Antifouling activity of meroditerpenoids from the marine brown alga halidrys siliquosa

Article abstract of DOI:10.1021/np070110k

Nine tetraprenyltoluquinol-related metabolites (1-9) have been isolated from the organic extract of the temperate brown alga Halidrys siliquosa that exhibits antifouling properties. The planar structure of compounds 1-9 was elucidated on the basis of exte

Full text of DOI:10.1021/np070110k

Copyright 2008 by the American Chemical Society and the American Society of Pharmacognosy
Volume 71, Number 7
July 2008
Full Papers
Antifouling Activity of Meroditerpenoids from the Marine Brown Alga Halidrys siliquosa
Ge´rald Culioli,*,† Annick Ortalo-Magne´,† Robert Valls,‡ Claire Hellio,§ Anthony S. Clare,† and Louis Piovetti
Laboratoire des Mate´riaux a` Finalite´s Spe´cifiques (MFS), UniVersite´ du Sud Toulon-Var, AVenue de l’UniVersite´, BP 20132,
F-83957 La Garde Cedex, France, UMR 6180 CNRS “Chirotechnologies: Catalyse et Biocatalyse”, Groupe Se´paration, Identification et
Synthe`se, UniVersite´ Paul Ce´zanne, AVenue Escadrille Normandie-Niemen, SerVice 551, F-13397 Marseille Cedex 20, France, School of
Biological Sciences, King Henry Building, Portsmouth UniVersity, Portsmouth, PO1 2DY, U.K., and School of Marine Science and
Technology, Ridley Building, Newcastle UniVersity, Newcastle upon Tyne, NE1 7RU, U.K.
ReceiVed March 13, 2007
Nine tetraprenyltoluquinol-related metabolites (1-9) have been isolated from the organic extract of the temperate brown
alga Halidrys siliquosa that exhibits antifouling properties. The planar structure of compounds 1-9 was elucidated on
the basis of extensive spectroscopic analysis and by comparison of the data with those of related metabolites. Antifouling
and toxicity tests were conducted on these compounds: the most active (compounds 2, 6, and 9) inhibited both the
growth of four strains of bacteria (MICs < 2.5 µg/mL) and settlement of cyprids of Balanus amphitrite (EC50 < 5
µg/mL), the latter at nontoxic concentrations (LC50 > 5 µg/mL).
Microbial biofilms and marine fouling organisms, such as
barnacles, can cause substantial technical and economical problems
on man-made surfaces submerged in seawater. Due to new
regulations on toxic antifouling compounds, agents that are effective
against biofouling and are environmentally benign are urgently
needed. A number of cobiocides are currently under environmental
and legislative scrutiny, and restrictions on where they can be used
will continue to grow, especially in Europe.1
negative chemotaxis, altering the surface of the organisms, or finally
acting as biocides.6,7
In the course of our continuing search for bioactive metabolites
from marine algae, this report describes the chemical and biological
investigations of Halidrys siliquosa (L.) Lyngb. (family Sargas-
saceae, class Phaeophyceae). This brown alga, found along NE
Atlantic shores, had previously been shown to contain meroditer-
penes8 as well as phlorotannins9–11 and arsenosugars.12 Herein, we
describe the structure elucidation and bioactivity of five new
compounds [one chromene (1) and four linear meroditerpenoids
(2-5)] together with four known metabolites (6-9). Screening of
new natural products with antifouling activities is usually conducted
by a bioassay on the cosmopolitan barnacle Balanus amphitrite. In
this study, these compounds were tested on both B. amphitrite and
marine bacteria (Cobetia marina, Marinobacterium stanieri, Vibrio
fischeri, and Pseudoalteromonas haloplanktis). It is of high
importance to evaluate the bioactivity of new compounds against
a large diversity of fouling organisms, as agents with a broad
spectrum activity are expected for antifouling purposes.
Marine organisms are a rich source of bioactive substances, and
many of them are able to stay free from biofouling. Natural marine
antifouling compounds are among the most promising alternatives
to the chemicals commonly used in antifouling coatings.2–4 Sessile
organisms such as sponges, soft corals, and seaweeds are known
to elaborate chemical defense mechanisms against predation and
epibiont growth. The metabolites excreted might repel or inhibit
fouling organisms5 and can act enzymatically by dissolving the
adhesives, interfering with the metabolism of the fouling organisms,
inhibiting the attachment, metamorphosis, or growth, promoting
* To whom correspondence should be addressed. Tel: (33) 494 142 935.
Fax: (33) 494 142 168. E-mail: culioli@univ-tln.fr.
† Universite´ du Sud Toulon-Var.
Results and Discussion
H. siliquosa was collected by hand from Saint-Gue´nole´ (Brittany)
on the French Atlantic coast in May 2004. After extraction with
CHCl3/MeOH (2:1 then 1:2, v/v), the resulting crude extracts were
‡ Universite´ Paul Ce´zanne.
§ Portsmouth University.
Newcastle University.
10.1021/np070110k CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/05/2008

1122 Journal of Natural Products, 2008, Vol. 71, No. 7
Culioli et al.
Table 1. 13C NMR Data for Compounds 1-5, 8, and 9 (100 MHz, C6D6)
1
2
3
4
5
8
9
position
δC, mult.a
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
1
2
3
4
5
6
7
8
123.3, CH
130.4, CH
77.4, qC
30.7, CH2
127.6, CH
131.7, qC
55.1, CH2
197.9, qC
123.5, CH
159.1, qC
33.5, CH2
25.8, CH2
33.9, CH2
41.2, CH
214.6, qC
75.0, CH
122.5, CH
138.8, qC
25.7, CH3
18.5, CH3
16.8, CH3
25.1, CH3
16.7, CH3
147.3, qC
127.9, qC
113.5, CH
153.9, qC
114.4, CH
126.2, qC
16.7, CH3
55.2, CH3
30.9, CH2
127.4, CH
132.2, qC
55.3, CH2
197.9, qC
122.7, CH
158.2, qC
41.0, CH2
25.4, CH2
32.3, CH2
41.2, CH
213.7, qC
73.6, CH
122.6, CH
138.6, qC
25.7, CH3
18.4, CH3
17.7, CH3
19.2, CH3
16.7, CH3
147.4, qC
128.0, qC
113.6, CH
154.0, qC
114.5, CH
126.3, qC
16.6, CH3
55.3, CH3
30.8, CH2
127.5, CH
132.1, qC
55.3, CH2
197.9, qC
122.7, CH
157.8, qC
40.9, CH2
25.1, CH2
33.6, CH2
41.5, CH
214.0, qC
74.9, CH
122.4, CH
138.8, qC
25.7, CH3
18.4, CH3
16.5, CH3
19.2, CH3
16.7, CH3
147.4, qC
127.8, qC
113.6, CH
154.0, qC
114.5, CH
126.2, qC
16.6, CH3
55. 2, CH3
30.8, CH2
127.3, CH
132.2, qC
55.1, CH2
197.5, qC
123.3, CH
159.5, qC
33.8, CH2
26.1, CH2
30.2, CH2
36.7, CH
81.1, CH
201.2, qC
120.1, CH
158.2, qC
27.5, CH3
21.2, CH3
17.5, CH3
25.1, CH3
16.7, CH3
147.3, qC
127.8, qC
113.5, CH
154.0, qC
114.5, CH
126.2, qC
16.6, CH3
55.2, CH3
30.9, CH2
127.3, CH
132.2, qC
55.2, CH2
197.9, qC
122.6, CH
157.7, qC
41.2, CH2
25.4, CH2
29.7, CH2
36.6, CH
81.1, CH
201.0, qC
119.8, CH
158.6, qC
27.5, CH3
21.2, CH3
17.5, CH3
19.2, CH3
16.7, CH3
147.4, qC
128.1, qC
113.5, CH
154.0, qC
114.5, CH
126.4, qC
16.6, CH3
55.2, CH3
31.0, CH2
127.5, CH
132.1, qC
55.3, CH2
198.0, qC
122.7, CH
158.7, qC
41.4, CH2
25.6, CH2
34.1, CH2
36.5, CH
78.7, CH
200.9, qC
119.5, CH
158.8, qC
27.5, CH3
21.1, CH3
13.0, CH3
19.4, CH3
16.7, CH3
147.4, qC
127.9, qC
113.6, CH
153.9, qC
114.5, CH
126.2, qC
16.6, CH3
55.2, CH3
54.2, CH2
197.1, qC
124.6, CH
157.8, qC
41.3, CH2
25.5, CH2
29.7, CH2
36.9, CH
81.1, CH
200.9, qC
119.8, CH
158.6, qC
27.5, CH3
21.2, CH3
17.6, CH3
19.2, CH3
26.3, CH3
144.9, qC
121.7, qC
109.4, CH
154.1, qC
116.9, CH
126.6, qC
16.0, CH3
55.2, CH3
9
10
11
12
13
14
15
16
17
18
19
20
1′
2′
3′
4′
5′
6′
7′
4′-OMe
a Multiplicities inferred from DEPT and HSQC experiments.
Table 2. 1H NMR Data of Compounds 1-5, 8, and 9 (400 MHz, C6D6, J in Hz)
position
1
2
3
4
5
8
9
1
2
6.11 (br d, 10.0)
5.73 (d, 10.0)
2.79 (d, 14.5)
2.67 (d, 14.5)
5.83 (s)
3.32 (br d, 7.5)
5.31 (t, 7.5)
2.81 (s)
3.30 (d, 7.0)
5.32 (t, 7.0)
2.89 (s)
3.31 (t, 7.0)
5.34 (t, 7.0)
2.89 (s)
3.28 (d, 7.5)
5.27 (t, 7.5)
2.80 (s)
3.30 (t, 7.0)
5.30 (t, 7.0)
2.86 (s)
3.31 (t, 7.5)
5.33 (t, 7.0)
2.90 (s)
4a
4b
6
8a
8b
9
10a
10b
11
12
13
14
16
17
18
19
20
3′
5.79 (br s)
2.61 (m)
2.37 (m)
1.27 (m)
1.69 (m)
1.30 (m)
2.67 (m)
5.88 (s)
1.73 (t, 7.5)
5.89 (br s)
1.71 (t, 7.5)
5.78 (br s)
2.66 (m)
2.37 (m)
1.27 (m)
1.69 (m)
1.28 (m)
1.94 (m)
3.97 (br s)
5.84 (s)
1.72 (t, 7.0)
5.98 (s)
1.86 (t, 7.0)
1.66 (t, 7.5)
1.11 (m)
1.41 (m)
1.05 (m)
1.72 (m)
3.93 (br s)
1.13 (m)
1.69 (m)
1.11 (m)
2.41 (m)
1.15 (m)
1.46 (m)
1.10 (m)
2.46 (m)
1.11 (m)
1.32 (m)
1.15 (m)
1.75 (m)
3.93 (m)
1.31 (m)
1.56 (m)
1.29 (m)
1.74 (m)
4.07 (m)
5.00 (d, 9.5)
5.05 (d, 9.5)
1.52 (s)
1.65 (s)
0.98 (d, 7.0)
1.48 (s)
4.82 (d, 9.5)
4.94 (d, 9.5)
1.49 (s)
1.48 (s)
0.80 (d, 7.0)
2.08 (s)
4.82 (d, 9.5)
4.95 (d, 9.5)
1.49 (s)
1.53 (s)
0.92 (d, 7.0)
2.08 (s)
5.72 (s)
1.42 (s)
2.07 (s)
1.11 (d, 7.0)
2.05 (s)
5.85 (br s)
1.47 (s)
2.09 (s)
1.19 (d, 7.0)
1.49 (s)
1.64 (s)
6.70 (d, 3.0)
6.64 (d, 3.0)
2.18 (s)
5.74 (br s)
1.43 (s)
2.06 (s)
1.11 (d, 7,0)
2.05 (s)
1.67 (s)
6.70 (d, 3.0)
6.65 (d, 3.0)
2.20 (s)
5.76 (s)
1.42 (s)
2.06 (s)
0.71 (d, 7.0)
2.17 (s)
1.58 (s)
1.66 (s)
1.66 (s)
1.67 (s)
1.68 (s)
6.39 (d, 3.0)
6.65 (d, 3.0)
2.22 (s)
6.69 (d, 3.0)
6.63 (d, 3.0)
2.19 (s)
6.70 (d, 3.0)
6.64 (d, 3.0)
2.19 (s)
6.71 (d, 3.0)
6.64 (d, 3.0)
2.19 (s)
6.71 (d, 3.0)
6.65 (d, 3.0)
2.21 (s)
5′
7′
4′-OMe
3.35 (s)
3.44 (s)
3.45 (s)
3.45 (s)
3.44 (s)
3.45 (s)
3.44 (s)
concentrated and an initial fractionation was accomplished by
normal-phase column chromatography (CC) (EtOAc/n-hexane).
Subsequent iterative C18 reversed-phase HPLC resulted in the
isolation of the five new compounds 1-5 in addition to the four
known meroditerpenoids 6-9.
exhibited the presence of two meta-coupled aromatic protons (δΗ
6.65 and 6.39), two strongly coupled olefinic protons (δΗ 6.11 and
5.73), one methoxy group (δΗ 3.35), and five singlet (δΗ 2.22, 2.07,
2.05, 1.58, and 1.42) and one doublet (δΗ 1.11) methyl. The proton
sequence from H-1 to H-2 and the gHMBC correlations from H3-
7′ to C-1′, C-5′, and C-6′; H-5′ to C-6′, C-1′, C-4′, and C-3′; H-3′
to C-5′, C-4′, C-2′, C-1′, and C-1; and H3-20 to C-2, C-3, and C-4
allowed the proposal of the chromenol fragment a (Figure 1). The
deshielded nature of the signals associated with H2-4, H-6, and H3-
19 revealed the proximity of these atoms to a conjugated carbonyl
function (C-5, δC 197.1). Long-range gHMBC couplings from H2-4
to C-5 and C-6; H-6 to C-4, C-5, C-7, C-8, and C-19; and H3-19
to C-6, C-7, and C-8 allowed inclusion of the carbonyl-containing
residue c in the already defined fragment a. The geometry of the
Compound 1 was isolated as an optically active yellow oil. Its
molecular formula C28H38O5 was established by HREIMS (m/z
454.2722), indicating 10 degrees of unsaturation. The 13C NMR
data of 1 in C6D6 (Table 1) contained a total of 28 carbon signals,
which were identified by the assistance of DEPT spectra as seven
methyls, four sp3 methylenes, one sp3 methine, two oxygenated
sp3 carbons (one methyne and one quaternary), six sp2 methines,
and eight sp2 quaternary carbons including those of two ketone
1
carbonyls. The H NMR spectrum (Table 2) measured in C6D6

Antifouling ActiVity of Meroditerpenoids
Journal of Natural Products, 2008, Vol. 71, No. 7 1123
double bond at C-6 was assigned to be E on the basis of (i) the
upfield 13C NMR chemical shift of the vinyl methyl carbon at δC
19.2 (C-19)13,14 and (ii) the NOE correlation between H-6 and H2-
8. The two last degrees of unsaturation were attributed, using the
as yet unassigned carbon atoms, to a double bond and a supple-
mentary conjugated ketone in an acyclic isoprenoid side chain.
Combined analysis of 2D NMR data located this carbonyl and the
remaining hydroxyl group at C-13 and C-12, respectively, thus
completing the partial structure of 1 with the fragment e. The
absolute configuration of the asymmetric center at C-12 was
assigned as R by application of the modified Mosher’s ester
method15 (Experimental Section). Thus, the structure of 1 was
defined to be a chromene derivative containing cyclized hydro-
quinone and tetraprenyl moieties.
Figure 1. Partial structures a-f for compounds 1-5, 8, and 9 as
deduced by 2D NMR data.
The second isolated compound (2) was an optically active pale
yellow oil. The HREIMS of 2 established the molecular formula
C28H40O5, implying nine degrees of unsaturation. An initial
examination of the 13C NMR data revealed two carbonyl carbons,
12 carbons in the aromatic/olefinic region of the spectrum, and 14
sp3 carbons. These data were in agreement with a structure that
could consist of an aromatic ring joined to a diterpenoid side chain
containing three olefinic bonds and two carbonyl groups. Analysis
of the NMR data revealed that the aromatic part of 2 was a
disubstituted hydroquinone. The 1H NMR spectrum presented two
meta-coupled aromatic signals at δΗ 6.69 (H-3′) and 6.63 (H-5′)
that showed correlations with (i) the aromatic carbon signals at δC
113.5 (C-3′) and 114.4 (C-5′), respectively, in the gHSQC; (ii) a
benzylic methylene group (δΗ 3.32, H2-1), a methyl (δΗ 2.19, H3-
Figure 2. Possible tautomeric acyloin isomerization for compounds
1-5, 8, and 9.
isoprenoid units, a complete analysis of the 2D NMR data indicated
that 3 and 2 shared the same planar structure (fragment f).
1
7′), and a methoxyl (δΗ 3.44, H3-OMe) by H-1H gCOSY; and
Compound 4, isolated as an optically active oil, has the molecular
formula C28H40O5 (HREIMS). Close comparison of its spectroscopic
data with those of 2 and 3 led us to conclude that 3 and 4 were
diastereoisomers. From a biosynthetic point of view, it seemed more
likely that the stereochemistry variation occurs for the hydroxyl
group rather than for the secondary methyl. In addition, the
hypothesis that 3 and 4 could be epimers at C-13 was supported
by the possible occurrence of a tautomeric acyloin-type isomer-
ization (Figure 2).
(iii) all the aromatic carbons in addition to C-1 and C-7′,
respectively, by gHMBC. All these data, in addition to further
gHMBC correlations between H2-1 and C-1′, C-2′, C-3′, C-2, and
C-3 and between H-2 and C-2′, C-1, C-4, and C-20, were consistent
with an isoprene unit being attached to an O-methyltoluquinol
moiety (fragment b). The E geometry of the ∆2 double bond was
confirmed by spatial correlations between H2-1/H3-20 and H-2/H2-4
on the basis of the gNOESY experiment. The structure of the second
isoprenoid unit was determined to be quite similar to that of
compound 1 (fragment d instead of c). Differences displayed in
the NMR data could be attributed to modification of the geometry
Compound 5 was an optically active oil, with the molecular
formula C28H40O5 deduced by MS and NMR spectrometries.
Comparison of its NMR data with those of 1 and 2 clearly showed
it to have a planar structure constituted by fragments b, d, and e.
Surprisingly, NMR data and the [R]D value of compound 5 did not
match with those of a compound already described by Higgs and
Mulheirn,8 which shows the same planar chemical structure. As
discussed previously in the case of 3 and 4, these two compounds
are probably epimers at C-12.
1
of the double bond ∆6. Specifically, the H NMR signal of H3-19
shifted from δH 2.05 in 1 to 1.48 in 2, the 13C chemical shift of
C-19 shifted from δC 19.2 in 1 to 25.1 in 2, and gNOESY key
interactions between H3-19 and H-6 were consistent with the change
of the configuration of the ∆6 double bond from E in 1 to Z in 2.
The constitution of the rest of the side chain was elucidated initially
1
by H COSY correlations from H2-8 to H2-9 and H2-10, H2-10 to
The two metabolites 6 and 7 (geranylgeranyltoluquinol) were
identical to compounds previously reported from Cystoseira el-
egans16 and Stypopodium zonale,17 two Phaeophyceae species
belonging to the Sargassaceae and the Dictyotaceae families,
respectively.
H-11, H-11 to H3-18, H-13 to H-14, and H-14 to H3-16 and H3-17,
and by cross-peaks in the gHMBC spectrum from H2-8 to C-10,
H2-9 to C-11, H2-10 to C-11 and C-12, H-11 to C-18 and C-13,
H-13 to C-14 and C-15, and H-14 to C15, C-16, and C-17. Thus,
by the analysis of these 2D NMR data, the connectivity from C-8
to C-16 was fully established and the localization of a hydroxyl
group at C-13 and a ketone at C-12 was confirmed. The planar
structure of 2 was completed by the fragment f.
The remaining meroditerpenoids 8 and 9 were characterized as
12′-hydroxy-5,13′-dioxoisohalidrols. These compounds, previously
reported from a specimen of H. siliquosa collected from the coast
of Scotland (UK),8 showed data in agreement with a probable
epimeric relationship at C-12. For these two compounds, all NMR
measurements and assignments were recorded, and this revealed
that the original data (recorded in CD3OD for 13C) contained
incorrect assignments. Tables 1 and 2 report the completely assigned
Compound 3, molecular formula C28H40O5 (HREIMS), was
obtained as an optically active oil. Comparison of 13C and 1H NMR
data (Tables 1 and 2) for 3 with those of 2 showed slight differences
and pointed out the same O-methyltoluquinol structure (fragment
b) of these two isomers. The most significant changes in the NMR
spectra appeared for signals of carbons and protons of the second
1
13C and H NMR data for 8 and 9 in C6D6.
1
isoprenoid unit: the H signal of H-19 shifted from δH 1.48 for 2
Concerning the absolute configuration of the carbon C-12 (or
C-13) of compounds 2-5, 8, and 9, the modified Mosher’s
esterification method was conducted after methylation of the free
phenoxy group, but the results were equivocal. The same methodol-
ogy used by Iwashima et al.18 with related compounds was also
attempted in order to determine simultaneously the absolute
to 2.08 for 3, and in the 13C NMR spectrum, the signal of C-19
moved from δC 25.1 in 2 to 19.2 in 3. These shifts, as well as
NOE correlations between H-6 and H2-8, clearly showed 3 to have
an E geometry of the ∆6 carbon-carbon double bond (fragment
c), as for 1, instead of Z for 2 (fragment d). For the two last

1124 Journal of Natural Products, 2008, Vol. 71, No. 7
Culioli et al.
Table 3. Results of the Biological Assays for Compounds 1-6, 8, and 9 and for the Crude Extract (CE)
Cobetia marina
Marinobacterium stanieri
Vibrio fischeri
Pseudoalteromonas haloplanktis
antisettlement assays
toxicity tests
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
EC50 (µg/mL)
LC50 (µg/mL)
1
2
3
4
5
6
>100
0.5
5
>100
>100
1
>100
>100
0.5
>100
1
>100
1
>100
>100
>100
5
>100
5
>100
>100
>100
10
1
5
>100
>100
1
5
10
>100
>100
0.5
>100
>100
1
8
9
CE
10
2.5
5
10
1
5
25
2.5
5
10
1
10
>100
>100
>100
33
4
5
Chart 1
configuration of the stereocenters at C-8 and C-12 or at C-8 and
C-13. Unfortunately, this method did not give clear results.
From a chemotaxonomical perspective, it is interesting to note
that several Sargassum species (e.g., S. siliquastrum,19 S. autum-
nale,20 or S. micracanthum18) have the ability to produce structur-
ally similar metabolites to those biosynthesized by H. siliquosa.
This similarity is in agreement with recent taxonomic works in this
field: H. siliquosa, which was included originally in the Cystosei-
raceae family, is now merged, as well as all the Cystoseiracean
species, into the larger Sargassaceae family.21,22
Compounds 1-6, 8, and 9 and the crude extract (CE) were tested
for their potential biological activities against important species
involved in the colonization of marine surfaces (Table 3). The crude
extract gave a good level of bioactivity against all of the organisms
screened. Among the compounds tested, 2, 6, and 9 were active
against the growth of the four strains of bacteria and in antisettle-
ment assays at nontoxic concentrations. For these three compounds,
the effect of increasing concentration on settlement was determined:
the inhibition of settlement was complete at 5 µg/mL for 2, 25
µg/mL for 6, and 100 µg/mL for 9. We also examined whether
settlement inhibition was reversible following transfer to clean
seawater. After 24 h, settlement was not significantly different from
the control (data not shown).
It is interesting to note that the most effective compound (2) is
the major constituent of the crude extract. However, in order to
fully understand the ecological roles of this compound, it will be
necessary to determine its location and measure the amounts
released by the alga.23,24 The surface localization of metabolites
could indicate their potential to be used in surface-mediated
interactions.24 The next step will be to determine whether a
bioactive substance retains activity following incorporation into a
paint matrix and to run immersion tests in various geographical
locations.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 343 polarimeter, using a 10 cm microcell. IR spectra
were recorded with a Jasco model J-410 FT-IR spectrometer as KBr
plates (films). Mass spectra were carried out at 70 eV with a Varian
MAT 311 double-focusing mass spectrometer with reversed Nier-

Antifouling ActiVity of Meroditerpenoids
Journal of Natural Products, 2008, Vol. 71, No. 7 1125
Johnson BE geometry. NMR spectra were recorded on a Bruker Avance
400 MHz instrument. All chemical shifts were referenced to the residual
solvent peak.
Plant Material. Halidrys siliquosa was collected in May 2004 in
France (Saint-Gue´nole´, Brittany, France; 47°47′ N, 4°23′ W). A
specimen of the alga has been identified by Prof. B. de Reviers
(Muse´um National d’Histoire Naturelle (MNHN), Paris, France) by
comparison to the reference herbarium, and a voucher specimen (No.
PC0111849) was deposited in the Herbarium of MNHN.
5′), 6.673 (H-3′), 6.460 (H-1), 6.321 (H-14), 6.161 (H-6), 5.973 (H-2),
5.469 (H-12), 2.283 (H-7′), 2.209 (H-17), 2.152 (H-19), 1.777 (H-16),
1.662 (H-20), 1.127 (H-18). Selected signals of 1R: 1H NMR (C5D5N,
400 MHz) δΗ 6.805 (H-5′), 6.666 (H-3′), 6.459 (H-1), 6.398 (H-14),
6.141 (H-6), 5.969 (H-2), 5.489 (H-12), 2.282 (H-7′), 2.219 (H-17),
2.148 (H-19), 1.818 (H-16), 1.661 (H-20), 1.013 (H-18). ∆δH(1S -
1R): H-5′, +0.002 ppm; H-3′, +0.002 ppm; H-1, +0.001 ppm; H-14,
-0.077 ppm; H-6, +0.020 ppm; H-2, +0.004 ppm; H-12, -0.020 ppm;
H-7′, +0.001 ppm; H-17, -0.010 ppm; H-19, +0.004 ppm; H-16,
-0.041 ppm; H-20, +0.001 ppm; H-18, +0.114 ppm.
Extraction and Purification. The dried blades and thalli (450 g
dry wt) of H. siliquosa were crushed in a mill and then extracted at
room temperature with CHCl3/MeOH (2:1 then 1:2, v/v) to produce
after filtration 17.8 g of crude extract. This extract was then partitioned
in a mixture of MeOH/CHCl3/H2O (4:3:1, v/v/v) to yield 13.5 g of
organic phase. A portion of the organic extract (9 g) was subjected to
normal-phase CC over silica gel (Si60, 40-63 µm, Merck) using step-
gradient elution from n-hexane/EtOAc (9:1, v/v) to EtOAc and then
from EtOAc to MeOH, to yield 25 fractions each of 150 mL. 1H NMR
investigations and TLC analysis of these fractions indicated fractions
6 and 7 (eluted with n-hexane/AcOEt, 6:4), fractions 8 and 9 (eluted
with n-hexane/AcOEt, 5:5), and fraction 10 (eluted with n-hexane/
AcOEt, 4:6) to be of further interest. Fractions 6 and 7 were subjected
to further purification on reversed-phase HPLC (Merck Purospher Star
RP-18e 5 µm; 10 × 250 mm; 2 mL/min) eluting with MeCN/H2O (5:
1) to yield, respectively, 7 (4 mg) and 1 (8 mg). From fractions 8 and
9, compounds 2 (76 mg), 5 (11 mg), 3 (14 mg), 4 (9 mg), 9 (12 mg),
and 8 (9 mg) were purified by reversed-phase HPLC (eluent MeCN/
H2O, 4:1). Fraction 10 was purified by reversed-phase HPLC with
MeCN/H2O (7:3) as eluent to afford 6 (6 mg).
Antimicrobial Assays. The compounds were tested for inhibitory
activity against four strains of marine bacteria: Cobetia marina (ATTC
25374), Marinobacterium stanieri (ATCC 27130), Vibrio fischeri
(ATCC 7744), and Pseudoalteromonas haloplanktis (ATCC 14393).
The experiments were performed as previously described by Mare´chal
et al.25 Compounds (at concentrations of 0.5, 1, 2.5, 5, 10, 25, 50, and
100 µg/mL) were incubated with the bacteria (2 × 108 cells/mL) in
96-well plates (Merck) in MHB medium (Mueller Hinton broth, Sigma),
supplemented with NaCl (15 g/L), at 25 °C for 24 h. Each treatment
and the seawater control were replicated six times. Minimum inhibitory
concentrations (MICs), compared to the seawater control, were
determined by the microtiter broth dilution method.26
Larval Bioassays. Larval Culture. Adult barnacles were collected
from the pier pilings at the Duke Marine populations, North Carolina.
They were maintained at 22 °C, with aeration (14 h light/10 h dark
cycle), and fed on a daily diet of Artemia salina nauplii (7 nauplii/
mL).27 Release of larvae was obtained as previously detailed by Hellio
et al.28 Stage-II nauplii were used for toxicity assays. After 4 days,
cyprids were collected by filtration.27
Settlement Assays. Settlement tests were conducted in 24-well
microplates (Iwaki). Compounds were dissolved in 2 mL of seawater
in the following concentrations: 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µg/
mL. Then, 10 to 15 cyprids were added to wells. Each test was
performed in six replicates. Test plates were incubated at 28 °C (in
darkness), and results were recorded after 24 h incubation. Each larva
was examined under a dissecting microscope and its condition recorded
(dead, settled or swimming).29 Results are presented as 24 h EC50 values
()concentration of compound leading to 50% inhibition of settlement
in comparison with the seawater control), which were determined using
Sigma Plot 8.0.
Toxicity Tests. Toxicity tests were conducted on nauplii according
to Wu et al.30 Ten to fifteen stage-II nauplii were added to 2 mL of
solution in the wells of a 24-well (Iwaki) plate. Compounds were tested
at the same concentrations as for the settlement assays, with six
replicates of each treatment and the control.27 The number of swimming
and dead nauplii was recorded after 24 h exposure to the compounds.
The data are expressed as a 24 h LC50 ()concentration of extract that
produces 50% of mortality in comparison with the control), which was
determined using Sigma Plot 8.0.
Compound 1: yellow oil; [R]25 -11 (c 0.1, MeOH); UV (EtOH)
D
λmax (log ꢀ) 270 (3.7), 320 (3.5) nm; IR (film) νmax 3420, 2895, 1712,
1
1680, 1438 cm-1
;
13C NMR (100 MHz, C6D6), see Table 1; H NMR
(400 MHz, C6D6), see Table 2; HRMS m/z 454.2722 [M]+ (calcd for
454.2719).
Compound 2: clear yellow oil; [R]25 -74 (c 0.1, MeOH); UV
D
(EtOH) λmax (log ꢀ) 240 (4.4), 292 (3.2) nm; IR (film) νmax 3461, 2969,
2929, 2850, 1707, 1684, 1613, 1484, 1445, 1383, 1200 cm-1 13C NMR
;
(100 MHz, C6D6), see Table 1; 1H NMR (400 MHz, C6D6), see Table
2; HRMS m/z 456.2871 [M]+ (calcd for 456.2876).
Compound 3: clear yellow oil; [R]25 +40 (c 0.1, MeOH); UV
D
(EtOH) λmax (log ꢀ) 242 (4.2), 270 (2.9) nm; IR (film) νmax 3398, 2925,
1737, 1610, 1437, 1385 cm-1 13C NMR (100 MHz, C6D6), see Table
;
1; 1H NMR (400 MHz, C6D6), see Table 2; HRMS m/z 456.2869 [M]+
(calcd for 456.2876).
Compound 4: clear yellow oil; [R]25 -83 (c 0.1, MeOH); UV
D
(EtOH) λmax (log ꢀ) 239 (4.3), 270 (3.3) nm; IR (film) νmax 3399, 2926,
2855, 1737, 1678, 1612, 1439, 1384 cm-1 13C NMR (100 MHz, C6D6),
;
see Table 1; 1H NMR (400 MHz, C6D6), see Table 2; HRMS m/z
456.2869 [M]+ (calcd for 456.2876).
Compound 5: clear yellow oil; [R]25 -3 (c 0.1, MeOH); UV
D
Acknowledgment. The authors wish to thank Prof. B. De Reviers
(De´partement Syste´matique et Evolution, UMR 7138, MNHN, Paris,
France) for taxonomic identification of the algal material and lodgement
of the voucher specimen.
(EtOH) λmax (log ꢀ) 242 (3.9), 292 (2.8) nm; IR (film) νmax 3461, 2939,
1681, 1617, 1485, 1444, 1382 cm-1 13C NMR (100 MHz, C6D6), see
;
Table 1; 1H NMR (400 MHz, C6D6), see Table 2; HRMS m/z 456.2874
[M]+ (calcd for 456.2876).
Compounds 6 and 7. Data were in agreement with those reported
in the literature for these compounds.16,17
References and Notes
Compound 8: clear yellow oil; [R]25D -12 (c 0.1, MeOH); IR (film)
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ν
max 3399, 2925, 2854, 1736, 1618, 1438, 1384, 1240 cm-1 13C NMR
;
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sample and MTPA chloride evenly. The reaction NMR tubes were
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1
monitored by H NMR: the reaction was completed in approximately
5 h for the (S)-MTPA ester 1S and 3 days for the (R)-MTPA ester 1R.
1
Selected signals of 1S: H NMR (C5D5N, 400 MHz) δΗ 6.812 (H-

1126 Journal of Natural Products, 2008, Vol. 71, No. 7
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NP070110K