Oxidized Metabolites of 20-Hydroxyecdysone and Their Activity on

Article pubs.acs.org/jnp

Oxidized Metabolites of 20-Hydroxyecdysone and Their Activity on Skeletal Muscle Cells: Preparation of a Pair of Desmotropes with Opposite Bioactivities József Csábi,† Tusty-Jiuan Hsieh,‡ Feria Hasanpour,† Ana Martins,§,# Zoltán Kele,⊥ Tamás Gáti,∥ András Simon,∥ Gábor Tóth,∥ and Attila Hunyadi*,† †

Institute of Pharmacognosy, §Department of Medical Microbiology and Immunobiology, and ⊥Department of Medical Chemistry, University of Szeged, 6720 Szeged, Hungary ‡ Department of Genome Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, Republic of China ∥ Department of Inorganic and Analytical Chemistry, NMR Group, Budapest University of Technology and Economics, 1111 Budapest, Hungary S Supporting Information *

ABSTRACT: Increasing the activation of protein kinase B (Akt) has been suggested as a key signaling step in the nonhormonal anabolic activity of the phytoecdysteroid 20hydroxyecdysone (20E) in mammals. Base-catalyzed autoxidation of this compound was shown previously to yield interesting B-ring-modified analogues. Herein is reported a thorough study on this reaction, resulting in the preparation and complete NMR spectroscopic assignments of calonysterone (5) and its previously overlooked desmotropic pair (7), along with two new sensitive metabolites of 20E. The two isomers showed considerable stability in solution. Time dependency of the reaction for yield optimization is also presented; by means of analytical HPLC, the two desmotropes can reach a maximum combined yield of >90%. The activity of these compounds on Akt phosphorylation was tested in murine skeletal muscle cells. Compounds 2 and 5 showed more potent activity than 20E in increasing Akt activation, while compound 7 exerted an opposite effect. As such, the present study provides the first direct evidence for a pair of desmotropes exerting significantly different bioactivities.

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droxyecdysteroid derivative in an approximately 70% isolated yield.9 Despite some sporadic reports, the possible mechanism(s) of action of ecdysteroids in mammals still remains unresolved. Nevertheless, it is clear that these compounds do not bind to mammalian steroid hormone receptors,3 and Ca2+-influxmediated protein kinase B (Akt) activation was shown to be involved in their activity of increasing protein synthesis.10 Moreover, there is a possible link between ecdysteroids and the vitamin D system,11 which makes B-ring-modified analogues of 20E particularly interesting for related bioactivity studies. Our attention was turned toward the above-mentioned basecatalyzed autoxidation of 20E, and the effects of the products on Akt phosphorylation in skeletal muscle cells were evaluated in order to gain insight into their potential anabolic and antidiabetic activities.

hytoecdysteroids, plant-derived analogues of the insect molting hormone, have attracted much research interest for nearly 50 years. In view of the possible use of these compounds, rational drug design to develop new insecticides has been explored.1 They are also used as elicitors for transgenic ecdysteroid-inducible gene expression systems,2 and, as a result of their nonhormonal anabolic/adaptogenic activity in mammals, including humans,2,3 various health benefits have been attributed to these compounds. Considering that 20-hydroxyecdysone (20E), by far the most abundant phytoecdysteroid, is also commercially available in large quantities, its semisynthetic modifications have served as a reasonable strategy to obtain rare, minor natural ecdysteroids and/or new bioactive derivatives.4−6 Known semisynthetic modifications of 20E and other major ecdysteroids recently have been reviewed.7 Autoxidation of 20E in aqueous MeOH containing 2% NaOH was reported by Suksamrarn et al. for the synthesis of 9α,20-dihydroxyecdysone (35% yield) and calonysterone (29% yield), two rare minor phytoecdysteroids.8 A recent update for this reaction has been published by Savchenko et al., who found that autoxidation of the 2,3;20,22diacetonide derivatives of 20E or ponasterone A by 10% NaOH in MeOH led to the epimerization at C-5 together with 9αhydroxylation to afford the corresponding (5α)-9α,20-dihy© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION First, autoxidation was conducted following the method of Suksamrarn et al., by stirring 20E for 3 h at room temperature (22−25 °C) in 70% aqueous MeOH containing 2% NaOH.8 As the authors did not describe their purification process, a routine Received: March 22, 2015

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DOI: 10.1021/acs.jnatprod.5b00249 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Spectroscopic Data for Compounds 2 and 4−7 (MeOH-d4) 2 no.

1

J (Hz)

1β

1.70

α 2

2.14 4.01

3

3.54

4β α

1.75 1.91

5

3.16

4 13

J (Hz)

36.1

3.04

dd; 14.7, 2.9

q; ∼3.0

70.8

1.40 3.96

ddd; 12.0, 4.2, 3.0 t; 12.2 dt; 12.2, 4.5 dd; 12.2, 3.4

72.5

24.7

C

23

1.66 1.29 1.80 1.43

24 25 26 27

1.21 3.32

1.19 1.20

70.2

1.35 4.00

3.52

ddd; 12.0,

73.5

3.52

2.54 3.16

4.5, 3.0 t; 12.0 dd; 12.0, 4.5

28.0

2.54 3.12

1.69

q; ∼3.0

70.3

1.45 4.00

q; ∼3.0

70.3

2.03 3.75

ddd; 12.0, 4.8, 3.0 t; 12.5 dd; 12.5, 4.8

73.7

3.51

73.5

27.6

2.54 3.10

ddd; 12.0, 4.8, 3.0 t; 12.5

27.5

1.79 1.36

dt; 13.0, 3.7 td; 13.0; 3.7

36.0

2.60

dd; 13.0, 3.7

49.6

dd; 13.5; 5.4 dd; 13.5; 7.1

49.2

2.65 2.30

37.9

2.58 2.08

30.0

2.29

50.4 18.3 19.8 77.9 21.2 78.5 27.5 42.5 71.3 29.0 29.9

6.52

dd; 3.0, 2.0

1.52 49.1 142.3 128.1

6.84

2.70 2.23 2.18

32.6

1.05 1.61

22.4 25.4 77.3 20.6 78.8

1.08 1.50

27.5

1.62 1.31 1.81 1.44

1.29 3.37 1.64 1.34 1.82 1.44 1.19 1.20

57.1

dd; 10.5, 2.0

42.4 71.4 29.0 30.0

1.99 48.8 142.6 127.8

2.71 2.24 1.99

1.27 3.38

1.18 1.20

32.7 dd; 11.0, 7.8 s s s dd; 10.5, 1.8

56.5 18.3 27.6 77.3 20.5 78.7 27.3 42.4 71.4 28.9 30.0

s s

s

dd; 7.4, 2.5

68.0

199.2 144.4 119.5 136.3 41.8 126.8

42.6

2.69 48.8 82.5 33.3

2.54 1.99 1.99 1.75 2.24

t; 9.0

48.6

0.93 1.51

s s

1.22 3.35

s dd; 10.5, 1.8

18.5 28.5 78.0 21.0 78.6

22.9

1.67 1.28 1.81 1.44 1.19 1.20

37.7

3.86

2.20

dt; 11.5, 5.5

C

68.7

6.00

dt; 11.5, 5.5

13

ddd; 12.0, 4.0, 3.0 q; ∼ 3,0

2.58

21.3

s s

42.0

2.65

46.1 86.9 31.4

s dd; 10.5, 1.8

dd; 14.3, 3.0

dd; 7.1, 5.4

2.09

dd, 9.2, 8.9 s s

2.31

J (Hz)

H

4.83

2.17

0.90 1.11

q; ∼3.0

1

43.0

C

144.5 181.2 133.1 168.7 42.5 25.4

α

18 19 20 21 22

dd; 14.2, 3.0

7 13

J (Hz)

144.4 181.5 124.8 165.5 42.3 25.5

29.9

1.65 2.00 1.71 2.43

2.42

1

H

C

144.3 181.3 127.1 164.6 43.2 68.3

1.72 1.83

α 16β α 17

41.7

13

133.3

α 12β

1.93

6

J (Hz)

133.2

2.09

13 14 15β

1

H

C

134.9

202.9 126.0 159.9 76.0 43.6 29.1

s

5 13

48.7

6 7 8 9 10 11β

5.83

1

H

H

27.4 42.5

s s

71.4 29.1 29.8

6.86

dd; 3.7, 2.4

48.1 144.3 135.3

2.74 2.33 2.10

33.1 dd; 11.0, 7.6

57.4

1.10 1.14

s s

1.27 3.39

s dd; 10.5, 1.8

20.6 30.9 77.2 20.4 78.7

1.63 1.33 1.82 1.45 1.19 1.21

27.4 42.4

s s

71.4 28.9 30.0

Subsequent small-scale experiments were performed, and the amount of 20E was monitored by TLC throughout the autoxidation process. An overnight reaction was found preferable. Following this, a more gentle purification procedure was applied: the neutralized reaction mixture was subjected directly to centrifugal partition chromatography (CPC) using a mixture of EtOAc−MeOH−H2O in the ascending mode (i.e., normal-phase separation). The crude, neutralized reaction mixture was also tested by HPLC, and, even though a much less complex mixture was detected than before, the compositions of the combined CPC fractions after vacuum evaporation at 40 °C did not correspond to that of the reaction outcome. The main constituents of certain fractions completely disappeared and apparently were converted to calonysterone

procedure was carried out for the isolation. Thus, after carefully neutralizing the pH by acetic acid, the mixture was evaporated under reduced pressure at 40 °C, and separation of the products was initiated by column chromatography over silica gel. Even though a few major products could be observed by TLC, the mixture was highly complex and did not reproduce the previous work.8 Moreover, a significant amount of unchanged 20E also remained. A multistep procedure including repeated column chromatographic and HPLC purification steps was needed to obtain four compounds: 9α,20-dihydroxyecdysone (1), (5α)-9α,20-dihydroxyecdysone (2), 25-hydroxydacryhainansterone (3), and 11α-hydroxycalonysterone (4), a new ecdysteroid. However, all of them were obtained in very low yields (see Experimental Section, reaction 1). B



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Figure 1. Structures of 20E and the isolated compounds 1−7.

For compound 3, the measured 1H and 13C NMR chemical shifts (in methanol-d4) were identical with NMR data reported previously for 25-hydroxydacryhainansterone.15 In order to facilitate understanding of the structural relationships between compounds 4−7, the structure elucidation of these compounds is discussed starting with compound 5. The APCIMS spectrum of compound 5 showed a [M + H]+ peak at m/z 477, and HRMS revealed the molecular formula C27H40O7. Thus, two new double bonds were incorporated into the steroid ring system as compared to the parental compound 20E. The appearance of a CO signal at δC 181.5 indicated its cross-conjugated arrangement. The HMBC correlations of H319 (δH 1.50/δC 42.3 and δH 1.50/δC 43.0, respectively) were used to assign the quaternary C-10 and H2C-1 methylene moieties. In addition, the signals at δH 1.50/δC 133.2 and 1.50/ 165.3 supported the presence of quaternary sp2 C atoms at positions C-5 and C-9, which together demonstrated the B ring as a Δ5,6-7-one-Δ8,9 chromophore. The HMBC cross-peak H318/C-14 (δH 1.08/δC 142.6) revealed the splitting of the OH14α group and the occurrence of a Δ14,15 CCH ethylene moiety. Compound 5 was identified as calonysterone (first isolation: Canonica et al.,16 first synthesis: Suksamrarn et al.17), but the available NMR data were reported in pyridine-d5 or dimethyl sulfoxide-d6 without complete 1H and 13C assignments.18 Although Khalig-uz-Zaman et al.19 reported a few assigned 1H NMR signals and the complete 13C NMR spectrum obtained in methanol-d4 for 5, their assignments were not supported by confirmatory NMR experiments. Utilizing edited HSQC, HMBC, and selective ROESY experiments on compound 5 to provide its complete 1H and 13 C NMR assignments (see Table 1), it was found that 10 out of the 27 13C NMR signals were assigned erroneously in the above-mentioned publication.19 Compound 4 exhibited 1H and 13C NMR spectroscopic data similar to those of calonysterone (5), except for the presence of an additional secondary OH group evident from the 1H and 13 C NMR spectra (δH 4.83 dd; δC 68.3 ppm). The detected δH 4.83/δH 1.05 (Hβ-11/H3-18) and δH 4.83/δH 1.61 (Hβ-11/H319) ROESY cross-peaks, respectively, revealed an α arrangement and the C-11 attachment of this OH substituent, leading to the conclusion that compound 4 is 11α-hydroxycalonysterone.

(5), one of the original target compounds that was otherwise not detected prior to the CPC purification (see Experimental Section, reaction 2). In an attempt to isolate the detected sensitive intermediates of 5, further changes were introduced into the purification process of the reaction. Thus, following the CPC separation of the carefully neutralized aqueous solution of the reaction products, the fractions were evaporated under a nitrogen stream, peaks were collected during the subsequent HPLC purifications under argon, and each compound obtained was dried under a nitrogen stream (see Experimental Section, reaction 3). Two further compounds, 6 and 7, were obtained this way, and, surprisingly, only traces of 5 were detected throughout the entire procedure. Structure elucidation of the products was performed by means of LRMS, HRMS, and 1D and 2D NMR spectroscopy.12,13 In the case of compound 1, the molecular formula of C27H44O8 was established by means of HRMS, and the assignment of the 1H and 13C NMR spectra revealed this compound to be identical with the natural product 9α,20dihydroxyecdysone. The chemical shifts correlated well with the values reported earlier for this compound.14 The HRMS of compound 2 revealed the same molecular formula, C27H44O8, as found for compound 1. In order to determine the complete 1H and 13C NMR assignments of compound 2, the DEPTQ, ed-HSQC, HMBC, and 1D selective TOCSY (irradiated at H-5, H-17, and H-22) and selective ROESY (irradiated at H3-19 and H-5) spectra were measured. The strong H3-19/Hβ-4 ROESY (δH 1.11/δC 1.75) response, in addition to the H-5/Hα-1 and H-5/Hα-3 (δH 3.16/δC 2.14, δH 3.16/δC 3.54, respectively) correlations, supported a trans A/B ring junction, whereas the H3-19/Hβ-11 (δH 2.09) and H3-19/ H3-18 (δH 0.91) steric proximities supported the retained B/C ring junction. Hence, the structure of compound 2 was established as the 5α-epimer of 1. Savchenko et al. reported recently the stereoselective synthesis of 2, (20R,22R)2β,3β,9α,14α,20,22,25-heptahydroxy-5α-cholest-7-en-6-one),9 but their 1H NMR chemical shifts, reported in pyridine-d5, were quite different from the present values (see Table 1) due to the strong anisotropic effect of this solvent. C



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Compound 6 also showed 1H and 13C NMR spectra similar to those of calonysterone (5). The molecular formula of C27H42O8Na [M + Na]+ was established by means of HRMS, so the molecule was found to consist of one more H2O molecule than 5. The most characteristic changes in the NMR spectra, as compared to that of 4, were the absence of signals for the Δ14,15 CCH ethylene moiety. In the HMBC spectrum, the cross-peak H3-18/C-14 (δH 0.93/δC 82.5) revealed the presence of a OH-14α group. Differentiation of the α/β position of the diastereotopic methylene hydrogens of the skeleton was achieved through the use of selective onedimensional ROESY experiments irradiating the H3-18, H3-19, and H3-21 atoms, respectively. For compound 7, a molecular formula of C27H40O7Na [M + Na]+ was established using HRMS, and it was determined that this molecule has the same elemental composition as calonysterone (5). Compound 7 was established as including all structural features of compound 3 (25-hydroxydacryhainansterone) from the NMR spectra,15 but instead of an OH-14α group, a Δ14,15 CCH ethylene moiety was present, as confirmed by the HMBC cross-peak between H3-18 and C-14 (δH 1.10/δC 144.3). In addition to this, the absence of the H-7 signal indicated that the OH group is connected to C-7 (144.4 ppm). A cis junction of the A/B rings was evident by considering the strong H3-19/Hβ-5 ROE interaction, whereas the assignment of the α/β position of the diastereotopic methylene hydrogens of the skeleton was revealed by onedimensional selective ROESY measurements irradiating the H318 and H3-19 atoms. In Table 1, the complete NMR assignments are provided for compounds 2 and 5, which were not available before, as well as for the three new compounds, 4, 6, and 7. Structures of the starting material 20E and compounds 1−7 are shown in Figure 1. As compounds 5 and 7 were obtained as protomers of each other, the possibility of a tautomeric interconversion occurring between these substances was investigated. Both compounds were found to be stable not only in neutral but also in acidic (pH = 3) and basic (pH = 8) media at room temperature. On the other hand, at 80 °C, the aqueous solution of 7 was found to yield 5 slowly, so that after 4 days the two compounds were present at an around 1:1 ratio. A similar heating of compound 5, however, led to the appearance of only trace amounts of 7, suggesting that the known form of calonysterone (5) is more stable than its newly identified analogue. On the basis of the above, compounds 5 and 7 represent a very rare case of isomeric compounds occurring as desmotropes. Desmotropy or desmotropism, a type of tautomerism where both forms may be isolated, is closely linked with tautomers in the solid state,20 and it is unusual to find two isolated desmotropes that are stable without the detectable coexistence of the other tautomer when dissolved in the same solvent. Further studies on the formation of these compounds revealed that the key step determining which compound forms is the elimination of water from compound 6. Thus, at pH = 8, 6 quantitatively dehydrated to 7, while 5 is yielded quantitatively from 6 at pH = 3. The formation of each isolated product over various reaction times was studied in three independently performed autoxidation reactions by HPLC at 0.5, 1, 2, 3, 4, 5, 6, 15, 24, and 48 h, and the results of this experiment are summarized in Figure 2.

Figure 2. Amounts of products of 20E obtained at various times as determined by means of analytical HPLC. Error bars represent SEM values from three independent experiments. Nonlinear regression was performed using the one-phase decay (20E, 1−3) or the log(Gaussian) (6 and 7) exponential models of GraphPad Prism 5. The sum of 6 and 7 and that of their regression curves (dashed green line) represent the possible total yields of this desmotropic pair.

As seen from Figure 2, 20E suffers a relatively quick decomposition with a half-life of 1.39 h. Compound 1, reported previously as a main product under similar conditions,8 was found as a minor component in the present work, with a maximum detected amount of 0.84%. Compound 2, its 5αepimer, was present instead in a significant amount, increasing over a 48 h period, leading to a plateau of 24.5% based on the regression curve. On considering the different results obtained by Suksamrarn et al.,8 one can only assume that temperature and/or the amount of methanol might play important roles in the stereoselectivity at C-5, since the ambient temperature used was 30−32 °C in the previous work, and no information was provided on the ratio of the “aqueous methanol” as solvent.8 The diacetonide derivative of compound 2 has been reported as the main product of a similar reaction of 20E 2,3;20,22diacetonide.9 Interestingly, 5α-20E was not detected by HPLC at any time of the reaction, suggesting that 9α-hydroxylation might be the first step followed by the epimerization at C-5. On using appropriate standards obtained from previous work, the presence of 9β,20-dihydroxyecdysone and polypodine B (5β,20-dihydroxyecdysone) was also investigated by HPLC, and neither was detected. Nevertheless, the formation of the corresponding 5α-epimers of these compounds cannot be excluded. Compound 3 was found to be a minor product, and, due to the very low amounts available from reaction 1 and subsequent decomposition, no compound 4 was available as a reference standard for monitoring the time dependence of its formation. Compound 6 was hypothesized previously as an intermediate to 5,8 which was confirmed herein. It was shown by the compositional changes during the reaction that an alkaline pH leads to the elimination of the OH-14 group of compound 6 to yield 7, the desmotropic form of 5. Compound 5 itself can be obtained with a maximum yield of ca. 40% at around 6−7 h by setting the pH as slightly acidic (pH 6−6.5) with a weak acid such as acetic acid prior to evaporation under nitrogen. Following the previously published observations on the increase in the complexity of the product mixture upon bubbling oxygen through the solvent,8 it can be hypothesized that the reaction could be performed in a shorter period of time, but, due to the gradual decomposition of compounds 6 and 7, a careful monitoring is suggested prior to such an D



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Scheme 1. Proposed Reaction Mechanism for the Formation of Compounds 1−7a

a

(a) Intermediates hypothesized previously,8 (b) new compounds, dashed arrows: minor products.

Altogether, compounds 2 and 5 showed favorable activities over that of 20E, suggesting their possible more potent anabolic, antidiabetic, and antiapoptotic (cytoprotective) activities as well. Moreover, as an unexpected outcome of our study, the two desmotropes 5 and 7 showed significant differences in their bioactivity. In fact, when both were applied at 30 μg/mL, 7 exerted an opposite effect of that of 5 and decreased the phosphorylation of Akt as compared to the control. This finding highlights that these two tautomers can be stable even in a biological environment. To the best of our knowledge, this provides the first direct proof of a pair of desmotropes exhibiting different bioactivities. The significant differences in the activities of compounds 5 and 7, as well as the potent activity of compound 2 on Akt phosphorylation, are of interest due to the fundamental role of this kinase in cellular metabolism and survival. Considering that the apoptosis of β-cells plays a crucial role in the pathologic mechanism of type II diabetes,21 and that decreased Akt kinase activity is connected to insulin resistance via impaired translocation of the glucose transporter GLUT4,22 compounds 2 and 5 have both anabolic and antidiabetic potential. Moreover, hyperactivation of the pro-survival kinase Akt, which is generally up-regulated in cancer, has been suggested as a potential new anticancer target, since it can effectively sensitize cancer cells to oxidative apoptosis.23,24 As such, compounds 2 and 5 are also potential candidates in combination studies with pro-oxidant anticancer agents, which may lead to reduced toxicity issues attributed to existing drugs that target the PI3K/Akt pathway.24 On the other hand, the negative influence on Akt activation makes 7 a valuable new probe for exploring the potential anticancer effects of ecdysteroid derivatives.5,6 Considering the acceptable to good yields that can be obtained from the autoxidation of the abundant, commercially available, and inexpensive 20E, largescale in vivo studies on these compounds are recommended.

attempt. Based on the present results, the reaction mechanism shown in Scheme 1 may be proposed. With the exception of compound 4, which was not available in sufficient amounts, all isolated products were tested for their capacity to influence the Akt phosphorylation in C2C12 myotubes, with the results shown in Figure 3.

Figure 3. Activity of compounds 1−3 and 5−7 and 20E on the Akt phosphorylation in murine skeletal muscle cells. Quantification of Western blots was performed by ImageJ; error bars represent SEM; **: p < 0.01, ***: p < 0.001 by one-way ANOVA followed by Bonferroni’s post hoc test; *: p < 0.05 by one-way ANOVA performed in a planned comparison of 5 and 7 to the control (C) by using Dunnett’s post hoc test; n = 2−5.

Although it is known that 20E can increase the Akt phosphorylation leading to a marked increase in the protein synthesis in the same skeletal muscle cell line used in this study,10 this could not be detected in the present experimental setup. There are major differences between our protocol and that previously utilized for 20E: we used much higher doses than Gorelick-Feldman et al. and, in our case, the cells were not serum-starved overnight but pretreated with the ecdysteroid.10 In order to evaluate this phenomenon, we have compared the two protocols by testing the dose dependency of the activities of 20E and compounds 2 and 5, and fundamental differences were found (see Supporting Information). Future studies on this phenomenon are necessary in order to better understand the bioactivity of ecdysteroids.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Boetius apparatus and are uncorrected. Optical rotation was determined with a PerkinElmer 341 polarimeter. 1H E



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(500.1 MHz) and 13C (125.6 MHz) NMR spectra were recorded at room temperature on a Bruker 500 Avance III NMR spectrometer equipped with cryogenic probe head and on a Bruker Avance 500 NMR spectrometer. Amounts of approximately 0.5−5 mg of compounds 1−7 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 (methanol-d4: δC = 49.1 and δH = 3.31 ppm). Pulse programs of all experiments (1H, 13C, DEPTQ, DEPT-135, sel-TOCSY (mixing time: 80−120 ms), sel-ROE (300 ms), gradient-selected (gs) 1H,1H-COSY, ROESY, edited gs-HSQC, gs-HMBC (optimized for 10 Hz)) were taken from the Bruker software library. For 1D measurements, 64K data points were used to yield the FID. For 2D measurements, the sweep width in F2 was 4000 Hz; all data points (t2 × t1) were acquired with 2K × 256. For F1, linear prediction was applied to enhance the resolution. Most 1H assignments were accomplished using general knowledge of chemical shift dispersion with the aid of the proton− proton coupling pattern (1H NMR spectra). The NMR signals of the products were assigned by one- and two-dimensional NMR methods using widely accepted strategies.12,13 Atmospheric pressure ionization (APCI) MS spectra were recorded on an API 2000 triple quadrupole tandem mass spectrometer (AB SCIEX, Foster City, CA, USA), used in the positive-ion mode. HRMS data were recorded on a WatersMicromass Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA) equipped with an electrospray ion source, which was used in the positive-ion mode. HPLC was performed on a gradient system of two JASCO PU2080 pumps connected to a JASCO MD-2010 Plus photodiode-array detector. Starting Material. 20-Hydroxyecdysone isolated from the roots of Cyanotis arachnoidea was purchased from Shaanxi KingSci Biotechnology Co., Ltd. (Shanghai, People’s Republic of 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. Reaction 1. A 1.70 g aliquot of 20E was dissolved in 50 mL of 70% MeOH(aq) (Merck), and 1.0 g of NaOH was added. The mixture was stirred for 3 h at room temperature, and the reaction was stopped by neutralizing the pH with concentrated acetic acid. The solvent was evaporated under reduced pressure at 40 °C. The dry residue was subjected to column chromatography (CC) over silica gel 60 (63−200 μm), eluting with ethyl acetate (22 fractions) and EtOAc−EtOH− H2O (80:2:1, v/v/v) (77 fractions). Fractions 48−70 and 71−99 were combined and evaporated to obtain dry residues of 0.24 g (A1) and 0.13 g (A2), respectively. Sample A1 was subjected to reversed-phase CC on Lichroprep RP-18 (40−63 μm) using a stepwise gradient of 30%, 35%, 40%, 45%, and 50% aqueous methanol (seven fractions each). Fractions 8−23 were combined and repeatedly purified on the same column with the same gradient; from fraction 10, compound 2 (10 mg) was obtained by crystallization from EtOAc−MeOH (2:1, v/ v). The combined fractions 11−18 were purified by HPLC on a Zorbax XDB-C8 (5 μm, 9.4 × 250 mm) column with an isocratic system of 17% aqueous acetonitrile at a flow rate of 3 mL/min, to yield compounds 1 (2.3 mg) and 4 (1.4 mg). Sample A2, obtained from the first silica gel column, was subjected to reversed-phase CC, using a gradient of 30%, 35%, 40%, and 50% MeOH(aq) (34, five, five, and five fractions, respectively) as above. Fractions 21−28, eluted with 30% MeOH, were combined and subjected to preparative TLC on Kieselgel 60 F254 plates (Merck) with a CH2Cl2−MeOH−benzene (25:5:5, v/v/ v) solvent system, to obtain compound 3 (5 mg). When using benzene, appropriate laboratory safety measures were taken. As also demonstrated by the needs of the complex isolation procedure and the very low yields, reaction 1 led to a highly complex mixture, making it inappropriate for a larger scale preparation of any of the above products. Reaction 2. A 120 mg aliquot of 20E was dissolved in a mixture of 1 mL of MeOH (Merck) and 8 mL of water, and 100 mg of NaOH dissolved in 1 mL of water was added, so that the reaction was performed in 10% MeOH(aq). The reaction mixture was stirred for 1 day at room temperature, and the reaction was stopped by neutralizing the pH with a 9.6% aqueous solution of acetic acid (Merck). The reaction mixture was fractionated by centrifugal partition chromatog-

raphy (Armen Spot CPC 250 mL, Armen Instrument, Saint Ave, France), in ascending mode, with a solvent system of EtOAc− MeOH−H2O (20:20:1, v/v/v), and 20 mL fractions were collected. The combined CPC fractions were evaporated at 40 °C, dissolved in 20% aqueous acetonitrile, and investigated by HPLC on a gradient system (acetonitrile−water) on a Kinetex XB-C18 column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Further purification of the combined CPC fractions by HPLC was scheduled 1 week later, but by that time the chief constituents of two of the fractions were completely converted to calonysterone (5). Reaction 3. A 120 mg aliquot of 20E was dissolved, and 100 mg of NaOH was added as described above. The reaction mixture was stirred for 8 h at room temperature, and the reaction was stopped by neutralizing the pH as above. The reaction mixture was purified over HPLC (35% methanol(aq), 3 mL/min) using a Zorbax XDB-C8 column (5 μm, 9.4 × 250 mm) to yield compound 6 (10.0 mg). Following this, 120 mg of 20E was reacted with NaOH using the same reaction setup as above, with the reaction mixture stirred for 15 h at room temperature and the reaction stopped by neutralizing the pH as above. The reaction mixture was fractionated by centrifugal partition chromatography the same way as described above for reaction 2. Fraction 4 was further purified over HPLC (30% acetonitrile(aq), 3 mL/min) using a Zorbax XDB-C8 column (5 μm, 9.4 × 250 mm) to yield compound 7 (4.4 mg). It should be noted that these yields do not correspond to the total amounts available from this reaction setup; in order to facilitate the work, HPLC purification was performed at once until sufficient amounts were obtained for structure elucidation and yield optimization for subsequent reactions (see below, longitudinal study of the reaction). (5α)-9α,20-Dihydroxyecdysone (2): white crystals; mp 273−275 °C; [α]25D −20.0 (c 0.2, MeOH); for NMR data, see Table 1; for NMR and UV spectra, see Supporting Information. 11α-Hydroxycalonysterone (4): amorphous, transparent solid; experimental data are limited due to the very low yield obtained and subsequent decomposition; for NMR data, see Table 1; for NMR spectra, see Supporting Information. Calonysterone (5): white crystals; mp 234−235 °C; [α]25D +26.0 (c 0.2, MeOH); APCIMS m/z 477 [M + H]+, 459 [M + H − H2O]+, 441 [M + H − 2H2O]+, 423 [M + H − 3H2O]+, 405 [M + H − 4H2O]+; for NMR data, see Table 1; for NMR and UV spectra, see Supporting Information. 14,15-Dihydro-14α-hydroxycalonysterone (6): yellow crystals; mp 145−147 °C; [α]27D +12.0 (c 0.3, MeOH); HRMS C27H42O8Na [M + Na]+, 517.2777, found 517.2778; for NMR data, see Table 1; for NMR and UV spectra, see Supporting Information. Isocalonysterone (7): yellow crystals; mp 140−142 °C; [α]25D −81.0 (c 0.2, MeOH); HRMS C27H40O7Na (M + Na+), 499.2672, found 499.2668; APCIMS m/z 477 [M + H]+, 459 [M + H − H2O]+, 441 [M + H − 2H2O]+, 423 [M + H − 3H2O]+, 405 [M + H − 4H2O]+; for NMR data, see Table 1; for NMR and UV spectra, see Supporting Information. Compound Purity. Purity testing was performed by HPLC, utilizing aqueous acetonitrile solvent systems on a Kinetex XB-C18 column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Integration was performed automatically by ChromNav software 1.16.02 (slope sensitivity: 100 μV/s, minimal area: 1000 μV·s), and purity was determined after manually deleting the solvent peak. For chromatograms and UV spectra, see Supporting Information. Compound 4 possessed an acceptable purity as visible from the NMR spectra (see Supporting Information); due to subsequent decomposition, however, this compound could neither be tested for its bioactivity nor utilized for the quantitative determination for yield optimization. Compounds 1−3 and 5−7 possessed a purity of >98% by means of HPLC at wavelengths of their corresponding UV absorption maxima. Longitudinal Study of Compound Autoxidation. A 120 mg (0.25 mmol) aliquot of 20E was dissolved, and 100 mg of NaOH was added as above (reaction 3), in three replicates. The reaction mixture was stirred for 2 days, and samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 15, 24, and 48 h. All samples from the three independently performed reactions were analyzed by HPLC-DAD after neutralizing the pH with F



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a 9.6% aqueous solution of acetic acid. A gradient system of aqueous acetonitrile was used on a Kinetex XB-C18 column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Concentrations of the compounds in each sample were determined from the AUC values of the corresponding peaks at the maximum absorption wavelength (λmax) of the compound (247, 228, 228, 304, 222, 260, and 360 nm for compounds 20E, 1−3, and 5−7, respectively). Series dilutions with known concentrations of the previously isolated compounds served as calibration. Relative amounts of the compounds as compared to the initial amount of 20-hydroxyecdysone are presented in Figure 2. Bioactivity Testing. Mouse C2C12 skeletal myoblasts (BCRC#60083) were purchased from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Taiwan). The cells were seeded in 6 cm dishes and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin solution in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. After reaching 100% confluence, C2C12 myoblasts were cultured in differentiation medium (DMEM containing 450 mg/dL D-glucose and 10% horse serum), and the medium was changed every 2 days. The cells became skeletal myotubes after 8 days of differentiation. Then, the culture medium was changed to 10% FBS high-glucose DMEM with or without 30 μg/mL of each test compound. The medium was removed after 24 h, and its glucose content was determined in order to calculate the compounds’ effect on the glucose consumption. No significant activities were found (data not presented). Fresh medium containing the same concentration of compound was then added to the cells that were incubated for further 2 h for testing the activity on AKT. The cells were then lysed with 700 μL of 1× sample buffer (62.5 mM Tris-HCl, pH 6.8; 10% glycerol; 2% SDS; 50 mM DTT; 0.0025% bromophenol blue), sonicated for 10−15 s, and heated to 95−100 °C for 5 min. For analysis of proteins, the cell lysate was loaded and separated on 10% SDS-polyacrylamide gels. Proteins were then transferred to PVDF membranes and detected using phosphorylated and total Akt antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA).

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acknowledge a bilateral mobility grant from the Hungarian Academy of Sciences and the NSC, Taiwan (NKM-113/2015 and MOST 104-2911-I-037-501). Technical assistance from I. Hevérné Herke is greatly appreciated.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00249. One- and two-dimensional NMR spectra of compounds 2, 4, and 5−7, HPLC chromatograms, and UV spectra of compounds 1−3 and 5−7, as well as a comparison of results from our bioactivity testing protocol with those from the protocol published previously for 20E (PDF)

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REFERENCES

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Corresponding Author

*Tel: +3662546456. Fax: +3662545704. E-mail: hunyadi.a@ pharm.u-szeged.hu. Present Address #

Synthetic Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Temesvári krt. 62, 6726 Szeged, Hungary. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Council (NSC), Taiwan (NSC 101-2314-B-037-033), and it was carried out within the framework of COST Action CM1407 (Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery). The authors G


Oxidized Metabolites of 20-Hydroxyecdysone and Their Activity on

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