Benzoic acid derivatives from Piper species and their antiparasitic activity

Article abstract of DOI:10.1021/np800104p

Piper glabratum and P. acutifolium were analyzed for their content of main secondary constituents, affording nine new benzoic acid derivatives (1, 2, 4, 5, 7, and 10-13), in addition to four known compounds (3, 6, 8, and 9). Their structures were elucidat

Full text of DOI:10.1021/np800104p

1538
J. Nat. Prod. 2008, 71, 1538–1543
Benzoic Acid Derivatives from Piper Species and Their Antiparasitic Activity
Ninoska Flores,†,‡ Ignacio A Jime´nez,† Alberto Gime´nez,‡ Grace Ruiz,‡ David Gutie´rrez,‡ Genevieve Bourdy,‡ and
Isabel L. Bazzocchi*,†
Instituto UniVersitario de Bio-Orga´nica “Antonio Gonza´lez”, UniVersidad de La Laguna, AVenida Astrof´ısico Francisco Sa´nchez 2,
La Laguna, 38206 Tenerife, Canary Islands, Spain, and Instituto de InVestigaciones Fa´rmaco Bioqu´ımicas, Facultad de Ciencias
Farmace´uticas y Bioqu´ımicas, UniVersidad Mayor de San Andre´s, AVenida SaaVedra 2224, Miraflores, La Paz, BoliVia
ReceiVed February 14, 2008
Piper glabratum and P. acutifolium were analyzed for their content of main secondary constituents, affording nine new
benzoic acid derivatives (1, 2, 4, 5, 7, and 10-13), in addition to four known compounds (3, 6, 8, and 9). Their structures
were elucidated on the basis of spectroscopic data. Riguera ester reactions and optical rotation measurements established
the new compounds as racemates. In the search for antiparasitic agents, the compounds were evaluated in Vitro against
the promastigote forms of Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum. Among the evaluated
compounds, methyl 3,4-dihydroxy-5-(3′-methyl-2′-butenyl)benzoate (7) exhibited leishmanicidal effect (IC50 13.8-18.5
µg/mL) against the three Leishmania strains used, and methyl 3,4-dihydroxy-5-(2-hydroxy-3-methylbutenyl)benzoate
(1), methyl 4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)benzoate (3), and methyl 3,4-dihydroxy-5-(3-methyl-2-butenyl)
benzoate (7) showed significant trypanocidal activity, with IC50 values of 16.4, 15.6, and 18.5 µg/mL, respectively.
Parasitic diseases such as leishmaniasis, malaria, and trypano-
somiasis have a significant impact in developing countries, affecting
hundreds of millions of people and which are the cause of a
mortality rate of several million per year.1 The existing chemo-
therapy for these diseases is not satisfactory due to its lack of
effectiveness and also the toxicity associated with long-term
treatments using empirically discovered drugs. Drug resistance and
different strain sensitivity to the available drugs are drawbacks for
clinically accessible chemotherapy. Therefore, in the absence of
vaccines, new chemotherapies are needed urgently to help in the
prevention and control of these parasitic diseases.2
methyl-3-butenyl)benzoate (3),11 methyl 4-hydroxy-3-(methyl-2-
butenyl)benzoate (6),12 4-hydroxy-3,5-bis(3-methyl-2-butenyl)ben-
zoic acid (8),13 and 4-methoxy-3,5-bis(3-methyl-2-butenyl)benzoic
acid (9),13 by comparison of their spectroscopic data with reported
data. In order to determine the configuration of the stereogenic
center of the new compounds, Riguera’s method,14 a modified
Mosher ester procedure, was applied to compounds 1 and 10, which
together with the optical rotation measurements established the new
compounds as racemates. We have investigated the isolated
compounds for their leishmanicidal, trypanocidal, and antiplasmo-
dial activities.
In spite of the great advances in modern medicine in recent
decades, plants still make an important contribution to health care,
and natural products have provided the inspiration for most of the
active ingredients in medicines.3 The genus Piper is widely
distributed in the tropical and subtropical regions of the world, and
species belonging to this genus are included in folklore medicine
of Latin America.4 In Bolivia, where resorting to medicinal plants
represents a primary health care measure of the native population,
the leaves of several species of Piper are used for the treatment of
parasitic diseases.5
Phytochemical investigations of Piper species have led to the
isolation of several classes of physiologically active compounds,4
such as flavanones and dihydrochalcones isolated from P. host-
mannianum with antiplasmodial activity6 and those isolated from
P. elongatum7 and P. rusbyi8 with leishmanicidal activity. Neo-
lignans from P. regnellii with trypanocidal activity have also been
reported.9 The evaluation of the essential oil from the leaves of P.
acutifolium against Callosobruchus maculates (a stored grain insect
pest) is the only study described for this species.10
As part of our research aiming to uncover antiparasitic com-
pounds from Bolivian Piper species, we have carried out a
phytochemical analysis of the leaves of Piper glabratum and P.
acutifolium. We now report the isolation and structural elucidation
of nine new benzoic acid derivatives (1, 2, 4, 5, 7, and 10-13) by
means of 1H and 13C NMR spectroscopic studies, including
homonuclear (COSY) and heteronuclear correlation experiments
(HSQC and HMBC). In addition, four known compounds were
isolated and were identified as methyl 4-hydroxy-3-(2-hydroxy-3-
Results and Discussion
Repeated chromatography of the dichloromethane extracts of the
leaves of P. glabratrum and P. acutifolium on Sephadex LH-20
and silica gel yielded nine new prenylated 4-hydroxybenzoic acids
(1, 2, 4, 5, 7, and 10-13).
Compound 1 was isolated as a white, amorphous solid and
showed the molecular formula C13H16O5 by analysis of its HREIMS
spectrum (m/z 252.1014, calcd 252.0998). The IR spectrum
indicated the presence of hydroxy (3409 cm-1) and carbonyl groups
(1692 cm-1) and an aromatic ring (1602 and 770 cm-1). The H
1
NMR (Table 1) spectrum exhibited signals for two aromatic protons
at δ 7.33 (d, J ) 1.9 Hz) and 7.48 (d, J ) 1.9 Hz) and two singlets
at δ 1.81 and 3.85 assigned to one methyl group attached to a double
bond and one methoxy group, respectively. The presence of an
oxymethine proton at δ 4.42 (dd, J ) 2.1, 8.5 Hz) associated with
the singlets at δ 4.88 and 5.99 suggested a hydroxylated isoprene
chain with a terminal double bond. The 13C NMR (Table 1)
spectrum showed signals for 13 carbon atoms, assigned as one
carbomethoxy (δ 167.2), six aromatic carbons (δ 114.9, 122.3,
124.6, 125.2, 145.5, 147.3), two signals corresponding to a double
bond at δ 145.9 (s) and 111.5 (t), one oxymethine carbon (δ 78.3),
one methoxy (δ 51.9), one aliphatic methylene (δ 37.7), and one
methyl (δ 18.1) carbon, suggesting a 3,4,5-trisubstituted benzoic
acid derivative with an isoprene chain. The assignments of the 1H
and 13C NMR data were based on 2D experiments and comparison
with reported data for known prenylated benzoic acid derivatives.11
The connectivity between the aromatic and aliphatic moieties was
revealed by analysis of the HMBC experiment (Figure 1A).
Therefore, correlation of signals at δ 7.48 (H-2)/7.33 (H-6) with
167.2 (CO2Me) indicated that the carboxyl group was attached to
C-1. The position of the isoprene side chain on the aromatic ring
* To whom correspondence should be addressed. Tel: +34-922-318594.
Fax: +34- 922-318571. E-mail: ilopez@ull.es.
† Universidad de La Laguna.
‡ Universidad Mayor de San Andre´s.
10.1021/np800104p CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/20/2008

Benzoic Acid DeriVatiVes from Piper Species
Journal of Natural Products, 2008, Vol. 71, No. 9 1539
was determined by a cross-peak of the signal at δH 4.42 (H-2′) and
δC 125.2 (C-5) and correlation between the signal at δH 2.99
(H-1′) and δC 124.6 (C-6)/147.3 (C-4). Moreover, the hydroxy group
was placed at C-2′ on the side chain, in agreement with the HMBC
correlations observed from the proton signals at δH 4.88, 5.99
(H-4′), and 1.81 (Me-5′) to the carbon signal at δ 78.3 (C-2′). These
data established its structure as methyl 3,4-dihydroxy-5-(2-hydroxy-
3-methyl-3-butenyl)benzoate.
derivative with an isoprene side chain. The NMR spectra indicated
that 5 is the 2-O-acetyl derivative of 1, as the presence of the
O-acetyl signals (δH 2.10 and δC 21.0, 171.9) and a less shielded
position of the signal at δΗ 5.20 assignable to H-2′ that appeared
in 1 at δH 4.42 were the most notable differences. Therefore, the
structure of 5 was determined as methyl 3-[2-(acetoxy)-3-methyl-
3-butenyl]-4,5-dihydroxybenzoate.
The structure of compound 7 was elucidated by physical methods
(Table 1), showing it to be related to 1, with the absence of the
secondary hydroxy group at C-2′ and isomerization of the double
bond on the side chain being the major differences. The isoprene
The structure of compound 2, with the molecular formula
C14H18O5 (HREIMS), was elucidated by physical methods, includ-
ing IR, UV, and 1D and 2D NMR data (Table 1). The 1H and 13
C
1
NMR data showed that 2 was related to compound 1, with the
presence of an ethoxy group [δH 4.32 (2H, J ) 7.2, Hz, H-1′′),
1.37 (3H, t, J ) 7.2 Hz, H-2′′) and δC 14.1 (C-2′′), 60.4 (C-1′′)]
chain was confirmed by the presence of signals in the H NMR
spectrum at δ 1.78 (6H, s), 3.38 (2H, d, J ) 7.2 Hz, H-1′), and
5.32 (1H, t, J ) 7.2 Hz, H-2′) and signals in the 13C NMR spectrum
at δ 17.9 (C-4′), δ 25.8 (C-5′), 29.2 (C-1′), 121.8 (C-2′), and 134.9
(C-3′). The linkage position of the isoprene side chain to the
aromatic ring was determined by an HMBC experiment, in which
correlations between the signals of H-2′/C-5 and H-1′/C-6, C-3′
were observed. Moreover, the complete assignment of the NMR
data was made by analysis of 2D NMR experiments and comparison
with those reported for related compounds,12 which established the
structure of 7 as methyl 3,4-dihydroxy-5-(3-methyl-2-butenyl)ben-
zoate.
1
instead of a methoxy group as found in 1. Assignments of the H
and 13C NMR signals performed by extended 2D NMR methods
confirmed the structure of 2 as ethyl 3,4-dihydroxy-5-(2-hydroxy-
3-methyl-3-butenyl)benzoate.
The HREIMS of compound 4 gave a molecular ion at m/z
250.1196, corresponding to the molecular formula C14H18O4, and
it was found to have a similar structure to 2 by comparison of their
NMR data (Table 1). The main difference observed was the loss
of the oxygenated aromatic carbon signal (δC 146.7) and the
presence of an additional aromatic methine with signals at δH 6.92
and δC 116.9. These data and 2D NMR experiments defined the
structure of 4 as ethyl 4-hydroxy-3-(2-hydroxy-3-methyl-3-bute-
nyl)benzoate.
Compound 10 was isolated as an amorphous powder. Its
molecular formula was established as C17H22O4 by analysis of the
HREIMS spectrum (m/z 290.1537, calcd 290.1518). The IR
spectrum indicated the presence of hydroxy (3370 cm-1), carboxylic
Compound 5, with the molecular formula C15H18O6 (HREIMS),
was shown by its IR, UV, and 1H and 13C NMR data (Table 1) and
2D NMR experiments to be a 3,4,5-trisubstituted benzoic acid
acid (3520-2670, 1680 cm-1), and aromatic (1601, 776 cm-1
)
groups. The 1H NMR (Table 2) spectrum exhibited signals for two
aromatic protons at δ 7.68 (d, J ) 1.3 Hz) and 7.79 (d, J ) 1.3

1540 Journal of Natural Products, 2008, Vol. 71, No. 9
Flores et al.
Hz), characteristic of a 1,3,4,5-tetrasubstituted aromatic ring.
Additional signals included three vinyl methyl groups as singlets
at δ 1.74, 1.76, and 1.83, two aliphatic methylenes at δ 2.86 (1H,
dd, J ) 1.3, 11.0 Hz), 2.99 (1H, dd, J ) 6.5, 11.0 Hz), and 3.38
(2H, d, J ) 5.4 Hz), one oxymethine proton at δ 4.43 (dd, J ) 1.3,
6.5 Hz), and three vinylic protons, two of them as singlets at δ
4.90 and 5.02 and the other as a triplet at δ 5.34 (2H, J ) 5.4 Hz),
indicating the presence of two double bonds in the side chains.
The 13C NMR (Table 2) spectrum exhibited signals for 17 carbon
atoms, showing in the low-field region one carboxylate carbon at
δ 170.1, six aromatic carbons (δ 121.8, 124.9, 129.5, 130.7, 131.5,
and 158.9), signals corresponding to two double-bond carbons (δ
111.2, 121.7, 133.2, and 146.2), and one secondary oxymethine
carbon at δ 77.9. These data indicated that compound 10 is a 3,4,5-
trisubstituted benzoic acid derivative with two isoprene chains. The
connectivity between aromatic and aliphatic moieties was revealed
by analysis of the HMBC spectrum (Figure 1B), showing correla-
tions between the signals at δH 7.68 (H-2)/7.79 (H-6) and δC 170.1
(CO2H), which indicated that the carboxyl group was attached to
C-1. The placements of the two isoprene side chains were based
on the long-range 3JH,C correlations, H-1′/C-4, C-6, C-3′ and H-1′′/
C-2, C-4, C-3′′. Analysis of the COSY, HSQC, and HMBC
experiments allowed full assignment of the 1H and 13C NMR
signals, and the structure of 10 was determined as 4-hydroxy-3-
(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)benzoic acid.
Figure 1. 1H-13C long-range connectivities (HMBC) for com-
pounds 1 (A) and 10 (B).
Treatment of those with (R)-(-)-R-methoxyphenylacetic acid
(MPA) afforded two monoester derivatives (16 and 17, respectively)
in approximately 1:1 ratio for each reaction. Thus, 1 and 10 were
each confirmed to be an equimolecular racemic mixture. Riguera
esterifications have not been performed for 2, 4, 5, 11, and 13 due
to the paucity of material. However, since their structures are closely
related to those of 1 and 10, and their specific rotations were close
to 0, it is assumed that these compounds are also racemic mixtures.
In the search for new antiparasitic natural compounds, all the
isolated compounds from the two Piper species studied, except for
compounds 2, 4, and 5 due to the small amounts obtained, were
investigated for their leishmanicidal, trypanocidal, and antimalarial
properties. The results of the evaluation of the compounds against
promastigote forms of Leishmania amazonensis, L. braziliensis, and
L. donoVani (Table 3) showed compound 7 as the most active, with
IC50 values of 18.2, 13.8, and 18.5 µg/mL, respectively. Compounds
1, 3, 6, 8, and 10 showed poor leishmanicidal activity (IC50
34.1-89.5 µg/mL), while compounds 9 and 11-13 were inactive
(IC50 > 100 µg/mL). Pentamidine was used as positive control (IC50
10 µg/mL). Regarding the trypanocidal activity (Table 3), com-
pounds 1, 3, and 7 showed significant activity (IC50 16.4, 15.6,
and 18.5 µg/mL, respectively) in comparison with benznidazole,
used as positive control (IC50 7.4 µg/mL), while the rest were
inactive (IC50 > 20 µg/mL). The compounds were also evaluated
for their antiplasmodial activity against a strain of chloroquine-
sensitive Plasmodium falciparum, and an IC50 > 5 µg/mL was
considered as inactive. Compounds 6 and 7 exhibited weak activity,
as the IC50 values were 2.8 and 4.1 µg/mL, respectively (Table 3).
Chloroquine was used as positive control (IC50 > 0.1 µg/mL).
Taking into account the results of the biological activities evaluated,
a preliminary structure-activity relationship revealed that the
prenylated 4-hydroxybenzoic acid derivatives with one side chain
(1, 3, 6, and 7) were substantially more active as potential
antiparasitic agents than those with two side chains (8, 9, 10, and
11).
Compound 11 was identified as 3-(2-hydroxy-3-methyl-3-bute-
nyl)-4-methoxy-5-(3-methyl-2-butenyl)benzoic acid, since its 1H and
13C NMR data (Table 2) were similar to those of compound 10.
The difference was confined to the presence of the signals at δH
3.82, δC 60.8 and 170.1, corresponding to a carboxymethyl group
on C-1.
Compound 12 exhibited the same molecular formula as 11.
Comparison of their NMR data indicated the modification of one
isoprene chain as the most notable difference, revealing the presence
of a chain with a trans double bond, two methyl groups, and a
tertiary hydroxy group. The HMBC experiment showed as the most
relevant connectivities on this side chain those of the two vinylic
protons at δH 6.56 (H-2′)/6.86(H-1′) with δC 130.7 (C-3) and
correlation of the signals at δH 1.46 (Me-4′, Me-5′) with δC 71.1
(C-3′). COSY, HSQC, and HMBC experiments permitted the
complete assignment of all the protons and carbons as shown in
Table 2. This evidence allowed definition of the structure of 12 as
3-[(1E)-3-hydroxy-3-methyl-1-butenyl)-4-methoxy-5-(3-methyl-2-
butenyl)benzoic acid.
The IR absorption bands (3442, 3494-2776, and 1699 cm-1
)
of compound 13 indicated the presence of hydroxy and carboxylic
acid groups, respectively. Resonances in the 1H and 13C NMR
spectra were assigned by the COSY, HSQC, and HMBC experi-
ments as shown in Table 2, which suggested a prenyl 3,4,5-
trisubstituted benzoic acid structure for 13. The NMR signals due
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 automatic polarimeter in CHCl3 at 20 °C, and
the [R]D are given in 10-1 deg cm2 g-1. UV spectra were obtained on
a JASCO V-560 spectrophotometer in absolute EtOH. IR (film) spectra
were measured on a Bruker IFS 55 spectrophotometer. NMR spectra
were recorded on a Bruker Avance 400 NMR spectrometer, and
chemical shifts are shown in δ (ppm) with TMS as an internal reference
and coupling constants in Hz. EIMS and HREIMS were obtained on a
Micromass Autospec spectrometer. Silica gel 60 (particle size 15-40
and 63-200 µm, Machery-Nagel) and Sephadex LH-20 (Pharmacia
Biotech) 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-HOAc (1:4:20). All solvents used were
analytical grade (Panreac). The optically pure (R)-(-)-R-methoxyphe-
nylacetic acid (MPA) was purchased from Sigma, whereas the other
reagents were purchased from Aldrich and used without further
purification.
to nine hydrogens and 10 carbons were observed in its 1H and 13
C
NMR spectra, whereas the molecular weight was found to be m/z
306 by the EIMS spectrum, suggesting that 13 has a symmetric
structure. On the basis of these findings the molecular formula was
deduced to be C17H22O5 (HREIMS). All the spectral data and
connectivity found in the HMBC experiment are compatible only
with the structure of 4-hydroxy-3,5-bis(2-hydroxy-3-methyl-3-
butenyl)benzoic acid for compound 13.
The specific rotations ([R]D) of the new compounds 1, 2, 4, 5,
10, and 13 were found to be close to 0, which suggested they were
racemates. Riguera esters14 prepared from derivatives 14 and 15
of compounds 1 and 10, respectively, confirmed this suggestion.
Theoretically, the Riguera reaction of an optically pure compound
will result in a single ester derivative being formed. Prior to
preparing the Riguera ester derivatives, the phenolic hydroxy and
carboxylic acid groups of compounds 1 and 10 were protected with
a methyl moiety, yielding derivatives 14 and 15, respectively.
Plant Material. Leaves of P. glabratum were collected in Ixiamas,
Abel Iturralde Province, La Paz, Bolivia, in October 1998. Leaves of
P. acutifolium were collected in south Yungas Province, La Paz, Bolivia,

Benzoic Acid DeriVatiVes from Piper Species
Journal of Natural Products, 2008, Vol. 71, No. 9 1541
Table 1. 1H-13C NMR (δ, CDCl3, J in Hz in parentheses) Dataa of Compounds 1, 2, 4, 5, and 7
1
2
4
5
7
a
a
a
a
a
δH
7.48 d (1.9)
δC
δH
δC
δH
δC
δH
δC
121.4 s
124.7 d 7.49 d (1.8) 114.5 d
δH
δC
1
2
3
4
5
6
122.3 s
114.9 d 7.51 d (2.0)
145.5 s
147.3 s
125.2 s
122.4 s
114.6 d 7.75 d (2.1)
146.7 s
146.5 s
124.9 s 6.92 d (8.4)
122.1 s
133.1 d 7.32 d (1.9)
125.0 s
160.0 s
116.9 d
122.2 s
122.5 s
146.9 s
143.6 s
143.1 s
146.8 s
127.3 s
7.33 d (1.9)
124.6 d 7.35 d (2.0)
124.2 d 7.85 dd (2.1, 8.4)
37.5 t 2.85 dd (2.2, 14.8)
2.99 dd (8.5, 14.8)
78.4 d 4.42 dd (2.2, 8.5)
145.7 s
130.2 d 7.46 d (1.9)
37.8 t 2.97 d (6.4)
114.2 d 7.43 d (1.8) 123.9 d
34.2 t 3.38 d (7.2) 29.2 t
1′ 2.84 dd (2.1,14.7)
2.99 dd (8.5, 14.7)
2′ 4.42 dd (2.1, 8.5)
3′
4′ 4.88 s 5.99 s
5′ 1.81 s
37.7 t
2.85 dd (2.0, 14.4)
3.01 dd (8.8, 14.4)
78.3 d 4.44 dd (2.0, 8.8)
145.9 s
77.9 d 5.20 t (6.4)
145.9 s
111.2 t 4.83 s 4.90 s
18.1 q 1.83 s
78.1 d 5.32 t (7.2) 121.8 d
140.9 s
114.2 t 1.78 s
17.8 q 1.78 s
134.9 s
17.9 q
25.8 q
111.5 t
4.90 s 5.01 s
111.4 t 4.89 s 5.01 s
17.9 q 1.82 s
18.1 q 1.82 s
a Data are based on DEPT, HSQC, and HMBC experiments.
Table 2. 1H-13C NMR (δ, CDCl3, J in Hz in parentheses) Dataa of Compounds 10-13
10
11
12
13
a
a
a
a
δH
δC
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
1′
121.8 s
131.5 d
124.9 s
158.9 s
129.5 s
130.7 d
37.9 t
124.8 s
130.9 d/
131.9 s
161.1 s
135.2 s
130.9 d/
36.5 t
124.9 s
126.9 d
130.7 s
160.1 s
135.4 s
130.6 d
120.4 d
7.76 s/
121.7 s
132.4 d/
126.9 s/
159.6 s
126.9 s/
132.4 d/
37.6 t/
7.68 d (1.3)
7.83 s
7.85 s
2.87 dd (8.6, 13.8)
2.95 dd (4.2, 13.8)
4.35 dd (4.2, 8.6)
7.82 d (1.3)
7.79 d (1.3)
8.07 d (1.3)
6.86 d (16.2)
7.76 s/
2.86 dd (1.3, 11.0)
2.99 dd (6.5, 11.0)
4.43 dd (1.3, 6.5)
2.93 d (6.5)/
2′
3′
4′
5′
1”
2”
3”
4”
5”
77.9 d
146.2 s
111.2 t
17.9 q
28.6 t
121.7 d
133.2 s
17.5 q
25.6 q
75.8 d
146.8 s
110.9 t
17.8 q
28.0 t
121.8 d
133.3 s
17.7 q
25.5 q
6.56 d (16.2)
139.8 d
71.1 s
4.38 d (6.5)/
76.8 d/
146.3 s/
110.9 t/
17.9 q/
37.6 t/
4.90 s 5.02 s
1.83 s
3.38 d (5.4)
5.34 t (5.4)
4.87 s 5.01 s
1.84 s
3.41 d (7.1)
5.29 t (7.1)
1.46 s/
29.6 q/
29.6 q/
28.1 t
4.87 s/ 5.00 s/
1.82 s/
1.46 s/
3.38 d (5.4)
5.27 t (5.4)
2.93 d (6.5)/
4.38 d (6.5)/
121.9 d
133.1 s
17.6 q
25.5 q
76.8 d/
146.3 s/
110.9 t/
17.9 q*
1.74 s
1.76 s
1.75 s
1.77 s
1.74 s/
1.74 s/
4.87 s/ 5.00 s/
1.82 s/
a Data are based on DEPT, HSQC, and HMBC experiments. / Overlapping signals.
Table 3. Antiparasitic Activity of Compounds 1, 3, 6, and 7-13 in Vitro on Leishmania spp., Trypanosoma cruzi, and Plasmodium
falciparum
IC50 ( SD (µg/mL)a
compound
L. amazonensis
L. braziliensis
L. donoVani
T. cruzi
P. falciparum
1
3
6
7
8
10
89.5 ( 0.8
78.2 ( 1.4
74.4 ( 2.7
18.2 ( 0.7
45.3 ( 1.6
67.6 ( 6.4
>100
81.9 ( 0.6
74.4 ( 0.4
74.4 ( 1.9
13.8 ( 0.5
34.1 ( 0.5
67.6 ( 3.4
78.8 ( 2.2
10.0 ( 0.7
81.9 ( 0.6
86.9 ( 1.0
62.5 ( 1.1
18.5 ( 0.2
40.0 ( 1.4
67.3 ( 2.5
>100
16.4 ( 0.8
15.6 ( 0.7
>20
18.5 ( 0.3
>20
>20
>20
7.4 ( 0.5
>10
10.0 ( 0.9
2.8 ( 0.6
4.1 ( 0.5
>10
>10
>10
12
controlb
10.0 ( 0.7
10.0 ( 0.7
0.1 ( 0.02
a Data are expressed as mean standard deviation of three determinations. b Pentamidine, benznidazole, and chloroquine were used as the reference
drugs for leishmanicidal, trypanocidal, and antiplasmodial tests, respectively.
in June 1998. Voucher specimens (GB-1877 and GB-1643, respectively)
are deposited in the Herbario Nacional de La Paz, Universidad Mayor
de San Andre´s, La Paz, Bolivia, and identified by Dr. R. Callejas,
Herbarium of the Antioquia University, Colombia.
(2.4 mg). Fraction IV (209 mg) was subjected to gel permeation column
chromatography on Sephadex LH-20 (60 × 2 cm), using CH2-
Cl2-MeOH (1:1) as eluent, to yield seven fractions (A-D). Fraction
B
(42 mg) was subjected to preparative TLC eluted with
Extraction and Isolation. The dry leaves (200 g) from P. glabratum
were crushed and extracted with EtOH-H2O (70:30, v/v) at room
temperature for 48 h. Evaporation of the solvent under reduced pressure
provided 35 g of crude extract, which was partitioned into a
CH2Cl2-H2O (1:1, v/v) solution, yielding the organic (19.2 g) and
aqueous (0.9 g) extracts. The organic extract was fractionated by VLC
on silica gel with gradient systems of increasing polarity of
n-hexane-EtOAc (0-100%), yielding eight fractions (1-8). Fraction
4 (1120 mg) was subjected to flash silica gel column chromatography
eluted with n-hexane containing increasing concentrations of Me2CO,
yielding five fractions (I-V). Fraction II (125 mg) was chromato-
graphed on a silica gel column, using a gradient from n-hexane to
EtOAc to yield five fractions (A-E). Fraction D (39 mg) was applied
to preparative TLC (CH2Cl2-Me2CO 9:1) to yield 3 (16.6 mg) and 4
n-hexane-Et2O (6:4), yielding 1 (15.5 mg), 2 (2.2 mg), and 3 (1.5
mg). Fraction 5 (4.7 g) was subjected to gel permeation column
chromatography on Shephadex LH-20 (1.30
×
4 cm), with
CH2Cl2-MeOH (1:1) as eluent, affording eight fractions (I-VIII).
Fraction III (923 mg) was subjected to flash silica gel column
chromatography, using a gradient elution from CH2Cl2 to Me2CO,
providing five fractions (A-E). Among them, fraction B (116.5 mg)
was rechromatographed on a silica gel column, eluted with n-hexane
containing increasing amounts of CH2Cl2 to give 5 (3.2 mg) and 6
(12.9 mg). Compounds 1 (13 mg) and 7 (19.5 mg) were obtained from
fraction D (58 mg) after preparative TLC, using CH2Cl2-Me2CO (9:
1) as eluent.
Leaves (400 g) of P. acutifolium were extracted with EtOH using a
Soxhlet apaparatus for 48 h. Evaporation of the solvent under reduced

1542 Journal of Natural Products, 2008, Vol. 71, No. 9
Flores et al.
pressure provided 55 g of crude extract, which was partitioned into a
CH2Cl2-H2O (1:1, v/v) solution, yielding the organic (38 g) and
aqueous (1.1 g) extracts. The organic extract was fractionated by VLC
on silica gel (63-200 mesh, 12 × 15 cm) and eluted with gradient
systems of increasing polarity of n-hexane to EtOAc to afford nine
fractions (1-9). Further flash column chromatography of fraction 5
(3.2 g) on silica gel, using a gradient elution from n-hexane to Et2O,
yielded seven fractions (I-VII). Compound 10 (7 mg) was obtained
by purification of fraction V (28 mg) after preparative TLC, using
n-hexane-Me2CO (8:2) as eluent. Fraction VII (26 mg) was subjected
to preparative TLC (n-hexane-Et2O, 3:7) to yield compounds 11 (6.8
mg) and 12 (10.1 mg). Analysis of fraction 7 (1.6 g), which was applied
to a flash silica gel column and eluted with CH2Cl2 containing increasing
volumes of Me2CO, afforded eight fractions (I-VIII). Fraction VII (117
mg) was subjected to silica gel column chromatography using a gradient
mixture of EtOAc in CH2Cl2, resulting in four fractions (A-D).
Compound 13 (6.7) was obtained from fraction D (30 mg) after
preparative TLC, using CH2Cl2-EtOAc (7:3) as eluent.
(100), 173 (55), 159 (39), 91 (50), 69 (53); HREIMS m/z 290.1537
(calcd for C17H22O4, 290.1518).
(()-3-(2-Hydroxy-3-methyl-3-butenyl)-4-methoxy-5-(3-methyl-2-
butenyl)benzoic acid (11): white, amorphous solid; [R]20 -4.7 (c
D
0.2, CHCl3); UV (EtOH) λmax (log ε) 207 (4.5), 242 (4.6) nm; IR (film)
1
νmax 3518-2694, 3469, 2927, 1694, 1603, 1205, 1002, 756 cm-1; H
NMR (CDCl3, 400 MHz) δ 3.82 (3H, s, OCH3), for other signals, see
Table 2; 13C NMR (CDCl3, 125 MHz) δ 60.8 (q, OCH3); 170.1 (s,
COOH), for other signals, see Table 2; EIMS m/z 304 (M+, 12), 286
(21), 247 (70), 232 (92), 199 (88), 149 (84), 91 (85), 71 (100); HREIMS
m/z 304.1608 (calcd for C18H24O4, 304.1596).
3-[(1E)-3-Hydroxy-3-methyl-1-butenyl]-4-methoxy-5-(3-methyl-
2-butenyl)benzoic acid (12). amorphous solid; [R]20 +1.0 (c 0.1,
D
CHCl3); UV (EtOH) λmax (log ε) 229 (4.1) nm; IR (film) νmax
3829-2790, 3433, 2970, 2928, 1696, 1601, 1380, 1265, 1202, 999,
757 cm-1; 1H NMR (CDCl3, 400 MHz) δ 3.76 (3H, s, OCH3), for other
signals, see Table 2; 13C NMR (CDCl3, 125 MHz) δ 61.0 (q, OCH3),
171.2 (s, COOH), for other signals, see Table 2; EIMS m/z 304 (M+,
12), 286 (83), 271 (44), 245 (47), 231 (75), 217 (100), 199 (83), 173
(69), 128 (63), 91 (53), 59 (59); HREIMS m/z 304.1675 (calcd for
C18H24O4, 304.1675).
(()-Methyl 3,4-dihydroxy-5-(2-hydroxy-3-methyl-3-butenyl)ben-
zoate (1): white, amorphous solid; [R]20 +1.2 (c 0.35, CHCl3); UV
D
(EtOH) λmax (log ε) 214 (3.4), 266 (4.5), 298 (4.6) nm; IR (film) νmax
1
3409, 2953, 1692, 1602, 1439, 1311, 1228, 901, 770 cm-1; H NMR
(()-4-Hydroxy-3,5-bis(2-hydroxy-3-methyl-3-butenyl)benzoic acid
(13): white, amorphous solid; [R]20D -5.0 (c 0.1, CHCl3); UV (EtOH)
(CDCl3, 400 MHz) δ 3.09 (1H, br s, OH), 3.85 (3H, s, OMe), 5.93
(1H, br s, OH), 9.20 (1H, br s, OH), for other signals, see Table 1. 13
C
λmax (log ε) 213 (4.6), 253 (4.7) nm; IR (film) νmax 3494-2776, 3442,
2925, 2854, 1699, 1605, 1455, 1378, 1292, 1201, 773 cm-1; 1H NMR
(CDCl3, 400 MHz) δ, see Table 2; 13C NMR (CDCl3, 125 MHz) δ
170.2 (s, COOH), for other signals, see Table 2; EIMS m/z 306 (M+,
5), 288 (7), 270 (14), 236 (48), 218 (100), 203 (32), 173 (24), 130
(18), 69 (57); HREIMS m/z 306.1521 (calcd for C17H22O5, 306.1467).
Preparation of Derivatives 14 and 15. To a solution of compound
1 (16 mg) or 10 (7 mg) in acetone (2 mL) were added K2CO3 (45 mg)
and dimethyl sulfate (0.05 mL), and the reaction was stirred for 72 h
at room temperature. The reaction mixture was concentrated to remove
the organic solvent. Water (5 mL) was added and the product was
extracted using CH2Cl2 (3 × 5 mL). The organic layer was dried over
MgSO4, filtered, and concentrated to yield an oil, which was purified
in preparative TLC (CH2Cl2-acetone, 9:1) to afford 14 (12 mg) and
15 (5 mg), respectively.
NMR (CDCl3, 125 MHz) δ 51.9 (q, OCH3), 167.2 (s, COOCH3), for
other signals, see Table 1; EIMS m/z 252 (M+, 28), 234 (55), 219 (81),
203 (19), 182 (100), 150 (47), 123 (18); HREIMS m/z 252.1014 (calcd
for C13H16O5, 252.0998).
(()-Ethyl 3,4-dihydroxy-5-(2-hydroxy-3-methyl-3-butenyl)ben-
zoate (2): amorphous solid; [R]20D -4.8 (c 0.12, CHCl3); UV (EtOH)
λmax (log ε) 218 (4.9), 265 (5.0), 299 (5.1) nm; IR (film) νmax 3433,
2923, 2853, 1712, 1601, 1448, 1303, 1221, 1056, 767 cm-1; 1H NMR
(CDCl3, 400 MHz) δ 1.37 (3H, t, J ) 7.2 Hz, H-2′′), 4.32 (2H, q, J )
7.2, Hz, H-1′′), for other signals, see Table 1; 13C NMR (CDCl3, 125
MHz) δ 14.1 (q, C-2′′), 60.4 (t, C-1′′), 166.2 (s, COOCH2CH3), for
other signals, see Table 1; EIMS m/z 266 (M+, 61), 248 (69), 233 (57),
221 (30), 196 (95), 167 (100), 149 (67), 123 (41), 57 (36); HREIMS
m/z 266.1154 (calcd for C14H18O5, 266.1154).
(()-Methyl 3,4-dimethoxy-5-(2-hydroxy-3-methyl-3-butenyl)ben-
(()-Ethyl 4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)benzoate
zoate (14): amorphous solid; [R]20 +0.4 (c 0.9, CHCl3); IR (film)
D
(4): white, amorphous solid: [R]20 +3.9 (c 0.2, CHCl3); UV (EtOH)
D
νmax 3459, 2953, 1693, 1602, 1439, 1311, 1228, 901, 772 cm-1; H
1
λmax (log ε) 260 (4.5) nm; IR (film) νmax 3409, 2923, 1710, 1690, 1607,
NMR (CDCl3, 400 MHz) δ 1.82 (3H, s, H-5′), 2.19 (1H, br s, OH),
2.81 (1H, dd, J ) 2.2, 14.7 Hz, H-1′), 2.96 (1H, dd, J ) 8.4, 14.7 Hz,
H-1′), 3.89 (3H, s, OCH3), 3.90 (6H, s, 2 × OCH3), 4.29 (1H, dd, J )
2.2, 8.4 Hz, H-2′), 4.84 (1H, s, H-4′), 4.96 (1H, s, H-4′), 7.49 (1H, s),
7.55 (1H, s); 13C NMR (CDCl3, 125 MHz) δ 17.8 (q, C-5′), 36.6 (t,
C-1′), 51.9 (q, OCH3), 55.6 (q, OCH3), 60.4 (q, OCH3), 75.8 (d, C-2′),
110.7 (t, C-4′), 111.8 (d, C-2), 124.6 (d, C-6), 125.2 (s, C-1), 132.0 (s,
C-5), 146.8 (s, C-3′), 151.1 (s, C-3), 152.1 (s, C-4), 166.5 (s, COOMe);
EIMS m/z 280 (M+, 4), 262 (37), 247 (32), 210 (100), 195 (51), 172
(40), 151 (15), 91 (11); HREIMS m/z 280.1309 (calcd for C15H20O5,
280.1311).
1284, 1180, 1018, 772 cm-1; 1H NMR (CDCl3, 400 MHz) δ 1.37 (3H,
t, J ) 7.1 Hz, H-2′′), 4.32 (2H, q, J ) 7.1 Hz, H-1′′), for other signals,
see Table 1; 13C NMR (CDCl3, 125 MHz) δ 14.2 (q, C-2′′), 60.4 (t,
C-1′′), 166.4 (s, COOCH2CH3), for other signals, see Table 1; EIMS
m/z 250 M+, 6), 232 (4), 217 (3), 205 (11), 180 (100), 151 (41), 134
(24), 107 (23), 71 (10); HREIMS m/z 250.1196 (calcd for C14H18O4,
250.1205).
(()-Methyl 3-[2-(acetoxy)-3-methyl-3-butenyl)-4,5-dihydroxyben-
zoate (5): amorphous solid; [R]20 -1.4 (c 0.2, CHCl3); UV (EtOH)
D
λmax (log ε) 213 (4.4), 265 (4.5), 298 (4.6) nm; IR (film) νmax 3391,
2953, 2926, 1712, 1603, 1441, 1375, 1308, 1233, 1020, 768 cm-1; 1H
NMR (CDCl3, 400 MHz) δ 2.10 (3H, s, H-2′′), 3.86 (3H, s, OCH3),
for other signals, see Table 1; 13C NMR (CDCl3, 125 MHz) δ 21.0
(q, C-2′′), 51.8 (q, OCH3), 166.9 (s, COOCH3), 171.9 (s, C-1′′), for
other signals, see Table 1; EIMS m/z 294 (M+, 2), 234 (78), 219 (100),
203 (21), 182 (34), 174 (51), 157 (43), 129 (21); HREIMS m/z 294.1107
(calcd for C15H18O6, 294.1103).
(()-Methyl 4-methoxy-3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-
methyl-2-butenyl)benzoate (15): amorphous solid; [R]20D -3.2 (c 0.3,
CHCl3); IR (film) νmax 3469, 2928, 1692, 1603, 1405, 1002, 775 cm-1
;
1H NMR (CDCl3, 400 MHz) δ 1.76 (3H, s, H-4′′), 1.78 (3H, s, H-5′′),
1.85 (3H, s, H-5′), 2.20 (1H, br s, OH), 2.87 (1H, dd, J ) 8.6, 13.8
Hz, H-1′), 2.97 (1H, dd, J ) 4.2, 13.8 Hz, H-1′), 3.41 (2H, d, J ) 7.1
Hz, H-1′′), 3.82 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.35 (1H, dd, J )
4.2, 8.6 Hz, H-2′), 4.88 (1H, s, H-4′), 5.03 (1H, s, H-4′), 5.30 (1H, t,
J ) 7.1 Hz, H-2′′), 7.79 (1H, s, H-2), 7.80 (1H, s, H-6); 13C NMR
(CDCl3, 125 MHz) δ 17.6 (q, C-4′′), 17.8 (q, C-5′), 25.5 (q, C-5′′),
28.1 (t, C-1′′), 36.5 (t, C-1′), 51.9 (q, OCH3), 60.8 (q, OCH3), 75.9 (d,
C-2′), 110.9 (t, C-4′), 121.9 (d, C-2′′), 123.8 (s, C-1), 130.9 (2 × d,
C-2, C-6), 131.9 (s, C-3), 133.3 (s, C-3′′), 135.2 (s, C-5), 146.8 (s,
C-3′), 161.1 (s, C-4), 167.3 (s, COOCH3); EIMS m/z 318 (M+, 5), 300
(19), 285 (14), 248 (100), 233 (25), 210 (22), 151 (12), 91 (5); HREIMS
m/z 318.1694 (calcd for C19H26O4, 318.1674).
Methyl 3,4-dihydroxy-5-(3-methyl-2-butenyl)benzoate (7): amor-
phous solid; UV (EtOH) λmax (log ε) 217 (4.3), 266 (4.4), 295 (4.5)
nm; IR (film) νmax 3471, 3371, 2923, 1692, 1604, 1440, 1303, 1237,
1
1010, 768 cm-1; H NMR (CDCl3, 400 MHz) δ 3.83 (3H, s, OCH3);
5.98 (1H, br s, OH), for other signals, see Table 1; 13C NMR (CDCl3,
125 MHz) δ 52.0 (q, OCH3), 167.3 (s, COOCH3), for other signals,
see Table 1; EIMS m/z 236 (M+, 100), 221 (5), 205 (18), 180 (100),
148 (18); HREIMS m/z 236.1034 (calcd for C13H16O4, 236.1049).
(()-4-Hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-
butenyl)benzoic acid (10): amorphous solid; [R]20 -3.6 (c 0.2,
Preparation of Riguera’s Esters. A solution of (R)-MPA (8 mg),
14 (6 mg) or 15 (4 mg), DCC (10 mg), and a catalytic amount of DMAP
in dry CH2Cl2 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-hexane-EtOAc, 6:4) to give 16 (4 mg) and 17 (3 mg),
respectively.
D
CHCl3); UV (EtOH) λmax (log ε) 209 (4.4), 258 (4.5) nm; IR (film)
νmax 3520-2670, 3370, 3167, 2924, 2855, 1680, 1601, 1441, 1409,
1286, 1214, 776 cm-1; 1H NMR (CDCl3, 400 MHz), see Table 2; 13C
NMR (CDCl3, 125 MHz) δ 170.1 (s, COOH), for other signals, see
Table 2; EIMS m/z 290 (M+, 5), 272 (29), 257 (45), 217 (38), 199

Benzoic Acid DeriVatiVes from Piper Species
Journal of Natural Products, 2008, Vol. 71, No. 9 1543
(R)-r-Methoxy-r-phenylacetyl derivative of 14 (16): 1H NMR
(CDCl3, 400 MHz) δ 1.68 (3H, s, H-5′), 1.78 (3H, s, H-5′), 2.88 (2H,
m, H-1′), 2.97 (1H, dd, J ) 4.6, 13.8 Hz, H-1′), 2.09 (1H, dd, J ) 5.2,
14.2 Hz, H-1′), 3.28 (3H, s, OCH3, MPA), 3.36 (3H, s, OCH3, MPA),
3.86 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.92 (6H, s, 2 × OCH3), 3.93
(3H, s, OCH3), 3.95 (3H, s, OCH3), 4.64 (1H, s, H-4′), 4.68 (1H, s,
H-4′), 4.70 (1H, s, MPA), 4.72 (1H, s, MPA), 4.87 (1H, s, H-4′), 4.90
(1H, s, H-4′), 5.48 (2H, m, H-2′), 7.26-7.55 (14H, m).
parasitemia was calculated for each concentration of sample compared
to the control.18 IC50 values were determined graphically by plotting
concentrations versus percent inhibition. Chloroquine (0.1 µg/mL) was
used as a positive control. All tests were performed in triplicate.
Acknowledgment. This investigation was supported by International
Foundation of Sciences (Switzerland, grant F/3408-1) and DGCYT
CTQ2006-13376/BQU and PCI-Iberoame´rica (A/4954/06, AECI) projects.
International collaboration was supported by project X.5 “Bu´squeda,
Obtencio´n y Evaluacio´n de Nuevos Agentes Antiparasitarios” CYTED
(Iberoamerican Program of Science and Technology for Development,
Subprogram X).
(R)-r-Methoxy-r-phenylacetyl derivative of 15 (17): 1H NMR
(CDCl3, 400 MHz) δ 1.60 (3H, s, H-5′), 1.75 (6H, s, H-4′′), 1.77 (6H,
s, H-5′′), 1.78 (3H, s, H-5′), 2.86 (2H, m, H-1′), 2.96 (1H, dd, J ) 4.0,
13.2 Hz, H-1′), 3.08 (1H, dd, J ) 4.1, 13.4 Hz, H-1′), 3.29 (3H, s,
OCH3, MPA), 3.35 (3H, s, OCH3, MPTA), 3.42 (4H, d, J ) 6.9 Hz,
H-1′′), 3.80 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.91 (3H, s, OCH3),
3.92 (3H, s, OCH3), 4.64 (1H, s, H-4′), 4.67 (1H, s, H-4′), 4.69 (1H,
s, MPA), 4.71 (1H, s, MPA), 4.80 (1H, s, H-4′), 4.89 (1H, s, H-4′),
5.31 (2H, t, J ) 6.9 Hz, H-2′′), 5.46 (2H, m, H-2′), 7.25-7.49
(14H, m).
Leishmanicidal Activity. The in Vitro leishmanicidal activity was
evaluated against promastigote forms of Leishmania braziliensis 2903,
L. amazonensis PH8, and L. donoVani PP75 (all from IBBA, Instituto
Boliviano de Biolog´ıa de Altura), which were grown at 28 °C in
stationary culture and were seeded at 1 × 104/100 µL/well in 96-well
flat bottom microtriter plates in Leishmania medium based on RPMI
1640. Test samples or standard drugs dissolved in DMSO were added
at a further 100 µL/well to give a final concentration of 100 µg/mL
and serial dilutions thereof.15 Leishmanicidal activity was expressed
as IC50 values (the concentration of a compound that caused a 50%
reduction in parasite viability). Pentamidine (10 µg/mL) (Sigma-
Aldrich) was used as positive control.
References and Notes
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Rodr´ıguez, A. J. Essent. Oil Res. 1996, 8, 689–691.
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Trypanocidal Activity. Epimastigote forms of Trypanosoma cruzi
Tulahuen strain were cultivated at 26 °C in liver infusion tryptose
medium (LIT), supplemented (5%) with heat-inactivated (56 °C for
30 min) fetal calf serum (technically modified from Chataing et al.).16
Parasites in logarithmic growth phase were distributed in 96-well flat-
bottom microtiter plates at a concentration of 5 × 104/mL. Test samples
or standard drugs dissolved in DMSO were added at a further 100 µL/
well to give a final concentration of 100 µg/mL and serial dilutions
thereof.15 The activity was measured by optic counting with an inverted
microscope and comparison with control wells. Benznidazole (7.4 µg/
mL) was used as the reference drug for this assay. All assays were
carried out in triplicate.
Antiplasmodial Activity. F-32 Tanzania (chloroquine sensitive)
strains of Plasmodium falciparum (kindly provided by Dr. Fandeur,
Pasteur Institute, Cayenne, France) were cultured according to Trager
and Jensen17 on glucose-enriched RPMI 1640 medium, supplemented
with 10% human serum at 37 °C. After 24 h of incubation at 37 °C,
the medium was replaced by fresh medium supplemented with the
compound to be evaluated at three different concentrations (0.1, 1, and
10 µg/mL), and incubation was continued for a further 48 h. On the
third day of the test, a blood smear was taken from each well, and
(16) Chataing, B.; Concepcio´n, J. L.; Lobato´n, R.; Usubillaga, A Planta
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