Antiparasitic activity of prenylated benzoic acid derivatives from Piper species

Article abstract of DOI:10.1016/j.phytochem.2009.03.010

Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)- 4,5-dihydroxybenzoic acid, 3-[(2E,6E,1

Full text of DOI:10.1016/j.phytochem.2009.03.010

Phytochemistry 70 (2009) 621–627
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Antiparasitic activity of prenylated benzoic acid derivatives from Piper species
Ninoska Flores a,b, Ignacio A. Jiménez a, Alberto Giménez b, Grace Ruiz b, David Gutiérrez b,
Genevieve Bourdy b, Isabel L. Bazzocchi a,
*
a Instituto Universitario de Bio-Orgánica ‘‘Antonio González”, Universidad de La Laguna, Avda. Astrofísico, Francisco Sánchez 2, La Laguna,
38206 Tenerife, Canary Islands, Spain
b Instituto de Investigaciones Fármaco Bioquímicas, Facultad de Ciencias Farmacéuticas y Bioquímicas, Universidad Mayor de San Andrés,
Avda. Saavedra 2224, Miraflores, La Paz, Bolivia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2008
Received in revised form 4 March 2009
Available online 8 April 2009
Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded
three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadeca-
tetraenyl)-4,5-dihydroxybenzoic acid, 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-2,6,10,
14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid and 3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-tri-
methyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid, along with the known compounds, 4,5-
dihydroxy-3-(E,E,E-11-formyl-3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (arieianal),
3,4-dihydroxy-5-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 4-hydroxy-
3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 3-(3,7-dimethyl-2,6-octadie-
nyl)-4-methoxy-benzoic acid, 4-hydroxy-3-(3,7-dimethyl-2,6-octadienyl)benzoic acid and 4-hydroxy-
3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid. Their structures were elucidated on
the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY,
HSQC and HMBC) and comparison with data reported in the literature. Riguera ester reactions and optical
rotation measurements established the compounds as racemates. The antiparasitic activity of the com-
pounds were tested against three strains of Leishmania spp., Trypanosoma cruzi and Plasmodium falciparum.
The results showed that 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid exhibited potent and
Keywords:
Piper heterophyllum
P. aduncum
Prenylated benzoic acids
Leishmanicidal
Trypanocidal
Antiplasmodial
selective activity against L. braziliensis (IC50 6.5
3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic
and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate
antiplasmodial (IC50 3.2 g/ml) and trypanocidal (16.5 g/ml) activities, respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
l
g/ml), higher that pentamidine used as control. Moreover,
acid
l
l
1. Introduction
analysis for the genus suggests three major clades in Piper, repre-
senting three large geographical regions: America (1300 sp.), Asia
(600 sp.) and the South Pacific (100 sp.) (Jaramillo and Manos,
2001). Several Piper species have been included in the system of
traditional medicine in Latin America (Gupta, 1995). Therefore,
the leaves of Piper hispidum and P. elongatum are used as poultices
for healing wounds and to treat the symptoms of cutaneous leish-
maniasis ‘‘Uta” (Estevez et al., 2007), and the leaves of P. aduncum
are used for inflammation, and as antiseptic (Orjala et al., 1994).
Phytochemical studies of Piper species (Parmar et al., 1997) de-
scribe the isolation of metabolites with antifungal (Terreaux
et al., 1998; Lago et al., 2004), antibacterial (Ramji et al., 2002),
insecticidal (Siddiqui et al., 2004), cytotoxic (Chen et al., 2003)
and antioxidant (Yamaguchi et al., 2006) properties. Regarding
antiprotozoal metabolites from Piper species, flavanones (Portet
et al., 2007), chalcones and dihydrochalcones (Torres-Santos
et al., 1999; Hermoso et al., 2003; Flores et al., 2007), neolignans
(Luize et al., 2006) and alkaloids (Rukachaisirikul et al., 2004) have
been described.
Parasitic diseases, including leishmaniasis, malaria and chagas,
are serious problems for public health in the world, especially in
tropical and subtropical regions. In the absence of effective vac-
cines, chemotherapy still plays a critical role in treating these dis-
eases, but in many cases current therapies are inadequate and in
some the situation is deteriorating because of drug resistance.
Therefore, there is an urgent need to search for novel, effective
and safe drugs for the treatment of these diseases. One of the main
opportunities is through the discovery of new antiparasitic agents
from natural origins (Ioset, 2008).
The genus Piper (Piperaceae) is a pantropical group with nearly
2000 species, constituting an important element of montane and
lowland forests (Quijano-Abril et al., 2006). A recent phylogenetic
* Corresponding author. Tel.: +34 922 318594; fax: +34 922 318571.
E-mail address: ilopez@ull.es (I.L. Bazzocchi).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.03.010

622
N. Flores et al. / Phytochemistry 70 (2009) 621–627
Previous investigations into P. aduncum report essential oils
3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (ariei-
anal) (4) (Green et al., 1999), 3,4-dihydroxy-5-(E,E,E-3,7,11,15-tet-
ramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (5) (Seeram
et al., 1996), 4-hydroxy-3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-
2,6,10,14-tetraenyl)benzoic acid (6) (Maxwell and Rampersad,
1989), 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid
(7) (Baldoqui et al., 1999), 4-hydroxy-3-(3,7-dimethyl-2,6-octadie-
nyl)benzoic acid (8) (Seeram et al., 1996) and 4-hydroxy-3-(3-
methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid (9)
(Orjala et al., 1993).
(Rali et al., 2007; Vila et al., 2005), chromenes (Moreira et al.,
1998), prenylated benzoic acids (Orjala et al., 1993; Baldoqui
et al., 1999; Lago et al., 2004) and dihydrochalcones (Orjala et al.,
1994). However, the chemical constituents of P. heterophyllum
have not been described.
Several Piper species are claimed to be used in traditional med-
icine for the treatment of parasitic diseases, and benzoic acid deriv-
atives are frequent metabolites in this genus. However, a few
reports on the antiparasitic activity of this class of compounds have
been published (Lopes et al., 2008; Flores et al., 2008). As part of
our research into the Bolivian Piper species, we have carried out
a phytochemical analysis of the leaves of Piper heterophyllum and
P. aduncum. We report herein the isolation and structural elucida-
tion of three new prenylated benzoic acid derivatives (1–3), along
with six known compounds (4–9). Their structures were elucidated
by extensive spectroscopic analysis, and comparison with data re-
ported in the literature. Concerning the absolute configuration of
the stereogenic center in compounds 2 and 3, Riguera’s method
(Seco et al., 2004), a modified Mosher ester procedure, together
with optical rotation measurements established the new com-
pounds as racemates. In the search for new antiparasitic agents,
the compounds have been tested for their leishmanicidal, trypano-
cidal and antiplasmodial activities.
The molecular formula C27H36O6 assigned to compound 1 was
determined by analysis of the 13C NMR data associated with HR
ESI(+)MS. The IR spectrum indicated the presence of hydroxyl
group (3407 cmꢀ1), carboxyl acid (3520–2560 and 1681 cmꢀ1
)
and aromatic ring (1612 and 775 cmꢀ1). The 1H NMR (Table 1)
spectrum exhibited signals for two aromatic protons at d 7.44 (d,
J = 2.8 Hz) and d 7.47 (d, J = 2.8 Hz), suggesting a 1,3,4,5-tetrasub-
stituted aromatic ring in the molecule. The spectrum also dis-
played signals for four vinyl methyl groups at d 1.56, 1.58, 1.65
and 1.72; seven allylic methylene groups, six of them as multiplets
at d 2.05–2.13 (8H) and 2.19–2.28 (4H) and one occurred as a dou-
blet at d 3.36 (2H, J = 6.7 Hz). Additionally, signals for four olefinic
protons at d 5.11 (2H, m), 5.31 (1H, t, J = 6.7 Hz) and 6.84 (1H, t,
J = 7.6 Hz), were observed. The 13C NMR (Table 1) spectrum exhib-
ited signals for 27 carbon atoms, showing signals in the low-field
region corresponding to two carboxyl acids (d 171.3 and 172.6)
and six aromatic carbons (d 114.4, 120.2, 124.2, 127.5, 142.8 and
147.7), along with signals for eight olefinic and eleven sp3-hybrid-
ized carbons. All these data suggested that 1 is a benzoic acid
derivative with a geranylgeranyl chain. The assignment of the 1H
and 13C NMR data were based on 2D experiments, and comparison
with data reported for benzoic acid analogues (Green et al., 1999).
The connectivity of aromatic and aliphatic moieties was revealed
2. Results and discussion
Repeated chromatography of the dichloromethane extracts of
the leaves of P. heterophyllum and P. aduncum on silica gel and
Sephadex-LH-20, afforded three new prenylated hydroxybenzoic
acid derivatives (1–3). In addition, six known compounds were iso-
lated and identified by comparison of their spectral data with those
reported in the literature as 4,5-dihydroxy-3-(E,E,E-11-formyl-
Table 1
1H and 13C NMR (400 MHz) data for compounds 1–3 in CDCl3.
Position
1
2
3
a
b
a
b
a
b
dH
dC
dH
dC
dH
dC
1
2
3
4
5
6
120.2 s
124.2 d
127.5 s
147.7 s
142.8 s
114.4 d
171.3 s
28.0 t
120.4 s
123.7 d
127.2 s
147.4 s
143.0 s
114.4 d
171.3 s
27.7 t
123.1 s
123.8 d
127.3 s
147.7 s
143.1 s
114.3 d
171.3 s
27.8 t
7.47 d (2.8)
7.48 s
7.47 d (2.8)
7.44 d (2.8)
7.54 s
7.52 d (2.8)
COOH
10
3.36 d (6.7)
5.31 t (6.7)
3.42 d (5.2)
5.34 t (5.2)
3.38 d (6.3)
5.33 t (6.3)
20
121.7 d
136.5 s
39.2 t
121.2 d
136.9 s
39.0 t
121.4 d
136.7 s
39.0 t
30
40
2.05–2.13 m*
2.05–2.13 m*
5.11 m*
2.16–2.28 m*
2.16–2.28 m*
5.16 t (5.8)
2.05–2.16 m*
2.05–2.16 m*
5.15 t (6.9)
50
25.6 t
25.6 t
26.1 t
60
124.8 d
133.5 s
37.9 t
125.0 d
133.5 s
37.4 t
124.9 d
133.6 s
37.3 t
70
80
2.05–2.13 m*
2.19–2.28 m*
6.84 t (7.6)
2.16–2.28 m*
2.16–2.28 m*
6.79 t (7.2)
2.19–2.40 m
2.05–2.16 m*
7.13 t (6.4)
90
26.9 t
28.9 t
25.5 t
100
110
120
145.2 d
130.8 s
26.5 t
141.4 d
126.1 s
32.2 t
147.5 d
124.3 s
22.1 t
2.19–2.28 m*
2.53 dd (4.9, 13.1)
3.06 dd (3.8, 13.1)
5.20–5.27 m*
2.05–2.16 m*
130
140
150
160
170
180
190
200
2.05–2.13 m*
5.11 m*
27.3 t
75.0 d
123.1 d
139.8 s
25.5 q
15.9 q
15.8 q
172.0 s
18.2 q
1.70–1.78 m
4.68 dd (2.2, 7.8)
27.2 t
123.4 d
132.0 s
25.4 q
15.7 q
15.6 q
172.6 s
17.3 q
5.20–5.27 m*
81.9 d
141.8 s
113.1 t
15.8 q
15.9 q
167.8 s
17.9 q
1.65 s
1.72 s
1.58 s
1.76 s
1.74 s
1.63 s
4.96 s, 5.01 s
1.73 s
1.67 s
1.56 s
1.78 s
1.80 s
a
Chemical shifts d, in ppm relative to TMS, J in Hz in parentheses.
Data are based on Dept, HSQC, and HMBC experiments.
b
*
Overlapping signals.

N. Flores et al. / Phytochemistry 70 (2009) 621–627
623
by analysis of the HMBC spectrum (Fig. 1). Thus, correlation be-
tween signals at d 7.47 (H-2)/7.44 (H-6) and d 171.3 (COOH) indi-
cated one of the carboxyl group was attached to C-1, and the
position of the terpene side chain to the aromatic ring was deter-
mined by correlation of the signals at dH 7.47 (H-2) and dC 28.0
(C-10). Moreover, the second carboxyl group was placed at C-110
of the side chain, in agreement with the correlation observed be-
tween the signal at dH 6.84 (H-100) and dC 172.6 (C-190). In addition,
a broad multiplete methylene at d 2.19–2.28 correlated to both, the
vinylic carbon of the conjugated olefin (C-100) and the vinylic
methine of the terminal isoprene unit (C-140), establishing the C-
100, C-110 and C-190 as a conjugated system. The stereochemistry
at C-20 and C-60 within the geranylgeranyl residue was determined
as (E) on the basis of the 13C NMR chemical shifts of the vinyl
methyl groups C-170 (dC 15.7) and C-180 (dC 15.6), which both lie
Compound 2 was obtained as a colorless oil, and its molecular
formula C27H36O7 was determined by analysis of 13C NMR and HR
EIMS spectra. Comparison of its 1H and 13C NMR data (Table 1)
with those of compound 1 indicated the presence of an additional
secondary hydroxyl group with signals at dH 5.20–5.27 and dC 75.0
and the loss of a methylene group (dH 2.05–2.13 and dC 27.3) for
compound 2, as the most notable differences. These data sug-
gested
a prenylated 3,4,5-trisubstituted benzoic acid with a
hydroxylated geranylgeranyl chain for compound 2, which was
further confirmed by COSY, HSQC and HMBC experiments. The
cross-peak observed in the HMBC (Fig. 1) between the signals at
dH 5.20–5.27 (H-130) and dC 126.1 (C-110)/139.8 (C-150) located
the hydroxyl group at C-130 on the geranylgeranyl chain. In order
to determine the absolute configuration of the stereogenic center
at C-130, and prior to preparing the Riguera ester derivative, the
phenolic hydroxyls and carboxyl acids of compound 2 were pro-
tected with a methyl moiety, yielding derivative 10. Treatment
between 15 ppm as a result of the shielding
c-cis interactions.
However, no such interaction exists for the C-160, as it resonates
at a lower field (d 25.4) (Maxwell and Rampersad, 1989). The
geometry of the C-100, C-110-double bond was established as E on
of 10 with (R)-(-)-a-methoxyphenylacetic acid (MPA) afforded
the corresponding diastereomeric esters (11). The magnetic non-
equivalence of the signals assigned to the oxymethine and meth-
oxyl groups belonging to the chiral auxiliary established the very
low diastereomeric excess (3%). Therefore, this suggests com-
pound 2 is almost a racemic mixture. Accordingly, the structure
1
the basis of the H NMR chemical shift of the vinyl proton H-100
(dH 6.84), which was further supported by comparison with the
shifts reported for Z and E-2-methyl-2-butenoic acid and arieianal
(Green et al., 1999). All these data allowed full assignment for all
hydrogen and carbon atoms, and established the structure of com-
pound 1 as 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-
hexadecatetraenyl)-4,5-dihydroxybenzoic acid.
of
2 was assigned as 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-
3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl]-4,5-dihydroxyben-
zoic acid.

624
N. Flores et al. / Phytochemistry 70 (2009) 621–627
Fig. 1. Selected 1H–13C (HMBC) correlations for compounds 1–3.
Compound 3 resulted to be an isomer of compound 2, as indi-
are presented in Table 2. Compound 7 showed a high selectivity
against L. braziliensis (IC50 6.5 g/ml), even being more active than
pentamidine, used as positive control (IC50 10.0 g/ml). In addi-
tion, compound 9 exhibited significant activity with an IC50 of
17.8 g/ml against the three strains of Leishmania used. The other
assayed compounds showed slight or no activity with IC50 values
ranging from 27.0 to 100 g/ml. Regarding the trypanocidal activ-
ity, compound 9 proved to be active against epimastigote forms
of T. cruzi, with an IC50 of 16.5 g/ml, while the other compounds
showed IC50 > 20 g/ml. Benznidazole was used as positive con-
trol (IC50 7.4 g/ml). The compounds were also evaluated for their
antiplasmodial activity. All the compounds were inactive
(IC50 > 10 g/ml), except for compound 1, which exhibited a mod-
erate activity against P. falciparum with an IC50 of 3.2 g/ml, in
comparison with chloroquine, used as a positive control (IC50
0.1 g/ml).
The biological activity seems to be associated with shorter
side chains in the molecule. It is also apparent that the presence
of two isoprene moieties instead a single geranyl chain around
the benzoic acid moiety improve the activity (9 versus 8). More-
over, concerning the regiosubstitution of the aromatic ring, the
C-4 position plays an important role for the selectivity (7 versus
8). Among the longer chain compounds, an aldehyde group in
position C-110 makes the molecule more potent than the related
carboxyl moiety (4 versus 1). Furthermore, it is important to
point out that introduction of a hydroxyl group in positions C-
130 or C-140 in the chain increase the activity (2 versus 1 or 3
versus 4). On the other hand, a geranylgeranyl side chain and a
carboxyl acid as in 1 seems relevant for the antiplasmodial
activity.
cated by the molecular formula C27H36O7 determined by 13C
NMR and HR ESI(+)MS data. The 1H NMR spectrum (Table 1) dis-
played signals for three vinyl methyl groups (dH 1.67, 1.73 and
1.80); eight methylene groups, six of them as multipletes at dH
1.70–1.78 (2H), 2.05–2.16 (8H), 2.19–2.40 (2H), one as a doublet
at dH 3.38 (2H), and the other as two singlets (dH 4.96 and 5.01).
Additionally, signals for three olefinic protons at dH 5.15 (1H),
5.33 (1H), 7.13 (1H) as triplets and one methine proton character-
istic to a secondary hydroxyl group at dH 4.68, were observed.
Comparison of its 1H and 13CNMR data (Table 1) with those of com-
pound 2 suggested that one isoprene unit in the geranylgeranyl
chain is the most notable difference between both compounds.
The connectivity observed in the HMBC (Fig. 1) experiment of
the vinylic protons at dH 4.96, 5.01 (H-160) with dC 17.9 (C-200)/
81.9 (C-140), and the cross-peak between the signals at dH 4.68
(H-140) and dC 113.1 (C-160)/22.1 (C-120) located the hydroxyl
group on C-140, allylic to a terminal double bound. Analysis of
the COSY, HSQC and HMBC experiments permitted the complete
assignment of all the protons and carbons as shown in Table 1.
Riguera’s method have not been performed for 3 due to paucity
of material. However, since its structure is closely related to that
of 2, and its optical rotation was close to 0°, this compound must
also be almost racemic. Therefore, the structure of 3 was deter-
l
l
l
l
l
l
l
l
l
l
mined
as
3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-tri-
methyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid.
In the search for potential antiparasitic agents, all compounds
were assessed in vitro for their leishmanicidal (Leishmania spp.),
trypanocidal (Trypanosoma cruzi) and antiplasmodial (Plasmodium
falciparum) activities. The results obtained in the evaluation of the
prenylated benzoic acids against promastigote forms of three
strains of Leishmania, L. amazonensis, L. braziliensis and L. donovani
This is the second report of the antiparasitic properties of
prenylated benzoic acid derivatives (Lopes et al., 2008). This study
therefore points to the in vitro effectiveness of the antileishmanial
benzoic acid derivative 7, and the moderate activity of 9 and 1 as
antiplasmodial and trypanocidal agents, respectively. These find-
ings encourage further studies in order to evaluate a higher num-
ber of prenylated benzoic acid derivatives to perform SAR studies
focusing on obtaining more potent and selective antiparasitic
compounds.
Table 2
Leishmanicidal activity of compounds 2–9 in vitro on promastigote forms of
Leishmania spp.
Compounds
IC50 SD (l
g/ml)a
L. amazonensis
L. braziliensis
L. donovani
1
2
3
4
5
6
7
8
> 100
> 100
> 100
3. Experimental
63.1 1.0
64.2 0.5
77.0 0.5
56.1 3.6
56.8 0.9
50.1 0.9
27.0 4.1
17.8 1.1
10 0.7
40.7 0.8
39.0 1.5
68.5 1.5
33.7 0.5
44.5 0.9
6.5 0.4
27.0 1.9
17.8 0.4
10 0.7
46.8 1.1
43.9 1.6
53.9 0.1
42.9 2.5
41.8 2.6
50.3 1.1
27.0 1.5
17.8 0.7
10 0.7
3.1. General
Optical rotations were measured on a Perkin Elmer 241 auto-
matic polarimeter in CHCl3 at 20 °C. UV spectra were obtained on
a JASCO V-560 spectrophotometer in absolute EtOH. IR (film) spec-
tra were measured on a Bruker IFS 55 spectrophotometer. NMR
spectra were recorded in CDCl3 on a Bruker Advance 400 spectrom-
eter. The chemical shifts are given in d (ppm) with residual CDCl3
9
Controlb
a
Data are expressed as mean standard deviation of three determinations.
Pentamidine was used as positive control.
b

N. Flores et al. / Phytochemistry 70 (2009) 621–627
625
as internal reference and coupling constants in Hz. EIMS and HR
3.4. 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-
EIMS were obtained on a Micromass Autospec Spectrometer, and
HR ESIMS spectra were performed on LCT Premier XE Micromass
Electrospray spectrometer. Silica gel 60 (particle size 15–40 and
hexadecatetraenyl)-4,5-dihydroxybenzoic acid (1)
EtOh
Colorless oil; UV k
nm (loge): 213 (6.3), 258 (6.4); IR
max
63–200
l
m, Machery–Nagel) and Sephadex LH-20 (Pharmacia Bio-
vfmilmaxcmꢀ1: 3520–2560, 3407, 2925, 2854, 1681, 1612, 1442, 1380,
1300, 1216, 1196, 775; for 1H (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3) data, see Table 1. Positive HR ESIMS m/z
479.2415 (calc. for C27H36NaO6, 479.2410).
tech) were used for column chromatography, while silica gel 60
F254 (Machery–Nagel) was used for analytical and preparative
TLC. The spots were visualized by UV light and heating silica gel
plates sprayed with H2O–H2SO4–AcH (1:4:20). All solvents used
were analytical grade (Panreac). The optically pure (R)-(-)-
a
-
3.5. ( )-3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-
methoxyphenylacetic acid (MPA) was purchased from Sigma,
whereas the other reagents were purchased from Aldrich and used
without further purification.
2,6,10,14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid (2)
Colorless oil; ½a D20
ꢂ
: ꢀ3.5 (CHCl3, c 0.31); UV kEmtaOxh nm (log
e): 213
(6.3), 262 (6.4); IR vfmilmaxcmꢀ1: 3513–2593, 3309, 2925, 2854, 1731,
1679, 1604, 1442, 1302, 1215, 1016, 756; for 1H (400 MHz, CDCl3)
and 13C NMR (100 MHz, CDCl3) data, see Table 1. EIMS m/z (rel.
int.): 454 [M+ꢀH2O] (3), 436 (13), 410 (18), 392 (5), 377 (4), 287
(7), 271 (8), 232 (15), 205 (37), 161 (53), 123 (100), 93 (31), 81
(41). HR EIMS: [M+ꢀ18] 454.2355 (calc. for C27H34O6, 454.2350).
3.2. Plant material
Leaves of Piper heterophyllum Ruiz & Pav. (voucher specimen
GB-1922, March 1997) and Piper aduncum L. (voucher specimen
GB-1892, January 1997), were collected in Ixiamas, Abel Iturralde
Province, La Paz, Bolivia. Voucher specimens are deposited in the
Herbario Nacional de La Paz, Universidad Mayor de San Andrés,
La Paz, Bolivia, and were identified by Dr. R. Callejas, Herbarium
of the Antioquia University, Colombia.
3.5.1. Preparation of derivative 10
To a solution of compound 2 (5.6 mg, 0.012 mM) in acetone
(1 ml), potassium carbonate (24 mg) and dimethyl sulfate
(0.02 ml), were added and the reaction was stirred for 72 h at room
temperature. The reaction mixture was concentrated to remove
the organic solvent. Water (3 ml) was added and the product was
extracted using dichloromethane (3 ꢁ 3 ml). The organic layer
was dried over MgSO4, filtered and concentrated to yield an oil,
which was purified by preparative TLC (CH2Cl2/acetone, 9:1) to af-
ford 10 (3.5 mg, 0.0068 mM).
3.3. Extraction and isolation
The dry leaves (400.0 g) of P. heterophyllum were crushed and
extracted with EtOH, using a Soxhlet apparatus for 48 h. After
removing the solvent in vacuum, the extract was partitioned into
a CH2Cl2–H2O (1:1, v/v) solution, yielding the organic (50.0 g)
and aqueous (1.9 g) extracts. The soluble fraction in CH2Cl2 was
fractionated by vacuum–liquid chromatography (VLC) on silica
gel and eluted with gradient systems of increasing polarity of n-
hexane:EtOAc (0–100%), yielding five major fractions (A–E), com-
bined according to TLC detection. Fraction C (2.0 g) was submitted
to column chromatography (CC) on Sephadex LH-20 (1.30 ꢁ 4 cm),
using hexane:CHCl3:MeOH (2:1:1) as eluent affording five frac-
tions, C1–C5. Fraction C4 (130 mg) was purified by prep. TLC
(CH2Cl2:MeOH 5%) to give 6 (5 mg). Fraction D (4.5 g) was submit-
ted to flash CC over silica gel eluted with a gradient of n-hex-
ane:CH2Cl2 to give seven fractions D1–D7. Fraction D2 (547.0 mg)
was chromatographed using flash CC over silica gel (CH2Cl2:iso-
PrOH of increasing polarity 1–20%) to obtain six fractions,
D2.1–D2.6. Fraction D2.4 (63 mg) was purified by prep. TLC (n-hex-
ane:Et2O, 3:7) to give 2 (9.9 mg) and 3 (4.0 mg). Fraction D4
(178 mg) was submitted to prep. TLC (CH2Cl2:Et2O, 7:3) to afford
4 (8.4 mg) and 5 (12.0 mg). Fraction D5 (1.1 g) was subjected to
flash CC over silica gel (CH2Cl2:iso-PrOH of increasing polarity 1–
25%) to obtain six fractions D5.1 to D5.6. Compound 1 (6.6 mg)
was isolated from fraction D5.5 (25 mg) after purification by prep.
TLC (n-hexane:EtOAc, 1:1).
Leaves (154.0 g) of P. aduncum were crushed and extracted with
EtOH–H2O (70:30, v/v) at room temperature for 48 h. After remov-
ing the solvent in vacuum, the extract was partitioned in CH2Cl2-
H2O (1:1, v/v) solution, yielding the organic (7.6 g) and aqueous
(0.5 g) extracts. The organic extract was fractionated by VLC on sil-
ica gel and with gradient systems of increasing polarity n-hex-
ane:Et2O (0–100%), yielding six fractions (A–E). Fraction B (2.0 g)
was subjected to flash CC, which was eluted with Et2O containing
increasing amounts of EtOAc, to give three major fractions, B6
(156 mg), B8 (302 mg) and B9 (72 mg). B6 and B8 were submitted
to CC on Sephadex LH-20, using hexane:CHCl3:MeOH (2:1:1) as
eluent, yielding compounds 7 (20 mg) and 9 (33 mg), respectively.
Compound 8 (7 mg) was isolated from fraction B9 after purification
by prep. TLC (hexane:Et2O 1:1).
3.5.2. ( )-(2E,6E,10E)-12-[2,3-Dimethoxy-5-(methoxycarbonyl)
phenyl]-2-(2-hydroxy-4-methyl-3-pentenyl)-6,10-dimethyl-2,6,
10-dodecatrienoic acid (10)
film
max
½
a 2D0
ꢂ
: ꢀ2.9 (CHCl3, c 0.3); IR
v
cmꢀ1: 3520–2567, 2926, 2854,
1732, 1680, 1612, 1442, 1216, 775; 1H NMR spectral data
(400 MHz, CDCl3): d 1.62 (3H, s, H-180), 1.76 (3H, s, H-170), 1.77
(3H, s, H-160), 1.79 (3H, s, H-200), 2.04–2.25 (8H, m, H-40, H-50,
H-80 and H-90), 2.50 (1H, dd, J = 3.8, 12.6 Hz, H-120), 3.03 (1H, dd,
J = 6.1, 12.6 Hz, H-120), 3.39 (2H, d, J = 5.7 Hz, H-10), 3.88 (3H, s,
OMe), 3.91 (3H, s, OMe), 3.92 (3H, s, OMe), 5.16 (1H, t, J = 5.2 Hz,
H-60), 5.29 (1H, t, J = 5.6 Hz, H-20), 5.20–5.26 (2H, m, H-130 and
H-140), 6.70 (1H, t, J = 7.3 Hz, H-100), 7.47 (1H, s, H-2), 7.53 (1H, s,
13
H-6). C NMR spectral data (100 MHz, CDCl3): d 16.0 (q, C-180),
16.3 (q, C-170), 18.4 (q, C-200), 25.7 (q, C-160), 26.5 (t, C-50), 28.5
(t, C-10), 28.8 (t, C-90), 32.4 (t, C-120), 37.7 (t, C-80), 39.6 (t, C-40),
52.1 (q, COOMe), 55.9 (q, OMe-5), 60.6 (q, OMe-4), 74.3 (d, C-130),
111.2 (d, C-6), 122.4 (d, C-20), 123.8 (2 ꢁ d, C-140, C-2), 125.3 (d,
C-60), 125.4 (s, C-1), 126.6 (s, C-110), 131.1 (s, C-3), 133.5 (s, C-70),
136.2 (s, C-30), 139.5 (s, C-150), 140.1 (2 ꢁ s, C-5, C-100), 152.4 (s,
C-4), 167.0 (s, COOMe), 171.0 (s, C-190). EIMS m/z (rel. int.): 496
[M+ꢀH2O] (2), 468 (5), 465 (4), 394 (2), 350 (6), 287 (2), 205
(33), 123 (100), 93 (29), 81 (39). HR EIMS: [M+ꢀ18] 496.2827 (calc.
C30H40O6, 496.2820).
3.5.3. Preparation of Riguera0s esters
A
solution of (R)-MPA (8 mg, 0.0482 mM), 10 (3 mg,
0.0058 mM), 1,3-dicyclohexylcarbodiimide (6 mg) and a catalytic
amount of 4-dimethylaminopyridine in dry dichloromethane was
stirred at room temperature for 24 h, and the solvent was removed
to give a thick oil, which was purified by preparative TLC (n-hex-
ane-EtOAc, 6:4) to give 11 (2.1 mg, 0.0032 mM).
3.5.4. (R)-a-Methoxy-a-phenylacetyl derivative of 10 (11)
½
a 2D0
ꢂ
: ꢀ14.9 (CHCl3, c 0.1); 1H NMR spectral data (400 MHz,
CDCl3): d 1.63 (6H, s, H-180), 1.76 (6H, s, H-170), 1.78 (6H, s,

626
N. Flores et al. / Phytochemistry 70 (2009) 621–627
H-160), 1.79 (6H, s, H-200), 2.04–2.27 (16H, m, H-40, H-50, H-80 and
H-90), 2.47 (1H, dd, J = 3.7, 12.5 Hz, H-120), 2.52 (1H, dd, J = 3.8,
12.6 Hz, H-120), 3.01 (1H, dd, J = 6.0, 12.6 Hz, H-120), 3.04 (1H, dd,
J = 6.1, 12.5 Hz, H-120), 3.38 (4H, d, J = 5.7 Hz, H-10), 3.29 (3H, s,
OMe, MPA), 3.36 (3H, s, OMe, MPA), 3.86 (3H, s, OMe), 3.89 (3H,
s, OMe), 3.91 (6H, s, OMe), 3.93 (6H, s, OMe), 4.70 (1H, s, MPA),
4.71 (1H, s, MPA), 5.16–5.31 (6H, m, H-20, H-60 and H-140), 5.37
(2H, m, H-130), 6.70 (2H, m, H-100) 7.25–7.55 (14H, m, H-2 and
H-6, MPA). EIMS m/z (rel. int.): 662 [M+] (1), 616 (1), 496 (1), 410
(5), 287 (2), 205 (23), 161 (46), 123 (100. HR EIMS: [M+]
662.3461 (calc. for C39H50O9, 662.3455).
concentrations vs. percent inhibition. Chloroquine (0.1 lg/ml) was
used as a positive control. All test were performed in triplicate.
Acknowledgment
This investigation was supported by International Foundation
of Sciences (Sweden, Grant F/3408-1), DGCYT (CTQ2006-13376/
BQU), Program of Swedish Cooperation (UMSA-SIDA/TB-BRC,
ASD91) and PCI-Iberoamérica (A/010794/07, AECI) projects. Inter-
national collaboration was supported by project X.5 ‘‘Búsqueda,
Obtención
y Evaluación de Nuevos Agentes Antiparasitarios”
CYTED (Iberoamerican Program of Science and Technology for
Development, Subprogram X).
3.6. ( )-3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-trimethyl-
2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid (3)
Colorless oil; ½a D20
ꢂ
: + 5.2 (CHCl3, c 0. 16); UV kEmtaOxh nm (log
e): 212
max
Appendix A. Supplementary data
film
(6.2), 260 (6.3); IR
v
cmꢀ1: 3490–2592, 3360, 2925, 2854, 1714,
1679, 1630, 1444, 1379, 1194, 775 cmꢀ1; for 1H (400 MHz, CDCl3)
and 13C NMR (100 MHz, CDCl3) data, see Table 1. Positive HR ESIMS
m/z 495.2357 (calc. for C27H36NaO7, 495.2359).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2009.03.010.
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