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.
appeared to be less preferable. Introducing larger aromatic groups did not lead to a breakthrough, although further substituents on the aromatic ring (compounds 11, 13) were able to increase activity as compared to the
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.
References
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dioxolane derivatives, a novel group of MDR modulator agents. Magn. Reson. Chem. 2013, 51, 830−836.
10. Chou, T.-C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies.
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11. Pastan, I.; Gottesman, M.M.; Ueda, K.; Lovelace, E.; Rutherford, A.V.; Willingham, M.C. A retrovirus carrying an MDR1 cDNA confers
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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.
![Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004](https://i0.wp.com/pharmaceuticalintelligence.com/wp-content/uploads/2013/12/fig-2a-measurements-of-range-of-ca2total-average-ca2free-values-_page_004.jpg)
![Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1](https://i0.wp.com/pharmaceuticalintelligence.com/wp-content/uploads/2013/12/fig-2b-measurements-of-range-of-ca2-total-average-ca2free-values_edited-1.jpg)
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