Rapid, laser-induced conversion of 20-hydroxyecdysone-A follow-up study on the products obtained

Article abstract of DOI:10.1016/j.steroids.2014.07.016

We have recently reported the set-up of an experimental system for the laser-induced photochemical modification of bioactive substances, where two ecdysteroids, 20-hydroxyecdysone (20E) and its diacetonide derivative served as probes. As a direct continuation of our previous work, three new compounds together with five other ecdysteroid derivatives, have been identified from the novel, laser-induced photo-transformation reaction of 20E. The structures and NMR signal assignment were established by comprehensive one- and two-dimensional NMR spectroscopy supported by mass spectroscopy. Possible ways for the formation of each species is also discussed. Similar to their parental compound, the products obtained are potentially bioactive and worthy for further investigations; due to the low yields, however, a different approach for their higher scale production is suggested.

Full text of DOI:10.1016/j.steroids.2014.07.016

Steroids 89 (2014) 56–62
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Rapid, laser-induced conversion of 20-hydroxyecdysone – A follow-up
study on the products obtained
Wan-Chun Lai a,b,1, Balázs Dankó , József Csábi , Zoltán Kele , Fang-Rong Chang , Mihail L. Pascu ,
a,1
a
c
b
d
e
e
f,g
e,
⇑
a,
⇑
Tamás Gáti , András Simon , Leonard Amaral , Gábor Tóth , Attila Hunyadi
a
Institute of Pharmacognosy, University of Szeged, 6720 Szeged, Hungary
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC
Department of Medical Chemistry, University of Szeged, 6720 Szeged, Hungary
Laser Department, National Institute for Laser, Plasma and Radiation Physics, Bucharest, Romania
Department of Inorganic and Analytical Chemistry, NMR Group, Budapest University of Technology and Economics, 1111 Budapest, Hungary
Travel Medicine of the Center for Malaria and Other Tropical Diseases (CMDT), Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 1349-008 Lisbon, Portugal
Department of Microbiology and Immunobiology, University of Szeged, 6720 Szeged, Hungary
b
c
d
e
f
g
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently reported the set-up of an experimental system for the laser-induced photochemical
modification of bioactive substances, where two ecdysteroids, 20-hydroxyecdysone (20E) and its diaceto-
nide derivative served as probes. As a direct continuation of our previous work, three new compounds
together with five other ecdysteroid derivatives, have been identified from the novel, laser-induced
photo-transformation reaction of 20E. The structures and NMR signal assignment were established by
comprehensive one- and two-dimensional NMR spectroscopy supported by mass spectroscopy. Possible
ways for the formation of each species is also discussed.
Similar to their parental compound, the products obtained are potentially bioactive and worthy for fur-
ther investigations; due to the low yields, however, a different approach for their higher scale production
is suggested.
Received 26 February 2014
Received in revised form 23 June 2014
Accepted 25 July 2014
Available online 20 August 2014
Keywords:
Ecdysteroid
2
0-Hydroxyecdysone
Enone photochemistry
Laser flash photolysis
NMR
Ó 2014 Elsevier Inc. All rights reserved.
Stereochemistry
1
. Introduction
Ecdysteroids represent one of the large families of steroid hor-
resistance of murine leukemia cells expressing the human ABCB1
transporter [5,6]. Based on the anabolic activity and several poten-
tial (expected) health benefits, many food supplements and other
products containing mainly 20-hydroxyecdysone (20E), the most
common ecdysteroid of herbal origin, are available worldwide for
various indications.
Ecdysteroids are significantly different from the mammalian
steroid hormones, although they show some structural similarities
to vitamin D, which might serve as an explanation for most (if not
all) of the effects of these compounds in mammals [7]. Due to their
several hydroxyl groups around the regularly side-chain bearing
steroid skeleton, they are typically polar compounds, and a 7-en-
mones in Nature; these compounds play a crucial role at all stages
of arthropods’ development including embryogenesis, molting, egg
and spermium production and diapause [1]. Interestingly, ecdys-
teroids can also exert various rather beneficial effects in verte-
brates:
a
general strengthening, somewhat ‘‘Ginseng-like’’
activity has been attributed to them, which includes a non-hor-
monal anabolic action and a series of further metabolic effects usu-
ally referred to as adaptogenic [2,3]. Ecdysteroids also play a
(
rather unclear) role in cancer: muristerone A exerted anti-apopto-
tic activity on a colon carcinoma cell line in vitro [4], and, most
recently, a strong effect of certain natural and semi-synthetic
ecdysteroids was discovered on the efflux-mediated multi-drug
6-one (a,b-enone) chromophore group is usually present in their
B ring. This latter moiety confers upon these compounds a charac-
teristic UV absorption at around k = 240–250 nm, and allows them
to be subjects of photochemical transformations. Such an approach
can offer several advantages over that of the conventional chemical
methods in the semi-synthetic modification of ecdysteroids – due
to their typically complex hydroxylation patterns, certain selective
transformations of these compounds can be challenging. On the
⇑
Corresponding authors. Tel.: +36 62546456 (A. Hunyadi), +36 204646486
G. Tóth).
E-mail addresses: drtothgabor@t-online.hu (G. Tóth), hunyadi.a@pharm.
(
u-szeged.hu (A. Hunyadi).
other hand, photoreactions of
a,b-enones represent a major area
1
The first two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.steroids.2014.07.016
0
039-128X/Ó 2014 Elsevier Inc. All rights reserved.

W.-C. Lai et al. / Steroids 89 (2014) 56–62
57
of modern photochemistry, which allows various specific, regiose-
lective modifications that are hard or even impossible to achieve
by using classical chemical strategies [8].
reagent for visualization of bands. Yields represent the isolated
amounts. Low-resolution mass spectra were recorded on an API
2000 triple quadrupole tandem mass spectrometer (AB SCIEX, Fos-
ter City, CA, USA) in positive mode with an atmospheric pressure
chemical ionization (APCI) ion source. High-resolution electro-
spray-ionization mass spectra (HRESIMS) were recorded on a
Waters Micromass Q-TOF premier mass spectrometer (Waters,
Milford, MA, USA).
Unusual photochemical behavior of the enone moiety of 20E
irradiated by a mercury UV-lamp over a Pyrex filter (k > 290 nm)
was discussed by Canonica et al. [9,10]. The irradiation of a
ꢀ3
1
0
M water solution of 20E was performed under argon atmo-
sphere and resulted in a full conversion after 12 h. The authors
8
–14
found the main photoproducts to be the
-deoxy-20-hydroxyecdysone (15%) and two derivatives formed
by ring rearrangement (23% and 18%), while 14 -hydroperoxy-20-
D
ketone (35%),
14a
a
2.2. NMR spectroscopy
hydroxyecdysone was also present. The photochemical transfor-
mation of 20E in 500 mL of water with a Pyrex-filtered medium-
pressure Hg lamp under inert gas (argon) was also performed by
Harmatha et al. [11]. The production of additional derivatives such
1H (500.1 MHz) and 13C (125.6 MHz) NMR spectra were
recorded at room temperature on Bruker 500 Avance III equipped
with cryo probehead and on Bruker Avance 500 spectrometers.
Amounts of approximately 1–5 mg of the laser derived compounds
0
8–14
as 14-epi-20E and the 7–7 homo-dimer of the
D
ketone was
8–14
reported, whereas the monomeric
D
ketone could not be
4
were dissolved in 0.1 mL of methanol-d and transferred to 2.5 mm
detected. These authors suggested differences in reactant concen-
tration and/or the effect of oxygen present from air as possible
explanations for this phenomenon. Yield of the dimer increased
with the concentration of 20E; up to 19% at 1.2 mg/mL [11].
The use of lasers has some major advantages over the Hg lamp
based photo-reactors due to their much higher energy and optical
purity. With the use of lasers, integration of photochemistry and
flow chemistry, as well as miniaturization of photoreactions with
a lab-on-a-chip approach, are also possible [12].
Recently, we reported the set-up of a 266 nm laser system for
the rapid photochemical transformation of bioactive substances
by using 20E and its 2,3;20,22-diacetonide as first probes [13]. To
our best knowledge, laser-catalyzed photoreactions of ecdysteroids
have not been studied by any other groups. As a direct continua-
tion of our previous work, here we describe the products obtained
from the scale-up irradiation of 20E and discuss possible ways of
their formation.
Bruker MATCH NMR sample tube. Chemical shifts are given on the
d-scale and are referenced to the solvent (methanol-d
and d
4
: d
C
= 49.1
1
13
H
= 3.31 ppm). Pulse programs of all experiments ( H– C,
DEPTQ, DEPT-135, sel-TOCSY (mixing time: 80 ms, 120 ms), sel-
ROE (300 ms), sel-NOE (350 ms, 500 ms), gradient-selected (gs)
1
1
H, H-COSY, edited gs-HSQC, gs-HMBC (optimized for 10 Hz; in
case of compound 5 also 5 Hz), NOESY (350 ms, 500 ms)) were
taken from the Bruker software library. For 1D measurements,
6
4 K data points were used to yield the FID. For 2D measurements,
sweep width in F was 4000 Hz; all data points (t ) were
2
2
ꢁ t
1
acquired with 2 K ꢁ 256. For F1, linear prediction was applied to
1
enhance the resolution. Most H assignments were accomplished
using general knowledge of chemical shift dispersion with the
1
aid of the proton–proton coupling pattern ( H NMR spectra). The
1
13
H and C NMR data of the parent 20E and that of the new
compounds 2, 5 and 7 are compiled in Table 1.
2
. Experimental
2.3. Isolation of the irradiation products
2.1. General information
The irradiated mixture was fractionated by rotation planar
chromatography (RPC) with a five step gradient of n-Hexane:CH2-
The starting material 20E was previously isolated from the
2
Cl :MeOH (3:125:5–3:50:5) to yield fractions 1-1–5-5 represent-
plant Serratula wolffii [14]. Two milliliters of 10 mg/mL methanolic
solutions of 20E were irradiated with a 266 nm laser at 15 mJ
impulse energy in a 4 mL cuvette with continuous stirring, as
described previously, and a total amount of 400 mg mixture of
products was obtained from the repeated irradiations [13].
ing five fractions with each solvent composition. Fraction 4-1
(25.7 mg) was purified over RP-HPLC (50% MeOH aq, 3 mL/min)
to yield poststerone (1) (13.1 mg). Fraction 4-2 (16.1 mg) was puri-
fied by RP-HPLC (58% MeOH aq, 3 mL/min) to obtain compound 2
(1.5 mg). Fraction 4-3 (16.5 mg) was purified by RP-HPLC (50%
Solvent system compositions are given in v/v%. Rotational pla-
nar chromatography (RPC) was performed on a Chromatotron
equipment (Harrison Research, Palo Alto, CA, USA). Analytical
and preparative reverse phase HPLC was performed by using a gra-
dient system consisting of two Jasco PU-2080 HPLC pumps
equipped with a Jasco AS2055Plus autosampler connected to a Jas-
co MD-2010 Plus photodiode array (PDA) detector (Jasco Co.,
Tokyo, Japan). For the quantitative determination of the remaining
unchanged 20E within the mixture, a Zorbax SB-Aq (250 ꢁ 4.6 mm,
MeOH aq, 3 mL/min) to yield 14a,15a-epoxy-14, 15-dihydrosta-
chysterone B (3) (0.62 mg) and stachysterone B (4) (2.0 mg). The
residue from fraction 4-3 (4.3 mg) was further purified with RP-
HPLC (30% CH
(28.1 mg) was purified by RP-HPLC (21% CH
obtain 14 -hydroperoxy-20-hydroxyecdysone (6) (5.2 mg). Frac-
3
CN aq, 3 mL/min) to give (5) (1.5 mg). Fraction 5-2
3
CN aq, 3 mL/min) to
a
tion 5-3 (83.1 mg) was further fractionated by centrifugal partition
chromatography (Armen Spot CPC 250 mL, Armen Instrument,
Saint Ave, France) in descending mode with a solvent system of
5
l
m) column (Agilent Technologies, Santa Clara, CA, USA) was
CHCl
Fraction 14 (20.7 mg) was purified with RPC with a three steps gra-
dient system of EtOAc:EtOH:H O (80:2:1–80:10:4), to obtain com-
3 2
:MeOH:H O (10:7:3), and 20 mL fractions were collected.
used with a solvent system of 50% aqueous MeOH at a flow rate
of 1.00 mL/min detecting at k = 245 nm, with a five-points calibra-
2
2
tion (R = 0.9999). For isolation, a Phenomenex C18 (250 ꢁ 10 mm,
pound 7 (3.7 mg). Compound 8, 14-epi-20-hydroxyecdysone was
identified by HPLC-DAD by using authentic reference compound
previously isolated from plant source [14], and the order of magni-
tude of its amount was estimated based on the calibration line
obtained for 20E.
Each isolated compound possessed a purity of >95% by means of
RP-HPLC-PDA, by using 50% aqueous MeOH at 1 mL/min of flow
rate.
5 lm) semi-preparative column (Phenomenex, Torrance, CA, USA)
was used. Compound purities were tested on an Agilent XDB-C8
4.6 ꢁ 150 mm, 5 m) analytical column (Agilent Technologies,
Santa Clara, USA). The separation was also monitored with thin
layer chromatography (TLC) on silica gel 60 F254 (0.25 m; Merck,
Budapest, Hungary) and 60 RP-18 F254S (0.25 m; Merck, Buda-
pest, Hungary) and the gels sprayed with vanillin sulfuric acid
(
l
l
l

5
8
W.-C. Lai et al. / Steroids 89 (2014) 56–62
Table 1
1
H and 13C chemical shifts, multiplicities and coupling constants of compounds 2, 5 and 7 in reference to those of 20-hydroxyecdysone (20E), in ppm (MeOH-d
4
).
Atom No.
20E
H
C
2a
H
J (Hz)
C
5
H
J (Hz)
C
7
H
J (Hz)
C
1
b
1.43
1.80
3.84
3.95
1.76
1.72
2.38
–
5.81
–
3.15
–
1.72
1.80
1.88
2.14
–
37.5
1.68
1.73
3.68
3.88
2.03
1.37
2.65
–
7.19
–
2.55
–
1.63
1.74
2.28
1.55
–
37.2
1.54
1.63
3.89
3.98
1.78
1.64
2.46
–
–
–
3.27
–
1.66
1.90
1.59
2.17
–
dd; 13.4, 11.9
38.6
1.46
1.74
3.81
4.04
2.13
2.24
2.42
–
3.53
–
2.99
–
1.58
1.86
1.93
1.58
–
36.8
a
2
3
68.8
68.6
33.0
td; 12.5, 3.0
q; ꢂ3.0
68.5
70.1
35.6
ddd; 11.9, 4.8, 3.0
q; ꢂ3.0
68.8
68.6
32.1
ddd; 12.5, 4.1, 2.9
q; ꢂ2.9,
68.5
69.1
34.0
4
a
5
6
7
8
9
b
ddd; 14.4, 4.0, 3.0
ddd; 14.0, 4.5, 3.7
ddd; 14.5, 4.4, 2.9
51.9
dd; 12.7, 4.0
s
36.2
dd; 13.5, 4.5
49.6
dd; 13.6, 4.4
s
56.1
216.1
59.2
124.6
33.6
41.3
21.5
206.5
122.2
168.1
35.2
39.4
21.6
141.6
146.1
126.4
36.9
37.1
20.4
199.5
142.8
145.0
45.8
40.1
24.4
dd, 11.7, 7.2
1
1
a
1
a
1
1
1
a
1
0
1b
2b
32.6
38.6
36.1
37.0
td; 11.5, 5.5
3
4
5b
48.8
85.3
31.9
46.7
161.7
26.1
50.2
175.5
30.0
44.0
153.1
26.6
–
–
–
–
1.97
1.59
1.98
1.69
2.39
0.89
0.97
–
2.53
2.66
2.15
1.74
1.69
1.19
0.68
–
1.25
3.40
1.58
1.28
1.80
1.43
–
2.53
2.53
2.10
1.97
3.13
1.34
0.95
–
1.37
3.71
1.70
1.42
1.74
1.33
–
2.35
2.35
1.86
1.92
2.37
0.90
0.71
–
1.13
3.49
1.63
1.63
1.73
1.45
–
6b
21.6
22.6
20.5
23.1
a
1
1
1
2
2
2
2
7
50.6
18.1
24.5
78.0
21.2
78.5
27.5
55.8
21.3
22.9
77.6
20.9
78.2
27.3
41.9
24.5
23.6
93.1
20.3
77.6
26.8
dd; 8.0, 4.7
s
s
57.9
19.8
25.4
78.4
21.3
77.6
27.4
8
9
0
1
2
3
s
s
s
s
1.20
3.33
1.66
s
s
s
dd; 10.8, 1.7
dd; 10.0, 1.9
dd; 9.2, 2.7
1.29
2
4
1.80
42.5
42.3
42.0
41.5
1
–
.44
2
2
2
5
6
7
71.4
29.1
29.8
71.3
28.8
29.9
71.4
29.0
29.9
71.7
29.0
30.1
1.19
1.20
1.17
1.20
s
s
1.16
1.17
s
s
1.19
1.21
s
s
a
HC@O (d( H): 9.40, d(13C): 196.0).
1
2.4. Compound characterization data
Compound 5
Colorless amorphous solid (1.5 mg, 0.38%); APCI-MS: 477
+
+
+
1
13
Poststerone (1) [15]
White crystalline powder (13.1 mg, 3.28%); m.p. 238–240 °C;
(M+H ), 459 (M+H ꢀH
2
O), 441 (M+H ꢀ2H
2
O); for H and
C
NMR, see Table 1.
+
HRESIMS C21
H
30
O
5
Na, calcd 385.1991, found 385.1996 [M+Na] ;
H NMR in methanol-d : 0.62 (H-18), 0.96 (H-19), 1.44 (H -1),
-15) 1.74 (H -4), 1.80 (H -1), 1.82 (H -12),
-11), 2.00 (H -15), 2.16 (H-21), 2.23 (H
-12), 2.39 (H-5), 3.19 (H-9), 3.33 (H-17), 3.86 (H-2),
1
4
b
14a
-hydroperoxy-20-hydroxyecdysone (6) [11]
Colorless amorphous solid (5.2 mg, 1.3%); HRESIMS C27
Na, calcd 519.2934, found 519.2931 [M+Na] ; H and C NMR data
are identical with those published earlier [11].
1
1
1
3
.67 (H
.88 (H
6), 2.33 (H
.97 (H-3), 5.82 (H-7); C NMR: 17.6 (C-18), 21.7 (C-11), 22.3
b
-11), 1.70 (H
a
2
a
b
44 8-
H O
a
-16), 1.89 (H
a
b
b
-
+
1
13
a
13
(
4
6
C-16), 24.5(C-19), 31.1 (C-12), 31.6(C-21), 32.2 (C-15), 32.9 (C-
), 35.2 (C-9), 37.4 (C-1), 39.3 (C-10), 48.9 (C-13), 51.9 (C-5),
0.2(C-17), 68.5 (C-3), 68.7 (C-2), 85.1 (C-14), 122.6 (C-7), 166.6
(
(
20R,22R)-2b,3b,7
7)
Colorless amorphous solid (3.7 mg, 0.93%); HRESIMS C27H O
44 7-
a,20,22,25-hexahydroxy-5b-cholest-8,14-en-6-one
(
C-8), 206.3 (C-6), 212.6 (C-20).
1
13
Na, calcd 503.2985, found 503.2970; for H and C NMR, see
Table 1.
(
20R,22R)-2b,3b,20,22,25-hexahydroxy-5b-cholest-6, 8(14)-dien-6-
carbaldehyde (2)
3
. Results and discussion
44 6-
Colorless amorphous solid (1.5 mg, 0.38%); HRESIMS: C28H O
Na, calcd 499.3036, found 499.3040 [M+Na] ; for H and C NMR,
see Table 1.
+
1
13
As previously reported [13], laser irradiation of 20E resulted in a
complex mixture of photoproducts within 15 min at 2 mg/mL and
6
1
.5 mJ impulse energy, whereas an increase of the concentration to
0 mg/mL led to the need for significantly stronger (15 mJ) and
1
4
a
,15
a-epoxy-14, 15-dihydrostachysterone B (3) [16]
Colorless amorphous solid (0.62 mg, 0.16%); HRESIMS: C27
H
42-
longer (30 min) irradiation, further increase in concentration dras-
tically decreased the efficiency of photo-transformation. In order to
obtain a reasonable amount of starting material applicable for iso-
lation and structure elucidation of the products, the scale-up was
performed by repeated irradiations. In this manner, a total amount
of 400 mg irradiated material could be obtained. However, the con-
tinuous effort in optimizing the scale-up process, despite its mon-
itoring by TLC, resulted in a combined irradiated material that still
+
1
13
7
O Na, calcd 501.2828, found 501.2831 [M+Na] ; H and C NMR
data are identical with those we published earlier [16].
Stachysterone B (4)
42 6
Colorless amorphous solid (2 mg, 0.5%); HRESIMS C27H O Na,
+
1
13
calcd 485.2879, found: 485.2886 [M+Na] ; H and C NMR data
are identical with those we published earlier [16].

W.-C. Lai et al. / Steroids 89 (2014) 56–62
59
contained a significant amount of unchanged 20E. This remaining
quantity of unchanged 20E was determined by HPLC-DAD
3
H -18/C-14 response (1.19/161.7) proved the splitting of the par-
ent OH group and the presence of a quaternary carbon atom in this
position. Lastly, the HMBC responses of the formyl H atom (9.40) to
C-5, to C-6 (141.6) and C-7 (146.1) proved that the formyl group is
attached to C-6. The H-7 olefinic hydrogen atom (7.19 ppm) corre-
lated with C-5, C-8, C-9 and C-14 carbon atoms in the HMBC spec-
(k = 245 nm) to be 14%.
The irradiated mixture was subjected to purification by rota-
tional planar chromatography (RPC) followed by RP-HPLC (com-
pounds 1–6), or by centrifugal partition chromatography (CPC)
and a final purification step by RP-HPLC (compound 7). Beyond
those isolated compounds, a minor product, 14-epi-20-hydroxyec-
dysone (8) was identified by HPLC-DAD. By means of the calibra-
tion data for 20E, the amount of this compound in the irradiated
mixture was ca. 0.3%.
6
,7;8,14
trum, verifying the presence of a conjugated
D
-diene-moiety.
The H -9/H -2 and H-19/H -5 correlations in the NOESY spectrum
a
a
b
of 2 established the cis type junction of the A/B rings. We have pre-
viously reported an analog structure without the formyl moiety,
namely the 2b,3b,20R,22R,25-pentahydroxy-5b-cholest-6, 8 (14)-
dien isolated from the roots of S. wolffii [16].
The structures and NMR signals of the isolated products were
assigned by comprehensive one- and two-dimensional NMR meth-
ods using widely accepted strategies [17,18]. Structures of 20E and
compounds 1–8 are shown in Fig. 1.
The HRMS measurement of 3 indicated the molecular formula
of C27
42 7
H O , i.e. the splitting of two hydrogens from the parent
1
13
20E. Moreover, the H and C NMR chemical shifts perfectly
The H and 13C NMR spectra of 1 clearly referred to the splitting
1
agreed with those previously obtained for 14a,15a-epoxy-14, 15-
of the C22–C27 side-chain moiety, and the hydrogen atoms of the
dihydrostachysterone B [16].
The H and 13C NMR spectra of compound 4 confirmed that this
1
3
new CH C@O group (2.16s) exhibited a strong HMBC cross-peak
with the C-17 carbon atom (60.2 ppm) proving the connection of
compound is identical with stachysterone B [16].
+
this group to C-17, i.e. compound 1 is identical to poststerone
The APCI-MS spectrum of compound 5 showed the [M+H] peak
at m/z 477, and, even though HRMS data could not be obtained due
to decomposition of compound 5, the yielded APCI-MS spectrum of
13
[
19]. In order to achieve the complete 1H and C NMR signal
4
assignment in methanol-d , HSQC, HMBC, NOESY and COSY spectra
were utilized.
this product corresponds to the molecular formula of C27
40 7
H O , i.e.
44 6
In the case of compound 2, the molecular formula of C28H O
a molecule containing four less hydrogen atom than the parental
1
3
was established by means of HRMS, i.e. the molecule consists of
one more carbon and one less oxygen atom than the parent 20E.
The most characteristic changes in the NMR spectra, as compared
to that of 20E, indicated the disappearance of the O@CACH@C
group in the B ring and the presence of a new HC@O group
20E. In agreement with this, twenty-seven C signals were dis-
cernible in the DEPTQ spectrum indicating the presence of five
methyl, eight methylene, six methyne groups furthermore four
3
2
quaternary sp and two quaternary sp carbon atoms, one conju-
gated C@O (d 199.5) and one new OAC@O (d 175.5) moieties. Inte-
1
(
9.40s/196.0 ppm). To explore the position of the new formyl
group, the HMBC and HSQC correlations were utilized. The HMBC
cross-peaks of the H -19 atoms of the angular methyl group
0.68 ppm) indicated the HC-5 and HC-9 methine carbons, whereas
gration of the signals in the H NMR spectrum obtained in
4
methanol-d summed up to 37 H atoms. Considering the identical
3
d C-2, C-3 and C-25 chemical shifts of compound 5 and 20E, respec-
tively, all of these should have HO– substituents, and thus com-
pound 5 consists of 40 hydrogen atoms. The number of double
bond equivalents in compound 5 increased to eight; therefore this
(
1
the HSQC correlations gave the corresponding H chemical shifts,
6.2/2.65 ppm and 36.9/2.55 ppm, respectively. In addition, the
3
Fig. 1. Structures of 20E and the compounds isolated (1–7) or detected (8) after laser irradiation. 5a represents an alternative structure for compound 5 which was ruled out
by means of 2D and a set of selective 1D NOESY experiments.

6
0
W.-C. Lai et al. / Steroids 89 (2014) 56–62
compound contains one more ring than the parent 20E. To facili-
tate the understanding of the different rearrangements of 20E dur-
ing its photo-irradiation resulting structure 5, we applied the
specific atomic numbering of the parental compound to compound
one-dimensional NOESY experiments were performed, irradiating
H-9, H -18, H -19 and H -21, respectively in the latter cases. The
3
3
3
NOESY results were in accordance with structure 5 and ruled out
5a. The schematic stereo-structure of 5 with the atomic numbering
is depicted in Fig. 2A; the arrows indicate the detected characteris-
tic spatial proximities, whereas the structure of Fig. 2B shows the
refined stereochemistry obtained by PM3 semi-empirical calcula-
tion [20]. For a better visualization of the steric arrangement of
compound 5, the C(23)–C(27) side chain is replaced with R.
The HRMS measurement of 6 yielded molecular formula
1
13
5
(see Fig. 1). For the H and C signal assignment, we first iden-
1
tified the five singlets in the H NMR spectrum. The identification
of the geminal H C-26 (1.16/29.0) and H C-27 (1.17/29.9) groups
3
3
is straightforward owing to their mutual HMBC correlation. The
HMBC responses of these methyl groups assigned the quaternary
C-25 (71.4) and also the H
the 3.71/42.0, 3.71/26.8 and 3.71/20.3 cross-peaks revealed the
assignment of HC-22 (3.71/77.6), H C-23 (1.71; 1.42/26.8) and
the third angular methyl H C-21 (1.37/20.3). By utilizing the HMBC
correlations of H -21, the quaternary C-20 (93.1) and HC-17 (3.13/
1.9) were assigned, and the H-17/H C-18 cross-peak (3.13/24.5)
revealed the assignment of the fourth angular methyl group. The
HMBC correlations of H -19 0.95/49.6 and 0.95/45.8, respectively,
2
C-24 methylene (42.0). In addition,
27 44 8
C H O . Therefore, one oxygen atom is incorporated into the
1
13
2
parental 20E. The H and C NMR chemical shifts corresponded
to those previously published by Harmatha et al. for 14 -hydro-
3
a
3
peroxy-20-hydroxyecdysone [11], a known photoproduct of 20E.
The presence of the peroxy group in position 14 of compound 6
4
3
is straightforward considering the characteristic (d
paramagnetic shift of C-14 to d 96.6 ppm.
D
ꢂ 12 ppm)
3
assigned the HC-5 and HC-9 methines. For their unambiguous dif-
ferentiation selective one-dimensional TOCSY experiments were
utilized. Irradiation of the well-separated H-3 signal (3.98q,
J ꢂ 2.5 Hz) revealed the 7-membered spin system of the A-ring,
whereas excitation of the signal at 3.27 gave the 5-membered spin
Despite the unchanged molecular formula, C27
pound 7 as compared to that of 20E, significant differences could
H O of com-
44 7
1
3
1
be observed in the C and H NMR spectra. A comparison of the
chemical shifts with those of 20E strongly suggested the shift of
7
,8
8,14
the
D
double bond to the
D
position. The observed 10 ppm
system of H-9, H
ation of H-17 (3.13dd, J: 8.0; 4.7 Hz) assigned the 5-membered spin
system of H-17, H -16 and H -15 hydrogen atoms. To achieve an
unequivocal assignment of C-14, the HMBC correlations of the
-15 and the H -16 hydrogens were utilized. Their responses to
2
-11 and H
2
-12. A similar experiment, the irradi-
paramagnetic shift of d C(6)@O to 216.1, together with the absence
of the characteristic signals of @CH in position 7, revealed that this
3
1
2
2
carbon atom evolved to sp CH (d H-7 3.53s; d C-7 59.2). These H
1
3
and C chemical shifts also indicated the presence of one –OH
group attached to C-7. Considering the strong NOE contacts of
H
2
2
the signal at d 175.5 ppm revealed that the original OH substituted
quaternary sp C atom turned into a lactone O@CAO carbon atom.
3
the protons H -19–H-7 and also to H-5 (2.42dd) hydrogen atoms
detected in the selective one-dimensional NOESY spectrum, the b
position of these atoms is straightforward, i.e. the 7-OH group is
3
Despite the absence of the characteristic H-7 olefin signal, the d
1
99.5 ppm chemical shift of C-6 strongly indicated the presence of
in a position and the cis A/B ring-junction remained unchanged.
7
,8
2
a conjugated
D
-6-one moiety in the B ring. The HMBC correla-
Both of the two quaternary sp carbon signals at d 124.6 and d
153.1 exhibited strong responses to the H-9 and H-7 signals in
the HMBC spectrum, respectively, and their differentiation pro-
tions of the H-5 hydrogen to the O@C-6 and to the quaternary
C-7 atoms (d 142.8 ppm), in addition with the H-9/C-7 (3.27/
42.8) and H-9/C-8 (3.27/145.0) cross-peaks, completed the signal
assignment of the B ring. The C-7 signal exhibits d 20.6 ppm para-
magnetic, whereas C-8 d 23.1 ppm diamagnetic shift in compar-
@
1
vided the H
3
-18/C-14 (0.90/153.1) cross-peak. The detailed NMR
1
1
D
analysis combining two-dimensional H, H-COSY, -NOESY,
-
D
ROESY, edited gs-HSQC, gs-HMBC and one-dimensional sel-ROESY
and DEPTQ measurements allowed the complete structural assign-
ment of all hydrogen and carbon signals (see Table 1), and con-
firmed the suggested structure for compound 7. It should also be
noted, that the 7-deoxy analog of 7 has been reported by Canonica
et al. [9], and their NMR chemical shifts correlate well with our
data.
Unsurprisingly, our results show fundamental differences as
compared to the previous studies on the photo-transformation of
20E due to highly different experimental conditions. First of all,
the powerful laser impacted the solution at much higher energy
than previously used [9–11], and the wavelength was also differ-
ent: in our case an optically pure, 266 nm UV–light was used, in
contrast with the Pyrex-filtered (>300 nm) UV–light provided by
Hg lamps. Moreover, in our case, methanol was used as a solvent,
which is a better solvent for 20E than water and allows to reach
higher concentrations of starting material (and products). Last
but not least, our experimental set-up made it a challenge to
remove oxygen from the system and to provide an inert atmo-
sphere, therefore our conditions must have been more oxidative
than those of the previous studies.
Compound 1 was formed by an oxidative side chain cleavage
between C-20 and C-22, which, due to the large distance from
the B-ring chromophore, should be the result of rather inter- than
intra-molecular processes. At the employed wavelength and
energy, direct photolysis of methanol (which could result in a large
amount of strong oxidizing species) is unlikely [21]. Oxygen or
other radicals (i.e. OH radical originated from C-14, see below) pro-
vided by neighboring 20E molecules can however give a reasonable
explanation for the formation of 1. In the case of all other
ison with the corresponding values measured for the parental 20E,
revealing that the substituent on the @C-7 atom should be oxygen.
3
Surprisingly, a strong correlation between the H -18 methyl
hydrogens and the @C-8 carbon atom was also detected, providing
evidence of a contraction in the C-ring. In the course of the photo-
chemical transformation of 20E, a similar rearrangement was
reported by Canonica et al. [9,10]. They explained the contraction
by assuming that the initially produced O-6, C-8 diradical would
undergo a homolytic cleavage of the 13–14 bond, and that the
recombination of C-13 and C-8 radicals led to the five-member
C-ring. In our case (due to the high distances between possible
members of the new lactone ring), the homolytic cleavage of the
8
–14 bond also had to be taken into account. Following such a
cleavage, C-14 can reasonably form two possible lactone rings:
either with the 20-OH resulting a six-member lactone, or with
2
tures are feasible; in the case of compound 5, an H-22/C-7 correla-
tion; and in the case of 5a, an H-22/C-14 HMBC correlation should
be expected. However, probably due to spatial reasons, neither of
these correlations could be detected even in the case where the
HMBC measurement was optimized to 5 Hz long-range couplings
instead of 10 Hz. In the course of building-up the fourth and fifth
rings, the configuration around the C-20 and C-22 chirality centers
2-OH forming a seven-member lactone ring. A priori both struc-
should remain unchanged. Therefore, in structure 5, both H
and H-22 necessarily exist in the position, whereas in case of
a they are in a b position. To choose between the two alternative
structures and to elucidate the configuration of the stereogenic
centers, and to determine the or b position of the methylene
hydrogen atoms, two-dimensional and set of selective
3
C-20
a
5
a
a
a

W.-C. Lai et al. / Steroids 89 (2014) 56–62
61
Fig. 2. Stereo-structure and characteristic NOE steric proximities (see arrows) of compound 5 (A) and its refined structure by means of PM3 semi-empirical calculations (B).
The C(23)–C(27) side chain is replaced with R in both structures.
compounds, rearrangements of the O-6, 8-C bi-radical can be rec-
ognized. Compound 2 is particularly interesting in this regard
due to the formation of a carbon–carbon bond with methanol, pos-
sibly following the homolytic cleavage of the 14-OH. The leaving
OH radical can directly couple to the same 20E molecule’s remain-
ing radical delocalized between the 6-carbonyl and C-14 [9], lead-
ing to the formation of 7 or 8, oxidize another 20E molecule (to
form compound 1, for example), or react with the solvent metha-
nol. Oxidation of methanol by OH radicals has previously been
studied under atmospheric conditions [22], and the primary prod-
The 14-hydroperoxi derivative 6 can be formed by the previ-
ously suggested 14 -oxyl radical of 20E [9] and an OH radical; this
a
mechanism is also supported by the fact that low yields of this
compound were also found to appear in both previous studies,
despite the removal of the residual oxygen from their systems
[9,11]. Suggested mechanism for the formation of the three newly
reported compounds, 2, 5 and 7, is presented in Fig. 3.
Based on our observations on the chromatographic fingerprints,
there seems to be low chance for the presence of further major
products that would have been neglected during the systematic
isolation procedure. On the other hand, the low isolated yields
can only partially be explained with chromatographic overlapping:
a very large number of minor compounds were observed, certainly
accounting for a significant fraction of the total amount of the irra-
diated mixture.
2
uct CH OH radical seems to be the most likely species to couple to
C-6 forming compound 2 with the elimination of a water molecule.
Compound 5 represents a novel photo-rearrangement of ecdys-
teroids. Although closely related 13(14 ? 8)-abeo-steroids were
previously obtained upon UV irradiation of 20E [9], the conserved
7
,8
D
double bond of our product suggests a different chemical route
than that found before [9,10]. As a most likely intermediate, forma-
tion of an aldehyde is suggested at C-14, following the homolytic
cleavage of the 8–14 bond. The oxidative environment can serve
as a reasonable explanation for the subsequent lactone ring forma-
tion, while the tetrahydro-oxepine ring closure would possibly
require a secondary photoreaction.
4. Summary
The applied 266 nm laser irradiation of 20-hydroxyecdysone
provided a rather complex mixture of various photo-derived prod-
ucts within a short period of time, clearly showing how powerful
Fig. 3. Suggested mechanism for the formation of compounds 2, 5 and 7.

6
2
W.-C. Lai et al. / Steroids 89 (2014) 56–62
such a laser can be in the transformation of ecdysteroids. Unique,
new steroid derivatives (compounds 2 and 7) were obtained. In
the case of compound 2, a novel, unexpected carbon–carbon cou-
pling reaction took place between the ecdysteroid radical and
methanol. Performing this reaction with conventional chemical
methods is very difficult if not impossible. A very interesting,
new, supposedly secondary photo-product was formed by a ring
rearrangement followed by the formation of a lactone and a tetra-
hydro-oxepine ring (compound 5). Other isolated or detected com-
pounds (1, 3,4, 6 and 8) confirmed that the same products found
previously with Pyrex-filtered Hg-lamps can also be obtained in
much shorter time by using a laser. Based on the bioactivity of
the parental 20E, all obtained derivatives are of interest for testing
by various bioassays.
On the other hand, the complexity of the mixture made the iso-
lation a challenging task with low yields of products, and it appears
that a different experimental set-up would be needed for a more
convenient and more selective photo-transformation of 20E and
related compounds. The presence of oxygen (which could not be
excluded with the actual set-up) might have played a significant
role in the formation of some products. Dimensions of the cuvette
might have decreased some benefits coming from the homogenous
UV light: for example, despite the continuous stirring, 20E mole-
cules at the irradiated side had higher chances for undergoing
multi-photon processes than those of the opposite side. In addition
to these, a longer irradiation time could probably lead to several
secondary, tertiary, etc. photoproducts.
be found, in the online version, at http://dx.doi.org/10.1016/
j.steroids.2014.07.016.
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Appendix A. Supplementary data
[
Key one- and two-dimensional NMR spectra and HPLC-UV chro-
matograms for compounds 2, 5 and 7 are available as supporting
information. Supplementary data associated with this article can