Structure and synthesis of a progesterone homologue from the skin of the dorid nudibranch Aldisa smaragdina

Article abstract of DOI:10.1002/1099-0690(200205)2002:9<1500::AID-EJOC1500>3.0.CO;2-D

The short-side-chain steroid 24-norchol-4-ene-3,22-done (6) has been found to be the main metabolite of both skin and mucus of the mollusc Aldisa smaragdina. This compound, previously reported as a microbial degradation product of cholesterol, has never previously been isolated from natural sources. The absolute stereochemistry was determined by synthesis from commercial stigmasterol, and a full NMR characterisation is also reported. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200205)2002:9<1500::AID-EJOC1500>3.0.CO;2-D

FULL PAPER
Structure and Synthesis of a Progesterone Homologue from the Skin of the
Dorid Nudibranch Aldisa smaragdina
[
a]
[a,c]
[a]
[b]
Margherita Gavagnin,* Nicon Ungur,
Ernesto Mollo, Jos e´ Templado, and
Guido Cimino[
a]
Dedicated to the memory of Prof. G. Sodano
Keywords: Aldisa smaragdina / Natural products / Nudibranch molluscs / Steroids / Structure elucidation
The short-side-chain steroid 24-norchol-4-ene-3,22-dione (6)
has been found to be the main metabolite of both skin and
sources. The absolute stereochemistry was determined by
synthesis from commercial stigmasterol, and a full NMR
mucus of the mollusc Aldisa smaragdina. This compound, characterisation is also reported.
previously reported as a microbial degradation product of
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
cholesterol, has never previously been isolated from natural 2002)
Introduction
A large variety of unusual steroids have been isolated
from marine organisms, and especially sponges.[ Among
the most remarkable features encountered, extensive oxy-
genation and alkylation of the side chain and oxidative and
structural modification of the polycyclic core are especially
relevant. Several biosynthesis studies of steroids in sponges
have also been conducted.[
1,2]
3]
In contrast, only a few steroid compounds from marine
molluscs, and in particular from nudibranchs (Figure 1),
have been reported. Some examples are the oxygenated
sterols 1, from the aeolidacean species Cratena peregrina,
[4]
Flabellina affinis and Coryphella lineata, 2, from Diaulula
[
5]
[6]
sandiegensis, and 3, from Adalaria sp. Nudibranchs are
naked molluscs that, even though unprotected by the phys-
ical shield of a shell, appear to be free of predation. Their
survival is in fact assured by defensive strategies that in-
clude the use of chemicals, usually selectively located on the
skin and in the defensive mucous secretion.[
7,8]
In continuation of our research of the chemical ecology
[
9]
of marine molluscs, we have examined the skin metabol- Figure 1. Structures of steroidal compounds from nudibranchs
ites of the doridacean nudibranch Aldisa smaragdina Ortea,
P e´ rez & Llera, 1982. This species is known from Asturias
(
NW Spain) to Morocco, in the Azores, Madeira and the
[
a]
Canary Islands, and also occurs in the Alboran Sea (SW
Mediterranean). In a remarkable example of colour camou-
flage, this nudibranch is quite invisible in the field, due to
its red colour, the same as the sponge on which it lives. A
previous study on the related Pacific species Aldisa
cooperi[ resulted in the finding of two antifeedant steroids
(4 and 5) featuring cholic acid side chains. In this paper, we
Istituto di Chimica di Biomolecolare, CNR,
Via Campi Flegrei 34, Fabbr. 70, 80078 Pozzuoli (Na), Italy
Fax: (internat.) ϩ 39-081/804-1770
E-mail: mgavagnin@icmib.na.cnr.it
[
[
b]
c]
Museo Nacional de Ciencias Naturales, CSIC
J. Gutierrez Abascal 2, 28006 Madrid, Spain
Institutul de Chimie al Academiei de Stiinte a Republicii
Moldova
10]
str. Academiei 3, MD 2028 Chisinau, Republic of Moldova
1500
 WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0509Ϫ1500 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 1500Ϫ1504

A Progesterone Homologue from the Skin of the Dorid Nudibranch Aldisa smaragdina
FULL PAPER
report on the isolation and the synthesis of the unusual ster- methyl group (doublet at δ ϭ 1.12, J ϭ 7.0 Hz). In particu-
oid: 24-norchol-4-ene-3,22-dione (6), previously obtained lar, a structure derived from the 4-cholest-3-one skeleton
[
11]
by chemical synthesis
cholesterol,
product.
and by microbial conversion of with a short side chain was clearly indicated by detailed
[
12Ϫ14]
1
1
but never before reported as a natural analysis of 2D NMR spectra ( H- H COSY and HMQC
experiments). The position of the saturated carbonyl group
at C-22 was confirmed by diagnostic correlations of this
carbon atom (δ ϭ 212.5) with 23-H (δ ϭ 2.10), 20-H (δ ϭ
3
Results and Discussion
2.50) and 21-H
proton and carbon resonances, assigned as shown in
Two specimens (sizes 0.8 and 1.5 cm in length) were Table 1, were consistent with the structure 6 (Figure 1), a
3
(δ ϭ 1.12) in the HMBC spectrum. All
[
11]
caught by scuba divers off Cabo Cope (Murcia, SE Spain) 22-oxosteroid synthesised in 1945
and subsequently ob-
during November 1997 and immediately frozen at Ϫ20 °C. tained from cholesterol by microbial degradation.[
12Ϫ14]
No
A small quantity of mucus secreted by the animals was re- spectral characterisation was reported in previous papers.
covered and also frozen separately. A fragment of the red
Table 1. NMR spectroscopic data[a] of steroid 6
encrusting demosponge on which the animals were found,
identified as Phorbas fictitius, was also collected and frozen.
A further small specimen (0.5 cm in length) was collected
off the island of Alboran (SW Mediterranean) during July
1
996, within the framework of the Spanish oceanographic
campaign ‘‘Fauna IV’’. Later, all this material was trans-
ferred to ICMIB in Naples and chemically analysed. The
largest animal was carefully dissected into the mantle and
the digestive gland, which were separately extracted with
acetone. Because of their small size, the remaining two an-
imals were not dissected, but first immersed in acetone and
submitted to ultrasound vibration for 1 min. Only the meta-
bolites present in the skin (external part) were extracted by
this procedure. The solvent was removed and the animals
were homogenised with a pestle and again treated with acet-
one, so as to extract the digestive gland content (internal
part). The mucous secretion was directly treated with di-
ethyl ether. The sponge sample was also extracted with acet-
one. The ether-soluble portions of the mantle and digestive
gland acetone extracts, obtained in the two different ways,
were analysed by TLC, together with mucus extract, and
compared to the ether-soluble portion of the sponge acet-
one extract. One main UV-sensitive metabolite at R ϭ 0.35
f
(light petroleum ether/diethyl ether, 1:1) characterised both
the skin and the mucus, but was absent in the digestive
gland of the mollusc and in the sponge. The two external
part and mucus extracts were combined (11.2 mg) and frac-
tionated by silica gel chromatography (petroleum ether/di-
ethyl ether gradient). Pure compound 6 (1.5 mg) was ob-
tained from the fractions eluted with light petroleum ether/
diethyl ether (8:2).
[
a]
Bruker 500 MHz; CDCl
3
; chemical shifts (ppm) referred to
CHCl
atom.
DEPT sequence.
3
[
(δ ϭ 7.26) for proton and to CDCl (δ ϭ 77.0) for carbon
3
b]
[c]
Assignments by HMQC and HMBC experiments. By
[d]
1
1
[e]
Assignments by H- H COSY experiment.
J ϭ 10.0 Hz.
The molecular formula of C H O , deduced from
2
3
34
2
The configuration of the stereogenic centres in the poly-
cyclic core, believed to be the same as in the 4-cholest-3-one
skeleton, was confirmed by diagnostic NOEs (Figure 2). In
HREIMS on the molecular ion at m/z ϭ 342, indicated
seven degrees of unsaturation. The presence of two oxo
functions, one of which was α,β-unsaturated, was suggested
Ϫ1
by two intense infrared bands at 1708 and 1650 cm and
1
3
confirmed by two signals in the C NMR spectrum at δ ϭ
12.5 and 199.4. A conjugated trisubstituted double bond
2
(
1
δ ϭ 5.73 in H NMR spectrum and δ ϭ 123.9 and 171.1
1
3
in C NMR spectrum) accounted for another degree of
1
unsaturation, the remainder being due to four rings. H and
13
C NMR spectra showed signals supporting a steroid tet-
racyclic structure featuring three tertiary methyl groups
(singlets at δ ϭ 0.74, 1.18 and 2.10) and one secondary Figure 2. Selected NOEs in steroid 6
Eur. J. Org. Chem. 2002, 1500Ϫ1504
1501

M. Gavagnin, N. Ungur, E. Mollo, J. Templado, G. Cimino
FULL PAPER
Scheme 1. Synthesis of steroid 6 from stigmasterol (7)
particular, cross-peaks in the NOESY spectrum between 8- α,β-unsaturated double bond in the A-ring and the oxo
H and both 19-H and 18-H , as well as 18-H and 20-H, group at C-22, is an interesting homologue of progesterone.
3
3
3
indicated their β-orientation. A significant NOE was also
observed between 9-H and 14-H, which were α-oriented.
The configuration at C-20 was not determined at this Experimental Section
point: the common (20R) stereochemistry should be fa-
voured even though the opposite configuration has also General: Silica gel chromatography was performed with precoated
been reported for marine sterols.[15] In order to confirm this,
Merck F254 plates and Merck Kieselgel 60 powder. Optical rota-
tions were measured with a Jasco DIP 370 digital polarimeter, and
CD curves were recorded on a Jasco 710 spectropolarimeter. The
IR spectra were taken with a Bio-Rad FTS 7 spectrophotometer.
The UV spectra were obtained with a Varian DMS 90 spectropho-
however, the partial synthesis of 6 from commercial stig-
masterol (7) was carried out in four steps (Scheme 1). Stig-
masterol (7) was oxidised by Oppenauer reaction (cyclo-
hexanone, aluminium isopropoxide) to α,β-unsaturated ke-
tone 8 (93% yield), which was subsequently subjected to
1
13
tometer. H and C NMR spectra were recorded with WM 500
and DPX 300 MHz Bruker spectrometers in CDCl ; chemical shifts
are reported in ppm referred to CHCl as internal standard (δ ϭ
3
ozonolysis to give the oxo aldehyde 9 (92% yield[16]).[17]
A
3
1
13
Grignard reaction between 9 and MeMgI in Et O gave a 7.26 for H and δ ϭ 77.0 for C). EIMS and HREIMS spectra
2
mixture of epimeric alcohols 10 (85% yield[ ), which were were measured with TRIO 2000 VG Carlo Erba and on Kratos
16]
transformed by treatment with PDC into the corresponding MS50 instruments, respectively. Stigmasterol (7) was purchased
1
from Aldrich Chemical Co. (m.p., [α]
D
and H NMR spectroscopic
ketone (87% yield), the spectroscopic data (including the
CD profile) of which were identical with those of 6, clearly
indicating a (20R) stereochemistry for the natural com-
pound.
data were identical to those reported in the literature).[
18,19]
Biological Material: Aldisa smaragdina (two specimens, 0.8 and
1
.5 cm long) was collected off Cabo Cope (SE Spain) by hand
A defensive role is strongly suspected for steroid 6 in the (scuba), during November 1997 at a depth of 2Ϫ3 m and frozen at
nudibranch, due to its selective presence in the skin and in Ϫ20 °C. A small quantity of mucus secreted by the animals was
recovered and separately frozen at Ϫ20 °C. A sample of the sponge
the mucous secretion of the animal. The absence of 6 in the
P. fictitius was also collected and frozen at Ϫ20 °C. The other small
digestive gland of the mollusc and in the sponge Phorbas
specimen of A. smaragdina (0.5 cm long) was collected off the is-
fictitius, on which the animal lives, should rule out a dietary
origin for this compound. Analogously with A. cooperi,[
10]
land of Alboran, at a depth of 9 m, on July 27, 1996, during the
Spanish oceanographic campaign ‘‘Fauna IV’’. All the material was
transferred to ICMIB. The nudibranch and the sponge were identi-
fied by one of us (J. T.).
A. smaragdina might obtain compound 6 from a dietary
steroid precursor by degradation of the side chain, or might
biosynthesise it de novo. This latter hypothesis seems to be
favoured; no steroidal precursors of 6 exhibiting an oxidised
A-ring were in fact detected in the preyed-upon sponge by
chemical analysis. However, it is worth noting that com-
pound 6 is biogenetically related to steroid 4, from which it
might derive through β-oxidation and subsequent decarb-
oxylation.
Extraction and Purification Procedure: The largest individual of A.
smaragdina was dissected into the mantle and the digestive gland,
which were extracted separately with acetone (each 3 mL ϫ 3). The
two other individuals were immersed in acetone (5 mL) and ex-
tracted by use of ultrasound vibration for 1 min. Only the metabol-
ites present in the external part were extracted by this procedure.
The solvent was removed, and the animals were homogenised with
a pestle and again treated with acetone (5 mL ϫ 3). The sample of
In addition to the ecological aspect, the discovery of a
3
,22-dioxosteroid from a natural source is very intriguing, the sponge P. fictitius was also extracted with acetone (10 mL ϫ
due to the structural feature that this molecule, with the 3). The small quantity of mucus secreted by the mollusc, recovered
1502
Eur. J. Org. Chem. 2002, 1500Ϫ1504

A Progesterone Homologue from the Skin of the Dorid Nudibranch Aldisa smaragdina
FULL PAPER
during collection, was directly extracted with diethyl ether (10 mL
ϫ 3). The extract obtained was compared by TLC chromatography
or C-17), 49.4 (C-17 or C-20), 43.0 (C-13), 39.2 (C-12), 38.5 (C-
10), 35.6 (C-1 or C-8), 35.5 (C-8 or C-1), 33.9 (C-2), 32.8 (C-6),
31.9 (C-7), 27.0 (C-16), 24.5 (C-15), 20.9 (C-11), 17.3 (C-19), 13.4
(silica gel and light petroleum ether/diethyl ether in different ratios
ϩ
as eluent) to the ether-soluble portion of the sponge acetone extract
and to the ether-soluble portions of both the mollusc external and
the mollusc internal part acetone extracts, obtained either by treat-
ment of dissected sections or by ultrasound extraction. The external
(C-21), 12.3 (C-18). EIMS: m/z (%) ϭ 328 (73) [M ], 286 (30),
ϩ
ϩ
[M Ϫ CH
2
CO], 271 (20) [M Ϫ side chain], 229 (32), 124 (100).
328.2402, found 328.2394.
HREIMS: calcd. for C22
32 2
H O
part and mucus extracts were combined (11.2 mg) and submitted Compound 10: MeMgI in Et
2
O (3 , 50 µL) was added, at room
temperature and under Ar, to a stirred solution of aldehyde 9
(41.1 mg, 0.12 mmol) in 1.0 mL of anhydrous Et O. The reaction
mixture was stirred at room temp. for 1.5 h, and at the end of this
period poured into 10% H SO solution (5 mL) and extracted with
Et O (5 mL ϫ 3). The combined ethereal extract was washed with
O, a saturated solution of NaHCO and again H O, and was
finally dried with Na SO . After evaporation of the solvent, the
to silica gel column chromatography (petroleum ether/diethyl ether
gradient). The fractions eluted by light petroleum ether/diethyl
ether (8:2) were combined to give pure compound 6 (1.5 mg).
2
2
4
Spectroscopic Data for Natural 6: R
ether, 1:1) ϭ 0.35. IR (liquid film): ν˜ ϭ 1707, 1645 cm . UV
f
(light petroleum ether/diethyl
2
Ϫ1
H
2
3
2
(
MeOH): λmax (lg ε) ϭ 238 nm (3.48). CD (EtOH): [θ]217 ϭ 6200.
2
4
1
13
ϩ
H and C NMR: see Table 1. EIMS: m/z (%) ϭ 342 (30) [M ],
residue was chromatographed on a silica gel column (0.8 g, light
petroleum ether/diethyl ether, from 100:0 to 50:50), to give starting
ϩ
ϩ
3
1
00 (12) [M Ϫ CH
2
CO], 271 (18) [M Ϫ side chain], 229 (24),
342.2559,
47 (35), 124 (100). HREIMS: calcd. for C23
34 2
H O
compound 9 (9.3 mg, 23%) and alcohol 10 (mixture of the two
found 342.2552.
[16]
f
epimers at C-22) (28.2 mg, 85% ). R (light petroleum ether/ethyl
Ϫ1
acetate, 7:3) ϭ 0.20. IR (liquid film): ν˜ ϭ 1660 cm . UV (MeOH):
Compound 8: Oppenauer oxidation of stigmasterol (7) was con-
1
ducted under the conditions reported in the literature.[
20]
λ
max (lg ε) ϭ 239 nm (3.29). H NMR (300 MHz, signals of the
Freshly
predominant epimer): δ ϭ 5.72 (s, 1 H, 4-H), 3.94 (m, 1 H, 22-H),
distilled cyclohexanone (14.0 mL, 135.1 mmol) was added to a so-
lution of stigmasterol (7, 500.0 mg, 1.2 mmol) in anhydrous toluene
1
6
.19 (s, 3 H, 19-H
.3 Hz, 3 H, 23-H
3
), 1.16 (d, J ϭ 6.5 Hz, 3 H, 21-H
3
), 0.92 (d, J ϭ
1
3
3
), 0.74 (s, 3 H, 18-H ). C NMR (75.5 MHz,
3
(
125.0 mL) under argon. The solution was then distilled until tolu-
ene (18.0 mL) had been removed. Aluminium isopropoxide (1.12 g,
.5 mmol) in toluene (42.0 mL) was added dropwise (over 30 min)
signals of the predominant epimer): δ ϭ199.8 (C-3), 171.8 (C-5),
23.8 (C-4), 69.3 (C-22), 55.8 (C-14), 53.8 (C-9), 52.8 (C-17), 42.1
C-13), 41.8 (C-20), 39.7 (C-12), 38.6 (C-10), 35.7 (2 C, C-1 and C-
), 34.0 (C-2), 33.0 (C-6), 32.0 (C-7), 27.7 (C-16), 24.2 (C-15), 21.5
C-21), 21.1 (C-11), 17.4 (C-19), 12.0 (C-18), 11.3 (C-23). EIMS:
1
(
8
5
to the residue and the solution was again distilled to remove tolu-
ene. After 45 min, the reaction mixture was cooled, washed with
water and dried with Na SO . The solvent was evaporated and the
2 4
residue was purified by flash chromatography (silica gel, light pet-
roleum ether/diethyl ether, 95:5) to give crystalline compound 8
(
ϩ
ϩ
m/z (%) ϭ 344 (95) [M ], 326 (75) [M Ϫ H
2
O], 281 (44), 269 (35)
Ϫ side chain], 229 (65), 207 (90), 124 (100). HREIMS: calcd.
for C23 344.2715, found 344.2788.
ϩ
[M
36 2
H O
f
(462.1 mg, 93%). R (light petroleum ether/ethyl acetate, 7:3) ϭ
0
1
D
.62. M.p. 128Ϫ129 °C (light petroleum ether). [α] ϭ ϩ40.5 (c ϭ
Compound 6: PDC (30 mg) was added to a stirred solution of com-
pound 10 (20.0 mg, 0.06 mmol) in anhydrous CH Cl (1.0 mL) un-
der Ar. The reaction mixture was stirred at room temp. for 3 h and
was then filtered through SiO . The solvent was evaporated and
the residue (20.7 mg) was subjected to chromatography on a silica
gel column (0.4 g, light petroleum ether/diethyl ether, from 100:0
). IR (liquid film): ν˜ ϭ 1685 cmϪ1. UV (MeOH): λmax
.25, CHCl
3
1
2
2
(lg ε) ϭ 238 nm (3.32). H NMR (300 MHz): δ ϭ 5.72 (s, 1 H, 4-
H), 5.14 (dd, J ϭ 8.4, 15.5 Hz, 1 H, 22-H), 5.01 (dd, J ϭ 8.4,
4.7 Hz, 1 H, 23-H), 1.18 (s, 3 H, 19-H ), 1.01 (d, J ϭ 6.7 Hz, 3
H, 21-H ), 0.84 (d, J ϭ 6.4 Hz, 3 H, 26-H ), 0.80 (t, J ϭ 7.3 Hz, 3
H, H -29), 0.80 (d, J ϭ 7.1 Hz, 3 H, 27-H ), 0.72 (s, 3 H, 18-H ).
2
1
3
3
3
3
3
3
to 80:20), to give crystalline diketone 6 (17.4 mg, 87%). R
petroleum ether/ethyl acetate 7:3) ϭ 0.32. M.p. 203Ϫ204 °C (light
petroleum ether/diethyl ether, 1:1). [α] ϭ ϩ34.0 (c ϭ 0.12, CHCl ).
IR (liquid film): ν˜ ϭ 1663 cm , 1710. UV (MeOH): λmax (lg ε) ϭ
f
(light
1
3
C NMR (75.5 MHz): δ ϭ 199.7 (C-3), 171.8 (C-5), 138.1 (C-22),
29.4 (C-23), 123.7 (C-4), 55.9 (C-17 or C-14), 55.8 (C-14 or C-17),
3.8 (C-9), 51.2 (C-24), 42.2 (C-13), 40.5 (C-20), 39.5 (C-12), 38.6
1
5
D
3
Ϫ1
(C-10), 35.64 (C-1 or C-8), 35.56 (C-8 or C-1), 34.0 (C-2), 32.9 (C-
1
2
38 nm (3.50). CD (EtOH): [θ]217 ϭ 21800. H NMR (300 MHz):
δ ϭ 5.73 (s, 1 H, 4-H), 2.10 (s, 3 H, 23-H ), 1.18 (s, 3 H, 19-H ),
.12 (d, J ϭ 6.9 Hz, 3 H, 21-H ), 0.73 (s, 3 H, 18-H ). C NMR
75.5 MHz): δ ϭ 212.3 (C-22), 199.3 (C-3), 171.0 (C-5), 123.9 (C-
6
2
1
), 32.0 (C-7), 31.8 (C-25), 28.9 (C-16), 25.4 (C-28), 24.2 (C-15),
1.13 (C-26), 21.10 (C-21), 21.0 (C-11), 18.9 (C-27), 17.3 (C-19),
2.2 (C-29), 12.1 (C-18). EIMS: m/z (%) ϭ 410 (100) [M ], 367
3
3
1
3
1
(
3
3
ϩ
ϩ
ϩ
(60) [M Ϫ iPr], 298 (50), 271 (75) [M Ϫ side chain], 245 (42).
4
3
), 55.4 (C-14), 53.8 (C-9), 52.1 (C-17), 50.3 (C-20), 42.7 (C-13),
9.6 (C-12), 38.6 (C-10), 35.8 (C-8 or C-1), 35.7 (C-1 or C-8), 34.0
HREIMS: calcd. for C29 46O 410.3548, found 410.3540.
H
Compound 9: An O /O mixture was slowly bubbled, at Ϫ60 °C for
3
3
2
(C-2), 32.9 (C-6), 32.0 (C-7), 28.0 (C-23), 27.4 (C-16), 24.4 (C-15),
min, through a solution of ketone 8 (136.0 mg, 0.33 mmol) in 21.0 (C-11), 17.4 (C-19), 16.3 (C-21), 12.2 (C-18). EIMS: m/z (%) ϭ
ϩ
ϩ
ϩ
anhydrous CH
2
Cl
2
2
(7.2 mL). Me S (0.1 mL) was then added to the
2
342 (80) [M ], 300 (30) [M Ϫ CH CO], 271 (55) [M Ϫ side
reaction mixture, and the solution was stirred for 20 min. The solv- chain], 228 (34), 147 (55), 124 (100). HREIMS: calcd. for C23
34 2
H O
ent was evaporated and the residue (108.2 mg) was chromato-
graphed on an silica gel column (light petroleum ether/diethyl ether,
342.2559, found 342.2570.
from 99:1 to 95:5), to give starting compound 8 (24.1 mg, 18%)
and oxo aldehyde 9 (82.0 mg, 92%[ ). R
16]
(light petroleum ether/
f
Acknowledgments
ethyl acetate, 7:3) ϭ 0.33. M.p. 175Ϫ176 °C (light petroleum ether/
3
diethyl ether, 1:1). [α] ϭ ϩ56.5 (c ϭ 0.96, CHCl ). IR (liquid film): We are grateful to Mr. F. Castelluccio for technical help. We also
D
Ϫ1
ν˜ ϭ 1672 cm , 1720. UV (MeOH): λmax (lg ε) ϭ 239 nm (3.23). thank Mr. R. Turco for drawings. This work was partly funded
1
H NMR (300 MHz): δ ϭ 9.56 (d, J ϭ 3.2 Hz, 1 H, 22-H), 5.73 (s, by Pharmamar (contract ‘‘Bioactive Marine Metabolites’’), by the
1
0
H, 4-H), 1.18 (s, 3 H, 19-H
3
), 1.12 (d, J ϭ 6.9 Hz, 3 H, 21-H
3
), Italian-Spanish bilateral project ‘‘Studio Biologico e Chimico di
Invertebrati Bentonici da Coste Mediterranee e Atlantiche’’, and
by the Spanish project REN2000-0890/GLO.
13
3
.75 (s, 3 H, 18-H ). C NMR (75.5 MHz): δ ϭ 205.0 (C-22), 199.8
(C-3), 171.3 (C-5), 123.8 (C-4), 55.1 (C-14), 53.7 (C-9), 50.8 (C-20
Eur. J. Org. Chem. 2002, 1500Ϫ1504
1503

M. Gavagnin, N. Ungur, E. Mollo, J. Templado, G. Cimino
FULL PAPER
[
[
12]
13]
V. Schwarz, P. Pihera, J. Protiva, R. Mickova, Collect. Czech.
Chem. Commun. 1984, 49, 2713Ϫ2719.
[
1]
D. J. Faulkner, Nat. Prod. Rep. 2001, 18, 1Ϫ49 and earlier art-
icles in this series.
J. Protiva, V. Schwarz, P. Pihera, Czech Patent no. CS235660,
[
2]
A. Aiello, E. Fattorusso, M. L. Menna, Steroids 1999, 64,
1
986.
687Ϫ714.
[14]
[15]
Jpn Kokai Tokkyo Koho, Japan Patent no. 56120698, 1981.
D. J. Vanderah, C. Djerassi, J. Org. Chem. 1978, 43,
[
[
3]
4]
C. Djerassi, C. J. Silva, Acc. Chem. Rev. 1991, 24, 371Ϫ378.
G. Cimino, S. De Stefano, S. De Rosa, G. Sodano, G. Villani,
Bull. Soc. Chim. Belg. 1980, 89, 1069Ϫ1073.
1
442Ϫ1448.
[16]
[17]
Yields are reported with allowance for recovered starting com-
pounds.
[
[
[
[
5]
6]
7]
8]
D. E. Williams, S. W. Ayer, R. J. Andersen, Can. J. Chem. 1986,
64, 1527Ϫ1529.
Racemisation at C-20 in aldehyde 9 could occur during the
workup. However, analysis of the NMR spectra of both the
crude ozonolysis mixture and the pure compound 9 ruled out
this possibility. Proton and carbon spectra recorded at different
times on the same sample showed signals attributable to one
epimer.
The Merck Index, An Encyclopedia of Chemicals, Drugs and
Biologicals, 12th ed. (Eds.: S. Budavari, M. J. O’Neil, A. Smith,
P. E. Heckelman, J. F. Kinnear), Merck & Co. Inc., Rahway,
NJ, 1996, pp. 8969.
T. Akihisa, Y. Matsubara, P. Ghosh, S. Thakur, N. Shimizu, T.
Tamura, T. Matsumoto, Phytochemistry 1988, 27, 1169Ϫ1172.
J. C. Orr, J. F. Templeton, H. Majgier-Baranowska, K. Marat,
J. Chem. Soc., Perkin Trans. 1 1994, 18, 2667Ϫ2670.
Received December 6, 2001
R. J. Stonard, J. C. Petrovitch, J. Andersen, Steroids 1980, 36,
81Ϫ86.
P. Karuso, in Bioorganic Marine Chemistry (Ed.: P. J. Scheuer),
Springer-Verlag, Berlin, 1987, pp. 31Ϫ60.
D. J. Faulkner, in Ecological Roles of Marine Natural Products
(
Ed.: V. J. Paul), Comstock Publishing Associates, Ithaca, New
[18]
York, 1992, pp.119Ϫ163.
[
9]
For recent reviews see: [9a] G. Cimino, A. Fontana, M. Gavag-
[9b]
nin, Curr. Org. Chem. 1999, 3, 327Ϫ372.
G. Cimino, M. L.
[
[
19]
20]
Ciavatta, A. Fontana, M. Gavagnin, in Bioactive Compounds
from Natural Sources (Ed.: C. Tringali), Taylor & Francis, Lon-
don, 2001, pp. 577Ϫ637.
[
[
10]
11]
S. W. Ayer, R. J. Andersen, Tetrahedron Lett. 1982, 23,
1039Ϫ1042.
W. Cole, P. L. Julian, J. Am. Chem. Soc. 1945, 67, 1369Ϫ1375.
[O01575]
1504
Eur. J. Org. Chem. 2002, 1500Ϫ1504