viability up to 10.3%,
whereas 11 only decreased it to 84.2%. However, the tested imidazolones (11 18) were found as
relatively safe in comparison to the reference doxorubicin (DOX), tested at 1 µM.
µM). Compound 18 was distinctly
µ
,
Figure 8. The viability of the HEK-293 cell line after incubation with compound 11 or 18 for 72 h.
DOX—doxorubicin. Control—1% DMSO in the cell culture media. The statistical significance was
evaluated by a one-way ANOVA, followed by Bonferroni’s comparison test (*** p < 0.001, **** p <
0.0001 compared to control).
3. Discussion
This study allowed an original group of modulators to be found for the T-lymphoma ABCB1 efflux
pump that may be helpful as potential “adjuvants” in order to improve the efficacy of cancer therapy.
The new group of morpholine-containing 5-arylideneimidazolones (7–20) distinctly differs from
typical Pgp-modulator structures that usually contain more than one hydrophobic/aromatic-terminate
fragment [43]. In the case of the imidazolones presented, a bulky hydrophobic area is concentrated at
only position 5 of the imidazolone, while the opposite ends, either at position 2 (
position 3 (14 20, group B), demonstrate a significant decrease in hydrophobic properties in favor of
alkaline ones, protonable to form HCl salts. Nevertheless, this deviation from the typical Pgp inhibitor
7–13, group A) or at
–

Molecules 2020, 25, 2258
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structure allowed some compounds to have a high potency to inhibit the dye-substrate efflux, in
comparable range with verapamil, or even higher. The mutual structural diversity within the considered
series (7–20), however, seems to be responsible for various kinds of forces of expected biological activity,
and, therefore, allows us to postulate some qualitative structure–activity relationship (SAR).
The main structural difference consists of the two ways of substitution topology at positions 2
and 3, i.e., the unsubstituted position 3 (group A) and the presence of a NH2 group at position 2 and
alkyl-linked morpholine at position 3 (group B). Furthermore, the modifications performed give the
possibility to estimate a role for the activity of chemical moieties, as follows: (i) The aromatic area
within both groups (A and B), (ii) the morpholine-containing moieties (group A), and finally (iii) the
length of alkyl linker at position 3 (group B).
As topology is concerned, the most active dye efflux inhibitors (11 and 18) represent both groups
(A and B), and there are either inactive or slightly active compounds found in both topological groups
(12, 13 vs. 15–17, Table 1). Thus, both kinds of topology (A and B) seem to equally contribute in the
desirable Pgp modulatory action. This has also been confirmed by our molecular modeling results that
indicated energetically favorable and mutually similar binding modes in the verapamil binding site for
representative members of both A (11, 12) and B (18).
In terms of insight into the role of the three modified moieties, at least three main modes of the
biological action should be considered, i.e., (1) the potency to inhibit 123 rhodamine efflux in the
accumulation assay, (2) the substrate “force” of compounds, observed as a difference in the cytotoxic
action between PAR and MDR T-lymphoma cells, and (3) the substrate properties seen as the ability
to increase the intrinsic ATPase activity of Pgp. In this context, the fluorene derivative containing
the propyl-linked morpholine moiety (18, group B) was found as the best agent, displaying both the
dye-inhibition in the accumulation assay and the substrate potency in the Pgp ATPase test, noticeably
higher than the reference verapamil. Furthermore, the Pgp-substrate properties of 18 can be confirmed
by an almost two-times-higher cytotoxicity toward PAR T-lymphoma cells in comparison to MDR ones.
On the other hand, the p-phenoxybenzylidene derivative with the piperidine-morpholine moiety
at position 2 (11, group A) was even slightly more potent than 18 to inhibit the dye-substrate efflux,
while the substrate potency of 11 was significantly weaker in either the Pgp ATPase- or the PAR vs.
MDR T-lymphoma cytotoxicity assays. Despite this, the compound was less toxic than 18 for HEK-293
cells and, therefore, could be the most proper among the investigated imidazolones for development
in search for new “adjuvants” of anticancer therapy.
Although the two compounds (11 and 18) were the most promising therapeutically, this
comprehensive study allowed us to also recognize other members that undoubtedly influence the
ABCB1-efflux pump and can contribute to SAR analysis. Among them, the anthracene derivative with
the propyl-linked morpholine moiety at position 2 (19, group B) displayed the strongest substrate
properties in the PAR/MDR T-lymphoma cytotoxicity assays (Table 3), while it did not act as an
efflux pump inhibitor in the accumulation assay (Table 2). This compound (19) differs from 18 only
within the aromatic area, i.e., possesses a slightly bigger and “more aromatic”, compared to fluorene,
anthracene group. This bulky and stiffened steric hindrance may be responsible for “a defeat” in the
competition with rhodamine 123 to be expelled by the ABCB1-pump.
Despite the different effects of imidazolones (
out, some structural features, which benefit the dye efflux inhibition, can be captured. In
the group with unsubstituted nitrogen at position 3 ( 13, group A), the piperidine-morpholine
substituent at imidazolone position 2 ( 10 13) is distinctly more favorable than either the longer
piperazine-ethyl-morpholine ( ) or the shorter directly substituted morpholine ( ). The compounds
and ) did not show any syndrome of action toward the ABCB1-efflux pump, either as substrates or
7–20), observed depending on the tests carried
7–
8,
–
9
7
(7
9
as inhibitors. The influence of the piperidine-morpholine scaffold is, however, closely associated with
the aromatic substituents at the 5-imidazolone. Thus, the flexible p-phenoxy-phenyl moiety can be
identified as the most profitable (11), and compared to the significantly less active m-phenoxy-phenyl
analog (10), it is supposed that not only steric but also electronic properties can be responsible for

Molecules 2020, 25, 2258
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the considered biological actions, with a preference for the electro-donating substituent at position
para. In group A, the tricyclic aromatic moieties seem to be profitable, too (12 13), in particular, the
,
fluorene moiety present in compound 12, while a slight ABCB1-modulatory effect was also found
for the anthracene derivative (13). These results are not surprising, as the fluorene moiety is also a
beneficial motif of our earlier efflux pump inhibitors, coming from different chemical groups [21,22,25].
The role of the fluorene motif is even more exposed in group B, being a trait of the most active
agent (18). Apart from the role of aromatic area, the group B with topology resulting from the Dimroth
rearrangement allows us to analyze the role of the spacer between the imidazolone and the morpholine
fragments. Results of the whole biological screening indicate that the compounds affecting the efflux
pump (15–19) include the three-carbon linker, while the two-carbon analog of 15, compound 14, was
inactive along the biological studies. Unlike group A, topology B does not seem to discriminate
between para (17) and meta (16) positions in the case of the phenyloxy-phenyl aromatic area, and even
some predominant influence of meta-substitution could be observed in both the accumulation and
cytotoxicity assays, respectively (16 vs. 17, Tables 2 and 3).
Summing up, the performed SAR analysis allowed us to recognize chemical moieties that could
be beneficial for the action on the cancer ABCB1-efflux pump, i.e., the fluorene, phenoxyphenyl,
or anthracene as aromatic moieties of 5-arylideneimidazolone on the one hand, and either the
morpholine-piperidine at position 2 or the propyl-linked morpholine at position 3 of the imidazolone
core, on the other.
4. Materials and Methods
4.1. Chemistry
Reagents were purchased from Alfa Aesar (Karlsruhe, germany) or Sigma Aldrich
(Darmstadt, germany) or Acros Organics (Geel, Belgium). Reaction progress was verified using
thin-layer chromatography (TLC), which was carried out on 0.2 mm Merck silica gel 60 F254 plates.
Spots were visualized by UV light. Melting points (m.p.) were determined using MEL-TEMP II
apparatus (LD Inc., Long Beach, CA, USA) and are uncorrected. 1H-NMR and 13C-NMR spectra
were obtained on a Mercury-VX 300 Mz spectrometer (Varian, Palo Alto, CA, USA) in DMSO-d6.
Chemical shifts in 1H-NMR spectra were reported in parts per million (ppm) on the δ scale using the
solvent signal as an internal standard. Data are reported as follows: Chemical shift, multiplicity (s,
singlet; br. s, broad singlet; d, doublet; d def.—doublet deformed; t, triplet; t def.—triplet deformed; m,
multiplet), coupling constant J in Hertz (Hz), number of protons, proton position (Ar—aromatic moiety
at position 5, Pp—piperidine, Pip—piperazine, Mor—morpholine). Mass spectra were recorded on
a UPLC-MS/MS system consisting of a Waters ACQUITY®®UPLC®® (Waters Corporation, Milford,
MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem
quadrupole). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged
ethyl hybrid) C18 column; 2.1
BEH C18 VanGuard precolumn (Waters Corporation, Milford, MA, USA); 2.1
particle size. The column was maintained at 40 ◦C and eluted under gradient conditions from 95% to
0% of eluent A over 10 min, at a flow rate of 0.3 mL
min−1. Eluent A: Water/formic acid (0.1%, v/v);
eluent B: Acetonitrile/formic acid (0.1%, v/v). Chromatograms were made using a Waters e PDA
×
100 mm, and 1.7
µ
M particle size, equipped with an Acquity UPLC
×
5 mm, and 1.7
µM
·
λ
detector. Spectra were analyzed in the 200–700 nm range with a 1.2 nm resolution and sampling rate
of 20 points/s. MS detection settings of the Waters TQD mass spectrometer were as follows: Source
temperature 150 ◦C, desolvation temperature 350 ◦C, desolvation gas flow rate 600 L −1, cone gas flow
h
·
100 L h
−1, capillary potential 3.00 kV, cone potential 40 V. Nitrogen was used for both nebulizing and
·
drying gas. The data were obtained in a scan mode ranging from 50 to 1000 m/z in 0.5 s time intervals.
The data acquisition software was MassLynx V 4.1 (Waters Corporation, Milford, MA, USA). Retention
times (tR) are given in minutes. The UPLC/MS purity of all final compounds was determined (%).
Synthesis of intermediates 21–32 and final products 16, 19 has been described earlier [21,22,44–46].

Molecules 2020, 25, 2258
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General Procedure for Synthesis of Final Products (7–15,17–18,20)
(Z)-5-arylidene-2-(methylthio)-3H-imidazol-4(5H)-one (2–5 mmol) and primary or secondary
amine derivative (2.5–6 mmol) were heated for 15 min without solvent in an oil bath with controlled
temperature (120–130 ◦C). Then, ethanol was added and the mixture was heated for 5–7 h. After that,
it was mixed for the next 20 h. Conversion into a hydrochloride form was performed using gaseous
hydrochloride acid. When necessary, purification was performed using crystallization from ethanol.
(Z)-5-(Biphen-4-ylmethylene)-2-morpholino-3H-imidazol-4(5H)-one hydrochloride (7)
(Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (27) (4 mmol, 1.18 g) and
morpholine (6 mmol, 0.52 g) were used. Yellow solid. Yield 63.24%; mp 248–252 ◦C. C20H20ClN3O2
1
MW 369.84. LC/MS
δ
±
: Purity 96.25% tR = 5.98, (ESI) m/z [M+H] 334.15. H-NMR (DMSO-d6, ppm):
8.01-7.99 (d def., 2H, Ar-2,6-H), 7.72–7.69 (d def., 5H, Ar-3,5,2‘,6‘-H, N3-H), 7.49-7.44 (t def., 2H,
Ar-3‘,5‘-H), 7.39–7.34 (m, 1H, Ar-4‘-H), 6.60 (s, 1H, C=CH), 3.72–3.70 (m, 8H, Mor). 13C NMR (75 MHz,
dmso) [ppm]: 140.12, 139.15, 131.05, 129.40, 127.92, 126.91, 65.98, 45.33, 40.79, 40.50, 40.22, 39.94,
δ
39.66, 39.38, 39.09.
(Z)-5-(Biphen-4-ylmethylene)-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one hydrochloride (8)
(Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (27) (4 mmol, 1.18 g) a◦nd
4-morpholinopiperidine (6 mmol, 1.02 g) were used. Yellow solid. Yield 59.01%; mp 267–269 C.
1
C25H29ClN4O2 MW 452.98. LC/MS
(DMSO-d6, ppm):
±
: Purity 100.00% tR = 4.54, (ESI) m/z [M+H] 417.23. H-NMR
δ
11.71 (s, 1H, NH+), 7.98-7.95 (d def., 2H, Ar-2,6-H), 7.75-7.70 (t def., 4H,
Ar-3,5,2‘,6‘-H), 7.49-7.45 (t def., 2H, Ar-3‘,5‘-H), 7.39-7.35 (m, 1H, Ar-4‘-H), 6.69 (s, 1H, CH=C), 4.97–2.74
(m, 13H, Mor, Pp-2,4,6-H), 2.31-2.27 (d def., 2H, Pp-3,5-Hb), 1.91-1.79 (m, 2H, Pp-3,5-Ha). 13C-NMR
(75 MHz, dmso) δ [ppm]: 140.15, 138.90, 130.92, 129.39, 127.88, 126.89, 66.99, 60.96, 49.74, 40.79, 40.51,
40.23, 39.95, 39.67, 39.38, 39.10, 27.85.
(Z)-5-(Biphen-4-ylmethylene)-2-(4-(2-morpholinoethyl)piperazin-1-yl)-imidazol-4(5H)-one
hydrochloride (9)
(Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (27) (2 mmol, 0.59 g) and
4-(2-morpholinoethyl)piperazine (2.5 mmol, 0.5 g) were used. Cream solid. Yield 59.81%; mp
305–307 ◦C. C26H32ClN5O2 MW 482.02. LC/MS
±
: Purity 100.00% tR = 4.60, (ESI) m/z [M+H] 446.27.
8.12-8.10 (d def., 2H, Ar-2,6-H), 7.71-7.66 (m, 5H, Ar-3,5,2‘,6‘-H, N3-H),
7.48-7.43 (t def., 2H, Ar-3‘,5‘-H), 7.38-7.33 (t def., 1H, Ar-4‘-H), 6.46 (s, 1H, C=CH), 3.88 (s, 6H, Mor-2,6-H,
N-CH2), 3.70-2.60 (m, 1H, Mor-3,5-H, N-CH2-CH2, Pip). 13C-NMR (75 MHz, dmso)
[ppm]: 140.14,
1H-NMR (DMSO-d6, ppm):
δ
δ
131.03, 129.40, 126.90, 66.65, 56.16, 55.21, 54.10, 52.82, 40.80, 40.51, 40.23, 39.95, 39.67, 39.38, 39.10.
(Z)-5-(3-Phenoxybenzylidene)-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one hydrochloride (10)
(Z)-5-(3-Phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (28) (4 mmol, 1.24 g) a◦nd
4-morpholinopiperidine (6 mmol, 1.02 g) were used. Cream solid. Yield 89.26%; mp 289–292 C.
C25H29ClN4O3 MW 468.98. LC/MS
±
: Purity 99.33% tR = 4.60, (ESI) m/z [M+H] 433.18. 1H-NMR
(DMSO-d6, ppm):
δ
11.80 (s, 1H, NH+), 7.79-7.61 (m, 1H, N3-H), 7.45-7.35 (m, 5H, Ar-2,5-H, Ar-3‘,4‘,5‘-H),
7.23–7.13 (m, 1H, Ar-6-H), 7.12-7.03 (m, 2H, Ar-2‘,6‘-H), 7.02-6.88 (m, 1H, Ar-4-H), 6.51 (s, 1H, CH=C),
4.21–4.17 (d.def, 2H, Mor-2,6-Hb), 3.95-3.93 (d.def, 2H, Mor-2,6-Ha), 3.58-3.22 (m, 4H, Pp-2,6-H), 3.03
(s, 5H, Pp-4-H, Mor-3,5-H), 2.27-2.23 (d def., 2H, Pp-3,5-Hb), 2.03-1.70 (m, 2H, Pp-3,5-Ha). 13C NMR
(75 MHz, dmso)
δ [ppm]: 157.98, 156.33, 138.24, 130.43, 130.05, 125.36, 124.28, 120.34, 117.99, 117.30,
110.39, 66.99, 60.90, 49.71, 40.78, 40.50, 40.21, 39.93, 39.65, 39.37, 39.08, 27.78.

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(Z)-5-(4-Phenoxybenzylidene)-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one hydrochloride (11)
(Z)-5-(4-Phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (29) (4 mmol, 1.24 g) a◦nd
4-morpholinopiperidine (5 mmol, 0.85 g) were used. Yellow solid. Yield 77.80%; mp 249–251 C.
C25H29ClN4O3 MW 468.98. LC/MS
±
: Purity 96.12% tR = 4.45, (ESI) m/z [M+H] 433.25. 1H-NMR
(DMSO-d6, ppm):
δ
11.34 (s, 1H, NH+), 8.14-7.98 (d def., 2H, Ar-2,6-H), 7.46-7.32 (t def., 2H, Ar-3‘,5‘-H),
7.19-7.09 (t def., 1H, Ar-4‘-H), 7.06-6.91 (m, 4H, Ar-3,5-H, Ar-2‘,6‘-H), 6.33 (s, 1H, CH=C), 4.31 (s, 2H,
Mor-2,6-Hb), 3.85 (s, 4H, Mor-2,6-Ha, Pp-2,6-Hb), 3.55-2.83 (m, 7H, Pp-2,6-Ha, Pp-4-H, Mor-3,5-H),
2.30–2.03 (m, 2H, Pp-3,5-Hb), 1.65 (s, 2H, Pp-3,5-Ha). 13C NMR (75 MHz, dmso)
δ [ppm]: 156.80, 156.21,
132.13, 131.80, 130.51, 124.06, 119.25, 118.73, 110.88, 66.97, 60.97, 56.46, 49.72, 40.78, 40.49, 40.21, 39.93,
39.65, 39.37, 39.08, 27.86, 19.00.
(Z)-5-((9H-Fluoren-2-yl)methylene)-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one
hydrochloride (12)
(Z)-5-((9H-Fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one (30) (5 mmol, 1.55 g)
and 4-morpholinopiperidine (6 mmol, 1.02 g) were used. Orange solid. Yield 44.39%; mp 274–276 ◦C.
C26H29ClN4O2 MW 464.99. LC/MS
±
: Purity 97.41% tR = 4.65, (ESI) m/z [M+H] 429.19. 1H-NMR
(DMSO-d6, ppm):
δ
11.37 (s, 1H, NH+), 8.24 (s, 1H, Ar-1-H), 8.12-8.09 (d def., 1H, Ar-4-H), 7.88-7.85 (d
def., 3H, Ar-5,8-H, N3-H), 7.57-7.55 (d def., 1H, Ar-3-H), 7.38-7.27 (m, 2H, Ar-6,7-H), 6.42 (s, 1H, CH=C),
4.35 (s, 2H, Ar-CH2), 4.07–3.62 (m, 6H, Mor-2,6-H, Pp-2,6-Hb), 3.62–3.20 (m, 2H, Pp-2,6-Ha), 3.11–3.03
(m, 5H, Pp-4-H, Mor-3,5-H), 2.22 (s, 2H, Pp-3,5-Hb), 1.72 (s, 2H, Pp-3,5-Ha). 13C-NMR (75 MHz, dmso)
δ
[ppm]: 185.01, 143.79, 141.08, 126.93, 125.51, 120.64, 67.15, 60.56, 49.74, 40.79, 40.51, 39.30, 39.09,
38.79, 27.94.
(Z)-5-(Anthracen-10-ylmethylene)-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one
hydrochloride (13)
(Z)-5-(Anthracen-10-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (31) (4 mmol, 1.22 g)
and 4-morpholinopiperidine (5 mmol, 0.85 g) were used. Yellow solid. Yield 38.48%; mp 303–305 ◦C.
C27H29ClN4O2 MW 477.00. LC/MS
±
: Purity 97.60% tR = 4.22, (ESI) m/z [M+H] 441.22. 1H-NMR
(DMSO-d6, ppm): 9.75 (s, 1H, N3-H), 8.63-8.54 (d def., 1H, Ar-9-H), 8.12–8.00 (m, 4H, Ar-1,4,5,8-H),
δ
7.54-7.45 (m, 4H, Ar-2,3,6,7-H), 7.11 (s, 1H, C=CH), 3.93-3.78 (m, 4H, Mor-2,6-H), 3.60-2.70 (m, 9H,
Pp-2,4,6-H, Mor-3,5-H), 2.25–2.02 (m, 2H, Pp-3,5-Hb), 1.58 (s, 2H, Pp-3,5-Ha).
(Z)-5-(Biphen-4-yl)-2-amino-3-(2-morpholinoethyl)-imidazol-4(5H)-one hydrochloride (14)
(Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (27) (4 mmol, 1.18 g) and
2-morpholinoethan-1-amine (5 mmol, 0.65 g) were used. Yellow solid. Yield 33.10%; mp 266–270 ◦C.
C22H25ClN4O2 MW 412.91. LC/MS
(DMSO-d6, ppm): 7.97 (s, 2H, NH2-hydantoin), 7.80–7.66 (m, 5H, Ar-2,3,5,6-H, Ar-2‘-H), 7.54-7.44 (t
def., 3H, Ar-3‘,5‘,6‘-H), 7.41-7.32 (m, 1H, Ar-4‘-H), 6.60 (s, 1H, CH=C), 4.26-2.97 (m, 12H, Mor-2,3,5,6-H,
N3-CH2-CH2). 13CNMR (75 MHz, dmso)
[ppm]: 140.12, 130.99, 130.89, 129.39, 127.88, 126.92, 66.64,
±
: Purity 98.26% tR = 4.60, (ESI) m/z [M+H] 377.22. 1H-NMR
δ
δ
53.66, 40.79, 40.51, 40.22, 39.94, 39.66, 39.38, 39.09.
(Z)-5-(Biphen-4-ylmethylene)-2-amino-3-(3-morpholinopropyl)-imidazol-4(5H)-one hydrochloride (15)
(Z)-5-(Biphen-4-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (27) (4 mmol, 1.18 g) and
3-morpholinopropan-1-amine (6 mmol, 0.87 g) were used. Yellow solid. Yield 56.93%; mp 248–252 ◦C.
C23H27ClN4O2 MW 426.94. LC/MS
±
: Purity 97.42% tR = 4.39, (ESI) m/z [M+H] 391.17. 1H-NMR
(DMSO-d6, ppm): 7.86 (s, 2H, NH2-hydantoin) 7.89-7.65 (t def., 5H, Ar-2,3,5,6-H, Ar-2‘-H), 7.52-7.45
δ
(t def., 3H, Ar-3‘,5‘,6‘-H), 7.40-7.33 (t def., 1H, Ar-4‘-H), 6.77 (br. s, 1H, CH=C), 3.98-2.80 (m, 12H, Mor,
N3-CH2-CH2-CH2), 2.13-2.00 (m, 2H, N3-CH2-CH2).

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(Z)-5-(4-Phenoxybenzylidene)-2-amino-3-(3-morpholinopropyl)-imidazol-4(5H)-one
hydrochloride (17)
(Z)-5-(4-Phenoxybenzylidene)-2-(methylthio)-3H-imidazol-4(5H)-one (29) (4 mmol, 1.24 g) and
3-morpholinopropan-1-amine (6 mmol, 0.87 g) were used. Cream solid. Yield 70.62%; mp 210–212 ◦C.
1
C23H27ClN4O3 MW 442.94. LC/MS
(DMSO-d6, ppm):
±
: Purity 100.00% tR = 4.32, (ESI) m/z [M+H] 407.26. H-NMR
δ
11.36 (s, 1H, NH+), 9.37 (s, 1H, NH), 7.79 (br. s, 2H, NH2-hydantoin), 7.47-7.37
(t def., 3H, Ar-2,6-H, Ar-3‘-H), 7.25-7.14 (t def., 1H, Ar-5‘-H), 7.11-6.97 (m, 5H, Ar-3,5-H, Ar-2‘,4‘,6‘-H),
6.76 (br. s, 1H, CH=C), 3.88 (s, 4H, Mor-2,6-H), 3.67-2.87 (m, 8H, Mor-3,5-H, N3-CH2-CH2-CH2), 2.05
(s, 2H, N3-CH2-CH2).13C NMR (75 MHz, dmso)
δ [ppm]: 156.84, 156.10, 132.09, 130.50, 124.03, 119.17,
118.74, 66.57, 53.71, 40.78, 40.49, 40.21, 39.93, 39.65, 39.36, 39.08.
(Z)-5-((9H-Fluoren-2-yl)methylene)-2-amino-3-(3-morpholinopropyl)-imidazol-4(5H)-one
hydrochloride (18)
(Z)-5-((9H-Fluoren-2-yl)methylene)-2-(methylthio)-3H-imidazol-4(5H)-one (30) (5 mmol, 1.55 g)
and 3-morpholinopropan-1-amine (6 mmol, 0.87 g) were used. Orange solid. Yield 63.90%; mp
261–263 ◦C. C24H27ClN4O2 MW 438.95. LC/MS
±
: Purity 100.00% tR = 4.46, (ESI) m/z [M+H] 403.20.
11.30 (br. s, 1H, NH+), 9.41 (s, 1H, NH), 8.18–7.91 (m, 5H, NH2-hydantoin,
Ar-1,4,5-H), 7.90–7.70 (m, 1H, Ar-8-H), 7.69-7.56 (m, 1H, Ar-3-H), 7.53–7.29 (m, 2H, Ar-6,7-H), 6.85 (s,
1H, CH=C), 4.06–2.53 (m, 14H, Ar-9-H, Mor, N3-C 2-CH2-C 2), 2.07 (s, 2H, N3-CH2-C
2). 13C-NMR
(75 MHz, dmso) [ppm]: 143.85, 143.49, 141.36, 127.25, 125.58, 120.50, 66.59, 53.74, 40.78, 40.50, 40.22,
1H-NMR (DMSO-d6, ppm):
δ
H
H
H
δ
39.94, 39.66, 39.37, 39.09, 36.76.
(Z)-5-(Phenanthren-9-ylmethylene)-2-amino-3-(3-morpholinopropyl)-imidazol-4(5H)-one
hydrochloride (20)
(Z)-5-(Phenanthren-9-ylmethylene)-2-(methylthio)-3H-imidazol-4(5H)-one (32) (4 mmol, 1.22 g)
and 3-morpholinopropan-1-amine (5 mmol, 0.72 g) were used. Yellow solid. Yield 25.62%; mp
238–240 ◦C. C25H27ClN4O2 MW 450.96. LC/MS
±
: Purity 95.34% tR = 4.40, (ESI) m/z [M+H] 415.23.
9.52 (s, 1H, NH2-hydantoin), 8.96-8.79 (m, 2H, Ar-4,5-H), 8.39–8.00
(m, 3H, Ar-1,8,10-H), 7.84-7.62 (m, 4H, Ar-2,3,6,7-H), 7.42 (s, 1H, CH=C), 3.89 (s, 4H, Mor-2,6-H),
3.67–2.89 (m, 8H, Mor-3,5-H, N3-C 2-CH2-C 2), 2.19–1.96 (m, 2H, N3-CH2-C
2). 13C NMR (75 MHz,
dmso) [ppm]: 131.69, 130.38, 130.33, 129.68, 129.16, 127.61, 127.53, 127.40, 123.94, 123.25, 66.58, 53.75,
1H-NMR (DMSO-d6, ppm):
δ
H
H
H
δ
40.78, 40.49, 40.21, 39.93, 39.65, 39.37, 39.08.
4.2. Biological Assays in Mouse T-Lymphoma Cells
4.2.1. Cell Lines
L5178 mouse T-cell lymphoma cells (PAR) (ECACC Cat. No. 87111908, obtained from FDA,
Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described by
Cornwell et al. [47]. The ABCB1-expressing cell line L5178Y (MDR) was selected by culturing the
infected cells with colchicine. The parental L5178 mouse T-cell lymphoma cells and the L5178Y human
ABCB1-transfected subline were cultured in McCoy’s 5A medium (Sigma-Aldrich, St Louis, MO, USA)
supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St Louis, MO, USA), 200 mM
L-glutamine (Sigma-Aldrich, St Louis, MO, USA), and a penicillin-streptomycin (Sigma-Aldrich, St
Louis, MO, USA) mixture in concentrations of 100 U/L and 10 mg/L, respectively. The cell lines were
incubated at 37 ◦C, in a 5% CO2, 95% air atmosphere.
4.2.2. Evaluation of Rhodamine 123 Retention by Flow Cytometry
The potency of the tested compounds as inhibitors of the ABCB1 efflux pump was determined using
a fluorescence-based detection system, described previously in the literature [23
,26]. Verapamil was
applied as a reference inhibitor of the ABCB1 transporter. The parental (PAR) and multidrug-resistant

Molecules 2020, 25, 2258
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(MDR) mouse T-lymphoma cells were adjusted to a density of 2
×
106/mL, re-suspended in serum-free
McCoy’s 5A medium, and distributed in 0.5 mL aliquots into Eppendorf centrifuge tubes. The tested
compounds (1 and 10 L, from a stock solution of 1 mM) were added at different concentrations
(2 and 20 M final concentrations, respectively), and the samples were incubated for 10 min at room
temperature. Subsequently, 10 L (with a final concentration of 5.2 M) of rhodamine 123 was added
µ
µ
µ
µ
to the samples and the cells were incubated for 20 min at 37 ◦C, washed twice, and re-suspended in
0.5 mL phosphate-buffered saline (PBS) for analysis. The fluorescence intensity of the cell population
was measured with a Partec CyFlow flow cytometer (Partec, Munster, germany). Verapamil was used
as a positive control at 20 µM final concentration in the rhodamine 123 exclusion experiments. The
mean fluorescence intensity (%) was calculated for the treated MDR and PAR mouse T-lymphoma cells
as compared to the untreated cells. The fluorescence activity ratio (FAR) was calculated based on the
following equation, which relates the measured fluorescence values:
MDRtreated/MDRcontrol
parentaltreated/parentalcontrol
FAR =
; Quotient = 100 × (FARcompound/FARverapamil
)
4.2.3. Cytotoxicity and Antiproliferative Effect
Cytotoxicity and antiproliferative assays were performed following the procedure described
in the literature [23 48 49]. Parental and multidrug-resistant mouse T-lymphoma cells were used to
,
,
determine the effect of the arylideneimidazolones on the growth of cells. The effects of increasing
concentrations of arylideneimidazolones on cell growth were tested in 96-well flat-bottomed microtiter
plates. The compounds were diluted in a volume of 100 µL of medium.
In the case of the mouse T-lymphoma cells, the two-fold serial dilutions were prepared in 100
of McCoy’s 5A, horizontally. The parental (PAR) and multidrug-resistant (MDR) mouse T-lymphoma
cells were adjusted to a density of 1
104 cells (for cytotoxic assay) and 6 103 (for antiproliferative
assay) in 100 L of McCoy’s 5A medium and were added to each well, with the exception of the
medium control wells.
The culture plates were incubated at 37 C for 24 h (for cytotoxicity assay) and 72 h (for
antiproliferative assay); at the end of the incubation period, 20 L of MTT (thiazolyl blue tetrazolium
bromide, Sigma) solution (from a stock solution of 5 mg/mL) were added to each well. After incubation
µL
×
×
µ
◦
µ
at 37 ◦C for 4 h, 100
µ
L of sodium dodecyl sulfate (SDS) (Sigma) so◦lution (10% in 0.01 M HCI) were
added to each well and the plates were further incubated at 37 C overnight. Cell growth was
determined by measuring the optical density (OD) at 540/630 nm with a Multiscan EX ELISA reader
(Thermo Labsystems, Cheshire, WA, USA). Inhibition of the cell growth was determined according to
the formula below:
"
#
ODsample − ODmedium control
IC50 = 100 −
× 100
ODcell control − ODmedium control
Results have been expressed in terms of IC50, defined as the inhibitory dose that reduces the growth
of the cells exposed to the tested compounds by 50%, each representing the mean of a minimum of
three independent experiments.
4.3. Intrinsic Activity Toward Pgp In Vitro
The luminescent Pgp-Glo
influence on P-glycoprotein activity was purchased from Promega (Madison, WI, USA). The assay
was performed in triplicate as described previously [27 29]. Compounds 11 12, and 18 (100 M) were
incubated with Pgp membranes for 40 min at 37 ◦C. The references, Pgp-stimulator verapamil (VER)
and Pgp-negative compound caffeine (CFN), were incubated at 200 and 100 M, respectively. For basal
P-gp activity calculation, the membranes were incubated with 100 M of Na3VO4. The luminescence
™ Assay System used for determination of the arylideneimidazolone
–
,
µ
µ
µ
signal was measured by a microplate reader EnSpire PerkinElmer (Waltham, MA, USA). The statistical
significances were calculated using graphPad Prism 8.0.1 software.

Molecules 2020, 25, 2258
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4.4. Molecular Modeling
4.4.1. Homology Modeling
The homology model of human P-glycoprotein was constructed by multiple comparative modeling
methods. The X-ray structures of murine Pgp: 4Q9H, 4XWK, and 4M1M, due to 87% sequence identity
and high diffraction resolution (3.4, 3.5, and 3.8 Å, respectively) were chosen as templates. The sequence
of human Pgp multidrug transporter (accession no. P08183) was retrieved from the UniProtKB/TrEMBL
database [42]. The multiple sequence alignment was performed by a PROMALS server [50] and
the result in the suitable format was used to build the homology model using MODELLER 9.21
software [51]. Out of 1000 models generated, three models with the lowest DOPE (Discrete Optimized
Protein Energy) scores were selected for further investigations and refined by minimization of energy
in the Schrodinger Suite software [52]. Analysis of the Ramachandran plots for all three models
allowed us to choose the most beneficial homology model showing the highest number of residues
in the most favored regions (1077; 93.7%) and the lowest number of residues in disallowed regions
(4; 0.3%), with a DOPE score 153,884.80 (Figure S1a, Supplementary). This model was subjected to
−
further structure validation using PROCHECK [53], ProSA [54], and QMEAN [55
S2 and S3, Supplementary).
,56] software (Figures
The topology and the secondary structure of the linker region (amino acids 630–698) were separately
predicted using the HMMTOP transmembrane topology prediction server [57]. The additional helix
was detected for residues Asp679–Asp689 of the linker. The modelled chain was incorporated into the
selected and validated homology model derived by MOLDELLER. The Ramachandran plot computed
by the PROCHECK program for the modified structure showed: 94.2% (1083) of residues in the
most favored regions, 4.8% (55) of residues in the additional allowed regions, 0.8% (9) of residues
in the generously allowed regions, and 0.3% (3) residues in the disallowed regions. Based on these
results, the model was considered to meet the requirements of quality and reliability and selected for
molecular docking studies.
4.4.2. Induced Fit Docking of Verapamil and Synthesized Compounds
Verapamil was used as a reference compound for analysis of interactions between the investigated
compounds and the human Pgp. The possible protonation and tautomeric states, as well as all
stereoisomers for the verapamil structure, were generated with the LigPrep module and optimized by
conformational search using the options of the MacroModel module implemented in the Schrodinger
Suite (Monte Carlo multiple minimization method, OPLS2005 force field, minimization with Truncated
Newton Conjugate gradient option until the root-mean-square of conjugate-gradient was below
0.05 kJ
·
mol , 12, and 18 were subjected to the same procedure of ligands
−1 Å−1) [58]. Compounds 11
·
preparation as verapamil. Both positively charged and neutral forms of the ligands were used in
further calculations.
The protein was prepared for docking in Protein Preparation Wizard (addition of hydrogen
atoms, assignment of protonation states of residues, and optimization of hydrogen bond network,
minimization of the protein energy using OPLS2005 force field) [52]. Flexible receptor docking was
performed using the induced fit protocol as implemented in the Schrodinger Suite [59].
For verapamil, the receptor grid was assigned as a box with the size of 25 Å, built around a
centroid of residues of Pgp known from mutagenesis experiments to interact with verapamil, described
by Loo et al. [36–38]. For the investigated arylideneimidazolone derivatives 11, 12, and 18, the
receptor grid box was set to include the whole binding cavity formed by the transmembrane region
of the human Pgp, as defined for the P08183 sequence in the UniProt database [42] (residues of TM1:
45–71, TM2: 113–132, TM3: 183–207, TM4: 209–228, TM5: 288–310, TM6: 327–348, TM7: 708–729, TM8:
751–775, TM9: 825–847, TM10: 852–874, TM11: 931–953, TM12: 970–991).
In the first step, glide docking of ligands was conducted with van der Waals radii scaling by a
factor of 0.5. The side chains of residues within 6 Å of ligand poses were predicted and optimized by

Molecules 2020, 25, 2258
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Prime. Initially docked ligands were re-docked into the resulting ligand–protein complexes with glide
XP precision. The final ligand–protein complexes were ranked and analyzed according to the obtained
docking score, XP glide score (XP gScore), and induced fit docking score (IFD Score) values (Tables S1
and S2, Supplementary).
For the graphic presentation of the selected structures with the highest docking scores, representing
individual clusters of poses, PyMOL software [60] was used.
4.5. Safety In Vitro
The human embryonic kidney HEK-293 (ATCC CRL-1573) cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA,
USA) according to previously described protocols [27
in 96-well plates at a concentration of 1
104 cells/well and incubated for 24 h at 37 ◦C in
5% CO2 atmosphere to reach 50% of confluence. Compounds 11 12, and 18 were diluted into
fresh growth medium and added to the cells at the final concentrations of 0.1–100 M. The
positive control DOX (1 M) was also added and the cells were incubated for 72 h. The MTS
–29]. For safety assay, cells were seeded
×
,
µ
µ
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent
(CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) was
added next to each well and incubated for 4 h. The absorbance was measured using a microplate
reader (EnSpire, PerkinElmer, Waltham, MA USA) at 490 nm to determine the cells’ viability. The
statistical significances were calculated using graphPad Prism 8.0.1 software.
5. Conclusions
In this study, a new series of fourteen 2-amine-5-arylideneimidazolones (7–20), representing
two different topologies (A and B), as potential inhibitors of the main cancer MDR protein, i.e.,
ABCB1 efflux pump, was investigated. The compounds were designed and synthesized within a
3–4 step synthesis, involving Knoevenagel condensation, S-methylation, and reaction with amines
proceeding via Dimroth rearrangement in the case of primary amines. The comprehensive biological
screening in vitro, including: the rhodamine 123 accumulation-, cytotoxicity-, and proliferation assays
in T-lymphoma cells with either a parental or multidrug resistance level of ABCB1, has indicated
that most of the series affected the efflux pump, either as efflux inhibitors and/or as substrates. The
structure–activity relationship analysis has shown the fluorene-, phenoxy-phenyl-, or anthracenyl
aromatic moieties, as well as the morpholine-piperidine or the propyl-linked morpholine, with respect
to the topology, as the benefit to affect this MDR efflux pump. The studies on potential mechanisms of
action for the most active modulators (11, 12, and 18), using the luminescence Pgp-Glo™ Assay in vitro
and computer-aided docking to human Pgp protein, have confirmed the Pgp substrate properties,
and the competitive action with the rhodamine 123 to the binding site for verapamil within the efflux
pump, as the most probable mechanism of the dye efflux inhibition.
This study allowed us to find two of the most active modulators, i.e., (Z)-5-(4-phenoxybenzylidene)
-2-(4-morpholinopiperidin-1-yl)-imidazol-4(5H)-one (11) and (Z)-5-((9H-fluoren-2-yl)methylene)-2-amino
-3-(3-morpholinopropyl)-imidazol-4(5H)-one (18), that have shown a significantly stronger efflux
inhibitory action than verapamil. Both compounds were less toxic than doxorubicin toward human
cells, but 11 was the safer one. Thus, phenoxybenzylidene derivative 11 seems to be the best candidate
for extended studies in search for “adjuvants” able to improve the efficacy of cancer therapy, while
further chemical modifications in order to improve the safety of 18 are required.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/9/2258/s1.
Spectral data for compounds (1HNMRs and 13CNMRs), Figure S1: (a) Ramachandran plot for the most beneficial
B008 homology model (DOPE –153,884.80); (b) Ramachandran plot for model B008 with a linker incorporated,
Figure S2: Evaluation of the top-ranked hPgp homology model B008 by ProSA server, Figure S3: Evaluation of the
top-ranked hPgp homology model B008 by QMEAN Swiss Model server, Figure S4: Comparison of the induced fit
docking results for the reference pose of verapamil in the neutral form and in the protonated form (top view).
The neutral form of verapamil and surrounding residues (side chains only) is marked in pink; the protonated

Molecules 2020, 25, 2258
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form of the drug and surrounding residues is marked in green. H-bonds between the protein and the ligands
are marked by the dashed line, Table S1: Comparison of docking results for top-ranked poses of the neutral and
protonated form of verapamil, Table S2: Comparison of the induced fit docking results for top-ranked poses of the
imidazolone derivatives 11, 12, and 18 (in the neutral form).
Author Contributions: A.K. (Aneta Kaczor) performed most of the syntheses; J.H. supervised and partly
contributed in the syntheses; M.N., A.K. (Annamária Kincses) and g.S. performed assays in T-lymphoma cell
lines; g.L. performed intrinsic activity toward ABCB1 and safety studies in vitro; E.S. performed molecular
modeling; A.K. (Aneta Kaczor), E.S., g.S. and J.H. have written the MS. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded from Ministry of Science and Higher Education budget funds for science
in 2017–2020, as a research project within “Diamond grant” no. 0169/DIA/2017/46 and Jagiellonian University
Medical College grants, grant number N42/DBS/000070 and N42/DBS/000027. M.N. was supported by EFOP
3.6.3-VEKOP-16-2017-00009.
Acknowledgments: Authors thank Maria Kaleta and Piotr Mazur for their irreplaceable support in the
synthetic work.
Conflicts of Interest: The authors declare no conflict of interest.
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