Antiplasmodial metabolites isolated from the marine octocoral Muricea austera

Article abstract of DOI:10.1021/np060007f

Bioassay-guided fractionation of the MeOH extract from the octocoral Muricea austera collected in the Pacific coast of Panama led to the isolation of eight compounds, including three tyramine derivatives (1-3), two steroidal pregnane glycosides (4, 5), and three sesquiterpenoids (6-8). Compounds 2-5 are new natural products, and their structures were determined on the basis of their spectroscopic data (HRMS, 1D and 2D NMR, and CD studies). The antiprotozoal activities of the natural compounds 1-8 as well as those of a series of synthetic glycosides (11-22) and tyramine derivatives (23-35) were evaluated in vitro against a drug-resistant Plasmodium falciparum and intracellular form of Trypanosoma cruzi.

Full text of DOI:10.1021/np060007f

© Copyright 2006 by the American Chemical Society and the American Society of Pharmacognosy
Volume 69, Number 10
October 2006
Full Papers
Antiplasmodial Metabolites Isolated from the Marine Octocoral Muricea austera
Marcelino Gutie´rrez,† Todd L. Capson,*,‡ He´ctor M. Guzma´n,‡ Jose´ Gonza´lez,§ Eduardo Ortega-Barr´ıa,§ Emilio Quin˜oa´,† and
Ricardo Riguera*,†
Departamento de Qu´ımica Orga´nica, Instituto de Acuicultura y Unidad de RMN de Biomole´culas Asociada al CSIC, UniVersidad de Santiago
de Compostela, E-15782 Santiago de Compostela, Spain, Smithsonian Tropical Research Institute, 2072 Balboa, Ancon, Republic of Panama,
and Institute of AdVanced Scientific InVestigations and High Technology SerVices, Secretariat for Science and Technology, Clayton, Panama
ReceiVed January 4, 2006
Bioassay-guided fractionation of the MeOH extract from the octocoral Muricea austera collected in the Pacific coast
of Panama led to the isolation of eight compounds, including three tyramine derivatives (1-3), two steroidal pregnane
glycosides (4, 5), and three sesquiterpenoids (6-8). Compounds 2-5 are new natural products, and their structures
were determined on the basis of their spectroscopic data (HRMS, 1D and 2D NMR, and CD studies). The antiprotozoal
activities of the natural compounds 1-8 as well as those of a series of synthetic glycosides (11-22) and tyramine
derivatives (23-35) were evaluated in vitro against a drug-resistant Plasmodium falciparum and intracellular form of
Trypanosoma cruzi.
Marine organisms are an extremely rich source of novel and
bioactive natural products.1 Among them, gorgonian octocorals
constitute one of the more interesting groups due to their ability to
synthesize a variety of compounds with chemical structures that
have no equivalent in the terrestrial environment.2 Recent studies
carried out at Coiba National Park (CNP) in the Republic of Panama
revealed that this region possesses a great diversity of octocoral
species.3 Although the natural products chemistry of marine
invertebrates in CNP remains largely unexplored, some efforts are
being directed to increase the knowledge of the chemistry and
biology of these organisms.4-6 As a part of these efforts, we are
exploring the chemistry of some sponges and gorgonian octocorals
collected from CNP using a variety of biological assays for drug
discovery against cancer, malaria, leismaniasis, and Chagas’ disease.
In this article we communicate the isolation and the antiprotozoal
activity of compounds 1-8 isolated from the octocoral Muricea
austera (Verrill, 1869; Plexauridae) collected from CNP. Com-
pounds 2-5 are new natural products, and their structures were
established on the basis of their HRMS, NMR spectroscopic data,
and CD studies. Preliminary studies related to the antiprotozoal
activity of the active natural products were carried out using a series
of glycosides and tyramine derivatives obtained through synthesis.
Results and Discussion
Specimens of M. austera were collected from CNP during an
expedition of the Smithsonian Tropical Research Institute. The
MeOH crude extract of M. austera showed in vitro activity against
chloroquine-resistant P. falciparum and was subjected to activity-
guided fractionation using solvent partition and vacuum liquid
chromatography (VLC), followed by silica gel flash chromatography
and normal-phase HPLC purification to yield compounds 1-8. The
structures of the new metabolites 2-5 were deduced using HRESI-
TOF MS, NMR (1H, 13C, DEPT, COSY, HSQC, HMBC, NOE,
1
and H decoupling) analysis, and CD studies. Compounds 17 and
6-88 were identified by comparison of their spectroscopic data with
those described in the literature.
25
The optically active compound 2 ([R]D -26.8) analyzed for
* To whom correspondence should be addressed. (R.R.) Tel/Fax:
+34981591091. E-mail: ricardo@usc.es. (T.L.C.) Tel: +507212.8039.
Fax: +507 212.8148. E-mail: capsont@si.edu.
† University of Santiago de Compostela.
C21H35NO2 by HRESI-TOFMS and 13C NMR. Five degrees of
unsaturation were calculated from the molecular formula. NMR
spectroscopic data of compound 2 allowed the construction of two
substructures. In the first, an intense broad singlet at δH 1.25 ppm
(14 H) and two methyl groups at δH 0.87 (3H) and δH 0.88 (3H)
‡ Smithsonian Tropical Research Institute.
§ Institute of Advanced Scientific Investigations and High Technology
Services.
10.1021/np060007f CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/22/2006

1380 Journal of Natural Products, 2006, Vol. 69, No. 10
Gutie´rrez et al.
and 62.4 ppm, respectively, suggested the presence of a pentose
sugar unit in the molecule. A deshielded methyl group at δH 2.18
and a carbonyl carbon at δC 170.8 corresponded to an acetate group
3
that was attached to C-3′ of the sugar unit on the basis of the J
HMBC correlation observed between one of the sugar methines
1
(H-3′, 5.09 ppm) and the acetate carbonyl. An analysis of the H
NMR coupling constants in addition to the data provided by COSY,
HMBC, and proton decoupling experiments allowed the sugar to
be identified as 3′-O-acetyl-â-D-arabinopyranose. The remaining
21 carbon atoms and the intense EIMS peak observed at m/z 282
(100%) matched with the C21 pregnane steroidal aglycon,12 which
has also been identified in other Muricea species.13 The attachment
of the sugar moiety to C-3 of the aglycon was deduced from the 3J
HMBC correlation between the anomeric proton (H-1′, 5.06 ppm)
and C-3 (77.8 ppm). Overall, the data provided by COSY, HSQC,
and HMBC correlations and MS allowed the assignment of the
structure of compound 4 as 3′-O-acetyl-3â-pregna-5,20-dienyl-â-
D-arabinopyranoside.
Figure 1. Selected 2D NMR correlations of compounds 2 (a) and
3 (b).
Compound 5 was consistent with C28H42O6 by HRESI-TOFMS
and 13C NMR. This compound displayed the same molecular
formula as 4, similar NMR spectroscopic features, and the same
fragmentation pattern in the EIMS. These similarities suggested
that 4 and 5 were isomers. After a detailed examination of 1D and
2D NMR spectroscopic data of 5, we observed that the only
difference between these compounds was the position of the acetate
clearly suggested the presence of a fatty acid moiety. On the other
hand, the AA′XX′ system formed by two doublets at 7.03 (2H, d,
J ) 8.4 Hz) and 6.78 ppm (2H, d, J ) 8.4 Hz), indicative of a
p-disubstituted aromatic ring and the presence of a pair of
methylenes mutually coupled at δH 2.73 (H-7′, t, J ) 6.8 Hz) and
3.48 (H-8′, dd, J ) 6.8, 12.0 Hz), together with a deuterium
interchangeable amide proton at δH 5.44, revealed a tyramine unit.
Both substructures were confirmed by fragments at m/z 120 (100%)
and 214 (78%) observed in the EIMS spectra.
The five degrees of unsaturation were accounted for by the
aromatic ring of the tyramine moiety and the amide carbonyl.
Therefore, the fatty acid residue must be formed by a saturated
C12 carbon chain. The carbon at δC 14.1 (C-12) showed the
characteristic chemical shift for a terminal CH3 in a fatty acid chain,
while the remaining methyl group at δC 19.6 (C-13) was attached
to C-3 on the basis of COSY experiments and its 2,3J HMBC
correlations with carbons C-2, C-3, and C-4 (Figure 1a). The
diasterotopic character of the methylene protons at δH 2.12 and
1.86 (H-2a and H-2b) indicated their proximity to a chiral center,
and their position was defined by their 2,3J HMBC correlations with
carbons C-1 and C-13 (Figure 1a). Thus, compound 2 was identified
as N-[2-(4-hydroxyphenyl)ethyl]-3-methyldodecanamide.
1
group on the arabinose unit. Through the analysis of H NMR
coupling constants, COSY, HMBC, and proton decoupling experi-
ments, we identified the sugar in 5 as 4′-O-acetyl-â-D-arabinopy-
ranose. Thus, compound 5 was identified as 4′-O-acetyl-3â-pregna-
5,20-dienyl-â-D-arabinopyranoside.
A base hydrolysis of the acetate groups in 4 and 5 was carried
out separately using LiOH in THF-H2O. In both cases, compound
9 was obtained, confirming the isomeric nature of 4 and 5. In order
to determine the absolute configuration of the sugar in 4 and 5, we
carried out circular dichroism (CD) studies on derivative 10
(obtained by perbenzoylation of compound 9 with benzoyl chloride
in pyridine) and the synthetic standard 12 (prepared from com-
mercial L-arabinopyranose). The CD spectra of glycoside 10 showed
a Davidov splitting identical with the standard 12 but with the
opposite chirality, indicating that the sugars were enantiomeric.
Given that the absolute configuration of the synthetic standard (12)
was “L”, we assigned the absolute configuration of the sugar in
10, and therefore in the natural products 4 and 5, as “D”.
Compound 3 was consistent with an empirical formula of C22H31-
NO2 by HRESI-TOFMS and 13C NMR and consistent with 8
1
degrees of unsaturation. H and 13C NMR spectroscopic data of
The hexane fraction obtained by solvent partition of the crude
extract showed antiplasmodial activity and was subjected to VLC
silica gel chromatography and normal-phase HPLC purification to
yield the germacrenes 6 and 7 and the elemane sesquiterpene 8;
these were identified by comparison of their spectroscopic data with
those described in the literature.8
compound 3 had some similarities with those of 2, indicating that
3 was also a tyramine fatty acid derivative. Five degrees of
unsaturation can be accounted for by the tyramine moiety and the
amide carbonyl. The remaining unsaturations were accounted for
by three carbon-carbon double bonds formed by six olefinic carbon
atoms at 126.9, 127.9, 128.5, 128.8, 128.9, and 132.1 ppm located
on the fatty acid chain. The spin systems formed by H-2, H-3, H-4,
H-5 and H-12, H-13, H-14 were assigned through COSY experi-
ments and their 2,3J HMBC correlations (Figure 1b). The cis-
geometries of the three double bonds of the polyunsaturated C14
fatty acid residue were assigned by 13C NMR, since the resonances
observed for the olefinic carbons (C-5, C-6, C-8, C-9, C-11, and
C-12) as well as those of the allylic methylenes (C-7 and C-10)
showed the typical δC values for a methylene-interrupted cis double-
bond system.9-11 The positions of the remaining carbons were
defined by comparison of their experimental δC with those
calculated theoretically following the method described by Sandri
et al.9
The antiplasmodial activities of the natural products 1-8 were
evaluated in vitro against chloroquine-resistant P. falciparum.14
Compounds 1-6 showed moderate antiplasmodial activity, with
IC50 values between 11 and 38 µg/mL, while compound 8 showed
no measurable activity (Table 1). The antiplasmodial activity of
the glycosides 4 and 5 (IC50 ) 32 and 38 µg/mL) was increased in
their peracetylated derivative 11 (IC50 ) 16 µg/mL). Since a
glycoside with the same aglycon as 4 and 5 but with L-galactose
instead of D-arabinose4 was shown to be inactive against P.
falciparum (unpublished results), we proceeded to the synthesis and
evaluation of the antiplasmodial activity of the arabinopyranosides
12-18. In this series, only the perbenzoylated compounds 12 and
13 were active against P. falciparum (Table 1), compound 13 (IC50
) 10 µg/mL) being more active than the natural arabinopyranosides
4 and 5. We also tested the antiplasmodial activity of simple sugar
derivatives with the same stereochemistry as that of D-arabinopy-
ranose, such as the D-fucosides 19 and 20 and D-galactosides 21
and 22. Curiously, compounds 19-21 displayed antiplasmodial
Compound 4 analyzed for C28H42O6 by HRESI-TOFMS and 13
C
NMR. DEPT and HSQC experiments revealed four quaternary
carbons, 11 methines, 10 methylenes, and three methyl groups.
Signals observed for an acetal carbon at 97.5 ppm joined by three
oxygen-bonded methines and one methylene at 73.0, 68.3, 67.2,

Antiplasmodial Metabolites from Muricea austera
Journal of Natural Products, 2006, Vol. 69, No. 10 1381
Table 1. Activity of Compounds 1-22 against
AMX-500, respectively (d-chloroform as internal standard). HREIMS
were measured on a Micromass Autospec mass spectrometer. CC was
performed on Si gel 60, 70-230 mesh for VLC and 230-400 mesh
for flash columns. Semipreparative HPLC purification was carried out
on a Waters apparatus equipped with a refractive index detector and a
spherisorb 10 µm Si semiprep column (10 × 250 mm).
Chloroquine-Resistant Plasmodium falciparum and Intracellular
a
Trypanosoma cruzi
P.
P.
falciparum
T. cruzi
falciparum
T. cruzi
compd
IC50 (µM) IC50 (µM) compd IC50 (µM) IC50 (µM)
Biological Material. Specimens of M. austera (voucher C5) were
collected by scuba in Coiba National Park at 3-10 m depth in August
2002. The samples were frozen immediately after collection and stored
at -80 °C until extraction. Voucher specimens are maintained for
reference at the Smithsonian Tropical Research Institute. Specimen C5
was consistent with the original description for M. austera (Verrill,
1869):16 large reddish or yellowish brown colonies, up to 30 cm in
height and thicker dichotomous branches 6-12 mm in diameter, with
occasional branching anastomosis near the base, and rough surface.
Calyces are prominently conical, having an acute lower lip, and made
by coarse and large light orange spindle sclerites (1.25-1.70 mm in
length) encompassing the polyp aperture. Sclerites are predominantly
reddish, with some white spindles. Largest sclerites are blunt and stout
(0.65-1.45 mm), mostly oblong or oval in shape covered by rough
warts, and smaller sclerites are short, irregular and thorny spindles
(0.15-0.30 mm).
1
2
3
4
5
6
7
8
36
45
38
67
80
42
ND
>50
ND
ND
28
>50
>50
ND
96
101
173
ND
ND
ND
ND
28
12
13
14
15
16
17
18
19
20
21
22
35
21
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
43
36
29
>50
9
10
11
chloroquine
nifurtimox
0.07
11
a ND ) not determined
Table 2. Activity of Compounds 23-35 against
Chloroquine-Resistant Plasmodium falciparuma
Extraction and Isolation. The octocoral M. austera was cut into
small pieces and extracted with MeOH and CH2Cl2. The extracts were
pooled and concentrated under reduced pressure in a rotary evaporator
to yield 21.0 g of crude extract, which was subjected to solvent partition,
affording four fractions (n-hexane, CH2Cl2, n-butanol, and H2O), which
were submitted to biological assays. The antiplasmodial activity was
located in the n-hexane and CH2Cl2 fractions. The active CH2Cl2
fraction (2.2 g) was subjected to activity-guided fractionation using
VLC eluted with CH2Cl2-MeOH mixtures of increasing polarity (from
3% to 67% of MeOH), yielding five fractions. Fraction 2 (517 mg)
was purified by two successive Si gel flash chromatographies (column
A, CH2Cl2-MeOH, 2% to 9% MeOH, four fractions; column B, CH2-
Cl2-MeOH, 2% MeOH, seven fractions). The sixth fraction from
column B (165 mg) was subjected to normal-phase HPLC purification
(isooctane-EtOAc, 1:1, 1 mL/min, isocratic elution), yielding com-
pounds 1 (12 mg), 2 (5 mg), and 3 (6 mg). The seventh fraction from
column B was subjected to Si gel column chromatography (column C,
CH2Cl2-acetone, 6% to 50% acetone, eight fractions) to yield
compounds 4 (2 mg) and 5 (3 mg) in fractions 8 and 9, respectively.
The n-hexane fraction (6.3 g), also active in the antiplasmodial assay,
was subjected to VLC (3% to 50% of EtOAc in n-hexane), yielding
eight fractions. Fraction 2 was subjected to normal-phase HPLC
purification (2.5% EtOAc in isooctane, 1 mL/min, isocratic elution),
yielding compounds 6 (10 mg), 7 (6 mg), and 8 (2 mg).
compound
P. falciparum, IC50 (µM)
23
24
25
26
27
28
29
30
31
32
33
34
160
>50
>50
64
>50
72
47
34
62
24
117
>50
17
35
chloroquine
0.07
a ND ) not determined.
activity, while the perbenzoylated methyl â-D-galactoside 22 was
inactive (Table 1). The whole series of natural and synthetic
compounds mentioned above were also tested for their activity
against intracellular T. cruzi (Table 1).15 In this assay, glycosides
4 and 5 and the elemane 6 displayed biological activity (46, 48,
and 45 µg/mL, respectively). The antitrypanosomal activity of
glycosides 4 and 5 was increased when these compounds were
peracetylated (compound 11 showed an IC50 ) 15.9 µg/mL).
Given the antiplasmodial activity displayed by natural tyramine
derivatives (1-3), we proceeded to synthesize and evaluate the
antiplasmodial activity of compounds 23-35 (Table 2). Of this
series, the derivatives with a fatty acid moiety (28-31) showed
antiplasmodial activity very similar to those of their natural
analogues. Variations in the structure and activity of the natural
and synthetic tyramides suggested that increasing the number of
carbons of the fatty acid chain produces an increase in potency
(28-30), while the presence of polar groups on the fatty acid chain
decreases potency (31). On the other hand, the introduction of
bromine atoms on the tyramine aromatic ring (35) produced an
increase in the antiplamodial activity. Finally, the activity displayed
by compound 32 (IC50 ) 8 µg/mL) indicated that the change in
the position of the amide bond (see 29 for comparison purposes)
also produces an increase in antimalarial activity.
N-(4-Hydroxyphenethyl)-3-methyldodecanamide (2): oil; [R]25
D
-26.8 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.03 (2H, d, J )
8.4 Hz, H-3′, H-5′), 6.78 (2H, d, J ) 8.4 Hz, H-2′, H-6′), 5.44 (1H, bt,
NH), 3.48 (2H, dd, J ) 12.0, 6.8 Hz, H-8′), 2.73 (2H, t, J ) 6.8 Hz,
H-7′), 2.12 (1H, m, H-2a), 1.87 (1H, m, H-3), 1.86 (1H, m, H-2b),
1.26 (2H, m, H-4), 1.25 (14H, bs, H-5 to H-11), 0.87 (3H, m, H-13),
0.88 (3H, m, H-12); 13C NMR (CDCl3, 100 MHz) δ 172.9 (C, C-1),
154.7 (C, C-1′), 130.4 (C, C-4′), 129.8 (2 CH, C-3′, C-5′), 115.5 (2
CH, C-2′, C-6′), 44.7 (CH2, C-2), 40.7 (CH2, C-8′), 36.8 (CH2, C-4),
34.8 (CH2, C-7′), 31.8 (CH2, C-10), 30.7 (CH, C-3), 29.8* (CH2, C-5),
29.7* (CH2, C-6), 29.6* (CH2, C-7), 29.3* (CH2, C-8), 26.9* (CH2,
C-9), 22.7 (CH2, C-11), 19.6 (CH3, C-13), 14.1 (CH3, C-12); EIMS
m/z 330 (0.4), 282 (2), 214 (12), 157 (6), 129 (1), 120 (100), 106 (23),
91 (3), 77 (4), * these signals may be interchanged; HRESI-TOFMS
m/z 356.2562 (calcd for C21H35NO2Na, 356.2560).
(5Z,8Z,11Z)-N-(4-Hydroxyphenylethyl)tetradeca-5,8,11-triena-
mide (3): oil; 1H NMR (CDCl3, 400 MHz) δ 7.02 (2H, d, J ) 8.6 Hz,
H-3′, H-5′), 6.79 (2H, d, J ) 8.6 Hz, H-2′, H-6′), 5.49 (1H, bt, NH),
5.35 (6H, m, H-5, H-6, H-8, H-9, H-11, H-12), 3.48 (2H, dd, J ) 12.0,
7.0 Hz, H-8′), 2.78 (2H dd, J ) 11.8, 5.8 Hz, H-7), 2.78 (4H dd, J )
11.8, 5.8 Hz, H-10), 2.73 (2H, t, J ) 7.0 Hz, H-7′), 2.14 (2H, m, H-2),
2.08 (2H, m, H-4), 2.04 (2H, m, H-13), 1.68 (2H, m, H-3), 0.96 (3H,
t, J ) 7.6 Hz, H-14); 13C NMR (CDCl3, 100 MHz) δ 173.2 (C, C-1),
154.8 (C, C-1′), 132.1 (CH, C-12), 130.2 (C, C-4′), 129.7 (2 CH, C-3′,
C-5′), 128.9 (CH, C-5), 128.8 (CH, C-6), 128.5 (CH, C-9), 127.9 (CH,
C-8), 126.9 (CH, C-11), 115.5 (2 CH, C-2′, C-6′), 40.8 (CH2, C-8′),
36.1 (CH2, C-2), 34.7 (CH2, C-7′), 26.6 (CH2, C-4), 25.59 (CH2, C-10),
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured on a JASCO DIP-360 polarimeter using a 1 dm path length cell.
CD spectra were obtained in MeCN in a JASCO J-720 spectropola-
rimeter. NMR spectra were recorded in d-chloroform at 250, 400, or
500 MHz using a Bruker DPX-250, Varian Inova-400, and Bruker

1382 Journal of Natural Products, 2006, Vol. 69, No. 10
Gutie´rrez et al.
Chart 1
25.52 (CH2, C-7), 25.49 (CH2, C-3), 20.54 (CH2, C-13), 14.3 (CH3,
C-14); EIMS m/z 341 [M]+ (0.6), 192 (1), 120 (100), 107 (38), 77
(10); HRESI-TOFMS m/z 364.2231 (calcd for C22H31NO2Na, 364.2250).
3′-O-Acetyl-3â-pregna-5,20-dienyl-â-D-arabinopyranoside (4): white
powder; [R ]25D -98.3 (c 0.515, CHCl3); 1H NMR (CDCl3, 500 MHz)
δ 5.76 (1H, ddd, J ) 16.2, 10.9, 7.7, H-20), 5.36 (1H, d, J ) 5.2 Hz,
H-6), 5.09 (1H, dd, J ) 3.2, 10.1 Hz, H-3′), 5.06 (1H, d, J ) 3.8 Hz,
H-1′), 4.99 (1H, d, J ) 16.2 Hz, H-21a), 4.96 (1H, d, J ) 10.9 Hz,
H-21b), 4.03 (1H, bs, H-4′), 3.95 (1H, m, H-2′), 3.95 (1H, m, H-5′a),
3.67 (1H, dd, J ) 12.5, 2.1 Hz, H5′b), 3.52 (1H, m, H-3), 2.36 (1H,
ddd, J ) 13.0, 4.9, 2.1 Hz, H-4a), 2.23 (1H, m, H-4b), 2.18 (3H, s,
H-7′), 2.01 (1H, m, H-7a), 1.95 (1H, m, H-17), 1.81 (1H, m, H-16a),
1.89 (1H, m, H-1a), 1.88 (1H, m, H-2a), 1.71 (1H, m, H-8), 1.69 (2H,
m, H-12), 1.67 (1H, m, H-15), 1.61 (1H, m, H-2b), 1.56 (1H, m, H-16b),
1.55 (1H, m, H-7b), 1.52 (2H, m, H-11a), 1.48 (1H, m, H-11b), 1.19
(1H, m, H-15b), 1.07 (1H, m, H-1b), 1.02 (1H, m, H-14), 1.02 (3H, s,
H-19), 0.97 (1H, m, H-9), 0.61 (3H, s, H-18); 13C NMR (CDCl3, 125
MHz) δ 170.8 (C, C-6′), 140.16 (C, C-5), 139.8 (CH, C-20), 122.2
(CH, C-6), 114.5 (CH2, C-21), 97.5 (CH, C-1′), 77.8 (CH, C-3), 73.0
(CH, C-3′), 68.3 (CH, C-4′), 67.2, (CH, C-2′), 62.4 (CH2, C-5′), 55.9
(CH, C-14), 55.3 (CH, C-17), 50.4 (CH, C-9), 43.4 (C, C-13), 38.4
(CH2, C-4), 37.4 (CH2, C-12), 37.3 (CH2, C-1), 36.8 (C, C-10), 31.9
(CH, C-8), 31.9 (CH2, C-7), 29,7 (CH2, C-2), 27.2 (CH2, C-16), 24.8
(CH2, C-15), 21.1 (CH3, C-7′), 20.6 (CH2, C-11), 19.4 (CH3, C-19),
12.7 (CH3, C-18); COSY (selected) H-3 (H-2a, H-2b, H-4a, H-4b),
H-6 (H-7), H-20 (H-21a, H21b, H-17), H-1 (H-2′), H-2′ (H-1′, H-3′),
H-3′ (H-2′, H-4′), H-4′ (H-3′, H-5a′, H-5b′), H-5a′ (H-5b′, H-4′), H-5b′
(H-5a, H-4′); HMBC H-18 (C-12, C-13, C-14), H-19 (C-1, C-5, C-9,
C-10), H-21 (C-17), H-1′ (C-3, C-5′, C-3′), H-2′ (C-4′), H-3′ (C-2′,
C-6′), H-5′ (C-1), H-7′(C-6′); EIMS m/z 396 [M - HOAc - H2O]
(0.01), 342 (2), 282 (100), 267 (22), 213 (9), 175 (5), 115 (3), 91 (2);
EIMS m/z 396 [M - HOAc - H2O] (0.01), 342 (2), 282 (100), 267
(22), 213 (9), 175 (5), 115 (3), 91 (2); HRESI-TOFMS m/z 497.2846
(calcd for C28H42O6Na, 497.2874).

Antiplasmodial Metabolites from Muricea austera
Journal of Natural Products, 2006, Vol. 69, No. 10 1383
4′-O-Acetyl-3-pregna-5,20-dienyl-â-D-arabinopyranoside (5): white
powder; [R ]25D -205.0 (c 0.16, CHCl3); 1H NMR (CDCl3, 500 MHz)
δ 5.76 (1H, ddd, J ) 16.5, 10.9, 7.8, H-20), 5.36 (1H, d, J ) 5.0 Hz,
H-6), 5.15 (1H, d, J ) 3.4 Hz, H-4′), 5.05 (1H, d, J ) 3.9 Hz, H-1′),
4.98 (1H, d, J ) 16.5 Hz, H-21a), 4.95 (1H, d, J ) 10.9 Hz, H-21b),
3.92 (1H, m, H-3′), 3.91 (1H, m, H-5′a), 3.78 (1H, dd, J ) 9.6, 3.9
Hz, H-2′), 3.71(1H, dd, J ) 13.0, 2.1 Hz, H-5′b), 3.49 (1H, m, H-3),
2.36 (1H, ddd, J ) 13.1, 4.7, 2.1 Hz, H-4a), 2.23 (1H, m, H-4b), 2.15
(3H, s, H-7′), 2.03 (1H, m, H-7a), 1.95 (1H, m, H-17), 1.87 (1H, m,
H-1a), 1.88 (1H, m, H-2a), 1.81 (1H, m, H-16a), 1.71 (2H, m, H-12),
1.71 (1H, m, H-8), 1.69 (1H, m, H-15a), 1.59 (1H, m, H-11a), 1.58
(1H, m, H-2b), 1.57 (1H, m, H-7b), 1.56 (1H, m, H-16b), 1.49 (1H,
m, H-11b), 1.19 (1H, m, H-15b), 1.08 (1H, dd, J ) 12.1, 3.6 Hz, H-1b),
1.02 (3H, s, H-19), 1.01 (1H, m, H-14), 0.96 (1H, m, H-9), 0.61 (3H,
s, H-18); 13C NMR (CDCl3, 125 MHz) δ 170.8 (C, C-6′), 139.9 (C,
C-5); 139.6 (CH, C-20), 122.1 (CH, C-6), 114.5 (CH2, C-21), 97.0
(CH, C-1′), 77.9 (CH, C-3), 69.8 (CH, C-3′), 71.2 (CH, C-4′), 69.2,
(CH, C-2′), 60.9 (CH2, C-5′), 55.9 (CH, C-14), 55.3 (CH, C-17), 50.4
(CH, C-9), 43.4 (C, C-13), 38.7 (CH2, C-4), 37.4 (CH2, C-12), 37.3
(CH2, C-1), 36.9 (C, C-10), 32.0 (CH, C-8), 32.0 (CH2, C-7), 29,6 (CH2,
C-2), 27.3 (CH2, C-16), 24.9 (CH2, C-15), 21.2 (CH3, C-7′), 20.7 (CH2,
C-11), 19.5 (CH3, C-19), 12.8 (CH3, C-18); COSY (selected) H-3 (H-
2a, H-2b, H-4a, H-4b), H-6 (H-7), H-20 (H-21a, H-21b, H-17), H-1′
(H-2′), H-2′ (H-1′, H-3′), H-3′ (H-2′, H-4′), H-4′ (H-3′, H-5a′, H-5b′),
H-5a (H-5b′, H-4′), H-5b′ (H-5a′, H-4′); HMBC H-4 (C-2, C-3, C-5,
C-6, C-10), H-6 (C-4, C-8, C-10), H-18 (C-12, C-13, C-14, C-17), H-19
(C-1, C-5, C-9, C-10), H-21 (C-17, C-20), H-1′ (C-3, C-3′), H-3′ (C-
1′, C-4′), H-5′(C-1′, C-3′, C-4′), H-7′ (C-6′); EIMS m/z 396 [M - HOAc
- H2O] (0.01), 342 (9), 300 (4), 282 (100), 267 (41), 213 (24), 175
(40), 115 (56), 91 (36), 43 (64); HRESI-TOFMS m/z 497.2858 (calcd
for C28H42O6Na, 497.2874).
Antiplasmodial Assay. The antiplasmodial activity was determined
in a chloroquine-resistant P. falciparum strain (W2) utilizing a novel
microfluorimetric assay to measure the inhibition of the parasite growth
based on the detection of the parasitic DNA by intercalation with
PicoGreen.14 P. falciparum was cultured according to the methods
described by Trager and Jensen.17 The parasites were mantained at 2%
hemeatocrit in flat-bottom flasks (75 mL) with RMPI 1640 medium
(GibcoBRL) supplemented with 10% human serum.
Intracellular T. cruzi Assay. The recombinant Tulahuen clone C4
of Trypanosoma cruzi that expresses â-galactosidase (â-Gal) as a
reporter enzyme was used in the assay (Buckner et al., 1996).15 The
method is based on the growth inhibition effect of test samples on
trypomastigote, the intracellular form of the parasite, infecting Vero
cells. The resulting color from the cleavage of chlorophenol red-â-D-
galactoside (CPRG) by â-Gal expressed by the parasite was determined
in a Benchmark plate reader (BIO-RAD) employing a 570 nm
wavelength filter. Antitrypanosomal activity was expressed as IC50,
which is a measure of the concentration of test substance that inhibits
50% parasite growth on duplicate samples, as compared with an
untreated control. Assays were conducted at 37 °C under an atmosphere
of 5% CO2 and 95% air mixture.
Acknowledgment. We thank the Ministerio de Educacio´n y Ciencia
(MEC) and the Xunta de Galicia for financial support for this research
(grant numbers CTQ2005-05296/BQU, SAF2003-08765-C03-01,
PGIDT02BTF20902PR, PGIDT03PXIC20908PN, and PGIDIT04PXIC-
20903PN). Funding was also provided by the Smithsonian Tropical
Research Institute, the AVINA Foundation, and the International
Cooperative Biodiversity Groups program (grant numbers 1U01
TW01021-01 and 1U01 TW006634-01) from the National Institutes
of Health, National Science Foundation, and the United States Depart-
ment of Agriculture. We thank C. Guevara for field assistance, O.
Breedy for assistance with the taxonomy, and the crew members from
the R/V Urraca´ for logistical support.
Supporting Information Available: General procedures for methan-
olysis and for the synthesis of sugar esters and tyramine derivatives;
experimental details for compounds 10-17, 19-22, 24-35; and CD
spectra of compounds 10 and 12. This material is available free of
charge via the Internet at http://pubs.acs.org.
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