An unusual halogenated meroditerpenoid from Stypopodium flabelliforme: Studies by NMR spectroscopic and computational methods

Article abstract of DOI:10.1016/j.phytochem.2009.07.017

Meroditerpenoids, 2-[2′(E)-3′,7′,11′,15′-tetramethylhexadec-2-en-1′-yl]-6-methyl-1,4-benzohydroquinone diacetate and 4′-chlorostypotriol triacetate, along with eight known compounds isolated from the dichloromethane extract of the brown alga Stypopodium f

Full text of DOI:10.1016/j.phytochem.2009.07.017

Phytochemistry 70 (2009) 1315–1320
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
An unusual halogenated meroditerpenoid from Stypopodium flabelliforme: Studies
by NMR spectroscopic and computational methods
*
Carlos Areche, Aurelio San-Martín , Juana Rovirosa, Jorge Soto-Delgado, Renato Contreras
Dpto. de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Meroditerpenoids, 2-[20(E)-30,70,110,150-tetramethylhexadec-2-en-10-yl]-6-methyl-1,4-benzohydroqui-
none diacetate and 40-chlorostypotriol triacetate, along with eight known compounds isolated from
the dichloromethane extract of the brown alga Stypopodium flabelliforme after peracetylation are
reported. One of them, 2-(1-oxo-hexadecyl)-1,3,5-trihydroxybenzene, is described for the first time
within this genus. Structural elucidation was carried out on the basis of spectroscopic data and theoret-
ical studies using GIAO/DFT analysis at B3LYP/6-31G(d) and mPW1PW91/6-31G(d) levels of theory for 40-
chlorostypotriol. This isomer is the first metabolite from the Stypopodium genus possessing one halogen
atom.
Article history:
Received 5 November 2008
Received in revised form 12 June 2009
Available online 19 August 2009
Keywords:
Stypopodium flabelliforme
Dictyotaceae
Seaweeds
Meroditerpenoids
GIAO/DFT
Ó 2009 Elsevier Ltd. All rights reserved.
Stypotriol
1. Introduction
halogenated moiety. Besides, a comparison between experimental
and theoretical chemical shifts was carried out for one of them in
Marine brown algae of the genus Stypopodium (Dictyotaceae,
Phaeophyta) are localized mainly in tropical and subtropical seas.
In Chile, Stypopodium flabelliforme is found in Easter Island. From
the genus Stypopodium, several secondary metabolites with differ-
ent biological activities have been reported (Gerwick and Fenical,
1981; Gonzalez et al., 1982; Gerwick et al., 1985; Rovirosa et al.,
1992, 1994; Gil et al., 1995; Depix et al., 1998; Wessels et al.,
1999; Dorta et al., 2002; Sabry et al., 2005). Those activities are
related to their unique phytochemical structures such as
chromenes, plastoquinones and polycyclic meroditerpenoids.
However, some are unstable to air oxidation, such as stypotriol
and tetraprenylhydroquinones (Gerwick and Fenical, 1981).
On the other hand, the gauge invariant atomic orbitals/density
functional theory (GIAO/DFT) approach has been used for calcula-
tion of chemical shifts for a variety of natural products. This meth-
od is very helpful to assign NMR spectroscopic data of rigid
molecular structures, as well as for determinations of relative con-
formations of flexible compounds (Bassarello et al., 2003; Da-Silva
and Cunha-Neto, 2005; Chini et al., 2008; Braddock and Rzepa,
2008).
order to illustrate the reliability of the method as a valuable aid
to the assignment of 7.
2. Results and discussion
Fresh algae were extracted with CH2Cl2, with the extract so ob-
tained concentrated under reduced pressure. Because of the insta-
bility of polyprenylhydroquinones, stypotriol and free phenols, the
CH2Cl2 extract was immediately acetylated with Ac2O/Py. Ten
compounds were thus isolated as acetates from S. flabelliforme
using chromatographic methods, and these metabolites were
grouped as tetraprenylhydroquinones 1–2, polycyclic meroditerp-
enoids 3–8, one chromene 9 and one polyketide 10, respectively,
Fig. 1. The polyketide, compound 10, was previously isolated from
the brown alga Lobophora papenfussii (Dictyotales) (Gerwick and
Fenical, 1982) but is reported here for the first time as a S. flabelli-
forme metabolite.
Compound 2 was obtained as pale yellow oil. It was character-
ized as 2-[20(E)-30,70,110,150-tetramethylhexadec-2-en-10-yl]-6-
methyl-1,4-benzohydroquinone diacetate. Its molecular formula
(C31H50O4) was determined on the basis of HREIMS analysis. Its
1H NMR spectrum (Table 1) displayed main proton signals at dH
6.98 (1H, d, J = 2.4, H-3), 6.77 (1H, d, J = 2.4, H-5), 5.30 (1H, t, J =
7.1, H-20), 3.23 (2H, d, J = 7.1, H-10) and 1.92 (3H, s, Me-7), along
with five methyl groups in the side-chain at dH 1.51 (3H, s, Me-
200), 0.88 (3H, d, J = 6.8, Me-190), 0.86 (9H, d, J = 6.8, Me-160, 170
and 180), indicating that 2 is a prenylated hydroquinone. The
In the course of our studies for bioactive metabolites from algae
from Chile, we re-investigated the brown alga S. flabelliforme. Here-
in, we report the structural elucidation using 1D and 2D NMR spec-
troscopy of two new meroditerpenoids including an unusual
* Corresponding author. Fax: +56 2 2713888.
E-mail address: aurelio@uchile.cl (A. San-Martín).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.07.017

1316
C. Areche et al. / Phytochemistry 70 (2009) 1315–1320
16
CH3
7'
CH3
17'
OAc
CH3
CH3
18C' H3
CH3
20'
19'
18
CH3
17
CH33
O
H3C
16'
CH3
6
1'
3'
7'
11'
1
3
3'
R3
1
5
1'
5'
10 H
14
8
R
R2
H
R1
OAc
OAc
1
2
H3C CH3
19
20
CH3
CH3
CH3
CH3
CH3
H3C
R1
R2
OAc
OAc OAc
OAc OAc
OAc OAc
R3
H
R
H
OAc
OAc
OAc
=O
5
H
6
7
8
Cl
H
OAc
CH3
CH3
CH3
O
CH3
O
CH3
CH3
CH3 CH3
OAc
CH3
CH3
H
H
AcO
AcO
H
3
H3C CH3
9
CH3
CH3
O
OAc
O
CH3 CH3
OAc
H
H
AcO
OAc
AcO
H
4
10
H3C CH3
Fig. 1. Structure of compounds isolated from the brown seaweed Stypopodium flabelliforme.
Table 1
1H and 13C NMR (HMQC), 1H–1H COSY, and HMBC spectroscopic data of 2 (C6D6).
atomaria (Tziveleka et al., 2005). However, some exceptions were
observed in the 13C NMR spectroscopic data; for instance one aro-
matic moiety was at dC 148.5 (C-4), 145.5 (C-1), 134.9 (C-2), 131.6
(C-6), 121.2 (C-5), 120.2 (C-3) and two acetate groups were at dC
168.0 and 167.5 instead of two quinone carbonyls. A combination
of HMQC and HMBC experiments support this interpretation. The
configuration of the carbon–carbon double bond was proposed to
be E on the basis of their chemical shifts (mainly 13C NMR), when
compared with prenylquinone data from literature (Tziveleka
et al., 2005; Mori et al., 2005; Seo et al., 2006). Compound 2 has
previously been synthesized in investigations of the biosynthesis
H
d
1H (J, Hz)
d
13C (DEPT)
HMBC
1
2
3
4
5
6
–
–
145.5 s
134.9 s
120.2 d
148.5 s
121.2 d
131.6 s
16.1 s
–
–
6.98 d (2.4)
–
6.77 d (2.4)
–
C-1; C-4; C-10
–
C-1; C-4; C-7
–
C-5; C-6
7
1.92 s
10
3.23 d (7.1)
5.3 t (7.1)
–
28.8 t
C-1; C-2; C-3; C-40
C-2; C-10; C-40; C-200
–
20
121.5 d
137.4 s
39.9 t
25.4 t
36.7 t
30
40
1.92a
C-30; C-50; C-60; C-200
of
a-tocopherols in spinach chloroplasts (Inoue et al., 1974).
50
1.41 m; 1.26 m
1.27 m; 1.22 m
1.37 m
60
C-80
C-190
According to the Dictionary of Natural Products on CD-ROM, com-
pound 2 has not been previously reported as a natural product.
Compound 7 was obtained as white powder. The 1H NMR spec-
trum showed signals for one methine proton at dH 4.51 (1H, dd,
J = 12.0, 5.0, H-14), one pair of doublets at dH 3.10 and 2.68 (1H,
d, J = 16.4, H-1), three acetate methyl groups at dH 2.31 (3H, s),
2.26 (3H, s) and 2.10 (3H, s), one aromatic methyl at dH 2.25 (3H,
s, Me-70) and five aliphatic methyl groups at dH 0.93 (3H, s, Me-
17), 0.87 (3H, s, Me-18), 0.84 (3H, s, Me-20), 0.83 (3H, s, Me-19)
and 0.72 (3H, d, J = 6.4, Me-16). 13C NMR spectroscopic analysis,
including DEPT 135, established the presence of ten quaternary
carbons, four methines, seven methylenes, six methyl groups and
three acetate groups (Table 2). These analysis and chemical shift
comparisons (data assigned by HMQC) were comparable to those
previously reported for 6 (Gerwick and Fenical, 1981; Rovirosa
et al., 1992). The main difference from 6 is the absence of C-40 aro-
matic proton and addition of a chlorine atom in 7. The above anal-
ysis was confirmed by HREIMS and 13C NMR spectroscopic analysis
thereby suggesting a molecular formula C33H45ClO7 and therefore
bearing 11 degrees of unsaturation. The EI mass spectrum indi-
cated molecular ion [M]+Å isotopic peaks at m/z 588 and 590. The
70
32.7 d
37.4 t
80
1.28 m; 1.12 m
90
1.22a
24.9 t
100
110
120
130
140
150
160
170
180
190
200
OAc
OAc
COCH3
COCH3
1.07 mb
1.37 m
37.6 tb
32.9 d
37.5 tb
24.6 t
C-180
C-100; C-120
1.33 mb
1.36 m; 1.21 m
1.13 m
39.4 t
C-130; C-160; C-170
1.48 dq (6.8)
0.86 d (6.8)
0.86 d (6.8)
0.86 d (6.8)
0.88 d (6.8)
1.51 s
1.73 s
1.79 s
–
–
27.9 d
22.5 qc
22.4 qc
19.5 q
19.6 q
15.7 q
20.2 q
19.5 q
168.0 s
167.5 s
C-160; C-170
C-140; C-150
C-140; C-150
C-110
C-70; C-80
C-20; C-30; C-40
–
–
–
–
a
Hidden signals.
b,c
Assignments may be interchanged.
carbon signals in the 13C NMR spectrum were similar to that of a
well-known tetraprenyl toluoquinone derivative from Taonia

C. Areche et al. / Phytochemistry 70 (2009) 1315–1320
1317
Table 2
1H and 13C NMR (HMQC), and HMBC spectroscopic data of 7 (CDCl3).
CH3
O
CH3
C
d
1H (J, Hz)
d
13C
HMBC
CH3
CH3
(DEPT)
Cl
1
3.10 d (16.4);
2.68 d (16.4)
–
32.8 t
C-2; C-3; C-7;
H
C-10; C-20; C-60
AcO
OAc
2
3
4
5
6
7
8
9
97.4 s
37.0 d
31.0 t
20.5 t
51.3 d
42.4 s
32.8 t
17.5 t
55.0 d
–
C-1
H
AcO
1.38 m
H3C CH3
1.46 m; 1.15 m
1.68 m; 1.41 m
1.52 d (12.6)
–
1.01 dd (15.1; 6.6)
1.47 m; 1.12 m
0.86 ma
Fig. 2. The main correlations in the HMBC spectrum of compound 7.
C-7; C-11
–
C-9; C-17
C-7; C-10; C-11
C-11; C-12;
C-14; C-15; C-18
–
(C-2), 42.4 (C-7) and 37.0 (C-3); proton signal dH 2.25 (3H, s, H-
70) with carbon resonances dC 157.5 (C-20), 126.8 (C-40) and 115.5
(C-30). This established the position of the chlorine atom at C-40.
Analysis of the NOESY spectrum confirmed that 7 had the same rel-
ative configuration as stypotriol.
10
11
12
13
–
37.0 s
38.1 t
23.7 t
1.75 t (13.1; 3.3); 1.45 t (13.1)
1.58 t (13.1; 3.3); 1.31 m
C-10; C-18
C-14; C-15
Finally, the theoretical 13C NMR chemical shifts were calculated
using B3LYP and mPW1PW91 functionals with a 6-31G(d) basis set
including solvent effect for the isomers of 7: 50-chlorostypotriol tri-
acetate (7a) and 60-chlorostypotriol triacetate (7b). The results are
summarized in Table 3, and Fig. 3 shows optimized structures. We
have thus studied changes in different positions of the chlorine
atom in the aromatic ring of 7. The changes in the chemical shift
for different positions represent the main information leading to
theoretical NMR parameter assignments. The theoretical chemical
shift for C-40 are dC 130.3 and 128.4 for 7; 142.1 and 141.9 for 7a
and 139.1 and 138.7 for 7b at B3LYP/6-31G(d) and mPW1PW91/
6-31G(d) level of theory, respectively. The same trend is observed
for other carbons of the aromatic rings. Thus, the difference be-
14
15
16
17
18
19
4.51 dd (12.0; 5.0)
–
0.72 d (6.4)
0.93 s
0.87 s
0.83 s
0.84 s
–
–
–
–
–
80.8 d
37.8 s
C-13; C-15; C-19; C-20
–
C-2; C-3; C-4
C-2; C-6; C-7; C-8
C-10; C-11; C-12
C-10; C-14; C-15; C-20
C-10; C-14; C-15; C-19
–
–
–
–
–
15.5 q
17.1 q
16.5 q
27.9 q
16.3 q
118.7 s
157.5 s
115.5 s
126.8 s
132.1 s
136.9 s
12.9 q
21.3 q
20.3 q
20.2 q
171.0 s
168.3 s
167.2 s
20
10
20
30
40
50
60
–
–
70
2.25 s
2.10
2.31
2.26
–
C-20; C-30; C-40
OAc
OAc
OAc
COCH3
COCH3
COCH3
–
–
–
–
–
–
tween theoretical and experimental values
D
d = |dexp ꢀ dcalc| in
13
the C NMR spectra of C-40 is shown as being
DdC about 2.6 and
3.5 at 7, 31.3 and 35.1 at 7a and 13.3 and 12.3, respectively. The
large differences for 7a and 7b isomers predict that structure 7
has the best agreement between experimental and theoretical val-
ues. This is essentially a similarity index. In summary, our results
show that GIAO/DFT calculations on the optimized structures indi-
cate that isomer 7 is the one that agrees with the experimental 13C
NMR chemical shift.
–
–
a
Hidden signal.
isotopic pattern (3:1), as well as the mass fragments, confirmed the
presence of one chlorine atom. The main connectivity in the HMBC
spectrum showed the following 1H, 13C long-range correlations
(Fig. 2): proton signals dH 3.10 (1H, d, H-1), 2.68 (1H, d, H-1) with
carbon resonances dC 157.5 (C-20), 136.9 (C-60), 118.7 (C-10), 97.4
In order to establish that halogenation of compound 7 was not
an artifact arising from the isolation procedure, additional assays
Table 3
Calculated 13C NMR chemical shifts of 7 and its isomers in CDCl3 (only for dihydrobenzofurane rings).
C
7a
36.3
7aa
36.5
7ba
36.8
7b
36.2
7ab
36.2
7bb
36.4
Exp.
32.8
1
2
98.1
115.8
151.2
111.4
130.3
127.8
133.2
13.9
98.4
115.4
152.4
108.4
142.1
112.5
136.2
11.8
97.7
119.0
150.9
109.3
139.1
128.2
123.6
11.2
94.2
116.1
151.4
111.2
128.4
126.7
133.3
14.0
94.6
115.3
152.7
108.3
141.9
110.7
136.0
11.8
94.0
119.2
151.0
109.4
138.7
127.8
122.2
11.4
97.4
118.7
157.5
115.5
126.8
132.1
136.9
12.9
10
20
30
40
50
60
70
a
d using B3LYP/6-31G(d) level.
d using PW1PW91/6-31G(d) level.
b

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C. Areche et al. / Phytochemistry 70 (2009) 1315–1320
Fig. 3. Optimized geometries of 7, 7a and 7b.
were carried out (see experimental) but with no formation of 7
being observed. Similar experiments were carried out with stypot-
riol triacetate and the results were the same: no reaction was ob-
served in all of them. Taken together, these data indicated 7 is a
natural product.
(J) in hertz. The pulse conditions were as follows: for the 1H
NMR spectrum, spectrometer frequency (SF) = 400.13 MHz, acqui-
sition time (AQ) = 2.045 s, relaxation delay (RD) = 1.0 s, 30° pulse
width = 6.5 ls, spectral width (SW) = 8012.8 Hz, line broadening
(LB) = 0.3 Hz, Fourier transform (FT) size = 32K; for the 13C NMR
spectrum, SF = 100.61 MHz, AQ = 1.2 s, RD = 2.0 s, 30° pulse
width = 14.0 ls, SW = 23980.8 Hz, LB = 1.0 Hz, FT size = 32K; for
the H–H COSY spectrum, AQ = 0.13 s, RD = 1.0 s, SW = 4006.4 Hz,
3. Concluding remarks
FT size = 1024 ꢁ 1024; for the HMBC spectrum, AQ = 0.13 s,
RD = 1.0 s, SW = 4006.4 (1H) and 30191.03
(
13C) Hz, FT
Halogenated metabolites are mainly known from marine organ-
isms, prokaryotes and several higher plants. It is well accepted that
the halometabolites isolated from marine invertebrates in many
cases originate from incorporation of algal food. Seaweeds provide
a rich source of structurally diverse secondary metabolites being
mainly halogenated. Their functions are in defense against herbi-
vores, fouling organisms and pathogens but they also play a role
in reproduction, protection from UV radiation and as allelopathic
agents. However, the variations in production of secondary metab-
olites in seaweeds is a function of life cycle and is genetically and
environmentally controlled (Hay, 1996; Soares et al., 2003; Ianora
et al., 2006). The occurrence of halogenated compounds is unusual
in brown seaweed and there is no report of their presence in the
Stypopodium genus.
size = 1024 ꢁ 2048, and 7.7 Hz long-range coupling constant; for
the HMQC spectrum, parameters were very similar to those used
in the HMBC experiments, except for the 145 Hz one-bond cou-
pling constant. The NOESY spectrum was performed with eight
scans, AQ = 0.106 s, RD = 1.0 s, SW = 4789.2 Hz, FT size = 1024 ꢁ
1024 and LB = 0.0 Hz. IR spectra were recorded on a Vector 22
FT-IR spectrometer with mass spectra acquired using a Thermo
Finnigan MAT 95XP model spectrometer. Optical rotations were
obtained in CHCl3 on a Polartronic E polarimeter and UV spectra
were recorded on a Shimadzu UV160 spectrophotometer.
4.2. Computational details
4. Experimental
DFT calculations were carried out using the B3LYP and
mPW1PW91 exchange-correlation functional, together with the
standard 6-31G(d) basis set (Hehre et al., 1986; Lee et al., 1988).
The 13C NMR chemical shifts were calculated using gauge invariant
atomic orbitals (GIAO) method at the same level (Wolinski et al.,
1990). Solvent effects were evaluated by performing single-point
B3LYP/6-31G(d) and mPW1PW91/6-31G(d) calculations at the
gas-phase stationary points involved in the reaction using the
polarizable continuum model (PCM) of Tomasi’s group (Tomasi
and Persico, 1994; Barone et al., 1998). A dielectric constant value
4.1. General experimental procedures
TLC was performed on Kieselgel 60 GF254 using n-hexane/
EtOAc (7:3 and 1:1 v/v) as mobile phase. TLC spots were visualized
by spraying the chromatograms with H2SO4–MeOH (5:95, v/v) and
heating at 120 °C for 3 min. Column chromatography (CC) was per-
formed over Merck Kieselgel 60, particle size 0.063–0.200 mm. All
solvents were dried and purified before use according to standard
procedures. Measurements of NMR spectra of compounds used a
Bruker Avance AM-400 spectrometer equipped with 5 mm probes.
All compounds were individually dissolved in CDCl3 (1.0 ml) or
C6D6 containing tetramethylsilane (TMS) as internal standard.
Chemical shifts (d) were reported in ppm and coupling constants
was used for CHCl3 (e = 4.9). Relative chemical shifts were esti-
mated by using the corresponding TMS shielding calculated at
the same theoretical model and using PCM method to solvent ef-
fect. All calculations were carried out with the Gaussian 03 suite
of programs (Frisch et al., 2004). All calculated data of the 13C

C. Areche et al. / Phytochemistry 70 (2009) 1315–1320
1319
MeOH
NMR chemical shifts can be obtained upon request from the
authors.
CHCl3). UV ½kꢂmax nm (log
e)
228 (4.0). HREIMS: calcd. for
C31H50O4 (M+Å): 486.3709, found: 486.3718. FT-IR
2948, 1731, 1460, 1385, 1240, 1110.
m
max cmꢀ1: 2970,
4.3. Plant material
4.6. Compound 7
The brown alga, Stypopodium flabelliforme, was collected inter-
tidally near the Hanga Roa in Easter Island (South Pacific), V Regió-
n, Chile, in 2005 at a depth of 5–10 m. A voucher specimen (No.
0053) was deposited in the Museo Nacional De Historia Natural,
Santiago, Chile and its identity was confirmed by Prof. M. Eliana
Ramirez from the Museo de Historia Natural de Santiago, Chile.
40-Chlorostypotriol triacetate: white powder. ½a 2D0
ꢂ
ꢀ5.0° (c
MeOH
0.004, CHCl3). UV ½kꢂmax nm (log
e
) 289 (3.5). HREIMS: calcd. for
C33H45ClO7 (M+Å): 588.2854, found: 588.2859. EI-MS: m/z (rel.
int.%): 588 [M]+Å/[M+2]+Å (6/2), 553 (2), 546 (100/33), 504 (21/7),
486 (8/3), 257 (39/13), 187 (14), 135 (8), 95 (8), 69 (9). FT-IR
m
max cmꢀ1: 2950, 1771, 1735, 1475, 1367, 1245, 1100.
4.4. Extraction and isolation
4.7. Hydrolisis of stypotriol triacetate (6)
Wet specimens (2.0 kg) were frozen for transportation and later
repeatedly extracted with CH2Cl2 (3 ꢁ 4 l). After filtration, the com-
bined extracts were concentrated under reduced pressure. Then,
the CH2Cl2 extract (60.5 g) was subsequently acetylated with
Ac2O/Py. The extract (60 g) was subjected to flash chromatography
on silica gel (n-hexane/EtOAc/MeOH in gradient from 10:1:0 to
0:1:1 v/v/v) to produce nine fractions 1–9. Fraction 1 (20 g), was
next subjected to silica gel CC and eluted with EtOAc–n-hexane
(1:9, v/v), with fractions of interest being further purified by silica
gel CC with a n-hexane/EtOAc gradient (0% up to 100% EtOAc) to
yield sargaol acetate 9 (200 mg) (Numata et al., 1992), 2-(geranyl-
To a stirred solution of stypotriol acetate 6 (200 mg) in MeOH
(20 ml), K2CO3 (catalytic amount) was added. The reaction mixture
was stirred at room temperature under N2 for 24 h, then rapidly
concentrated and kept under N2 atmosphere. Immediately, FeCl3
(catalytic amount) was added to a stirred solution of stypotriol in
CH2Cl2 (10 ml) under N2. The resulting suspension was heated until
reflux began, thus being maintained for one week and following
the product was rapidly acetylated (Ac2O/Py). The resulting mix-
ture was then diluted with H2O (20 ml) and filtered to yield a solid
following solvent removed. The latter was re-dissolved in Et2O for
analysis. The same procedure without FeCl3 was done as well. No
halogenation of stypotriol was observed.
geranyl)-6-methyl-1,4-benzohydroquinone diacetate
(Gerwick and Fenical, 1981), 2-[20(E)-30,70,110,150-tetramethylhexa-
dec-2-en-10-yl]-6-methyl-1,4-benzohydroquinone diacetate
1 (70 mg)
2
(7 mg), epitaondiol diacetate 3 (2 g) (Sanchez-Ferrando and San-
Martin, 1995) and a mixture of three compounds (1.0 g). An aliquot
(150 mg) of this mixture was further separated by silica gel CC
impregnated with AgNO3 (1:9) with n-hexane/EtOAc as system sol-
vent to give stypodiol diacetate 5 (30 mg) (Gerwick and Fenical,
1981; Abad et al., 1998), isoepitaondiol diacetate 4 (25 mg) (Rovi-
rosa et al., 1992) and 3 (50 mg).
Acknowledgements
This investigation was subsidised by ‘‘Proyecto Anillo ACT-38”
and FONDECYT Grant No. 1070715. JSD acknowledges MECESUP
fellowship No. 0408.
Fraction 2 (5 g) was applied to a silica gel column, this being
eluted with EtOAc–n-hexane (1:4, v/v), with fractions of interest
subjected to Sephadex LH-20 CC using MeOH to separate fatty
acids, chlorophylls and pigments. Then, various fractions were fur-
ther purified by silica gel CC, using a n-hexane/EtOAc gradient (0%
up to 100% EtOAc) to afford 750 mg of a mixture and stypotriol tri-
acetate 6 (800 mg) (Gerwick and Fenical, 1981). The mixture was
subjected successively to further silica gel CC (n-hexane/EtOAc gra-
dient of 10% up to 100% EtOAc) and Sephadex LH-20 (n-hexane/
CH2Cl2/MeOH: 8/1/1 v/v/v) to afford 40-chlorostypotriol triacetate
7 (2 mg) and 2-(1-oxo-hexadecyl)-1,3,5-trihydroxybenzene triace-
tate 10 (30 mg) (Gerwick and Fenical, 1982).
Fraction 3 (12 g) was subjected to silica gel CC, eluted with
EtOAc–n-hexane (3:7, v/v), and then passed through a Sephadex
LH-20 column using MeOH to separate chlorophyll. The fraction
containing the meroditerpenoids applied to a silica gel CC (n-hex-
ane–EtOAc gradient of 10% up to 100%) to afford 6 (5 g) and 14-
ketostypotriol diacetate 8 (300 mg) (Rovirosa et al., 1994).
Fraction 4 (9 g) was subjected to silica gel CC, eluted with
EtOAc–n-hexane (1:1, v/v), and contained pigments such as chloro-
phylls as evidenced by 1H NMR and UV analysis.
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4.5. Compound 2
2-[20(E)-30,70,110,150-Tetramethylhexadec-2-en-10-yl]-6-methyl-1,
4-benzohydroquinone diacetate: pale yellow oil. ½a D20
ꢂ
+2.0° (c 0.01,

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