Side-chain cleaved phytoecdysteroid metabolites as activators of protein kinase B

Article abstract of DOI:10.1016/j.bioorg.2018.10.049

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete ¹H and ¹³C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures.

Full text of DOI:10.1016/j.bioorg.2018.10.049

Accepted Manuscript
Side-chain cleaved phytoecdysteroid metabolites as activators of Protein Kinase
B
Halima Meriem Issaadi, József Csábi, Tusty-Jiuan Hsieh, Tamás Gáti, Gábor
Tóth, Attila Hunyadi
PII:
S0045-2068(18)30769-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.049
YBIOO 2583
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 July 2018
10 September 2018
24 October 2018
Please cite this article as: H. Meriem Issaadi, J. Csábi, T-J. Hsieh, T. Gáti, G. Tóth, A. Hunyadi, Side-chain cleaved
phytoecdysteroid metabolites as activators of Protein Kinase B, Bioorganic Chemistry (2018), doi: https://doi.org/
1
0.1016/j.bioorg.2018.10.049
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Side-chain cleaved phytoecdysteroid metabolites as activators of Protein
Kinase B
a
a
b
c
a,d,
Halima Meriem Issaadi , József Csábi , Tusty-Jiuan Hsieh , Tamás Gáti , Gábor Tóth *,
a,e,
Attila Hunyadi *
a
Institute of Pharmacognosy, Interdisciplinary Excellence Center, University of Szeged,
Eötvös str. 6, 6720 Szeged, Hungary;
b
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan,
Republic of China;
c
Servier Research Institute of Medicinal Chemistry (SRIMC), Záhony utca 7, H-1031
Budapest, Hungary;
d
NMR Group, Department of Inorganic and Analytical Chemistry, University of Technology
and Economics, Szt. Gellért Sq. 4, H-1111 Budapest, Hungary;
e
Interdisciplinary Centre for Natural Products, University of Szeged, Eötvös str. 6, 6720
Szeged, Hungary
*
Corresponding Author: Tel: +3662546456. Fax: +3662545704.
E-mail: hunyadi.a@pharm.u-szeged.hu (A.H.) and drtothgabor@t-online.hu (G.T.)
1

Abstract
Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in
mammals, including humans, through a partially revealed mechanism of action involving the
activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain
cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats.
Here we report the semi-synthetic preparation of a series of side-chain cleaved
ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells.
Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative
side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation
1
13
of poststerone. The complete H and C NMR spectroscopic assignments of the new
compounds are presented. Among the tested compounds, 9 could activate Akt stronger than
poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-
hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the
expectable in vivo formation of such compounds should contribute to the bioactivity of herbal
preparations containing ecdysteroid mixtures.
Keywords: Ecdysteroids; in vivo metabolism; Hypervalent iodine chemistry; Base-catalyzed
autoxidation; Akt phosphorylation; NMR; Stereo structure
2

1
. Introduction
The global phenomenon of population ageing directs an increasing attention towards
natural approaches to maintain health and strength in general, and supplementation with
herbal adaptogens and/or natural anabolic agents provides an attractive way to achieve this.
Phytoecdysteroids are among the most popular options in this regard.
Ecdysteroids are a large and diverse family of polyhydroxylated sterols; there are more
than 500 representatives of these compounds currently known to occur in Nature [1].
Concerning their chemistry, most of them possess a C27 carbon skeleton derived
biosynthetically from cholesterol. However, the number of carbon atoms can vary.
Ecdysteroids occur as C27, C28, or C29 compounds if the whole sterol-type side chain is
present, or they can be C19, C21, or C24 compounds if a partial or complete metabolic side-
chain cleavage takes place between C17-C20, C20-C22 or C24-C25, respectively. Most of the
naturally occurring phytoecdysteroids possess a 7-en-6-one chromophore group in the ring B.
The A/B ring junction is normally cis, whereas the B/C and C/D ring junctions are almost
always trans [2].
Ecdysteroids were initially discovered as the molting hormones of insects [3], but soon
after they were also isolated from Plant Kingdom, where their concentration can reach as
much as 1,000-fold higher than that typically found in arthropods [4]. Phytoecdysteroids can
reach as much as 2-3% of the dry weight in some species (e.g. seeds of Rhaponticum
carthamoides and stalks of Diploclisia glaucescens, inflorescences of Serrulata inermis and
roots of Cyanotis arachnoidea) [5]. It is important to note that while plants’ ecdysteroid
composition is typically dominated by a few major ecdysteroids (usually 20-
hydroxyecdysone; 20E), a complex cocktail of minor compounds is generally present whose
bioactivity needs to be taken into account when ecdysteroid-containing herbal preparations
are used.
It is generally accepted that phytoecdysteroids do not interact with the vertebrate
steroid hormone system. Still, they exert numerous beneficial effects on mammals (e.g.
anabolic, adaptogenic, antidiabetic, hypolipidemic, hepatoprotective, etc.), while their acute
toxicity is negligible [4-10]. Phytoecdysteroids have long been known for their complex
bioactivity that could be referred to as a broad-spectrum, general strengthening effect. Several
in vitro and in vivo studies confirmed the anabolic activity of ecdysteroids that is
characterized by an increase in growth and skeletal muscle mass [11-12], as well as in the
fiber size through muscle-specific effects [13] in different animals. The exact mechanism of
action behind this anabolic activity is only partially understood, and needs further
3

clarification. Recently, Gorelick-Feldman et al. found that 20E (1) caused a hasty elevation in
intracellular calcium levels, leading to a maintained activation of protein kinase B (Akt) and
to an increased protein synthesis in a mouse skeletal muscle cell line (C2C12) [14]. Protein
kinase B and its downstream effectors, inter alia, represent a major signaling pathway that
controls protein turnover in skeletal muscles, and this function appears to be maintained in
ageing muscles as well [15-16]. This role makes it an appropriate target to test ecdysteroids
for their anabolic “strengthening” potential. Activity on this pathway can also provide
indirect information on the capacity of ecdysteroids to promote muscle gain and/or prevent
muscle loss.
The possible role of in vivo metabolites of ecdysteroids in the observed bioactivities
has long been part of related scientific discussion. Recent studies of Kumpun et al. identified
several metabolites in the urine and feces of mice treated by an intraperitoneal injection of
2
0E (1) [17]. Side chain cleavage between C-20 and C-22, and dehydroxylation at C-14 were
identified as major metabolic steps, thus yielding poststerone (7), 14-deoxy-20-
hydroxyecdysone and 14-deoxypoststerone, which then undergo a reductive metabolism in
several further steps [17]. We have recently found that poststerone (7) acts as a potent
anabolic agent on rat skeletal muscles in vivo, implying that it plays an important role in the in
vivo anabolic activity of its parental compound 20E (1) (unpublished results; manuscript by
Csábi et al. is currently under evaluation at Sci. Rep., first submission: 26.04.2018).
Accordingly, it seems reasonable to assume that side-chain cleaved metabolites of
phytoecdysteroids other than 20E (1) might also arise in vivo, and that these metabolites are
also valuable bioactive compounds.
The base-catalyzed autoxidation of 20E (1), first described by Suksamrarn et al. in
1
994 [18], was recently found by our research group to yield several oxidized derivatives
exerting stronger activity on the Akt phosphorylation in murine skeletal muscle cells as
compared to their parental compound 20E (1) [19]. This, together with the in vivo relevance
of poststerone (7), led us to rise the following objectives for the present study: (i) to prepare a
series of new C-21 ecdysteroid derivatives by using oxidative side-chain cleavage on various
phytoecdysteroids or through the autoxidation of poststerone (7), and (ii) to evaluate the
compounds’ activity on the protein kinase B activation in murine skeletal muscle cells.
2
2
2
. Results and discussion
.1 Chemistry
.1.1. Preparation of ecdysteroids 7-12 through oxidative side-chain cleavage
4

Oxidative side-chain cleavage of different phytoecdysteroids (1-6) was performed by
using either the hypervalent iodine reagent [bis(trifluoroacetoxy)iodo]benzene (PIFA) or
(
diacetoxyiodo)benzene (PIDA).
Poststerone (7) was previously published to form when oxidizing 20E (1) with NaIO
4
(
yield not reported) [20] or with Jones reagent (62% yield) [21]. Our recently published
procedure, using PIFA for the same oxidation, could provide compound 7 in a yield of 57.8%
22]. In the present study, we report an update to this reaction: when using PIDA in a large-
[
scale reaction followed by purification with flash chromatography, poststerone (7) could be
obtained in a decent isolated yield of 81.41%.
In order to obtain side-chain cleaved analogs (i.e. likely major in vivo metabolites) of
other phytoecdysteroids, the reaction was also carried out on natural ecdysteroids with
different substituents on the steroid skeleton, namely 2-deoxy-20-hydroxyecdysone (2),
ajugasterone C (3), polypodine B (4), and dacryhainansterone (5) with the use of PIFA, and
calonysterone (6) with the use of PIDA as reagent. Since these compounds were available in
lower amounts than 20E (1), smaller scale reactions were performed. Preparative reverse-
phase HPLC (RP-HPLC) was applied to obtain compounds 8 and 9, rotation planar
chromatography (RPC) was used to obtain compounds 10 and 11, and centrifugal partition
chromatography (CPC) was utilized to obtain compound 12. Structures of the starting
materials and their respective analogs are presented in Figure1.
5

R1
OH
H
R2
H
R3
H
R1
OH
H
R2
H
R3
H
1
2
3
4
7
8
9
H
H
H
H
OH
H
OH
H
OH
H
OH
H
OH OH
10 OH OH
5
11
6
12
Figure 1. Preparation of ecdysteroid derivatives through oxidative side-chain cleavage
For comparison purposes, reactions first performed with the use of PIFA as reagent
were also carried out also by using PIDA instead. Less complex mixtures were obtained this
way, and the expected products were obtained with the following yields: 8 (68.34%), 9
(
63.57%), 10 (71.23%) and 11 (82.71%). Based on these comparative experiments, we could
conclude that the use of PIDA as reagent instead of PIFA provides higher yields of the desired
C-21 ecdysteroids. We hypothesize that the reason for this is decomposition due to by-product
TFA that strongly acidifies the reaction medium when using PIFA, while acetic acid released
by PIDA provides milder conditions leading to less complex mixtures and higher final yields
of the target compounds.
2
.1.2. Preparation of autoxidized derivatives 12-18 of poststerone (7)
Considering our previous observations on the significant bioactivity changes
connected to certain autoxidized derivatives 20E (1) [19], our second aim was to prepare
similar derivatives from poststerone (7), which would likely represent in vivo relevant
metabolites of the corresponding analogs obtained from 20E (1). The base-catalyzed
autoxidation of poststerone (7) was carried by dissolving the starting material in methanol -
6

water (9:1, v/v) and subsequently NaOH was added to the mixture as a catalyst. The reaction
mixture was stirred for 4 hours, stopped by neutralizing the pH with 9.6% acetic acid,
extracted through silica and dried. The dry residue was fractionated by centrifugal partition
chromatography (CPC) in ascending mode, with a biphasic solvent system composed of ethyl
acetate - water - methanol (20:20:1, v/v/v). Combined fractions were further purified by RP-
HPLC to obtain compounds 12-18. Structures of the synthesized derivatives (13-18) are
presented in Figure 2.
13
14
15
16
17
18
Figure 2. Structure of the isolated compounds 13-18
In order to allow biological testing of the isolated products, larger amounts were
required. To achieve this, a set of small-scale reactions were performed, and the degradation
of poststerone (7) and the formation of its oxidized derivatives (12-18) were monitored by
normal phase (NP-TLC). As a result, we found that a longer reaction time of ca. 7-9 hours is
preferable to increase the yield of the targeted compounds. Thus, the same reaction was
conducted for 7 hours using 500 mg of poststerone (7), followed by the same purification
steps as before. Yields of the compounds 13, 15, 16 and 18 increased by 2.46, 1.59, 4.81, 1.10
times respectively. On the other hand, compound 17 was not detected anymore after 4 hours.
2
.2 Structure elucidation
Structure elucidation of the products was performed by means of comprehensive one-
and two-dimensional NMR methods using widely accepted strategies [23-24], and we
1
13
1
established their complete H and C signal assignment. Most H assignments were
1
1
accomplished using general knowledge of chemical shift dispersion with the aid of the H- H
1
1
coupling pattern ( H NMR spectra). H NMR chemical shifts of overlapped signals were
7

identified utilising 1D selective ROESY (Rotating frame Overhauser Enchancement
Spectroscopy) responses, or from 1D selective TOCSY experiments, or by 2D HSQC and
HMBC experiments, as well as.
Compounds 7, 9 and 11 (poststerone, 11α-hydroxypoststerone and 9,11-
didehydropoststerone, respectively) [22,25-26], were previously isolated and fully
1
13
characterized by our research group; their H and C NMR chemical shifts correlated well
with the values reported earlier. HPLC chromatograms and UV spectra of compounds 7, 9
and 11 are available as Supplementary Information (Figures S46, S48, and S50, respectively).
1
13
The H and C NMR chemical shifts of the eight new compounds (8, 10, 12, 14 - 18)
together with the data of poststerone as reference (7) are compiled in Table 1 and 2.
8

1
Table 1. H chemical shift of compounds 7, 8, 10, 12, 14 - 18 in methanol-d
4
and 18 also in
a
dimethyl sulfoxide-d
6
(18 )
a
Atom no.
7
8
10
12
14
15
16
17
18
18
1
α
β
α
β
1.80
1.44
3.86
-
3.97
1.74
1.74
2.39
5.82
-
3.19
1.89
1.67
2.33
1.82
1.70
2.00
1.88
2.23
3.33
0.62
0.96
2.16
1.62 1.74*
1.47 1.74*
1.38
2.44
4.01
-
3.53
3.13
2.54
-
2.17
1.71
4.01
-
3.55
1.91
1.75
3.17
5.84
-
1.46
2.32
4.01
-
3.51
3.11
2.53
-
2.03
1.70
3.77
-
3.88
1.38
1.80
2.62
-
1.93
1.62
3.48
-
3.94
1.79
1.70
2.49
3.33
2.69
-
2.41
2.41
1.80
2.30
5.45
-
2.31
2.90
3.04
0.78
1.01
2.21
1.96
2.21
-
1.84
2.08
-
2
1.81
1.66
3.98
1.81
1.57
2.44
5.81
-
3.96
-
4.00
2.08
1.78
-
-
-
3
4
3.79
2.16
1.81
2.43
5.76
-
3.61
1.98
1.61
2.29
5.64
-
α
β
5
7
α
β
5.87
-
-
-
-
-
-
-
-
-
9
1 α
β
2 α
β
5 α
β
6 α
β
7
8
9
3.25
1.81
1.63
2.32
1.79
1.69
2.02
1.90
2.24
3.34
0.63
0.96
2.14
3.23
1.89
1.73
2.33
1.83
1.69
2.00
1.88
2.25
3.33
0.63
0.91
2.15
-
-
-
1
1
1
1
2.73
2.65
1.74
2.33
6.81
-
2.38
2.98
3.00
0.80
1.50
2.22
a
1.80 2.63*
2.07 2.63*
6.04
-
2.52
2.73
6.79
-
2.46
3.01
3.11
0.82
1.13
2.23
2.06
1.81
2.35
1.82
1.62
1.94
1.89
2.24
3.33
0.64
1.05
2.16
1.97
1.62
2.21
1.71
1.50
1.84
1.72
2.09
3.20
0.50
0.93
2.10
2.36
1.78
1.75
1.97
1.87
2.25
3.35
0.65
1.10
2.16
2.26
1.93
2.63
2.00
1.90
2.25
3.16
0.66
1.50
2.17
1
1
1
2
1
*
5
Signals that are accidentally isochrones. OH signals of compound 18: 2-OH 6.63 s; 3-OH
.00 d 3.9 Hz; 14-OH 4.49 s.
9

1
3
a
Table 2. C chemical shifts of compounds 7, 8, 10, 12, 14-18 in methanol-d
dimethyl sulfoxide-d
4
and 18 in
6
.
a
Atom no.
7
8
10
12
14
15
16
0
38.1
18
39.2
18
37.8
1
2
3
4
5
6
7
8
9
37.4
68.7
68.5
32.9
51.9
30.0
29.1
65.6
33.3
52.4
34.3
68.5
70.3
36.2
39.5
72.8
73.7
27.6
36.1
70.8
72.5
24.7
42.1
70.3
73.5
27.5
37.7
68.7
68.0
35.9
49.7
70.0 109.0 107.8
68.6
33.2
54.1
72.3
35.1
53.1
70.1
34.0
51.2
80.4 133.6
49.0 133.7
206.3 206.2 202.3 144.2 202.7 144.6 199.1 215.0 204.8 202.4
122.6 122.4 120.1 181.2 125.3 180.9 144.8 39.8 122.6 120.8
166.6 167.1 166.1 124.7 158.8 133.0 118.7 123.8 153.8 152.4
35.2
39.3
21.7
31.1
48.9
85.1
32.2
22.3
60.2
17.6
24.5
35.3
37.7
22.1
31.4
49.4
85.3
32.1
22.4
60.3
17.6
24.5
39.1 165.5
76.2 168.2 137.1 137.4
83.2
48.9
31.5
28.7
80.7
47.3
29.7
26.9
47.8
84.9
29.9
20.6
57.9
17.3
20.3
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
45.5
22.7
31.2
49.0
84.9 140.3
32.1 127.4
22.2
60.1
17.6
17.1
42.2
25.6
36.7
48.1
43.7
29.1
28.4
49.0
86.4
31.7
22.1
60.1
17.6
19.8
42.6
25.4 125.9
28.4
49.2
82.1 142.2 147.3
33.7 134.1 120.1
23.8
58.5
18.1
28.6
41.9
44.3
23.8
37.0
40.9
48.0
47.5 49.65
87.3
31.2
22.2
59.7
18.4
21.0
33.6
63.9
18.3
27.6
33.9
64.5
20.0
30.9
32.7
66.0
17.8
29.6
212.6 212.5 212.5 211.9 212.4 213.4 211.4 211.6 212.5 209.3
31.6 31.6 31.6 31.6 31.6 31.7 31.4 31.5 31.5 31.1
The side-chain cleavage of phytoecdysteroids (1-6) took place between the two
hydroxylated carbon atoms C-20 and C-22, and led to the formation of a CH -C=O group
attached to C-17 as it is straightforward from the H -21 signals around 2.16 ppm, and the
C-21 and C-20 peaks at ~31 and ~212 ppm, respectively (see Table 1 and 2). It should be
3
3

1
3
mentioned that the C signals of the steroid skeleton of the starting compounds showed only
moderate changes upon side chain cleavage.
Compound 8 presented similar UV spectrum as its parental compound, 2-Deoxy-20-
hydroxyecdysone (2), indicating no change in the chromophore. By means of MS, molecular
+
weight of compound 8 was determined as m/z = 347 [M + H] , suggesting that the side-chain
was successfully cleaved between C20-22, resulting in the molecular formula C21
H
30
4
O .
HPLC chromatogram and UV spectrum of compound 8 are available as Supplementary
1
Information: Figure S47, together with the NMR spectra: Figure S1 - Figure S6. In the H
spectrum taken at 27 °C, some signals of the A/B rings appeared rather broadened, but when
raising the temperature up to 50 °C they became sharp, and the bandwidth at half maximum
of the H-3 signal changed from 21 Hz to 11 Hz, whereas the broad coalescence-like H-9
4
3
signal turned into a ddd multiplet with J(H-9,H-7) = 2.5 Hz, J(H-9,H-11) = 6.8 Hz and
10

3
13
J(H-9,H-11) = 11.9 Hz couplings. Similar line-broadening was observed with the
signals. Even at 50 °C, in the edited DEPTQ spectrum (Figure S3), from the five CH atoms
upside signals), only four: C-7, C-3, C-17 and C-5 appeared, whereas C-9, around 35 ppm,
C
(
remained under the noise level due to the strong broadening. The exact C-9 = 35.5 ppm
value was established from the H-9/C-9 cross-peak of HSQC spectrum (Figure S4). Utilizing
the edited HSQC spectrum (Figure S5), the assignment of the two broad methylene signals
(
C-11 = 22.1, C-1 = 30.0 ppm) can also be verified. The HMBC spectrum (Figure S6)
2
,3
provided H/C correlations ( J(H,C) over two and three bonds, supporting the structure and
the assignment even for the quaternary carbon atoms. Considering the inserted H -19/C and
-18/C section of HMBC, the cross-peaks of H -19 methyl hydrogens unambiguously
assigned the quaternary C-10, tertiary C-5 and C-9, and secondary C-1 signals, and the H -18
3
H
3
3
3
hydrogens identified the quaternary C-13 and C-14, tertiary C-17 and secondary C-12 signals.
Differentiation between the / positions of hydrogen atoms in the poststerone core, and also
confirming the cis-type junction of the A/B rings were achieved through the one-dimensional
selective ROESY experiments with irradiation of the H
3
-18 and H
3
-19 hydrogen atoms
(
Figure S3). We have previously proven that, in case of studying ecdysteroids at 500 MHz
frequency, this approach provide a sensitivity that strongly exceeds that of the analogous
selective NOE (Nuclear Overhauser-Enhancement) experiment [27].
While in case of compound 8 we observed a temperature dependent signal
broadening/sharpening, indicating a slow interconversion of different conformers, such a
phenomenon did not occur in the case of poststerone (7). Such a different behavior of these
compounds is likely due to the fact that in compound 7 the A ring of the 5 cis-linked A/B
steroid frame predominantly appears in a chair conformation with equatorial HO-2 and axial
HO-3 groups. Despite of the axial position of HO-3 group, the equatorial HO-2 substituent
stabilizes this conformer. In compound 8, however, there is only one unfavorable axial HO-
group, allowing the appearance of such twisted-boat conformer(s) where the substituent is no
more axial (see Figure S5). The slow interconversion of these conformers could explain the
1
13
reported coalescence in the H and C spectra.
The UV spectrum of compound 10 was practically identical with that of its parental
compound, polypodine B (4), indicating no change in the chromophore. By means of MS, its
+
molecular weight was established as m/z = 401 [M + Na] , suggesting that the side-chain was
cleaved between C20-22, resulting in a molecular formula of C21
H
30
6
O . The HPLC
chromatogram along with the UV spectrum of compound 10 is available as Supplementary
11

Information: Figure S49, together with NMR spectra: Figure S7 - Figure S14. In the
hydrogen and carbon spectra, taken at 27 °C, no line-broadening were observed. The H-9 =
4
3
3
.23 ppm signal appeared as ddd multiplet with J(H-9,H-7) = 2.5 Hz, J(H-9,H-11) = 6.8
3
Hz and J(H-9,H-11) = 11.5 Hz couplings. In accordance with the expected structure of 10,
3
the DEPTQ spectrum (see Figure S8) revealed the signals of three CH
3
, six CH
2
, four sp CH,
3
one =CH, and seven quaternary carbon atoms. Among the quaternary carbons, four sp and
2
three sp hybrids could be identified. An inspection of the routine HSQC spectrum (Figure
S10 and S11) showed that the diastereotopic H-1 and H-1 atoms are accidentally
1
3
isochrones, whereas, due to the moderate resolution in the C range, an adequate assignment
of the C-11 and C-16 signals (22.7/22.2 ppm) is not possible. By utilizing the band-selective
version of the two-dimensional HSQC measurement in the corresponding area (37-21 ppm)
(
Figure S12), a perfect separation of the overlapping cross-peaks (H-11/C-11: 1.89/22.7
and H-16/C-16: 1.88/22.2) was accomplished. Differentiation between the / positions
of hydrogen atoms of the methylene groups in the steroid core was achieved through one-
dimensional selective ROESY experiments (Figure S9). Irradiation of the H-9 = 3.23 ppm
signal resulted ROE responses on H-2, H-4 and H-12, respectively, and this proved not
only their signal assignment but also the retained cis-type junction of the A/B rings. Inserting
the one-dimensional selROE on H-9 spectrum (see red line in Figure S13) into the band-
selective HSQC convincingly demonstrated the unambiguous differentiation of methylene
groups in position 11 and 17, despite their rather similar chemical shifts. The HMBC
2
,3
spectrum (Figure S14) provided the H/C correlations ( J(H,C). Considering the H
-18/C section of HMBC (shown in Figure S14), the cross-peaks of the H -19 methyl
hydrogens unambiguously assigned signals of the quaternary C-5 and C-10, the tertiary C-9,
and the secondary C-1, and the H -18 hydrogens assigned the quaternary C-13 and C-14,
3
-19/C and
H
3
3
3
tertiary C-17 and secondary C-12 signals.
Compound 12 retained the characteristic UV spectrum of its parental compound
calonysterone (6). The structural formula of compound 12 was established as C21
H
26
5
O by
+
means of HRMS ([M+H] calculated: 359.18530, found: 359.18602). HPLC chromatogram
and UV spectrum of compound 12 are available as Supplementary Information: Figure S51
together with NMR spectra: Figure S15 - Figure S18. To facilitate the evaluation of the
HSQC spectrum (Figure S17) we have inserted the one-dimensional selTOCSY spectrum
with irradiation on H-12 (see green line), from which the separately appearing H-11/H-11
signals (2.73/2.65 ppm) assigned C-11 at 25.6 ppm. To verify the C-4 = 27.6 ppm
12

assignment, we also utilized selROE on H -19 (see red line), where the ROE response at 2.54
3
ppm unambiguously marked out H-4.
Compound 13 showed a slight hypsochromic shift in its UV spectrum as compared to
that of poststerone (7), rising the suspicion that it might be a 9-hydroxylated derivative
expectable to form analogously to our previous results on the autoxidation of 20E (1) [19].
+
The molecular formula was established as C21
H O
30 6
by means of HRMS ([M+H] calculated:
1
13
3
79.21206, found: 379.21210). The H and C NMR spectra revealed this compound to be
the analog of the natural product 9α,20-dihydroxyecdysone. The chemical shifts correlated
well with the values of C-1 to C-16 reported earlier for this compound [28]. The H -21
signal at 2.17 ppm, C-21 and C-20 peaks, at 31.6 and 212.3 ppm, respectively, demonstrate
the presence of the CH -C=O group attached to C-17. HPLC chromatogram and UV spectrum
3
3
of compound 13 is available as Supplementary Information (Figure S52).
The UV spectrum of compound 14 was similar to that of 13, and its molecular formula
+
was also established as the same, C21
H
30
O
6
, by means of HRMS ([M+H] calculated:
3
79.21206 found: 379.21246). HPLC chromatogram and UV spectrum of compound 14 are
available as Supplementary Information: Figure S53 together with NMR spectra: Figure S19
Figure S23. In the DEPTQ measurement (Figure S20) the C-9 signal remained under the
noise level. The H -19/C-9 HMBC cross-peak (1.10/76.2 ppm, see Figure S23) identified the
C-9 signal whose high chemical shift of C-9 = 76.2 ppm proved the OH substituent at this
position. The selROE experiments (Figure S21) on H -19 (see green line) and on H-5 (purple
-
3
3
line) revealed the trans type A/B ring-junction and the H-5 configuration, evidencing that is
this compound is the H-5 epimer of compound 13.
Compound 15 exhibited a characteristic UV spectrum similar to that previously found
for 14,15-dihydro-14α-hydroxycalonysterone, a product of the autoxidation of 20E (1) [19].
The molecular formula of compound 15 was established as C21
H
28
6
O by means of HRMS
+
(
[M+H] calculated: 377.19641, found: 377.19621). HPLC chromatogram and UV spectrum
of compound 15 are available as Supplementary Information: Figure S54 together with NMR
spectra: Figure S24 - Figure S28. The H -21 signal at 2.17 ppm, C-21 and C-20 peaks, at
1.6 and 213.4 ppm, respectively, verified that the CH -C=O group attached to C-17 was
retained. The appearance of a C=O signal at 180.9 ppm indicated its cross-conjugated
arrangement. The HMBC (Figure S28) correlations of H -19 (1.50/42.1 and 1.50/42.6,
3
3
3
3
respectively) assigned the quaternary C-10 and H
2
C-1 methylene moieties, furthermore the
2
1
.50/133.7 and 1.50/168.2 responses proved the presence of quaternary sp C= atoms in
13

5
,6
8,9
positions C-5 and C-9, which together showed the B ring as a ∆ -7-one-∆ chromophore.
1
13
The  H and  C values of the A/B rings (see Table 1 and 2) of 15 correlate well with the
corresponding values of 12, revealing that both compounds contain the same structural
moieties at this region. On the other hand, the HMBC cross-peak H
3
-18/C-14 (0.66/82.1) of
14,15
compound 15 revealed that, instead of the 
C=CH ethylene moiety (see structure 12 in
Figure 2), the HO-14 group of the parental poststerone 7 remained intact. Differentiation
between the α/β positions of the diastereotopic methylene hydrogens of the skeleton was
achieved by selective one-dimensional ROESY experiments (Figure S26), irradiating the H
8 and H -19 atoms, respectively.
Compound 16 showed a characteristic UV spectrum similar to that previously found
3
-
1
3
for isocalonysterone, the desmotropic pair of calonysterone (6), forming through the
autoxidation of 20E (1) [19]. The molecular formula of compound 16 was established as
+
C
21
H
26
O
5
, the same as that of compound 12 ([M+H] calculated: 359.18530, found:
3
59.18592). HPLC chromatogram and UV spectrum of compound 16 are available as
Supplementary Information: Figure S55 together with NMR spectra: Figure S29 - S30. The
1
13
H and C (APT) NMR spectra revealed this compound to be the 17-acetyl analog of
isocalonysterone and the chemical shifts correlated well with the values of C-1 to C-16
reported earlier for isocalonysterone [19], moreover the signals of the 17 CH -C=O group
-21 2.23 ppm, C-21 and C-20 peaks, at 31.4 and 211.4 ppm,
3
were measured at H
3
respectively.
In case of compound 17, the UV spectrum did not provide relevant structural
information. The molecular formula of this compound was established as C21
H
28
4
O by means
+
of HRMS ([M+H] calculated: 345.20603, found: 345.20683). The number of double bond
equivalents in compound 17 increased to eight, indicating that this compound contains one
1
3
more double bound than its parent compound 7. Twenty one C signals were discernible in
the DEPTQ spectrum of 17 (Figure S32) indicating the presence of three methyl, six
3
2
3
methylene, four sp CH methyne and one sp =CH groups, along with two quaternary sp and
2
five quaternary sp carbon atoms, the latter including two non-conjugated C=O (δ 215.0 and
1
2
11.6 ppm) moieties. Integration of the signals in the H NMR spectrum (Figure S31)
obtained in methanol-d summed up to 26 H atoms. Considering the nearly identical δC-2 and
4
δC-3 chemical shifts of compound 17 and the parental 7, both of these should have HO-
substituents, and thus compound 17 contains 28 hydrogen atoms, coherently with the HRMS
results. The HMBC (Figure S35) correlations of H -19 (1.01/38.1, 1.01/44.3 and 1.01/54.1,
3
14

respectively) assigned the H
2
C-1 methylene, the quaternary C-10 and HC-5 moieties,
2
furthermore the 1.01/137.4 responses proved the presence of quaternary sp =C atom in
position C-9. The H
3
-18 HMBC correlations assigned C-12, C-13 and C-16 atoms at 37.0,
7.5 and 65.0 ppm, respectively. Moreover, the 1.01/147.3 (H -18/C-14) correlation revealed
C=CH ethylene moiety in the D-ring of the steroid skeleton. The H-
4
3
1
4,15
the presence of a 
8
,9
1
5/C-8 HMBC cross-peak established the  position of the second conjugated double bond.
HMBC correlations of H-5 (2.49 dd, 12.8 and 4.0 Hz) verified the assignment of O=C-6
215.0 ppm) and H C-7 (39.8 ppm) signals, respectively. Differentiation between the α/β
hydrogens of the skeleton was achieved by selective one-dimensional ROESY experiments
Figure S33), irradiating the H -18 and H -19 atoms. Checking the literary data revealed that
(
2
(
3
3
compound 17 was formerly reported by Galbrait and his co-workers [29] in 1969 as a
1
decomposition product of compound 7 after refluxing it in 0.5 N HCl in ethanol. Limited H
NMR data in CDCl
assigned. Here we provide a complete H and C NMR signal assignment for this compound
in methanol-d . HPLC chromatogram and UV spectra (Figure S56), and the NMR spectra of
3
are given, only the three methyl signals and the allyl hydrogen were
1
13
4
compound 17 (Figure S31 - S35) are available as Supplementary Information.
Compound 18 also did not show analogous UV spectrum to any of the known
autoxidized metabolites of 20E (1) [19]. HPLC chromatogram and UV spectra of compound
1
8 are available as Supplementary Information (Figure S57). According to our NMR
1
protocol, first we performed the H, DEPTQ, selROE, HSQC and HMBC measurements in
methanol-d
4
(Supplementary Information: Figure S36 – S40). From these, we managed to
1
3
1
assign the C and H signals of 18 in this solvent, certainly without the OH peaks. Taking
into account the anomalous C-2 = 109.0 ppm chemical shift of this quaternary carbon atom,
attachment of a second oxygen substituent (e.g. -OH, -OOH, -OC) to C-2 was suspected. In
analogous 9-OH substituted ecdysteroids (e.g. 13 and 14) the value of chemical shift for C-9
is expected around 76 ppm, but now in compound 18 it is C-9 = 83.2 ppm, suggesting the
presence of an -OOH or -OC substituent. To overcome this uncertainty, we evaporated the
methanol solvent and all NMR investigations were subsequently performed in dimethyl
sulfoxide-d
6
(Supplementary Information: Figure S41 - S45). In the DEPTQ spectrum (see
3
Figure S42), we identified the signals of three CH
3
, six CH
2
, three sp CH, one =CH, and
3
2
eight quaternary carbon atoms including five sp and one sp carbons, and two C=O groups.
1
1
There are only three H signals (6.63s, 4.49s and 5.00d (3.9 Hz) without J(CH) correlation in
the HSQC spectrum, proving the presence of three hydroxyl groups in compound 18. The H
3
-
15

2
,3
1
8/C and H -19/C HMBC correlations over two and three bonds ( J(H,C) provided the
3
unequivocal assignment of C-12, C-13, C-14, and C-17 atoms, and C-1, C-5, C-9, and C-10
atoms, respectively (Figure S45). The assignment of the detected hydroxyl signals to HO-2,
HO-14 and HO-3 groups is straightforward on the basis of their HMBC cross-peaks. Whereas
3
the HO- signal at 6.63s shows J(H,C) correlation with C-1 (37.8 ppm), this hydroxyl group is
obviously connected to the quaternary C-2 (107.8 ppm). The second oxygen atom attached to
this extremely deshielded C-2 forms a bridge to C-9 resulting a new five-membered ring in
compound 18. The second singulet HO-14 hydroxyl signal (4.49 s) showed cross-peaks not
2
only with C-14, but also with the quaternary sp C-8 (152.4 ppm), revealing the presence of
7
,8
13
the 6-one- moiety. The H-7/C-5 and H-7/C-9 correlations completed the C assignment.
This way the molecular formula should be C21
H
28
O (DBE = 8), which was then subsequently
6
+
supported by HRMS ([M+H] calculated: 377.19586, found: 377.19625). One-dimensional
selective ROESY experiments allowed differentiation between the / hydrogen atoms in the
steroid core, and also proved the cis-type junction of the A/B rings (Figure S43). To achieve
1
an exact H assignment even in case of strongly overlapping signals, the selTOCSY
experiment was the method of choice. Irradiation on H-17 (3.20 ppm) resulted selectively the
1
H signals of the D-ring (see the green line inserted into the edHSQC spectrum of 18 in
Figure S44), whereas experiment on H-12 (2.21 ppm) showed the spin system of the C-ring
(
inserted red line).
When comparing the oxidized products obtained from the base-catalyzed autoxidation
of 20E (1) [19] with those obtained in this study from poststerone (7), the formation of both
similar and different structures can be observed. Compound 12 was also one of the reaction
products, even though with a lower yield than from the straightforward side-chain cleavage of
calonysterone (6). Compounds 12-16 are the cleaved side-chain analogues of previously
isolated compounds from the base-catalyzed autoxidation of 20E (1), while 17 and 18
represent the results of previously unobserved oxidative transformations.
2
.3. Bioactivity on the Akt phosphorylation
The compounds were tested for their capacity to influence the Akt phosphorylation in
murine C2C12 skeletal myotubes. The observed activities at 10 µM concentration are shown
in Figure 3.
16

Con
17
14
15
13
18
12
16
11
10
8
9
7
p - AKT –
T - AKT –
GAPDH –
c
4
00
150
100
c
c
300
c
b
a
b
a
200
50
100
0
0
C 17 14 15 13 18 12 16 11 10
8
9
7
C 17 14 15 13 18 12 16 11 10 8 9 7
Figure 3. Activity of compounds 7 - 18 as compared to the control (C) on the Akt
phosphorylation in murine skeletal muscle myotubes. Quantification of Western blots was
performed by ImageJ; error bars represent SEM from four parallel experiments. a: p<0.05; b:
p<0.01; c: p<0.001
With the exception of the side-chain cleaved derivatives of 2-deoxy-20-
hydroxyecdysone (2), ajugasterone C (3) and polypodine B (4) (i.e. compounds 8, 9 and 10,
respectively), all tested compounds demonstrated the ability to increase the activation of Akt.
In particular, the side-chain cleaved analogs of dacryhainansterone (5), calonysterone (6) and
isocalonysterone (i.e. compounds 11, 12 and 16, respectively) showed the strongest activity in
this regard. All of these compounds express higher degree of unsaturation due to further
conjugation extended to their D and/or C rings. This suggests that further double bonds
conjugated with those present in the ecdysteroid B-ring may positively impact the
bioactivities of these compounds in mammalian cells, and that such compounds might have
stronger anabolic, antidiabetic and anti-apoptotic activities than the more saturated ones
including the abundant 20E (1) and its known in vivo metabolites [15]. On the other hand,
when a separate experiment was performed to test poststerone (7) in comparison with 20E (1),
it was found a weaker activator of Akt (293.3 ± 68.3 % vs. 537.1 ± 25.3 % activation at 10
µM, respectively, n=6. Interestingly, however, poststerone (7) was also active at as low as 10
nM concentration (263.4 ± 69.6 % activation, n=6), where 20E (1) was already inactive. For
the dose-dependency observed in this experiment, see Supplementary Information, Figure
S58.
17

The well-known role of Akt in driving cancer [30] should also be taken into account
when interpreting such bioactivity results. At this point it is impossible to make a sound
judgment whether or not a stronger in vitro activation of this protein kinase would have in
vivo relevance, and particularly if this would confer ecdysteroid metabolites any pro-
cancerous risk. Current experiences with the apparent safety of ingesting large (up to several
grams) doses of ecdysteroids by sportsmen at least do not seem to raise concerns, even though
long-term safety of such a practice has never been studied in humans. Nevertheless, our
results clearly show that side-chain cleaved metabolites of ecdysteroids accompanying 20E
(
1) within plant extracts have all the potential to significantly contribute to the complex
bioactivity.
3
. Conclusions
By using two different chemical strategies (oxidative side-chain cleavage of various
phytoecdysteroids and autoxidation of poststerone), a set of semi-synthetic side-chain cleaved
ecdysteroids was obtained from acceptable to good yields. In particular, poststerone could be
obtained with an isolated yield of as high as 81.35%. Comprehensive NMR spectroscopic
techniques allowed a complete signal assignment for each new compound. Several of the new
compounds are derivatives of known phytoecdysteroids with an intact side-chain. Since the
side-chain cleavage is a known metabolic step of 20E in rodents, the prepared compounds can
be assumed as potential in vivo metabolites of the corresponding phytoecdysteroids.
The compounds showed the ability to increase the activation of protein kinase B in
C2C12 myotubes in murine skeletal muscle cells. Our results suggest that, similarly to our
most recent results with poststerone, the metabolic step of side-chain cleavage leads to
compounds with altered, and apparently increased bioactivity. Such compounds can
reasonably be expected to significantly contribute to the overall activity observed after the
consumption of preparations (e.g. food supplements) containing a variety of
phytoecdysteroids. Moreover, these metabolites are potentially valuable leads towards
possible ecdysteroid-inspired drug discovery initiatives.
4
4
. Materials and methods
.1. Starting materials
2
0-Hydroxyecdysone (1) with a purity of 90%, originated from the roots of Cyanotis
arachnoidea was purchased from Shaanxi KingSci Biotechnology Co., Ltd. (Shanghai,
18

China). A purification step was performed by recrystallization from ethyl acetate - methanol
2:1, v/v) to reach a 97.8% purity by means of RP-HPLC.
-Deoxy-20-hydroxyecdysone (2) was previously isolated by our research group from
(
2
Silene italica ssp. nemoralis [31], ajugasterone C (3), polypodine B (4) were obtained from
our previous isolation from Serratula wolffii [32]. Compounds 2, 3 and 4 possessed a purity
of > 95% by means of RP-HPLC. Dacryhainansterone (5) and calonysterone (6) were isolated
in our laboratory with a purity of 99.1% and 99.7%, respectively, from a commercial extract
of Cyanotis arachnoidea [33].
4
.2 General methods
All reagents and solvents were obtained from commercially available sources and
were used without further purification.
Reactions’ progress were monitored by NP-TLC performed on silica plates (Silica gel
6
0F254, E. Merck, Germany), by using the solvent system composed of ethyl acetate -
ethanol - water (12:1:0.5, v/v/v). Visualizations were performed under UV light λ = 254 nm
and after spraying with vanillin/sulfuric acid reagent under λ = 365 nm.
1
2
Compounds were fractionated and/or purified by using different chromatographic
techniques. RP-HPLC for semi-preparative purifications were carried out on an Agilent 1100
series (Waters Co., Milford, MA, USA) connected to a Jasco UV-2075 detector (Jasco Co.,
Tokyo, Japan). RP-HPLC for preparative purifications were performed on a Waters 600 Pump
connected to a Waters 2487 Dual λ Absorbance Detector (Waters Co., Milford, MA, USA).
Rotation planar chromatography was completed on a Chromatotron device (Harrison
Research, Palo Alto, CA, USA). Flash chromatography was achieved by means of a
®
CombiFlash Rf + apparatus (Teledyne Isco, Lincoln, USA) with an 80 g HP Silica
RediSep® Rf Gold column (Teledyne Isco, Lincoln, USA). Centrifugal Partition
Chromatography was performed on an Armen Spot CPC 250 ml (Armen Instrument, Saint
Ave, France) system composed of an Armen Spot Prep II equipment and an Armen Spot CPC
multilayer coil separation column, and controlled by the Armen Glider CPC software.
Optimization of the CPC separation was through following well-established general concepts
published in the literature [33-36].
Purity of the obtained compounds was determined by RP-HPLC analyses on a system
of two Jasco PU-2080 pumps, a Jasco AS-2055Plus intelligent sampler connected to a
JASCO LC-Net II/ADC equipped with a Jasco MD-2010 Plus PDA detector (Jasco Co.,
Tokyo, Japan). Turbo ion spray tandem MS of compounds 8 and 10 were recorded on a API
2
000 triple quadrupole tandem mass spectrometer (AB SCIEX, Foster City, CA, USA) in
19

positive mode equipped with ESI ion source. HRMS data for compounds 12-18 were
recorded on a Waters Acquity I-Class UHPLC system (Waters Co., Milford, MA, USA)
coupled with a Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer equipped with
HESI ion source (Thermo Fisher Scientific, Scoresby, Australia).
1
13
H (500.1 MHz) and C (125.6 MHz) NMR spectra of compounds 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 0.7-5
mg of compounds were dissolved in 0.1 ml of methanol-d
MATCH NMR sample tube (Bruker). Chemical shifts are given on the -scale and are
referenced to the solvent (methanol-d : δ = 49.1 and δ = 3.31 ppm). NMR data of
compound 18 were also determined in dimethyl sulfoxide-d (δ = 39.5 and δ = 2.50 ppm).
Pulse programs of all experiments (one-dimensional H, C, DEPTQ, DEPT-135, APT, sel-
4
and transferred to 2.5 mm Bruker
4
C
H
6
C
H
1
13
1
1
TOCSY, sel-ROE (mix: 300 ms), two-dimensional H, H-COSY, gs-HSQC, edited gs-HSQC,
gs-HMBC and band-selective HSQC and band-selective HMBC) were taken from the Bruker
software library.
4
4
.3. Synthesis
.3.1 Synthesis of compounds 7-12 through oxidative side-chain cleavage of
phytoecdysteroids 1-6
Synthesis of poststerone (7). 20-hydroxyecdysone (1) was used as starting material
for the synthesis of poststerone (7). 5.0 g of 20E (1) was dissolved in 400 ml methanol, 1
equiv. of PIDA was added, and the solution was stirred at room temperature for 45 min. The
reaction mixture was then neutralized with a 5% aqueous solution of NaHCO , and the
3
solvent was evaporated at 40°C under vacuum. The residue was dissolved in methanol, and 12
g of silica powder was added. The solvent was evaporated under vacuum, and the dried
extract adsorbed onto silica (‘dried solid sample loading’ technique) was transferred to a
universal Rf cartridge (Teledyne Isco, Lincoln, USA). For the purification, flash
chromatography was used with a mobile phase of n-hexane - dichloromethane (6:4, v/v;
solvent A), and dichloromethane - methanol (1:1, v/v; solvent B), and a 40 min gradient
program of 0 - 30% B in A was applied at a flow rate of 60 ml/min. Compound 7 was
obtained with an isolated yield of 81.41% (3.07 g).
General procedure for the synthesis of compounds 8-11. Compounds 2-5 were used
as starting materials for the synthesis of compounds 8, 9, 10 and 11 respectively. Each starting
material (2-5) was dissolved in methanol to a final concentration of 100 mM (2, 5: 46 mg; 3:
20

4
8 mg; 4: 50 mg), and 1.2 equiv. of PIFA was added. The mixtures were stirred for 60 min at
room temperature. The reactions were stopped by neutralizing the pH by using a 5% aqueous
solution of NaHCO , followed by evaporation to dryness. Each residue was dissolved in ethyl
3
acetate and filtered through silica, then dried. Compounds 8 and 9 were purified over
preparative RP-HPLC applying a Kinetex XB-C18 (5 µm, 250 x 21.2 mm) column with 50%
aqueous methanol as eluent at a flow rate of 10 ml/min, detecting at 254 nm. Compounds 8
and 9 were isolated at 56.4% (19.5 mg) and 62.6% (22.7 mg) yields, respectively.
Compounds 10 and 11 were purified over RPC using a four-step gradient elution with
cyclohexane - ethyl acetate (1:1 and 1:5, v/v) and ethyl acetate - ethanol (12:1 and 6:1, v/v),
with 80 ml for each solvent, and 23 fractions were collected. Compounds 10 and 11 were
obtained at 60.2% (21.6 mg) and 59.5% (22.7 mg) yield, respectively.
Synthesis of compound 12. Calonysterone (6) was used as starting material for the
synthesis of compound 12. 50 mg of calonysterone (6) was dissolved in 12.5 ml methanol, 1
equiv. of PIDA was added, and the solution was stirred at room temperature for 60 min. The
reaction was stopped by adjusting the pH of the final mixture to neutral with a 5% aqueous
solution of NaHCO . After evaporation of the solvent under nitrogen, the residue was purified
3
by CPC in ascending mode with a biphasic solvent system composed of n-hexane - ethyl
acetate - methanol - water (3:10:3:10, v/v/v/v). The solvent system offered the following
separation characteristics: a very short settling time (17s), the volume ratio of the upper and
lower phases was 0.86, and the retention volume ratio Sf was 0.68. The equipment optimized
parameters were as follows: constant pressure of 86 bars, flow rate of 10 ml/min, and rotation
speed of 2600 rpm. The purification was performed through a single injection and an
altogether 94.42% of the initial weight was recovered after the separation. Compound 12 was
obtained with a yield of 51.8% (19.5mg).
4
.3.2. Synthesis of compounds 12-18 through base-catalyzed autoxidation of poststerone
(
7)
The base-catalyzed autoxidation of poststerone (7) was performed by dissolving 450
mg of poststerone (7) in a mixture of 10 ml of MeOH and 80 ml of water, then 500 mg of
NaOH dissolved in 10 ml of water was added. The reaction mixture was stirred for 4 hours at
room temperature, and the reaction was stopped by neutralizing the mixture with a 9.6%
aqueous solution of acetic acid. After evaporating the solvent under nitrogen stream, the
residue was pre-purified by solid-phase extraction through silica using methanol as eluent,
then dried.
21

The reaction mixture was subsequently fractionated by CPC in ascending mode, by using the
biphasic solvent system previously developed in our laboratory for the separation of 20E (1)
and its oxidized derivatives [19]; it was composed of ethyl acetate - water - methanol
(
20:20:1, v/v/v). The equipment optimized parameters were settled as follows: constant
pressure of 80 bars, flow rate of 10 ml/min and rotation speed of 2900 rpm. The fractionation
was performed through four consecutive injections, and an altogether 95.43% of the initial
weight was recovered after the separation. The CPC fractions were combined based on their
NP-TLC fingerprints, evaporated under vacuum at 40 °C, dissolved in methanol and
investigated by RP-HPLC on a Kinetex XB C-18 (5 μm, 250 × 4.6 mm) column with an
isocratic solvent system of 23% aqueous acetonitrile at a flow rate of 1 ml/min. Fractions 6, 7
and combined fractions 11-13 were selected for additional purification steps. Fraction 6 was
further purified over semi-preparative RP-HPLC with 30% aqueous acetonitrile at a flow rate
of 3 ml/min using a Gemini C18 (5 μm, 250 × 10 mm) column to yield compounds 12 (1.5
mg, 0.34%) and 16 (3.76 mg, 0.84%). Fraction 7 was further purified over semi-preparative
RP-HPLC with 27% aqueous acetonitrile at a flow rate of 3 ml/min using a Gemini C18 (5
μm, 250 × 10 mm) column to yield compound 17 (10.61 mg, 2.48%). The CPC combined
fractions 11-13 were further purified over semi-preparative RP-HPLC with 20% aqueous
acetonitrile at a flow rate of 3 ml/min using a Luna Phenyl-Hexyl (5 μm, 250 × 10 mm)
column to yield compounds 13 (10.2 mg, 2.17%), 14 (5.7 mg, 1.21%), 15 (11.3 mg, 2.42%)
and 18 (4.6 mg, 1.00%).
In order to obtain more amounts of the above compounds, the reaction was repeated
by using another 500 mg of poststerone (7) that was stirred for 7 hours, and the work-up
procedure consisted of the same purification steps as detailed above. The isolated yields in
this case were as follows: compound 13 (27.9 mg, 5.34%), compound 14 (3.04 mg, 0.58%),
compound 15 (20 mg, 3.85%), compound 16 (20 mg, 4.04%) and compound 18 (5.72 mg,
1
.1%).
4
.4. 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-well plates and maintained in
high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum
and 1% penicillin/streptomycin solution in a humidified atmosphere of 95% air and 5% CO
2
at 37 °C. After reaching 100% confluence, C2C12 myoblasts were cultured in differentiation
22

medium (DMEM containing 4500 mg/L 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 serum free normal-glucose DMEM with or without
1
0 µM of each test compound. After 2 h, the cells were lysed with 500 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 AKT
activation, 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).
Acknowledgements
A.H. acknowledges the János Bolyai fellowship of the Hungarian Academy of Sciences and
the Kálmán Szász Prize. The authors wish to thank Mr. Attila Csorba (Institute of
Pharmacognosy, University of Szeged) for the HRMS measurements.
Funding
This work was supported by the National Research, Development and Innovation Office,
Hungary (NKFIH; K119770), and by the Ministry of Science and Technology, Taiwan
(
MOST 104-2314-B-037-067). The authors acknowledge support from the Ministry of
Human Capacities, Hungary grant 20391-3/2018/FEKUSTRAT, the EU-funded Hungarian
grant EFOP-3.6.1-16-2016-00008, and GINOP-2.3.2-15-2016-00012, and from a bilateral
mobility grant from the Hungarian Academy of Sciences and the Ministry of Science and
Technology, Taiwan (MOST 107-2911-I-037-502).
References
1
. R. Lafont, J. Harmatha, F. Marion-Poll, L. Dinan, I.D. Wilson, Ecdybase: a free
ecdysteroid database. http://ecdybase.org/, 2002 → continuously updated (accessed 05
July 2018).
2
. L. Dinan, Phytoecdysteroids: biological aspects, Phytochemistry, 57 (2001) 325-339.
https://doi.org/10.1016/S0031-9422(01)00078-4.
23

3
. M.J. Klowden, Signaling systems in Physiological Systems in Insects in: Academic Press
Ed.), Physiological Systems in Insects Elsevier Science & Technology, London, 2013,
(
pp. 1-87.
4
5
. L. Dinan, The Karlson Lecture. Phytoecdysteroids: what use are they?, Arch. Insect
Biochem. Physiol. 72 (2009) 126-141. https://doi.org/10.1002/arch.20334
. G. Laekeman, A. Vlietinck, Phytoecdysteroids: Phytochemistry and Pharmacological
Activity in: K.G. Ramawat, J.M. Merillon (Eds.), Natural products: phytochemistry,
botany and metabolism of alkaloids, phenolics and terpenes, Springer, Berlin, 2013, pp.
3
827-3850.
6
. M. Báthori, N. Tóth, A. Hunyadi, A. Márki, E. Zádor, Phytoecdysteroids and anabolic-
androgenic steroids-structure and effects on humans, Curr. Med. Chem. 15 (2008) 75-91.
https://doi.org/10.2174/092986708783330674.
7
8
. Q. Chen, Y. Xia, Z. Qiu, Effect of ecdysterone on glucose metabolism in vitro, Life Sci.
7
8 (2006) 1108-1113. https://doi.org/10.1016/j.lfs.2005.06.031.
. P. Kizelsztein, D. Govorko, S. Komarnytsky, A. Evans, Z. Wang, W.T. Cefalu, I. Raskin,
0-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice
model, Am. J. Physiol. Endocrinol. Metab. 296 (2009) E433-E439. https://doi.org/
0.1152/ajpendo.90772.2008.
2
1
9
1
. T.A. Kutepova, V.N. Syrov, Z.A. Khushbaktova, Z. Saatov, Hypoglycemic activity of the
total ecdysteroid extract from Ajuga turkestanica, Pharm. Chem. J. 35 (2001) 608-609.
https://doi.org/10.1023/A:1015145811009.
0. K. Hamden, F. Ayadi, K. Jamoussi, H. Masmoudi, A. Elfeki, Therapeutic effect of
phytoecdysteroids rich extract from Ajuga iva on alloxan induced diabetic rats liver,
kidney
and
pancreas,
BioFactors,
33
(2008)
165-175.
https://doi.org/10.1002/biof.5520330302
1
1
1
1. I. Todorov, Y. Mitrokin, O. Efremova, L. Sidorenko, The effect of ecdysterone on the
biosynthesis of proteins and nucleic acids in mice, Pharm. Chem. J. 34 (2000) 455-458.
https://doi.org/10.1021/jf073059z.
2. J. Gorelick-Feldman, D. MacLean, N. Ilic, A. Poulev, M.A. Lila, D. Cheng, I. Raskin,
Phytoecdysteroids increase protein synthesis in skeletal muscle cells, J. Agric. Food.
Chem. 56 (2008) 3532-3537. https://doi.org/10.1021/jf073059z.
3. N. Tóth, A. Szabó, P. Kacsala, J. Héger, E. Zádor, 20-Hydroxyecdysone increases fiber
size in a muscle-specific fashion in rat, Phytomedicine, 15 (2008) 691-698.
https://doi.org/10.1016/j.phymed.2008.04.015.
24

2
+
1
1
1
4. J. Gorelick-Feldman, W. Cohick, I. Raskin, Ecdysteroids elicit a rapid Ca flux leading
to Akt activation and increased protein synthesis in skeletal muscle cells, Steroids, 75
(
2010) 632-637. https://doi.org/10.1016/j.steroids.2010.03.008.
5. Schiaffino, S., Mammucari, C., 2011. Regulation of skeletal muscle growth by the IGF1-
Akt/PKB pathway: insights from genetic models. Skeletal muscle. 1, 4. https://doi.org/
1
0.1186/2044-5040-1-4.
6. M. Sandri, L. Barberi, A.Y. Bijlsma, B. Blaauw, K.A. Dyar, G. Milan, C. Mammucari, C.
G.M. Meskers, G. Pallafacchina, A. Paoli, D. Pion, M. Roceri, V. Romanello, A.L.
Serrano, L. Toniolo, L. Larsson, A.B. Maier, P. Muñoz-Cánoves, A. Musarò, M. Pende,
C. Reggiani, R. Rizzuto, S. Schiaffino, Signalling pathways regulating muscle mass in
ageing skeletal muscle. The role of the IGF1-Akt-mTOR-FoxO pathway,
Biogerontology, 14 (2013) 303-323. https://doi.org/10.1007/s10522-013-9432-9.
7. S. Kumpun, J.P. Girault, L. Dinan, C. Blais, A. Maria, C. Dauphin-Villemant, B.
Yingyongnarongkul, A. Suksamrarn, R. Lafont, The metabolism of 20-hydroxyecdysone
in mice: relevance to pharmacological effects and gene switch applications of
1
ecdysteroids,
J.
Steroid
Biochem.
Mol.
Biol.
126
(2011)
1-9.
https://doi.org/10.1016/j.jsbmb.2011.03.016.
1
1
8. A. Suksamrarn, P. Ganpinyo, C. Sommechai, Base-catalyzed autoxidation of 20-
hydroxyecdysone: synthesis of calonysterone and 9,20-dihydroxyecdysone, Tetrahedron
Lett. 35 (1994) 4445-4448. https://doi.org/10.1016/S0040-4039(00)73380-6.
9. J. Csábi, T.J. Hsieh, F. Hasanpour, A. Martins, Z. Kele, T. Gáti, A. Simon, G. Tóth, A.
Hunyadi, Oxidized metabolites of 20-hydroxyecdysone and their activity on skeletal
muscle cells: Preparation of a pair of desmotropes with opposite bioactivities, J. Nat.
Prod. 78 (2015) 2339-2345. https://doi.org/ 10.1021/acs.jnatprod.5b00249.
2
2
2
0. J.B. Siddall, D.H.S. Horn, E.J. Middleton, Synthetic studies on insect hormones. The
synthesis of a possible metabolite of crustecdysone (20-hydroxyecdysone), Chem.
Commun. 0 (1967) 899-900. https://doi.org/ 10.1039/C19670000899.
1. R.Q. Petersen, R.C. Cambie, G.B. Russell, Jones oxidation of 20-hydroxyecdysone
(
crustecdysone),
Aust.
J.
Chem.
46
(1993)
1961-1964.
https://doi.org/10.1071/CH9931961.
2. Hunyadi, A., Csábi, J., Martins, A., Molnár, J., Balázs, A., Tóth, G., 2017. Backstabbing
P-gp: side-chain cleaved ecdysteroid 2,3-dioxolanes hyper-sensitize MDR cancer cells to
doxorubicin
without
efflux
inhibition.
Molecules.
22,
199.
https://doi.org/10.3390/molecules22020199.
25

2
2
2
3. H. Duddeck, W. Dietrich, G. ꢀꢁth, Structure Elucidation by Modern NMR, Springer-
Steinkopff-Verlag Heidelberg, 1998.
4. E. Pretsch, G. ꢀꢁth, M.E. Munk, M. Badertscher, Computer-Aided Structure Elucidation.
Spectra Interpretation and Structure Generation, Wiley-VCH: Weinheim, 2002.
5. A. Hunyadi, G. Tóth, A. Simon, M. Mák, Z. Kele, I. Máthé, M. Báthori, Two new
ecdysteroids from Serratula wolffii, J. Nat. Prod. 67 (2004) 1070-1072. https://doi.org/
1
0.1021/np030544p.
2
2
2
6. A. Martins, N. Tóth, A. Ványolós, Z. Béni, I. Zupkó, J. Molnár, M. Báthori, A. Hunyadi,
Significant activity of ecdysteroids on the resistance to doxorubicin in mammalian cancer
cells expressing the human ABCB1 transporter, J. Med. Chem. 55 (2012) 5034-5043.
https://doi.org/ 10.1021/jm300424n.
7. M. Vágvölgyi, A. Martins, Á. Kulmány, I. Zupkó, T. Gáti, A. Simon, G. Tóth, A.
Hunyadi, Nitrogen-containing ecdysteroid derivatives vs. multi-drug resistance in cancer:
preparation and antitumor activity of oximes, oxime ethers and a lactam, Eur. J. Med.
Chem. 144 (2018) 730-739. https://doi.org/10.1016/j.ejmech.2017.12.032.
8. Z. Pongrácz, M. Báthori, G. ꢀꢁth, A. Simon, M. Mák, I. Máthé, 9α,20-
Dihydroxyecdysone, a new natural ecdysteroid from Silene italica ssp. nemoralis, J. Nat.
Prod. 66 (2003) 450-451. https://doi.org/10.1021/np0205194.
2
3
9. M.N. Galbraith, D.H.S. Horn, E.J. Middleton, The structure of podecdysone B, a new
phytoecdysone, J. Chem. Soc. 0 (1969) 402-403. https://doi.org/10.1039/C29690000402.
0. P.S. Mundi, J. Sachdev, C. McCourt, K. Kalinsky, AKT in cancer: new molecular
insights and advances in drug development, Br. J. Clin. Pharmacol. 82 (2016) 943-956.
https://doi.org/10.1111/bcp.13021.
3
3
3
1. N. Tóth, A. Simon, G. Tóth, Z. Kele, A. Hunyadi, M. Báthori, 26-Hydroxylated
ecdysteroids from Silene viridiflora, J. Nat. Prod. 71 (2008) 1461-1463.
https://doi.org/10.1021/np800139q.
2. A. Hunyadi, A. Gergely, A. Simon, G. Tóth, G. Veress, M. Báthori, Preparative-scale
chromatography of ecdysteroids of Serratula wolffii Andrae, J. Chromatogr. Sci. 45
(
2007) 76-86. https://doi.org/10.1093/chromsci/45.2.76.
3. H.M. Issaadi, Y.C. Tsai, F.R. Chang, A. Hunyadi, Centrifugal partition chromatography
in the isolation of minor ecdysteroids from Cyanotis arachnoidea, J. Chromatog. B, 1054
(
2017) 44-49. https://doi.org/10.1016/j.jchromb.2017.03.043.
26

3
4. J.B. Friesen, J.B. McAlpine, S.N. Chen, G.F. Paulin, Countercurrent separation of natural
products: an update, J. Nat. Prod. 78 (2015) 1765-1796.
https://doi.org/10.1021/np501065h.
3
3
5. J. Cazes, Encyclopedia of Chromatography 2004 Update Supplement, Marcel Dekker,
Inc., New York, 2003.
6. M. Bojczuk, D. Żyżelewicz, P. Hodurek, Centrifugal partition chromatography - a review
of recent applications and some classic references, J. Sep. Sci. 40 (2017) 1597-1609.
https://doi.org/10.1002/jssc.201601221.
27

Highlights
Ecdysteroids undergo side-chain cleavage during their in vivo metabolism.
Through oxidative chemistry, several new ecdysteroid metabolites were prepared.
The new compounds were characterized with complete NMR signal assignment.
The compounds showed the ability to increase the activation of protein kinase B.
Ecdysteroids with higher degree of unsaturation showed stronger bioactivity.
28

Graphical abstract
29