Obtusifoliol and related steroids from the whole herb of Euphorbia chamaesyce

Article abstract of DOI:10.1016/S0031-9422(99)00041-2

Two new ergostane-type steroids were isolated together with two known compounds, obtusifoliol and 4α,14α-dimethyl-5α-ergosta-7,9(11),24(28)- trine-3β-ol, from the whole herb of Euphorbia chamaesyce. The structures of the new compounds were established as 3β-hydroxy-4α,14α-dimethyl-5α- ergosta-8,24(28)-dien-7-one and 3β-hydroxy-4α,14α-dimethyl-5α-ergosta- 8,24(28)diene-7,11-dione on the basis of chemical and spectral evidence.

Full text of DOI:10.1016/S0031-9422(99)00041-2

Phytochemistry 51 (1999) 457±463
Obtusifoliol and related steroids from the whole herb of
Euphorbia chamaesyce
Reiko Tanaka, Kazuaki Kasubuchi, Shunji Kita, Shunyo Matsunaga*
Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 20 April 1998; received in revised form 5 October 1998
Abstract
Two new ergostane-type steroids were isolated together with two known compounds, obtusifoliol and 4a,14a-dimethyl-5a-
ergosta-7,9(11),24(28)-trine-3b-ol, from the whole herb of Euphorbia chamaesyce. The structures of the new compounds were
established as 3b-hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-dien-7-one and 3b-hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-
diene-7,11-dione on the basis of chemical and spectral evidence. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Euphorbia chamaesyce; Euphorbiaceae; Steroids; 3b-Hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-dien-7-one; 3b-Hydroxy-4a,14a-
dimethyl-5a-ergosta-8,24(28)-diene-7,11-dione
1. Introduction
spirosupinanes from supinenolone E via epoxidation
of the D8-double bond and subsequent cleavage of the
epoxy ring involving transformation of the C-7/C-8
bond to the C-9 position (Tanaka, & Matsunaga,
1991). These results aroused our phytochemical and
biological interest in the constituents of Euphorbia
chaemaesyce L., native to the torrid zone of the world
and now becoming naturalized in Japan. Recently, we
Previously, it was reported that Euphorbia supina
Ra®n., an annual weed native to North America and
introduced into Japan, contained two unusual fernane
triterpenoids named spirosupinanonediol [7(849)abeo-
9S-D:C-friedo-B':A'-neogrammaceran-8-one-3S,7S-diol
(Matsunaga et al., 1984) and neospirosupinanetrione
[7(8 4 9)abeo-9R-D:C-friedo-B':A'-neogammaceran-
3,7,8-trione] (Tanaka, & Matsunaga, 1991), together
with 30 other triterpenoids including ®ve oxygenated
fernanes named supinenolones A (3b,7a-dihydroxy-
isolated
3,4-seco-oleana-4(23)-18-dien-3-oic
acid
(Tanaka, Ida, Kita, Kamisako, & Matsunaga, 1994)
and 3,4-seco-8bH-ferna-4(23),9(11)-dien-3-oic acid
(Tanaka, Ida, Kita, Kamisako, & Matsunaga, 1996),
as well as butyrospermol, cycloart-23Z-en-3b,25-diol,
lupeol, glutinol, 11a,12a-oxidotaraxerol, 3b-hydroxy-
30-nor-lupan-20-one and 3b-hydroxymulti¯or-8-en-7-
one, from the methylene chloride extract of the whole
herb of E. chamaesyce. Further examination of this
extract led to the isolation of two known and two new
steroids and this paper deals with their structural eluci-
dation.
fern-8-en-11-one),
B
(3b,11b-dihydroxyfern-8-en-7-
one), C (3b-hydroxyfern-8-en-7,11-dione), D (fern-8-
en-3,7,11-trione) and E (3b-hydroxyfern-8-en-7-one)
(Tanaka, & Matsunaga, 1989, 1991) and ®ve 3,4-seco-
adiananes named espinendiols A and B, espinenoxide,
trisnor-isoespinenoxide and espinanoxide (Tanaka,
Matsunaga, Ishida, & Shingu, 1989). Plausible biosyn-
thetic pathways were also proposed to the above two
2. Results and discussion
* Corresponding author. Tel.: +81-726-90-1084; fax: +81-726-90-
1084.
E-mail address: matunaga@oysun01.oups.ac.jp (S. Matsunaga)
The known compounds were con®rmed as obtusifo-
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00041-2

458
R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
liol
[4a,14a-dimethyl-5a-ergosta-8,24(28)-dien-3b-ol]
secondary methyl groups, an isopropyl group (dH
1.025, 1.030 (each 3H, d) and 2.25 (1H, sept.)), nine
methylene groups, four methine groups, three sp3 qua-
ternary carbons, a hydroxymethine group (dH 3.15
(1H, ddd); dC 75.3 (d)), a terminal methylene group
(dH 4.66 (1H, d) and 4.72 (1H, s); dC 106.0 (t) and
156.7 (s)) and a conjugated enone including a tetrasub-
stituted double bond (dC 139.5 (s), 164.8 (s) and 198.1
(s)). Acetylation of 3a a€orded a monoacetate (3b), in
which the carbinol methine proton signal was shifted
to d 4.39 (ddd). In the EIMS spectrum, 3a exhibited
fragment peaks due to ions a±d and j±m, along with
peaks which had lost one atom of hydrogen from ions
a and b and two atoms of hydrogen from ions c, d and
j±m, indicating it to have the same side chain 1a and
2a (see Section 3). Moreover, a peak corresponding to
ion i was observed as a predominant peak at m/z
247.1681 [C16H23O2]+, together with peaks attribu-
table to ions e, f and g at m/z 301, 288 and 273, re-
(1a) and 4a,14a-dimethyl-5a-ergosta-7,9(11),24(28)-
trien-3b-ol (2a), respectively, based on the physical and
spectral data of the corresponding acetates, 1b and 2b,
which were in good agreement with literature values
(Gonzalez, Breton, & Garcia, 1958; Gonzalez, Breton,
Dergado Martin, & Fraga, 1972; Itoh, Kikuchi,
Shimizu, Tamura,
& Matsumoto, 1981; Akihisa,
Yokota, Takahashi, Tamura, & Matsumoto, 1989;
Akihisa, Kokke, Yokota, Tamura, & Matsumoto,
1990). An assignment of the 13C NMR signals of 2a is
shown in Table 2 and is based on 1H±1H COSY,
HMQC, HMBC and NOESY analyses.
Compound 3a was assigned the molecular formula
C30H48O2 (HREIMS). The UV and IR spectra showed
absorption bands for a hydroxyl group, a conjugated
enone (lmax 253 nm) and a terminal methylene group.
The 1H and 13C NMR and DEPT spectra (Tables 1
and 2) revealed signals of three quaternary and two

R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
459
Table 1
500 MHz 1H NMR spectra data of 3a, 3b, 4a, 4b and 4c
H
3a
3b
4a
4b
4c
1a
1.41 m
1.44 m
1.16 m
1.18 m
1.18 m
1b
2a
2b
1.87 dt (13.1, 3.0)
1.92 ddt (12.4, 4.8, 3.0)
1.60 m
1.87 dt (13.0, 3.5)
1.97 m
1.57 m
2.85 dt (13.5, 3.8)
1.89 m
1.61 m
2.86 dt (13.5, 3.8)
1.93 m
1.60 m
2.85 dt (13.7, 3.8)
1.94 m
1.61 m
3a
4b
5a
3.15 ddd (11.3, 9.5, 4.8)
1.46 m
1.55 ddd (11.6, 13.6, 3.7) 1.62 m
4.39 ddd (11.0, 11.0, 4.8) 3.14 ddd (11.0, 11.0, 4.8) 4.40 ddd (11.0, 11.0, 4.8) 4.39 ddd (11.0, 11.0, 4.8)
1.68 m
1.54 m
1.45 m
1.75 m
1.54 ddd (15.2, 11.1, 3.1) 1.53 ddd (15.0, 11.5, 3.0)
1.76 m
6a
6b
2.48 dd (16.5, 3.7)
2.19 dd (16.5, 13.6)
2.39 ddd (21.0, 7.5, 3.0)
2.31 m
2.48 dd (16.5, 3.7)
2.51 dd (15.2, 3.1)
2.28 dd (15.2, 15.2)
±
2.52 dd (15.2, 3.1)
2.29 dd (15.2, 15.2)
±
2.53 d (15.0, 3.0)
2.28 dd (15.0, 15.0)
2.18 dd (16.5, 13.5)
2.37 ddd (21.0, 6.0, 4.0)
2.27 m
11a
11b
12a
12b
15a
15b
16a
16b
17a
18
±
±
±
±
1.79 m
1.79 m
1.79 m
1.79 m
2.77 dd (16.2, 1.1)
2.64 d (16.2)
2.14 m
2.79 dd (15., 1.2)
2.65 d (15.8)
2.13 m
2.78 d (16.0, 0.8)
2.62 d (16.0)
2.16 ddd (12.5, 10.0, 2.5)
1.75 m
2.05 m
1.73 m
2.06 m
1.73 m
1.75 m
2.01 m
1.74 m
2.01 m
1.98 m
1.34 m
1.98 m
1.34 m
2.02 m
1.42 m
1.36 m
1.70 m
1.36 m
1.70 m
1.47 m
0.68 s
1.45 m
0.68 s
1.70 m
0.80 s
0.82 s
1.30 s
1.40 m
0.82 s
1.32 s
1.42 m
19
1.19 s
1.40 m
1.20 s
1.39 m
1.31 s
1.39 m
20b
21
0.94 d
1.16 m
0.96 d (6.5)
1.14 m
1.58 m
0.93 d (6.5)
1.17 m
1.59 m
0.93 d (6.5)
1.16 m
1.57 m
0.89 d (6.4)
1.26 m
1.79 m
22
22
1.58 m
1.88 m
2.10 m
23
23
1.89 m 1.89 m
2.12 ddd (15.0, 11.0, 4.0) 2.11 m
1.88 m
2.11 m
2.39 ddd (17.0, 9.5, 6.5)
2.49 dd (17.0, 10.0, 5.0)
2.62 septet (6.9)
1.09 d (6.9)
1.09 d (6.9)
±
25
26
2.25 septet (6.9)
1.025 d (6.9)a
1.030 d (6.9)a
4.66 d (1.4)
4.72 s
2.23 septet (7.0)
1.025 d (7.0)a
1.030 d (7.0)a
4.66 d (1.4)
4.72 s
2.23 septet (6.9)
2.23 septet (6.9)
1.026 d (6.9)a
1.031 d (6.9)a
4.66 d (1.2)
4.72 s
1.025 d (6.9)a
1.030 d (6.9)a
4.66 d (1.2)
4.73 s
27
28a
28b
30
±
0.99 d (6.2)
0.93 s
0.86 d (6.2)
0.93 s
2.06 s
1.02 d (6.4)
1.19 s
0.88 d (6.4)
1.20 s
2.07 s
0.88 d (6.4)
1.19 s
2.06 s
32
OAc
±
±
a Assignments may be interchangeble vertically.
nm). The IR, H and 13C NMR spectra (Tables 1 and
2) revealed the presence of three quaternary and two
secondary methyl groups, an isopropyl group (dH
1.025 and 1.030 (each 3H, d), 2.23 (1H, sept.)), eight
methylene groups, four methine groups, a hydroxy-
methine group (dH 3.14 (1H, ddd); dC 75.4 (d)), a
terminal methylene group (nmax 888 cm 1; dH 4.66
(1H, d) and 4.73 (1H, s); dC 106.2 (t) and 156.4 (s)), a
tetrasubstituted double bond (dC 151.2 and 151.5 (each
s)) and two carbonyl carbons (nmax 1673 cm 1; dC
201.5 and 202.6 (each s)). Acetylation of 4a yielded an
acetate (4b), in which the carbinol methine proton sig-
nal was shifted to d 4.40 (1H, ddd). The EIMS spec-
trum of 4a also exhibited fragment peaks due to ions
a±d and k±m, along with peaks which had eliminated
one atom of hydrogen from ions a and b and two
atoms of hydrogen from ions c, d and j±m, indicating
it to have the same side chain as 3a (see Section 3). A
peak attributable to [ion j (side chain part)±CH4]+
1
spectively. These data suggested 3a to be an analogue
of obtusifoliol attached to a keto group not at C-11
but at C-7. This assumption was supported by the 2D
1H±1H COSY, HMQC, HMBC and J-resolved spectra.
The HMBC analysis (Fig. 1) indicated 3a to have the
gross structure of 7-oxo-obtusifoliol. The NOESY data
(Fig. 1) exhibited cross correlations for H-3a (with H-
1a and H-5a), Me-30 (with H-3a, H-5a and H-6a),
Me-19 (with H-1b, H-4b, H-6b, H-11b and Me-18),
Me-18 (with H-12b, H-15b, H-20 and Me-21), Me-21
(with H-17a, H-23a and H-23b) and Me-32 (with H-
15a and H-17a), indicating 3a to have a 3b-hydroxyl
group and the same 20R-orientation of the side chain
moiety as obtusifoliol (1a) (Akihisa et al., 1990; Nes et
al., 1998). Hence, 3a was 3b-hydroxy-4a,14a-dimethyl-
5a-ergosta-8,24(28)-dien-7-one.
Compound 4a, obtained as pale yellow needles, was
assigned the molecular formula C30H46O3 (HREIMS).
The UV spectrum showed absorption bands for a
transoid ene-dione chromophore (lmax 205 and 270
was observed as
a base peak at m/z 109.1026

460
R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
Table 2
125 MHz 13C NMR spectral data of compounds 2a, 3a, 3b, 4a, 4b and 4c
C
2a
3a
3b
4a
4b
4c
1
34.7 t
31.3 t
76.0 d
39.7 d
45.5 d
26.5 t
119.3 d
143.0 s
143.3 s
36.5 s
117.0 d
37.9 t
43.8 s
50.4 s
31.5 t
27.9 t
50.9 d
15.7 q
20.7 q
36.3 d
18.5 q
39.4 t
31.3 t
156.8 s
33.8 d
34.2 t
30.7 t
75.3 d
39.1 d
46.7 d
39.1 t
198.1 s
139.5 s
164.8 s
38.8 s
24.5 t
30.1 t
44.9 s
47.8 s
31.9 t
28.7 t
48.9 d
15.7 q
17.7 q
36.4 d
18.8 q
34.9 t
31.2 t
156.7 s
33.7 d
33.9 t
28.7 t
76.7 d
36.4 d
46.7 d
39.1 t
197.6 s
139.7 s
164.4 s
38.7 s
24.5 t
30.1 t
44.9 s
47.8 s
32.0 t
26.9 t
48.9 d
15.8 q
17.7 q
36.1 d
18.8 q
34.9 t
31.6 t
156.7 s
33.7 d
33.3 t
30.9 t
74.5 d
38.2 d
47.2 d
38.2 t
201.5 s
151.2 s
151.5 s
38.6 s
202.6 s
51.5 t
49.0 s
47.5 s
32.0 t
27.3 t
49.0 d
16.8 q
16.4 q
36.1 d
18.6 q
34.7 t
31.1 t
156.4 w
33.8 d
33.0 t
26.9 t
77.3 d
35.2 d
47.2 d
38.2 t
201.1 s
151.3 s
151.3 s
38.4 s
202.5 s
51.5 t
49.0 s
47.6 s
32.1 t
27.3 t
49.0 d
16.8 q
16.3 q
36.2 d
18.6 q
34.7 t
31.1 t
156.4 s
33.8 d
33.0 t
26.9 t
77.3 d
35.1 d
47.2 d
38.1 t
201.0 s
151.3 s
151.3 s
38.4 s
202.4 s
51.5 t
49.0 s
47.5 s
32.0 t
27.2 t
49.1 d
16.8 q
16.3 q
35.8 d
18.3 q
29.7 t
29.7 t
215.0 s
40.9 d
18.4 q
18.4 q
±
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
21.9a
22.0a
q
q
21.8a
22.0a
q
q
21.8a
22.0a
q
q
21.8a
22.0a
q
q
21.8a
22.0a
q
q
27
28
106.0 t
15.1 q
25.6 q
±
106.0 t
14.4 q
25.1 q
±
106.0 t
14.5 q
25.1 q
21.2 q
170.7 s
106.2 t
14.8 q
26.0 q
±
106.2 t
14.8 q
26.0 q
21.2 q
170.7 s
30
32
14.8 q
26.0 q
21.2 q
170.7 s
OCOMe
OCOMe
±
±
±
a Assignments in some column are interchangeable.
[C8H13]+, although it appeared as an inconspicuous
peak in 3a. Furthermore, two characteristic peaks ten-
tatively assigned to ions h and i' were observed at m/z
275 [C17H23O3]+ and 263.1655 [C16H23O3]+, respect-
ively, as well as peaks due to ions e, f and g. Together
with these results, the HMBC data (Fig. 2) indicated
that 4a must be 7,11-dioxo-obtusifoliol. The complete
structure was obtained by the following experiments.
The NOESY spectrum provided cross correlations
(Fig. 2) for Me-30 (with H-3a, H-5a and H-6a), Me-19
(with H-1b, H-2b, H-4b and Me-18), Me-18 (with H-
12b, H-20 and Me-21), Me-21 (with H-17a, H-23a and
H-23b) and Me-32 (with H-15a and H-17a), indicating
4a to have a 3b-hydroxyl group and the same 20R-
orientation of side chain as obtusifoliol (1a) (Akihisa
et al., 1990; Nes et al., 1998). Oxidation of 1a with
chromium trioxide in acetic acid furnished 3b-acetoxy-
4a,14a-dimethyl-5a-ergosta-8,24(28)-diene-7,11-dione,
together with almost the same amount of 3b-acetoxy-
3b-hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-diene-
7,11-dione.
This is the ®rst report for the isolation of 3a and 4a
in the literature, although the 24(28)-dihydro-deriva-
tives of 3b and 4b had previously been prepared from
1a (Barrera, Breton, Dergado Martin, & Gonzalez,
1967).
3. Experimental
3.1. General
M.p.'s: uncorr.; optical rotations: CHCl3, unless
otherwise noted. UV: EtOH; IR: KBr discs; 1H (500
MHz) and 13C NMR (125 MHz): CDCl3 with TMS as
int. standard; EIMS (probe): 70 eV; CC: Kieselgel 60
(70±230 mesh, Merck) and alumina 90 (70±230 mesh,
Merck). Reversed phase MPLC: Cosmosil-40 ODS
C18-PREP (Nacalai Tesque Co.); equipped with
Yamazen SS 50-1296 pump; TLC and prep. TLC:
silica gel HF254 and PF254 (Merck).
4a,14a-dimethyl-5a-cholest-8-ene-7,11,24-trione (4c).
The former product was identical in all respects with
4b prepared from 4a. Hence, 4a was proved to be

R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
461
Fig. 1. HMBC (plain arrow) and selective NOESY (dashed arrow) correlations of 3a.
3.2. Isolation of compounds
phous gummy product (1.54 g) from frs eluted with n-
hexane±C6H6 (2:1±1:1). Rechromatography of the pro-
duct over silica gel (150 g) CC yielded an amorphous
gum (141 mg) from the fr. eluted with n-hexane±
EtoAc (7:1), which was subjected to reversed phase
MPLC using Cosmosil-40 C18 (ODS) column. Elution
with MeOH a€orded obtusifoliol (1a), 72 mg, m.p.
142±1448C (MeOH±CHCl3), [a]2D3 +738 (c 0.34) (lit.
(Gonzalez et al., 1972) m.p. 145±1468, [a ]D +748);
Details on isolation of 3,4-seco-oleana-4(23),18-dien-
3-oic acid, 3,4-seco-8bH-ferna-4(23),9(11)-dien-3-oic
acid and eight known constituents separated from the
CH2Cl2 extract of the whole herb of Euphorbia cha-
maesyce L. (5.56 kg) by preliminary silica gel CC, were
described previously (Tanaka et al., 1994, 1996).
Continuous CC of residue I (32.29 g) yielded an amor-
Fig. 2. HMBC (plain arrow) and selective NOESY (dashed arrow) correlations of 4a.

462
R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
EIMS: m/z 426 [M]+, from fr. No.'s: 9±12 (each fr.:
30 ml). Acetylation of 1a (30 mg) with Ac2O/C5H5N
(1:1, 3 ml) furnished the corresponding acetate (1b), 30
mg, m.p. 106±1078C (MeOH±CHCl3), [a ]2D3 +738 (lit.
(Gonzalez et al., 1972) m.p. 107±1088C; [a]D +798; lit.
(Itoh et al., 1981) m.p. 111±1138C), EIMS: m/z 468
[M]+. Further elution of the column with the same
H2O]+, 371 (1, ion a) [M±C5H9]+, 370 (2, ion a±H),
357 (2, ion b) [M±C6H11]+, 356.2707 (13, ion b±H,
calc. for C24H36O2: 356.2713), 343 (16, ion c) [M±
C7H13]+, 341.2472 (29, ion c±2H, calc. for C23H33O2:
341.2479), 315 (24, ion d) [M±C9H17]+, 313.2179 (30,
ion d±2H, calc. for C21H29O2: 313.2166), 301 (30, ion
e) [C20H29O2]+, 288 (20, ion f) [C19H28O2]+, 273.1832
(18, ion g, calc. for C18H25O2: 273.1853), 247.1681 (83,
ion i, calc. for C16H23O2: 247.1697), 222.1615 (14)
[C14H22O2]+, 215 (19), 187 (11), 175 (19), 173 (18),
135 (18), 125 (5, ion j, side chain) [C9H17]+, 123 (11,
ion j±2H), 121 (27), 97 (8, ion k) [C7H13]+, 95 (16, ion
k±2H), 83 (13, ion l) [C6H11]+, 81 (14, ion l±2H), 69
(30, ion m) [C5H9]+ and 67 (10, ion m±2H).
solvent
a€orded
4a,14a-dimethyl-5a-ergosta-
7,9(11),24(28)-trien-3b-ol (2a), 55 mg, m.p. 152.5±
1548C (MeOH±CHCl3), [a]2D3 +468 (c 0.13); H NMR:
1
d 0.58 (3H, s, Me-18), 0.90 (3H, s, Me-32), 0.92 (3H,
d, J=6.4Hz, Me-21), 0.94 (3H, s, Me-19), 1.00 (3H, d,
J=6.4Hz, Me-30), 1.028 (3H, d, J=6.9 Hz, Me-26),
1.033 (3H, d, J=6.9Hz, Me-27), 3.12 (1H, ddd,
J=11.0, 10.9, 4.8 Hz, H-3a), 4.67 (1H, d, J=1.2 Hz,
H-28a), 4.72 (1H, s, H-28b), 5.39 (1H, d, J=6.2 Hz,
H-11), 5.42 (1H, d, J=6.6 Hz, H-7); 13C NMR: see
Table 2; EIMS: m/z 424 [M]+, from fr. 6. Acetylation
of 2a (6 mg) with Ac2O/C5H5N (1:1, 1 ml) a€orded
the corresponding acetate (2b), 6 mg, m.p. 123±
124.58C (MeOH±CHCl3), (lit. (Gonzalez et al., 1958)
m.p. 123±1248C), EIMS; m/z 466 [M]+. All the above
physical and spectral data for 1a (Gonzalez et al.,
1972), 1b (Gonzalez et al., 1972; Itoh et al., 1981;
Akihisa et al., 1989; Akihisa et al., 1990) and 2b
(Gonzalez et al., 1958) were in good agreement with
those already reported in the literature, respectively. A
residue (18.92 g) eluting between residues III and IV
on the preliminary silica gel CC of the CH2Cl2 extract
(fr. No.'s: 106±116, each fr.: 1 l), was fractionated by
silica gel (800 g) CC to yield a gummy product (280
mg) from the fr. No.'s 55±82 eluted with CHCl3 (each
fr.: 200 ml) (Tanaka et al., 1994). Further CC of the
gum with 10% AgNO3 impregnated silica gel (20 g)
a€orded a crystalline solid (159 mg) from the frs eluted
with C6H6±CHCl3 (1:1), which was puri®ed by prep.
TLC (plate: 20Â20 cm; solvent: CHCl3±MeOH, 50:1)
to furnish compound 3a (103 mg). We also reported
that alumina CC of residue IV (41.02 g) a€orded three
known triterpenes including cycloart-23Z-ene-3b,25-
diol (Tanaka et al., 1996). Continuous fractionation of
this column a€orded compound 4a, 31 mg, from the
frs eluted with C6H6±CHCl3 (5:1).
3.4. Acetylation of 3a
A soln of compound 3a (21.5 mg) in Ac2O/C5H5N
(1:1, 2 ml) was kept at room temp. overnight. The
reaction mixture was poured into ice water and the
resulting precipitate was extracted with Et2O. Evapn
of the neutral Et2O layer yielded a solid, which was
puri®ed by prep. TLC (plate: 20 Â 20 cm; solvent:
CHCl3±MeOH, 100:1) to a€ord the corresponding
acetate (3b), 18.5 mg, m.p. 148.5±1508C (MeOH±
CHCl3), [a]2D3 +288 (c 0.18); HREIMS: m/z 482.3743
(C32H50O3 requires 482.3757); UV lmax (nm): 253 (e
9600); IR nmax cm 1: 3077 (.C±H), 2963, 2876, 1729
and 1246 (OAc), 1649 (>C.C±C.O), 1641 and 886
(>C.CH2), 1468, 1421 (±CH2CO), 1374, 1335, 1194
and 1027; 1H and 13C NMR: see Tables 1 and 2;
EIMS m/z (rel. int.): 482 (41) [M]+, 467.3531 (100)
[M±Me]+, 422.3522 (4) [M±HOAc]+, 413 (3, ion a±H),
399 (8, ion b), 398.2809 (13, ion b±H, calc. for
C26H38O3: 398.2819), 385 (19, ion c), 383.2605 (28, ion
c-2H, calc. for C25H35O3, 383.2584), 357 (26, ion d),
355 (30, ion d±2H), 343 (30, ion e), 330 (19, ion f),
315.1931 (18, ion g, calc. for C20H27O3, 315.1959),
289.1782 (55, ion i, calc. for C18H25O3, 289.1802),
264.1713 (10) [C16H24O3]+, 215 (23), 187 (10), 175
(16), 173 (16), 135 (18), 123 (16, ion j±2H) [side chain,
2H]+, 121 (25), 97 (8, ion k), 95 (18, ion k±2H), 83
(12, ion l), 81 (16, ion l±2H), 69 (31, ion m) and 67
(12, ion m±2H).
3.3. 3b-Hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-
dien-7-one (3a)
3.5. 3b-Hydroxy-4a,14a-dimethyl-5a-ergosta-8,24(28)-
diene-7,11-dione (4a)
Needles, m.p. 149±1508C (MeOH±CHCl3), [a]D23
+338 (c 0.48); HREIMS: m/z 440.3655 (C30H48O2
requires 440.3652); UV lmax (nm): 253 (e 5000) (conj.
enone); IR nmax cm 1: 3436 (OH), 3077 (.C±H), 2965,
2932, 1662 (>C.C±C.O), 1640 and 886 (>C.CH2),
1456, 1418 (±CH2CO), 1374, 1335, 1269, 1243, 1193
and 1024; 1H and 13C NMR: see Tables 1 and 2;
EIMS m/z (rel.int.): 440 (51) [M]+, 425.3423 (100,
calc. for C29H45O2: 425.3417) [M±Me]+, 422 (2) [M±
Pale yellow needles, m.p. 118±1208C (MeOH±
CHCl3), [a]2D3 +1028 (c 0.92); HREIMS: m/z 454.3449
(C30H46O3 requires 454.3445); UV lmax (nm): 205 and
270 (e 4500 and 6200) (transoid ene-dione); IR nmax
cm 1: 3392 (OH), 3033 (.C±H), 2965, 2931, 2871,
1673 (O.C±C.C±C.O), 1643 and 888 (>C.CH2),
1459, 1430 and 1421 (±CH2CO), 1377, 1345, 1232,
1207, 1017 and 947; 1H and 13C NMR: see Tables 1

R. Tanaka et al. / Phytochemistry 51 (1999) 457±463
463
and 2; EIMS m/z (rel. int.): 454 (55) [M]+, 436.3342
(7, calc. for C30H44O2: 436.3339) [M±H2O]+, 385 (2,
ion a), 384 (2, ion a±H), 371 (12, ion b), 370.2525 (46,
ion b±H, calc. for C24H34O2: 370.2507), 357.2430 (8,
ion c, calc. for C23H33O3: 357.2428), 355 (2, ion c-2H),
329 (11, ion d), 327 (4, ion d±2H), 315 (8, ion e),
302.1883 (35, ion f, calc. for C19H26O3: 302.1880), 289
(4), 287 (4, ion g) [C18H23O3]+, 275 (13, ion h)
[C17H23O3]+, 263.1655 (22, ion i', calc. for C16H23O3:
263.1646), 241 (11), 236 (10), 187.0767 (19)
[C12H11O2]+, 123 (11, ion j±2H), 121 (10), 109.1026
(100, ion j±CH4, calc. for C8H13: 109.1017), 97 (8, ion
k), 95 (13, ion k±2H), 83 (17, ion l), 81 (15, ion l±2H),
69 (43, ion m) and 67 (12, ion m±2H).
8,24(28)-diene-7,11-dione (4b), as pale yellow needles,
m.p. 138±1398C (MeOH±CHCl3), [a ]2D3 +898 (c 0.30),
12 mg, EIMS: m/z 496 [M]+, TLC: Rf 0.56 (plate:
0.25 mm; solvent: CHCl3±MeOH 100:1) and 3b-acet-
oxy-4a,14a-dimethyl-5a-cholest-8-ene-7,11,24-trione
(4c), 11 mg, as an amorphous pale yellow powder,
[a]2D3 +1008 (c 0.16); TLC: Rf 0.34 (plate: 0.25 mm;
solvent: CHCl3±MeOH: 100:1), UV lmax (nm): 219
and 268 (e 5300 and 5500); IR nmax cm 1: 2963, 2876,
1733 and 1246 (OAc), 1712 (C.O), 1673 (O.C±C.C±
C.O), 1467, 1429 and 1420 (CH2CO), 1377, 1100 and
1030; H and 13C NMR: see Tables 1 and 2; EIMS m/
1
z (rel. int.): 498 (44) [M]+, 438 (11) [M±HOAc]+, 413
(7, ion b), 399 (7, ion c), 371 (6, ion d), 370 (10, ion
d±H), 357 (5, ion e), 355 (6, ion e±2H), 344 (7, ion f),
331 (6), 329 (6, ion g), 317 (20 ion h), 305 (10, ion i'),
304 (13), 289 (11), 241 (13), 127 (20, ion j) [C8H15O]+,
85 (12, ion l), 83 (18, ion l±2H) and 71 (100, ion m).
The former product was identi®ed by direct compari-
son (m.p., co-TLC, [a]D, UV, IR, 1H and 13C NMR
and EIMS) with 4b derived from compound 4a.
3.6. Acetylation of 4a
A soln of compound 4a (13 mg) in Ac2O/C5H5N
(1:1, 2 ml) was kept at room temp. overnight. Work
up as described above yielded a crystalline mass, which
was puri®ed by prep. TLC (plate: 20Â20 cm; solvent:
CHCl3±MeOH, 100:1) to furnish the corresponding
acetate (4a), 12 mg, m.p. 137.5±1398C (MeOH±
CHCl3), [a]D23 +898 (c 0.33); IR nmax cm 1: 3034, 1641
and 888 (>C.CH2), 2963, 2893, 1736 and 1243 (OAc),
1677 (O.C±C.C±C.O), 1457, 1430 (±CH2CO), 1376,
Acknowledgements
The authors are grateful to Mr. Katsuhiko Minoura
and Mrs. Mihoyo Fujitake of this University for
NMR and MS measurements.
1363, 1205 and 1029; H and 13C NMR: see Tables 1
1
and 2; EIMS (rel. int.): m/z 496 (33) [M]+, 481 (2)
[M±Me]+, 436 (4) [M±HOAc]+, 426 (2, ion a±H), 413
(14, ion b), 412 (23, ion b±H), 399 (4, ion c), 397 (3,
ion c±2H), 371 (9, ion d), 369 (3, ion d±2H), 352 (6)
[412-HOAc], 344 (33, ion f), 331 (5, ion g), 317 (13,
ion h) [C19H25O4]+, 305 (20, ion i'), 289 (9), 278 (10),
256 (10), 241 (14), 187 (25), 123 (18, ion j±2H), 121
(17), 109 (100, ion j, CH4), 97 (14, ion k), 95 (28, ion
k±2H), 83 (33, ion l), 81 (42, ion l±2H), 69 (75, ion m)
and 67 (20, ion m±2H).
References
Akihisa, T., Kokke, W. C. M. C., Yokota, T., Tamura, T., &
Matsumoto, T. (1990). Phytochemistry, 29, 1647.
Akihisa, T., Yokota, T., Takahashi, N., Tamura, T., & Matsumoto,
T. (1989). Phytochemistry, 28, 1219.
Barrera, J. B., Breton, J. L., Dergado Martin Jr, & Gonzalez, A. G.
(1967). Annales de Fisica y Quimica, 63B, 191.
Gonzalez, A. G., Breton, J. L., & Garcia, P. A. (1958). Annales de
Fisica y Quimica, 54B, 93.
3.7. Synthesis of 4b and 4c from 1b
Gonzalez, A. G., Breton, J. L., Dergado Martin, J., & Fraga, B. M.
(1972). Annales de Quimica, 68, 203.
A soln of CrO3 (33.3 mg) in HOAc (5 ml) contain-
ing 3 drops of H2O was gradually added into a soln of
obtusifoliol acetate (1b) (30 mg) in HOAc (15 ml)
under stirring at 808C and then the reaction was con-
tinued for 4 h. After cooling, a few drops of 5%
NaHSO3 were added into the mixture to destroy excess
CrO3. Evapn of HOAc in vacuo gave a residue, which
was dissolved in CHCl3 (20 ml) and the organic layer
was washed with sat. NaHCO3 and H2O and dried
(Na2SO4). Removal of the solvent in vacuo yielded a
yellow residue (31 mg) showing two spots on a TLC
plate. Separation of the residue by prep. TLC
(plate: 20 Â 20 cm, 0.5 mm; solvent: CHCl3±MeOH,
100:1), furnished 3b-acetoxy-4a,14a-dimethyl-5a-ergosta-
Itoh, T., Kikuchi, Y., Shimizu, N., Tamura, T., & Matsumoto, T.
(1981). Phytochemistry, 20, 1929.
Matsunaga, S., Morita, R., Ishida, T., Inoue, M., Shigi, M., &
Miyamae, A. (1984). Journal of the Chemical Society, Chemical
Communications, 1984, 1128.
Nes, W. D., Koike, K., Jia, Z., Sakamoto, Y., Satu, T., Nikaido, T.,
& Grin, J. F. (1998). Journal of American Chemical Society,
120, 5870.
Tanaka, R., & Matsunaga, S. (1989). Phytochemistry, 28, 3149.
Tanaka, R., & Matsunaga, S. (1991). Phytochemistry, 30, 293.
Tanaka, R., Ida, T., Kita, S., Kamisako, W., & Matsunaga, S.
(1994). Phytochemistry, 36, 129.
Tanaka, R., Ida, T., Kita, S., Kamisako, W., & Matsunaga, S.
(1996). Phytochemistry, 41, 1163.
Tanaka, R., Matsunaga, S., Ishida, T.,
Tetrahedron Letters, 30, 1661.
& Shingu, T. (1989).