Dolabellanes with antibacterial activity from the brown alga Dilophus spiralis

Article abstract of DOI:10.1021/np1006586

Seventeen diterpenes featuring the dolabellane skeleton (1-17) were isolated from the organic extracts of the brown alga Dilophus spiralis. Seven compounds are new natural products (1, 3, 5, 6, 11, 14, 15) and eight are structurally revised (2, 4, 7-10, 12, 13), among which three are reported for the first time from a natural source (4, 9, 10). The structure elucidation and the assignment of the relative configurations of the isolated natural products were based on detailed analyses of their spectroscopic data. The structure of metabolite 10 was confirmed by single-crystal X-ray diffraction analysis, whereas the absolute configurations of compounds 2, 4-10, 12, and 13 were determined using the modified Mosher's method on the semisynthetic product 18 and chemical interconversions. The antibacterial activities of compounds 1-18 were evaluated against six strains of Staphylococcus aureus, including multidrug- and methicillin-resistant variants.

Full text of DOI:10.1021/np1006586

ARTICLE
pubs.acs.org/jnp
Dolabellanes with Antibacterial Activity from the Brown Alga Dilophus
spiralis
Efstathia Ioannou,† Antonio Quesada,‡ M. Mukhlesur Rahman,§ Simon Gibbons,§ Constantinos Vagias,^,†
and Vassilios Roussis*,†
†Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens,
Panepistimiopolis Zografou, Athens 15771, Greece
‡Department of Didactics of Sciences, University of Jaꢀen, PO 23071, Jaꢀen, Spain
§Department of Pharmaceutical and Biological Chemistry, School of Pharmacy,
University of London, 29-39 Brunswick Square, London WC1N 1AX, U.K.
S Supporting Information
b
ABSTRACT: Seventeen diterpenes featuring the dolabellane skeleton
(1-17) were isolated from the organic extracts of the brown alga
Dilophus spiralis. Seven compounds are new natural products (1, 3, 5, 6,
11, 14, 15) and eight are structurally revised (2, 4, 7-10, 12, 13),
among whichthreeare reportedforthefirst time from a natural source (4, 9,
10). The structure elucidation and the assignment of the relative
configurations of the isolated natural products were based on detailed analyses of their spectroscopic data. The structure of
metabolite 10 was confirmed by single-crystal X-ray diffraction analysis, whereas the absolute configurations of compounds 2, 4-10,
12, and 13 were determined using the modified Mosher's method on the semisynthetic product 18 and chemical interconversions.
The antibacterial activities of compounds 1-18 were evaluated against six strains of Staphylococcus aureus, including multidrug- and
methicillin-resistant variants.
rown algae of the family Dictyotaceae are widely distributed
in the tropical and subtropical waters of the world, found
and MeOH, and the organic extracts were subsequently sub-
jected to a series of chromatographic separations to allow for the
isolation of compounds 1-17.
B
mainly in the Atlantic, Pacific, and Indian Oceans, the Caribbean
and Mediterranean Seas, and the Sea of Japan. They have been
the subject of extensive studies in the last five decades, having
yielded almost 500 new secondary metabolites to date. The majority
of these natural products are sesquiterpenes and diterpenes of normal
or mixed biosynthesis, often exhibiting antibacterial, antiviral, cyto-
toxic, algicidal, antifouling, antifeedant, and/or ichthyotoxic activity.1,2
In the course of our continuing research aimed at the isolation
of bioactive natural products from marine organisms found along
the coastlines of Greece, we undertook a thorough investigation
of the chemical composition of Dilophus spiralis (Montagne) Hamel
(syn. ligulatus). Previously, we reported the isolation and struc-
tural characterization of five new dolastanes, one new 2,6-cyclo-
xenicane, and several known metabolites from D. spiralis.3,4 Herein,
we describe the isolation and structure elucidation of 17 dola-
bellanes (1-17) from the same algal specimens and the evalua-
tion of their antibacterial activities against six strains of Staphylococcus
aureus, some of which are resistant, via multidrug efflux. Seven
compounds are new natural products (1, 3, 5, 6, 11, 14, 15) and
eight are structurally revised (2, 4, 7-10, 12, 13), among which three
are reported for the first time from a natural source (4, 9, 10).
Compounds 1-3, isolated as oils, displayed molecular ion
peaks at m/z 286 (EIMS), corresponding to C20H30O. The
absorption band at 1735 cm-1 in their IR spectra indicated the
presence of a carbonyl group, while their 13C NMR spectra
revealed 20 carbon signals, which were assigned to five quater-
nary carbon atoms, four methines, seven methylenes, and four
methyls, as determined by DEPT experiments. The structural
1
elements displayed in the H and 13C NMR spectra of 1-3
(Tables 1 and 2) included four methyl groups on quaternary
carbons (δH/C 0.91/17.5, 1.48/15.8, 1.54/18.1, and 1.77/18.2
for 1; δH/C 1.09/18.3, 1.51/15.7, 1.53/16.5, and 1.70/22.8 for 2;
δH/C 0.93/21.9, 1.66/23.5, 1.63/17.2, and 1.76/25.0 for 3), one
1,1-disubstituted double bond (δH/C 4.81, 4.93/113.5, δC 144.7
for 1; δH/C 4.64, 4.91/112.1, δC 144.7 for 2; δH/C 4.58, 4.91/
113.3, δC 148.0 for 3), two trisubstituted double bonds (δH/C
4.70/122.8, δC 134.6 and δH/C 4.86/125.3, δC 136.1 for 1; δH/C
5.12/122.8, δC 136.6 and δH/C 4.84/128.3, δC 133.0 for 2; δH/C
4.69/122.4, δC 137.4 and δH/C 5.13/125.4, δC 134.9 for 3), and
a ketone functionality (δC 221.8 for 1; δC 222.8 for 2; δC 226.3
for 3). Because the carbonyl group and the three carbon-carbon
double bonds accounted for four of the six degrees of unsaturation,
’ RESULTS AND DISCUSSION
Specimens of the brown alga D. spiralis, collected on Elafo-
nissos Island, Greece, were exhaustively extracted with CH2Cl2
Received: September 16, 2010
Published: December 29, 2010
r
Copyright
2010 American Chemical Society and
213
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Δ7 double bonds were determined as 3E,7E on the basis of the
NOE interactions of H-2R/H3-16, H-2β/H-3, H-3/H3-15, H-7/
H-9β, and H-9R/H3-17. This was further supported by the fact
that C-16 and C-17 resonated at lower frequencies (δC 15.8 and
18.1, respectively). In contrast, the intense NOE enhancement of
H-11/H-12 displayed in the NOESY spectra of 2 and 3, in con-
junction with the observed NOE cross-peaks of H-2R/H-11,
H-2β/H3-15, H-12/H-20R, and H3-19/H-20β for 2 and of H-3/
H-11, H-3/H-13R, H-12/H-13R, H-12/H3-19, H-13β/H-20β,
H3-15/H-20β, and H3-19/H-20R for 3, suggested the trans
fusion of the two rings and the cis- and trans-orientation of
H-12 in relation to H-11 and H3-15, respectively. The geometries
of the Δ3 and Δ7 double bonds in 2 were determined as 3E,7E on
the basis of the NOE interactions of H-2R/H3-16, H-2R/H3-17,
H-2β/H-3, H-3/H-7, and H-3/H3-15, as in the case of 1, which
was further supported by the fact that C-16 and C-17 resonated
at lower frequencies (δC 15.7 and 16.5, respectively). However,
the geometries of the Δ3 and Δ7 double bonds in 3 were determined
as Z and E, respectively, on the basis of the NOE cross-peaks of
H-3/H3-16, H2-6/H3-17, H-7/H-9β, H-7/H-10β, and H-10β/
H3-15. This was further verified by the fact that C-16 and C-17
resonated at higher (δC 23.5) and lower (δC 17.2) frequencies,
respectively. On the basis of the above-mentioned data, meta-
bolite 1 was identified as (1R,3E,7E,11S,12R)-14-oxo-3,7,18-
dolabellatriene, 2 as its epimer at C-12, and 3 as the geometrical
isomer of 2 at Δ3. The spectroscopic and physical characteristics
of 2 were identical to those of a previously reported dolabellane,
although its structure was established as that of compound 1.5,6 A
closer examination of the details of that work revealed that the
relative configuration of C-12 had been erroneously assigned, based
on rather tenuous evidence prior to the introduction of 2D NMR
experiments. Possible reasons for the misassignment of the
relative configuration of C-12 might include a misinterpretation
of the results of the lanthanide-induced shift experiments used to
determined the relative orientation of H3-15 and the isopropenyl
group and the fact that the relative orientation of H-11 and H-12
was established on the basis of their coupling constant alone
without the use of NOE correlations.
Compound 4, obtained as a yellowish oil, displayed a profile
closely resembling those of metabolites 1-3. Its spectroscopic
and physical characteristics were the same as those of a semisyn-
thetic product reported in the literature, whose structure had
been mistakenly assigned, concerning the relative configuration
of C-12, on the basis of the original misassignment regarding
compound 2.7 Indeed, the NOE enhancements of H-2R/H-11,
H-2β/H3-15, H-11/H-12, H-12/H-20R, and H3-19/H-20β, as
in the case of 2, suggested the trans fusion of the two rings and
indicated that H-12 was cis- and trans-oriented relative to H-11 and
H3-15, respectively. Thus, metabolite 4 was identified as the 14-deoxo
derivative of 2, reported for the first time as a natural product.
Compound 5, isolated as a colorless oil, had the molecular
formula C20H32O, as calculated from the HRFABMS measure-
ments. Analysis of the spectroscopic data of 5 (Tables 1 and 2)
showed a high degree of similarity with metabolite 4. In agree-
ment with the molecular formula, it was clear that the difference
was the presence of one hydroxy group. This was verified from
the signals of an oxygenated methine (δH/C 3.90/81.8) evident
in the 1H and 13C NMR spectra, as well as the absorption band at
3370 cm-1 in the IR spectrum. The hydroxy group was placed at
C-14 due to the heteronuclear correlations of C-14 with H-11,
H-12, H2-13, and H3-15. The relative configurations of the
stereogenic centers C-1, C-11, and C-12 and the geometries of
the molecular structures of 1-3 were determined as bicyclic.
Analyses of their 2D NMR spectra resulted in the establishment
of the same planar structure, suggesting that the three compounds
were stereoisomers. Specifically, the long-range coupling between
H3-19 and H2-20 observed in the COSY spectrum indicated the
presence of an isopropenyl group, whereas the correlations
of C-18 with H-12 and H3-19, as well as of C-12 with H3-19 and
H2-20 in the HMBC spectrum, fixed its position. The cross-peaks
of H-11/H-12 and H-12/H2-13 observed in the COSY spectrum,
in combination with the HMBC correlations of C-1, C-12, and
C-14 with H-11 and H2-13, identified the five-membered ring.
Furthermore, the correlations of C-3 and C-4 with H2-2, H2-5,
and H3-16, of C-7 and C-8 with H2-6, H2-9, and H3-17, and of
C-1 and C-11 with H2-2 and H2-10 displayed in the HMBC
spectrum, in conjunction with the COSY correlations of H2-2/
H-3, H2-5/H2-6, H2-6/H-7, H2-9/H2-10, and H2-10/H-11,
concluded the assignment of the 11-membered ring. Finally,
the HMBC correlations of H3-15 with C-1, C-11, and C-14 placed
the aliphatic methyl on C-1. The relative configurations of the
stereogenic centers and the geometries of the double bonds of
metabolites 1-3 were assigned on the basis of interactions observed
in their NOESY spectra. The NOE enhancements of H-11/
H3-19, H-12/H3-15, H-12/H-20β, and H3-19/H-20R evident in
the NOESY spectrum of 1 suggested the trans fusion of the two
rings and indicated that H-12 was trans- and cis-oriented relative
to H-11 and H3-15, respectively. The geometries of the Δ3 and
214
dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222

Journal of Natural Products
ARTICLE
1
Table 1. H NMR Data (400 MHz, CDCl3) of Compounds 1, 3-6, and 18
position
2
1 (J in Hz)
3 (J in Hz)
1.95, m
4 (J in Hz)
5 (J in Hz)
6 (J in Hz)
18 (J in Hz)
R
2.35, dd (14.6, 6.6)
1.93, dd (14.6, 6.6)
4.70, dd (6.6, 6.6)
R
2.19, m
1.67, m
5.13, dd
(11.4, 4.4)
2.22, m
2.05, m
2.28, m
2.03, m
4.84, m
R
2.23, m
1.72, m
5.10, dd
(10.8, 3.8)
2.19, m
2.05, m
2.28, m
2.03, m
4.87, dd
(10.8, 1.8)
2.10, m
1.77, m
1.34, m
1.23, m
1.93, m
R
2.30, m
1.66, m
5.09, dd
(11.1, 4.0)
2.21, m
2.08, m
2.27, m
2.05, m
4.86, m
R
2.10, m
β
β
β
β
β
1.79, m
3
4.69, dd
(11.6, 2.9)
5.09, dd (8.4, 6.4)
5
a
2.08, m
a
2.29, m
1.78, m
2.19, m
a
a
a
2.12, m
b
a
2.01, m
b
b
a
b
a
b
a
6
2.17, m
a
2.22, m
b
2.11, m
b
b
b
b
2.08, m
7
4.86, dd (7.5, 7.5)
5.13, dd
4.83, dd (9.8, 4.2)
(7.7, 7.7)
9
R
β
a
2.03, m
1.75, m
1.49, m
1.37, m
1.99, m
R
β
R
β
2.15, m
1.80, m
1.62, m
1.18, m
2.55, ddd
a
2.11, m
1.88, m
1.31, m
1.23, m
1.74, m
a
a
2.11, m
1.85, m
1.33, m
1.22, m
1.92, m
a
2.05, m
1.87, m
1.36, m
1.18, m
1.84, m
b
a
b
a
b
a
b
a
10
11
12
13
b
b
b
b
b
(8.9, 7.9, 1.5)
2.87, dd
(8.9, 8.9)
2.53, ddd
2.61, ddd
2.90, ddd
(9.9, 7.6, 7.6)
1.95, m
2.85, ddd
(9.5, 7.4, 7.4)
2.02, m
2.56, ddd
(11.3, 11.3, 7.9)
2.36, dd (18.5, 7.9)
(10.0, 6.8, 6.8)
1.64, m
(12.4, 7.8, 6.4)
1.99, ddd
a
R
2.31, dd (18.2, 8.9)
a
a
a
R
(12.4, 6.4, 6.4)
1.64, ddd
b
2.23, dd (18.5, 11.3)
β
2.48, d (18.2)
b
1.56, m
b
1.65, m
b
1.64, m
β
(12.4, 12.4, 9.8)
3.72, dd (9.8, 6.4)
14
a
1.52, m
1.42, m
1.06, s
3.90, dd (6.3, 6.3)
4.91, dd (6.0, 4.2)
b
15
16
17
19
20
0.91, s
0.93, s
1.03, s
1.10, s
1.52, s
1.51, s
1.72, s
4.64, brs
4.85, brs
2.03, s
0.96, s
1.48, s
1.66, s
1.51, s
1.52, s
1.50, s
1.54, s
1.63, s
1.51, s
1.53, s
1.49, s
1.77, s
1.76, s
1.70, s
1.70, s
1.71, s
R
4.81, brs
4.93, brs
R
4.91, brs
4.58, brs
R
4.64, brs
4.82, brs
R
4.63, brs
4.82, brs
R
R
4.68, brs
4.87, brs
β
β
β
β
β
β
OAc
the double bonds at C-3 and C-7 were established by analysis of
the key correlations displayed in the NOESY spectrum of 5, in
accordance with those of 4. The NOE enhancements of H-2β/
H-14 and H-14/H3-15 suggested a cis-relationship between H-14
and H3-15 and determined the relative configuration of C-14
as S*. Therefore, metabolite 5 was identified as the 14S-hydroxy
derivative of 4.
incorrectly assigned, concerning the relative configuration of
C-12, on the basis of the original misassignment regarding com-
pound 2.5,6 Inspection of the NOESY spectra of 7-9 revealed
cross-peaks of H-2R/H-12, H-2β/H-3, H-3/H3-15, H-11/H-12,
H-12/H-20R, and H3-19/H-20β, thus determining the same
relative configurations for C-1, C-11, and C-12 as in 2-6.
Metabolite 9 is reported for the first time as a natural product.
Compounds 10 and 11, isolated as white crystals and a colorless
oil, respectively, displayed molecular ion peaks at m/z 302 (EIMS),
corresponding to C20H30O2. The structural elements displayed
in the 1H and 13C NMR spectra of 10 and 11 (Tables 3 and 4)
exhibited a high degree of similarity with those of metabolite 7. In
agreement with the molecular formula, it was obvious that they
were both isomers of the latter. Analyses of their 2D NMR
spectra resulted in the establishment of the same planar structure,
implying that 10 and 11 were stereoisomers. In this case, the
trisubstituted double bond remained between carbons C-3 and
C-4, as in 1-3, whereas the epoxide function was placed between
carbons C-7 and C-8, on the basis of the HMBC correlations of
C-5, C-6, C-8, and C-9 with H-7 and both C-7 and C-8 with H2-6,
H2-9, and H3-17. The relative configuration and the geometry of
the double bond of both metabolites were assigned on the basis
of interactions observed in their NOESY spectra. The intense
NOE enhancement of H-11/H-12 established the same relative
Compound 6, with the molecular formula C22H34O2, as deduced
from the HRFABMS measurements, was obtained as a colorless
1
oil. Its H and 13C NMR spectra (Tables 1 and 2) were rather
similar to those of 5, with the most prominent difference being
the replacement of the hydroxy group by an acetoxy group. The
shift of H-14 to higher frequencies (δH 4.91), in conjunction with
the presence of an acetoxy group, as suggested by an ester
carbonyl (δC 171.0) and an acetoxy methyl (δH/C 2.03/21.2),
was indicative of the acetylation of the hydroxy group at C-14.
The correlations observed in the homo- and heteronuclear
experiments supported the proposed structure of 6 as the acetyl
derivative of 5. The relative configuration and the geometry of
the double bonds of 6, found in accordance with those of 5, were
established by analysis of its NOESY spectrum.
Compounds 7-9, obtained as oils, exhibited spectroscopic
and physical characteristics consistent with those of dolabellanes
already reported in the literature, whose structures had been
215
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Journal of Natural Products
ARTICLE
Table 2. 13C NMR Data (50 MHz, CDCl3) of Compounds 1, 3-6, and 18
position
1
3
4
5
6
18
1
53.0,
35.2,
C
50.6,
38.0,
C
46.5,
43.4,
125.8,
134.5,
39.9,
24.4,
127.3,
133.9,
37.7,
24.4,
41.8,
51.0,
28.5,
42.1,
24.3,
15.6,
16.7,
146.9,
23.5,
111.2,
C
48.3,
35.0,
124.2,
135.4,
39.9,
24.4,
126.8,
134.4,
36.8,
26.3,
42.0,
46.0,
37.6,
81.8,
22.3,
15.5,
17.7,
146.4,
23.3,
112.4,
C
49.2,
35.2,
124.4,
135.4,
39.8,
24.9,
127.3,
133.9,
37.3,
24.4,
41.8,
47.1,
35.0,
83.3,
22.6,
15.6,
17.0,
145.7,
23.3,
112.2,
171.0,
21.2,
C
48.9,
40.0,
124.5,
134.9,
39.8,
24.6,
127.2,
134.1,
38.2,
24.1,
41.4,
44.4,
37.7,
80.3,
16.9,
15.7,
16.4,
145.6,
23.3,
112.1,
C
2
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH2
CH
C
3
122.8,
134.6,
39.3,
122.4,
137.4,
31.7,
4
5
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
6
25.0,
25.2,
7
125.3,
136.1,
37.1,
125.4,
134.9,
36.0,
8
9
CH2
CH2
CH
CH
CH2
C
CH2
CH2
CH
CH
CH2
C
CH2
CH2
CH
CH
CH2
CH2
CH3
CH3
CH3
C
CH2
CH2
CH
CH
CH2
CH
CH3
CH3
CH3
C
CH2
CH2
CH
CH
CH2
CH
CH3
CH3
CH3
C
CH2
CH2
CH
CH
CH2
CH
CH3
CH3
CH3
C
10
11
12
13
14
15
16
17
18
19
20
OAc
OAc
29.9,
23.8,
43.3,
41.7,
49.9,
41.4,
42.0,
47.1,
221.8,
17.5,
226.3,
21.9,
CH3
CH3
CH3
C
CH3
CH3
CH3
C
15.8,
23.5,
18.1,
17.2,
144.7,
18.2,
148.0,
25.0,
CH3
CH2
CH3
CH2
CH3
CH2
CH3
CH2
CH3
CH2
C
CH3
CH2
113.5,
113.3,
CH3
1
Table 3. H NMR Data (400 MHz, CDCl3) of Compounds 9-11 and 13-15
position
2
9 (J in Hz)
10 (J in Hz)
11 (J in Hz)
13 (J in Hz)
14 (J in Hz)
15 (J in Hz)
R
1.41, dd (14.2, 11.0)
1.78, dd (14.2, 2.4)
2.89, dd (11.0, 2.4)
R
2.15, m
2.15, m
a
2.11, m
a
2.09, m
1.73, m
4.93, m
5.20, d (15.9)
β
β
1.83, dd (13.1, 3.9)
5.38, dd (11.4, 3.9)
b
1.67, m
b
3
4
5
4.73, m
4.98, dd (7.2, 7.2)
5.13, dd (15.9, 7.8)
2.03, m
R
β
R
β
2.14, m
2.27, m
a
2.53, m
2.08, m
2.08, m
a
1.52, m
1.24, m
b
a
1.88, dd (14.0, 7.4)
2.03, m
b
a
1.34, m
6
2.32, m
R
1.56, m
a
2.13, m
a
2.14, m
2.10, m
4.95, m
2.11, m
1.83, m
1.64, m
1.27, m
1.67, m
2.12, m
2.16, m
β
1.90, m
b
1.44, m
b
2.10, m
b
b
2.08, m
7
9
5.03, brd (10.7)
2.00, m
2.72, brd (10.0)
2.01, m
2.88, m
4.86, dd (6.7, 6.7)
2.12, m
4.98, dd (9.8, 5.4)
1.99, m
R
β
a
R
a
2.02, m
a
R
β
a
2.19, m
β
1.28, m
b
R
β
1.55, m
b
a
1.57, m
10
1.39, m
1.42, m
1.51, m
1.50, m
1.42, m
b
1.28, m
1.37, m
b
b
1.23, m
11
12
13
1.95, m
2.14, m
2.52, m
1.52, m
1.72, m
1.65, m
1.33, m
1.44, m
1.37, m
0.97, s
1.37, m
2.77, ddd (11.7, 7.0, 7.0)
2.03, m
2.94, ddd (7.9, 7.7, 7.7)
2.40, m
2.89, m
2.27, m
a
a
2.54, m
a
a
1.68, m
1.42, m
1.57, m
1.43, m
0.97, s
a
b
a
1.76, m
b
1.57, m
b
2.29, dd (18.4, 9.0)
b
a
b
a
1.50, m
14
4.84, dd (7.1, 3.2)
1.72, m
b
b
b
1.41, m
15
16
17
18
19
20
1.29, s
1.23, s
1.56, s
1.08, s
1.64, s
1.27, s
0.96, s
1.67, s
1.33, s
0.85, s
1.54, s
1.48, s
0.91, d (6.8)
1.46, s
1.53, s
1.55, s
1.85, m
0.93, d (6.8)
0.97, d (6.6)
1.70, s
1.73, s
1.79, s
1.21, s
1.23, s
1.70, s
R
4.63, brs
4.87, brs
2.01, s
R
4.69, brs
4.96, brs
R
4.95, brs
4.61, brs
R
4.68, brs
4.70, brs
β
β
β
β
OAc
216
dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222

Journal of Natural Products
ARTICLE
Table 4. 13C NMR Data (50 MHz, CDCl3) of Compounds 9-11 and 13-15
position
9
10
11
13
14
15
1
46.7,
35.6,
63.9,
62.0,
38.8,
24.2,
126.5,
133.8,
37.1,
23.2,
42.0,
47.7,
34.3,
83.1,
22.5,
15.9,
16.3,
145.0,
22.8,
112.1,
170.9,
21.2,
C
52.8,
37.1,
122.1,
136.9,
37.9,
23.6,
64.9,
61.2,
36.2,
23.3,
41.9,
43.3,
42.0,
222.3,
18.0,
15.9,
19.1,
144.6,
23.4,
113.4,
C
50.1,
38.0,
122.8,
137.5,
27.7,
25.1,
62.5,
62.2,
35.0,
23.7,
42.5,
40.9,
46.9,
224.7,
21.3,
23.8,
21.7,
147.0,
25.2,
114.2,
C
46.7,
38.7,
124.6,
133.0,
39.5,
24.9,
126.5,
135.3,
39.2,
31.5,
41.7,
60.2,
26.4,
41.1,
23.2,
16.4,
16.5,
73.3,
26.5,
30.8,
C
44.5,
41.8,
124.2,
133.8,
39.0,
24.3,
124.4,
135.9,
37.7,
23.0,
45.6,
87.3,
30.5,
39.5,
24.3,
15.6,
17.4,
35.0,
18.7,
17.9,
C
45.1,
136.5,
132.7,
38.5,
35.3,
27.8,
127.2,
133.2,
40.5,
24.9,
55.1,
54.1,
27.3,
39.8,
20.7,
22.6,
16.6,
147.5,
18.5,
110.4,
C
2
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH2
CH
C
CH
CH
CH
CH2
CH2
CH
C
3
4
5
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
6
7
8
9
CH2
CH2
CH
CH
CH2
CH
CH3
CH3
CH3
C
CH2
CH2
CH
CH
CH2
C
CH2
CH2
CH
CH
CH2
C
CH2
CH2
CH
CH
CH2
CH2
CH3
CH3
CH3
C
CH2
CH2
CH
C
CH2
CH2
CH
CH
CH2
CH2
CH3
CH3
CH3
C
10
11
12
13
14
15
16
17
18
19
20
OAc
OAc
CH2
CH2
CH3
CH3
CH3
CH
CH3
CH3
CH3
CH3
CH3
C
CH3
CH3
CH3
C
CH3
CH2
C
CH3
CH2
CH3
CH2
CH3
CH3
CH3
CH2
CH3
configurations for C-1, C-11, and C-12 as in 7. The NOE
interactions of H-3/H-7, H-7/H3-15, H-11/H3-17, and H3-16/
H3-17 for 10 and the cross-peaks of H-7/H-10β, H-10β/H3-15,
H-11/H3-17, and H3-16/H3-17 for 11 suggested that the oxyge-
nated methine H-7 was cis- and trans-oriented relative to H3-15
and H3-17, respectively, and established the relative configura-
tion of C-7 and C-8 as 7S*,8S*. The geometry of the double bond
at C-3 was established as E in 10 on the basis of the NOE
enhancements of H-2R/H3-16, H-2β/H-3, and H-3/H3-15,
which was further supported by the fact that C-16 resonated at
lower frequencies (δC 15.9). On the contrary, the geometry of
the Δ3 double bond was established as Z in 11 on the basis of the
NOE enhancements of H-3/H-11 and H-3/H3-16, which was
further verified by the fact that C-16 resonated at higher
frequencies (δC 23.8). Thus, the geometrical isomers 10 and
11 were identified as positional isomers of 7. The proposed
structure of 10 was confirmed by single-crystal X-ray diffraction
analysis (Figure 1).8 The spectroscopic and physical character-
istics of compound 10 were identical to those of a semisynthetic
product reported in the literature, whose structure had been
erroneously assigned concerning the relative configuration of
C-12 on the basis of the original misassignment regarding com-
pound 2, while the relative configurations of C-7 and C-8 were
not established.5
Compound 12, obtained as a yellowish oil, possessed spectro-
scopic and physical characteristics congruent with those of a
previously reported dolabellane, whose structure had been
incorrectly assigned concerning the relative configuration of
C-12 due to the overlap of H-7 and H-12 and the misinterpreta-
tion thereafter of the NOE correlations observed.9 The NOE
enhancements of H-2R/H3-16, H-2β/H-3, H-3/H-7, H-3/H3-
15, H-7/H3-15, H-11/H-12, H-12/H-20R, and H3-19/H-20β,
more easily distinguishable in 1D NOE experiments, indicated
the same relative configurations for C-1, C-7, C-8, C-11, and
Figure 1. ORTEP drawing of compound 10. Displacement ellipsoids
are shown at 30% probability.
C-12 and the geometry of the Δ3 double bond as in the case
of 10.
Compound 13, isolated as a colorless oil, displayed spectro-
scopic and physical characteristics identical to those of a pre-
viously reported dolabellane, whose structure had been erroneously
assigned, concerning the relative configuration of C-12, on the
basis of the original misassignment regarding compound 2.7 As in
the case of metabolites 2-12, the NOE enhancement of H-11/
H-12 indicated the same relative configurations for C-1, C-11,
and C-12.
Compound 14, with the molecular formula C20H34O, as
deduced from the HRFABMS measurements, was obtained as a
1
colorless oil. The structural characteristics evident in the H
and 13C NMR spectra included three singlet methyls (δH/C
0.97/24.3, 1.48/15.6, and 1.55/17.4), two doublet methyls
217
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Journal of Natural Products
ARTICLE
Scheme 1. Chemical Interconversions Performeda Correlating Compounds 2, 4-10, 12, 13, and 18
a Reagents and conditions: (i) NaBH4, MeOH, 1 h; (ii) NaBH4, EtOH, 2 h; (iii) Ac2O, pyridine, overnight; (iv) m-CPBA, benzene, 45 min; (v) Ac2O,
pyridine, 70 ꢀC, 16 h; (vi) POCl3, pyridine, 0 ꢀC, 20 min; (vii) N2H4 H2O, N2H4 2HCl, TEG, 130 ꢀC, 1.5 h, KOH, 170 ꢀC, 2 h; (viii) m-CPBA,
3
3
benzene, 30 min.
(δH/C 0.93/18.7 and 0.97/17.9), two trisubstituted double
bonds (δH/C 4.93/124.2, δC 133.8 and δH/C 4.95/124.4, δC
135.9), and an oxygenated quaternary carbon (δC 87.3). The
spectroscopic data of 14 (Tables 3 and 4) closely resembled
those of 13. In this case, the hydroxy group was placed at C-12, as
indicated by the HMBC correlations of C-12 with H-11, H2-13,
H3-19, and H3-20 and the COSY cross-peaks of H-18 with both
H3-19 and H3-20. The geometries of the Δ3 and Δ7 double
bonds were determined as 3E,7E due to the fact that C-16 and
C-17 resonated at lower frequencies (δC 15.6 and 17.4, re-
spectively). On the basis of the interaction of H-11/H3-19
observed in the NOESY spectrum, measured in C6D6 because
there was partial overlapping of key NMR signals in CDCl3, the
relative configurations of C-1, C-11, and C-12 were established as
1R*,11R*,12R*.
Figure 2. ΔδS-R values (ppm) for the C-14 MTPA derivatives of 18 in
CDCl3.
Compound 15, obtained as a colorless oil, displayed an ion
peak at m/z 272.2495 (HRFABMS), corresponding to C20H32
and consistent with [M]þ. Analysis of the spectroscopic data of
15 (Tables 3 and 4) showed a high degree of similarity with
metabolite 4, and with an identical molecular formula, it was
determination of the relative configurations of C-1, C-11, and
C-12 as 1R*,11S*,12R*. Furthermore, the NOE enhancements of
H-2/H-11, H-2/H3-16, H-11/H3-17, H-3/H-7, and H-3/H3-15
established the relative configuration of C-4 as R* and the
geometries of the double bonds at C-2 and C-7 as 2E,7E. The
latter conclusion was also supported by the large coupling
constant of H-2/H-3 (15.9 Hz) and the fact that C-17 resonated
at lower frequencies (δC 16.6).
Reduction of metabolite 2 yielded both epimeric alcohols at
C-14 (5 and 18), while acetylation of metabolite 5 afforded 6.
Furthermore, epoxidation of metabolite 4 yielded monoepoxides
8 and 12 (Scheme 1). Semisynthetic compounds 5, 6, 8, and 12
were identical in all respects to the natural products, whereas 18
was not detected as a natural product during the chromato-
graphic separations. In the previous reports on compounds 2, 4,
7-10, and 13, several chemical interconversions were used to
1
obvious that the two were isomers. In the H NMR spectrum
three singlet methyls (δH 0.85, 1.46, and 1.70), one doublet
methyl (δH 0.91), an exomethylene group (δH 4.68 and 4.70),
and three olefinic methines (δH 4.98, 5.13, and 5.20) were
evident, suggesting that the difference between the two mole-
cules was the replacement of a trisubstituted double bond by a
1,2-disubstituted one. The 1,2-disubstituted double bond was
placed between C-2 and C-3 on the basis of the COSY cross-
peaks of H-2/H-3, H-3/H-4, H-4/H2-5, and H-4/H3-16 and the
correlations of C-1 and C-15 with H-2, as well as of C-3, C-4, and
C-5 with H3-16. Inspection of the NOESY spectrum of 15
revealed the interaction of H-12/H3-15, which led to the
218
dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222

Journal of Natural Products
ARTICLE
Table 5. Antibacterial Activitiesa of Compounds 1-18
compound
ATCC 25923
EMRSA-15
EMRSA-16
RN4220
SA1199B
XU212
1
inactiveb
inactiveb
inactiveb
64
inactiveb
inactiveb
inactiveb
128
inactiveb
inactiveb
inactiveb
inactiveb
128
inactiveb
128
inactiveb
inactiveb
inactiveb
inactiveb
128
2
16
inactiveb
3
4
16
128
5
128
64
8
64
inactiveb
32
64
6
inactiveb
inactiveb
inactiveb
32
inactiveb
inactiveb
inactiveb
128
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
64
inactiveb
inactiveb
inactiveb
128
7
128
8
32
inactiveb
64
9
32
10
inactiveb
inactiveb
inactiveb
32
inactiveb
inactiveb
128
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
64
inactiveb
inactiveb
inactiveb
32
inactiveb
inactiveb
inactiveb
64
11
12
4
13
32
8
14
8
8
8
8
16
inactiveb
16
inactiveb
15
inactiveb
inactiveb
inactiveb
inactiveb
16
64
64
16
64
2
64
64
64
17
inactiveb
4
inactiveb
2
inactiveb
4
inactiveb
2
inactiveb
4
18
norfloxacin
0.5
0.5
128
0.5
32
8
a Expressed as MIC (in μg/mL). b MIC > 128 μg/mL.
correlate them, and it was proven that the relative configurations
of the asymmetric centers C-1, C-11, and C-12 remained un-
changed (Scheme 1).5,7 In the present study it was shown using
the observed NOE enhancements that the relative configuration
of C-12 of these compounds should be inverted, a fact that was
also verified through the single-crystal X-ray diffraction analysis
of 10. The absolute configuration of 18 was determined by
application of a modified Mosher's method.10 When 18 was
treated with (R)- and (S)-MTPA chloride, the secondary hy-
droxy group at C-14 reacted to give the (S)- and (R)-MTPA
derivatives (18a and 18b), respectively. The 1H NMR chemical
described dolabellanes featuring an isopropenyl group,12,13 re-
vealed that the chemical shift of C-19 is very characteristic of the
orientation of methine H-12 relative to H-11 and H3-15. Specifically,
when H-12 is cis- and trans-oriented in relation to H-11 and H3-
15, respectively, C-19 resonates at higher frequencies (δC 22.5 to
25.5), whereas when H-12 is trans- and cis-oriented relative to
H-11 and H3-15, respectively, C-19 resonates at lower frequen-
cies (δC 18.0 to 19.5).
Compounds 1-18 were evaluated for their antibacterial
activities against a panel of six strains of Staphylococcus aureus.
These included a standard laboratory strain (ATCC 25923), two
epidemic MRSA strains (EMRSA-15 and EMRSA-16), a macro-
lide-resistant variant (RN4220), and two multi-drug-resistant
effluxing strains (SA1199B and XU212). According to the results
of the antibacterial activity assessment (Table 5), the most active
compound against all tested bacterial strains was the semisyn-
thetic alcohol 18, with MIC values in the range 2-4 μg/mL.
Interestingly, metabolite 5, which is the epimer of 18 at C-14,
exhibited moderate activity against strain EMRSA-16 with a MIC
value of 8 μg/mL, but only weak activity against the other strains,
with MIC values ranging from 32 to 128 μg/mL. Metabolite 14
was moderately active against all tested strains, with MIC values
in the range 8-16 μg/mL, while compounds 2, 4, 12, 13, and 16
displayed moderate activity only against strain EMRSA-16, with
MIC values between 4 and 16 μg/mL, but weak or no activity
against the other strains. Thus, it seems that strain EMRSA-16 is
fairly susceptible to dolabellane diterpenes. It is worth noting that
the majority of the dolabellanes that demonstrated antibacterial
activity against the tested strains possessed a hydroxy group,
whereas the presence of a ketone functionality at C-14 rendered
the dolabellanes inactive.
1
shifts of 18a and 18b were assigned by analysis of H NMR,
HSQC, and COSY spectra. The calculation of the ΔδS-R values,
shown in Figure 2, clearly defined the absolute configuration of
C-14 as R and, subsequently, on the basis of its relative config-
uration, established the absolute configuration of 18 as depicted.
Because compounds 2, 4-10, 12, 13, and 18 were clearly correlated
through the chemical interconversions described above, the
absolute configurations of 2, 4-10, 12, and 13 are also as shown.
The absolute configurations of metabolites 1, 3, 11, 14, and 15
were not determined, but on the basis of biogenetic considera-
tions they are expected to be the same.
In addition to metabolites 1-15, two known natural products
were isolated and identified as (1R,2E,4R,7E,11S,12R)-18-hy-
droxy-2,7-dolabelladiene (16) and (1R,2E,4R,7E,10S,11S,12R)-
10-acetoxy-18-hydroxy-2,7-dolabelladiene (17) by comparison
of their spectroscopic and physical characteristics with those
reported in the literature.11 Even though the absolute configura-
tions of 16 and 17 were not established, they are expected to be
the same as those of 1-15 on the basis of a common biogenetic
1
route. Furthermore, the H and 13C NMR chemical shifts for
compounds 4, 9, 10, and 13 are presented in Tables 1-4, supple-
menting the relevant literature, since only a few characteristic 1H
NMR resonances were available.
’ EXPERIMENTAL SECTION
Comprehensive examination of the spectroscopic data for
compounds 1-12, 15, and 18, as well as for other previously
General Experimental Procedures. Optical rotations were
measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell.
219
dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222

Journal of Natural Products
ARTICLE
UV spectra were obtained on a Shimadzu UV-160A spectrophotometer.
IR spectra were obtained on a Paragon 500 Perkin-Elmer spectrometer.
NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400
spectrometers. Chemical shifts are given on a δ (ppm) scale using TMS
as internal standard. The 2D experiments (HSQC, HMBC, COSY,
NOESY) were performed using standard Bruker pulse sequences. High-
resolution mass spectrometric data were provided by the University of
Notre Dame, Department of Chemistry and Biochemistry, Notre Dame,
IN, USA. Low-resolution EI mass spectra were measured on a Hewlett-
Packard 5973 mass spectrometer. Column chromatography separations
were performed with Kieselgel 60 (Merck). HPLC separations were
conducted using a CECIL 1100 Series liquid chromatography pump
equipped with a GBC LC-1240 refractive index detector, using the
following columns: (i) Spherisorb S10W (Phase Sep, 25 cm ꢀ 10 mm),
(ii) Econoshpere Silica 10u (Grace, 25 cm ꢀ 10 mm), and (iii) Chiralcel
OD 10 μm (Daicel Chemical Industries Ltd., 25 cm ꢀ 10 mm). TLC was
performed with Kieselgel 60 F254 (Merck aluminum support plates), and
spots were detected after spraying with 15% H2SO4 in MeOH reagent
and heating at 100 ꢀC for 1 min. The lyophilization was carried out in a
Freezone 4.5 freeze-dry system (Labconco).
afford nine fractions (A4a-A4i). Fraction A4b (10% EtOAc in cyclohexane,
46.1 mg) was purified by normal-phase HPLC, using cyclohexane/
EtOAc (98:2) as eluent, to yield 1 (0.4 mg), 2 (12.8 mg), 8 (2.2 mg),
12 (0.4 mg), and 13 (1.2 mg). Fraction A4c (20% EtOAc in cyclohexane,
812.3 mg) was subjected to gravity column chromatography on silica gel,
using cyclohexane with increasing amounts of EtOAc, followed by EtOAc
with increasing amounts of MeOH as the mobile phase, to afford 23
fractions (A4c1-A4c23). Fractions A4c2 (1% EtOAc in cyclohexane,
174.3 mg), A4c3 (1% EtOAc in cyclohexane, 129.8 mg), and A4c4 (1%
EtOAc in cyclohexane, 9.9 mg) were separately purified by normal-phase
HPLC, using n-hexane/EtOAc (97:3) and subsequently n-hexane/
2-propanol (99.5:0.5) as eluent, to yield 1 (1.4 mg), 2 (144.8 mg),
3 (2.7 mg), 8 (2.4 mg), and 12 (7.8 mg). Fractions A4c10 (2% EtOAc
in cyclohexane, 17.0 mg), A4c11 (3% EtOAc in cyclohexane, 10.8 mg),
A4c12 (5% EtOAc in cyclohexane, 18.7 mg), A4c13 (7% EtOAc in
cyclohexane, 47.0 mg), A4c14 (10% EtOAc in cyclohexane, 46.6 mg),
A4c15 (12% EtOAc in cyclohexane, 138.5 mg), A4c16 (20% EtOAc in
cyclohexane, 13.3 mg), and A4c17 (20% EtOAc in cyclohexane, 24.7 mg)
were separately purified by normal-phase HPLC, using cyclohexane/
EtOAc (90:10) and subsequently n-hexane/2-propanol (90:10, 87:13,
and 82:18) as eluent, to afford 5 (16.3 mg), 7 (27.2 mg), 9 (13.6 mg), 10
(17.6 mg), 11 (5.2 mg), and 16 (0.9 mg). The MeOH residue (32.8 g)
was subjected to vacuum column chromatography on silica gel, using
cyclohexane with increasing amounts of EtOAc, followed by EtOAc with
increasing amounts of MeOH as the mobile phase, to yield 14 fractions
(B1-B14). Fraction B1 (10% EtOAc in cyclohexane, 51.0 mg) was purified
by normal-phase HPLC, using n-hexane (100%) as eluent, to afford 4 (13.3
mg). Fraction B3 (20% EtOAc in cyclohexane, 361.0 mg) was repeatedly
purified by normal-phase HPLC, using cyclohexane/EtOAc (90:10)
and subsequently n-hexane/2-propanol (86:14 and 83:17) as eluent, to
yield 2 (3.3 mg), 5 (9.0 mg), 9 (5.4 mg), 11 (3.8 mg), 16 (2.4 mg), and
17 (0.9 mg).
Plant Material. Specimens of Dilophus spiralis were collected by
hand on Elafonissos Island (GPS coordinates 36ꢀ300 N, 22ꢀ580 E), south
of Peloponnese, Greece, at a depth of 0.1-1 m, in April 2004. A voucher
specimen of the alga has been deposited at the Herbarium of the
Department of Pharmacognosy and Chemistry of Natural Products,
University of Athens (ATPH/MO/159).
Extraction and Isolation. Specimens of the freeze-dried alga
(272 g) were exhaustively extracted with CH2Cl2 and subsequently with
MeOH at room temperature. Evaporation of the solvents in vacuo
afforded two dark green oily residues. The CH2Cl2 residue (9.2 g) was
subjected to vacuum column chromatography on silica gel, using cyclo-
hexane with increasing amounts of EtOAc, followed by EtOAc with
increasing amounts of MeOH as the mobile phase, to yield 15 fractions
(A1-A15). Fractions A1 (100% cyclohexane, 131.4 mg) and A2 (10%
EtOAc in cyclohexane, 18.3 mg) were separately and repeatedly purified
by normal-phase HPLC, using n-hexane (100%) as eluent, to afford
4 (39.6 mg) and 15 (0.9 mg). Fraction A3 (20% EtOAc in cyclohexane,
1.17 g) was further fractionated by gravity column chromatography on
silica gel, using cyclohexane with increasing amounts of EtOAc as the
mobile phase, to yield 21 fractions (A3a-A3u). Fraction A3b (1%
EtOAc in cyclohexane, 355.7 mg) was subjected to gravity column
chromatography on silica gel, using cyclohexane with increasing
amounts of CH2Cl2, followed by CH2Cl2 with increasing amounts of
EtOAc as the mobile phase, to afford 11 fractions (A3b1-A3b11). Fractions
A3b8 (100% CH2Cl2, 55.6 mg) and A3b9 (50% EtOAc in CH2Cl2,
195.6 mg) were separately purified by normal-phase HPLC, using
cyclohexane/EtOAc (99:1) and subsequently n-hexane/EtOAc (99:1)
as eluent, to yield 2 (185.6 mg), 3 (2.8 mg), and 6 (1.9 mg). Fractions
A3c (1% EtOAc in cyclohexane, 162.9 mg) and A3d (1% EtOAc in
cyclohexane, 55.3 mg) were separately purified by normal-phase HPLC,
using n-hexane/EtOAc (98:2 and subsequently 99:1) as eluent, to afford
1 (1.1 mg), 2 (96.5 mg), 3 (1.5 mg), 8 (29.1 mg), and 14 (0.8 mg).
Fractions A3i (2% EtOAc in cyclohexane, 81.7 mg) and A3j (2% EtOAc
in cyclohexane, 28.2 mg) were purified separately by normal-phase HPLC,
using cyclohexane/EtOAc (95:5) as eluent, to yield 16 (21.2 mg).
Fraction A3l (10% EtOAc in cyclohexane, 81.9 mg) was subjected to
gravity column chromatography on silica gel, using cyclohexane with
increasing amounts of EtOAc as the mobile phase, to afford 10 fractions
(A3l1-A3l10). Fraction A3l6 (6% EtOAc in cyclohexane, 7.6 mg) was
purified by normal-phase HPLC, using cyclohexane/EtOAc (90:10) as
eluent, to yield 5 (1.4 mg). Fraction A4 (30% EtOAc in cyclohexane,
3.58 g) was further fractionated by vacuum column chromatography on
silica gel, using cyclohexane with increasing amounts of EtOAc, followed
by EtOAc with increasing amounts of MeOH as the mobile phase, to
(1R,3E,7E,11S,12R)-14-Oxo-3,7,18-dolabellatriene (1): colorless oil;
[R]2D0 -76 (c 0.09, CHCl3); UV (CHCl3) λmax (log ε) 245.5 (2.96) nm;
1
IR (thin film) νmax 2971, 2916, 1735, 1275 cm-1; H NMR data, see
Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int %) 286
(22), 271 (22), 253 (10), 243 (6), 228 (6), 213 (9), 203 (7), 189 (43),
175 (23), 161 (22), 147 (28), 135 (42), 121 (42), 107 (85), 91 (87), 79
(79), 67 (100), 53 (53); HRFABMS m/z 286.2303 [M]þ (calcd for
C20H30O, 286.2297).
(1R,3Z,7E,11S,12S)-14-Oxo-3,7,18-dolabellatriene (3): yellowish oil;
[R]2D0 -98 (c 0.09, CHCl3); UV (CHCl3) λmax (log ε) 241.8 (2.28) nm;
IR (thin film) νmax 2958, 2920, 1734, 1454 cm-1; 1H NMR data, see
Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int %) 286
(11), 271 (15), 253 (3), 243 (6), 229 (4), 217 (7), 203 (8), 189 (28), 175
(19), 163(55), 150 (75), 135 (100), 121 (39), 107 (57), 93(57), 81 (49),
67 (48), 55 (34); HRFABMS m/z 287.2366 [M þ H]þ (calcd for
C20H31O, 287.2375).
(1R,3E,7E,11S,12S)-3,7,18-Dolabellatriene (4): yellowish oil; [R]D20
þ41.0 (c 0.10, CHCl3); UV (CHCl3) λmax (log ε) 243.8 (2.57) nm; IR
1
(thin film) νmax 2930, 2854, 1645, 1454 cm-1; H NMR data, see
Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int %) 272
(38), 257 (26), 243 (4), 229 (33), 215 (10), 203 (12), 189 (35), 175
(43), 161 (54), 147 (63), 135 (75), 121 (90), 107 (93), 93 (100), 81
(73), 79 (72), 67 (67), 55 (47); HRFABMS m/z 272.2493 [M]þ (calcd
for C20H32, 272.2504).
(1R,3E,7E,11S,12S,14S)-14-Hydroxy-3,7,18-dolabellatriene (5): colorless
oil; [R]2D0 þ44 (c0.06, CHCl3); UV (CHCl3) λmax (log ε) 244.0 (2.48) nm;
IR (thin film) νmax 3370, 2936, 2360, 1290 cm-1;1HNMRdata,seeTable1;
13C NMR data, see Table 2; EIMS 70 eV m/z (rel int %) 288 (5), 270 (12),
255 (22), 227 (17), 213 (12), 201 (15), 191 (38), 173 (25), 163 (41), 145
(51), 135 (76), 121 (81), 107 (94), 95 (86), 81 (100), 67 (64), 55 (69);
HRFABMS m/z 288.2479 [M]þ (calcd for C20H32O, 288.2453).
220
dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222

Journal of Natural Products
ARTICLE
(1R,3E,7E,11S,12S,14S)-14-Acetoxy-3,7,18-dolabellatriene (6): colorless
oil; [R]2D0 þ29 (c 0.07, CHCl3); UV (CHCl3) λmax (log ε) 241.4
(2.31) nm; IR (thin film) νmax 3389, 2926, 2360, 1279 cm-1; 1H NMR
data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int
%) 288 (8), 270 (14), 255 (12), 245 (6), 227 (13), 220 (14), 205 (12),
189 (21), 173 (17), 163 (56), 149 (40), 135 (79), 121 (78), 107 (92), 95
(84), 81 (100), 67 (63), 55 (70); HRFABMS m/z 288.2426 [M]þ
(calcd for C20H32O, 288.2453).
Epoxidation of 4. A solution of m-chloroperbenzoic acid (20.0 mg)
in benzene (1 mL) was added dropwise to a solution of compound
4 (20.0 mg) in benzene (2 mL), and the mixture was left under constant
stirring at room temperature for 30 min. The reaction was quenched by
the addition of 10% Na2SO3 (3 mL), and the mixture was partitioned
between the aqueous and the organic layer. The organic layer was
washed with 5% NaHCO3 and subsequently H2O. After evaporation of
the organic layer in vacuo, the residue was purified by normal-phase HPLC,
using n-hexane/2-propanol (99.75:0.25) as eluent, to afford 8 (6.3 mg)
and 12 (5.9 mg).
Acetylation of 5. Compound 5 (2.8 mg) was treated with Ac2O
(1 mL) in pyridine (1 mL) and left under constant stirring at 70 ꢀC for
16 h. The reaction was quenched by the addition of H2O (1 mL), and the
mixture was evaporated in vacuo. The residue was purified by normal-
phase HPLC, using cyclohexane/EtOAc (99:1) as eluent, to obtain
6 (2.3 mg).
Preparation of MTPA Derivatives of 18. Compound 18
(3.3 mg) was treated with (R)-MTPA chloride (5 μL) in freshly
distilled dry pyridine (1 mL) and left under constant stirring at room
temperature for 16 h. The reaction was quenched by the addition of
H2O (1 mL) and CH2Cl2 (3 mL), and the mixture was partitioned
between the aqueous and the organic layer. After evaporation of the
organic layer in vacuo, the residue was purified by normal-phase
HPLC, using cyclohexane/EtOAc (95:5) as eluent, to give the (S)-
MTPA derivative (18a, 3.2 mg). The (R)-MTPA derivative (18b,
2.4 mg) was prepared with (S)-MTPA chloride and purified in the
same manner.
1
(2.27) nm; IR (thin film) νmax 2967, 2920, 1734, 1538 cm-1; H
NMR data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z
(rel int %) 330 (1), 315 (1), 288 (1), 270 (50), 255 (46), 241 (8), 227
(31), 213 (17), 201 (20), 187 (32), 173 (42), 159 (55), 145 (69), 133
(93), 119 (100), 105 (69), 91 (67), 81 (54), 67 (42), 55 (43);
HRFABMS m/z 330.2535 [M]þ (calcd for C22H34O2, 330.2559).
(1R,3S,4S,7E,11S,12S,14S)-14-Acetoxy-3,4-epoxy-7,18-dolabelladiene
(9): yellowish oil; [R]2D0 þ47.8 (c 0.25, CHCl3); UV (CHCl3) λmax
(log ε) 243.0 (2.18) nm; IR (thin film) νmax 2962, 2907, 1734, 1243 cm-1
;
1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z
(rel int %) 346 (1), 328 (8), 286 (61), 271 (28), 268 (27), 253 (25), 243
(19), 228 (33), 213 (29), 201 (41), 187 (51), 173 (47), 159 (62), 145
(79), 133 (100), 119 (92), 105 (96), 91 (81), 79 (49), 67 (25), 55 (31);
HRFABMS m/z 346.2488 [M]þ (calcd for C22H34O3, 346.2508).
(1R,3E,7S,8S,11S,12S)-7,8-Epoxy-14-oxo-3,18-dolabelladiene
(10): colorless crystals; [R]2D0 -66.4 (c 0.19, CHCl3); UV (CHCl3)
λmax (log ε) 241.5 (2.24) nm; IR (thin film) νmax 2966, 2925, 2859,
1733, 1457 cm-1; H NMR data, see Table 3; 13C NMR data, see
1
Table 4; EIMS 70 eV m/z (rel int %) 302 (39), 284 (78), 269 (41), 241
(19), 233 (14), 227 (16), 215 (31), 205 (24), 187 (57), 173 (45), 163
(92), 159 (57), 150 (58), 145 (63), 135 (90), 119 (75), 105 (94), 91
(100), 79 (69), 67 (58), 55 (50); HRFABMS m/z 303.2325 [M þ H]þ
(calcd for C20H31O2, 303.2324).
(1R,3Z,7S,8S,11S,12S)-7,8-Epoxy-14-oxo-3,18-dolabelladiene (11):
colorless oil; [R]D20 -13.0 (c 0.25, CHCl3); UV (CHCl3) λmax (log ε)
242.5 (2.16) nm; IR (thin film) νmax 2935, 1733, 1275 cm-1; 1H NMR
data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel
int %) 302 (14), 284 (63), 269 (22), 256 (11), 241 (15), 215 (25), 205
(20), 187 (58), 173 (40), 163 (96), 159 (55), 150 (53), 145 (68), 135
(80), 119 (80), 105 (97), 91 (100), 79 (68), 67 (55), 55 (49);
HRFABMS m/z 303.2330 [M þ H]þ (calcd for C20H31O2, 303.2324).
(1R,3E,7E,11R,12R)-12-Hydroxy-3,7-dolabelladiene (14): colorless
oil; [R]2D0 þ16 (c 0.09, CHCl3); UV (CHCl3) λmax (log ε) 242.5
(2.23) nm; IR (thin film) νmax 3330, 2958, 2877, 1276 cm-1; 1H NMR
(S)-MTPA Derivative of 18 (18a): 1H NMR (400 MHz, CDCl3)
δ 7.53 (2H, m, Ar-H), 7.39 (3H, m, Ar-H), 5.07 (1H, dd, 9.2, 6.0 Hz,
H-3), 4.97 (1H, dd, 9.1, 7.1 Hz, H-14), 4.89 (1H, brs, H-20a), 4.79 (1H,
dd, 9.5, 4.1 Hz, H-7), 4.66 (1H, brs, H-20b), 3.55 (3H, s, OMe), 2.65
(1H, m, H-12), 2.24 (1H, m, H-6a), 2.18 (1H, m, H-5a), 2.17 (1H, m,
H-2R), 2.15 (1H, m, H-13β), 2.08 (1H, m, H-6b), 2.07 (1H, m, H-5b),
2.02 (1H, m, H-9a), 1.90 (1H, m, H-2β), 1.89 (1H, m, H-9b), 1.88 (1H,
m, H-11), 1.81 (1H, m, H-13R), 1.70 (3H, s, H-19), 1.50 (3H, s, H-16),
1.47 (3H, s, H-17), 1.34 (1H, m, H-10a), 1.16 (1H, m, H-10b), 0.83
(3H, s, H-15).
1
data, see Table 3; 13C NMR data, see Table 4; H NMR (400 MHz,
C6D6) δ 5.03 (1H, m, H-3), 5.02 (1H, m, H-7), 2.15 (1H, m, H-9a), 2.14
(1H, m, H-2a), 2.09 (2H, m, H-6), 2.05 (2H, m, H-5), 1.90 (1H, ddd,
13.8, 12.3, 1.9 Hz, H-9b), 1.80 (1H, dd, 14.9, 5.2 Hz, H-2b), 1.75 (1H, m,
H-18), 1.71 (1H, m, H-10a), 1.67 (1H, m, H-11), 1.64 (1H, m, H-14a),
1.54 (1H, m, H-13a), 1.53 (3H, s, H-17), 1.46 (3H, s, H-16), 1.39 (1H,
m, H-14b), 1.30 (1H, m, H-13b), 1.29 (1H, m, H-10b), 1.07 (3H, s,
H-15), 0.93 (3H, d, 6.7 Hz, H-20), 0.88 (3H, d, 6.8 Hz, H-19); EIMS 70
eV m/z (rel int %) 290 (1), 272 (12), 257 (10), 247 (3), 229 (25), 216
(4), 201 (5), 189 (18), 175 (9), 161 (30), 149 (20), 135 (100), 121 (71),
107 (54), 93 (49), 81 (31), 67 (27), 55 (20); HRFABMS m/z 290.2629
[M]þ (calcd for C20H34O, 290.2610).
(R)-MTPA Derivative of 18 (18b): 1H NMR (400 MHz, CDCl3)
δ 7.51 (2H, m, Ar-H), 7.39 (3H, m, Ar-H), 5.10 (1H, dd, 9.3, 6.6 Hz,
H-3), 4.92 (1H, dd, 8.9, 7.0 Hz, H-14), 4.86 (1H, brs, H-20a), 4.80 (1H,
dd, 8.5, 3.7 Hz, H-7), 4.62 (1H, brs, H-20b), 3.52 (3H, s, OMe), 2.65
(1H, ddd, 13.3, 7.1, 7.1 Hz, H-12), 2.24 (1H, m, H-6a), 2.21 (1H, m,
H-2R), 2.17 (1H, m, H-5a), 2.14 (1H, m, H-13β), 2.08 (1H, m, H-6b),
2.07 (1H, m, H-5b), 2.02 (1H, m, H-9a), 1.90 (1H, m, H-2β), 1.89 (1H, m,
H-9b), 1.88 (1H, m, H-11), 1.68 (1H, m, H-13R), 1.67 (3H, s, H-19),
1.51 (3H, s, H-16), 1.47 (3H, s, H-17), 1.34 (1H, m, H-10a), 1.16
(1H, m, H-10b), 0.94 (3H, s, H-15).
Single-Crystal X-ray Analysis of 10. Compound 10 crystallized
after slow evaporation of a saturated solution of EtOAc/CHCl3 (1:1) as
colorless blocks. Single-crystal X-ray diffraction data were collected at
120 K on a Nonius Kappa CCD diffractometer with graphite-mono-
chromated Mo KR radiation (λ = 0.71073 Å) using the Nonius Collect
Software. The space group was determined on the basis of the systematic
absences and confirmed by the successful structure solution and refinement.
The structure was solved by direct methods and refined based on F2 using
the WINGX package. All non-hydrogen atoms were refined with
anisotropic thermal parameters, whereas all hydrogen atoms were
located in the calculated positions and refined in a rigid group model.
(1R,2E,4R,7E,11S,12R)-2,7,18-Dolabellatriene (15): colorless oil;
[R]2D0 -21 (c 0.03, CHCl3); UV (CHCl3) λmax (log ε) 242.2 (2.53)
1
nm; IR (thin film) νmax 2948, 2920, 1538 cm-1; H NMR data, see
Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int %) 272
(32), 257 (25), 243 (5), 229 (68), 215 (10), 201 (13), 190 (25), 175
(73), 161 (47), 147 (71), 133 (60), 121 (67), 107 (100), 93 (83), 81
(74), 67 (57), 55 (50); HRFABMS m/z 272.2495 [M]þ (calcd for
C20H32, 272.2504).
Reduction of 2. Compound 2 (48.0 mg) was treated with NaBH4
(50.0 mg) in MeOH (10 mL) and left under constant stirring at room
temperature for 1 h. The reaction was quenched by the addition of H2O
(3 mL), and the mixture was evaporated in vacuo. The residue was
purified by normal-phase HPLC, using cyclohexane/EtOAc (90:10) as
eluent, to obtain 5 (7.3 mg) and 18 (31.2 mg).
(1R,3E,7E,11S,12S,14R)-14-Hydroxy-3,7,18-dolabellatriene (18): colorless
oil; [R]2D0 þ12.0 (c 0.10, CHCl3); UV (CHCl3) λmax (log ε) 244.0
221
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Journal of Natural Products
ARTICLE
In the absence of atoms with significant anomalous scattering, the absolute
configuration of 10 was indeterminate.
’ REFERENCES
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Crystallographic Data of 10: C20H30O2, M = 302.4, 0.52 ꢀ 0.24 ꢀ
0.22 mm, T = 120(2) K, orthorhombic, space group P212121(#19) with
a = 7.7877(11) Å, b = 13.9979(13) Å, c = 15.5830(16) Å, V = 1698.73(3)
Å3, Z = 4, Z0 = 1, Dcalcd = 1.183 Mg/m3, μ = 0.074 mm-1, F(000) = 664,
2θmax = 55.00ꢀ, 16 541 collected reflections, 3866 independent reflections
(Rint = 0.0467), R1 = 0.0450, wR2 = 0.0939, GoF = 0.994 for 2997 reflections
(201 parameters) with I> 2σ(I), R1 = 0.0722, wR2 = 0.1080, GoF = 0.994 for
all 3866 reflections, max./min. residual electron density þ0.182/-0.179 e Å3.
Evaluation of Antibacterial Activity. Standard strain ATCC
25923 and strain XU212, which possesses the gene encoding the TetK
tetracycline efflux protein, were provided by Dr. E. Udo. Strain SA1199B,
which possesses the gene encoding the NorA quinolone efflux protein,
was a generous gift of Prof. G. W. Kaatz. Strain RN4220, which possesses
the gene encoding the MsrA macrolide efflux protein, was provided by
Dr. J. Cove. The epidemic methicillin-resistant strains EMRSA-15 and
EMRSA-16 were obtained from Dr. P. Stapleton. All strains were
cultured on nutrient agar and incubated for 24 h at 37 ꢀC prior to the
determination of minimum inhibitory concentration (MIC) values. Com-
pounds 1-18 were dissolved in DMSO and subsequently diluted
in Mueller-Hinton broth (MHB) to give a starting concentration of
512 μg/mL. Bacterial inocula equivalent to the 0.5 McFarland turbidity
standard were prepared in normal saline for each strain and diluted to
a final inoculum density of 5 ꢀ 105 cfu/mL. MHB supplemented with
10 mg/L Mg2þ and 20 mg/L Ca2þ (125 μL/well) was dispensed into
wells 1-11 of each row of 96-well microtiter plates. The compound
solution (125 μL) was added to the first well of each row and was serially
diluted across the row, leaving well 11 empty for growth control. The
final volume was dispensed into well 12, which being free of MHB or
inoculum served as the sterility control. The inoculum (125 μL/well)
was added to wells 1-11 of each row, and the microtiter plates were
incubated for 18 h at 37 ꢀC. The lowest concentration at which no
bacterial growth was observed was recorded as the MIC. The observa-
tion was confirmed by the addition of a 5 mg/mL methanolic solution of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
20 μL/well) and further incubation for 20 min at 37 ꢀC. Bacterial growth
was indicated by a color alteration from yellow to dark blue. Norfloxacin
was used as a positive control. The highest concentration of DMSO
remaining after dilution (3.125% v/v) caused no inhibition of bacterial
growth. All samples were tested in triplicate. Culture media were obtained
from Oxoid, whereas all other chemicals were obtained from Sigma-Aldrich.
(7) Amico, V.; Currenti, R.; Oriente, G.; Piattelli, M.; Tringali, C.
Phytochemistry 1981, 20, 848–849.
(8) Crystallographic data (excluding structure factors) for com-
pound 11 have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication no. CCDC 759848. Copies
of the data can be obtained, free of charge, on application to CCDC,
12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44-(0)1223-336033
or e-mail: deposit@ccdc.cam.ac.uk).
(9) Duran, R.; Zubia, E.; Ortega, M. J.; Salva, J. Tetrahedron 1997, 53,
8675–8688.
(10) Seco, J. M.; Qui~noꢀa, E.; Riguera, R. Chem. Rev. 2004, 104, 17–117.
(11) Ireland, C.; Faulkner, D. J. J. Org. Chem. 1977, 42, 3157–3162.
(12) Ramírez, M. C.; Toscano, R. A.; Arnason, J.; Omar, S.;
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(13) Wang, Y.; Harrison, L. J.; Tan, B. C. Tetrahedron 2009, 65,
4035–4043.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
compounds 1-18, NOESY spectra of compounds 1-15 and 18,
NMR data in tabular form of compounds 2, 7, 8, and 12, and CIF
data for the crystal structure of metabolite 10. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. Tel/Fax: þ30-
210-7274592. E-mail: roussis@pharm.uoa.gr.
Notes
^Deceased on May 12, 2010.
’ ACKNOWLEDGMENT
This study was partially supported by a “Kapodistrias” grant
from the University of Athens.
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dx.doi.org/10.1021/np1006586 |J. Nat. Prod. 2011, 74, 213–222