Backstabbing P-gp: Side-chain cleaved ecdysteroid 2,3-dioxolanes hyper-sensitize MDR cancer cells to doxorubicin without efflux inhibition

Article abstract of DOI:10.3390/molecules22020199

P-glycoprotein (P-gp, ABCB1) over-expression, causing a multi-drug resistant (MDR) phenotype, is a major problem in cancer chemotherapy that urgently requires novel approaches. Our previous studies showed certain ecdysteroid derivatives as promising chemo-sensitizers against MDR and non-MDR cancer cell lines while also exerting mild to moderate inhibition of P-gp function. Here we report the preparation of a set of substituted 2,3-dioxolane derivatives of poststerone, a known in vivo metabolite of 20-hydroxyecdysone (20E). In contrast with previously studied ecdysteroid dioxolanes, the majority of the new compounds did not inhibit the efflux function of P-gp. Nevertheless, a strong, dose dependent sensitization to doxorubicin was observed on a P-gp transfected cancer cell line and on its susceptible counterpart. We also observed that the MDR cell line was more sensitive to the compounds' effect than the non-MDR. Our results showed for the first time that the chemo-sensitizing activity of ecdysteroids can be fully independent of functional efflux pump inhibition, and suggest these compounds as favorable leads against MDR cancer.

Full text of DOI:10.3390/molecules22020199

molecules
Article
Backstabbing P-gp: Side-Chain Cleaved Ecdysteroid
,3-Dioxolanes Hyper-Sensitize MDR Cancer Cells to
Doxorubicin without Efflux Inhibition
2
Attila Hunyadi 1 *, József Csábi , Ana Martins , Joseph Molnár , Attila Balázs 4
,2,
1
3,†
3
and Gábor Tóth 4
1
Institute of Pharmacognosy, University of Szeged, 6720 Szeged, Hungary; csjoco88@gmail.com
Interdisciplinary Centre for Natural Products, University of Szeged, Eötvös str. 6, 6720 Szeged, Hungary
Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sq. 9, 6720 Szeged,
2
3
Hungary; anasfmartins@gmail.com (A.M.); molnar.jozsef@med.u-szeged.hu (J.M.)
NMR Group, Department of Inorganic and Analytical Chemistry, Budapest University of Technology and
4
Economics, Szt. Gellért Sq. 4, H-1111 Budapest, Hungary; balazs_attila_gyorgy@ymail.com (A.B.);
drtothgabor@t-online.hu (G.T.)
*
†
Correspondence: hunyadi.a@pharm.u-szeged.hu; Tel.: +36-62-546-456
Current address: Synthetic Systems Biology Unit, Institute of Biochemistry, Biological Research Centre,
Temesvári krt. 62, 6726 Szeged, Hungary.
Academic Editor: Constantinos M. Athanassopoulos
Received: 2 December 2016; Accepted: 18 January 2017; Published: 25 January 2017
Abstract: P-glycoprotein (P-gp, ABCB1) over-expression, causing a multi-drug resistant (MDR)
phenotype, is a major problem in cancer chemotherapy that urgently requires novel approaches.
Our previous studies showed certain ecdysteroid derivatives as promising chemo-sensitizers against
MDR and non-MDR cancer cell lines while also exerting mild to moderate inhibition of P-gp function.
Here we report the preparation of a set of substituted 2,3-dioxolane derivatives of poststerone,
a known in vivo metabolite of 20-hydroxyecdysone (20E). In contrast with previously studied
ecdysteroid dioxolanes, the majority of the new compounds did not inhibit the efflux function
of P-gp. Nevertheless, a strong, dose dependent sensitization to doxorubicin was observed on a P-gp
transfected cancer cell line and on its susceptible counterpart. We also observed that the MDR cell
line was more sensitive to the compounds’ effect than the non-MDR. Our results showed for the first
time that the chemo-sensitizing activity of ecdysteroids can be fully independent of functional efflux
pump inhibition, and suggest these compounds as favorable leads against MDR cancer.
Keywords: ecdysteroid metabolite; poststerone acetonide; ABCB1 efflux transporter; cancer;
multi-drug resistance; chemo-sensitization; combination therapy
1
. Introduction
Ecdysteroids have a broad range of bioactivities in mammals, as extensively reviewed
elsewhere [
these compounds can exert a potent chemo-sensitizing activity in various multi-drug resistant [
as well as drug susceptible [ ] cancer cell lines. In particular, ecdysteroid dioxolanes such as
0-hydroxyecdysone 2,3;20,22-diacetonide were found by our group to effectively potentiate the
in vitro antitumor activity of several chemotherapeutic agents including doxorubicin, paclitaxel,
and vincristine, which was, however, not the case for cisplatin [ ]. The strong synergistic action of
1–3]. In addition to these, it has recently been revealed that less polar derivatives of
4–7]
6–8
2
6
certain ecdysteroids with one or more of the above-mentioned chemotherapeutics was confirmed on
a broad range of cancer cell lines of various origin, including human breast (MCF-7 and its sub-cell
line MCF-7Dox adapted to doxorubicin), prostate (PC3, LNCaP), epidermal (KB-3-1 and its sub-cell
Molecules 2017, 22, 199; doi:10.3390/molecules22020199
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Molecules 2017, 22, 199
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line KB-C-1 adapted to colchicine), and neuroblastoma (SH-SY5Y), as well as two murine lymphoma
cell lines (L5178 and its sub-cell line L5178MDR transfected to express the human ABCB1 transporter,
commonly referred to as P-glycoprotein or P-gp) [4–8].
Over the last few years that have passed since the initial discovery of the chemo-sensitizing
activity of these compounds [
relationships. In this endeavor, several important observations were made including the most recently
reported effect of fluoride substitution on the activity [ ]. In particular, the presence of apolar groups
at positions 2 and 3 was found of outmost importance for the chemo-sensitizing potential, while such
a substituent at the 20,22-diol appears to be of much less or if any significance [ ]. Considering that
the most abundant natural ecdysteroid, 20-hydroxyecdysone (20E) is known to undergo a side-chain
cleavage between C-20 and C-22 during its in vivo metabolism to yield poststerone ( ) [ ], it seems
4], our research group has been pursuing related structure-activity
7
5
1
9
reasonable to speculate that similar side-chain cleaved metabolites can also be formed from other
ecdysteroids with a non-substituted diol at this position. Considering that such an in vivo metabolic
alteration can apply to potential antitumor ecdysteroid derivatives containing apolar substituents on
the 2,3-diol, the aim of the present study was to prepare such derivatives from poststerone (
investigate their bioactivity as potential chemo-sensitizing agents on the L5178 cell line and on its P-gp
transfected counterpart, L5178MDR
1) and to
.
2
. Results and Discussion
2
.1. Chemistry
Oxidative side-chain cleavage of 20E was achieved by using a common hypervalent iodine
reagent (PIFA), and the subsequent purification by centrifugal partition chromatography (CPC) led
to a relatively good, 57.8% isolated yield of poststerone ( ). Following this, our aim was to prepare
various substituted dioxolane rings fused to the ecdysteroid A-ring by utilizing different aldehydes or
ketones reacting with the 2,3-diol similarly to our previously applied strategy [ ]. Structure elucidation
1
5
by NMR, performed as previously published in detail [10], revealed the chemical structures presented
in Figure 1.
20
O
O
18
R1
R2
Me
H
17
1
9
2
3
4
6
7
8
9
Me
Et
HO
HO
R1
R2
O
22
2
3
H
O
OH
H
O
OH
O
n-Pr
n-Bu
i-Bu
Me
Et
H
O
H
H
H
1
Me
Et
Me
Et
HO
O
O
H
O
OH
H
5
10 Et
Figure 1. Chemical structures of compounds 1–10.
To facilitate the comparison of NMR data of products and the parental
1, we measured and
assigned the NMR signals of poststerone also in MeOH-d , in which solvent they were not available
4
1
13
before [11]. The corresponding H- and C-NMR shifts are compiled in Tables 1 and 2, respectively.

Molecules 2017, 22, 199
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1
Table 1. H-NMR chemical shifts of compounds 1–10, in ppm, in MeOH-d .
4
Atom No.
1
2 a
3
4
5
6
7
8
9
10 a
1
α
2
3
4
α
5
7
β
1.44
1.80
3.86
3.97
1.74
1.74
2.39
5.82
3.19
1.67
1.89
1.82
2.33
2.00
1.70
2.23
1.88
3.33
0.62
0.96
2.16
-
1.24
1.18
2.01
4.23
4.13
2.02
2.02
2.25
5.81
2.99
1.67
1.88
1.81
2.32
2.00
1.69
2.25
1.88
3.32
0.62
0.96
2.15
0.98
1.68
-
1.17
2.00
4.23
4.13
2.02
2.02
2.25
5.80
2.99
1.67
1.88
1.81
2.32
2.02
1.69
2.26
1.90
3.32
0.62
0.96
2.15
0.97
1.46
1.65
-
1.44
1.94
4.00
5.17
1.78
1.78
2.22
5.83
3.21
1.69
1.89
1.83
2.35
2.01
1.69
2.26
1.89
3.33
0.63
0.99
2.16
0.99
1.69
2.40
-
1.18
2.01
4.22
4.13
2.02
2.02
2.25
5.80
2.99
1.66
1.87
1.81
2.32
2.01
1.69
2.25
1.89
3.33
0.62
0.96
2.15
0.93
1.39
1.40
1.67
4.94
-
1.23
1.98
4.27
4.31
1.98
1.98
2.23
5.80
2.97
1.66
1.87
1.80
2.32
1.99
1.69
2.24
1.88
3.33
0.61
0.96
2.15
0.98
0.98
1.83
1.63
1.30
-
1.26
2.00
4.29
4.27
1.98
1.98
2.25
5.80
2.97
1.66
1.86
1.81
2.32
2.00
1.68
2.25
1.89
3.34
0.62
0.97
2.15
1.42
-
1.23
1.99
4.29
4.34
1.99
1.99
2.24
5.80
2.97
1.65
1.86
1.81
2.32
2.00
1.68
2.25
1.88
3.32
0.61
0.95
2.15
0.90
1.73
-
1.25
4.28
4.31
4.29
4.32
β
5.80
2.98
5.80
2.96
9
1 β
α
2 β
α
5 β
α
1
1
1
1
2.32
2.32
6 β
α
1
7
8
9
3.32
0.62
0.96
2.15
1.47
-
3.32
0.61
0.96
2.15
0.95
1
1
2
1
1
R
R2
-
1.32
-
4.90
-
4.95
-
-
0.92
1.62
1.28
-
0.88
a
Only the characteristic 1H chemical shifts were assigned for compounds
2 and 10, considering that these
1
2
compounds contain identical R and R groups, hence diastereomer pairs at C-22 were not distinguishable.
Table 2. 13C-NMR chemical shifts of compounds 1–10, in ppm, in MeOH-d4.
Atom No.
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
37.4
68.7
68.5
32.9
51.9
206.3
122.6
166.6
35.2
39.3
21.7
31.1
48.9
85.1
32.2
22.3
60.2
17.6
24.5
212.6
31.6
-
38.8
73.6
73.2
27.8
52.6
205.5
122.3
165.9
35.9
39.0
21.8
31.2
49.1
85.0
32.1
22.3
60.2
17.6
24.1
212.6
31.6
109.6
28.9
-
39.5
72.9
75.0
27.8
52.6
205.3
122.4
166.0
36.3
38.8
21.9
31.2
49.2
85.0
32.1
22.3
60.2
17.6
24.1
212.5
31.6
106.7
8.80
29.5
-
39.5
72.8
75.0
27.8
52.6
205.3
122.3
166.0
36.3
38.7
21.9
31.2
49.2
85.0
32.1
22.3
60.2
17.6
24.1
212.5
31.6
105.7
14.5
18.8
38.7
-
38.5
67.2
71.6
30.5
52.8
205.2
122.5
166.8
35.4
39.3
21.7
31.2
48.8
85.1
32.2
22.3
60.2
17.6
24.5
212.5
31.6
175.1
14.1
19.6
37.3
-
39.6
72.8
75.0
27.8
52.6
205.3
122.3
166.0
36.3
38.8
21.9
31.2
49.1
85.0
32.1
22.3
60.2
17.6
24.1
212.5
31.6
105.9
14.4
23.8
27.7
36.7
-
39.0
73.2
72.6
27.8
52.8
205.5
122.3
166.0
36.1
38.9
21.8
31.2
49.4
85.1
32.1
22.3
60.2
17.6
24.2
212.5
31.6
111.4
24.9
24.9
26.1
51.8
24.4
-
39.0
73.2
73.6
27.8
52.6
205.4
122.3
165.9
36.0
38.9
21.8
31.2
49.2
85.0
32.1
22.3
60.2
17.6
24.1
212.5
31.6
111.7
25.7
-
38.94
73.2
72.8
27.8
52.6
205.5
122.3
165.9
36.0
38.9
21.8
31.2
49.2
85.0
32.1
22.3
60.2
17.6
24.1
212.5
31.6
111.5
9.5
39.1
72.8
73.2
27.8
52.6
205.4
122.3
165.9
36.0
38.9
21.8
31.2
49.0
85.0
32.1
22.3
60.2
17.6
24.2
212.5
31.6
113.5
9.07
31.7
-
1
1
1
1
1
1
1
1
1
1
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
1
R
-
35.7
-
R2
-
26.7
-
-
-
-
9.40
33.4
23.6
-
9.11
29.6
A comparison of the
C-2 and
δ
H-2 and
δ
H-3 (3.86 and 3.97 ppm), moreover the corresponding
δ
, with the corresponding H and 13C
1
δ
C-3 (68.7 and 68.5 ppm) values of the parental
1

Molecules 2017, 22, 199
4 of 10
chemical shifts obtained for the dioxolane derivatives (4.22–4.29 and 4.13–4.34 ppm) and (72.8–73.6 and
7
5
2.6–75.0 ppm) shows a pronounced deshielding. With the exception of the low-yield 3-acyl compound
, an unexpected by-product of compound likely formed through the oxidation of an intermediate,
4
the targeted dioxolane derivatives were obtained. Similar to our previous observation with analogous
derivatives of 20E [ 10], whenever an asymmetrically substituted dioxolane ring was formed, the
C-22 atom of the 2,3-dioxolane ring turns into a new stereogenic center. Steric effects caused the
larger substituent on C-22 to be situated preferentially in the -position [ 10] except for the present
case of compounds and where the two possible 22-epimers were obtained in nearly identical
5,
β
5,
8
9
yields. Their differentiation was then elucidated by one-dimensional Rotating frame Overhauser
Enhancement (ROE) experiments, because this experiment in our case was much more effective than
the analogous selective Nuclear Overhauser Enhancement (NOE) experiment. As shown in Figure 2,
selective irradiation on the CH signal (1.28 ppm) of compound
8
resulted in strong ROESY responses
3
on H-2 and H-3 signals, therefore their cis
α positions as well as the R configuration of C-22 was
unequivocally established.
1
0
7.6
.61
CH3
31.6
2.15
H3C
1
.65
0.95
1.81
H
H
2
12.5
O
CH3
49.2
2.00H
3
1.2
1
.99
1.86 H
36.0
60.2
21.8
H 2.25
3
9.94
H
2
.32H
1
.23H
38.9
32.1
2
2.3
8
5.0 H
1.68
H
2.24
165.9
2.97H
9
.5
35.7
H
52.6
3.32 H 1.88
OH
0
.99 1.73
122.3
205.5
7
3.2
O
CH3 CH2
1
4.29
H
H
5.80
11.5
O
2
7.8
2
3.6
7
2.8
H
CH3
O
H 1.99
H4.34
1
.28
Figure 2. Stereostructure and one-dimensional selective Rotating frame Overhauser Enhancement
2
Spectroscopy (ROESY) responses (irradiated at R : δ = 1.28 ppm) of compound 8. Blue numbers refer
1
13
to H chemical shifts; black numbers give the C δ values.
2
.2. Bioactivity
The poststerone derivatives
2–10 represented an ideal set of compounds to investigate the effect
of a lacking side-chain on the chemo-sensitizing activity, and to study the relevant structure-activity
relationships around C-2,3 in comparison with those concluded from our previous studies [4,5].
2
.2.1. Cytotoxic Activity and Functional Inhibition of P-Glycoprotein
Evaluation of the cytotoxicity of compounds 10 showed that, with the exception of compound 7,
the 50% inhibitory concentration (IC ) was higher than 75 M, both on the L5178 and the L5178MDR
0.8 and 55.4 4.9 M against L5178 and
1
–
µ
50
cell lines. Compound
L5178MDR, respectively.
7
had an IC50 equal to 59.6
±
±
µ
The inhibitory effect on the efflux function was studied by measuring the compounds’ activity
on the intracellular accumulation of rhodamine 123, a well-known P-gp substrate fluorescent dye,
within the L5178MDR cells. The efflux pump inhibitor tariquidar was used as positive control.
Each compound was dissolved in DMSO whose final concentration (2%) was also evaluated for
any effect on the retention of the fluorochrome, but no effect of the solvent was observed. The values
were normalized to the fluorescence (FL) of rhodamine accumulated by L5178 and L5178MDR cells
such as: rhodamine 123 accumulation in the L5178 cells that do not overexpress the human P-gp,
was considered as 100% P-gp inhibition while accumulation of Rhodamine 123 in L5178MDR cells, that
do overexpress the human P-gp, was considered as 0% inhibition. The obtained results are compiled
in Table 3.

Molecules 2017, 22, 199
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Table 3. Functional inhibition of P-gp by compounds 1–10. a
Inhibition (%)
Inhibition (%)
Compound
Compound
2
µM
0.31
0.08
20 µM
0.43
0.64
2 µM
−0.06
3.08
−0.19
−0.32
−0.30
20 µM
2.32
56.36
0.19
1
2
3
4
6
7
8
9
10
0.24
0.29
0.03
3.85
−0.16
5
−0.19
0.03
0.11
a
Positive control: 50 nM of tariquidar (109.59% inhibition), negative control: 2% DMSO (−0.42% inhibition).
Compound
activity of 20-hydroxyecdysone 2,3;20,22-diacetonide (20.91% inhibition at 20
data published in [ ]); none of the other ecdysteroids exerted any significant inhibitory effect on the
7
was the only one showing an effect in this bioassay, with approximately twice the
µM, recalculated from
4
efflux function of P-gp. This indicates that lacking a side-chain significantly decreases this activity of
apolar ecdysteroid derivatives, while a bulky 2,3-substituent, such as the methyl-isobutyl substituted
dioxolane ring in compound 7, can restore the P-gp inhibitory effect.
2
.2.2. Combination Studies with Doxorubicin
Considering the negligible cytotoxic activity of each of the compounds, we evaluated the capacity
of the compounds to potentiate the effect of doxorubicin. The effect of 10 or 25 µM of each compound
was studied on the IC50 value of doxorubicin on both cell lines in order to allow evaluation of the
effect of the missing side-chain on the chemo-sensitizing activity by comparing our new compounds’
activity with that of the non-substituted poststerone (
0-hydroxyecdysone 2,3;20,22-diacetonide (20DA); results of this study are presented in Figure 3.
It is noteworthy, that all compounds showed a dose dependent chemo-sensitizing activity on both
cell lines. With the exception of poststerone, 9, and 10, sensitization of the L5178 cells to doxorubicin
was higher than 2-fold on the cells compared to when doxorubicin was applied alone, at least at the
higher concentration (25 M). On this cell line, the strongest activity was exerted by compounds 6–8,
1) as well as with our previous lead compound
2
2,
µ
each of which was significantly stronger than the diacetonide of 20E (20DA) when tested in a planned
comparison by one-way ANOVA followed by Bonferroni’s post hoc test.
Concerning the transfected MDR cell line L5178MDR, compounds
strong chemo-sensitizing activity, even though they did not inhibit the efflux function of P-gp
in contrast with 20DA’s mild inhibition). As seen from Figure 3C, these compounds have up to
–7 times stronger sensitizing effect on the MDR cells than on the parental cells, indicating that
2–6 and 8–10 showed remarkably
(
5
overexpression of P-gp by MDR cells induces a significant collateral sensitivity to the chemo-sensitizing
activity of ecdysteroids. Exploiting collateral sensitivity—i.e., an evolutionary disadvantage connected
to the MDR phenotype making such cells paradoxically hypersensitive to certain agents other than
the ones they have become resistant to—appears to be a highly attractive strategy for overcoming
MDR-related therapeutic difficulties [12]. A number of compounds have been identified that are
able to exploit the fitness cost of resistance through various mechanisms [13]. In our case, further
studies are necessary to clarify which mechanisms can be responsible for the observed MDR selective
chemo-sensitizing activity, nevertheless, it appears clear that it can be completely separated from a
functional efflux pump inhibition.
According to the structure-activity relationships (SARs), even though the acyl compound 5 was
also active, the dioxolane derivatives were found favorable in this regard. Among these, a clear
tendency was observed for gradually increasing the chemo-sensitizing activity on both cell lines when
increasing the size of the β-substituent on the 2,3-dioxolane ring (in the order of compounds 2, 3,
4
2
, and
6
). Moreover, from the activities of compounds
8
and
9
α
it can also be seen that, in case of
-position (as in compound ) for a
2-epimers, the larger substituent is preferred to be in the 22
8
stronger sensitizing activity particularly on the non-MDR cell line. This extends our previous findings
for the SAR observed at this position of side-chain bearing ecdysteroid dioxolanes [ ] also to this series
5

Molecules 2017, 22, 199
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of compounds. Interestingly, however, the same difference between the bioactivites of compounds
and was not observed on the L5178MDR cells, which resulted in the higher MDR-selectivity of
8
9
compound
compound
9
as compared to
8. In contrast with the other compounds investigated in this study,
7
could inhibit the efflux of rhodamine 123 by P-gp. Nevertheless, this compound showed a
great sensitizing activity on both cell lines. It could completely reverse the resistance of the MDR cells
at as low as 10 M concentration, and at the higher dose (25 M) it decreased the IC50 of doxorubicin
from 0.41 and 11.8 M (L5178 and L5178MDR, respectively) to 0.12 and 0.17
respectively), highlighting its potential interest as a chemo-sensitizing agent.
µ
µ
µ
µM (L5178 and L5178MDR,
0
.5
.4
.3
.2
.1
.0
L5178
10 μM
A
2
5 μM
0
0
0
0
0
0
0
Dox 20DA
1
2
3
4
5
6
7
8
9
10
1
5
B
C
L5178MDR
10 μM
2
5 μM
10
5
0.0
0
Dox 20DA
1
2
3
4
5
6
7
8
9
10
1
2
1
2
1
0 μM 1.53 1.09 1.19 1.25 1.57 1.47 1.92 2.22 2.18 1.22 1.70
5 μM 2.41 1.41 1.78 2.80 2.41 2.07 2.73 3.50 3.62 1.60 1.77
0 μM 10.11 0.89 3.11 4.32 6.81 1.74 7.57 27.26 4.54 5.30 7.42
5 μM 22.84 1.57 6.68 8.54 13.58 3.94 15.05 70.00 8.45 8.05 12.17
0 μM 6.61 0.82 2.61 3.46 4.34 1.18 3.94 12.28 2.08 4.34 4.36
L5178
Fold
sensitization
towards Dox
L5178MDR
MDR
selectivity 25 μM 9.48 1.11 3.75 3.05 5.63 1.90 5.51 20.00 2.33 5.03 6.88
Figure 3. Chemo-sensitizing activity of compounds
the IC50 value of doxorubicin (Dox) alone or in combination with 10 or 25
,3;20,22-diacetonide (20DA) or compounds 10. The dashed line marks the threshold of two-fold
sensitization. With the exception of 10 M of compound (either cell line), and , and (L5178),
1–10 on the two lymphoma cell lines. (A,B) Show
µM of 20-hydroxyecdysone
2
1–
µ
1
2,
3
9
all IC50 values differ from that found for Dox alone at p < 0.01 by means of one-way ANOVA followed by
Dunnett’s post-hoc test; IC50 values and levels of significance are provided as supporting information
(
Table S1); (
certain concentration (10 or 25
to when it was used alone; MDR selectivity refers to the ratios of the IC50 values on the resistant and
sensitive cell line: MDR selectivity = IC (L5178 )/IC (L5178).
C
) Fold sensitization on both cell lines is presented, corresponding to how many times a
µM) of the tested compound decreased the IC50 of Dox as compared
5
0
MDR
50
3
. Materials and Methods
3
.1. General Information
The compound 20-hydroxyecdysone (20E) was purchased from Shaanxi KingsSci Biotechnology
Co., Ltd. (Shanghai, China) at 90% purity and recrystallized from ethyl acetate/methanol (2:1, v/v)
to reach a purity of 97.8% by means of HPLC–DAD. Reverse phase HPLC was performed on a system
of two Jasco PU-2080 pumps connected to a Jasco MD-2010 Plus photodiode-array detector (Jasco Co.,
Tokyo, Japan). Normal phase HPLC was performed on a Waters 600 Pump connected to a Waters
2
487 Dual
λ Absorbance Detector (Waters Co., Milford, MA, USA). Mass spectra were recorded on an
API 2000 triple quadrupole tandem mass spectrometer (AB SCIEX, Foster City, CA, USA) in positive

Molecules 2017, 22, 199
7 of 10
mode with atmospheric pressure chemical ionization (APCI) ion source except for compounds
which were measured with electron-spray ionization (ESI).
8–10,
1H- (500.1 MHz) and 13C- (125.6 MHz) NMR spectra were recorded at room temperature on a
Bruker Avance spectrometer and on Avance-III spectrometer (Bruker Biospin Co., Karlsruhe, Germany)
equipped with a cryo probehead. Regarding the compounds, amounts of approximately 1–5 mg
were dissolved in 0.1 mL of methanol-d and transferred to 2.5 mm Bruker MATCH NMR sample
4
tube (Bruker). Chemical shifts are given on the
δ-scale and are referenced to the solvent (MeOH-d :
4
1
13
δ
= 49.1 and = 3.31 ppm). Pulse programs of all experiments ( H, C, DEPTQ, DEPT-135,
δ
H
C
sel-TOCSY, sel-ROE, edited gs-HSQC and gs-HMBC) were taken from the Bruker software library.
The NMR signals of the product were assigned by comprehensive one- and two-dimensional NMR
1
methods using widely accepted strategies [14
,
15]. Most H assignments were accomplished using
general knowledge of chemical shift dispersion with the aid of the proton-proton coupling pattern
1
(
H-NMR spectra).
3
.2. Synthetic Procedure
Poststerone (1) was synthesized from 20E as follows: 2.0 g (4.17 mmol) of 20E was dissolved in
5
0.0 mL of methanol, and 2.7 g (1.5 equiv.) of [bis(trifluoroacetoxy)iodo]benzene (PIFA, Sigma-Aldrich,
Budapest, Hungary) was added, and the reaction mixture was stirred at room temperature for 1 h.
On completion, the mixture was quenched with 5% aqueous solution of NaHCO (Reanal Plc.,
3
Budapest, Hungary) and evaporated to dryness. The residue was dissolved in ethyl acetate, filtered
through silica (Merck, Darmstadt, Germany) and dried in vacuo. The product was purified by
centrifugal partition chromatography (Armen Spot CPC 250 mL, Armen Instrument, Saint Ave, France)
in ascending mode with a solvent system of ethyl acetate/water/methanol (20:20:1, v/v/v), and 20 mL
fractions were collected. Corresponding fractions were combined and dried to give poststerone (1),
(
(
871.6 mg, 57.8%).
Compounds
2
–
10 were synthesized from poststerone (
) was dissolved in 10 mL of methanol, then the reagent was added to the
: propionaldehyde, 5 mL; and : butyraldehyde, 5 mL; : valeraldehyde,
: methyl isobutyl ketone, 5 mL; and : methyl ethyl ketone, 5 mL; 10: 3-pentanone, 5 mL).
Catalytic amounts of p-toluenesulfonic acid (Sigma-Aldrich) were added, and the mixture was stirred
at room temperature for two days in case of compound and one week in case of compounds 10
1) according to the followings: 60 mg
0.166 mmol) of poststerone (
: acetone, 20 mL;
1
3
solution (
mL;
2
4
5
6
5
7
8
9
2
3–
.
Then the reaction mixture was quenched with 5% aqueous solution of NaHCO and diluted with
3
water. The mixture was concentrated by vacuum distillation until only water was present, and the
aqueous solution was extracted three times with methylene chloride. The combined organic layers
were dried with anhydrous Na SO and evaporated to dryness. The products were isolated by
2
4
rotational planar chromatography on silica gel with appropriate eluents composed of ethyl acetate
and ethanol. Compounds and were further purified by semi-preparative HPLC using isocratic
elution with aqueous methanol (63%, 70%, 68%, 75% for compounds , and , respectively,
and 65% for compounds and ) at a flow rate of 3.0 mL/min, by utilizing an Agilent Eclipse XDB-C8
Agilent Technologies, Santa Clara, CA, USA) (9.4 mm 250 mm, 5 m) column. The yields were as
follows: (39.2 mg, 58.8%), (16.7 mg, 25.1%), (10.0 mg, 14.5%), (7.3 mg, 10.2%), (7.6 mg, 10.7%),
(1.5 mg, 2.0%), 8 (8.9 mg, 12.9%), 9 (7.0 mg, 10.2%), 10 (16.2 mg, 22.7%).
3
5–9
3,
5,
6
7
8
9
(
×
µ
2
3
4
5
6
7
3
.3. Compound Characterization Data
◦
1
Poststerone (1
): White crystals; m.p. 238–240 C; for H- and 13C-NMR data, see Tables 1 and 2,
+
+
respectively; APCI-MS: 363 [M + H] , 345 [M + H − H O] , 327, 309.
2
◦
1
2
2,22-Dimethyl-2,3-O-methylidene-poststerone (2
): White crystals; m.p. 185–186 C; for H- and 13C-NMR
+
+
data, see Tables 1 and 2, respectively; APCI-MS: 403 [M + H] , 385 [M + H − H O] , 359, 345, 327.
2

Molecules 2017, 22, 199
8 of 10
◦
1
22β-Ethyl-2,3-O-methylidene-poststerone (3
): White crystals; m.p. 82–83 C; for H- and 13C-NMR data,
+
+
see Tables 1 and 2, respectively; APCI-MS: 403 [M + H] , 385 [M + H − H O] , 359, 345, 327.
2
◦
1
22β-Propyl-2,3-O-methylidene-poststerone (4
): White crystals; m.p. 76–77 C; for H- and 13C-NMR data,
+
+
see Tables 1 and 2, respectively; APCI-MS: 417 [M + H] , 399 [M + H − H O] , 395, 377, 345, 327.
Poststerone 3-butyrate (5): White crystals; m.p. 95–97; for H- and C-NMR data, see Tables 1 and 2,
2
1
13
+
+
respectively; APCI-MS: 433 [M + H] , 415 [M + H − H O] , 391, 377, 345, 327.
2
◦
1
22
β
-Butyl-2,3-O-methylidene-poststerone (
6
): White crystals; m.p. 162–163 C; for H- and 13C-NMR
+
+
data, see Tables 1 and 2, respectively; APCI-MS: 431 [M + H] , 413 [M + H
−
H O] , 377, 359, 345, 327.
2
◦
1
2
2
β
-Isobutyl-22
α
-methyl-2,3-O-methylidene-poststerone (
7
): White crystals; m.p. 195–197 C; for H- and
C-NMR data, see Tables 1 and 2, respectively; APCI-MS: 445 [M + H] , 427 [M + H
09, 391, 377, 359, 345, 327.
1
3
+
+
−
H O] , 423,
2
4
◦
1
2
2
α
-Ethyl-22
β
-methyl-2,3-O-methylidene-poststerone (
8
): White crystals; m.p. 193–194 C; for H- and
1
3
+
+
C-NMR data, see Tables 1 and 2, respectively; ESI-MS: 449 [M + CH O] , 417 [M + H] .
4
◦
1
2
2
β
-Ethyl-22
α
-methyl-2,3-O-methylidene-poststerone (
9
): White crystals; m.p. 88–89 C; for H- and
1
3
+
+
C-NMR data, see Tables 1 and 2, respectively; ESI-MS: 449 [M + CH O] , 417 [M + H] .
4
1
2
2,22-Diethyl-2,3-O-methylidene-poststerone (10): White crystals; m.p. 183–185 C; for H- and 13C-NMR
◦
+
+
data, see Tables 1 and 2, respectively; ESI-MS: 463 [M + CH O] , 431 [M + H] .
4
3
.4. Cytotoxicity Assay
Cytotoxicity activities on the L5178 and L5178
cell lines were performed as described
MDR
4
before [
6
]. Briefly, 10 cells/well were incubated with serial dilutions of each compound (n = 3)
◦
in McCoy’s 5 A medium (Sigma-Aldrich) for 48 h at 37 C, 5% CO . Then, 3-(4,5-dimethylthiazol-2-yl)-
2
2
,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well at a final concentration
of 0.5 mg/mL per well and after 4 h of incubation, 100 L of sodium dodecyl sulfate (SDS) 10%
Sigma-Aldrich) in 0.01 M HCl was added to each well. Plates were further incubated overnight and
the optical densities were read at 540 and 630 nm using an ELISA reader (Multiskan EX, Thermo
Labsystem, Milford, MA, USA). Fifty percent inhibitory concentrations (IC ) were calculated using
µ
(
50
non-linear regression curve fitting of log (inhibitor) vs. response and variable slope with a least
squares (ordinary) fit of GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).
IC50 values were statistically evaluated by one-way ANOVA followed by Dunnett’s (with doxorubicin
applied alone as control column) or Bonferroni’s post hoc test (with planned comparisons between the
activities of compounds of particular interest).
3
.5. Sensitization of L5178 and L5178MDR Cells to Doxorubicin
Sensitization assay was performed in the same way as the cytotoxicity assay described above.
However, in this assay doxorubicin was serially diluted in the 96 well plate and compounds
1–10
were added to each well at fix concentrations of 10 M or 25 M. In addition to the medium and cell
µ
µ
control, wells that contained only cells and compound at the studied concentration were also included
to assure that the concentrations used did not affect the cell growth by themselves.
3
.6. Evaluation of P-glycoprotein Function through Rhodamine 123 Accumulation Assay
Inhibition of efflux function was evaluated using rhodamine 123, a fluorescent dye, whose
6
retention inside the cells was evaluated by flow cytometry [
4]. Briefly, 2
×
10 cells/mL were treated
with 2 and 20
µ
M of each compound. After 10 min incubation, rhodamine 123 (Sigma-Aldrich) was
◦
added to a final concentration of 5.2
µ
M and the samples were incubated at 37 C in a water bath
for 20 min. Samples were centrifuged (Heraeus Labofuge 400, Thermo Fisher Scientific, Waltham,
MA, USA) (2000 rpm, 2 min) and washed twice with phosphate buffer saline (PBS, Sigma). The final

Molecules 2017, 22, 199
9 of 10
samples were re-suspended in 0.5 mL PBS and its fluorescence measured with a Partec CyFlow flow
cytometer (Partec, Münster, Germany). Tariquidar was kindly provided by Dr. Milica Pesic from
the Institute for Biological Research Sinisa Stankovic, Belgrade, Serbia, and it was used at 50 nM as
positive control.
4
. Conclusions
Through the synthesis and bioactivity testing of a series of poststerone 2,3-dioxolanes, we have
shown for the first time that structure-activity relationships connected to the chemo-sensitizing activity
of ecdysteroids can in fact be separated from those describing their inhibitory effect on P-gp function.
On the one hand, this strongly supports our previous assumption, namely that a mechanism(s) other
than a functional P-gp inhibition must primarily be responsible for the chemo-sensitizing activity of
less-polar ecdysteroid derivatives, and P-gp inhibition, if any, is rather a side-effect to this. On the
other hand, side-chain cleaved ecdysteroid 2,3-dioxolanes, originated from the in vivo ecdysteroid
metabolite poststerone, are hereby suggested as promising, non-P-gp inhibitor MDR selective adjuvant
agents for further development.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/22/
1
2
/199/s1. Figure S1: H-NMR spectrum of compound
8
, Figure S2: DEPTQ spectrum of compound
, Figure S4: DEPTQ spectrum of compound , Figure S5: Confirmation of the
at C-22, Table S1: Cytotoxicity of doxorubicin alone and in combination with 10 or
5 µM of compounds 1–10 on the L5178 and L5178MDR cell lines.
8, Figure S3:
1
H-NMR spectrum of compound
stereochemistry of compound
9
9
9
2
Acknowledgments: This work was supported by the National Research, Development and Innovation Office,
Hungary (NKFIH; K119770). Networking contribution by the COST Action CM1407 “Challenging organic
syntheses inspired by nature—from natural products chemistry to drug discovery” and support from the
Foundation for Cancer Research Szeged is acknowledged. A.H. acknowledges support from the János Bolyai
fellowship of the Hungarian Academy of Sciences and from the Kálmán Szász prize.
Author Contributions: A.H. conceived and designed the experiments and wrote the main manuscript text;
J.C. and A.M. performed the experiments; A.H., J.C. and A.M. analyzed the data; J.M. provided cell lines and
laboratory facilities for bioactivity testing; A.B. and G.T. performed NMR studies and analyzed related data.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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Sample Availability: Samples of compounds 1–10 are available from the authors.
©
2017 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 (http://creativecommons.org/licenses/by/4.0/).