Larry H Bernstein, MD, Reviewer and Content Advisor
Stephen Williams, PhD, Cancer Editor
http://pharmaceuticalintelligence.com/2013-12-24/larryhbern/
Ecdysteroid-Dioxolanes-as-MDR-Modulators-in Cancer
This article is a presentation on drug research and development in cancer therapeutics
introducing structure activity relationships of a novel class of oncotherapeutic drugs –
ecdysteroid dioxolanes as MDR modulators. Ecdysteroids are the molting hormones
of insects, and they have nonsteroidal activity in mammals. However, they have been
found to have an effect on certain derivatives on the ABCB1 transporter mediated
multidrug resistance (MDR) of a transfected murine leukemia cell line. The following
study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone.
Synthesis and Structure-Activity Relationships of
Novel Ecdysteroid Dioxolanes as MDR Modulators in Cancer
Ana Martins 1,2,†,*, József Csábi 3,†, Attila Balázs 4, Diána Kitka 1,
Leonard Amaral 5, József Molnár 1, András Simon 6, Gábor Tóth 6
and Attila Hunyadi 3,*
1 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged Hungary;
2 Unidade de Parasitologia e Microbiologia Médica, Institute of
Hygiene and Tropical Medicine, Universidade Nova de Lisboa,
Lisbon, Portugal
3 Institute of Pharmacognosy, Faculty of Pharmacy, University
of Szeged, Szeged, Hungary;
Ubichem Research Ltd., Budapest, Hungary;
4 Center for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Universidade Nova
de Lisboa, Lisbon,
Portugal;
5 Department of Inorganic and Analytical Chemistry, Budapest
University of Technology and Economics, Budapest, Hungary;
*correspondence; E-Mails: martins.a@pharm.u-szeged.hu (A.M.);
hunyadi.a@pharm.u-szeged.hu (A.H.);
Molecules 2013, 18, 15255-15275;
http://dx.doi.org/10.3390/molecules181215255
Keywords:
ecdysteroids; 20-hydroxyecdysone; acetonide; dioxolane;
stereochemistry; cancer; multi-drug resistance;
P-glycoprotein; ABCB1 transporter; efflux pump
Abstract:
Ecdysteroids, molting hormones of insects, can exert several mild,
- non-hormonal bioactivities in mammals,
including humans. In a previous study, we found a significant effect of
- derivatives on the ABCB1 transporter mediated multi-drug resistance
of a transfected murine leukemia cell line. In this paper, we present
- a structure-activity relationship study of the apolar dioxolane
derivatives of 20-hydroxyecdysone.
Semi-synthesis and bioactivity of a total of 32 ecdysteroids,
including 20 new compounds, is presented, supplemented
- with their complete 1H- and 13C-NMR signal assignment.
1. Introduction
Ecdysteroids represent a large family of steroid hormones that play a crucial role in
arthropods’physiology. The most abundant representative of these compounds,
- 20-hydroxyecdysone (20E), regulates
- the reproduction,
- embryogenesis,
- diapause and
- molting of arthropods [1].
Their role in plants is still to be fully understood, but it had been suggested that
they have importance in
- several plants as defensive agents against non-adapted herbivores [2].
An estimated 5%–6% of the terrestrial plant species
- accumulate detectable levels of ecdysteroids, among which
- Ajuga, Serratula and Silene spp.,
- containing high amounts of these compounds, are
- good sources of ecdysteroids of herbal origin [3].
Ecdysteroids generally retain the cholesterol-originated side-chain, typically
- contain 27–29 carbon atoms and
- are substituted with 4–8 hydroxyl groups.
- their A/B ring junction is usually cis, and
- a characteristic 7-en-6-one
(α,β-unsaturated ketone) chromophore group is present in their B-ring [4].
Due to their significantly different structure as compared to the
vertebrate steroid hormones, these compounds
- have no hormonal effects in humans [5].
On the other hand, a number of beneficial
- metabolic effects are attributed to them [4–6], which has
- encouraged the production and worldwide marketing of food supplements,
- mainly containing the isolated ecdysteroid compound 20E [6].
In our recent studies, we found that certain ecdysteroid derivatives significantly
- decrease the resistance of a multi-drug resistant (MDR) murine leukemia cell line
- expressing the human ABCB1 transporter to doxorubicin,
- a chemotherapeutic agent and a
- substrate of the ABCB1 transporter, and
we discussed the possible mechanisms that might be involved in this activity [7].
Based on the observed structure-activity relationships
- of the isolated and semi-synthesized ecdysteroids,
- 20-hydroxyecdysone 2,3;20,22-diacetonide (1)
- the acetonide moiety is generally utilized as
- a protecting group for vicinal diols
(which needs a strong acidic environment for removal), - and it is also an important structural element of certain drugs,
such as triamcinolone acetonide (
not a pro-drug for triamcinolone but
has),
having
different pharmacological and pharmacokinetical properties [8].
Based on our previous work,
we have synthesized
- additional dioxolane derivatives and
- thoroughly discussed their structure elucidation and stereochemistry [9].
the study reported herein we present the
- synthesis,
- structure and
- MDR-modulating activity
of 32 ecdysteroid dioxolanes, including 20 new derivatives, and
- provide insights on
- their structure-activity relationships
2. Results and Discussion
2.1. Semi-Synthesis
Having a common protecting group of vicinal diols, the acetonide,
- 32 compounds containing one or two dioxolane rings were
- synthesized from 20-hydroxyecysone
- with various aldehydes and ketones
- in the presence of phosphomolybdic acid.
A summary of the reactions performed and their product structures are presented
in Figure 1.
Figure 1. Semi-synthetic transformations of 20E
and structures of the products obtained.
Substituents of the reagent oxo-compound (X1/X2 and X3/X4)
- typically correspond to R1/R2 and R2/R3, respectively,
except for compounds 18 and 19, where the reagent was methyl-ethyl ketone. C-15
was obtained as a side product in the synthesis of 23.1H- and 13C-NMR data of the
new compounds are presented in Tables 1–3. To facilitate the comparison between
- the NMR signals of structurally analogous hydrogen and carbon atoms
- in the different dioxolane compounds,
we applied a special numbering system
- for the central atoms (C-28 and C-29) of
- the 2,3- and 20,22-dioxolane structures.
Compounds containing similar number of carbon atoms are presented
in one table, and
- compounds with the highest structural similarity are presented
in neighbouring columns.
Table 1. 1H- and 13C-NMR shifts of compounds 4, 6, 22, 30, 33, 9 and 10; in ppm, in methanol-d4.
(go to source)
Table 2. 1H- and 13C-NMR shifts of compounds 16–20 and 26–27; in ppm, in methanol-d4.
(go to source)
Table 3. 1H- and 13C-NMR shifts of compounds 11–14; 31 and 32; in ppm, in methanol-d4.
(go to source)
As published before [9], the 20,22-diol moiety of 20E
- is more reactive than the 2,3-diol, probably
- due tothe free rotation of the 20,22-bond of 20E that
- allows the 20,22-dioxolane ring to form with less strain.
This allowed us to selectively obtain the 20,22-mono-dioxolane derivatives 2–14,
or, depending on the amount of reagent and the reaction time, the
- 2,3;20,22-bis-homo-dioxolanes 17 and 21–25.
By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde or ketone
- could be coupled to position 2,3, resulting in
- several bis-hetero-dioxolane derivatives 26–33.
For this, however, gradually decreasing reactivity with the increase of
- the size of the reagent was a limiting factor: larger aldehydes or ketones
(mainly those containing a substituted aromatic ring) - could not be coupled at the 2,3-position.
The 2,3-monodioxolane derivatives also appeared to be present
- as minor side-products of the reactions,
and as a consequence of their low amount, only one such compound (C-15) was isolated
and studied. To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:
- after protecting the 20,22-diol with phenylboronic acid,
- the 2,3-acetonide could be prepared, and
- removal of the 20,22 protecting group
afforded the desired 2,3-monoacetonide in a one-pot procedure.
In the case of the reactions with aldehydes or asymmetric ketones, the new
- C-28 and C-29 central atoms of the dioxolane rings are stereogenic centers
- two possible diastereomers can be formed at both diols.
Their configuration was elucidated by
- two-dimensional ROESY or
- selective one-dimensional ROESY experiments,
e.g., in the doubly substituted dioxolane derivative 22
(R1 = R4 = n-Bu, R2 = R3 = H) the unambiguous differentiation of the
- 1H and 13C signals of the two n-butyl groups
was achieved in the following way (see Figure 2).
Figure 2. Stereostructure of 22.
Red arrows indicate the detected ROESY steric proximities, the blue numbers
give the characteristic 1H, and the black numbers the 13C chemical shifts.
Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by
- the H-2/C-28 and H-3/C-28 HMBC correlations, and
- that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29 cross peak
The selective ROESY experiment irradiating at 4.93 ppm
- showed contacts with the Hα-2 and Hα-3 atoms
- proving the α position of the R2 = H atom.
The ROESY response obtained irradiating H = R3 signal (δ = 4.91) on H-22 (δ = 3.64 ppm)
- revealed their cis arrangement and the R configuration around C-29.
- assignments of the signals of the two n-butyl groups R1 and R4
- were achieved by selective TOCSY experiments
(irradiation at δ = 4.93 and 4.91, respectively).
- were achieved by selective TOCSY experiments
In case of the C-28-epimers, typically an approximately 1:1 yield was obtained, and a good
separation was achieved by simple chromatographic methods (see below). On the other hand,
possibly due to steric reasons,
- the longer chain of the reagent was highly selective in the α-position
- in the 20,22-dioxolane moiety.
This selectivity was, however, decreased in cases
- when larger moieties were present in the reagent,
such as substituted aromatic rings, resulting in the appearance of the other epimers
These epimer pairs (compounds 11-12 and 13-14) required high-performance liquid
chromatography (HPLC) for their successful separation. C-10 was isolated by HPLC
as a minor product from the preparation of C-9; this compound, considering
- the vicinal coupling constant of the olefinic hydrogen atoms
(J = 11.8 Hz) contains a Z double bond,
and most likely originated from an impurity in the trans-cinnamic aldehyde reagent used.
C-18 and C-19 were the only cases where one of the dioxolane rings was formed with
- the elimination of ethanol instead of water, losing an
- ethyl group from the reagent methyl ethyl ketone.
2.2. Anti-Proliferative Effect of Ecdysteroid Derivatives on
PAR and MDR Mouse Lymphoma Cells
The anti-proliferative activity of the derivatives was determined by
- incubation of each of the cell lines with
- serial dilutions of the compounds.
Inhibitory concentrations (IC50) were calculated and are presented in Table 4.
Table 4. IC50 values of the ecdysteroid derivatives and fluorescence activity ratio (FAR)
values determined in presence of 2 and 20 μM of compound. IC50—inhibitory concentration
(concentration of compound that inhibits 50% of cell growth); IC50 values are presented as
the average of 3 independent experiments ± the standard error of the mean (SEM);
*— the compound showed cytotoxicity at this concentration and it was not possible to
calculate the FAR value; FAR values of the positive control verapamil (20.4 μM) and the negative
control DMSO (0.2%) were 5.73 and 0.72, respectively.
As seen from the table, several compounds
- exert much lower anti-proliferative activity on the MDR cell line
as compared to the parental one,
while other compounds show similar activities on both cell lines.
2.3. Inhibition of the ABCB1 Pump of MDR Mouse Lymphoma Cells
(Rhodamine 123 Accumulation Assay)
Accumulation of rhodamine 123 by MDR mouse lymphoma cells
was evaluated by flow cytometry
- in the presence of the newly described compounds
- in order to study their capacity to inhibit the ABCB1 pump and
- therefore prevent the efflux of the dye,
which was consequentially retained inside the MDR cell.
Parental mouse lymphoma cells were used as control
- for dye retention inside the cell
- while MDR cells alone do not retain rhodamine 123 at
the concentration employed.
The efflux pump inhibitor (EPI) verapamil was used as positive control.
All the compounds were dissolved in DMSO, which was also evaluated for
- any effect on the retention of the fluorochrome.
DMSO concentration in the assay was 0.2%. For each compound,
- the fluorescence activity ratio (FAR), which measures
- the amount of rhodamine 123 accumulated by the cell
- in presence of the compound was calculated as follows:
FAR = (FLMDRtreated/FLMDRuntreated)/(FLPARtreated/FLPARuntreated) (1)
where FL is the mean of the fluorescence. The obtained results are shown by Table 4.
As seen from the table, the compounds
- showed marked differences according to their capacity to inhibit the efflux of rhodamine 123 in this bioassay:
from the practically inactive (compounds 5, 7, 9, 10, 12 and 20) to the very strong (compounds 6, 8, 14 and 25),
various activities were observed.
Most interestingly, these results did not always conform to those obtained from the combination studies, for example,
no significant differences can be observed
- between the combination indices of compounds 20 and 25, and
- compound 3, very weak in this assay, was able to act in a rather significant synergism with doxorubicin (see below).
These observations seem to support our initial theory, that
- these compounds are not or not exclusively acting as EPIs
but other mechanisms may also be involved in their activity [7].2.4. Combination Studies: Effect of Ecdysteroid Derivatives on the Activity of Doxorubicin on MDR Mouse Lymphoma Cells
Effect of the newly synthesized derivatives was evaluated on checkerboard 96-cell plates with different concentrations of
doxorubicin and compound after 48 h of incubation of the cells, similarly to our previous approach [7]. Combination
indices for the different constant ratios of compound vs.doxorubicin were determined by using the CompuSyn software to plot four to five data points to each ratio. CI values were calculated by means of the median-effect equation [10], where
CI < 1, CI = 1, and CI > 1 represent
- synergism,
- additive effect (i.e., no interaction), and
- antagonism, respectively.
The CI values are presented on Table 5. Combination index plots (or Fa-CI plots, where Fa is the fraction affected) were
also generated for each compound using serial deletion analysis in order to determine variability of the data [10]. An example
of Fa-CI plot is given by Figure 3 for compounds 1, 5 and 15.
Figure 3. Fraction affected (Fa) vs. combination index (CI) value plot for compounds 5 and 15, in comparison with the original lead compound 1.
Table 5.
Combination index (CI) values at different drug ratios (compound vs. doxorubicin, respectively) at 50, 75 and 90% of growth inhibition (ED50, ED75
and ED90, respectively); CIavg— weighted average CI value; CIavg = (CI50 + 2CI75 + 3CI90)/6. CI < 1, CI = 1, and CI > 1 represent
- synergism,
- additivity, and
- antagonism, respectively.
Dm, m, and r represent antilog of the x-intercept, slope, and
- linear correlation coefficient of the median-effect plot, respectively.
As seen from Table 5, all compounds acted synergistically with doxorubicin and their behavior followed our previous observation,
Error bars represent 95% confidence intervals by means of serial deletion analysis performed with the CompuSyn software.
The 2,3-mono-dioxolane derivative 15 represents significantly
- stronger synergism with doxorubicin than the corresponding 20,22-dioxolane derivative 5 at practically all activity levels, and above Fa = 0.7 (which, in case of cancer, matters the most [10])
- it is also stronger than compound 1.
- in case of all ecdysteroids there seems to be an “ideal” compound vs. doxorubicin ratio
- where the strongest synergistic effect occurs.
- in case of all ecdysteroids there seems to be an “ideal” compound vs. doxorubicin ratio
Based on the variability of the mono-, homo-di- and hetero-di-substituted compounds, as well as
- that of the coupled substituents at R1–R4, several novel structure-activity relationships (SARs) were observed.
According to this, we followed our previous approach [7]—for each compound, the strongest activity by
- means of the weighted average CI values was primarily considered for comparison,
regardless of the compound vs. doxorubicin ratio where this activity was found.
- the 2,3-dioxolane moiety is far more important for a strong activity, than the one at positions 20,22. compound 15, monosubstituted at position 2,3, was the only ecdysteroid derivative that was able to exert a stronger activity at its best ratio than our original lead, the diacetonide
compound 1 (Figure 3). - A very interesting SAR was revealed by comparing the activity of the C-28 and C-29 epimer pairs:
at C-28, the larger substituent needs to take the α‐position (24 vs. 25), while at C-29 the β-position
for a stronger activity (cf. 11 vs. 12 and 13 vs. 14). - As concerns the 20,22-monodioxolanes, increasing the length of the side chains coupled to C-29 lead to a significant increase in the synergistic activity with doxorubicin
till the length of three carbon atoms (compound 3), however a longer alkyl substituent (compound 4)
case when a non-substituted phenyl group was present (compound 7).Addition of a β-methyl group to C-29 could significantly improve the activity as compared to that of
- the 29α-phenyl substituted derivative (cf. 8 vs. 7, respectively).
The observed structure-activity relationships are summarized in Figure 4.
Figure 4. SAR summary for compounds 1–33.
“Greater than” symbols denote stronger synergistic activities, i.e., lower weighted average CI values
when applied together with doxorubicin.
3. Experimental
3.1 General Information
The starting material 20E (90%, originated from the roots of Cyanotis arachnoidea) was purchased
from Shaanxi KingSci Biotechnology Co., Ltd. (Shanghai, China), and further purified by crystallization
from ethyl acetate–methanol (2:1, v/v), so that purity of 20E utilized for the semi-syntheses was 97.8%,
by means of HPLC-DAD, maximum absorbance within the range of 220–400 nm. Mono- and disubstituted
ecdysteroid dioxolanes were synthesized as published before [9]. Briefly, the starting compound was
reacted with the aldehyde or ketone to be coupled to positions 20,22 and/or 2,3 in the presence of
phosphomolybdic acid (Lach-Ner, Neratovice, Czech Republic) at room temp. for 5–60 min depending on the target compound. The reaction was terminated by neutralizing the pH with a 5% aqueous solution of NaHCO3 (Merck, Munich, Germany), methanol was evaporated until only water was present, and the
product(s) were extracted with methylene chloride. Column chromatography (CC), rotational planar
chromatography (RPC) and/or crystallization was used for purification, as detailed below. Solvent system
compositions are given in v/v%. For RPC, a Chromatotron device (Harrison Research, Palo Alto)
was used. The separation was monitored with thin layer chromatography (TLC) on silica gel 60 F254
(0.25 μm, Merck). HPLC purification of compounds 9–14 was performed on a gradient system of two
Jasco PU2080 pumps connected to a Jasco MD-2010 Plus photodiode – array detector, on a Zorbax
XDB-C8 column (5 μm, 9.6 × 250 mm) at a flow rate of 3 mL/min. Mass spectra were recorded on an
API 2000 triple quadrupole tandem mass spectrometer (AB SCIEX, Foster City, CA) in positive mode with
atmospheric pressure chemical ionization (APCI) ion source except for compound 29 which was measured
with electron-spray ionization (ESI). 1H- (500.1) and 13C- (125.6) MHz NMR spectra were recorded at room temperature on an Avance 500 spectrometer (Bruker, Billerica, MA). For the examples of compounds
3, 5, 7, 8, 15, 21, 23–25, 28 and 29, structure elucidation of ecdysteroid dioxolanes by comprehensive one-
and two-dimensional NMR methods has recently been discussed in detail elsewhere, including experimental
details for the aforementioned compounds [9]. Regarding the new compounds, amounts of approximately
1–10 mg were dissolved in 0.1 mL of methanol-d4 and transferred to a 2.5 mm Bruker MATCH NMR sample
tube. Chemical shifts are given on the δ-scale and are referenced to the solvent (MeOH-d4: δC = 49.1 and δH =
3.31 ppm). Pulse programs of all experiments (1H, 13C, DEPTQ, DEPT-135, sel-TOCSY, sel-ROE, sel-NOE,
gradient-selected (gs) 1H, 1H-COSY, edited gs-HSQC, gs-HMBC, ROESY) were taken from the Bruker software
library. Most 1H assignments were accomplished using general knowledge of chemical shift dispersion with
the aid of the proton-proton coupling pattern (1H-NMR spectra).
3.2. Semi-Synthesis and Purification of Monosubstituted Ecdysteroid Dioxolane Derivatives 2–16
20E was dissolved in methanol (10 mL, Merck) to a final concentration of 100 mM or 25 mM in case of compounds
9, 10, 13, 14, and the corresponding reagent (3: butyraldehyde, 4: valeraldehyde, 5: 3-pentanone, 6: methyl isobutyl
ketone, 10 equivalents each; 7: benzaldehyde, 5 g; 8: acetophenone, 6 g; 9, 10: cinnamaldehyde, 11, 12: vanillin, 13, 14:
4-benzyloxybenzaldehyde, 10 equivalents each; 15: 3-pentanone, 100 equivalents; (compound 15 was obtained from the
synthesis of 25, see below) was added to the solution. Phosphomolybdic acid (1.00 g) was added (except in the case of the synthesis of 9 and 10, when 0.50 g were added) and the mixture was stirred at room temp. for 10 min (except for 7: 5 min, 8: 60 min, 15: 30 min). In the case of compound 16, 20E was dissolved in methanol (10 mL) to a final
concentration of 100 mM, and after adding phenylboronic acid (1 equivalent), the mixture was stirred for 30 min.
Acetone (500 equivalents) and phoshomolybdic acid (0.5 g) were added to the mixture, and after 1 h stirring a solution
of NaOH and H2O2 was added in order to remove the phenyl-boronate group. Then, the reaction was worked up as
described above. Compounds 3, 4, 7, 8, a mixture of 9-10, and compounds 15 and 16 were obtained from RPC on silica
gel with appropriate solvent systems of ethyl acetate-ethanol-water (3, 4) or cyclohexane-ethyl acetate (7, 8, 9-10, 15, 16).
The purification of compounds 11-12 and 13-14 started with CC by using solvent systems of ethyl acetate-ethanol-water.
Isomer pairs 9-10, 11-12 and 13-14 were isolated by RP-HPLC (9, 10: 75% CH3OH aq., 3 mL/min; 11, 12: 70% CH3OH
aq., 3 mL/min; 13, 14: 80% CH3OH aq., 3 mL/min). Compounds 2, 5 and 6 were recrystallized from acetonitrile without
chromatographic purification. The yields were:
2 (236.6 mg, 45.43%), 3 (116.2 mg, 21.7%), 4 (142.8 mg, 26.0%), 5 (183.5 mg, 33.4%), 6 (71.9 mg, 25.2%), 7 (292.5 mg, 51.4%),
8 (196.8 mg, 33.8%), 9 (27.0 mg, 18.5%), 10 (13.9 mg, 9.4%), 11 (156.3 mg, 25.4%), 12 (67.0 mg, 10.9%), 13 (67.3 mg, 39.9%),
14 (33.7 mg, 20.0%), 15 (27.4 mg, 5.0%), 16 (13.3 mg, 10.2%).
3.3. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 17–25 in One-Step
20E (17–20: 200 mg; 21–25: 480 mg) was dissolved in methyl-ethyl ketone (20 mL, compounds 17–20) or methanol (10 mL)
and the reagent was added to the solution (21: butyraldehyde, 100 equivalents, 22: valeraldehyde, 100 equivalents, 23: 3-pentanone,
100 equivalents, 24, 25: benzaldehyde, 5 g). Phosphomolybdic acid was added (17–20: 20 mg; 21–25: 0.50 g), and the mixture was
stirred at room temperature for 5 (17–20, 24–25) or 30 (21–23) min. The reactions were worked up as described above, and the
products were isolated by RPC using the appropriate n-hexane-acetone (17–20) or cyclohexane-ethyl acetate-ethanol (21–25) solvent
systems. As a side-product of the reaction of 20E with methyl ethyl ketone, 20 was obtained as a 20,22-onodioxolane derivative. The
yields were:
17 (15.5 mg, 6.3%), 18 (4.9 mg, 2.1%), 19 (8.4 mg, 3.6%), 20 (4.46 mg, 2.0%) 21 (242.4 mg, 41.2%), 22 (134.5 mg, 21.8%),
23 (42.3 mg, 6.9%), 24 (36.1 mg, 5.5%), 25 (43.8 mg, 6.7%).
3.4. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 26–33 in Two-Steps
Previously obtained 20,22-monosubstituted compounds (2, 20.7 mg; 3, 40.0 mg; 5, 40.7 mg; 6, 50.0 mg; 7, 57.0 mg; 8, 87.3 mg; 2, 104.0 mg)
were dissolved in methyl ethyl ketone (2 mL, 26 and 27) or in methanol (5 mL) and the reagent (28–32: acetone, 500 equivalents; 33: butyraldehyde,
500 equivalents) was added to the solution. Phosphomolybdic acid (26, 27: 20 mg; 28–32: 0.5 g) was added to the solution, and the mixture was
stirred at room temperature for 5 (26, 27) or 60 (28–33) min. The reactions were terminated and the products were purified as described above for
the disubstituted derivatives. The yields were: 26 (5.1 mg, 23.1%), 27 (5.1 mg, 23.1%), 28 (10.9 mg, 25.4%), 29 (15.8 mg, 36.2%), 30 (15.5 mg, 28.9%),
31 (24.8 mg, 40.6%), 32 (38.7 mg, 41.5%), 33 (53.0 mg, 46.2%).
3.5. Further Experimental Data for the New Compounds
(see Archival supplement)
3.6. Preparation of the Compounds for the Bioassays
Each compound was dissolved in 99.5% DMSO (Sigma, Munich, Germany). In each protocol DMSO was always tested
as solvent control and no activity was observed.
3.7. Cell Lines
Two mouse lymphoma cell lines were used in this work: a parental (PAR) cell line, L5178 mouse T-cell lymphoma cells (ECACC
catalog no.87111908, U.S. FDA, Silver Spring, MD); and a multi-drug resistant (MDR) cell line derived from PAR by transfection
with pHa MDR1/A retrovirus [11]. MDR cell line was selected by culturing the infected cells with 60 μg/L colchicine. Both cell
lines were cultured in McCoy’s 5A medium supplemented with 10% heat inactivated horse serum, L-glutamine, and antibiotics
(penicillin and streptomycin) at 37 °C and 5% CO2 atmosphere [12].
Medium, horse serum, and antibiotics were purchased from Difco (Detroit, MI).
3.8. Anti-proliferative Assay
Anti-proliferative activities on PAR and MDR cell lines were performed as described before [7]. Briefly, 6 × 103 cells/well were
incubated with serial dilutions of each compound (n = 3) in McCoy’s 5 A medium for 72 h at 37 °C, 5% CO2. Then, MTT (Sigma) [13]
was added to each well at a final concentration of 0.5 mg/mL per well) and after 4 h of incubation, 100 μL of SDS 10% (Sigma) in
0.01 M HCl was added to each well. Plates were further incubated overnight and optical density at 540 and 630 nm using an ELISA
reader (Multiskan EX, Thermo Labsystem, Milford, MA). Fifty percent inhibitory concentrations (IC50) were calculated using 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, San Diego, CA,).
3.9. Inhibition of ABCB1 Pump of MDR Mouse Lymphoma Cells (Rhodamine 123 Accumulation Assay)
Inhibition of ABCB1 was evaluated using rhodamine 123, a fluorescent dye, which retention inside the cells was evaluated by flow cytometry (14).
Briefly, 2 × 106 cells/mL were treated with 2 and 20 μM of each compound. After 10 min incubation, rhodamine 123 (Sigma) was added to a final
concentration of 5.2 μM and the samples were incubated at 37 °C in water bath for 20 min. Samples were centrifuged (2,000 rpm, 2 min) and washed
twice with phosphate buffer saline (PBS, Sigma). The final samples were re-suspended in 0.5 mL PBS and its fluorescence measured with a Partec CyFlow flow cytometer (Partec, Münster, Germany). Verapamil (Sanofi-Synthelabo, Budapest, Hungary) at 20.4 μM was used as positive control.
3.10. Combination Assays
The combined activity of doxorubicin (Teva, Budapest, Hungary) and the ecdysteroids was determined using the checkerboard microplate method, as
described before [7]. Briefly, 5 × 104 cells/well were incubated with doxorubicin and the compound to be tested for 48 h at 37 °C under 5% CO2. Cell
viability rate was determined through MTT staining, as described above. The interaction was evaluated using the CompuSyn software (CompuSyn Inc.,
Paramus, NJ) at each constant ratio of compound vs. doxorubicin (M/M), and combination index (CI) values were obtained for 50%, 75%, and 90% of
growth inhibition.
4. Conclusions
In the present study, we have prepared 32 semi-synthetic derivatives of 20-hydroxy- ecdysone,following our previously observed structure-activity relationships on the strong synergistic activity of ecdysteroid dioxolanes with doxorubicin on a murine MDR cancer cell line expressing the human ABCB1
transporter. By utilizing the different reactivity of the 2,3 and 20,22 vicinal diol moieties, various bis-homo- and bis-hetero-dioxolanes were synthesized,
as well as several 20,22- and two 2,3-monodioxolane derivatives. In addition to these, two epimer pairs were also obtained. Twenty compounds are reported for the first time; their chemical structures were thoroughly investigated by comprehensive 1 and 2D-NMR methods, based on which complete signal
assignments are provided. The compounds showed mild to very strong synergistic effects with doxorubicin against the aforementioned MDR cancer cell line, and the diversity of the substituents allowed us to observe several new structure-activity relationships. Among these, the importance of the 2,3-dioxolane substitution and the observations concerning the role of stereochemistry at C-28 and C-29 are the most interesting results. Apparently, ecdysteroids can be engineered to become strong MDR modulators only by decreasing the polarity at the A-ring, while the polar side-chain can be kept, providing the possibility for designing such compounds with a reasonable water solubility and high drug-likeness.
Considering the high importance of the 2,3-dioxolane group in our compounds and the fact that exactly this part is the most sensitive to an acidic environment,
per os application of these compounds requires an appropriate formulation; development of such delivery systems is currently in process, investigation on their activity against MDR cancer xenografts is going to be reported in the near future.
Acknowledgments
The authors acknowledge the support from the European Union co-funded by the European Social Fund (TÁMOP 4.2.2/B-10/1-2010-0012,
TÁMOP 4.2.2.A-11/1/KONV-2012-0035) and the Fundação para a Ciência e a Tecnologia (FCT), Portugal (PEsT-OE/SAU/UI0074/2011). A. Martins was supported by the grant SFRH/BPD/81118/2011, FCT, Portugal. The work presented here was performed within the framework
of COST Action CM1106, Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells. The authors thank Nikoletta Jedlinszki for
the mass spectroscopic measurements, Imre Ocsovszki, supported by the grant TÁMOP-4.2.1/B-09/KONV-2010-0005, for the flow cytometry measurements and Ibolya Hevérné Herke for the semi-synthetic preparation and purification of compounds 17–20.
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–33 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
Archival Supplement
29α-Butyl-20,22-O-methylidene-20-hydroxyecdysone (4): white needle-like crystals; mp 197–199 °C; for 1H- and 13C-NMR data, see
Table 1; APCI-MS, m/z (Irel, %): 549 [M+H]+, 531 [M+H-H2O]+, 445, 427, 409.
29α-I-butyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (6): white needle-like crystals; mp 198–199 °C; for 1H- and 13C-NMR
data, see Table 1; APCI-MS, m/z (Irel, %): 563 [M+H]+, 545 [M+H-H2O]+, 445, 427, 409.
29α-E-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (9): white solid; mp. 161–163 °C; for 1H- and 13C-NMR data, see Table 1,
in addition to this, the vicinal coupling constant of the olefinic hydrogen atoms (J = 16.0 Hz) proved the E configuration of the double bond;
APCI-MS, m/z (Irel, %): 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.
29α-Z-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (10): white solid; mp 138–140 °C; for 1H- and 13C-NMR data, see
Table 1; APCI-MS, m/z (Irel, %): 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.
29α-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (11): white solid; mp 163–165 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.
29β-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (12): white solid; mp 157–159 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.
29α-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (13): white solid; mp 144–146 °C; for 1H- and 13C-NMR data, see
Table 3; APCI-MS, m/z (Irel, %): 675 [M+H]+, 445, 427, 409.
29β-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (14): white solid; mp 139–141 °C; for 1H- and 13C-NMR data, see
Table 3; APCI-MS, m/z (Irel, %): 675 [M+H]+, 445, 427, 409.
20-Hydroxyecdysone 2,3-acetonide (16): white solid; mp 124–126 °C; for 1H- and 13C-NMR data, see Table 2; APCI-MS, m/z (Irel, %):
553 [M+H+MeOH]+, 535, 503, 485, 467, 409.
28α,29α-Diethyl-28β,29β-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (17): white solid; mp 98–100 °C; for 1H- and
13C-NMR data, see Table 2; APCI-MS, m/z (Irel, %): 589 [M+H]+, 571 [M+H-H2O]+, 499, 481, 409.
28α,29α-Dimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (18): white solid; mp 99–101 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 561 [M+H]+, 543 [M+H-H2O]+, 499, 481, 409.
28β,29β-Dimethyl-29α-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (19): white solid; mp 79–81 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 561 [M+H]+, 543 [M+H-H2O]+, 471, 453, 409.
29α-Ethyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (20): white solid; mp 140–142 °C; for 1H- and 13C-NMR data, see
Table 2; APCI-MS, m/z (Irel, %): 535 [M+H]+, 517 [M+H-H2O]+, 445, 427, 409.
28β,29α-Dibutyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (22): transparent crystals; mp 186–187 °C; for 1H- and 13C-NMR
data, see Table 1; APCI-MS, m/z (Irel, %): 617 [M+H]+, 599 [M+H-H2O]+, 513, 495, 409.
28β,29,29-Trimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (26): white solid; mp 100–102 °C; for 1H- and 13C-NMR data,
see Table 2; APCI-MS, m/z (Irel, %): 547 [M+H]+, 517, 499, 467, 409.
28α,29,29-Trimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (27) white solid; mp 360–362 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 575 [M+H]+, 557 [M+H-H2O]+, 499, 481, 409.
28,28,29β-Trimethyl-29α-i-buthyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (30): transparent solid; mp 114–115 °C; for 1H-
and 13C-NMR data, see Table 1; APCI-MS, m/z (Irel, %): 603 [M+H]+, 585 [M+H-H2O]+, 485, 467, 409.
28,28-Dimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (31): white solid; mp 114–117 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 641 [M+H+MeOH]+, 623, 517, 485, 467, 409.
28,28,29β-Trimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (32): white solid; mp 126–128 °C; for 1H- and 13C-
NMR data, see Table 3; APCI-MS, m/z (Irel, %): 623 [M+H]+, 605 [M+H-H2O]+, 485, 467, 409.
28β-Propyl-29,29-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (33): transparent solid; mp 109–111 °C; for 1H- and 13C-
NMR data, see Table 1; APCI-MS, m/z (Irel, %): 575 [M+H]+, 557 [M+H-H2O]+, 499, 481.
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