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New Sesquiterpenes from the Red Alga Laurencia microcladia

Article in Tetrahedron · August 2007

DOI: 10.1016/j.tet.2007.05.051

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 Tetrahedron 63 (2007) 7606–7611

New sesquiterpenes from the red alga Laurencia microcladia

Maria Kladi,a Constantinos Vagias,a Panagiota Papazafiri,b Giovanni Furnari,c
Donatella Serioc and Vassilios Roussisa,*
a
Division of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Athens,
Panepistimiopolis Zografou, Athens 15771, Greece
b
Department of Animal and Human Physiology, School of Sciences, University of Athens, Panepistimiopolis Zografou,
Athens 15784, Greece
c
Dipartimento di Botanica dell, Universit
a di Catania,Via A. Longo, 19, 95125 Catania, Italy
Received 8 March 2007; revised 24 April 2007; accepted 10 May 2007
Available online 17 May 2007

Abstract—Three new aromatic sesquiterpenes (1, 2, and 4), one new dimeric sesquiterpene of the cyclolaurane-type (3), one sesquiterpene
alcohol of bisabolene type (8) along with three previously reported metabolites (5–7), were isolated from the organic extracts of Laurencia
microcladia, collected from the Chios island in the North Aegean Sea. The structures of the new natural products, as well as their relative
stereochemistries, were established by means of spectral data analyses, including 2D experiments. The cytotoxicity of the isolated metabolites
was evaluated against five human tumor cell lines.
Ó 2007 Elsevier Ltd. All rights reserved.

1. Introduction During the course of our ongoing investigations toward the

isolation and biological evaluation of compounds from ma-
Red algae of the genus Laurencia (Ceramiales, Rhodomela- rine organisms of the Greek seas,14–16 we studied specimens
ceae) are unique for their ability to biosynthesize a wide of Laurencia microcladia K€utzing, collected off the coasts of
variety of secondary metabolites with diverse structural fea- Chios island. In this report we describe the isolation and
tures depending on the species and localities.1 The chemistry structure elucidation of five new metabolites (1–4 and 8)
of halogenated compounds from Laurencia is a very interest- along with the known metabolites dibromophenol (5), (+)-
ing area of research and it never fails to offer the possibility a-isobromocuparene (6), and ()-a-bromocuparene (7), all
of discovering new compounds with novel structures and of which were obtained from the non-polar fractions of the
properties.2 The vast majority of Laurencia metabolites, so organic extracts of L. microcladia.
far, includes sesquiterpenes,3,4 diterpenes,5 triterpenes,6
and C15-acetogenins.7 Most species of Laurencia biosyn- An assessment of their cytotoxicity was performed on the
thesize a characteristic major metabolite or a class of com- following human tumor cell lines: HT29 (derived from colo-
pounds that are not commonly widely distributed within rectal adenocarcinoma), MCF7 (derived from a mammary
the genus.8 Chemical studies based on cultured and field- adenocarcinoma), PC3 (derived from a prostate adenocarci-
collected materials of several species have revealed that noma), HeLa (derived from cervix adenocarcinoma), and
the synthesis of halogenated secondary metabolites is not A431 (derived from epidermoid carcinoma).17
affected by environmental factors.9 Thus, secondary meta-
bolite chemistry can serve as an important tool for taxonom-
ical studies in the genus Laurencia especially since many 2. Results and discussion
species appear morphologically very similar.10 Moreover
a number of halogenated metabolites have been shown to L. microcladia was collected from the island of Chios and
possess antibacterial, antifungal,11 insecticidal12 activities, the CH2Cl2/MeOH extract of the freeze-dried alga was sub-
as well as worth noting cytotoxicity against mammalian jected to a series of vacuum column chromatography (VCC)
cells.13 on silica gel and normal phase high pressure liquid chroma-
tography (HPLC), using mixtures of cyclohexane/EtOAc as
the mobile phase, to yield compounds 1–8 in pure form.
Keywords: Laurencia microcladia; Sesquiterpenes; Cytotoxic activity.
* Corresponding author. Tel./fax: +30 210 7274592; e-mail: roussis@ Compound 1, after HPLC purification, was isolated as color-
pharm.uoa.gr less oil. The molecular formula C15H18OBrI was deduced

0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.05.051
 M. Kladi et al. / Tetrahedron 63 (2007) 7606–7611 7607

from HRFABMS data in combination with the NMR spectra

(a) (m); 1.82 (b) (m)

(Tables 1 and 2). The LREIMS peaks at m/z 420/422 [M]+,
with relative intensities 1/0.9 79Br/81Br, indicated the pres-
ence of one bromine atom. The intense absorption at vmax
3424 cm1 showed the presence of a hydroxyl functionality

(b) (br s)
(a) (br s)
in the molecule. The 13C NMR and DEPT experiments

(a) (m);
(b) (m)
allowed the determination of seven quaternary, two methine,

(br s)
three methylene, and three methyl carbon atoms. The 1H and

(m)

(m)

(m)
(m)
(s)

1.67 (s)
1.60 (s)
13
C NMR spectra displayed resonances for one aromatic

1.67

4.86
5.13
(dd, 9.9, 8.9) 5.31
2.03
2.31

1.72
2.16

2.12
2.13
5.13
—

—
methyl (dH/C 2.61/29.8), two quaternary methyls (dH/C

8
1.28/18.7 and dH/C 1.37/22.4), one aromatic proton (dH/C
7.70/132.4), one methine (dH/C 1.09/24.2), cyclopropyl pro-

(a) (m);
(b) (m)

(b) (m)
(a) (m)
tons (dH/C 0.48, 0.52/16.2), and two methylenes (dH/C 1.61,
1.92/25.2 and dH/C 2.20, 1.19/35.1). All protonated carbons

(s)
(s)

(s)
(s)
(s)
(s)

1.40 (s)
2.29 (s)
and their protons were assigned by the COSY and HMQC

4.04
2.18
2.49
1.94
2.26

7.07
7.07

7.07
7.07
0.59
1.06
—
—

—
experiments. The structure elucidation was assisted by anal-

7
yses of the HMBC experiments. The correlation in the

2.18 (a) (m); 2.50

1.58 (a) (m); 2.69

4.44 (dd, 9.2, 9.2)
HMBC experiments, between H-14 (dH 1.37) with C-1 (dC
49.1), C-5 (dC 35.1) and the aromatic carbon C-6 (dC

7.25 (d, 7.2)

7.11 (d, 8.2)

(d, 8.2)
(d, 7.2)
134.4) confirmed the position of H-14 on C-1. The correla-

(b) (m)

(b) (m)

(s)
(s)

1.27 (s)
2.32 (s)
tion of signals at dH 1.28 (H-12) with 29.6 (C-2), 24.2

7.11
7.25
0.63
1.08
(C-3), and 16.2 (C-13) secured the position of H-12 on C-2.

—
—

—
6
Comparison of the NMR data of 1 with reported values for
laurinterol,18,19 led to the assignment of the structure as

(b) (dddd, 12.0, 12.0, 7.5, 4.1)

1.61 (a) (dd, 12.0, 7.9); 1.92
the iodo-laurinterol. The shift of the aromatic methyl group

1.22 (a) (m); 2.21 (b)

(d 2.61/29.8) in lower fields compared to laurinterol (d 2.27/

0.47 (dd, 7.8, 4.9); 0.51

1.08 (ddd, 7.8, 4.9, 3.0)
22.2) supported the position of the iodine on C-8. Further-

All spectra were recorded in CDCl3. Chemical shifts are expressed in parts per million. J values in parentheses are in hertz.
more, an HMBC correlation between H-11 with C-1 (dC
49.1) and C-6 (dC 134.4) confirmed the relative positions

(dd, 13.4, 7.5)

(dd, 4.9, 3.0)

of the substituents on the aromatic ring. The strong NOE cor-
H (d)

relations between H-12/H-14 with H-5b and H-3 with H-4b

7.68 (s)
1.28 (s)

1.38 (s)
2.51 (s)
5.76 (s)
determined the relative stereochemistry at C-1, C-2, and
1

—
—

—
—
—
—

—
C-3. Consideration of the above data led to the determination
5

of the structure as 8-iodo-laurinterol. As far as we know, this

1.23 (a) (m); 2.17 (b)
1.09 (ddd, 7.7, 4.7, 4.0)

is only the third iodinated sesquiterpene from the genus

Laurencia, besides 10-bromo-7-hydroxy-11-iodolaurene
and a related iodo ether A that have been isolated from
1.61 (a) (m);
1.92 (b) (m)

(dd, 13.5, 8.0)

Laurencia nana Howe.20

0.54 (m)
7.80 (s)
1.31 (s)

1.39 (s)
1.95 (s)
4.81 (s)
Compound 2, a bromo sesquiterpene ether, was purified by
means of HPLC and was also isolated as colorless oil. Com-
—
—

—
—
—
—

—
3

bination of its 13C NMR data and HRFABMS measurements

(a) (d, 10.4);
(b) (d, 10.4)

suggested a molecular formula of C15H17OBr3. The

LREIMS peaks at m/z 450/452/454/456 [M]+, with relative
(a) (m);
(b) (m)

(b) (m)
(a) (m)

(d, 7.1)

intensities 1.0/3.0/2.9/0.9, indicated the presence of three

Table 1. 1H NMR data (400 MHz) of compounds 1–3 and 5–8

bromine atoms. The IR absorption at 1240 cm1 and the ab-

(m)

(s)

(s)
(s)

sence of an absorption band for hydroxyl or carbonyl groups

1.86

2.02
2.17
(a) (m); 2.20 (b) (dd, 13.7, 8.2) 1.69
1.89

7.20
0.72
3.52
3.71
1.35
2.50
—

—
—
—
—

indicated that the oxygen atom was involved in an ether link-

2

age. The 13C NMR spectrum of 2 (Table 2) exhibited signals

for 15 carbons with the multiplicities of the carbon signals
(b) (dddd, 12.2, 11.7, 8.2, 4.4)

determined from the DEPT spectrum as: seven quaternary,

two methine, three methylene and three methyl carbon
atoms. The 1H and 13C NMR spectra displayed resonances
(a) (dd, 12.2, 8.3);

for one aromatic methyl (dH/C 2.50/23.2), one quaternary

(a) (dd, 7.8, 4.9);
(ddd, 7.8, 4.9, 3.9)

(b) (dd, 4.9, 3.9)

methyl (dH/C 1.35/20.1), one secondary methyl (dH/C 0.72/

6.7), one aromatic proton (dH/C 7.20/126.9), and two protons
on carbon bearing bromine at dH/C 3.52, 3.71/34.8. With six
degrees of unsaturation, the structure was suggested to
contain besides the aromatic ring, two other rings, one of
(s)
(s)

(s)
(s)
(s)

which incorporates the ether linkage and the other a five-

1.09
1.61
1.92
1.19

7.70
1.28
0.48
0.52
1.37
2.61
–OH 5.59
—
—

—
—
—
—

membered carbocyclic ring. The 1H and 13C NMR data of

1

the compound 2 were similar to the reported values for the

No

10
11
12
13

14
15

known metabolite bromoether A, isolated previously from

1
2
3
4

6
7
8
9
7608 M. Kladi et al. / Tetrahedron 63 (2007) 7606–7611

Laurencia glandulifera.20,21 On the basis of the absence of

a second aromatic proton and the molecular formula,
compound 2 was suggested to be the bromo analogue of

H-14, H-15
H-14, H-15
HMBC bromoether A. The chemical shift of the aromatic methyl
group resonating at d 2.50/23.2, at lower field compared to

H-10

H-14
H-1
H-1
H-1
H-9

H-9
bromoether A (d 2.27/22.5) and the HMBC correlation be-
8

tween H-11 (d 7.20) and C-1 (d 45.3) supported the position

of the bromine atom on C-8. The correlation of signals at
C (d)

d 0.72 (H-12) with 45.3 (C-1), 43.3 (C-2), and 87.1 (C-3)
23.3

37.7
72.4
32.3
27.3

30.9
27.5

25.7
17.9
133.8
118.4

154.0
108.3

124.3
131.0
as well as that at d 1.35 (H-14) with 45.3 (C-1), 43.3
13

(C-2), 36.8 (C-4), 41.1 (C-5), and 129.9 (C-6) confirmed

the position of the methyl groups H-12 and H-14 on C-2 and
C-1, respectively. The relative stereochemistry of 2 was as-
C (d)

143.9
127.4
128.2

127.4
48.4
47.4
62.1

135.5
128.2
33.1
36.5

22.4
20.9
25.1
20.8
signed on the basis of NOESY experiments. In view of the
13
7

above-mentioned data, the proposed structure for metabolite

2 is shown in Figure 1.
C (d)
48.4
47.7
63.6
31.2
33.5

22.2
20.8
25.3
20.6
143.0
126.5
128.6
135.4
128.6
126.5

Compound 3 was purified by means of HPLC separation and

was isolated as colorless oil. Both 13C NMR data and
13
6

HRFABMS measurements supported the molecular formula

of C30H36O2Br2. The LREIMS showed [M]+ peaks at m/z
C (d)

586/588/590 with intensities 1.0/2.0/1.0, indicating the pres-

135.1
150.1
116.0
134.3
113.4
131.1
49.0
29.5
24.2
25.2
35.0

18.6
16.1
22.3
24.1

ence of two bromine atoms. The presence of a hydroxyl

13
5

group was evident from the IR band at 3538 and

3409 cm1. The 13C NMR spectrum of 3 (Table 2) exhibited
H-4a, H-12, H-14

H-11, H-15, –OH

signals for 15 carbons, with the multiplicities of the carbon

signals determined from the DEPT spectrum as: seven qua-
H-5b, H-12
H-11, H-14

H-11, H-14

H-11, H-15

ternary, two methine, three methylene, and three methyl

carbon atoms. The 1H and 13C NMR spectra displayed reso-
HMBC

H-14

H-11
H-15

H-12

nances for one aromatic proton (dH/C 7.80/132.7), one aro-

3

matic methyl group (dH/C 1.95/19.3), two tertiary methyl

groups (dH/C 1.31/17.8 and 1.39/21.8), cyclopropyl protons
(dH/C 0.54/15.6), one methine (dH/C 1.09/23.3), two methyl-
C (d)
48.1
29.3
23.3
24.3
34.6

17.8
15.6
21.8
19.3
135.0
151.8
121.5
134.5
115.4
132.7

enes (dH/C 1.92, 1.61/24.3 and 1.23, 2.17/34.6), one ex-

13

changeable proton (dH 4.81), and six aromatic carbons (dC

115.4, 121.5, 132.7, 134.5, 135.0, 151.8). The spectral data
of 3 were similar to the known dimeric sesquiterpene of
H-2, H-11, H-12, H-14

the cyclolaurane type, isolated previously from L. microcla-

All spectra were recorded in CDCl3. Chemical shifts are expressed in parts per million.

dia.17 The correlations observed in the HMBC spectrum

between H-11 (dH 7.80) with C-1 (dC 48.1), C-6 (dC
135.0), C-10 (dC 115.4), and C-7 (dC 151.8), supported the
H-12, H-14

H-11, H-15
H-11, H-15
Table 2. 13C NMR (50.3 MHz) and HMBC data of compounds 1–3 and 5–8

fusion of the phenolic rings of the two laurinterol molecules,

HMBC

H-12
H-14
H-14
H-14
H-11

H-15
2

which is ortho to the methyl. The absence of a NOE effect

between the methyls H-12 and H-14 (in contrast to the
known dimeric sesquiterpene), along with the NOE effect
between H-12 and H-5b, suggested an inversion of configu-
C (d)

ration at C-1. In view of the above-mentioned data, the pro-

45.3
43.3
87.1
36.8
41.1

34.8
20.1
23.2
6.7
129.9
148.6
114.9
136.0
112.4
126.9

posed structure for metabolite 3 is shown in Figure 1.

13

Compound 4 was also purified by means of HPLC separation

H-4a, H-12, H-14
H-5b, H-12, H-14

and was isolated as colorless oil. The compound was shown

to be a hydrocarbon with the molecular formula C15H20 by
H-5b, H-12

LREIMS at m/z 200 [M]+. The 1H NMR spectrum exhibited

H-11, H-15
OH-C-7

two 2H doublets at d 7.06 (d, J¼7.9 Hz, 2H) and d 7.25 (d,
HMBC

H-14
H-14

H-15

H-15

H-12

J¼7.9 Hz, 2H), indicating the presence of a 1,4-disubstituted

1

benzene ring, together with an aromatic methyl resonance at

d 2.29 (s, 3H). The remaining observable resonances con-
sisted of two exocyclic methylene protons at d 4.80 and
C (d)
49.1
29.6
24.2
25.2
35.1

96.5

18.7
16.2
22.4
29.8
134.4
159.6

137.5
113.3
132.4

5.06 (s, 1H each), a quaternary methyl group at d 1.42 (s,

3H), and a secondary methyl group at d 1.12 (d, J¼7.0 Hz,
13

3H) coupled to a deshielded allylic proton at d 2.65 (dtt,

J¼8.5, 7.0, 2.9 Hz, 1H). With an unsaturation degree of
No

10
11
12
13
14
15

six, the structure was suggested to contain one aromatic

1
2
3
4
5
6
7
8
9
 M. Kladi et al. / Tetrahedron 63 (2007) 7606–7611 7609

Br Br Br 14
5 4
14 14 10 116 1
2 3 14
5 4
10 11 5 10 11 5 9 10 11
9 6 4 9 6 4 15 8 7 6
15 1 15 1 13 15 9 1
8 7 2
3
8 7 2 3 12 8 7 2 3
H OH
12
13 Br OH 13
I OH 12
13 Br 12
O Br

1 2 3 4
Br 9

14 14 14 OH
10 11 5 4 10 11 5 1011 5 4
15 9 6 1 4 8 4
15 9 6 1 15 9 6 1 10 3
8 7 2 3 8 7 2 3 8 7 2
3 5
Br Br 6 2
11 12 7
Br OH 12 13 12
H 12 H 1
13 13

13
14 15
5 6 7 8

Figure 1. Metabolites isolated from L. microcladia.

ring, one five-membered ring, and an exocyclic methylene. were olefinic resonating at d 154.0, 133.8, 131.0, 124.3,
The protons of 4 were assigned by 1H–1H COSY experi- 118.4, and 108.3 ppm, indicating the presence of three
ments and the position of H-13 was determined from corre- double bonds. In addition, the 1H NMR spectrum displayed
lations between H-13/H-3, H-13/H-4a, and H-13/H-4b. The a pair of singlets characteristic for an exocyclic methylene at
isolation of metabolite 4 in minute amounts as well as its dH 4.86 (br s, H-9a) and 5.13 (br s, H-9b), two olefinic pro-
instability, made impossible 13C NMR measurements. Con- tons at dH 5.31 (m, H-3) and 5.13 (br s, H-12), as well as
sidering the above-mentioned data, the proposed structure three vinyl methyl groups at dH 1.60 (3H, s) and 1.67 (6H,
for metabolite 4 is shown in Figure 1. In the past, compound s). With an unsaturation degree of four, the structure was
4 has been mentioned as an intermediate for the synthesis of suggested to contain, besides the three double bonds, one
the known metabolite laurene.22 ring. All protonated carbons and their protons were matched
precisely by COSY and HMQC experiments. Comparison of
Compound 5 after purification by HPLC was isolated as col- the spectral data (Tables 1 and 2) for 8 with literature
orless oil and identified by comparison of its 1H NMR and values,25,26 suggested that metabolite 8 possessed the skele-
MS spectra with previously reported data as being dibromo- ton of b-bisabolene. The main difference was the presence
phenol.23 Extensive analyses of the 13C NMR, HMQC, and of an hydroxyl group attached to a quaternary carbon. The
HMBC spectra allowed 13C NMR assignments for the dibro- position of the hydroxyl group at C-5 was confirmed by
mophenol, and this is the first report of metabolite 5 from the HMBC correlation of the protons of the exocyclic meth-
L. microcladia. ylene H-9 (dH 4.86, 5.13) and C-5 (dC 72.4). Moreover the
correlations between H-1/C-2, H-1/C-3, H-1/C-4 and H-
Compound 6 was purified following chromatographic sepa- 14,H-15/C12,C-13 confirmed the positions of the methyl
rations and was isolated as colorless oil. It was identified on groups. In view of the above mentioned data, metabolite 8
the basis of its 1H NMR and MS spectra and comparison is the hydroxyl derivative of b-bisabolene.
with previously reported data as being (+)-a-isobromo-
cuparene.24 Extensive analyses of the 13C NMR, HMQC, Metabolites 1–3 and 5–7 were evaluated for their cyto-
and HMBC spectra allowed the 13C NMR assignments for toxicity against five human tumor cell lines. The results of
compound 6, reported for the first time as L. microcladia the cytotoxic activity of the tested compounds after 48 h of
metabolite. incubation are given in Table 3. All compounds were found
to be deprived of significant cytotoxic activity. Compounds 1
Compound 7 was isolated as colorless oil following HPLC and 5 exhibited relatively higher cytotoxicity against all
purification and was identified by comparison of its 1H tested cell lines. In combination with results obtained in
NMR and MS spectra and with literature values as being the past with similar metabolites it seems that the presence
()-a-bromocuparene.24 Extensive analyses of the 13C of the aromatic hydroxyl group is increasing the cytotoxicity
NMR, HMQC, and HMBC spectra allowed the 13C NMR
assignments for compound 7, reported for the first time as Table 3. In vitro cytotoxic activities of metabolites 1–3 and 5–7
L. microcladia metabolite.
Compound Cell line
Compound 8 was purified by HPLC and was isolated as col- HT29 MCF7 PC3 HeLa A431
orless oil. The molecular formula for 8 was established as IC50 (mM)
C15H24O by combinations of HRFABMS and the NMR
experiments. The IR spectrum displayed absorptions for 1 78.4 86.3 88.5 81.4 92.7
2 170.5 167.9 183.9 174.4 178.8
a tertiary hydroxyl group (3409 cm1, broad) and an exo- 3 >300 >300 >300 >300 >300
methylene group (3010 and 1654 cm1). The 13C NMR 5 98.7 104.1 75.2 114.6 93.4
spectrum along with the DEPT experiments showed the 6 130.4 177.6 191.2 204.3 198.4
presence of four quaternary, two methine, six methylene, 7 287.3 265.4 271.3 240.8 277.4
and three methyl carbon atoms. Among the carbons, one For clarity, SEM values are not included in the table, but they were less than
was bonded to oxygen resonating at d 72.4 ppm and six 5% of the mean in all the cases.
7610 M. Kladi et al. / Tetrahedron 63 (2007) 7606–7611

on the tested cell lines, whereas the significantly more bulky pure compound 3 (0.7 mg). Fraction VI (5% EtOAc)
dimer appears deprived of any cytotoxicity. Based on these (53.6 mg) was subjected to normal phase HPLC chromato-
preliminary results a structure–activity study might help in graphy, using n-hexane/EtOAc (96/4) as mobile phase to
the future synthesis of related compounds possessing selec- yield pure compound 8 (0.8 mg).
tivity to tumor cells.
3.3.1. Compound 1. Colorless oil; [a]20 D 20 (c 0.04,
3. Experimental CH2Cl2); UV lCH max
2 Cl2
(log 3): 242.6 (3.1), 263.0 (2.6),
292.0 (2.8), 303.4 (2.6) (nm); IR (CH2Cl2) nmax 3424,
3.1. General experimental procedures 3020, 1030, 1630, 1470 cm1; HRFABMS (m/z): 419.9611
[M]+ (calcd for C15H18O79BrI 419.9586), Dm/m¼59
Optical rotations were measured using a Perkin–Elmer (ppm); NMR data (CDCl3), see Tables 1 and 2; EIMS
model 341 polarimeter and a 10-cm cell. UV spectra were 70 eV, m/z (rel int. %): 420/422 [M]+ (20/19), 405/407
determined in spectroscopic grade CH2Cl2 and CHCl3 on [MCH3]+ (44/40), 352/354 (65/60), 325/327 (14/15), 300
a Shimadzu UV-160A spectrophotometer. IR spectra were (31), 173 (88), 128 (77), 115 (100), 77 (76), 41 (66).
obtained using a Paragon 500 Perkin–Elmer spectrophoto-
meter. NMR spectra were recorded using a Bruker AC 200 3.3.2. Compound 2. Colorless oil; [a]20 D +33 (c 0.09,
and Bruker DRX 400 spectrometers. Chemical shifts are CH2Cl2); UV lCH 2 Cl2
max (log 3): 234 (2.9), 270 (2.1), 295.4
given on a d (ppm) scale using TMS as an internal standard. (2.3) (nm); IR (CH2Cl2) nmax 1634, 1565, 1381, 1240,
The 2D experiments (1H–1H COSY, HMQC, HMBC, 1169, 1041 cm1; HRFABMS (m/z): 449.8835 [MH]+
NOESY) were performed using standard Bruker micropro- (calcd for C15H17O79Br3 449.8829), Dm/m¼13 (ppm);
grams. High-resolution mass spectral data were provided NMR data (CDCl3), see Tables 1 and 2; EIMS 70 eV, m/z
by the University of Notre Dame, Department of Chemistry (rel int. %): 450/452/454/456 [M]+ (33/100/97/30), 371/
and Biochemistry, Notre Dame, Indiana, USA. EIMS data 373/375 [MBr]+ (42/77/40), 315/317/319 (40/75/40),
were recorded on a Hewlett Packard 5973 Mass Selective 292/294 (64/61), 279 (70), 251 (21), 238 (20), 197 (17),
Detector. VCC separation was performed with Kieselgel 128 (25), 107 (77), 55 (60).
60H (Merck), and TLC was performed with Kieselgel 60
F254 aluminum support plates (Merck) and spots were de- 3.3.3. Compound 3. Colorless oil; [a]20 D 10.0 (c 0.04,
tected with 15% H2SO4 in MeOH reagent. HPLC separation CH2Cl2); UV lCH 2 Cl2
max (log 3): 240.0 (3.5), 280 (3.2) (nm); IR
was conducted using a Pharmacia LKB 2248 model equip- (CH2Cl2) nmax 3538, 3409, 2955, 2866, 1706, 1605, 1493,
ped with a refractive index detector RI GBC LC-1240 and 1448, 1392, 1380, 1268, 1066, 1042, 848 cm1; HRFABMS
a Spherisorb HPLC normal phase column, 25 cm10 mm, (m/z): 586.1087 [M]+ (calcd for C30H36O279Br2 586.1084),
S10W, 64340 plates/meter. Dm/m¼5 (ppm); NMR data (CDCl3), see Tables 1 and 2;
EIMS 70 eV, m/z (rel int. %): 586/588/590 [M]+ (29/62/31),
3.2. Plant material 571/573/575 (31/64/33), 518/520/522 (53/100/51), 503/505/
507 (29/60/35), 450/452/454 (14/27/15), 294/296 (17/16),
L. microcladia was collected by hand at Vroulidia bay, Chios 279 (28), 109 (41), 67 (32).
island in the North Aegean Sea, Greece, at a depth of 0.5–
1 m in May of 2002. A specimen is kept at the Herbarium 3.3.4. Compound 4. Colorless oil; [a]20 D +40.0 (c 0.04,
of the Laboratory of Pharmacognosy and Chemistry of CHCl3); UV lCHCl
max
3
(log 3): 292.6 (1.6) (nm); IR (CHCl3)
Natural Products, University of Athens (ATPH/MO/151). nmax 2960, 2927, 2869, 1653, 1512, 1451, 1370,
1143 cm1; 1H NMR data (CDCl3): 7.06 (d, J¼7.9 Hz,
3.3. Extraction and isolation 2H), 7.25 (d, J¼7.9 Hz, 2H), 2.29 (s, 3H, H-15), 4.80 and
5.06 (s, 1H each, H-12a and H-12b), 1.42 (s, 3H, H-14),
L. microcladia was initially freeze-dried (291.4 g dry 1.12 (d, J¼7.0 Hz, 3H, H-13), 2.65 (dtt, J¼8.5, 7.0,
weight) and then exhaustively extracted with mixtures of 2.9 Hz, 1H, H-3), 2.11 (m, 2H, H-4a, H-5a), 1.80 (m, 1H,
CH2Cl2/MeOH (3/1) at room temperature. The extract was H-4b), 1.66 (m, 1H, H-5b); EIMS 70 eV, m/z (rel int. %):
concentrated to give a dark green residue (12.0 g), which 200 [M]+ (63), 185 (75), 171 (13), 158 (41), 143 (100),
later was subjected to vacuum column chromatography 129 (28), 115 (22), 105 (31), 91 (19), 77 (13).
(VCC) on silica gel, using cyclohexane with increasing
amounts (10%) of EtOAc as mobile phase and finally 3.3.5. Compound 5. Colorless oil; [a]20 D +10.0 (c 0.04,
MeOH. Fraction A (100% cyclohexane) (307.8 mg) was CHCl3); UV lCHCl
max
3
(log 3): 292.4 (2.3) (nm); IR (CHCl3)
subjected to normal phase HPLC chromatography, using nmax 3485, 3393, 1651, 1616, 1558, 1385, 1288, 1155,
100% n-hexane as mobile phase to yield pure compounds 1022 cm1; NMR data (CDCl3), see Tables 1 and 2; EIMS
1 (1.2 mg), 4 (0.4 mg), and 5 (1.5 mg). Fraction C (20% 70 eV, m/z (rel int. %): 372/374/376 [M]+ (31/62/32), 357/
EtOAc in cyclohexane) (27.3 mg) was subjected to normal 359/361 (51/100/55), 315/317/319 (15/32/18), 304/306/
phase HPLC chromatography, using 100% n-hexane as 308 (45/85/45), 252/254 (26/23), 212/214 (12/12), 199
mobile phase to yield pure compounds 2 (0.9 mg), 6 (15), 173 (31), 158 (16), 115 (25), 91 (17), 77 (18).
(9.8 mg), and 7 (4.8 mg). Fraction D (30% EtOAc in cyclo-
hexane) (2.8 g) was further purified by gravity column on 3.3.6. Compound 8. Colorless oil; [a]20 D 2.5 (c 0.04,
silica gel using cyclohexane with increasing amounts (1%) CHCl3); UV lCHCl
max
3
(log 3): 285 (2.29) (nm); IR (CHCl3)
of EtOAc as mobile phase. Fraction V (4% EtOAc) nmax 3409, 3010, 1654, 1629, 1275 cm1; HRFABMS
(4.4 mg) was subjected to normal phase HPLC chromato- (m/z): 220.1844 [M]+ (calcd for C15H24O 220.1828),
graphy, using 100% n-hexane as mobile phase to yield Dm/m¼72 (ppm); NMR data (CDCl3), see Tables 1 and
 M. Kladi et al. / Tetrahedron 63 (2007) 7606–7611 7611

2; EIMS 70 eV, m/z (rel int. %): 220 (1), 202 (13), 187 (10), 2. Vairappan, C. S. Biomol. Eng. 2003, 20, 255–259.
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This study was partially supported by the 01ED146 35, 3057–3058.
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References and notes 28. Alley, M. C.; Scudiero, A. D.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.;
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