Xenimanadins A-D, a family of xenicane diterpenoids from the Indonesian soft coral Xenia sp.

Article abstract of DOI:10.1016/j.tet.2008.01.120

Four novel xenicane diterpenoids, xenimanadins A-D (1-4), characterized by the unusual 2,6-dimethoxytetrahydropyran functionality, have been isolated from the Indonesian soft coral Xenia sp., together with three known xeniolides. The stereostructure of these metabolites has been established through extensive interpretation of NMR data and application of the modified Mosher method. Xenimanadins were tested against tumor cell lines, revealing details about the cytotoxic potential of xenicane diterpenoids.

Full text of DOI:10.1016/j.tet.2008.01.120

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3141e3146
www.elsevier.com/locate/tet
Xenimanadins AeD, a family of xenicane diterpenoids
from the Indonesian soft coral Xenia sp.
Ernesto Fattorusso a, Adriana Romano a, Orazio Taglialatela-Scafati a,
M. Janib Achmad b,y, Giorgio Bavestrello c, Carlo Cerrano d
,
*
a Dipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
b Faculty of Fisheries and Marine Sciences, Sam Ratulangi University, 95115 Manado, Indonesia
c Dipartimento di Scienze del Mare, Universita` Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
d Dipartimento per lo Studio del Territorio e delle sue Risorse, Universita` di Genova, Corso Europa 26, 16132 Genova, Italy
Received 6 December 2007; received in revised form 16 January 2008; accepted 31 January 2008
Available online 3 February 2008
Abstract
Four novel xenicane diterpenoids, xenimanadins AeD (1e4), characterized by the unusual 2,6-dimethoxytetrahydropyran functionality, have
been isolated from the Indonesian soft coral Xenia sp., together with three known xeniolides. The stereostructure of these metabolites has been
established through extensive interpretation of NMR data and application of the modified Mosher method. Xenimanadins were tested against
tumor cell lines, revealing details about the cytotoxic potential of xenicane diterpenoids.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Xenia soft corals; Indonesia; Diterpenes; Structure elucidation; NMR
1. Introduction
Soft corals belonging to genus Xenia (order Alcyonacea,
OHC
family Xeniidae) are prolific producers of diterpenoid meta-
bolites, which are collectively called xenicanes.1 Among these
molecules, a remarkable group, structurally characterized by
a pyran ring fused to a nine-membered carbocyclic ring to
build a 2-oxabicyclo[7.4.0]tridecane system, represents an in-
teresting class of bioactive metabolites exhibiting antitumor2,3
and antibacterial4 activities. These metabolites have been clas-
sified according to the structure of the six-membered ring (ring
A). In this way, starting from 1977, with the isolation of the
first member of this class,5 four families have been identified
(Fig. 1): xenicins,5 xenialactols,6 xeniolides A7 and xeniolides
B.7 It has been hypothesized that these molecules were derived
Putative precursor
OHC
E or
Z (iso- series)
O
A
O
O
O
O
OR
O
OR
Xenialactols
Xenicins
Xeniolides B
Xeniolides A
Figure 1. The four classes of pyran-cyclononane xenicane diterpenoids and
their postulated precursor.
* Corresponding author. Tel.: þ39 081 678509; fax: þ39 081 678552.
E-mail address: scatagli@unina.it (O. Taglialatela-Scafati).
biogenetically from GGPP through a common precursor,
namely the nine-membered dialdehyde derivative reported in
Figure 1. Some molecules maintaining an opened ring A
y
Present address: Universitas Khairun, Kampus II, Kelurahan Gambesi,
97719 Ternate, North Moluccas, Indonesia.
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.120

3142
E. Fattorusso et al. / Tetrahedron 64 (2008) 3141e3146
16
15
polarity. Theobtainedfractions werefurtherpurified byHPLCto
H3CO
H3CO
17
yield seven pure xenicane diterpenoids: the new xenimanadins
AeD (1e4, Fig. 2) and the known blumiolide C (5),10 xeniolide
F (6),11 and deoxyoxidoisoxenioliode A (7).10 Compounds 5e7
have been identified by comparison of obtained spectroscopic
data with those reported in the literature.10,11
12
5
H
H
7
H3CO
18
H3CO
4
4a
11a
3
O
O
O
H
1
OH
OH
H
H
10
H3CO
The ESI-MS (positive ions) spectrum of xenimanadin A (1)
exhibited a pseudo-molecular ion peak at m/z 415 [MþNa]þ,
while the molecular formula C23H36O5, implying six unsatura-
H3CO
19
2
1
tion degrees, was assigned to 1 by means of HREIMS. The 13
C
H3CO
H3CO
NMR spectrum of 1 (CDCl3, Table 2) showed 22 signals (the
resonances of two carbons being coincident), eight of which
were located in the sp2 region of the spectrum. The 1H
NMR spectrum of 1 (CDCl3, Table 1), exhibited a series of
well resolved signals between dH 4.40 and 6.70, three methyl
singlets between dH 3.10 and 3.60, a series of multiplets
between dH 1.50 and 2.60 and, finally, three methyl singlets
at dH 1.70 (3H) and 1.30 (6H). All these proton signals were
associated with those of the directly linked carbons by utiliz-
ing an HSQC experiment. Obtained information indicated the
presence of three methoxy groups, two dioxymethines (dH
4.47, dC 104.5; dH 5.46, dC 100.0), and four double bonds
H
H
H3CO
H3CO
O
O
H
O
O
H
H
H3CO
H3CO
4
3
Figure 2. Xenimanadins AeD (1e4).
HO
HO
H
H
O
O
O
O
OH
Table 1
1H NMR data of xenimanadins AeD (1e4) in CDCl3 at 500 MHz
O
H
H
Position
1
2
3
4
HO
5
6
dH, mult.,
J in Hz
dH, mult.,
J in Hz
dH, mult.,
J in Hz
dH, mult.,
J in Hz
H
1
4.47, d, 8.8
3.42, s
4.53, d, 8.8
3.45, s
4.48, d, 8.8
3.43, s
4.71, d, 8.5
3.45, s
O
1-OMe
3
O
H
O
5.46, br s
3.59, s
5.50, br s
3.60, s
5.48, br s
3.59, s
5.51, br s
3.54, s
H
3-OMe
4a
2.30, br dd,
10.6, 4.5
1.88, m
2.37, br dd,
10.6, 4.5
1.94, m
2.32, br dd,
10.6, 4.5
1.92, m
1.61, m
2.20, m
1.12, m
2.89, dd,
11.4, 2.7
1.50a
2.31, br dd,
10.6, 4.5
1.71, m
1.71, m
2.87, m
2.24, m
5.93, s
7
Figure 3. The known blumiolide C (5), xeniolide F (6), and deoxyoxidoisox-
eniolide A (7).
5
1.53, m
2.15a
2.13a
5.17, br d, 7.0
1.67, m
2.16, m
6
1.14, m
2.88, d, 7.2
(e.g., azamilides8) or exhibiting a contracted ring A (e.g.,
xeniaethers9 and xeniaphyllanes6) have also been found.
We have recently analyzed a specimen of Xenia sp. collected
along the coasts of Manado (Indonesia), and the present paper
describes the isolation and the structural elucidation of seven
xenicane diterpenoids from this source. Four of these metabo-
lites are new compounds showing an unusual 2,6-dialkoxypyran
system, and we propose to designate this class of xenicanes with
the name xenimanadin (xenimanadins AeD, 1e4, Fig. 2). The
three additional isolated metabolites are known compounds
belonging to the class of xeniolide A (5e7, Fig. 3).
8
9
4.74, br t, 7.0
3.78, m
1.45a
10
2.53, dd,
14.4, 7.0
2.35, br d,
14.4
2.66, dd,
13.5, 7.0
2.46, br d,
13.5
2.43, br t,
10.8
2.26, m
3.32, s
3.32, s
2.51, m
5.92a
11a
12
1.69, dd,
10.6, 8.8
6.08, d, 11.3
2.08, dd,
10.6, 8.8
6.07, d,
11.3
2.13, dd,
10.6, 8.8
6.04, d,
11.3
13
6.63, dd,
15.8, 11.3
5.61, d, 15.8
6.60, dd,
15.8, 11.3
5.63, d,
15.8
6.58, dd,
15.8, 11.3
5.60, d,
15.8
6.43, dd,
15.8, 11.3
5.66, d,
15.8
2. Results
14
15-OMe
16
17
3.17, s
1.30, s
3.17, s
1.30, s
3.15, s
1.26, s
3.14, s
1.26, s
Specimens of Xenia sp. were collected in the Bunaken Marine
Park of Manado (North Sulawesi, Indonesia) and extracted
repeatedly with methanol. The methanol extract was partitioned
between water and ethyl acetate to yield a brown organic frac-
tion, which was subsequently chromatographed by MPLC over
silica gel column using n-hexane/EtOAc mixtures of increasing
1.30, s
1.70, s
1.30, s
1.42, s
1.30, s
1.37, s
1.30, s
1.81, s
18
19
4.94, br s
4.91, br s
5.12, br s
5.01, br s
4.98, br s
4.91, br s
5.08, br s
5.02, br s
a
Overlapped with other signals.

E. Fattorusso et al. / Tetrahedron 64 (2008) 3141e3146
3143
(four of the eight sp2 carbons are monoprotonated, one is di-
MeO
protonated, and three are unprotonated). Consequently, taking
into account the degrees of unsaturation implied by molecular
formula, xenimanadin A (1) must be a bicyclic compound.
The COSY spectrum of 1 disclosed the presence of three
spin systems, (A)e(C), depicted in bold in Figure 4: moiety
(A) includes a sequence of three double bond methines; moi-
ety (B) starts with the dioxymethine at dH 4.47 and terminates
with the relatively deshielded methylene at dH 2.15 and 2.13;
moiety (C) includes the fourth double bond methine, which is
coupled with a carbinolic proton (dH 4.47; dC 68.0), in turn
12
1
13
3
4a
6
H
H
MeO
11a
OH
H
8
O
H
H
MeO
Figure 5. Diagnostic ROESY correlations detected for xenimanadin A (1).
constant JH-11a/H-4a¼10.6 Hz, supporting a pseudo-diaxial
orientation. ROESY cross-peaks H-1/H-4a and 1-OMe/H-11a
supported the trans ring junction and pointed also to the trans
orientation of H-1 and H-11a; accordingly, the coupling
constant JH-1/H-11a¼8.8 Hz is in agreement with a pseudo-
diaxial orientation. The relative orientation of H-3 was inferred
by the ROESY cross-peak of 3-OMewith H-4a, indicative of the
trans relationship between H-3 and H-4a. The relative configu-
ration at the remaining chiral carbon of the molecule, located at
C-9, was suggested by a cross-peak between H-11a and H-9,
indicative of the cis orientation of these two protons on the
nine-membered ring.
The presence of a secondary alcohol group in the structure
of xenimanadin A (1), offered us the possibility of upgrading
the above determined relative stereochemistry to the absolute
one, through application of the modified Mosher’s method.12
For this aim, two aliquots of 1 were treated with (R)- and
(S)-MTPA chloride in dry pyridine to give the corresponding
(S)-1a and (R)-1b MTPA esters. The pattern of Dd (SꢀR)
values (see Fig. 6) allowed us to establish the R configuration
at C-9 and, consequently, to deduce the complete stereostruc-
ture of xenimanadin A (1) as reported in Figure 2.
Stereostructural determination of the remaining xenimana-
dins BeD (2e4) followed the same approach above detailed
for xenimanadin A (1). Accurate inspection of COSY, HSQC,
and g-HMBC spectra was instrumental to deduce their planar
structures and the signal assignments reported in Tables 1 and
2, while, as above, stereochemical details were deduced on the
basis of scalar coupling constants and spatial couplings. For
these molecules, the absolute configuration has not been
determined, however, their structures have been drawn in
Figure 2 by extending the absolute configuration above deduced
for xenimanadin A (1). For brevity, here we will discuss only the
significant differences between each of these molecules and
coupled with an allylic methylene (dH 2.35 and 2.53).
2,3
A series of key
J
correlations evidenced by the
H,C
g-HMBC (gradient-selected heteronuclear multiple bond coher-
ence) spectrum (Fig. 4) allowed us to assemble the above
skeletal fragments and, including also the remaining uncoupled
moieties, to draw the planar structure of xenimanadin A (1). In
particular, the three correlation peaks of H3-18 (dH 1.70) allowed
us to join moieties (B) and (C); analogously, the three cross-
peaks exhibited by the exocyclic double bond protons at dH
4.91 and 4.94 (H2-19) indicated the presence of the cyclononene
ring. Going on, valuable information came from the g-HMBC
cross-peaks of the two dioxymethine protons: the 2,3J cross-
peaks of H-3 with C-1, C-4, C-4a, and C-12 indicated the struc-
ture of ring A, its junction to the nine-membered ring, and the
linkage site of moiety (A). Attachment of this moiety to the
dimethyl-bearing C-15 was deduced by the cross-peaks
exhibited by the methyl protons H3-16/H3-17. Finally, carbons
linking the three methoxy groups were identified on the basis
of the 3J cross-peak of the corresponding methyl protons.
Once the gross structure of xenimanadin A (1) was in hand,
our next task was the definition of the configuration at the five
asymmetric carbons and at the three double bonds. For this
aim, analysis of coupling constants and information arising
from a ROESY experiment (Fig. 5) proved to be particularly
valuable. The Z geometry of the D4,12 double bond was deduced
on the basis of the ROESY cross-peaks H-3/H-13 and H-12/
H-4a; analogously, the E geometry of D7,8 double bond was
indicated by the ROESY cross-peak H-8/H2-6. As for the double
bond D13,14, its E geometry was easily deduced by considering
the large value of JH-13/H-14 (15.8 Hz). The relative arrangement
of the four asymmetric carbons around the six-membered ring
was deduced on the basis of the following scalar and spatial
couplings. The trans junction of the two rings was suggested
by the ROESY correlation H-11a/H-5b and by the coupling
H3CO
H3CO
H3CO
H
-0.01
H3CO
-0.02
F3C
(A)
-0.06O
OMe
O
)
*
H3CO
18
+ 0.10
+ 0.07
H
H3CO
H3CO+0.03
O
+ 0.08
+ 0.06
O
OH
O
OH
1a S-MTPA ester
1b R-MTPA ester
(C)
H3CO
19
H3CO
1H-1H COSY
Figure 4. COSY and 2,3J H/C HMBC correlations of xenimanadin A (1).
1H-13C HMBC
Figure 6. Application of the modified Mosher’s method for secondary alcohols
on the MTPA esters of xenimanadin A (1a and 1b). Dd (dSꢀdR) are given in
parts per million.

3144
E. Fattorusso et al. / Tetrahedron 64 (2008) 3141e3146
Table 2
13C NMR data of xenimanadins AeD (1e4) in CDCl3 (125 MHz)
same epoxide geometry as 2 (cross-peaks H3-18/H2-9 and
H-8/H-4a).
Position
1
2
3
4
The molecular formula of xenimanadin D (4), C23H34O5 by
HREIMS, includes two hydrogen atoms less than that of
xenimanadin A (1). Analysis of the 13C NMR spectrum of 4
(CDCl3, Table 2) suggested that a conjugated ketone group
(signal at dC 203.3) could account for the additional unsatura-
tion degree. Combined inspection of COSY, HSQC, and
g-HMBC spectra suggested that all the differences between
compounds 3 and 1 consisted in the replacement of the
secondary alcohol with the ketone group, locating it at C-9.
Accordingly, the sp2 methine H-8 and H2-10 resonated, both
downfield shifted, as singlets at dH 5.93 (1H) and dH 3.32
(2H), respectively. The g-HMBC correlations H-8/C-9, H-8/
C-10, and H2-19/C-10 proved to be particularly helpful to
define the structure of this moiety, while the ROESY
cross-peak of H3-18 with H-8 indicated the Z geometry of
the double bond.
dC, mult.
dC, mult.
dC, mult.
dC, mult.
1
1-OMe
104.5, d
57.2, q
100.0, d
55.5, q
139.5, s
51.3, d
36.0, t
104.4, d
57.0, q
99.7, d
55.3, q
135.9, s
51.6, d
36.1, t
104.4, d
57.1, q
99.7, d
55.2, q
135.5, s
51.7, d
35.4, t
100.0, d
57.7, q
97.9, d
54.8, q
138.7, s
50.6, d
32.6, t
3
3-OMe
4
4a
5
6
40.5, t
40.6, t
40.6, t
30.3, t
7
133.5, s
131.5, d
68.0, d
47.2, t
60.5, s
67.0, d
69.0, d
44.9, t
59.9, s
62.4, d
27.8, t
146.6, s
130.0, d
203.3, s
51.6, t
8
9
10
11
11a
12
13
14
15
15-OMe
16
17
18
19
33.9, t
150.5, s
56.1, d
128.7, d
126.2, d
140.6, d
75.2, s
150.0, s
54.8, d
129.0, d
126.1, d
141.3, d
75.8, s
50.6, q
26.0, q
26.0, q
17.6, q
114.4, t
139.0, s
54.3, d
128.8, d
126.1, d
141.1, d
75.7, s
50.6, q
26.2, q
30.1, q
18.3, q
105.5, t
143.4, s
53.3, d
128.7, d
126.8, d
141.2, d
74.9, s
50.5, q
26.1, q
26.1, q
27.4, q
116.0, t
50.9, q
26.1, q
26.1, q
18.0, q
112.7, t
3. Discussion
Xenimanadins AeD build an interesting group of diterpe-
noids and they can be considered as a fifth family of pyran-
cyclononane derivatives in the xenicane class (see Fig. 1).
Their 2,6-dimethoxytetrahydropyran functionality appears to
be strictly reminiscent of the structure of the postulated dialde-
hyde precursor, which, notably, has never been isolated to
date. The presence of this functionality could raise the ques-
tion whether compounds 1e4 are actually artifacts produced
during the extraction process with methanol. Unfortunately,
due to the lack of material, we cannot unambiguously shed
light on this issue by repeating the extraction with a different
solvent. However, the absence in our extract of stereoisomers
at C-1 and/or C-3 makes the formation of 1e4 unlikely during
the extraction procedure. In addition, the following literature
evidence gives strong support to the genuine natural origin
of xenimanadins AeD. (i) A xenicane dialdehyde (8, Fig. 7)
derivative possessing a rearranged nine-membered ring has
been recently isolated from Xenia umbellata.13 Interestingly,
xenimanadin A. These differences are restricted to the function-
alities of the nine-membered ring, and, accordingly, H/C reso-
nances of the six-membered ring and of the di-unsaturated
side chain resulted to be almost coincident (see Tables 1 and 2).
Xenimanadin B (2) is closely related to xenimanadin A, in-
deed its molecular formula exhibits only one oxygen atom
more (C23H36O6). Preliminary inspection of 1H and 13C
NMR spectra of 2 (Tables 1 and 2) revealed the absence of
the double bond D7,8, while the unsaturation degree was guar-
anteed by an epoxide group (dC-7 60.5; dH-8 2.88, dC-8 67.0).
The COSY spectrum of 2 showed the coupling of the epoxide
doublet (dH 2.88) with a carbinolic proton at dH 3.78, which, in
turn, was coupled with H2-10 protons (dH 2.66 and 2.46).
g-HMBC correlations (H3-18 with C-6, C-7 and C-8; H2-19
with C-10, C-11, and C-11a) allowed us to attach this segment
to moiety (B), thus unveiling the planar structure of this addi-
tional xenimanadin. The ROESY cross-peak H-8/H-6a
revealed the geometry of the epoxide ring, while the spatial
coupling H-8/H-4a connected this geometry with that of the
asymmetric centers belonging to the six-membered ring.
Finally, the spatial coupling H-11a/H-9 indicated the cis orien-
tation of these two protons.
OH
H
O
H
H
H
O
O
Xenimanadin C (3) showed the same molecular formula of
xenimanadin A (C23H36O5). COSY and HSQC spectra of 3
revealed that, also in this case, the subunit C-7/C-10, including
an epoxide group followed by two methylenes in sequence (dH
1.50 and 1.45, dH 2.43 and 2.26), accounted for all the struc-
tural differences of 3 with 1. The g-HMBC couplings H2-19/
C-10, H3-18/C-6, H3-18/C-7, and H3-18/C-8 allowed the
attachment of this subunit to the remaining parts of the mole-
cule, thus building up the xenimanadin C (3) planar structure.
ROESY spectrum revealed that compound 3 possessed the
8
OH
OH
H
H
H3CO
AcO
O
H3CO
H3CO
H
H
H3CO
9
10
Figure 7. A rearranged xenicane dialdehyde derivative (8) and two known
methoxylated xenicanes (9 and 10).

E. Fattorusso et al. / Tetrahedron 64 (2008) 3141e3146
3145
this molecule was shown to be unreactive toward MeOH,
which was used during the purification procedure. (ii) A mem-
ber of the xenimanadin family had been found before the pres-
ent work, namely compound 9 (Fig. 7),14 absent in our sample.
Notably, this compound was obtained by using a completely
methanol-free procedure (extraction in CH2Cl2; chromatogra-
phies in hexane/EtOAc mixtures),14 thus demonstrating that
Xenia soft corals are able to biosynthesize these methoxylated
derivatives. (iii) This ability is confirmed by the recent isola-
tion of compound 10 (Fig. 7) from Xenia elongata,2 also in
this case obtained through a methanol-free procedure.
Compounds 5 and 7 are cytotoxic against P-388 (mouse
lymphocytic leukemia) tumor cell line with IC50 of 0.2 and
4.7 mg/mL, respectively.10 On the contrary, xenimanadins
AeD (1e4) proved to be non-cytotoxic (IC50>20 mg/mL)
against several cell lines (including P-388). Comparison of
structures and activities of xenimanadin D (4) and blumiolide
C (5) clearly indicates that ring A must exert a key role to
determine the cytotoxic potentialities of these compounds.
was purified by analytical HPLC using n-hexane/EtOAc 85:15
as eluent, flow rate 0.8 mL/min, affording xenimanadin C (3,
1.5 mg, tR 11 min). A second fraction (n-hexane/EtOAc, 7:3)
was purified by analytical HPLC using n-hexane/EtOAc 7:3
as eluent, flow rate 0.8 mL/min, affording xenimanadins B (2,
3.8 mg, tR 18 min) and D (4, 3.9 mg, tR 10 min). The fraction
obtained by eluting with n-hexane/EtOAc 6:4 was purified by
analytical HPLC using n-hexane/EtOAc 65:35 as eluent, flow
rate 0.8 mL/min, yielding xenimanadin A (1, 6.0 mg, tR
9 min). The fraction obtained by eluting with n-hexane/EtOAc
1:1 was purified by analytical HPLC using n-hexane/EtOAc
1:1 as eluent, flow rate 0.8 mL/min, affording deoxyoxido-
isoxeniolide A (7, 1.0 mg, tR 10 min). The fraction obtained
by eluting with n-hexane/EtOAc 4:6 was purified by analytical
HPLC using n-hexane/EtOAc 1:1 as eluent, flow rate 0.8 mL/
min, to afford blumiolide C (5, 2.1 mg, tR 15 min). Finally,
the fraction obtained by eluting with EtOAc was purified
by analytical HPLC using n-hexane/EtOAc 1:9 as eluent,
flow rate 0.8 mL/min, to afford xeniolide F (6, 1.9 mg, tR
11 min).
4. Experimental
4.2.1. Xenimanadin A (1)
Colorless amorphous solid; [a]2D2 ꢀ4 (c 0.11, CHCl3); IR
4.1. General experimental procedures
1
(KBr) nmax 3415, 1608, 1240 cmꢀ1; For H and 13C NMR
Optical rotations (CHCl3) were measured at 589 nm on a
PerkineElmer 192 polarimeter. 1H (500 MHz) and 13C
(125 MHz) NMR spectra were measured on a Varian INOVA
spectrometer. Chemical shifts were referenced to the residual
(CDCl3) see Tables 1 and 2, respectively; ESI-MS (positive
ions) m/z 415 [MþNa]þ, 431 [MþK]þ; HREIMS m/z
374.2468 [MꢀH2O] (calcd for C23H34O4 374.2452).
1
solvent signal (CDCl3: dH 7.26, dC 77.0). Homonuclear H
4.2.2. Xenimanadin B (2)
Colorless amorphous solid; [a]2D2 þ8 (c 0.15, CHCl3); IR
connectivities were determined by the COSY experiment.
One-bond heteronuclear 1He13C connectivities were deter-
mined with the HSQC experiment. Two- and three-bond
1He13C connectivities were determined by gradient-HMBC
1
(KBr) nmax 3425, 1612, 1236 cmꢀ1; For H and 13C NMR
(CDCl3) see Tables 1 and 2, respectively; ESI-MS (positive
ions) m/z 431 [MþNa]þ, 447 [MþK]þ; HREIMS m/z
390.2418 [MꢀH2O] (calcd for C23H34O5 390.2406).
1
experiments optimized for a 2,3J of 9 Hz. Through-space H
connectivities were evidenced by using a ROESY experiment
with a mixing time of 500 ms. ESI-MS spectra were per-
formed on a LCQ Finnigan MAT mass spectrometer. Medium
4.2.3. Xenimanadin C (3)
Colorless amorphous solid; [a]2D2 þ24 (c 0.12, CHCl3); IR
1
pressure liquid chromatography was performed on a Buchi
¨
(KBr) nmax 1632, 1230 cmꢀ1; For H and 13C NMR (CDCl3)
apparatus using a silica gel (230e400 mesh) column; HPLC
were achieved on a Knauer apparatus equipped with a refrac-
tive index detector and analytical LUNA (Phenomenex) SI60
(250ꢁ4 mm) columns.
see Tables 1 and 2, respectively; ESI-MS (positive ions) m/z
415 [MþNa]þ, 807 [2MþNa]þ; HREIMS m/z 361.2390
[MꢀOCH3] (calcd for C22H33O4 361.2379).
4.2.4. Xenimanadin D (3)
Colorless amorphous solid; [a]2D2 þ6 (c 0.20, CHCl3); IR
4.2. Collection, extraction, and isolation
1
(KBr) nmax 1720, 1640, 1215 cmꢀ1; For H and 13C NMR
Specimens of Xenia sp. (40.0 g, dry weight) were collected in
March 2006 in the Bunaken Marine Park of Manado along the
coasts of the small island of Siladen (North Sulawesi, Indonesia)
at a depth of 2e5 m. A small voucher sample is deposited at the
Dipartimento per lo Studio del Territorio e delle sue Risorse,
(CDCl3) see Tables 1 and 2, respectively; ESI-MS (positive
ions) m/z 413 [MþNa]þ, 429 [MþK]þ; HREIMS m/z
390.2397 (calcd for C23H34O5 390.2406).
4.3. Preparation of MTPA esters of xenimanadin A
`
Universita di Genova. Xenia sp. was extracted (3ꢁ3 L) with
methanol at room temperature to obtain a brown material, which
was partitioned between water and EtOAc to yield a brown
organic fraction (1.16 g). This was subsequently chromato-
graphed by MPLC over silica gel (230e400 mesh) column
(750ꢁ25 mm), using a linear gradient system from n-hexane/
EtOAc 8:2 to EtOAc. The first fraction (n-hexane/EtOAc, 8:2)
To a solution of xenimanadin A (1) (1.0 mg) in 0.3 mL of
dry pyridine an excess of (R)- or (S)-MTPA chloride was
added and the mixture left at room temperature for 12 h under
stirring. The reaction mixture was then diluted with ether and
washed with H2O and saturated NaCl aqueous solution. The
organic layer was dried over Na2SO4, concentrated under

3146
E. Fattorusso et al. / Tetrahedron 64 (2008) 3141e3146
reduced pressure, and purified by HPLC (eluent n-hexane/
EtOAc 9:1) to give (S)-MTPA ester 1a (0.8 mg, 52% yield)
and R-MTPA ester 1b (0.7 mg, 45% yield), respectively.
Acknowledgements
This research was partially conduced during the Master
Course ‘Tropical Marine Biodiversity and Natural Products’
`
of the Universita Politecnica delle Marche. Mass and NMR
spectra were recorded at the ‘Centro di Servizio Interdiparti-
4.3.1. (S)-MTPA ester of xenimanadin A
1
Colorless oil. H NMR (CDCl3, 500 MHz): dH 7.55e7.51
`
mentale di Analisi Strumentale’, Universita di Napoli ‘Feder-
ico II’. The assistance of the staff is acknowledged.
(3H, MTPA phenyl, m), 7.43e7.41 (2H, MTPA phenyl, m),
6.60 (1H, H-13, dd, J¼15.8, 11.3 Hz), 6.09 (1H, H-12, d,
J¼11.3 Hz), 5.91 (1H, H-9, t, J¼7.0 Hz), 5.60 (1H, H-14, d,
J¼15.8 Hz), 5.43 (1H, H-3, s), 5.17 (1H, H-8, d, J¼7.0 Hz),
4.89 (1H, H-19, s), 4.87 (1H, H-19, s), 4.43 (1H, H-1, d,
J¼8.8 Hz), 3.61 (3H, MTPA-OMe, s), 3.59 (3H, 3-OMe, s),
3.42 (3H, 1-OMe, s), 3.17 (3H, 5-OMe, s), 2.52 (1H, H-10,
dd, J¼14.0, 7.0 Hz), 2.32 (1H, H-4a, overlapped), 2.30 (1H,
H-10, overlapped), 2.13e2.09 (2H, H-6, m), 1.90e1.88 (1H,
H-5, m), 1.79 (3H, H-18, s), 1.71 (1H, H-11a, dd, J¼10.6,
8.8 Hz), 1.56e1.54 (1H, H-5, m), 1.30 (6H, H-16 and H-17,
s). ESI-MS (positive ions) m/z 631 [MþNa]þ.
References and notes
1. Blunt, J. W.; Copp, B. R.; Hu, W.-P.; Munro, M. H. G.; Northcote, P. T.;
Prinsep, M. R. Nat. Prod. Rep. 2007, 24, 31 and previous reports of the
same series.
2. Andrianasolo, E. H.; Haramaty, L.; Degenhardt, K.; Mathew, R.; White,
E.; Lutz, R.; Falkowski, P. J. Nat. Prod. 2007, 70, 1551.
3. Cheng, Y.-B.; Jang, J.-Y.; Khalil, A. T.; Kuo, Y.-H.; Shen, Y.-C. J. Nat.
Prod. 2006, 69, 675.
4. Bishara, A.; Rudi, A.; Goldberg, I.; Benayahu, Y.; Kashman, Y.
Tetrahedron 2006, 62, 12092.
5. Vanderah, D. J.; Steudler, P. A.; Ciereszko, L. S.; Schmitz, F. J.; Ekstrand,
J. D.; Van Der Helm, D. J. Am. Chem. Soc. 1977, 99, 5780.
6. Kashman, Y.; Groweiss, A. J. Org. Chem. 1980, 45, 3814.
7. Kashman, Y.; Groweiss, A. Tetrahedron Lett. 1978, 4833.
8. Iwagawa, T.; Takahiro, M.; Okamura, H.; Nakatani, M. Tetrahedron 1996,
52, 13121.
4.3.2. (R)-MTPA ester of xenimanadin A
1
Colorless oil. H NMR (CDCl3, 500 MHz): dH 7.58e7.54
(3H, MTPA phenyl, m), 7.43e7.41 (2H, MTPA phenyl, m),
6.60 (1H, H-13, dd, J¼15.8, 11.3 Hz), 6.10 (1H, H-12, d,
J¼11.3 Hz), 5.92 (1H, H-9, t, J¼7.0 Hz), 5.60 (1H, H-14, d,
J¼15.8 Hz), 5.43 (1H, H-3, s), 5.23 (1H, H-8, d, J¼7.0 Hz),
4.81 (2H, H-19, s), 4.41 (1H, H-1, d, J¼8.8 Hz), 3.59 (3H,
3-OMe, s), 3.56 (3H, MTPA-OMe, s), 3.40 (3H, 1-OMe, s),
3.17 (3H, 5-OMe, s), 2.42 (1H, H-10, dd, J¼14.0, 7.0 Hz),
2.32 (1H, H-4a, dd, J¼10.6, 4.5 Hz), 2.23 (1H, H-10, d,
J¼14.0 Hz), 2.13e2.10 (2H, H-6, m), 1.90e1.88 (1H, H-5,
m), 1.81 (3H, H-18, s), 1.68 (1H, H-11a, dd, J¼10.6,
8.8 Hz), 1.56e1.54 (1H, H-5, m), 1.30 (6H, H-16 and H-17,
s). ESI-MS (positive ions) m/z 631 [MþNa]þ.
9. Iwagawa, T.; Amano, Y.; Hase, T.; Shiro, M. Tetrahedron 1995, 51,
11111.
10. El Gamal, A. A. H.; Chiang, C.-Y.; Huang, S.-H.; Wang, S.-K.; Duh, C.-Y.
J. Nat. Prod. 2005, 68, 1336.
´
11. Anta, C.; Gonzalez, N.; Santafe, G.; Rodriguez, J.; Jimenez, C. J. Nat.
´
Prod. 2002, 65, 766.
12. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991, 113, 4092.
13. El-Gamal, A. A. H.; Wang, S.-K.; Duh, C.-Y. Org. Lett. 2005, 7, 2023.
14. Duh, C.-Y.; El-Gamal, A. A. H.; Chiang, C. Y.; Chu, C.-J.; Wang, S.-K.;
Dai, C.-F. J. Nat. Prod. 2002, 65, 1882.