Bisabolane and chamigrane sesquiterpenes from the soft coral

Pseudopterogorgia rigida
Panagiota Georgantea, Efstathia Ioannou, Constantinos Vagias
1
, Vassilios Roussis *
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou,
Athens 15771, Greece
1. Introduction
Gorgonian octocorals (Phylum Coelenterata, Subclass Octocor-
allia, Order Gorgonacea) have been the subject of numerous
chemical investigations which have yielded mainly sesquiter-
penes, diterpenes and highly functionalized steroids. Several of
these metabolites have exhibited cytotoxic, antiproliferative,
antibacterial, antiviral, anti-inammatory, antiplasmodial and
insecticidal activities (Blunt et al., 2013; MarinLit Database, 2013).
Species of the genus Pseudopterogorgia have proven to be a rich
source of secondary metabolites, mostly sesquiterpenes and
diterpenes. Among them, the family of the diterpene glycosides
pseudopterosins represents a characteristic example of bioactive
natural products that have found application in the cosmetics
industry and are candidates as anti-inammatory and analgesic
drugs in advanced preclinical trials (Newman and Cragg, 2004).
In continuation of our studies aiming at the isolation of new
bioactive marine metabolites, we began a detailed chemical
investigation of the organic extract of the soft coral Pseudopter-
ogorgia rigida Bielshowsky, which, thus far, has led to the isolation
of three representatives from the rare class of bisabolane dimers
(Georgantea et al., 2013). Herein, we report the isolation and
structure elucidation of eight new sesquiterpenes with chami-
grane (1) and bisabolane (28) carbon skeletons, along with 19
previously reported compounds (927). Among them, seven
metabolites (11, 13, 14, 17, 19, 22 and 23) were previously
described as semisynthetic compounds and are reported for the
rst time as natural products (Fig. 1).
2. Results and discussion
Specimens of the gorgonian P. rigida were initially freeze-dried
and then exhaustively extracted at room temperature with
mixtures of CH
2
Cl
2
/MeOH to afford a dark orange oily residue.
The organic extract was subjected to several steps of chro-
matographic separations to yield compounds 127.
Compound 1, puried by means of HPLC, was obtained as a
yellowish oil. The pseudomolecular ion at m/z 251.1649 [M+H]
+
observed in the HR-ESIMS spectrum suggested the molecular
formula C
15
H
22
O
3
, indicating ve degrees of unsaturation. The
1
H
NMR spectrum (Table 1) exhibited signals for an olenic proton at
d
H
6.59, an oxygenated methine at d
H
3.68, a vinylic methyl at d
H
1.98, two aliphatic methyls on quaternary carbons at d
H
0.91 and
0.85 and an aliphatic methyl on a tertiary carbon at d
H
0.74. The
13
C
NMR spectrum and DEPT experiments (Table 2) revealed 15
signals, including two carbonyls at d
C
199.9 and 199.7, two sp
2
carbon atoms resonating at d
C
149.8 and 140.2 and an oxygenated
methine at d
C
72.3. The presence of one carboncarbon double
bond and two carbonyls, in combination with the ve degrees of
Phytochemistry Letters 8 (2014) 8691
A R T I C L E I N F O
Article history:
Received 27 December 2013
Received in revised form 6 February 2014
Accepted 10 February 2014
Available online 26 February 2014
Keywords:
Pseudopterogorgia rigida
Sesquiterpenes
Bisabolane
Chamigrane
A B S T R A C T
A chemical investigation of the organic extract of the tropical soft coral Pseudopterogorgia rigida afforded
27 sesquiterpenes, among which one chamigrane (1) and seven bisabolanes (28) are new natural
products. The structures of compounds 18 were deduced by analysis of their NMR and MS data. The full
assignment of the spectroscopic data of four aromatic bisabolanes previously reported as semisynthetic
products (11, 19, 22, 23) is reported.
2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
* Corresponding author. Tel.: +30 210 7274592; fax: +30 210 7274592.
E-mail address: roussis@pharm.uoa.gr (V. Roussis).
1
Deceased on May 12, 2010.
Contents lists available at ScienceDirect
Phytochemistry Letters
j o u r n al h omep ag e: ww w. el s evi er . co m/ l oc at e/ p hyt ol
http://dx.doi.org/10.1016/j.phytol.2014.02.006
1874-3900/ 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
unsaturation, suggested that metabolite 1 was a bicyclic sesqui-
terpene. In the COSY spectrum evident were the cross-peaks of H-7
with both H
3
-14 and H
2
-8, of H
2
-8 with H
2
-9 and of H
2
-9 with H-
10, as well as the long range correlation of H
3
-15 with the olenic
H-2. In the HMBC spectrum both aliphatic H
3
-12 and H
3
-13
presented strong heteronuclear interactions with the oxygenated
C-10 and the quaternary carbons C-11 (d
C
42.2) and C-6 (d
C
59.6).
Furthermore, H
3
-14 displayed correlations with C-6, C-7 and C-8,
thus concluding a six-membered aliphatic ring. The vinylic methyl
H
3
-15 was correlated with C-2 (d
C
140.2), C-3 (d
C
149.8) and C-4 (d
C
199.7), while the olenic methine H-2 was correlated with C-1 (d
C
199.9). Finally, the HMBC correlations of C-1, C-4, C-6 and C-11
with H
2
-5 concluded a p-quinone ring and the spiro[5.5]
chamigane skeleton of 1. The relative congurations of the
asymmetric centers of compound 1 were proposed on the basis
of the interactions observed in the NOESY spectrum. Specically,
the NOE enhancements of H-5a (d
H
2.96)/H-10, H-5a/H
3
-13, H-5b
(d
H
2.65)/H
3
-14, H-7/H
3
-12 and H-10/H
3
-13 determined the
relative congurations at C-6, C-7 and C-10 as 6S*,7R*,10R*.
Compound 2, isolated as a yellowish oil, displayed a pseudo-
molecular ion peak at m/z 233.1538 (HR-ESIMS), corresponding to
C
15
H
21
O
2
and consistent with [M+H]
+
. The assigned molecular
formula C
15
H
20
O
2
indicated six degrees of unsaturation. The IR
spectrum showed a broad absorption band at 3400 cm
1
supporting the presence of a hydroxy group, while the absorption
band at 1192 cm
1
indicated an ether moiety in the molecule. The
1
H NMR spectrum (Table 1) exhibited signals for two aromatic
protons resonating at d
H
6.63 and 6.59, one olenic methine at d
H
5.29, an oxygenated methine at d
H
4.64, three vinylic methyls at d
H
2.15, 1.76 and 1.71 and a doublet methyl at d
H
1.26. The
13
C NMR
spectrum (Table 2) showed the presence of 15 carbon signals,
among which eight were sp
2
carbons and one was oxygenated. The
COSY cross-peaks of H-7/H
3
-14, H-7/H
2
-8, H
2
-8/H-9 and H-9/H-10
dened the only spin system in the molecule. The HMBC
Fig. 1. Structures of compounds 127 isolated from Pseudopterogorgia rigida.
P. Georgantea et al. / Phytochemistry Letters 8 (2014) 8691 87
correlations of H-2 with C-4, C-6 and C-15, of H-5 with C-1, C-3 and
C-7, of H
3
-15 with C-2, C-3 and C-4 and of OH-4 with C-3 and C-5
concluded the 1,2,4,5-tetrasubstituted aromatic ring, while the
correlations of H
3
-14 with C-6, C-7 and C-8 veried the connection
of the side chain at C-6. Taking into consideration the six degrees of
unsaturation, in combination with the presence of the aromatic
ring, the trisubstituted double bond and the ether ring, it was
suggested that the second ring was formed between C-1 and C-9.
The relative congurations of the chiral centers of 2 were assigned
on the basis of 1D NOE experiments. In particular, the strong NOE
interaction between H-7 and H-9 established the relative
congurations at C-7 and C-9 as 7R*,9R*. Therefore, metabolite 2
was identied as (9R*)-1,9-epoxy-4-hydroxy-a-curcumene.
Compound 3, obtained as a yellowish oil, had the molecular
formula C
15
H
20
O
2
, as deduced from the HR-ESIMS and
13
C NMR
spectra. Both
1
H and
13
C NMR spectra of 3 (Tables 1 and 2)
presented a close resemblance to those of compound 2. Analysis of
the 2D NMR spectra (HSQC, HMBC and COSY) suggested that 2 and
3 shared identical planar structures. Therefore, the difference
between them should have been stereochemical. In contrast to 2, a
strong NOE interaction between H-9 and H
3
-14 was observed for 3,
which suggested their coplanar orientation and dened the
relative congurations at C-7 and C-9 as 7R*,9S*. Thus, metabolite
3 was identied as the epimer of 2 at C-9, namely (9S*)-1,9-epoxy-
4-hydroxy-a-curcumene.
Compound 4 was obtained as a yellowish oil. Its molecular
formula was postulated to be C
17
H
28
O
2
by combined analysis of the
HR-ESIMS and
13
C NMR data, dictating four degrees of unsaturation.
The IR spectrum showed an absorption band at 1723 cm
1
indicative for an ester carbonyl. Eleven aliphatic carbons, including
ve methyls, ve methylenes and one methine, four olenic carbons,
including two methines and two quaternary carbons, a quaternary
oxygenated carbon and a carbonyl were revealedby the information
extracted from the
13
C NMR spectrum and DEPT experiments
(Table 2). The
1
H NMR spectrum (Table 1) displayed an isopropy-
lidene group (d
H
5.07, 1H; d
H
1.66, 3H; d
H
1.57, 3H), a vinylic methyl
(d
H
1.64, 3H), a doublet methyl (d
H
0.88, 3H), a second olenic
methine (d
H
5.21, 1H) and a deshielded methyl of an acetoxy group
(d
H
1.96, 3H). The COSY spectrum showed cross-peaks of H
2
-1 with
H-2, of H
2
-4 with H
2
-5, of H-7 with both H
3
-14 and H
2
-8, of H
2
-8 with
H
2
-9 and of H
2
-9 with H-10, along with the long range correlations of
H
3
-15 with both H-2 and H
2
-4 and of H-10 with H
3
-12 and H
3
-13.
Table 2
13
C NMR data (50 MHz, CDCl
3
) for compounds 18.
Position 1 2 3 4 5 6 7 8
1 199.9, C 148.5, C 148.4, C 30.5, CH
2
146.8, C 148.4, C 148.5, C 153.2, C
2 140.2, CH 118.9, CH 119.2, CH 117.6, CH 118.1, CH 118.3, CH 119.1, CH 116.5, CH
3 149.8, C 123.2, C 123.4, C 135.7, C 121.9, C 121.6, C 122.7, C 136.6, C
4 199.7, C 147.6, C 147.3, C 27.9, CH
2
147.2, C 147.3, C 148.5, C 121.5, CH
5 39.0, CH
2
113.5, CH 115.1, CH 27.4, CH
2
113.2, CH 112.8, CH 112.9, CH 126.6, CH
6 59.6, C 125.6, C 125.5, C 86.2, C 131.5, C 131.2, C 130.8, C 129.9, C
7 32.1, C 30.1, CH 28.7, CH 37.4, CH 32.6, CH 30.7, C 27.8, CH 32.3, CH
8 27.6, CH
2
38.3, CH
2
36.0, CH
2
31.8, CH
2
34.0, CH 34.9, CH
2
46.5, CH
2
33.8, CH
2
9 30.2, CH
2
73.3, CH 69.2, CH 26.6, CH
2
34.0, CH
2
31.8, CH
2
67.5, CH 31.4, CH
2
10 72.3, CH 125.4, CH 124.9, CH 124.5, CH 76.0, CH 77.3, CH 127.3, CH 78.0, CH
11 42.2, C 137.5, C 137.0, C 131.6, C 147.7, C 147.6, C 136.8, C 27.5, CH
12 15.5, CH
3
26.4, CH
3
26.3, CH
3
17.6, CH
3
18.0, CH
3
18.0, CH
3
26.1, CH
3
18.3, CH
3
13 23.6, CH
3
19.0, CH
3
18.9, CH
3
25.7, CH
3
110.8, CH
2
110.6, CH
3
18.7, CH
3
18.3, CH
3
14 17.9, CH
3
21.1, CH
3
24.7, CH
3
13.8, CH 21.8, CH
3
20.8, CH
3
21.9, CH
3
20.8, CH
3
15 15.5, CH
3
16.1, CH
3
16.0, CH
3
23.2, CH
3
15.4, CH
3
15.8, CH
3
15.9, CH
3
21.1, CH
3
OAc 170.5, C
OAc 22.3, CH
3
Table 1
1
H NMR data (400 MHz, CDCl
3
) for compounds 18.
Position 1 2 3 4 5 6 7 8
1 2.28, d (17.9),
2.10, d (17.9)
2 6.59, brs 6.59, s 6.60, s 5.21, m 6.55, s 6.59, s 6.67, s 6.59, s
4 2.40, m, 1.70, m 6.70, d (7.8)
5 2.96, d (17.6),
2.65, d (17.6)
6.63, s 6.53, s 1.86, m 6.55, s 6.56, s 6.57, s 7.01, d (7.8)
7 2.53, m 2.97, m 2.86, m 2.52, m 3.02, m 3.14, m 3.24, m 3.01, sextet (6.8)
8 1.52, m, 1.15, m 1.89, ddd (13.3,
5.7, 1.5), 1.54, m
1.90, ddd (13.7,
10.2, 5.9), 1.62,
ddd (13.7, 5.0, 2.4)
1.44, m, 1.02, m 1.56, m 1.63, m 1.82, m, 1.35, m 1.70, m, 1.56, m
9 1.74, m, 1.49, m 4.64, m 4.71, m 2.04, m, 1.86, m 1.56, m,
1.48, m
1.78, m,
1.45, m
4.02, ddd
(11.1, 8.4, 2.9)
1.52, m, 1.33, m
10 3.68, dd (11.2, 4.2) 5.29, d (8.8) 5.32, d (8.3) 5.07, m 4.09, m 4.14, m 5.15, brt (8.4) 3.38, ddd
(8.9, 5.8, 3.4)
11 1.65, m
12 0.85, s 1.76, s 1.76, s 1.57, s 1.66, s 1.66, s 1.65, brs 0.88, d (6.9)
13 0.91, s 1.71, s 1.72, s 1.66, s 4.94, brs,
4.83, brs
4.92, brs,
4.79, brs
1.47, brs 0.86, d (6.9)
14 0.74, d (6.5) 1.26, d (7.0) 1.31, d (7.0) 0.88, d (7.0) 1.18, d (7.0) 1.20, d (7.0) 1.26, d (7.0) 1.22, d (6.8)
15 1.98, brs 2.15, s 2.15, s 1.64, s 2.15, s 2.16, s 2.17, s 2.25, s
OAc 1.96, s
OH-4 4.26, brs 4.24, brs
P. Georgantea et al. / Phytochemistry Letters 8 (2014) 8691 88
The sequence of the carbons in the molecule was further supported
by the strong HMBC correlations between H
3
-14 and C-6, C-7 and C-
8, between H
3
-15 and C-2, C-3 and C-4, and between H
3
-12 and H
3
-
13 and C-10 and C-11. Finally, the acetoxy group was positioned at
the quaternary oxygenated carbon C-6 resonating at d
C
86.2. The
relative conguration of the stereogenic center C-6 could not be
assigned as the observed NOE cross-peaks were not considered
adequate evidence to safely distinguish between the two possible
isomers for 4, namely 6R*,7R* and 6S*,7R*. Thus, metabolite 4 was
identied as acetyl-b-bisabolol.
Compounds 5 and 6 displayed molecular ion peaks at m/z 250 in
their respective mass spectra, while their NMR spectra (Tables 1
and 2) exhibited characteristic signals for aromatic bisabolane
sesquiterpenes. In particular, their
1
H NMR spectra included
signals for two aromatic protons, an oxygenated methine, an
aromatic methyl and an aliphatic methyl on a tertiary carbon, as
well as a vinylic methyl and an exomethylene. Their spectroscopic
data revealed strong similarities with those of the C-10 epimeric
compounds 9 and 10. The main difference observed was the
presence of only two aromatic protons in para positions, as
deduced from the absence of coupling between them. The
correlations evident in their 2D NMR spectra supported the
proposed structures as the 4-hydroxy analogs of 9 and 10. Even
though metabolites 5 and 6 were isolated in pure form, it was not
possible as in the case of 9 and 10, to characterize their relative
congurations at C-10 by spectroscopic analysis.
Compound 7 was isolated as a yellowish oil. The molecular
formula C
15
H
22
O
3
was deduced from the HR-ESIMS and
13
C NMR
data. The
1
H NMR spectrum revealed an isopropylidene group (d
H
5.15, 1H; d
H
1.65, 3H; d
H
1.47, 3H), a doublet methyl on a benzylic
methine (d
H
1.26, 3H; d
H
3.24, 1H), two aromatic protons (d
H
6.67,
1H; d
H
6.57, 1H) and an aromatic methyl (d
H
2.17, 3H). In
comparison with the
1
H NMR data of curcuhydroquinone (12),
metabolite 7 displayed an additional signal at d
H
4.02 that
corresponded to the oxygenated methine H-9, as supported by the
COSY and HMBC correlations observed. It was not possible to
determine the relative conguration of 7 at C-9 on the basis of its
spectroscopic data. Therefore, metabolite 7 was identied as 9-
hydroxy-curcuhydroquinone.
Compound 8, isolated as a yellowish oil, was assigned the
molecular formula C
15
H
24
O
2
on the basis of its HR-FABMS and
13
C
NMR data. The
1
H NMR spectrum of 8 revealed signals for three
aromatic protons on a 1,2,4-trisubstituted aromatic ring at d
H
6.59,
6.70 and 7.01, one aromatic methyl group at d
H
2.25, one aliphatic
methyl on a tertiary carbon at d
H
1.22, two doublet methyls of an
isopropryl moiety at d
H
0.88 and 0.86 and one oxygenated methine
at d
H
3.38. Based on the COSY and HMBC correlations, compound 8
was identied as the 10-hydroxy derivative of 10,11-dihydrocur-
cuphenol (15).
Compounds 927 were identied by comparison of their
spectroscopic and physical characteristics with those reported in
the literature. Specically, compounds 9, 10 and 20 have been
isolated from sponges of the genus Arenochalina (Butler et al.,
1991), curcuhydroquinone (12) and curcuphenol (15) have been
obtained from the soft coral P. rigida (McEnroe and Fenical, 1978),
(7R*,9S*)-bisacumol (16) has been isolated from the plant Curcuma
xanthorrhiza (Uehara et al., 1989), a-curcumene (18) has been
reported from a variety of natural sources (Maurer and Grieder,
1977; Van Beek and Lelyveld, 1991), compound 21 has been
isolated from the plant Baccharis dracunculifolia (Fukuda et al.,
2006), a-pipitzol (24) and the isomeric b-pipitzol (25) have been
isolated from plants of the genus Perezia (Walls et al., 1965) and
()-loliolide (26) and (+)-loliolide (27) have been isolated in the
past from several plants, such as Lollium perenne (Hodges and
Porte, 1964) and Salvia divinorum (Valde s, 1986). Metabolites 11,
13, 14, 17, 19, 22 and 23 were previously described as
semisynthetic compounds (Green et al., 2011; Li et al., 2003;
Sugahara and Ogasawara, 1998; Thomas, 1980; Vig et al., 1980,
1983) and are reported for the rst time as natural products.
Analysis of the 1D and 2D NMR spectra allowed the assignment of
the
1
H and
13
C NMR chemical shifts for 11, 19, 22 and 23 for which
only a few characteristic
1
H NMR resonances had been reported.
On the basis of biosynthetic considerations the absolute
conguration of compounds 28 at C-7 is expected to be R. In
support of this, all known marine and terrestrial bisabolanes exhibit
a 7R conguration with the exception of sponge-derived analogs
which possess a 7S conguration (Harrison and Crews, 1997).
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer model 341
polarimeter with a 1 dm cell. UV spectra were acquired on a
Shimadzu UV-160A spectrophotometer. IR spectra were obtained
using a Paragon 500 Perkin-Elmer spectrometer. NMR spectra were
recorded on Bruker AC 200 and Bruker DRX 400 spectrometers.
Chemical shifts are given on the d (ppm) scale using TMS as
internal standard. The 2D experiments (HSQC, HMBC, COSY,
NOESY) were performed using standard Bruker pulse sequences.
High resolution FAB mass spectrometric data were provided by the
University of Notre Dame, Department of Chemistry and Biochem-
istry, Notre Dame, IN, USA. High resolution ESI mass spectrometric
data were provided by the National Hellenic Research Foundation,
Institute of Organic and Pharmaceutical Chemistry, Greece. Low
resolution EI mass spectra were measured on a Hewlett Packard
5973 mass spectrometer. Column chromatography separations
were performed with Kieselgel 60 or C
18
-RP silica gel (Merck).
HPLC separations were conducted on an Agilent 1100 liquid
chromatography system equipped with refractive index detector,
using the following columns: (i) Kromasil 100 C
18
5 mm (MZ-
Analysentechnik, 25 cm 8 mm), (ii) Nucleosil SP250/10 100-7
C
18
(Macherey-Nagel, 25 cm 10 mm), (iii) Econosphere C
18
5 mm
(Alltech, 25 cm 4.6 mm) and (iv) Chiralcel OD 10 mm (Daicel
Chemical Industries Ltd., 25 cm 10 mm). TLC were performed
with Kieselgel 60 F
254
(Merck aluminum support plates) and spots
were detected after spraying with 15% H
2
SO
4
in MeOH reagent and
heating at 100 8C for 1 min. The lyophilization was carried out in a
Freezone 4.5 freeze dry system (Labconco).
3.2. Animal material
Specimens of P. rigida were collected by scuba diving in the
Caribbean Sea, U.S.A., at a depth of 2030 m. A voucher specimen
has been deposited at the Herbarium of the Department of
Pharmacognosy and Chemistry of Natural Products, University of
Athens (ATPH/MO/57).
3.3. Extraction and isolation
Specimens of the freeze-dried gorgonian were exhaustively
extracted with mixtures of CH
2
Cl
2
/MeOH at room temperature.
Evaporation of the solvents in vacuo afforded a dark orange oily
residue (27.0 g) which was subjected to vacuum column chroma-
tography on silica gel, using cyclohexane with increasing amounts
of EtOAc, followed by EtOAc with increasing amounts of MeOH as
the mobile phase, to yield 10 fractions (110). The soluble in 95%
MeOH in H
2
O part of fraction 1 (100.0 mg) was puried by reversed
phase HPLC, using MeOH/H
2
O (95:5) as eluent, to yield 18 (22 mg).
A part of fraction 2 (8.6 g) was subjected to gravity column
chromatography on silica gel, using cyclohexane/EtOAc (95:5) as
the mobile phase, to afford 12 fractions (2A2L). The soluble in
P. Georgantea et al. / Phytochemistry Letters 8 (2014) 8691 89
80% MeOH in H
2
O part of fraction 2 C (10.0 mg) was puried by
reversed phase HPLC, using MeOH/H
2
O (80:20) as eluent, to yield 4
(1.5 mg). A part of fraction 2H (284.0 mg) was subjected to gravity
column chromatography on silica gel, using cyclohexane/EtOAc
(80:20) to afford 6 fractions (2H12H6). Fraction 2H5 (39.0 mg)
was puried by reversed phase HPLC, using MeOH/H
2
O (75:25) as
eluent, to afford 15 (17.8 mg), 24 (3.2 mg) and 25 (1.2 mg). Fraction
3 (1.0 g) was subjected to gravity column chromatography on silica
gel, using cyclohexane/EtOAc (95:5) as the mobile phase, to yield
12 fractions (3A3L). The soluble in 80% MeOH in H
2
O part of
fraction 3I (23.5 mg) was puried by reversed phase HPLC, using
MeOH/H
2
O (90:10) as eluent, to yield 11 (1.5 mg) and 19 (2.8 mg).
The soluble in 80% MeOH in H
2
O part of fraction 3K (50.0 mg) was
puried by reversed phase HPLC, using MeOH/H
2
O (80:20) as
eluent, to yield 21 (6.0 mg) and a mixture of the isomers 16
(2.5 mg) and 17 (1.0 mg) that was further puried by chiral HPLC
using n-Hex/i-Prop (95:5). Fraction 5 (1.24 g) was subjected to
reversed phase vacuum column chromatography, using H
2
O with
increasing amounts of MeOH as the mobile phase, to yield 14
fractions (5A5N). Fraction 5A (63.3 mg) was puried by reversed
phase HPLC, using MeOH/H
2
O (80:20) as eluent, to afford 1
(1.0 mg) and 12 (3.4 mg). Fraction 5B (90.4 mg) was puried by
reversed phase HPLC, using MeOH/H
2
O (75:25) as eluent, to afford
8 (3.8 mg). Fraction 5 C (46.0 mg) was puried by reversed phase
HPLC, using MeOH/H
2
O (72:25) as eluent, to yield 14 (2.2 mg), 22
(1.5 mg) and 23 (0.5 mg). The soluble in 50% MeOH in H
2
O part of
fraction 6 (22.0 mg) was puried by reversed phase HPLC, using
MeOH/H
2
O (70:30) as eluent, to yield 7 (2.1 mg) and 13 (1.1 mg).
The soluble in 60% MeOH in H
2
O part of fraction 6 (34.0 mg) was
puried by reversed phase HPLC, using MeOH/H
2
O (70:30) as
eluent, to yield 20 (0.8 mg) and a mixture of the isomers 9 (3.0 mg)
and 10 (1.1 mg) that was further puried by chiral HPLC using n-
Hex/i-Prop (90:10). Fraction 7 (487.0 mg) was subjected to gravity
column chromatography on silica gel, using cyclohexane/EtOAc
(70:30) as the mobile phase, to afford 11 fractions (7A7K).
Fraction 7F (50.0 mg) was puried by chiral HPLC, using n-Hex/i-
Prop (85:15) as eluent, to yield 2 (1.0 mg) and 3 (1.5 mg). Fraction
7G (69.0 mg) was puried by chiral HPLC, using n-Hex/i-Prop
(85:15) as eluent, to yield 5 (2.8 mg) and 6 (0.7 mg). The soluble in
50% MeOH in H
2
O part of fraction 7 K (4.0 mg) was puried by
chiral HPLC, using n-Hex/i-Prop (85:15) as eluent, to yield 26
(1.8 mg) and 27 (1.7 mg).
3.3.1. Compound 1
Yellowish oil; a
20
D
12.0 (c 0.108, CHCl
3
); UV (CHCl
3
) l
max
(log e) 211 (3.25) nm; IR (lm) n
max
3300, 2939, 2216, 1650 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2; EIMS (70 eV)
m/z (rel. int. %) 250 (1), 232 (16), 217 (5), 189 (8), 175 (9), 164 (16),
151 (100), 137 (10), 96 (11), 68 (13), 55 (10), 41 (14); HR-ESIMS m/z
251.1649 [M+H]
+
(calcd for C
15
H
23
O
3
, 251.1647).
3.3.2. Compound 2
Yellowish oil; a
20
D
30.4 (c 0.125, CHCl
3
); UV (CHCl
3
) l
max
(log e) 296 (3.20) nm; IR (lm) n
max
3400, 2926, 1419, 1376, 1192,
1017 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2;
EIMS (70 eV) m/z (rel. int. %) 232 (69), 217 (14), 202 (2), 189 (12),
175 (25), 163 (15), 150 (100), 135 (10), 121 (6), 107 (10), 91 (9), 77
(11), 67 (9), 53 (6), 41 (8); HR-ESIMS m/z 233.1538 [M+H]
+
(calcd
for C
15
H
21
O
2
, 233.1542).
3.3.3. Compound 3
Yellowish oil; a
20
D
+12.8 (c 0.125, CHCl
3
); UV (CHCl
3
) l
max
(log e) 297 (3.15) nm; IR (lm) n
max
3402, 2925, 1418, 1374, 1190,
1017 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2;
EIMS (70 eV) m/z (rel. int. %) 232 (68), 217 (14), 203 (2), 189 (12),
175 (26), 163 (15), 150 (100), 137 (8), 121 (6), 107 (10), 91 (9), 77
(11), 67 (10), 53 (6), 41 (8); HR-ESIMS m/z 233.1542 [M+H]
+
(calcd
for C
15
H
21
O
2
, 233.1542).
3.3.4. Compound 4
Yellowish oil; a
20
D
+26.7 (c 0.075, CHCl
3
); UV (CHCl
3
) l
max
(log e) 275 (2.63) nm; IR (lm) n
max
2930, 1723, 1447, 1368,
1266 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2;
EIMS (70 eV) m/z (rel. int. %) 204 (26), 189 (4), 161 (12), 147 (6), 134
(13), 119 (100), 105 (22), 93 (54), 82 (22), 69 (26), 55 (18); HR-
ESIMS m/z 265.2162 [M+H]
+
(calcd for C
17
H
29
O
2
, 265.2168).
3.3.5. Compound 5
Yellowish oil; a
20
D
7.5 (c 0.040, CHCl
3
); UV (CHCl
3
) l
max
(log e) 296 (2.93) nm; IR (lm) n
max
3406, 2965, 1446, 1259,
995 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2;
EIMS (70 eV) m/z (rel. int. %) 250 (2), 232 (3), 189 (1), 177 (3), 164
(4), 151 (100), 137 (4), 123 (3), 107 (3), 95 (4), 79 (5), 69 (4), 55 (3);
HR-FABMS m/z 250.1581 [M]
+
(calcd for C
15
H
22
O
3
, 250.1569).
3.3.6. Compound 6
Yellowish oil; a
20
D
+12.0 (c 0.025, CHCl
3
); UV (CHCl
3
) l
max
(log e) 295 (3.02) nm; IR (lm) n
max
3402, 2966, 1440, 1260,
997 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2;
EIMS (70 eV) m/z (rel. int. %) 250 (2), 230 (15), 215 (24), 202 (8), 187
(13), 177 (21), 161 (15), 151 (100), 137 (9), 122 (23), 105 (34), 91
(22), 81 (42), 69 (32), 53 (21), 41 (30); HR-FABMS m/z 250.1578
[M]
+
(calcd for C
15
H
22
O
3
, 250.1569).
3.3.7. Compound 7
Yellowish oil; a
20
D
18.0 (c 0.083, CHCl
3
); UV (CHCl
3
) l
max
(log e) 292 (3.13) nm; IR (lm) n
max
3370, 2929, 1432, 1173 cm
1
;
1
H NMR data, see Table 1;
13
C NMR data, see Table 2; EIMS (70 eV)
m/z (rel. int. %) 250 (8), 232 (42), 217 (12), 189 (11), 175 (28), 161
(17), 151 (100), 137 (14), 121 (8), 107 (14), 91 (18), 77 (25), 67 (17),
53 (16), 41 (23); HR-ESIMS m/z 249.1498 [MH]

(calcd for
C
15
H
21
O
3
, 249.1491).
3.3.8. Compound 8
Yellowish oil; a
20
D
23.0 (c 0.100, CHCl
3
); UV (CHCl
3
) l
max
(log e) 276 (2.81) nm; IR (lm) n
max
3302, 2948, 1164 cm
1
;
1
H
NMR data, see Table 1;
13
C NMR data, see Table 2; EIMS (70 eV) m/z
(rel. int. %) 236 (9), 218 (3), 203 (2), 175 (10), 161 (6), 148 (26), 135
(100), 121 (9), 91 (11), 55 (4); HR-FABMS m/z 235.1681 [MH]

(calcd for C
15
H
23
O
2
, 235.1698).
3.3.9. Compound 11
Yellowish oil; a
20
D
6.0 (c 0.100, CHCl
3
);
1
H NMR (400 MHz,
CDCl
3
) d 7.06 (4H, m, H-1, H-2, H-4, H-5), 4.88 (1H, brs, H-13a), 4.79
(1H, brs, H-13b), 3.99 (1H, t, J = 5.8 Hz, H-10), 2.63 (1H, m, H-7),
2.29 (3H, s, H-15), 1.63 (3H, s, H-12), 1.61 (1H, m, H-8a), 1.53 (1H,
m, H-8b), 1.51 (1H, m, H-9a), 1.38 (1H, m, H-9b), 1.21 (3H, d,
J = 7.0 Hz, H-14);
13
C NMR (50 MHz, CDCl
3
) d 147.9 (C, C-11), 144.5
(C, C-6), 135.8 (C, C-3), 128.1 (CH, C-1, C-2, C-4, C-5), 111.4 (CH
2
, C-
13), 76.1 (CH, C-10), 39.8 (CH, C-7), 34.4 (CH
2
, C-8), 33.6 (CH
2
, C-9),
23.1 (CH
3
, C-14), 21.7 (CH
3
, C-15), 17.9 (CH
3
, C-12).
3.3.10. Compound 19
Yellowish oil; a
20
D
17.0 (c 0.100, CHCl
3
);
1
H NMR (400 MHz,
CDCl
3
) d 7.08 (2H, m, H-1, H-5), 7.06 (2H, m, H-2, H-4), 3.31 (1H, m,
H-10), 2.64 (1H, sextet, J = 7.0 Hz, H-7), 2.30 (3H, s, H-15), 1.75 (1H,
m, H-8a), 1.58 (2H, m, H-8b, H-11), 1.41 (1H, m, H-9a), 1.23 (1H, m,
H-9b), 1.22 (3H, d, J = 7.0 Hz, H-14), 0.84 (3H, d, J = 6.8 Hz, H-12),
0.83 (3H, d, J = 6.8 Hz, H-13);
13
C NMR (50 MHz, CDCl
3
) d 143.9 (C, C-
6), 134.9 (C, C-3), 129.0 (CH, C-2, C-4), 126.8 (CH, C-1, C-5), 76.5 (CH,
C-10), 39.3 (CH, C-7), 34.2 (CH
2
, C-8), 33.1 (CH, C-11), 32.0 (CH
2
, C-9),
22.6 (CH
3
, C-14), 20.9 (CH
3
, C-15), 18.8 (CH
3
, C-12), 16.9 (CH
3
, C-13).
P. Georgantea et al. / Phytochemistry Letters 8 (2014) 8691 90
3.3.11. Compound 22
Yellowish oil; a
20
D
15.0 (c 0.067, CHCl
3
);
1
H NMR (400 MHz,
CDCl
3
) d 7.32 (1H, brs, OH-1), 6.91 (1H, d, J = 7.7 Hz, H-5), 6.70 (1H,
brs, H-2), 6.67 (1H, d, J = 7.7 Hz, H-4), 5.08 (1H, m, H-10), 3.94 (1H,
m, H-14a), 3.85 (1H, m, H-14b), 2.93 (1H, m, H-7), 2.26 (3H, s, H-
15), 2.02 (1H, brs, OH-14), 1.94 (2H, m, H-9), 1.81 (1H, m, H-8a),
1.71 (1H, m, H-8b), 1.65 (3H, s, H-12), 1.50 (3H, s, H-13);
13
C NMR
(50 MHz, CDCl
3
) d 154.4 (C, C-1), 137.6 (C, C-3), 131.9 (C, C-11),
129.1 (CH, C-5), 125.7 (C, C-6), 124.1 (CH, C-10), 121.6 (CH, C-4),
118.1 (CH, C-2), 68.1 (CH
2
, C-14), 43.1 (CH, C-7), 29.7 (CH
2
, C-8),
26.5 (CH
2
, C-9), 25.8 (CH
3
, C-12), 21.2 (CH
3
, C-15), 18.2 (CH
3
, C-13).
3.3.12. Compound 23
Yellowish oil; a
20
D
5.2 (c 0.058, CHCl
3
);
1
H NMR (400 MHz,
CDCl
3
) d 7.33 (2H, d, J = 7.8 Hz, H-1, H-5), 7.15 (2H, d, J = 7.8 Hz, H-
2, H-4), 5.17 (1H, d, J = 8.8 Hz, H-10), 4.19 (1H, m, H-9), 2.33 (3H, s,
H-15), 2.05 (1H, m, H-8a), 1.94 (1H, m, H-8b), 1.63 (3H, brs, H-12),
1.47 (3H, s, H-14), 1.39 (3H, brs, H-13);
13
C NMR (50 MHz, CDCl
3
) d
144.2 (C, C-6), 135.4 (C, C-3), 134.3 (C, C-11), 128.3 (CH, C-2, C-4),
127.3 (CH, C-10), 124.3 (CH, C-1, C-5), 75.0 (C, C-7), 67.7 (CH, C-9),
48.3 (CH
2
, C-8), 32.2 (CH
3
, C-14), 25.5 (CH
3
, C-12), 21.1 (CH
3
, C-15),
18.0 (CH
3
, C-13).
Acknowledgments
The authors thank Prof. W. Fenical (SIO, UCSD, U.S.A.),
coordinator of a National Science Foundation project, for allowing
V.R. and C.V. to participate and collect specimens of P. rigida during
a collection campaign in the Bahamas Islands aboard the R/V
Seward Johnson. This study was partially supported by a
Kapodistrias grant from the University of Athens.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2014.02.006.
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