Meroditerpenoids and derivatives from the brown alga Cystoseira baccata and their antifouling properties

Article abstract of DOI:10.1021/np8004216

The brown alga Cystoseira baccata harvested along the Atlantic coasts of Morocco yielded seven new meroditerpenoids (1-4) and derivatives (5-7), whose chemical structures were elucidated mainly by 2D NMR and mass spectrometry. Surprisingly, for all these compounds, which possess a bicyclo[4.3.0]nonane ring system, a trans fusion of the bicyclic system was deduced by stereochemical studies even though such compounds isolated from Cystoseira species are known to have a typical cis orientation for the bridgehead methyls. The antifouling and antibacterial activities of compounds 1-5 and 7 were evaluated, as well as their toxicity toward nontarget species. Compounds 4, 5, and 7 showed antifouling activities against growth of microalgae, macroalgal settlement, and mussel phenoloxidase activity, while being nontoxic to larvae of sea urchins and oysters.

Full text of DOI:10.1021/np8004216

1806
J. Nat. Prod. 2008, 71, 1806–1811
Meroditerpenoids and Derivatives from the Brown Alga Cystoseira baccata and Their
Antifouling Properties
Redouane Mokrini,† Mohammed Ben Mesaoud,‡ Mohammed Daoudi,‡ Claire Hellio,§ Jean-Philippe Mare´chal,
Mohamed El Hattab, Annick Ortalo-Magne´,† Louis Piovetti,† and Ge´rald Culioli*,†
Laboratoire MAPIEM, UniVersite´ du Sud Toulon-Var, AVenue de l’UniVersite´, BP 20132, F-83957 La Garde Cedex, France, Laboratoire de
Chimie Organique et Bioorganique, Faculte´ des Sciences Choua¨ıb Doukkali, El Jadida, Morocco, School of Biological Sciences, King Henry
Building, Portsmouth UniVersity, Portsmouth, PO1 2DY, U.K., Marine EnVironment ObserVatory of Martinique, 3 AVenue Condorcet,
F-97200 Fort de France, French West Indies, and Laboratoire des Plantes Aromatiques et Me´dicinales, De´partement de Chimie, Faculte´ des
Sciences, UniVersite´ de Blida, Algeria
ReceiVed July 11, 2008
The brown alga Cystoseira baccata harvested along the Atlantic coasts of Morocco yielded seven new meroditerpenoids
(1-4) and derivatives (5-7), whose chemical structures were elucidated mainly by 2D NMR and mass spectrometry.
Surprisingly, for all these compounds, which possess a bicyclo[4.3.0]nonane ring system, a trans fusion of the bicyclic system
was deduced by stereochemical studies even though such compounds isolated from Cystoseira species are known to have a
typical cis orientation for the bridgehead methyls. The antifouling and antibacterial activities of compounds 1-5 and 7 were
evaluated, as well as their toxicity toward nontarget species. Compounds 4, 5, and 7 showed antifouling activities against growth of
microalgae, macroalgal settlement, and mussel phenoloxidase activity, while being nontoxic to larvae of sea urchins and oysters.
Marine brown algae of the genus Cystoseira (family Sargas-
saceae, order Fucales) are widely distributed through the subtropics
with a prevalent center of speciation in the Mediterranean Sea.1
Previous chemical investigations have demonstrated that species
of this genus are prolific producers of diterpenoids, meroditerpe-
noids, and phlorotannins.2
tion of antifouling paints, toxicity tests were also run toward two
crucial indicator species (sea urchins and oysters).
As a part of our project focused on searching for new antifouling
compounds from seaweeds, we have investigated specimens of
Cystoseira baccata collected off the Atlantic coast of Morocco.
This species is commonly found along the NE Atlantic shores from
Ireland to Mauritania, and to date only a few studies have been
published on the chemical composition of C. baccata.3-6
In this paper, further investigation of this species led to the
isolation of compound 1, which has the same planar structure as
cyclo-1′-demethylcystalgerone (8a), a meroditerpenoid with a
bicyclic diterpenoid side chain first isolated from a related Mediter-
ranean species (Cystoseira algeriensis),7 but also described from
C. baccata.6 However, further exploration of the stereostructure
of 1 revealed a trans orientation of the two bridgehead methyls of
the hydrindane system instead of a cis orientation in the case of 8a
and all the cyclic meroditerpenes previously described from species
of the Cystoseira genus.2,8,9 In addition to 1, we report on the
isolation and structural characterization by spectroscopic analysis
(HRMS, UV, IR, 1D and 2D NMR) of six other novel C. baccata
metabolites (2-7) that possess the same bicyclic system with a
characteristic trans fusion. Relative configurations were determined
by 2D NOE experiments, while absolute configurations were
attributed with the help of CD measurements or using the modified
Mosher ester method.10,11 Compounds 1-5 and 7, together with
the crude extract of C. baccata, were screened for their potential
antifouling activities toward model species representative of mi-
crofouling (marine and terrestrial bacteria; microalgae) and mac-
rofouling species (macroalgae and invertebrates). Because the goal
was to find new environmentally friendly compounds for formula-
* 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.
‡ Universite´ d’El Jadida.
§ Portsmouth University.
Marine Environment Observatory of Martinique.
Universite´ de Blida.
10.1021/np8004216 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/04/2008

Meroditerpenoids from Cystoseira baccata
Journal of Natural Products, 2008, Vol. 71, No. 11 1807
Table 1. 13C NMR Data for Compounds 1-7 (100 MHz, CDCl3)
1
2
3
4
5
6
7
position
δC, mult.a
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
1
2
3
4
5
6
7
29.7 CH2
125.2 CH
134.0 qC
45.1 CH2
154.2 qC
41.4 CH2
44.5 qC
30.2 CH2
127.7 CH
135.2 qC
47.5 CH2
80.2 qC
123.6 CH
129.4 CH
78.9 qC
46.1 CH2
153.4 qC
43.4 CH2
44.8 qC
123.3 CH
130.2 CH
78.6 qC
46.2 CH2
153.3 qC
44.3 CH2
44.8 qC
30.7 CH3
205.5 qC
50.2 CH2
149.0 qC
43.1 CH2
44.8 qC
31.7 CH3
197.6 qC
114.9 CH
107.3 qC
36.7 CH2
46.7 qC
22.3 CH3
153.6 qC
44.4 CH2
44.5 qC
42.3 CH2
45.7 qC
8
9
34.9 CH2
18.8 CH2
29.3 CH2
54.9 qC
208.3 qC
132.1 qC
39.0 CH2
71.2 qC
29.6 CH3
30.3 CH3
21.0 CH3
22.5 CH3
16.3 CH3
146.1 qC
127.1 qC
112.9 CH
153.1 qC
113.9 CH
124.7 qC
16.3 CH3
55.6 CH3
35.5 CH2
19.3 CH2
28.7 CH2
58.2 qC
213.2 qC
54.4 CH
34.7 CH2
70.9 qC
28.9 CH3
30.9 CH3
22.4 CH3
23.2 CH3
18.3 CH3
146.3 qC
127.2 qC
113.0 CH
153.1 qC
114.0 CH
125.0 qC
16.3 CH3
55.6 CH3
35.0 CH2
18.8 CH2
29.3 CH2
54.8 qC
208.5 qC
133.0 qC
39.4 CH2
71.1 qC
29.5 CH3
30.7 CH3
20.9 CH3
22.4 CH3
27.6 CH3
144.2 qC
120.4 qC
109.0 CH
153.2 qC
116.5 CH
126.2 qC
15.8 CH3
55.7 CH3
35.0 CH2
18.8 CH2
29.4 CH2
54.9 qC
208.7 qC
133.2 qC
39.6 CH2
70.8 qC
28.8 CH3
31.4 CH3
20.9 CH3
22.5 CH3
27.0 CH3
144.1 qC
120.5 qC
109.1 CH
152.9 qC
116.6 CH
126.0 qC
16.0 CH3
55.7 CH3
34.8 CH2
18.8 CH2
29.2 CH2
54.8 qC
208.4 qC
130.6 qC
39.3 CH2
71.2 qC
29.2 CH3
30.7 CH3
21.1 CH3
22.6 CH3
35.0 CH2
18.8 CH2
29.2 CH2
55.0 qC
206.5 qC
133.2 qC
38.7 CH2
71.3 qC
29.5 CH3
30.5 CH3
21.1 CH3
22.5 CH3
33.5 CH2
19.9 CH2
28.9 CH2
45.9 qC
173.1 qC
153.5 qC
41.7 CH2
88.8 qC
28.7 CH3
28.3 CH3
23.1 CH3
23.6 CH3
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.
between Ha,b-6 and C-5, C-7, C-11, C-13, and C-19 allowed not
only the localization of the ketone and the second trisusbtituted
double bond but also the completion of the carbon skeleton of
substructure b as a bicyclo[4.3.0]nonane ring. Fragment c was
deduced by HMBC cross-peaks between Ha,b-14 and C-15, C-16,
and C-17, H3-16 and C-14, C-15, and C-17, and H3-17 and C-13,
C-15, and C-16. The planar structure of 1 was completed by the
linkage of these three fragments using additional HMBC cross-
peaks between Ha,b-14 and C-5, C-12, and C-13, Ha,b-4 and C-5,
C-6, and C-13, and Ha,b-6 and C-4. A detailed comparison of the
NMR data of 1 with those reported for cyclo-1′-demethylcystal-
gerone (8a)6,7 revealed the similarity of these molecules. However,
the NOESY spectrum distinguished these two compounds based
on spatial correlations of the bridgehead methyls (Figure 2), which
were found to have a trans orientation in 1 instead of cis in the
case of 8a. To our knowledge this is the first report of such a
structural feature in the Sargassaceae family. Finally, the geometry
of the double bond at C-2 was assigned to be E on the basis of (i)
the upfield 13C NMR chemical shift of the vinyl methyl carbon at
δC 16.3 (C-20)12,13 and (ii) the NOE correlations observed between
H-2 and H2-4 as well as between H2-1 and H3-20.
Results and Discussion
The lipophilic fraction derived from the crude extract of dried
and powdered C. baccata was purified by a combination of Si gel
column chromatography and reversed-phase semipreparative HPLC
to yield compounds 1-7.
Compound 1 was obtained as an optically active pale yellow oil
([R]25 +83) and displayed absorption bands for hydroxyl (3415
D
cm-1) and conjugated carbonyl functionalities (1658 and 1604
cm-1) in the IR spectrum. It gave an [M + Na]+ ion by HRESIMS
at m/z 463.2812, which suggested a molecular formula of C28H40O4
and, therefore, possessed nine degrees of unsaturation. The 1H NMR
data (recorded in CDCl3) indicated the presence of two meta-
coupled aromatic protons (δH 6.56 and 6.51), an olefinic triplet (δH
5.37, J ) 7.0 Hz), two D2O exchangeable singlets (δH 4.71 and
3.63), one methoxy group (δH 3.73), and six singlet methyls (δH
2.22, 1.69, 1.21, 1.12, 1.03, and 0.80). The 13C NMR and DEPT
spectra revealed the occurrence of 11 quaternary carbons (δC 208.3,
154.2, 153.1, 146.1, 134.0, 132.1, 127.1, 124.7, 71.2, 54.9, and
44.5), three methines (δC 125.2, 113.9, and 112.9), seven methylenes
(δC 45.1, 41.4, 39.0, 34.9, 29.7, 29.3, and 18.8), and, again, seven
methyl groups (δC 55.6, 30.3, 29.6, 22.5, 21.0, 16.3, and 16.3).
With nine degrees of unsaturation, 1 apparently contained two rings
besides the carbonyl, the two double-bond groups (one trisubstituted
and one tetrasubstituted), and the aromatic ring. The proton and
protonated carbon signals in the NMR spectra of 1 were unequivo-
cally assigned by the HSQC experiments (Tables 1 and 2), and
1H-1H COSY was then used to deduce extensive coupling systems.
Homonuclear coupling correlations from H3-7′ through H-5′, H-3′,
H2-1, and H-2 to H3-20 and Ha,b-4 allowed formulation of fragment
a (Figure 1), commonly found in Cystoseira-type metabolites.
Fragment b was delineated by sequential couplings between Hb-
6/H3-19/Ha-8 and H2-9/Ha,b-10/H3-18 as well as by HMBC cor-
relations from C-8 to H-9, C-9 to Hb-8, and C-10 to Hb-8 and H2-
9. HMBC correlations between H3-19 and C-7, C-8, and C-11, on
one hand, and between H3-18 and C-7, C-10, and C-11, on the
other, revealed the presence of a cyclopentane moiety in fragment
b. Further heteronuclear couplings between H3-18 and C-12 and
Compound 2 was isolated as an optically active, yellowish oil,
[R]25 +28, and had a molecular formula of C28H42O5 (m/z 458,
D
ESIMS), which was suggested by the HRESIMS of the M + Na
adduct. Its IR spectrum showed absorption bands at 3432 and 1694
cm-1, indicating the presence of both hydroxyl and nonconjugated
carbonyl groups. Overall, the 13C NMR features of 2 showed
similarity to those of 1 except the replacement of two quaternary
sp2-hybridized carbons by two sp3 carbons (a methine at δC 54.4
and a hydroxyl-bearing quaternary carbon at δC 80.2). On the basis
of the assignments of the NMR data (Tables 1 and 2) by the 1H-1H
COSY, HSQC, and HMBC experiments, the 1H NMR spectrum of
2 showed, by comparison with those of 1, that the chemical shift
values of Ha,b-4, Ha,b-6, and Ha,b-14 were shifted significantly upfield
and an additional methine triplet signal (δH 3.32, J ) 5.5 Hz, H-13)
was observed. These data suggested that the ∆5,13 double bond of
1 was replaced, in the case of 2, by its hydrated counterpart. This

1808 Journal of Natural Products, 2008, Vol. 71, No. 11
Mokrini et al.
Table 2. 1H NMR Data of Compounds 1-7 (400 MHz, CDCl3, J in Hz)
position
1
2
3
4
5
6
7
1a
1b
2
4a
4b
6a
3.35 (d, 7.0)
3.39 (dd, 16.0, 7.5)
3.31 (dd, 16.0, 7.5)
5.32 (t, 7.5)
2.52 (d, 14.5)
2.14 (d, 14.5)
2.12 (d, 14.0)
2.06 (d, 14.0)
1.62 (m)
6.28 (d, 10.0)
6.32 (d, 10.0)
5.37 (t, 7.0)
3.05 (d, 14.5)
2.98 (d, 14.5)
2.44 (d, 18.5)
2.21 (d, 18.5)
1.73 (m)
5.53 (d, 10.0)
2.78 (d, 14.0)
2.71 (d, 14.0)
2.63 (d, 18.5)
2.51 (d, 18.5)
1.67 (m)
5.61 (d, 10.0)
2.97 (d, 14.0)
2.53 (d, 14.0)
2.94 (d, 18.5)
2.24 (d, 18.5)
1.73 (m)
2.22 (s)
2.16 (s)
1.92 (s)
3.62 (d, 17.0)
3.42 (d, 17.0)
2.65 (d, 18.5)
2.10 (d, 18.5)
1.76 (m)
5.62 (br s)
2.57 (d, 18.0)
2.21 (d, 18.0)
1.74 (m)
3.40 (d, 18.0)
2.57 (d, 18.0)
1.70 (m)
6b
8a
8b
9
1.50 (m)
1.74 (m)
1.43 (m)
1.75 (m)
1.47 (m)
1.70 (m)
1.50 (m)
1.72 (m)
1.53 (m)
1.75 (m)
1.53 (m)
1.76 (m)
1.52 (m)
1.81 (m)
10a
10b
13
14a
14b
16
17
18
19
20
3′
1.97 (m)
1.46 (m)
1.98 (m)
1.27 (m)
3.32 (t, 5.5)
2.20 (dd, 15.0, 5.5)
1.75 (dd, 15.0, 5.5)
1.17 (s)
1.27 (s)
1.29 (s)
0.68 (s)
1.88 (s)
1.95 (m)
1.43 (m)
1.96 (m)
1.44 (m)
1.97 (m)
1.46 (m)
2.00 (m)
1.48 (m)
1.81 (m)
1.50 (m)
2.73 (d, 14.0)
2.46 (d, 14.0)
1.12 (s
1.21 (s)
1.03 (s)
0.80 (s)
1.69 (s)
6.51 (d, 3.0)
6.56 (d, 3.0)
2.22 (s)
2.67 (d, 14.5)
2.53 (d, 14.5)
1.13 (s)
1.23 (s)
0.97 (s)
0.82 (s)
1.42 (s)
6.38 (d, 3.0)
6.58 (d, 3.0)
2.15 (s)
2.71 (d, 14.0)
2.49 (d, 14.0)
1.08 (s)
1.29 (s)
0.99 (s)
0.81 (s)
1.40 (s)
6.39 (d, 3.0)
6.57 (d, 3.0)
2.15 (s)
2.60 (d, 14.0)
2.43 (d, 14.0)
1.12 (s)
1.24 (s)
1.05 (s)
2.72 (d, 14.5)
2.33 (d, 14.5)
1.10 (s)
1.22 (s)
1.13 (s)
2.50 (br d, 2.0)
1.44 (s)
1.41 (s)
1.04 (s)
0.82 (s)
0.86 (s)
0.87 (s)
6.52 (d, 3.0)
6.56 (d, 3.0)
2.21 (s)
5′
7′
4′-OMe
OH
3.73 (s)
4.71 (s) 3.63 (s)
3.73 (s)
4.76 (s)
3.73 (s)
3.58 (s)
3.74 (s)
4.02 (s)
3.85 (br s)
hypothesis was confirmed in particular by the HMBC correlations
from H-13 to C-4, C-5, C-6, C-12, C-14, and C-15. The MS data
(an extra H2O and eight degrees of unsaturation) together with the
IR and the NMR spectra were consistent with the occurrence of
fragment d in the chemical structure of 2 instead of b in the case
of 1. The relative stereochemistry of 2 was determined from NOE
enhancements observed in a NOESY experiment. NOE correlations
from H3-18 to Hb-6, Ha-8, Ha-10, and H-13 together with correla-
tions from H3-19 to Hb-4b, Ha-6, Hb-8, and Hb-10 suggested not
only the trans-fused nature of the hydrindane moiety but also the
relative stereochemistry at C-5 and C-13 by the fact that H-13,
H3-18, and the hydroxyl group at C-5 were all cofacial.
Compound 3 was obtained as a clear yellow oil, with [R]25D -6,
and showed IR absorption bands for hydroxyl (3421 cm-1) and
conjugated carbonyl (1657 and 1601 cm-1) groups. It showed an
HRESIMS [M + Na]+ ion at m/z 479.2767, suggesting a molecular
formula of C28H38O4 (10 degrees of unsaturation). 13C NMR analysis
revealed the presence of 10 olefinic carbons in addition to the
carbonyl, and hence, 3 possessed four rings. The 2D NMR
experiments including 1H-1H COSY, HSQC, and HMBC dis-
1
played, by comparison of their H and 13C NMR data (Tables 1
and 2), that 3 and 1 shared the same fragments b and c. Moreover,
as typical NMR signals were observed for the O-methyltoluquinol
moiety, features differing from 1 were easily localized in the first
isoprenoid unit using the HMBC spectrum. The main differences
were, in the 1H NMR data, the two signals of a pair of cis-coupled
olefinic protons at δH 6.28 (d, J ) 10 Hz, H-1) and 5.53 (d, J ) 10
Hz, H-2) and, in the 13C NMR spectrum, the occurrence of two
sp2 methine carbons at δC 123.6 (C-1) and 129.4 (C-2) and the
quaternary oxygenated carbon at δC 78.9 (C-3). Thus, the planar
structure of 3 was completed by the chromenol fragment e.
Furthermore, the NOESY spectrum showed characteristic cross-
peaks indicating, as for 1 and 2, a trans fusion of the bicyclic system
in fragment b. On the basis of the above observations, the structure
of compound 3 was established and assigned as the chromene
derivative of 1. Moreover, the CD data (negative Cotton effect
around 260-290 nm) established, by application of Crabbe´’s
rule,14,15 that the absolute configuration at C-3 was R.
Figure 1. Partial structures a-e for compounds 1-5 and 7 as
deduced by 2D NMR data.
Compound 4 (clear yellow oil, [R]25D +65) showed IR and NMR
spectral features very similar to those of 3, indicating that 4 was
an isomer of 3. This was further confirmed by HRESIMS (m/z
479.2735 [M + Na]+ for C28H38O4). A complete analysis of the
2D NMR data revealed that 4 possessed a planar structure identical
with those of 3. From a biosynthetic point of view, it seemed more
likely that the stereochemical variation occurred for the oxygen-
bearing quaternary carbon (C-3) rather than for asymmetric carbons
(C-7 and C-11) of the bicyclo[4.3.0]nonane ring system. This was
supported by NOE correlations, identical to those of 1-3 for the
bridgehead methyls of the hydrindane part, implying a trans junction
for 4. Finally, these data allowed the description of compound 4
Figure 2. Computer-generated model for compound 1 using MM2
force field calculations and key NOE correlations.

Meroditerpenoids from Cystoseira baccata
Journal of Natural Products, 2008, Vol. 71, No. 11 1809
as the C-3 epimer of 3. The S configuration of the chromene moiety
was corroborated by CD measurement (positive Cotton effect
around 260-290 nm), which was opposite that of 3. These two
compounds are probably artifacts formed from 1 during the
extraction or the isolation procedures.
Table 3. Results of the Antimacroalgal and Anti-invertebrate
Assays for Compounds 4, 5, and 7
IC50 (µg/mL)
compound Sargassum muticum UlVa intestinalis phenoloxydase
4
5
7
2.5
1
>100
>100
1
1
2.5
The molecular formula C16H26O2 of compound 5, which was
deduced by HRESIMS on the sodiated molecular ion at m/z 273,
suggested a smaller chemical structure than the four compounds
previously described. The IR spectrum indicated the presence of
hydroxyl (3428 cm-1) and conjugated carbonyl (1652 and 1612
cm-1) groups. From the point of view of NMR data, the absence
1
2.5
1
in the 13C NMR spectrum taken along with all observed H and
13C chemical shifts allowed for the assignment of 7 as a highly
conjugated derivative of 6 resulting from an intramolecular cy-
clization between C-15 and C-12 with loss of water.
1
of aromatic and olefinic signals in the H spectrum, together with
the occurrence of the signals at δC 208.4, 153.6, and 130.6, allowed
the attribution of the four degrees of unsaturation to one carbonyl,
one tetrasubstituted double bond, and two rings in the molecule. A
comparison of overall 1H and 13C NMR spectroscopic data (Tables
1 and 2) established that differences between 5 and 1, 3, and 4 lie
only in the lack of signals corresponding to the O-methyltoluquinol
moiety and the first isoprenoid unit (fragment a). Thus, the chemical
structure of 5 was constructed of fragments b and c and completed
by a methyl group (δC 22.3, C-4; δH 1.92, s, H3-4). This hypothesis
was supported by the long-range heteronuclear correlations of both
H3-4 with C-5, C-6, and C-13 and Ha,b-6 with C-4 in the HMBC
spectrum, thus establishing the structure of compound 5. Analysis
of the 2D NOESY data for 5 and similar optical rotation values
gave the relative configuration of the bridgehead quaternary carbons
as being consistent with those of 1.
Finally, in order to determine the absolute configuration of the
bridgehead carbons (C-7 and C-11) of the hydrindane moiety,
compound 1 was reduced with NaBH4 to give the alcohol 1a in
42% yield. The effectiveness of the reduction was checked by NMR.
The 1H NMR spectrum showed an additional signal at δH 3.81 (s,
H-12) due to the oxymethine proton at C-12, while the ketone signal
at δC 208.3 was replaced in the 13C NMR spectrum by an oxygen-
bearing methine carbon at δC 78.0. The absolute configuration of
this newly appearing asymmetric center at C-12 was assigned as R
by application of modified Mosher’s method10,11 (see experimental
data on δS - δR). The S configuration was then suggested for the
neighboring C-11 on the basis of NOESY cross-peaks between H-12
and H-18, while the presence of a trans fusion of the bicyclic system
allowed us to attribute an R configuration to C-7. The same
methodology was employed with 5, and its absolute configuration
was assumed to be 7 R, 11 S. The absolute stereochemistry of
compounds 2-4, 6, and 7 was not determined due to a limited
quantity of the isolated natural products. However, in consideration
of the similar biogenetic origin of 1-7, the absolute configurations
at C-7 and C-11 of 2-4, 6, and 7 are proposed as R and S,
respectively. In the case of 2, NOE correlations suggested that the
two other stereocenters at C-5 and C-13 are S and R, respectively.
To date, only two papers have been published describing
chemical investigations of terpenoids from C. baccata, leading to
isolation of a total of three metabolites: cyclo-1′-demethylcystal-
gerone (8a)6 and its two chromanol derivatives (9a and 9b).3 For
these compounds, primarily described by spectroscopic comparison
with cystalgerone (8b) and similar metabolites, the hypothesis of a
cis-fused bicyclic moiety has not been fully demonstrated. It seems
more likely that compounds 8a, 9a, and 9b possess the same
absolute configurations at C-7 and C-11 as found in 1-7.
Comparison of [R]D and NMR data of 1 with those of 8a (isolated
from C. baccata)6 had identical values, thus supporting this
hypothesis.
As regards the novelty of this trans-fused hydrindane carbon
skeleton in the Sargassaceae family, actual studies held in our
laboratory, on Mediterranean species of Cystoseira, are inclined to
favor the existence of several biosynthetic pathways. Indeed, both
cis and trans junctions of the cyclopentane ring have been recently
observed in cyclized meroditerpenoids isolated from a same sample
(M. El Hattab, A. Ortalo-Magne´, and G. Culioli, unpublished data).
Compounds 1-5 and 7 and the crude extract of C. baccata were
assayed for antifouling and antibacterial activities and in toxicity
tests. No activity, at the concentrations tested (MICs > 100 µg/
mL), were recorded on marine (Pseudoalteromonas elyakoVii,
Vibrio aestuarianus, and Polaribacter irgensii) or terrestrial bacteria
(Salmonella typhimurium, Escherichia coli, and Bacillus subtilis).
Among the three microalgae studied (Exanthemachrysis gayraliae,
Cylindrotheca closterium, and Pleurochrysis roscoffensis), only E.
gayraliae growth was inhibited by 5 with a MIC value of 1 µg/
mL. Results on antimacroalgal and anti-invertebrate activities were
promising for 4, 5, and 7 (see Table 3). None of the compounds
tested showed significant toxicity toward larvae of sea urchin
(Echinus esculentus) or oysters (Crassostrea gigas) (LC50 > 100
µg/mL).
Compound 6 was obtained as an optically active, clear yellow
oil ([R]25 +7). The molecular formula was determined to be
D
C18H28O3 by high-resolution ESIMS (m/z 315.1919, [M + Na]+),
suggesting five degrees of unsaturation. Comparison of the NMR
data of 6 with those of 1, 3, 4, and 5 showed the occurrence, for
all of these compounds, of the same bicyclo[4.3.0]nonane ring
system (fragment b) and the same fragment c. Additional signals
at δC 30.7 (CH3, C-2), 50.2 (CH2, C-4), and 205.5 (qC, C-3) in the
13C NMR spectrum of 6, together with appropriate HMBC
correlations between H3-2 and C-3 and C-4 and between Ha,b-4
and C-3, were consistent with the presence of a 2-ketopropyl group.
This structural feature was corroborated by an absorption band for
a nonconjugated carbonyl (1718 cm-1) in the IR spectrum of 6.
The connectivity between this moiety and the rest of the structure
was fully established by HMBC correlations of Ha,b-4 with C-5,
C-6, and C-13 and Ha,b-6 with C-4, and thus, all of these data
supported the proposed planar structure for 6. In fact, compound 6
was probably a product obtained during the treatment of the algal
material, or the extraction procedure, by oxidation of 1 at the level
of the ∆2 double bond. This hypothesis was in agreement with that
reported by Amico et al.,16 who obtained an equivalent byproduct
after ozonolysis of cystalgerone (8b).
Compound 7 showed an HRESIMS [M + Na]+ ion at m/z
297.1807 for a molecular formula of C18H26O2 (six degrees of
unsaturation). With one ketone carbonyl (δC 197.6) and four other
sp2 carbons (δC 173.1, 153.5, 114.9, and 107.3), the 13C NMR data
accounted for three of the six double-bond equivalents, leaving a
structure that contained three rings. In comparison with the NMR
1
data of 6, the main difference in the H NMR spectrum of 7 was
the replacement of a pair of doublets of nonequivalent methylene
protons (δH 3.62 and 3.42, Ha,b-4) by an olefinic singlet signal at
δH 5.62. This information was corroborated, in the 13C and the
HSQC spectra of 7, by the occurrence of the corresponding signal
for a sp2 methine at δC 114.9, and it implied a ∆4 double bond
instead of a ∆5 double bond in the case of 6. In the NOESY
spectrum, the occurrence of a correlation between H-4 and H2-14,
together with the absence of cross-peaks between H-4 and Ha,b-6,
defined the absolute configuration of this double bond as E. Finally,
the molecular formula (loss of 18 amu with respect to 6) suggested
the presence of an additional ring. The loss of a ketone resonance

1810 Journal of Natural Products, 2008, Vol. 71, No. 11
Mokrini et al.
Experimental Section
at room temperature. The reaction was quenched with H2O, and then
the mixture was extracted with Et2O (3 × 25 mL). The combined ether
extracts were dried over anhydrous MgSO4. The products were purified
using reversed-phase HPLC (Merck Purospher Star RP-18e 5 µm; 4.6
× 250 mm; 1 mL/min), with H2O/MeCN (20:80, v/v) as eluent, to
yield the pure alcohol 1a (2.1 mg).
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 343 polarimeter, using a 10 cm microcell. CD spectra
were obtained on a Jasco J-810 spectrophotometer. IR spectra were
recorded with a Jasco model J-410 FT-IR spectrometer as KBr plates
(films). 1D and 2D NMR spectra were obtained at 400 and 100 MHz
for 1H and 13C, respectively, on a Bruker Avance 400 MHz NMR
spectrometer in CDCl3. All chemical shifts were referenced to the
residual solvent peak observed at δH 7.26 and δC 77.0. High-resolution
mass spectra were carried out on an electrospray QTOF spectrometer
(Applied Biosystems, Model QStar). HPLC isolations were performed
using a Varian model Prostar 210 pump with RI monitoring (Varian
RI-4).
Plant Material. The marine brown alga Cystoseira baccata was
collected by hand at Sidi Bouzid near El Jadida on the Moroccan
Atlantic coast in December 2005 and was identified by two of the
authors (M.B.M. and M.D.). A voucher specimen is available from
G.C. as collection number CB-EJ-12/05.
Alcohol 1a: colorless oil; 1H NMR (CDCl3, 400 MHz) δH 6.56 (1H,
d, J ) 3.0 Hz, H-5′), 6.52 (1H, d, J ) 3.0 Hz, H-3′), 5.33 (1H, t, J )
7.0 Hz, H-2), 3.81 (1H, s, H-12), 3.73 (3H, s, H-4′OMe), 3.33 (2H,
brd, J ) 7.0 Hz, H-1), 3.12 (1H, d, J ) 14.5 Hz, H-4a), 2.92 (1H, d,
J ) 14.5 Hz, H-14a), 2.54 (1H, d, J ) 14.5 Hz, H-4b), 2.22 (3H, s,
H-6′Me), 2.12 (1H, d, J ) 14.5 Hz, H-14b), 2.05 (1H, m, H-10a), 1.95
(1H, d, J ) 17.0 Hz, H-6a), 1.83 (1H, d, J ) 17.0 Hz, H-6b), 1.74
(2H, m, H-9), 1.64 (3H, s, H-20), 1.54 (1H, m, H-8a), 1.39 (1H, m, H
8b), 1.30 (3H, s, H-17), 1.24 (1H, m, H-10b), 1.19 (3H, s, H-16), 0.97
(3H, s, H-19), 0.72 (3H, s, H-18); 13C NMR (CDCl3, 100 MHz) δC
153.1 (C, C-4′), 146.3 (C, C-1′), 135.9 (C, C-3), 135.4 (C, C-5), 129.3
(C, C-13), 127.3 (C, C-2′), 125.0 (C, C-6′), 123.6 (CH, C-2), 114.0
(CH, C-5′), 112.8 (CH, C-3′), 78.0 (CH, C-12), 71.5 (C, C-15), 55.6
(CH3, C-OMe), 47.1 (CH2, C-14), 46.7 (C, C-11), 44.9 (CH2, C-4),
41.1 (CH2, C-6), 40.8 (C, C-7), 36.7 (CH2, C-8), 31.5 (CH3, C-17),
30.0 (CH2, C-10), 29.9 (CH2, C-1), 28.7 (CH3, C-16), 24.8 (CH3, C-19),
22.4 (CH3, C-18), 19.1 (CH2, C-9), 16.2 (CH3, C-6’Me), 15.9 (CH3,
C-20).
Extraction and Purification. The shade-dried material (268 g dry
wt) was ground and extracted with mixtures of CHCl3/MeOH (2:1 then
1:1 and finally 1:2, v/v) at room temperature. The concentrated extracts
were combined and partitioned in a mixture of MeOH/CHCl3/H2O (4:
3:1, v/v/v) to yield, after solvent removal, 6.7 g of a dark brown
lipophilic extract. This extract was subjected to CC on silica gel (Si60,
40-63 µm, Merck) with a solvent gradient from n-hexane to EtOAc
and then from EtOAc to MeOH. The fraction eluting with 50% EtOAc
in n-hexane was further purified by repeated semipreparative HPLC
(Merck Purospher Star RP-18e 5 µm; 10 × 250 mm; 3 mL/min) using
H2O/MeCN (15:85, v/v) to afford five new compounds, 1 (9.8 mg), 3
(3.1 mg), 4 (3.7 mg), 5 (9.1 mg), and 7 (3.4 mg). From the fraction
eluted with EtOAC/n-hexane (60:40, v/v), two new metabolites, 2 (4.6
mg) and 6 (1.2 mg), were also purified by reversed-phase HPLC with
H2O/MeCN (20:80, v/v) as eluent.
Preparation of S- and R-MTPA Ester Derivatives of Compound
1a. Both (S)- and (R)-MTPA esters of 1a (1aS, 1aR) were obtained by
treatment of 1a (1.0 mg) with (R)- and (S)-MTPA chlorides (10 µL) in
dry pyridine (0.5 mL) catalyzed with DMAP and stirred at room
temperature overnight. The MTPA esters were purified using analytical
HPLC (Merck Purospher Star RP-18e 5 µm; 4.6 × 250 mm; 1 mL/
min), with H2O/MeCN (10:90, v/v) as eluent.
Selected signals of 1aS: 1H NMR (CDCl3, 400 MHz) δΗ 1.591 (H-
17), 1.373 (H-16), 0.614 (H-19), 0.462 (H-18). Selected signals of 1aR:
1H NMR (CDCl3, 400 MHz) δΗ 1.510 (H-17), 1.340 (H-16), 0.720
(H-19), 0.628 (H-18). ∆δH(1aS - 1aR): H-17, +0.081 ppm; H-16,
+0.033 ppm; H-19, -0.106 ppm; H-18, -0.166 ppm.
Compound 1: clear yellow oil; [R]25 +83 (c 0.1, MeOH); UV
D
(EtOH) λmax 300 nm (ε 2030), 330 nm (ε 520); IR (film) νmax 3415,
2968, 2931, 2881, 1658, 1604, 1484, 1460, 1379, 1354, 1199, 1144,
1060, 1010 cm-1
;
13C and H NMR (400 MHz, CDCl3), see Tables
1
Reduction of Compound 5 into the Corresponding Alcohol 5a.
A sample of 5 (5 mg) was treated as described above for compound 1.
Usual workup followed by reversed-phase HPLC afforded 5a (2.3 mg).
Alcohol 5a: colorless oil; 1H NMR (CDCl3, 400 MHz) δΗ 3.77 (1H,
brs, H-12), 2.83 (1H, d, J ) 14.5 Hz, H-14a), 2.08 (1H, d, J ) 14.5
Hz, H-14b), 2.05 (1H, d, J ) 17.5 Hz, H-6a), 2.04 (1H, m, H-10a),
1.91 (1H, d, J ) 17.5 Hz, H-6b), 1.76 (2H, m, H-9), 1.57 (1H, m,
H-8a), 1.42 (1H, m, H 8b), 1.66 (3H, s, H-4), 1.32 (3H, s, H-17), 1.27
(1H, m, H-10b), 1.20 (3H, s, H-16), 1.00 (3H, s, H-19), 0.75 (3H, s,
H-18); 13C NMR (CDCl3, 100 MHz) δC 134.5 (C, C-5), 126.9 (C, C-13),
78.5 (CH, C-12), 71.7 (C, C-15), 47.6 (CH2, C-14), 47.0 (C, C-11),
43.9 (CH2, C-6), 41.2 (C, C-7), 36.8 (CH2, C-8), 31.8 (CH3, C-17),
30.1 (CH2, C-10), 28.7 (CH3, C-16), 25.0 (CH3, C-19), 22.5 (CH3,
C-18), 21.4 (CH3, C-4), 19.3 (CH2, C-9).
1,2; HRESIMS m/z 463.2812 [M + Na]+ (calcd for C28H40O4Na,
463.2824).
Compound 2: clear yellow oil; [R]25 +28 (c 0.1, MeOH); UV
D
(EtOH) λmax 300 nm (ε 2290); IR (Film) νmax 3432, 3203, 2950, 2877,
1694, 1482, 1453, 1380, 1331, 1224, 1186, 1139, 1054 cm-1 13C and
;
1H NMR (400 MHz, CDCl3), see Tables 1 and 2; HRESIMS m/z
481.2904 [M + Na]+ (calcd for C28H42O5Na, 481.2930).
Compound 3: clear yellow oil; [R]25 -6 (c 0.1, MeOH); CD
D
(MeOH) λmax ∆ε274 -34.55; UV (EtOH) λmax 290 nm (ε 3070), 330
nm (ε 2240); IR (film) νmax 3421, 2966, 2927, 1657, 1601, 1469, 1377,
1193, 1143, 1059 cm-1 13C and 1H NMR (400 MHz, CDCl3), see
;
Tables 1 and 2; HRESIMS m/z 479.2767 [M + Na]+ (calcd for
C28H40O5Na, 479.2773).
Compound 4: clear yellow oil; [R]25 +65 (c 0.1, MeOH); CD
Preparation of S- and R-MTPA Ester Derivatives of Compound
5a. A solution of pure compound (2.3 mg) was divided into two parts,
then transferred into clean NMR tubes and dried under the stream of
N2 gas. Deuterated pyridine (0.6 mL) and (S)-(-)-MTPACl (4 µL) or
(R)-(+)-MTPACl (4 µL) were added successively to the NMR tube
under a N2 gas stream, and the tube was carefully shaken to mix the
sample and MTPA chloride evenly. The reaction in the NMR tubes
D
(MeOH) λmax ∆ε274 +2.50; UV (EtOH) λmax 275 nm (ε 1740), 330 nm
(ε 970); IR (film) νmax 3439, 2965, 2926, 1656, 1602, 1468, 1376, 1196,
1146, 1060 cm-1
;
13C and H NMR (400 MHz, CDCl3), see Tables 1
1
and 2; HRESIMS m/z 479.2735 [M + Na]+ (calcd for C28H40O5Na,
479.2773).
Compound 5: clear yellow oil; [R]25 +53 (c 0.1, MeOH); UV
D
1
was monitored by H NMR and was completed in approximately 6 h
(EtOH) λmax 280 nm (ε 1070); IR (film) νmax 3428, 2966, 2930, 2879,
1652, 1612, 1450, 1378, 1150 cm-1 13C and 1H NMR (400 MHz,
;
for the (S)-MTPA ester 5aS and 48 h for the (R)-MTPA ester 5aR.
Selected signals of 5aS: 1H NMR (C5D5N, 400 MHz) δΗ 1.711 (H-
4), 1.694 (H-17), 1.670 (H-16), 1.206 (H-19), 0.715 (H-18). Selected
CDCl3), see Tables 1 and 2; HRESIMS m/z 273.1822 [M + Na]+ (calcd
for C16H26O2Na, 273.1830).
1
Compound 6: clear yellow oil; [R]25D +7 (c 0.1, MeOH); IR (film)
νmax 3402, 2970, 2923, 1718, 1680, 1608, 1456, 1378, 1314, 1126,
signals of 5aR: H NMR (C5D5N, 400 MHz) δΗ 1.706 (H-2), 1.496
(H-17), 1.423 (H-16), 1.279 (H-19), 0.812 (H-18). ∆δH(5aS - 5aR):
H-4, +0.005 ppm; H-17, +0.198 ppm; H-16, +0.247 ppm; H-19,
-0.073 ppm; H-18, -0.097 ppm.
1038 cm-1 13C and 1H NMR (400 MHz, CDCl3), see Tables 1 and 2;
;
HRESIMS m/z 315.1919 [M + Na]+ (calcd for C18H28O3Na, 315.1936).
Compound 7: clear yellow oil; [R]25 -11 (c 0.1, MeOH); UV
Biological Assays. Biological assays of the compounds were
performed toward bacteria (marine and terrestrial), microalgae, mac-
roalgae, and marine invertebrates. Toxicity was checked against larvae
of sea urchins and oysters. All the compounds were tested on a range
of concentrations from 0.5 to 100 µg/mL (0.5, 1, 2.5, 5, 10, 25, 50,
and 100 µg/mL). Each experiment and controls were run in six replicates
and using two batches of organisms.
D
(EtOH) λmax 350 nm (ε 920), 270 nm (ε 350); IR (film) νmax 3433,
2966, 2931, 2879, 1715, 1684, 1607, 1547, 1449, 1376, 1332, 1277,
1258, 1181, 1130 cm-1 13C and 1H NMR (400 MHz, CDCl3), see
;
Tables 1 and 2; HRESIMS m/z 297.1807 [M + Na]+ (calcd for
C18H26O2Na, 297.1830).
Reduction of Compound 1 into the Corresponding Alcohol 1a.
Reduction of 1 (5 mg) was conducted in MeOH (10 mL) with an excess
of NaBH4 (10 mg), and the solution was then allowed to stand overnight
Antibacterial Assays. The compounds were tested for inhibitory
activities against three strains of marine bacteria [Pseudoalteromonas

Meroditerpenoids from Cystoseira baccata
Journal of Natural Products, 2008, Vol. 71, No. 11 1811
elyakoVii (ATCC 700519), Vibrio aestuarianus (ATCC 35048), and
Polaribacter irgensii (ATCC 700398)] and three strains of terrestrial
bacteria [Salmonella typhimurium (ATCC 35986), Escherichia coli
(ATCC 35049), and Bacillus subtilis (ATCC 55572)]. The experiments
were performed as previously described by Mare´chal et al.17 Com-
pounds were incubated at 25 °C for 48 h 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) for marine bacteria
and in TSM medium (trypsic soy broth, Sigma) for terrestrial bacteria.
Minimum inhibitory concentrations (MICs), compared to the control
(culture media), were determined by the microtiter broth dilution
method.18
Antimicroalgal Assays. The compounds were screened for antimi-
croalgal activities against three microalgae: Exanthemachrysis gayraliae
(AC 15), Cylindrotheca closterium (AC 170), and Pleurochrysis
roscoffensis (AC 238). Compounds were incubated (at 18 °C, for 48 h
under a light regime of 15:9 light:dark using 54 µmol photons/m2 ·s
cool-white fluorescent lamp) with the algae (0.4 µg/mL of chlorophyll
A) in F/2 medium19 as outlined by Plouguerne et al.20 After 48 h, the
relative optical density of the sample suspension was determined at
600 nm. Minimum inhibitory concentrations (MICs) compared to the
seawater control were determined by the method of Tsoukatou et al.21
Antimacroalgal Assays. The compounds were screened for anti-
macroalgal activities against two species: Sargassum muticum and UlVa
intestinalis. Compounds were incubated (at 18 °C, for 48 h under a
light regime of 15:9 light:dark using 54 µmol photons/m2 ·s cool-white
fluorescent lamp) with the algae (3000 spores/mL) in F/2 medium19 as
outlined by Hellio et al.22 After 48 h, the germination and attachment
rates were determined as stated by Hellio et al.22
Anti-invertebrate Assays. Compounds were tested for their inhibi-
tory activity of the phenoloxidase (PO) purified from Mytilus edulis
following the method of Hellio et al.23 Aliquots of pure enzyme were
incubated for 2 h with the test compounds. Then, the PO activity was
followed by monitoring the increase of absorbance at 475 nm using
L-Dopa (10 mM) as substrate. In comparison with the control,
percentages of inhibition of PO activity were determined for the test
compounds.24
Toxicity Tests. In order to determine the toxicity, larvae of sea
urchins (Echinus esculentus) and oysters (Crassostrea gigas) were
incubated with the compounds according to the procedures of Hellio
et al.25 Ten to fifteen larvae were added to 2 mL of solution in the
wells of a 24-well (Iwaki) plate. After 48 h exposure, the number of
viable larvae was recorded. The data are expressed as a 24 h LC50 ()
concentration of extract that produces 50% mortality in comparison
with the control), which was determined using Sigma Plot 8.0.
the editor and the anonymous referees for their valuable comments on
the manuscript.
Supporting Information Available: Tables T1-T7 with complete
NMR data together with all 1D and 2D NMR spectra of compounds
1-7. This material is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
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(17) Mare´chal, J.-P.; Culioli, G.; Hellio, C.; Thomas-Guyon, H.; Callow,
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(18) Amsterdam, D. In Antibiotics in Laboratory Medicine; Lorian, V.,
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(19) Guillard, R. R.; Ryther, J. H. Can. J. Microbiol. 1962, 8, 229–239.
(20) Plouguerne´, E.; Hellio, C.; Deslandes, E.; Ve´ron, B.; Stiger-Pouvreau,
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(21) Tsoukatou, M.; Hellio, C.; Vagias, C.; Harvala, C.; Roussis, V. Z.
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Acknowledgment. The authors are grateful to O. Thomas and M.
Mehiri (LCMBA, Universite´ de Nice Sophia-Antipolis, France) for CD
analysis. The authors express their gratitude to R. Valls (Universite´
Paul Ce´zanne, Marseilles, France) for having initiated this work and
for his invaluable scientific support. We would also like to acknowledge
(24) Zentz, F.; Hellio, C.; Valla, A.; De La Broise, D.; Bremer, G.; Labia,
R. Mar. Biotechnol. 2002, 4, 431–440.
(25) Hellio, C.; Bremer, G.; Pons, A. M.; Gal, Y. L.; Bourgougnon, N.
Appl. Microbiol. Biot. 2000, 54, 543–549.
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