Three new polyoxygenated steroids from two species of the South China Sea gorgonian Muricella flexuosa and Menella verrucosa Brundin

Article abstract of DOI:10.1002/hlca.200690073

Three new polyoxygenated steroids, muricesteroid (1), and menellsteroids A (2) and B (3), were isolated from two species of the South China Sea gorgonian Muricella flexuosa and Menella verrucosa BRUNDIN, respectively. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analysis, chemical methods and comparison with known related compounds.

Full text of DOI:10.1002/hlca.200690073

Helvetica Chimica Acta – Vol. 89 (2006)
813
Three New Polyoxygenated Steroids from Two Species of the South China Sea
Gorgonian Muricella flexuosa and Menella verrucosa BRUNDIN
by Wen Zhanga), Hui Huangb), Yu Dinga), Margherita Gavagninc), Ernesto Molloc), Guido Ciminoc),
and Yue-Wei Guo*a)
a) State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Bio-
logical Sciences, Chinese Academy of Sciences, Zu Chong Zhi Rd. 555, Zhangjiang Hi-Tech Park,
Shanghai 201203, P. R. China (phone: 86-21-50805813; e-mail: ywguo@mail.shcnc.ac.cn)
b) South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510000,
P. R. China
c) Istituto di Chimica Biomolecolare-CNR, I-80078 Napoli
Three new polyoxygenated steroids, muricesteroid (1), and menellsteroids A (2) and B (3), were iso-
lated fromtwo species of the South China Sea gorgonian Muricella flexuosa and Menella verrucosa
BRUNDIN, respectively. The structures of these new compounds were elucidated on the basis of extensive
spectroscopic analysis, chemical methods and comparison with known related compounds.
Introduction. – Gorgonians of the genera Muricella and Menella (both of them
belong to the family Paramuriceidae, order Gorgonacea, class Anthozoa) is prolific
in the South China Sea. A literature survey revealed that the chemical constituents
of the gorgonians of the genus Muricella have been intensively investigated. A variety
of secondary metabolites with different C-skeletons such as eunicellane diterpenoids
[1–3], 9,11-secosteroids [4][5], and a carotenoid [6] were reported fromvarious species
of this genus, and some of them exhibited potent cytotoxic activities against several
tumor cell lines [2][3][5], and toxic effects to brine-shrimp as well [2–6]. In contrast,
chemical studies on the genus Menella were relatively rare. In particular, no chemical
study has been done on the Hainan gorgonian Menella verrucosa BRUNDIN, except for
our recent work [7c] reporting five new uncommon guaiane lactones from the species.
Recently, in the course of our systematic studies on the chemical constituents of the
South China Sea gorgonians [7], we have made a collection of Muricella flexuosa off
Sanya, Hainan Province, China. Chemical investigation of the Et2
of the Me2CO extract from Muricella flexuosa resulted in the isolation of a new oxygen-
ated steroid, named muricesteroid (1). Further, a continuous chemical study on the
Et2O-soluble fraction of the Me2CO extract fromanother gorgonian, Menella verru-
O-soluble fraction
AHCTREUNG
A
ACHTREUNG
cosa, allowed to isolate two new polyhydroxylated steroids, named menellsteroids A
(2) and B (3), respectively. Herein, we describe the isolation and structural elucidation
of these new metabolites.
Results and Discussion. – Both gorgonians were collected off the coast of Xiaodong
Hai, Hainan Province, China, in December 2001, at a depth of 20 m. The animals were
immediately put at ꢀ208 and kept frozen until extraction. Frozen materials of Muricella
ꢀ 2006 Verlag Helvetica Chimica Acta AG, Zürich

814
Helvetica Chimica Acta – Vol. 89 (2006)
flexuosa were cut into small pieces and subsequently extracted with Me2
N
N
soluble portion fromthe Me 2CO extract was repeatedly subjected to column chromato-
AHCTREUNG
graphed (silica gel, Sephadex LH-20) to afford muricesteroid (1). In a similar manner,
menellsteroids A (2) and B (3) were obtained fromthe gorgonian Menella verrucosa.
All three compounds were highly oxygenated steroids possessing a cholestane skel-
eton. The structures of the new metabolites were elucidated by detailed analysis of their
spectroscopic data, chemical correlations, and comparison with those of closely related
known compounds, i.e., 4 and 5–12.
Muricesteroid (1) was obtained as a colorless glass. Its molecular formula C29H46O4,
deduced fromthe HR-ESI-MS exhibiting the pseudo-molecular ion at m/z 481.3294
1
([M+Na]+), indicated seven degrees of unsaturation. The H- and 13C-NMR spectra
(Table 1), HMQC, COSY and HMBC data (Fig. 1), and comparison with data of
known compounds (Table 2), established the structure of 1 as (20R,22R)-22-(acetyl-
oxy)-20-hydroxycholest-4-en-3-one. The absolute configurations (20R,22R) were con-
firmed by comparison of the 1H-NMR data of the 22-O-deacetyl derivative 1a with sim-
ilar compounds.
Analysis of the 13C-NMR and DEPT spectra (Table 1) of 1 assigned three of the seven degrees of
unsaturation to a C=C (d 123.8 (d), 171.4 (s)), one C=O (d 199.6 (s)), and an AcO moiety (d 21.1 (q),
172.5 (s)). Consequently, the remaining unsaturations were due to four rings. In addition, 13C-NMR
and DEPT spectra also supported the presence of 24 sp3 C-atoms (5 Me, 10 CH2, 6 CH, and 3 C), includ-
ing an oxygenated secondary C-atom(79.1, d) and a related tertiary C-atom(77.2, s).

Helvetica Chimica Acta – Vol. 89 (2006)
815
Table 1. NMRData a)b) for Muricesteroid (1). d in ppm, J in Hz.
d(H)
d(C)c)
d(H)
d(C)c)
CH2(1)
CH2(2)
1.66–1.71 (m),
2.02 (dt, J=13.8, 3.6)
2.31–2.36 (m),
35.6 (t)
CH2(15)
CH2(16)
1.27–1.34 (m),
1.64–1.71 (m)
1.19–1.25 (m),
1.82–1.89 (m)
1.43–1.46 (s)
0.91 (s)
23.8 (t)
22.0 (t)
34.0 (t)
2.41–2.46 (m)
C(3)
CH(4)
C(5)
199.6 (s)
123.8 (d)
171.4 (s)
32.9 (t)
CH(17)
Me(18)
Me(19)
C(20)
Me(21)
CH(22)
CH2(23)
55.3 (d)
13.6 (q)
17.3 (q)
77.2 (s)
20.8 (q)
79.1 (d)
27.8 (t)
5.73 (s)
1.20 (s)
CH2(6)
2.26 (dt, J=13.5, 2.0),
2.37–2.43 (m)
1.00–1.05 (m),
1.80–1.89 (m)
1.53–1.63 (m)
0.87–0.94 (m)
1.24 (s)
CH2(7)
31.9 (t)
4.80 (d, J=9.0)
1.18–1.24 (m),
1.36–1.44 (m)
1.12–1.19 (m),
1.12–1.19 (m)
1.45–1.55 (m)
0.87 (d, J=6.6)
0.88 (d, J=6.6)
2.10 (s)
CH(8)
CH(9)
C(10)
34.8 (d)
53.8 (d)
38.6 (s)
20.9 (t)
CH2(24)
35.7 (t)
CH2(11)
1.45–1.51 (m),
1.43–1.53 (m)
1.18–1.25 (m),
2.14 (dt, J=12.6, 2.4)
CH(25)
27.8 (d)
22.8 (q)
22.3 (q)
21.1 (q)
172.5 (s)
Me(26)d)
CH2(12)
40.1 (t)
Me(27)d)
MeCOOꢀC(22)
C(13)
CH(14)
43.4 (s)
55.8 (d)
MeCOOꢀC(22)
0.99–1.04 (m)
a) Bruker-DRX-400-MHz spectrometers, CDCl3, chemical shifts referred to CHCl3 (d(H) 7.26) and to
1
1
b
c
CDCl3 (d(C) 77.0). ) Assignments made by H, H COSY, HSQC, and HMBC. ) By DEPT sequence.
d) Signals may be interchanged.
Fig. 1. 1H,1H-COSY
(
)
and selected HMBC
(
) correlations of 1
Two downfield 1H-NMR signals were assigned to the protons of a trisubstituted C=C (d 5.73 (s)) and
a CHꢀO (d 4.80 (d, J=9.0)). Obviously, the Me s at d 2.10 should be assigned to the AcO, while the addi-
tional three Me s d 1.24, 1.20, and 0.91 and two d of an iPr group (d 0.88 and 0.89 (each J=6.6 Hz, 3 H))
were attributed to the Me groups of the cholestane skeleton. Finally, the m integrating for 24 H-atoms
1
between d 2.48 and 0.98 were due to 10 CH2 and 4 CH as established by HMQC experiments. H,1H-
COSY experiments established the proton sequence as depicted in Fig. 1. All these data strongly sug-
gested for 1 a cholest-4-enone framework substituted by both OH and AcO. Significant HMBC long-
range correlations HꢀC(17)/C(13), C(16), and C(18), Me(18)/C(12), C(13), and C(14), and Me(19)/
C(1), C(5), C(9), and C(10), unambiguously confirmed the suggested skeleton. The presence of a conju-
gated 4-en-3-one moiety was deduced from the typical d values of the trisubstituted C=C (d(H) 5.73 (s),
d(C) 123.8 (d) and 171.4 (s)) and C=O (d 199.6 (s)), and further confirmed by the long-range 1H,13C-cor-
relations CH2(1)/C(3) and C(5), CH2(2)/C(3) and C(10), as well as HꢀC(4)/C(6) and C(10). The obvious
1
correlations HꢀC(22)/CH2(23)/CH2(24)/HꢀC(25)/Me(26) and Me(27)) in the H,1H-COSY allowed to

816
Helvetica Chimica Acta – Vol. 89 (2006)
place the CHꢀO (d 4.80) at C(22). The distinguished long-range correlations HꢀC(22)/C(20), C(21) and
C(24) and d(C) 172.5 not only confirmed the above conclusion but also revealed the linkage between
C(22) and AcO. As a consequence, the remaining OH had to be connected to C(20) according to the
s of Me(21) (d 1.24) in the 1H-NMR spectrum. This assignment was supported by the long-range corre-
lations Me(21)/C(20), C(17), and C(22) in the HMBC spectrum( Fig. 1).
The absolute configuration at the chiral centers C(20) and C(22) of 1 was tentatively assigned as
1
(20R) and (22R), mainly by comparison of the H-NMR data of 1 and its 22-hydrolyzate 1a with those
of the related model compounds 5–8 and 9–12, respectively. Some selected 1H-NMR data of 1, 1a,
and 5–12 are listed in Table 2. As shown in Table 2, both the chemical shifts of Me(21) (d 1.24) and
HꢀC(22) (d 4.80) of 1 were in good agreement with those of 5 (d 1.23, 4.77), the (20R,22R)-isomer, sug-
gesting that the absolute configuration of C(20) and C(22) of 1 could be the same as that of compound 5.
To confirmthis suggestion, compound 1 was hydrolyzed to the expected 22-O-deacetyl derivative 1a. The
chemical shifts of Me(21) (d 1.52) and HꢀC(22) (d 3.75) of 1a were almost identical to those of 9 (d 1.51,
3.78) but different fromthose of the other three stereoisomers 10–12 (d 1.60 and 3.70 for 10, 1.44 and
3.84 for 11, and 1.32 and 4.02 for 12) [8], supporting the (20R,22R) configuration of 1.
Table 2. Selected 1H-NMRData for 1 and 1a and Comparison with Those of Model Compounds 5–12a).
d in ppm, J in Hz.
d(Me(21))
d(HꢀC(22))
d(Me(21))
d(HꢀC(22))
1b)
5b)
6b)
7b)
8b)
1.24 (s)
1.23 (s)
1.27 (s)
1.17 (s)
1.06 (s)
4.80 (d, J=9.0)
4.77 (d, J=9.0)
4.78 (d, J=9.0)
4.71 (d, J=9.0)
5.17 (d, J=9.0)
1ac)
9c)
10c)
11c)
12c)
1.52 (s)
1.51 (s)
1.60 (s)
1.44 (s)
1.32 (s)
3.75 (d, J=8.8)
3.78 (d, J=9.0)
3.70 (d, J=9.0)
3.84 (d, J=9.0)
4.02 (d, J=9.0)
c
a) Data reported in [8]. b) In CDCl3. ) In C5
C
G
To our surprise, compound 1 was degraded completely during storage in the NMR
tube. Interestingly, the main degradation product 1b displayed a similarly strong UV
absorption as 1 but was a less polar on TLC than 1. This suggested that the degradation
probably occurred at the polar parts of 1, i.e., in the side chain, while the conjugated
4-en-3-one unit remained intact. The structure of 1b was corroborated by spectroscopic
means.
The appearance of two new olefinic-proton signals at d 5.60 (br. s) and 5.57 (t, J=7.0 Hz) in 1b indi-
cated the formation of a new pair of C=C in the structure, implying elimination of both the OH and the
AcO groups. This conclusion was consistent with the absence of the AcO signal in the 1H-NMR spectrum
of 1b, and satisfied the molecular formula of C27H40O established by the pseudo-molecular-ion at m/z
403.2980 ([M+Na]+) in the HR-ESI-MS. Furthermore, the relatively downfield chemical shift value of
the two new olefinic protons (d 5.60, 5.57) suggested the formation of a conjugated unit which was further
confirmed by the strong absorption at l 229 nmin the UV spectrum. Me(21) was obviously attached to
the conjugated C=C moiety as shown by its typical downfield shift at d 1.76. The signal at d 5.57 was thus
assigned to the olefinic proton HꢀC(22) due to its coupling pattern (t, J=7.0 Hz), and the resonance at d
5.60 (br. s) to HꢀC(16).
Menellsteroid A (2) was obtained as optically active amorphous white powder. The
molecular formula C27H48O4 of 2 was established by the molecular ion peak at m/z
A
436.3544 (M+) in the HR-EI-MS, indicating four degrees of unsaturation assignable
to four rings of a cholestane skeleton. The structure of 2 was established as

Helvetica Chimica Acta – Vol. 89 (2006)
817
1
(3b,5a,6b,11b)-cholestane-3,5,6,11-tetrol by the H- and 13C-NMR spectra (Table 3),
COSY, HMBC, and NOESY data (Fig. 2), and comparison with the data of
(3b,5a,6b)-cholestane-3,5,6-triol [9].
Fig. 2. 1H,1H-COSY (
), selected HMBC (
), and key NOESY (
) correlations of 2
The IR spectrumof 2 implied the presence of OH groups (3443 cmꢀ1), in agreement with the pres-
ence of four oxygenated C-atoms at d 67.5 (d), 68.7 (d), 76.4 (d) and 76.8 (s) in the 13C-NMR spectrum
and three CHꢀO signals at d 4.19, 4.66, and 4.80–4.88 in the 1H-NMR spectrum( Table 3). Also, the 13C-
NMR and DEPT spectra revealed additional 23 sp3 C-signals (2 C, 6 CH, 10 CH2, 5 Me), which were
completely assigned to their corresponding proton signals by the HMQC experiment (Table 3). Analysis
of the 1H,1H-COSY data led to the two separated proton spin systems CH2(1) to CH2(4) and HꢀC(6) to
Me(26)/Me(27)), as shown in Fig. 2. Two significant HMBC correlations Me(19)/C(1), C(5), C(9), and
C(10), and Me(18)/C(12), C(13), C(14), and C(17) allowed to connect the two proton spin systems
and to establish the constitution of 2 (Fig. 2). The location of HOꢀC(11) was deduced fromthe proton
correlations HꢀC(9)/HꢀC(11)/CH2(12), and further confirmed by the diagnostic long-range correlations
HꢀC(11)/C(13) and CH2(12)/C(11). Careful comparison of the 13C-NMR data of 2 with those of choles-
tane-3b,5a,6b-triol [9] readily revealed that 2 is its 11-hydroxy analog. The a-configuration of HꢀC(11)
was deduced fromits coupling pattern (br. s) and supported by the observation of a distinct NOE cross-
peak between HꢀC(11) and HaꢀC(1). Moreover, the absence of NOE correlations between HꢀC(11)
and both Me(18) and Me(19) further confirmed this conclusion.
Menellsteroid B (3) was obtained as optically active amorphous white powder. The
HR-EI-MS data of 3 established the molecular formula C27H46O4 (m/z 434.3411),
1
resulting in 2 mass units less than 2. Careful comparison the H- and 13C-NMR data
of 3 with those of 2 (Table 3) revealed that 3 differs from 2 only in the nature of the
side chain. Analysis of a couple of new olefinic proton signals and comparison with
reported data established the structure of 3 as (3b,5a,6b,11b,22E)-cholest-22-ene-
3,5,6,11-tetrol.
New olefinic proton signals, an AB systemat d 5.34 (ddd, J=15.6, 7.1, 6.7 Hz, HꢀC(23)) and 5.26
(dd, J=15.6, 8.2 Hz, HꢀC(22)), were observed in 1H-NMR spectrumof 3, as compared to 2. The corre-
sponding C=C bond was positioned at C(22) due to the downfield shift of Me(21) (d 1.09 in 3 and 0.99 in
2). The (E)-configuration of this C=C bond was obviously deduced fromthe coupling constant
J(22,23)=15.6 Hz. Assignments of the 13C-NMR signals (Table 3) for the side chain of 3 were strongly
supported by comparison with reported data [7e][9a].
It may be worth to point out that among the polyhydroxylated steroids, an HOꢀ
C(11) group in b-configuration is quite rare. The related compound sarcoldesterol B

818
Helvetica Chimica Acta – Vol. 89 (2006)
Table 3. 1H- and 13C-NMRData a,b) for Menellsteroid A (2) and 13C-NMRData for Menellsteroid B (3).
d in ppm, J in Hz.
No.
2
3
d(H)
d(C) c)
33.2 (t)
d(C) c)
33.2 (t)
HaꢀC(1)
HbꢀC(1)
HaꢀC(2)
HbꢀC(2)
HꢀC(3)
2.13 (t, J=11.5)
2.34–2.38 (overlapped)
2.25–2.29 (m)
32.6 (t)
32.7 (t)
2.27–2.37 (m)
4.80–4.88 (m)
2.35–2.39 (m)
3.12 (dd, J=12.9, 11.5)
67.5 (d)
42.6 (t)
67.5 (d)
42.6 (t)
HaꢀC(4)
HbꢀC(4)
C(5)
76.8 (s)
76.4 (d)
37.1 (t)
76.8 (s)
76.4 (d)
37.1 (t)
HꢀC(6)
HaꢀC(7)
HbꢀC(7)
HꢀC(8)
HꢀC(9)
C(10)
HꢀC(11)
HaꢀC(12)
HbꢀC(12)
C(13)
HꢀC(14)
HaꢀC(15)
HbꢀC(15)
HaꢀC(16)
HbꢀC(16)
HꢀC(17)
Me(18)
4.19 (br. s)
2.29–2.35 (m)
2.17–2.21 (m)
2.64–2.75 (m)
2.18–2.22 (m)
28,3 (d)
49.1 (d)
40.2 (s)
68.7 (d)
50.3 (t)
28.4 (d)
49.2 (d)
40.2 (s)
68.7 (d)
50.2 (t)
4.66 (br. s)
1.46–1.51 (overlapped)
2.50 (dd, J=13.4, 2.4)
42.6 (s)
58.5 (d)
24.9 (t)
42.5 (s)
58.7 (d)
24.8 (t)
1.25–1.30 (m)
1.72–1.79 (m)
1.24–1.30 (m)
1.82–1.89 (m)
1.23–1.30 (m)
1.10–1.14 (m)
1.26 (s)
28.5 (t)
28.5 (t)
57.3 (d)
15.1 (q)
20.4 (q)
36.4 (d)
19.0 (q)
36.6 (t)
57.0 (d)
15.2 (q)
20.4 (q)
40.8 (d)
21.2 (q)
138.9 (d)
Me(19)
2.20 (s)
HꢀC(20)
1.38–1.44 (m)
0.99 (d, J=6.3)
1.00–1.06 (m)
1.00–1.06 (m)
1.15–1.23 (m)
1.36–1.44 (m)
1.11–1.18 (m)
1.11–1.18 (m)
1.45–1.56 (m)
0.89 (d, J=6.6)
0.89 (d, J=6.6)
Me(21)
HaꢀC(22)
HbꢀC(22)
HaꢀC(23)
HbꢀC(23)
HaꢀC(24)
HbꢀC(24)
HꢀC(25)
Me(26)d)
Me(27)d)
24.3 (t)
39.9 (t)
126.5 (d)
42.3 (t)
28.4 (d)
22.8 (q)
23.0 (q)
28.9 (d)
22.5 (q)
22.6 (q)
a) Bruker DRX-400-MHz spectrometer, C5
and to C5D5
N (d(C) 123.6, 135.8, 150.0). b) Assignments made by HMQC and HMBC. ) By DEPT
sequence. d) Signals may be interchanged.
A
N
c
A
ACHTREUNG
1
(4) [10c] with an a-positioned HOꢀC(11) exhibited different H-NMR data and cou-
pling pattern of HꢀC(11) (d 4.35 (m)) in comparison with those of 2 and 3 both reso-
nating as br. s at d 4.66.

Helvetica Chimica Acta – Vol. 89 (2006)
819
Polyoxygenated steroids with a 3b,5a,6b-trihydroxy moiety are frequently encoun-
tered in marine organisms, such as in sponges [9a][11], anthozoans [9b][10], and star-
fishes [12]. It was reported that sterols with the 3,5,6-trihydroxy moiety might arise bio-
genetically fromthe corresponding sterols with a C(5) =C(6) moiety [9a]. Further stud-
ies should be conducted to verify this hypothesis by biosynthetic experiments.
This research work was financially supported by the National Marine 863 Project (No.
2005AA624040), the Natural Science Foundation of China (No. 20572116), STCSM Projects (Nos.
04ZR14156 and 054307062), and CNR-Italy/CAS-China Joint Projects 2004/2007 and partly supported
by the State Key Program of Basic Research of China (No. 2004CB518905). We are indebted to Prof.
Ren-Lin Zhou of the South China Sea Institute of Oceanology, Chinese Academy of Sciences, for the
identification of the specimen.
Experimental Part
General. Column chromatography (CC): silica gel (Qing Dao Hai Yang Chemical Group Co.;
200–300 and 400–600 mesh). Anal. TLC: precoated silica gel plates (Yan Tai Zi Fu Chemical Group
Co.; G60 F-254). Optical rotation: Perkin-Elmer-341 polarimeter. UV Spectra: 756-CRT spectrophotom-
1
~
eter; lmax in nm. IR Spectra: Nicolet-Magna-FT-IR-750 spectrometer; nmax
in cmꢀ1. H- and 13C-NMR
Spectra: Bruker-DRX-400 spectrometer; at 400 (1H) and 100 MHz (13C); chemical shifts d in ppmrel.
to the residual CHCl3 (d(H) 7.26) or C5H5N (d(H) 7.20, 7.57, 8.73) signals for H, and CDCl3 (d(C)
1
77.0) or C
N
N
1H,1H-COSY, HMQC, HMBC, and NOESY experiments. MS: EI and HR-EI, Finnigan-MAT-95 mass
spectrometer; ESI and HR-ESI, Q-TOF-Micro-LC-MS-MS mass spectrometer; in m/z.
Animal Material. Both gorgonians Muricella flexuosa and Menella verrucosa BRUNDIN were collected
along the coast of Xiaodong Hai, Hainan Province, China, in December 2001, at a depth of 20 m. The
voucher specimens are available for inspection at the Institute of Materia Medica, SIBS-CAS.
Extraction and Purification. The frozen animals (dry weight 28 g for Muricella flexuosa and 209 g for
Menella verrucosa) were cut into small pieces and then extracted with acetone at r.t. The org. extracts
were evaporated to give residues which were partitioned between Et
N
N
were evaporated to give dark green residues: 301 g from Muricella flexuosa and 3.3 mg from Menella ver-
rucosa). The residues were fractionated by CC (silica gel, light petroleumether/acetone gradient), yield-
ing fractions containing crude steroids. The crude steroid fraction from Muricella flexuosa was subjected
to CC (Sephadex-LH-20): muricesteroid (1; 2.4 mg). The crude steroid fraction from Menella verrucosa
was purified by CC (Sephadex-LH-20), followed by reversed-phase HPLC (semi-prep. ODS-HG-5 (5 m,
250”10 mm), MeOH/H2O 3 :1, 2.0 ml/min): menellsteroids A (2; 5.3 mg) and B (3; 1.2 mg).
Muricesteroid (=(20R,22R)-22-(Acetyloxy)-20-hydroxycholest-4-en-3-one; 1). Colorless glass. 1H-
(400 MHz, CDCl3) and 13C-NMR (100 MHz, CDCl3): Table 1. HR-ESI-MS: 481.3311 ([C29
calc. 481.3294).
Hydrolysis of Muricesteroid (1). To 1 (0.2 mg) in dry MeOH (1.0 ml), dry Na2CO3 (3.0 mg) was
added, and the mixture was stirred at r.t. for 72 h. Having adjusted the pH to 7 with dil. HCl soln.
A
(quenching of the reaction), the mixture was extracted with Et2O: (20R,22R)-20,22-dihydroxycholest-
A
4-en-3-one (1a; 0.18 mg). Colorless glass. 1H-NMR (400 MHz, CDCl3): 5.73 (s, HꢀC(4)); 3.38 (d,
J=8.8, HꢀC(22)); 1.21 (s, Me(21)); 1.19 (s, Me(19)); 0.93 (s, Me(18)); 0.90, 0.89 (d, J=6.8, Me(26),
1
Me(27)). H-NMR (400 MHz, C5
A
G
1.17 (s, Me(19)); 1.02 (s, Me(18)); 0.93, 0.92 (d, J=6.7, Me(26), Me(27)). ESI-MS: 439.167 ([M+Na]+).
Cholesta-4,16,20(22)-trien-3-one (1b). Colorless oil. UV (MeOH) 203, 229, 274. 1H-NMR (400 MHz,
CDCl3): 5.74 (s, HꢀC(4)); 5.60 (br. s, HꢀC(16)); 5.57 (t, J=7.0, HꢀC(22)); 1.76 (s, Me(21)); 1.20 (s,
Me(19)); 0.98 (s, Me(18)); 0.88, 0.87 (d, J=6.7, Me(26), Me(27)). HR-ESI-MS: 403.2980 ([C
Na]+; calc. 403.2977).

820
Helvetica Chimica Acta – Vol. 89 (2006)
Menellsteroid
A
(=(3b,5a,6b,11b)-Cholestane-3,5,6,11-tetrol; 2). Amorphous white powder.
[a]2D0 =+6.4 (c=0.33, MeOH). IR (KBr): 3443, 2926, 2857. H- (400 MHz, C5
A
N
1
MHz, C
48O4+; calc. 436.3535).
Menellsteroid B (=(3b,5a,6b,11b,22E)-Cholest-22-ene-3,5,6,11-tetrol; 3). Amorphous white powder.
A
G
H
ACHTREUNG
[a]2D0 =+11.2 (c=0.11, MeOH). IR (KBr): 3447, 2931, 2856. H-NMR (400 MHz, C5
N
N
1
J=15.6, 7.1, 6.7, HꢀC(23)); 5.26 (dd, J=15.6, 8.2, HꢀC(22)); 4.80–4.88 (m, HꢀC(3)); 4.66 (br. s, Hꢀ
C(11)); 4.19 (br. s, HꢀC(6)); 3.12 (dd, J=12.9, 11.5, HbꢀC(4)); 2.50 (dd, J=13.4, 2.4, HbꢀC(12)); 2.20
(s, Me(19)); 1.27 (s, Me(18)); 1.09 (d, J=6.6, Me(21)); 0.89 (d, J=6.6, Me(26), Me(27)). 13C-NMR
(100 MHz, C
N
N
(C27H46
A
ACHTREUNG
REFERENCES
[1] Y. Kashman, Tetrahedron Lett. 1980, 21, 879.
[2] Y. Seo, J.-R. Rho, K. W. Cho, J. Shin, J. Nat. Prod. 1997, 60, 171.
[3] Y. Seo, K. W. Cho, S. Chung, J. Shin, Nat. Prod. Lett. 2000, 14, 197.
[4] Y. Seo, J. Shin, J.-I. Song, J. Nat. Prod. 1995, 58, 1291.
[5] Y. Seo, K. W. Cho, H. Chung, H.-S. Lee, J. Shin, J. Nat. Prod. 1998, 61, 1441.
[6] J.-R. Rho, Y. Seo, K. W. Cho, J.-I. Song, J. Shin, Bull. Korean Chem. Soc. 1996, 17, 529.
[7] a) W. Zhang, Y.-W. Guo, M. Gavagnin, E. Mollo, G. Cimino, Helv. Chim. Acta 2005, 88, 87; b) W.
Zhang, G.-M. Yao, Y.-W. Guo, Acta Crystallogr. Sect. E 2005, 61, 2884; c) W. Zhang, Y.-W. Guo, E.
Mollo, G. Cimino, Helv. Chim. Acta 2004, 87, 2919; d) W. Zhang, Y.-W. Guo, E. Mollo, G. Cimino,
Helv. Chim. Acta 2004, 87, 2341; e) W. Zhang, Y.-W. Guo, E. Mollo, A. Fontana, G. Cimino, J. Nat.
Prod. 2004, 67, 2083.
[8] H. Hikino, T. Okuyama, S. Arihara, Y. Hikino, T. Takemoto, H. Mori, K. Shibata, Chem. Pharm.
Bull. 1975, 23, 1458.
[9] a) G. Notaro, V. Piccialli, D. Sica, G. Corriero, J. Nat. Prod. 1991, 54, 1570; b) Q. Lu, D. J. Faulkner,
Nat. Prod. Lett. 1997, 10, 231.
[10] a) P. K. Datta, A. K. Ray, A. K. Barua, S. K. Chowdhuri, A. Patra, J. Nat. Prod. 1990, 53, 1347; b) I.
Kitagawa, M. Kobayashi, Z. Cui, Y. Kiyota, M. Ohnishi, Chem. Pharm. Bull. 1986, 34, 4590; c) A.
Umeyama, N. Shoji, M. Ozeki, S. Arihara, J. Nat. Prod. 1996, 59, 894.
[11] C. Delseth, R. M. K. Carlson, C. Djerassi, T. R. Erdman, P. J. Scheuer, Helv. Chim. Acta 1978, 61,
1470.
[12] E. Finamore, L. Minale, R. Riccio, G. Rinaldo, F. Zollo, J. Org. Chem. 1991, 56, 1146; M. Iorizzi, P.
Bryan, J. McClintock, L. Minale, E. Palagiano, S. Maurelli, R. Riccio, F. Zollo, J. Nat. Prod. 1995, 58,
653; A. J. Roccatagliata, M. S. Maier, A. M. Seldes, J. Nat. Prod. 1995, 58, 1941.
Received September 6, 2005