Stereochemical assignment of c-24 and c-25 of amarasterone a, a putative biosynthetic intermediate of cyasterone

Article abstract of DOI:10.1021/jo5005108

A C₂₉ phytoecdysteroid named amarasterone A (1) has been isolated from Cyathula capitata (Amaranthaceae), Leuzea carthamoides (Asteraceae), and Microsorum scolopendria (Polypodiaceae). We recently isolated amarasterone A from C. officinalis. Am

Full text of DOI:10.1021/jo5005108

Article
pubs.acs.org/joc
Stereochemical Assignment of C‑24 and C‑25 of Amarasterone A,
a Putative Biosynthetic Intermediate of Cyasterone
Yui Hirayama,† Keiko Okuzumi,† Hironori Masubuti,† Hidehiro Uekusa,† Jean-Pierre Girault,‡
and Yoshinori Fujimoto*,†
†Department of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan
‡Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite
́
Paris Descartes, UMR 8601. CNRS, 45, Rue
des Saints-Per
̀
es, 75270 Paris Cedex 06, France
S
* Supporting Information
ABSTRACT: A C29 phytoecdysteroid named amarasterone A (1) has been isolated from Cyathula capitata (Amaranthaceae),
Leuzea carthamoides (Asteraceae), and Microsorum scolopendria (Polypodiaceae). We recently isolated amarasterone A from C.
officinalis. Amarasterone A has been postulated as a biosynthetic intermediate of cyasterone in Cyathula sp. The stereochemistry
at the C-24 and C-25 positions of these amarasterone A samples was investigated by comparing the NMR spectroscopic data
with those of stereodefined model compounds, (24R,25S)-, (24R,25R)-, (24S,25S)-, and (24S,25R)-isomers of (20R,22R)-3β-
methoxystigmast-5-ene-20,22,26-triol (2a−d), which were synthesized in the present study. Amarasterone A isolated from
Cyathula officinalis was determined to be the (24R,25S)-isomer (1a), while amarasterone A from L. carthamoides was found to be
the (24R,25R)-isomer (1b). Amarasterone A from M. scolopendria was found to be a mixture of 1a and 1b. The biosynthesis of
cyasterone in Cyathula sp. is discussed on the basis of the identical C-24 configuration of sitosterol and amarasterone A.
established to be a biosynthetic precursor of cyasterone and 28-
epi-cyasterone in a feeding study using hairy roots of Ajuga
reptans var. atropurpurea.20 To our knowledge, this is the only
report clarifying the biosynthetic relationship between a C29
sterol and a C29 phytoecdysteroid. Notably, the C-24
configuration of clerosterol is the same as that of cyasterone;
thus the configuration at C-24 of clerosterol is not inverted
during the biosynthesis.
Our analysis of the sterol fraction of C. officinalis indicated
that the major sterols were sitosterol (59% of total sterol) and
stigmasterol ((22E,24S)-stigmasta-5,22-dien-3β-ol) (31%);
22,23-dihydrospinasterol ((24R)-stigmast-7-en-3β-ol) (4%)
and 24-methylcholesterol (5%) were also present.20,21 This
raised a question as to the identity of the sterol precursor of
cyasterone in Cyathula species. Radioactively labeled sitosterol
and fucosterol ((E)-stigmasta-5,24(28)-dien-3β-ol) were not
incorporated into cyasterone in C. capitata.22,23 We prepared
several lines of hairy roots of C. officinalis to find an effective
experimental system, but none of them were capable of
producing ecdysteroids (unpublished results).
INTRODUCTION
■
Cyasterone is a well-known C29 phytoecdysteroid first isolated
from Cyathula capitata (Amaranthaceae) by Japanese research-
ers in 1968.1 They further reported the isolation of amara-
sterone A (1), amarasterone B,2 capitasterone,3 and pre-
cyasterone4 from the same species and proposed potential
biosynthetic correlations (Figure 1). Cyasterone, 28-epi-
cyasterone, and 25,28-di-epi-cyasterone were isolated from C.
officinalis, and the structures of the former two ecdysteroids
were unambiguously established by X-ray crystallography.5,6
Interestingly, the occurrence of cyasterone has been reported in
various Ajuga species such as A. reptans of the Labiatae
family.7−14
In general, it can be assumed that a C29 phytoecdysteroid is
biosynthesized from a C29 plant sterol. This implies that the C-
24 configuration of the sterol precursor is the same as that of
the C29 phytoecdysteroid. For example, the C-24 configuration
of makisterone C (lemnisterone, podecdysone A, 24-ethyl-20-
hydroxyecdysone) is suggested to be R simply by analogy to the
C-24 stereochemistry of a typical C29 plant sterol, sitosterol,
although there has been no research reported concerning the
stereochemistry.15−17
The rather rare sterol, clerosterol ((24S)-stigmasta-5,25-dien-
Received: March 4, 2014
3β-ol), is a major sterol in Ajuga species,18,19 and this sterol was
© XXXX American Chemical Society
A
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Figure 1. Structures of ecdysteroids isolated from Cyathula species and their tentative biosynthetic relationships.
To elucidate the structure of a sterol that serves as the
precursor of cyasterone in Cyathula sp., we took an indirect
approach. As described above, amarasterone A is presumed to
be a biosynthetic precursor of cyasterone. It is therefore
reasonable to assume that amarasterone A from Cyathula
species should retain the C-24 configuration of the precursor
sterol, as the side chain of amarasterone A is functionalized only
at the C-20, C-22, and C-26 positions, most likely by the action
of P450 enzymes.24,25 Thus, it is critical to determine the C-24
configuration of amarasterone A.
Our recent isolation of amarasterone A from C. officinalis26
prompted us to pursue this stereochemical study. Furthermore,
amarasterone A was isolated as a mixture of two diastereomers
(designated as epimer 1 and epimer 2) from the fern
Microsorum scolopendria (Polypodiaceae).27 Unfortunately, the
NMR data for the epimer 1 and the epimer 2 were recorded in
D2O, which hindered comparison with our sample of
amarasterone A. These samples seemed to be isomeric at the
C-24 and/or C-25 positions from the available NMR data.27
Amarasterone A was also previously isolated from Leuzea
carthamoides (Asteraceae).28 The reported NMR data (in
CD3OD) were different from those of our material. Thus, it is
also worthwhile to determine the stereochemistry at C-24 and
C-25 of the amarasterone A samples to determine which
stereoisomers occur in plants.
Figure 2. Structures of synthesized stereodefined model compounds
2a−d.
with an organo metal species derived from a C7 fragment
halide. It has been reported that such a coupling reaction yields
the desired (20R,22R)-glycol product.29 Racemic 2-ethyl-3-
methylbut-3-enyl bromide in which the C-27 hydroxyl group
was masked as an olefin was used as the C7 unit. It was
anticipated that a mixture of the C-24 epimeric coupling
products would be separable. Hydroboration−oxidation of the
25-olefins would complete the synthesis of 2a−d (Figure 3).
Aldehyde 3 was prepared from pregnenolone in three
steps:30 protection of the C-3 hydroxyl group as the methyl
ether, addition of 2-lithio-1,3-dithiane, and hydrolysis of the
resulting adduct with NCS.31 ( )-2-Ethyl-3-methylbut-3-enyl
bromide (4) was synthesized from ethyl 3-methylcrotonate in
In the present study, we investigated the configuration of C-
24 and C-25 of amarasterone A samples and established that
only the (24R,25S)- and (24R,25R)-isomers have been
observed thus far. In addition, the sterol precursor for
cyasterone and the biosynthesis of cyasterone in Cyathula
species are discussed.
RESULTS AND DISCUSSION
■
The amount of amarasterone A available from C. officinalis (and
also from other plant sources) was very small; thus determining
the stereochemistry from a natural sample would be difficult.
We therefore pursued a strategy to synthesize stereodefined
model compounds and determine the configuration of
amarasterone A by comparing the NMR data. (20R,22S)-3β-
Methoxystigmast-5-ene-20,22,26-triols (2a−d) were selected as
model compounds (Figure 2).
Four diastereomers 2a−d could most likely be conveniently
synthesized by coupling the known 20-hydroxy-22-aldehyde 3
Figure 3. Synthesis of model compounds 2a−d.
B
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four steps. Reaction of the enolate generated by the action of
LDA with EtI gave ethyl 2-ethyl-3-methylbut-3-enoate.
Reduction of the ethylated ester with LiAlH4 afforded 2-
ethyl-3-methylbut-3-en-1-ol. The alcohol was converted to 2-
ethyl-3-methylbut-3-enyl bromide (4) via the tosylate.
Attempted coupling of aldehyde 3 with the Grignard reagent
prepared from the bromide did not afford the desired product
but instead yielded a product resulting from methyl migration
(22-hydroxy-3β-methoxy-24-norchol-5-en-20-one).29 The orga-
no lithium species prepared from the bromide with 2 equiv of t-
BuLi32 in pentane−ether at −78 °C gave a product resulting
from attack of the t-Bu anion on the aldehyde. When the
amount of t-BuLi was reduced to 1 equiv, the resulting C7
lithium species smoothly reacted with the aldehyde to give the
desired coupling product as a mixture of the C-24 epimers,
which were separated by MPLC using a silica gel column to
yield the less polar epimer 5 (32%) and the more polar epimer
6 (48%). Crystalline compound 6 was determined to have the
24S configuration by X-ray crystallography, as shown in the
ORTEP drawing (Figure 4). Compound 5 was thus the 24R-
epimer.
chemical shifts of the C-28 and C-23 signals (recorded in
CD3OD) of 2a′−d′ allowed 2a′/2d′ to be differentiated from
2b′/2c′, that is, the C-24/C-25 relative configuration. The C-28
signals of (24R,25R)/(24S,25S)-isomers (2b′/2c′) (δ 24.9 and
25.2, respectively) resonated at a lower field compared with
those of their (24R,25S)/(24S,25R)-counterparts (2a′/2d′) (δ
23.6 and 23.7, respectively), while the C-23 signals of 2b′/2c′
resonated at a higher field (δ 27.0 and 27.3, respectively)
compared with those of 2a′/2d′ (δ 28.3 and 28.4, respectively).
Thus, compounds 2a and 2b could be assigned as the
(24R,25S)- and (24R,25R)-isomers, respectively, and com-
pounds 2c and 2d were determined to be the (24S,25S)- and
(24S,25R)-isomers, respectively. Comparison of the other side
chain signals (C-24, C-25, C-26, C-27, and C-29) supported the
above stereochemical assignments (Table 1). The 25S
configuration of 2a was further verified by X-ray analysis of
2a (Figure 6). The configurations at C-24 and C-25 of 2a, 2b,
2c, and 2d were thus established as described above.
With the configuration of the model compounds 2a−d
having been established, the NMR data of 2a−d were
compared with those of natural amarasterone A samples.
1
Comparison of the H NMR data of amarasterone from C.
officinalis with those of 2a−d indicated that the natural
amarasterone is neither the (24R,25R)- nor the (24S,25R)-
isomer, as the signal of H3-27 (δ 0.93) was significantly
different from those of 2b and 2d (δ 0.77 and 0.80,
respectively). However, the unambiguous assignment of the
amarasterone sample to either the (24R,25S)- or the (24S,25S)-
isomer was problematic, although the 1H NMR values
compared more favorably with those of 2a than 2c. Similarly,
the amarasterone sample form L. carthamoides was determined
to be either the (24R,25R)- or the (24S,25R)-isomer.
However, comparison of the 13C NMR data allowed us to
assign the stereochemistry of amarasterone samples. The 13C
NMR values for the side chain of amarasterone A from C.
officinalis were very similar to those of 2a and significantly
different from the other three stereoisomers 2b−d (Table 1). A
similar comparison of the 13C NMR data of amarasterone A
from Leuzea carthamoides showed that they matched with those
Figure 4. ORTEP drawing of compound 6.
Hydroboration of compound 5 yielded a mixture of (24R)-
C-25 epimeric alcohols, which were separated by p-TLC into
the less polar 2a (36% from 5, Rf 0.35 with hexane/EtOAc 1:2)
and the more polar 2b (20%, Rf 0.33) as crystalline products.
Similar hydroboration of 6 yielded a mixture of (24S)-C-25
epimeric alcohols, which were separated by silica gel column
chromatography to give the less polar alcohol 2c (38%, Rf 0.53
with hexane/EtOAc 1:2) and the more polar alcohol 2d (21%,
Rf 0.28). Compounds 2a−d were sparingly soluble in D2O but
well soluble in CD3OD.
The configurations at C-25 of diastereomers 2a−d were
deduced from the comparison of their 13C NMR data with
those of the stereodefined 6β-methoxy-3,5-cyclo-5α-stigmast-5-
en-26-ols 2a′−d′ (Figure 5).33 It has been reported that the
1
of the (24R,25R)-isomer 2b. The H (Table 2) and 13C NMR
values for amarasterone A/epimer 1 and amarasterone A/
epimer 2 from M. scolopendria, as recorded in CD3OD solvent
in the present study, agreed with those of amarasterone A from
C. officinalis and those of amarasterone A from L. carthamoides,
respectively, thus establishing that the epimer 1 and epimer 2
correspond to the (24R,25S)- and the (24R,25R)-isomers.
CONCLUSIONS
■
The present study established that amarasterone A obtained
from C. officinalis (and presumably the amarasterone A
originally isolated from C. capitata) has a 24R,25S configuration
(1a). In contrast, amarasterone A from L. carthamoides has a
24R,25R configuration (1b). Furthermore, M. scolopendria was
found to contain both the (24R,25S)- and (24R,25R)-isomers
(1a and 1b). Until now, neither the (24S,25S)- nor the
(24S,25R)-isomer has been reported. This may reflect the
abundance of the major sterol sitosterol rather than its C-24
epimer in the plant kingdom, including the Amaranthaceae,
Asteraceae, and Polypodiaceae families. The occurrence of 1a
and 1b in M. scolopendria is reminiscent of the report of the
isolation of inokosterone from Achylanthes fauriei (Amarantha-
ceae), which is a C-25 epimeric mixture.34 It is conceivable that
the specificity of the C-26/C-27 hydroxylation depends on the
Figure 5. Structures of stereodefined sterols 2a′−d′.
C
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Table 1. 13C NMR Data (125 MHz) for 2a−d, 2a′−d′, and Amarasterone A in CD3OD
a
a
a
a
b
c
no.
2a
2a′
2b
2b′
2c
2c′
2d
2d′
1a
1b
1
38.3
29.0
82.0
39.7
141.8
122.8
33.0
32.7
51.7
38.0
22.1
41.6
44.3
58.2
23.0
25.1
56.2
14.0
19.8
78.1
20.7
75.8
34.1
39.8
39.1
66.1
14.7
23.8
12.1
55.8
38.3
29.0
82.0
39.7
141.8
122.8
33.0
32.7
51.7
38.0
22.1
41.6
44.3
58.2
23.0
25.1
56.2
14.0
19.8
78.1
20.7
75.6
32.1
37.9
37.6
66.7
11.5
25.4
12.3
55.8
38.4
29.0
82.0
39.7
141.8
122.8
33.0
32.7
51.7
38.0
22.1
41.6
44.4
58.2
23.2
25.1
56.2
14.0
19.8
78.2
20.6
76.1
33.4
41.1
37.9
66.1
14.3
25.4
12.8
55.8
38.3
29.0
82.0
39.7
141.8
122.8
33.0
32.7
51.7
38.0
22.1
41.7
44.4
58.3
23.2
25.1
56.2
14.0
19.8
78.0
20.7
74.6
33.7
38.4
37.6
67.2
11.5
23.1
12.8
55.8
37.4
68.7
68.5
32.9
51.8
206.4
122.1
168.0
35.1
39.3
21.5
32.5
37.4
68.7
68.5
32.9
51.8
206.4
122.1
167.9
35.1
39.3
21.5
32.5
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
MeO
e
e
48.6
48.7
85.2
31.8
21.5
50.3
18.0
24.4
78.0
20.9
75.9
34.1
39.7
39.1
66.1
14.8
23.7
12.1
85.2
31.8
21.5
50.3
18.1
24.4
78.0
20.9
75.7
32.0
37.9
37.6
66.8
11.5
25.4
12.3
28.3
43.1
40.0
66.7
13.6
23.6
12.5
27.0
42.5
38.5
66.9
27.3
43.2
38.7
66.8
13.5
25.2
12.6
28.4
42.8
38.7
67.0
d
d
12.3
24.9
12.7
23.7
d
d
13.0
13.0
a
b
Adopted from ref 33. Pertinent data are included in the table. Amarasterone A from C. officinalis. The data were identical with those of epimer 1
c
d
from M. scolopendria. Amarasterone A from L. leuzea. Adopted from ref 27. The data were identical with those of epimer 2 from M. scolopendria. An
HMBC correlation was observed from H3-27 to C-26 for 2d. The signals for C-27 and C-29 in ref 33 were interchanged. The signal, which was
e
overlapped by solvent, was assigned based on the HMBC correlation from H3-18.
Δ24(25) intermediate, even though it has been isolated from
Ajuga sp. and not from Cyathula sp.35 24-Dehydroprecya-
sterone is likely an intermediate placed between capitasterone
(most likely 24R) and precyasterone (presumably 24S) in
cyasterone (24S) biosynthesis. Thus, capitasterone should have
the same C-24 stereochemistry as amarasterone A1; therefore,
the C-24 configuration of precyasterone should be investigated.
The previously observed negligible conversion of sitosterol
into cyasterone is most likely due to insufficient uptake of the
sterol substrate, as the authors noted.22,23 It has been well-
documented that cholesterol is incorporated into a typical C27
phytoecdysteroid, 20-hydroxyecdysone, in plants.36−39 In
Figure 6. ORTEP drawing of compound 2a.
contrast, the biosynthetic study of C29 and C28 phyto-
plants and M. scolopendria hydroxylates both C-26 and C-27 in
non-regiospecific manner. We recommend the names of
amarasterone A1 for the (24R,25S)-isomer (1a) and amaraster-
one A2 for the (24R,25R)-isomer (1b) (Figure 7).
ecdysteroids has been hampered by the lack of a suitable
plant culture system. It is essential to develop an appropriate
culture system for Cyathula or other plant species to study the
hypothesis described above.
The finding that amarasterone A1 from C. officinalis has a
24R configuration, which is the same as that in sitosterol,
strongly suggests that sitosterol is a biosynthetic precursor of
amarasterone A in Cyathula sp. This implies that stereo-
chemical inversion at C-24 must occur in a reaction step
subsequent to the formation of amarasterone A1. One
possibility is that the Δ24(25) intermediate is involved in the
inversion mechanism. 24-Dehydroprecyasterone (the Δ24(25)
derivative of precyasterone) is possibly a candidate for the
EXPERIMENTAL SECTION
■
General. 1H and 13C NMR spectra were recorded at 500 MHz for
1H and 125 MHz for 13C, using CDCl3 or CD3OD as the solvent. For
some compounds, the spectra were recorded with a 400/100 MHz
spectrometer when it was specified. 1H chemical shifts are reported in
reference to the internal standard TMS (0.00 ppm) or the residual
proton signal of CD3OD (3.30 ppm). 13C chemical shifts are
referenced to CDCl3 (77.0 ppm) or CD3OD (49.0 ppm). HRMS
D
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a
1
Table 2. H NMR (500 MHz) Data for 2a−d and Amarasterone A in CD3OD
b
c
no.
2a
2b
2c
2d
1a
1b
18
19
21
22
26
0.90 (s)
1.01 (s)
1.18 (s)
0.90 (s)
1.01 (s)
1.17 (s)
0.90 (s)
1.01 (s)
1.18 (s)
0.91 (s)
1.01 (s)
1.19 (s)
0.88 (s)
0.96 (s)
1.17 (s)
0.89 (s)
0.97 (s)
1.18 (s)
3.40 (m)
3.41 (brd, 10.0)
3.38 (dd, 11.5, 7.0)
3.48 (dd, 11.0, 7.5)
0.77 (d, 7.0)
3.43 (brd, 10.5)
3.35 (dd, 10.5, 7.0)
3.54 (dd, 10.5, 5.5)
0.90 (d, 7.0)
3.43 (m)
3.43 (brd, 8.4)
3.35 (dd, 10.8, 7.6)
3.56 (dd, 10.8, 5.1)
0.93 (d, 6.9)
3.45 (dd, 10.4, 2.3)
3.40 (dd, 11.0, 6.8)
3.50 (dd, 11.0, 7.3)
0.79 (d, 7.0)
3.34 (dd, 10.5, 7.5)
3.55 (dd, 11.0, 5.0)
0.92 (d, 7.0)
3.40 (dd, 10.5, 7.0)
3.49 (dd, 10.5, 6.5)
0.80 (d, 7.0)
27
29
0.92 (t, 7.0)
0.92 (t, 7.0)
0.90 (t, 7.0)
0.91 (t, 7.0)
0.93 (t, 7.5)
0.94 (t, 6.4)
a
b
The chemical shifts not listed in the table are described in the Experimental Section. Amarasterone A from C. officinalis. The data were identical
c
with those of epimer 1 from M. scolopendria. Amarasterone A from L. leuzea. From ref 27. The data were identical with those of epimer 2 from M.
scolopendria.
(hexane/EtOAc 10:1) to afford pregnenolone methyl ether (2.4 g, 7.3
mmol, 73%): white crystals, mp 117−119 °C (hexane/AcOEt) (lit.40
124−125 °C); 1H NMR (CDCl3, 400 MHz) δ 0.63 (s, H3-18), 1.00 (s,
H3-19), 2.12 (s, H3-21), 2.53 (t, J = 9.0 Hz, H1-17) 3.06 (m, H-3), 3.36
(s, OCH3), 5.36 (m, H-6); 13C NMR (CDCl3, 100 MHz) δ 13.2, 19.3,
21.0, 22.8, 24.4, 28.0, 31.5, 31.8, 31.8, 36.9, 37.2, 38.6, 38.8, 43.9, 50.0,
55.5, 56.9, 63.7, 80.2, 121.2, 140.8, 209.4. Anal. Calcd for C22H34O2: C,
79.95; H, 10.37. Found: C, 79.85; H, 10.33.
n-BuLi (1.6 M n-hexane solution, 5.0 mL, 8.0 mmol) was added
Figure 7. Structures of naturally occurring amarasterone A.
dropwise to a solution of 1.3-dithiane (873 mg, 7.26 mmol) in dry
THF (9.5 mL) at −20 °C under N2, and the mixture was cooled to
−78 °C. The methyl ether (2.40 g, 7.26 mmol) in THF (25 mL) was
added, and the reaction was stirred for 30 min at the same
temperature. The solution was warmed to −50 °C for 1 h. Ether
and saturated NH4Cl (aq) were added, and the organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel (hexane/EtOAc 8:1
containing 10% CHCl3) to give the dithiane adduct (2.63 g, 5.85
(FAB) spectra were obtained on a double-focusing magnetic sector
mass spectrometer. IR spectra were recorded with a Fourier transform
(FT-IR) spectrometer. Optical rotations were measured on a
polarimeter using a 5 cm cell at approximately 25 °C. TLC and p-
TLC were performed on precoated silica gel 60 F254 glass plates (0.25
mm thickness). Silica gel 60 (spherical neutral, 40−100 μm) was used
for column chromatography. MPLC was carried out using a silica gel-
packed glass column.
1
mmol, 79%): white crystals (hexane/AcOEt), mp 185−186 °C; H
NMR (CDCl3, 400 MHz) δ 0.88 (s, 3H), 1.00 (s, 3H), 1.44 (s, 3H),
2.75−3.00 (m, 4H), 3.06 (m, 1H), 3.35 (s, 3H), 4.14 (s, 1H), 5.35
(brs, 1H); 13C NMR (CDCl3, 100 MHz) δ 13.3, 19.3, 20.9, 21.6, 23.7,
24.1, 26.0, 27.9, 30.8, 31.3, 31.5, 31.7, 36.8, 37.1, 38.6, 40.1, 42.9, 50.0,
55.1, 55.5, 56.8, 61.2, 76.7, 80.3, 121.4, 140.9. Anal. Calcd for
C26H42O2S2: C, 69.28; H, 9.39; S, 14.23; Found: C, 69.30; H, 9.68; S,
13.96.
Isolation of Amarasterone A1 (1a) from Cyathula officinalis.
The chips of roots and stems of Cyathula officinalis (300 g dry wt)
were ground using a mill. The residue was extracted with benzene
(900 mL) by heating at 80 °C for 1 h, and the benzene extract was
discarded. The defatted residue was extracted with methanol (900 mL)
twice by heating at 80 °C for 1 h. The combined methanol solutions
were concentrated in vacuo. The residue (45 g) was dissolved in n-
butanol, washed with brine, and concentrated in vacuo. The residue
was chromatographed on silica gel (CHCl3/MeOH 7:1−4:1) to give
an ecdysteroid-enriched fraction (101 mg). HPLC analysis (solvent,
MeOH/H2O 1:1; flow rate, 1.0 mL/min; UV detection, 243 nm) of
the fraction revealed a peak at 14.5 min, which was different from
cyasterone (11.2 min), 25,28-di-epi-cyasterone (12.3 min), and 28-epi-
cyasterone (13.4 min). HPLC separation of the peak afforded 1a (2.5
mg, 0.0084% based on the dried chips): amorphous solid: 13C NMR
A mixture of the dithiane adduct (1.2 g, 2.7 mmol) and N-
chlorosuccinimide (936 mg, 7.0 mmol) in CH2Cl2/H2O (10:1, 40
mL) was stirred for 3 h at room temperature under N2.31 Ether and
aqueous Na2S2O3 were added, and the separated organic layer was
washed with NaHCO3 and brine, then dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica gel
(hexane/EtOAc 8:1) to afford the aldehyde (647 mg, 67%): colorless
needles, mp 128−130 °C (hexane/AcOEt) (lit.41 168−170 °C); H
1
1
NMR (CDCl3, 400 MHz) δ 0.80 (s, 3H), 1.00 (s, 3H), 1.35 (s, 3H),
3.06 (m, 3H), 3.26 (brs, OH), 3.35 (s, 3H), 5.35 (br, 1H), 9.57 (s,
1H); 13C NMR (CDCl3, 100 MHz) δ 13.8, 19.3, 20.9, 22.1, 23.0, 24.1,
27.9, 31.4, 31.7, 36.9, 37.1, 38.6, 40.0, 43.3, 50.1, 55.5, 55.6, 56.5, 79.5,
80.3, 121.2, 140.9, 203.5. Anal. Calcd for C23H36O3: C, 76.62; H,
10.06. Found: C, 76.53; H, 10.35.
data, see Table 1; H NMR (CD3OD) δ 1.43 (t, J = 12.7 Hz, Ha-1),
1.78 (dd, J = 12.7, 4.4 Hz, Hb-1), 3.83 (ddd, J = 12.7, 4.4, 3.6 Hz, H-
2), 3.94 (m, H-3), 1.70−1.75 (m, Ha-4, Hb-4), 2.37 (dd, J = 12.7, 4.8
Hz, H-5), 5.80 (d, J = 2.4 Hz, H-7), 3.14 (m, H-9), 1.70 (m, Ha-11),
1.80 (m, Hb-11), 2.11 (ddd, J = 13.0, 13.0, 4.8 Hz, Ha-12), 1.87 (m,
Hb-12), 1.95 (m, Ha-15), 1.60 (m, Hb-15), 1.99 (m, Ha-16), 1.74 (m,
Hb-16), 2.35 (t, J = 10.0 Hz, H-17), 1.27−1.35 (m, Ha-23, Hb-23),
1.56 (m, H-24), 1.76 (m, H-25), 1.41−1.35 (m, Ha-28, Hb-28); the
2-Ethyl-3-methylbut-3-en-1-ol. n-BuLi (1.60 M n-hexane
solution, 20 mL, 32.0 mmol) was added dropwise to a solution of
diisopropylamine (4.5 mL, 32 mmol) in dry THF (40 mL) at −78 °C
under N2. The solution was stirred and allowed to warm to 0 °C and
then stirred at the same temperature for 1 h. A solution of ethyl 3-
methylcrotonate (3.3 mL, 24 mmol) in dry THF (6.0 mL) was added
dropwise to the LDA solution at −78 °C, and the mixture was stirred
for 30 min. EtI (2.6 mL, 32 mmol) was added dropwise, then the
mixture was allowed to warm to 0 °C for 45 min, and finally stirred for
30 min at room temperature. Ether and saturated aqueous NH4Cl
were added, and the separated organic layer was washed with 2 N HCl,
saturated aqueous NaHCO3, and brine, dried over Na2SO4, and
concentrated in vacuo to give the ethylated ester as a pale yellow oil
1
remainder of the H shifts are listed in Table 2; MS (FAB) m/z 509
[M + H]+.
(3β,20R)-22-Formyl-3-methoxypregn-5-en-20-ol (3). Sodium
hydride (60%, dispersion in paraffin oil) (800 mg, 20 mmol) was
added to a solution of pregnenolone (3.16 g, 10 mmol) in dry THF
(40 mL) at 0 °C under N2, and the mixture was warmed to 50 °C and
stirred for 30 min at the same temperature. MeI (6.4 mL, 15 mmol)
was added to the mixture, and stirring was continued for 2 h at the
same temperature. Ether and saturated NH4Cl (aq) were added, and
the organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica gel
E
dx.doi.org/10.1021/jo5005108 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(3.7 g, 98%): 1H NMR (CDCl3, 400 MHz) δ 0.89 (t, J = 7.5 Hz, 3H),
1.25 (t, J = 7.1 Hz, 3H), 1.60−1.84 (m, 2H), 1.75 (s, 3H), 2.91 (t, J =
7.5 Hz, 1H), 4.14 (dd, J = 7.1 Hz, 2H), 4.88 (m, 2H); 13C NMR
(CDCl3, 100 MHz) δ 12.0, 14.2, 20.2, 23.3, 54.9, 60.4, 113.6, 142.5,
173.8. Anal. Calcd for C9H16O2: C, 69.19; H, 10.32. Found: C, 69.15;
H, 10.32.
LiAlH4 (972 mg, 25.6 mmol) was added in several portions to a
solution of the ester (3.7 g, 23.7 mmol) in dry ether (45 mL) at 0 °C
under N2, and the mixture was then allowed to warm to room
temperature. After being stirred for 3 h at the same temperature, moist
ether and saturated aqueous NH4Cl were added to the reaction
mixture, and the organic layer was washed with aqueous NaHCO3 and
brine, dried over Na2SO4, and concentrated in vacuo to give the
alcohol as a pale yellow oil (2.4 g, 89%): 1H NMR (CDCl3, 400 MHz)
δ 0.87 (t, J = 7.5 Hz, 3H), 1.31−1.45 (m, 2H), 1.67 (s, 3H), 2.18 (m,
1H), 3.51 (m, 2H), 4.83 (brs, 1H), 4.95 (brs, 1H); 13C NMR (CDCl3,
100 MHz) δ 11.7, 18.7, 22.1, 51.8, 63.8, 113.8, 145.0. Anal. Calcd for
C7H14O: C, 73.63; H, 12.36. Found: C, 73.57; H, 12.62.
80.3, 111.6, 121.4, 140.9, 149.3; HRMS (FAB) m/z 459.3814 [M +
H]+ (calcd for C30H51O3, 459.3838).
Compound 6: colorless needles, mp 169 °C (MeOH); Rf 0.38
(hexane/EtOAc 4:1, developed twice); 1H NMR (CDCl3) δ 0.83 (t, J
= 7.5 Hz, 3H), 0.88 (s, 3H), 1.01 (s, 3H), 1.21 (s, 3H), 1.61 (s, 3H),
3.06 (m, 1H), 3.35 (s, 3H), 3.38 (brd, J = 10.5 Hz, 1H), 4.78 (brd, J =
2.0 Hz, 1H), 4.84 (brd, J = 2.5 Hz, 1H), 5.36 (brd, J = 5.0 Hz, 1H);
13C NMR (CDCl3, 125 MHz) δ 12.0, 13.5, 17.7, 19.4, 20.4, 21.0, 21.8,
23.9, 27.0, 28.0, 31.3, 31.8, 34.9, 36.9, 37.2, 38.7, 40.2, 43.2, 46.0, 50.2,
54.7, 55.6, 56.8, 73.5, 77.0, 80.3, 112.8, 121.4, 140.9, 146.6; HRMS
(FAB) m/z 459.3814 [M + H]+ (calcd for C30H51O3, 459.3838).
(3β,20R,22S,24R,25S)-3-Methoxystigmast-5-ene-20,22,26-
triol (2a) and (3β,20R,22S,24R,25R)-3-Methoxystigmast-5-ene-
20,22,26-triol (2b). BH3/THF (1 M solution in THF, 100 μL, 0.10
mmol) was added to a solution of the 25-ene 5 (25 mg, 55 μmol) at
room temperature, and the mixture was stirred for 2 h at the same
temperature. Water (100 μL), 3 M NaOH (100 μL), and 30% H2O2
(100 μL) were added, and the mixture was stirred for 30 min at 40 °C.
An extractive workup with CHCl3 yielded a crude product, which was
chromatographed on silica gel to give a 3:2 mixture of 2a and 2b (10
mg, eluting with hexane/EtOAc 4:1). The mixture was further
separated by p-TLC with benzene/EtOAc 4:3 as the developing
solvent (developed five times) to afford the less polar 2a (9.2 mg,
36%) and the more polar 2b (5.6 mg, 20%).
2-Ethyl-3-methylbut-3-enyl Bromide (4). TsCl (1.5 g, 7.9
mmol) was added to a solution of the alcohol (755 mg, 6.6 mmol) in
pyridine (2.0 mL) at 0 °C. The mixture was stirred for 2 h at the same
temperature. Ice chips were added, and the mixture was stirred for 5
min. Ether and saturated aqueous NH4Cl were added to the reaction
mixture, and the organic layer was washed with saturated aqueous
NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo to
Compound 2a: colorless needles, mp 168−170 °C (MeOH); Rf
0.35 (hexane/EtOAc 1:2); [α]D25 −8.0 (c 0.5, CHCl3); νmax (CHCl3)
1
give the tosylate (1.4 g, 78%) as a pale oil: H NMR (CDCl3, 400
1
3434, 3003, 2939, 2875 cm−1; H and 13C NMR (CD3OD) data, see
MHz) δ 0.80 (t, J = 7.3 Hz, 3H), 1.20−1.50 (m, 2H), 1.56 (s, 3H),
2.27 (m, 1H), 2.45 (s, 3H), 3.96 (m, 2H), 4.69 (brs, 1H), 4.83 (brs,
1H), 7.34 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H); 13C NMR
(CDCl3, 100 MHz) δ 11.2, 19.3, 21.6, 22.0, 47.6, 71.9, 113.7, 127.9,
129.7, 133.1, 142.9, 144.6. Anal. Calcd for C14H20O3S: C, 62.66; H,
7.51; S, 11.95. Found: C, 62.56; H, 7.50; S, 11.67.
LiBr (1.4 g, 16.1 mmol) was added to solution of the tosylate (1.4 g,
5.2 mmol) in dry acetone (7 mL), and the mixture was stirred at 60 °C
for 5 h. Ether and brine were added to the reaction mixture, and the
separated organic layer was washed with brine, dried over Na2SO4, and
concentrated on a rotary evaporator under reduced pressure (200
mmHg). The residue was passed through a short silica gel column with
hexane as an eluent. Removal of the solvent, as described above,
afforded the bromide 4 (655 mg, 3.7 mmol, 71%) as a pale yellow oil:
1H NMR (CDCl3, 400 MHz) δ 0.87 (t, J = 8.0 Hz, 3H), 1.42−1.60
1
Tables 1 and 2; compounds 2a−d exhibited common H signals at δ
3.05 (m, 1H) and 5.36 (m, 1H) for 3α-H and 6-H, respectively;
HRMS (FAB) m/z 477.3964 [M + H]+ (calcd for C30H53O4,
477.3944).
Compound 2b: colorless needles, mp 163−165 °C (MeOH); Rf
0.33 (hexane/EtOAc 1:2); [α]D25 −3.8 (c 0.5, CHCl3); νmax (CHCl3)
1
3434, 3003, 2939, 2875 cm−1; H and 13C NMR (CD3OD) data, see
Tables 1 and 2; HRMS (FAB) m/z 477.3953 [M + H]+ (calcd for
C30H53O4, 477.3944).
(3β,20R,22S,24S,25S)-3-Methoxystigmast-5-ene-20,22,26-
triol (2c) and (3β,20R,22S,24S,25R)-3-Methoxystigmast-5-ene-
20,22,26-triol (2d). Compound 6 (48 mg, 105 μmol) was subjected
to hydroboration and oxidation as described above to give the less
polar 2c (19.1 mg, 38%, eluting with CHCl3/EtOAc 3:1) and the more
polar 2d (10.3 mg, 21%, eluting with CHCl3/EtOAc 5:4) after silica
gel chromatography.
(m, 2H), 1.67 (s, 3H), 2.32 (m, 1H), 3.39 (d, J = 6.8 Hz, 2H), 4.78
(brs, 1H), 4.91 (brs, 1H); 13C NMR (CDCl3, 100 MHz) δ 11.7, 18.8,
24.5, 36.4, 50.9, 113.4, 144.5. Anal. Calcd for C7H13Br: C, 47.48; H,
7.40. Found: C, 47.57; H, 7.12.
Compound 2c: amorphous solid; Rf 0.53 (hexane/EtOAc 1:2);
25
[α]D +5.0 (c 0.04, CHCl3); νmax (CHCl3) 3434, 3003, 2939, 2875
1
cm−1; H and 13C NMR (CD3OD) data, see Tables 1 and 2; HRMS
(FAB) m/z 477.3953 [M + H]+ (calcd for C30H53O4, 477.3944).
Compound 2d: amorphous solid; Rf 0.28 (hexane/EtOAc 1:2);
(3β,20R,22S,24R)-3-Methoxystigmasta-5,25-diene-20,22-
diol (5) and (3β,20R,22S,24S)-3-Methoxystigmasta-5,25-diene-
20,22-diol (6). t-BuLi (1.60 M n-pentane solution, 2.68 mL, 4.29
mmol) was added to solution of 4 (760 mg, 4.29 mmol) in dry ether
(17 mL) and n-pentane (26 mL) under argon at −78 °C, and the
solution was stirred at the same temperature for 20 min. A solution of
3 (301 mg, 0.859 mmol) in dry ether (8.6 mL) and n-pentane (2.9
mL) was added dropwise to the organolithium solution at −78 °C.
The mixture was stirred at the same temperature for 5 min and diluted
with ether and saturated aqueous NH4Cl. The organic layer was
washed brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel (eluting with hexane/
EtOAc/CHCl3, 6:1:0.7) to obtain a mixture of 5 and 6 (335 mg).
Separation of the mixture by MPLC (eluting with hexane/EtOAc 5:1)
using a silica gel-packed column (LiChroprep Si-60, mesh 40−63,
Merck) gave the less polar 5 (125 mg, 32%) and the more polar 6
(189 mg, 48%).
Compound 5: colorless needles, mp 142−144 °C (MeOH); Rf 0.41
(hexane/EtOAc 4:1, developed twice); 1H NMR (CDCl3) δ 0.81 (t, J
= 7.5 Hz, 3H), 0.90 (s, 3H), 1.01 (s, 3H), 1.19 (s, 3H), 1.67 (s, 3H),
3.06 (m, 1H), 3.35 (s, 3H), 3.50 (brd, J = 10.5 Hz, 1H), 4.80 (brd, J =
1.0 Hz, 2H), 5.36 (brd, J = 5.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz)
δ 11.6, 13.5, 18.5, 19.4, 20.4, 21.0, 22.0, 24.0, 25.2, 28.0, 31.3, 31.8,
35.9, 36.9, 37.2, 38.7, 40.3, 43.2, 47.3, 50.2, 54.6, 55.6, 56.8, 75.3, 76.9,
25
[α]D +3.6 (c 0.5, CHCl3); νmax (CHCl3) 3434, 3003, 2939, 2875
1
cm−1; H and 13C NMR (CD3OD) data, see Tables 1 and 2; HRMS
(FAB) m/z 477.3943 [M + H]+ (calcd for C30H53O4, 477.3944).
X-ray Crystal Data of (3β,20R,22S,24S)-3-Methoxystigmasta-
5,25-diene-20,22-diol (6). Crystallographic data: colorless,
C30H50O3, FW 458.70, monoclinic, space group C2, a =
29.1632(11), b = 6.6782(3), c = 14.5294(6) Å, β = 98.666(1)°, V =
2797.4(2) Å3, Z = 4, Dcalcd = 1.089 g cm−3, T = 173 K, λ = 0.71075 Å,
μ = 0.068 mm−1, 13 745 measured reflections, 3461 independent
reflections, 310 parameters, 1 restraint, F(000) = 1016, R1 = 0.0360,
wR2 = 0.0950 [3213 I > 2σ(I)], R1 = 0.0388, wR2 = 0.0964 (all data),
max and min residual density 0.250 and −0.159 e·Å−3, and goodness
of fit (F2) = 1.052. The absolute structure was deduced from the
known chirality of pregnenolone, which was used for the synthesis.
Crystallographic data for the structure have been deposited with
CCDC (Deposition No. CCDC 978812).
X-ray Crystal Data of (3β,20R,22S,24R,25S)-3-Methoxy-
stigmasta-5,25-diene-20,22,26-triol (2a). Crystallographic data:
colorless, C31H56O5, FW 508.75, monoclinic, space group P21, a =
12.7540(4), b = 6.03911(17), c = 19.9264(7) Å, β = 95.3800(15)°, V =
1528.03(8) Å3, Z = 2, Dcalcd = 1.106 g cm−3, T = 173 K, λ = 0.71075 Å,
μ = 0.569 mm−1, 17 466 measured reflections, 5124 independent
F
dx.doi.org/10.1021/jo5005108 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
reflections, 325 parameters, 1 restraint, F(000) = 564, R1 = 0.0564,
wR2 = 0.1452 [3166 I > 2σ(I)], R1 = 0.0844, wR2 = 0.1772 (all data),
max and min residual density 0.234 and −0.149 e·Å−3, and goodness
of fit (F2) = 1.021. The absolute structure was deduced from the
known chirality of pregnenolone, which was used for the synthesis.
Crystallographic data for the structure have been deposited with
CCDC (Deposition No. CCDC 978811).
(17) Imai, S.; Hori, M.; Fujioka, S.; Murata, E.; Goto, M.; Nakanishi,
K. Tetrahedron Lett. 1968, 36, 3883−3886.
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ASSOCIATED CONTENT
■
S
* Supporting Information
(22) Boid, R.; Rees, H. H.; Goodwin, T. W. Biochem. Soc. Trans.
1974, 2, 1066−1070.
1H and 13C NMR spectra of amarasterone A (1a) isolated from
Cyathula officinalis and compounds 2a, 2b, 2c, and 2d, and CIF
files for compounds 2a and 6. This material is available free of
charge via the Internet at http://pubs.acs.org.
(23) Boid, R.; Rees, H. H.; Goodwin, T. W. Biochem. Physiol. Pflanz.
1975, 168, 27−40.
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4817−4825.
AUTHOR INFORMATION
Corresponding Author
*Tel: 81 3 5734 2241. Fax: 81 3 5734 2241. E-mail: fujimoto.y.
aa@m.titech.ac.jp.
(25) Alekseeva, L. I. Appl. Biochem. Microbiol. 2004, 40, 135−139.
(26) Preliminary results were presented at 92nd Annual Meeting of
the Chemical Society of Japan: Hirayama, Y.; Okuzumi, K.; Ohyama,
K.; Fujimoto, Y. March 2012; 2PC-042.
■
(27) Snogan, E.; Vahirua-Lechat, I.; Ho, R.; Bertho, G.; Girault, J. P.;
Notes
Ortiga, S.; Maria, A.; Lafont, R. Phytochem. Anal. 2007, 18, 441−450.
The authors declare no competing financial interest.
(28) Budes
2008, 73, 502−514.
́
̌ ̌ ́ ̌ ̌
ínsky, M.; Vokac, K.; Harmatha, J.; Cvacka, J. Steroids
ACKNOWLEDGMENTS
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2576−2583.
■
We thank Ms. Satsuki Itoh of Tokyo Institute of Technology
for the HRMS (FAB) measurements, and Ms. Chika Hisaoka
for her help in the early stage of this study. Thanks are also due
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17, 4565−4568.
(31) Jena, C. K.; Hoecker, J.; Woods, T. M.; Jessen, H. J.; Neuburger,
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́ ́ ́
to Professor Rene Lafont, Sorbonne Universite, Universite
Pierre et Marie Curie, IBPS-BIOSIPE, for providing pure
samples of the two epimers of amarasterone A (compounds 1a
and 1b) obtained from Microsorum scolopendria. We also
acknowledge Professor Dong-Lu Bai, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, for his kind gift
of a commercial package of Cyathula officinalis and
identification of the plant.
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