Antileishmanial activities of dihydrochalcones from piper elongatum and synthetic related compounds. Structural requirements for activity

Article abstract of DOI:10.1016/S0968-0896(03)00406-1

Two dihydrochalcones (1 and 2) were isolated from Piper elongatum Vahl by activity-guided fractionation against extracellular promastigotes of Leishmania braziliensis in vitro. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments. Derivatives 3-7 and 20 synthetic related compounds (8-27) were also assayed to establish the structural requirements for antileishmanial activity. Compounds 1-11 that proved to be more active that ketoconazol, used as positive control, were further assayed against promastigotes of Leishmania tropica and Leishmania infantum. Compounds 7 and 11, with a C₆-C₃-C₆ system, proved to be the most promising compounds, with IC₅₀ values of 2.98 and 3.65 μg/mL, respectively, and exhibited no toxic effect on macrophages (around 90% viability). Correlation between the molecular structures and antileishmanial activity is discussed in detail.

Full text of DOI:10.1016/S0968-0896(03)00406-1

Bioorganic & Medicinal Chemistry 11 (2003) 3975–3980
Antileishmanial Activities of Dihydrochalcones from Piper
elongatum and Synthetic Related Compounds.
Structural Requirements for Activity
Alicia Hermoso,a Ignacio A. Jimenez,b Zulma A. Mamani,a Isabel L. Bazzocchi,b,*
Jose E. Pinero,a Angel G. Ravelob and Basilio Valladaresa
a
´
Departamento de Parasitologıa, Ecologıa y Genetica, Facultad de Farmacia, Universidad de La Laguna, Avda. Francisco Sanchezs/n,
´
´
La Laguna, 38206 Tenerife, Canary Islands, Spain
´
b
´
Instituto Universitario de Bio-Organica Antonio Gonzalez, Universidad de La Laguna, Avda. Astrofısico Francisco Sanchez2,
´
´
La Laguna, 38206 Tenerife, Canary Islands, Spain
´
Received 31 March 2003; accepted 10 June 2003
Abstract—Two dihydrochalcones (1 and 2) were isolated from Piper elongatum Vahl by activity-guided fractionation against
extracellular promastigotes of Leishmania braziliensis in vitro. Their structures were elucidated by spectral analysis, including
homonuclear and heteronuclear correlation NMRexperiments. Derivatives 3–7 and 20 synthetic related compounds (8–27) were
also assayed to establish the structural requirements for antileishmanial activity. Compounds 1–11 that proved to be more active
that ketoconazol, used as positive control, were further assayed against promastigotes of Leishmania tropica and Leishmania
infantum. Compounds 7 and 11, with a C6–C3–C6 system, proved to be the most promising compounds, with IC50 values of 2.98
and 3.65 mg/mL, respectively, and exhibited no toxic effect on macrophages (around 90% viability). Correlation between the
molecular structures and antileishmanial activity is discussed in detail.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
resistance.3 Although alternative drugs or drug for-
mulations have been proved to be effective (e.g.,
amphotericin B liposomes and paramomycin ointment),
they present several drawbacks, such as their very high
cost and their scant availability.4 Therefore, the search
for novel, effective and safe drugs for the treatment of
the diseases has become a priority.5
Leishmaniasis is a group of prevalent diseases caused by
protozoan parasites belonging to the genus Leishmania.
This ailment affects some 12 million people in 88 coun-
tries with an annual incidence of about 2–3 million; it is
also considered that presently some 350 million of peo-
ple are at risk of infection.1 Recently, a dramatic
increase in the rate of Leishmania infections in human
immunodeficiency virus patients,2 together with the
development of drug resistance by the parasites,3 has
worsened this problem.
Recently, a series of synthetic6 and naturally occurring7
chalcone derivatives were reported to be potential
agents against Leishmania in a number of in vitro and in
vivo assays. Though a large number of synthetic com-
pounds has been tested, licochalcone A8 still remains
one of the few naturally occurring chalcones under
investigation. Thus, oxygenated chalcones, such as lico-
chalcone A, exhibit a strong antileishmanial activity
both in vitro and in vivo by interfering with the function
of the parasite mitochondria.9 The parasite fumarate
reductase has recently been proposed as the specific tar-
get for the action of chalcones.10 Although the use of
chalcones for the treatment of leishmaniasis may result in
the suppression of the immune system as an undesirable
Current therapies for the disease are still inadequate.
The recommended standard drugs for treatment are still
the pentavalent antimonial drugs Pestostam and Glu-
cantime, despite the requirement of long courses of
parenteral administration4 and increasing levels of
*Corresponding author. Tel.: +34-922-318576; fax: +34-922-318571;
e-mail: ilopez@ull.es
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00406-1

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A. Hermoso et al. / Bioorg. Med. Chem. 11 (2003) 3975–3980
side effect, a study of the structure–activity relationship6
revealed that it is possible to separate the antileishman-
ial and antilymphocyte properties of the chalcones.
20,60-Dihydroxy-40-methoxychalcone (DMC) isolated
from Piper aduncum inflorescences is reported to show a
significant activity in vitro against promastigotes and
amastigotes of Leishmania amazonensis, which was
improved by encapsulation in polymeric nanoparticles
in vitro and in vivo.11 However, in contrast to synthetic,
semisynthetic or plant-derived chalcones, only a few
dihydrochalcones have been investigated.
directed by activity-guided fractionation against promas-
tigote forms of L. braziliensis, gave the known compounds
1, 20,60-dihydroxy-40-methoxy-dihydrochalcone,14 and 2,
20,60,4-trihydroxy-40-methoxy-dihydrochalcone (asebo-
genin),14 as the active compounds, displaying IC50
values of 27.04 and 28.47 mg/mL, respectively, slightly
higher than that of ketoconazol (IC50 34.89 mg/mL),
used as positive control. Although molluscicidal and
antimicrobial activities have been previously reported
for compounds 1 and 2,14 no antiprotozoal activity has
been described. Their partial and total acetylated deri-
vatives (3–7) (Fig. 1) were prepared in order to study the
effect of the acetate groups on the activity. The structure
of derivatives 3–7, which were not previously described,
were determined by spectroscopic data (Tables 1 and 2),
including homonuclear and heteronuclear correlation
NMRexperiments (COSY, ORESY, HSQC and
HMBC).
Due to the limited availability of effective pharmaceu-
tical products, most people in areas where leishmaniasis
is endemic depend largely on traditional medicine.
Besides, traditionally, medicinal plants have already
provided valuable leads for potential antiparasitic com-
pounds.12 In Peru, two forms of leishmaniasis exist, the
cutaneous, ‘Uta’, which predominates in the Andes, and
the mucocutaneous form, ‘Espundia’, which pre-
dominates in the jungle, and both are attributed to
Leishmania braziliensis. The leaves of Piper elongatum
(Piperaceae), referred to as ‘matico’ or ‘mocco-mocco’,
are used by the inhabitants of Cusco, Peru, as a powder
to heal ulcers produced by the ‘Uta’.13
Furthermore, to investigate the relevance of the aro-
matic rings, and the substituents for the expression of
antileishmanial activity, we carried out a primary
screening for in vitro activity of a series of synthetic
related compounds (8–27, Fig. 2) against promastigotes
of L. braziliensis: compounds 8–15 having a C6–C3–C6
system (aryl-C3-aryl) with different substituents; com-
pounds 16–18 and 19–23, corresponding to related
To provide a scientific reason for the ethnomedicinal
use of P. elongatum, and as a part of a program in the
search for new antiprotozoal drugs, we carried out an
activity-guided fractionation of P. elongatum against
extracellular promastigotes of L. braziliensis in vitro.
Two dihydrochalcones (1 and 2)14 were isolated as the
active components. A primary screening for in vitro
antileishmanial activity of their derivatives 3–7 (Fig. 1)
and of 20 synthetic related compounds (8–27) (Fig. 2)
were also performed in order to establish the structural
requirements for antileishmanial activity. The most
active compounds, 1–11, were further assayed against
promastigotes of Leishmania tropica and Leishmania
infantum. The structure–activity relationships for anti-
leishmanial activity is discussed in detail.
Results and Discussion
Repeated chromatography on Si gel and Sephadex LH-20
of the EtOH extract of the aerial part of P. elongatum,
Figure 2. Chemical structures of synthetic compounds used in this
study.
Figure 1. Naturally dihydrochalcones (1 and 2) and its derivatives (3–
7) used in this study.

A. Hermoso et al. / Bioorg. Med. Chem. 11 (2003) 3975–3980
Table 1. 1H NMR(400 MHz) data ( d, CDCl3, J are given in Hz in parentheses) of 1–7
3977
Proton
1
2
3
4
5
6
7
H-2, H-6
H-3, H-5
H-4
7.19–7.31 ma
7.19–7.31 ma
7.19–7.31 ma
3.39 t (5.6)
3.02 t (5.6)
5.93 sa
7.00 d (8.3)
6.72 d (8.3)
7.21–7.32 ma
7.21–7.32 ma
7.21–7.32 ma
3.21 t (7.4)
3.02 t (7.4)
6.36 d (2.5)
6.17 d (2.5)
3.83 s
7.20–7.28 ma
7.20–7.28 ma
7.20–7.28 ma
3.05 t (5.3)
2.97 t (5.3)
6.57 sa
7.24 d (8.5)
7.00 d (8.5)
7.21 d (11.0)
7.02 d (11.0)
7.25 d (10.0)
7.01 d (10.0)
H-a
3.36 t (5.7)
2.86 t (5.7)
5.87 sa
5.87 sa
3.35 t (7.3)
3.00 t (7.3)
5.92 sa
5.92 sa
3.78 s
2.30 s
3.18 t (7.1)
3.00 t (7.1)
6.35 d (2.5)
6.16 d (2.5)
3.82 s
3.78 t (7.6)
3.39 t (7.6)
6.52 sa
6.52 sa
H-b
H-30
H-50
5.93 sa
6.57 sa
OCH3
Ac-4
Ac-20
Ac-60
3.79 s
3.58 s
3.80 s
3.77 s
2.29 s
2.29 s
2.16 sa
2.16 sa
2.15 sa
2.20 s
2.21 s
2.15 sa
aOverlapping signals.
Table 2. 13C NMR(100 MHz) data ( d, CDCl3) of 1–7a
of the parent 2, but also the cytotoxity was strongly
reduced. Besides, compounds 8–10 showed a high anti-
leishmanial activity (IC50<1 mg/mL), but also correlated
with a high toxicity on macrophages. On the other hand,
compound 11 with an IC50 of 3.65 mg/mL showed no
toxic effect on macrophages (91% viability at 3.6 mg/mL).
Position
1
2
3
4
5
6
7
C-1
C-2
C-3
C-4
C-5
C-6
C-a
C-b
141.5 s 132.6 s 140.9 s 141.4 s 139.3 s 138.5 s 136.9 s
128.3 db 129.4 db 128.1 db 128.4 db 129.5 db 129.2 db 129.5 db
128.5 dc 115.5 dc 128.5 dc 128.5 dc 121.4 dc 121.6 dc 121.7 dc
125.9 d 155.8 s 126.1 d 126.1 d 148.8 s 149.1 s 149.2 s
128.5 dc 115.5 dc 128.5 dc 128.5 dc 121.4 dc 121.6 dc 121.7 dc
128.3 db 129.4 db 128.1 db 128.4 db 129.5 db 129.2 db 129.5 db
45.5 t 46.1 t 44.7 t 45.4 t 45.5 t 44.6 t 32.0 t
30.5 t 30.0 t 29.8 t 29.7 t 29.9 t 29.7 t 29.7 t
Analysis of their structures allowed us to conclude that
the substitution of the methoxy group at C-40 by an
acetoxy group increased both the activity and toxicity (6
and 7 vs 8 and 9). However, replacement of the methoxy
group at C-40 by an O-tetra-acetyl-b-d-glucosyl group
increased the activity almost 3-fold without increasing
cytotoxity (4 vs 11). These results confirm the 2D-
C¼O 204.5 s 205.4 s 202.6 s 199.2 s 204.3 s 202.4 s 201.0 s
C-10
C-20
C-30
C-40
C-50
C-60
OCH3
Ac-4
104.7 s 105.5 s 108.2 s 120.6 s 104.8 s 108.3 s 121.4 s
165.5 sd 165.6 sd 166.5 s 149.4 sd 165.6 sd 166.6 s 150.2 sd
94.3 de 93.3 de 99.2 d 106.7 de 94.4 de 103.2 d 107.0 de
165.5 sd 164.9 s 164.8 s 161.2 s 163.3 s 165.5 s 160.3 s
94.3 de 93.3 de 103.1 d 106.7 de 94.4 de 99.3 d 107.0 de
165.5 sd 165.6 sd 152.4 s 149.4 sd 165.6 sd 152.4 s 150.2 sd
55.4 c 55.1 c 55.7 c 55.7 c 55.5 c 55.7 c 55.7 c
21.1 c 21.1 c 21.1 c
6
QSARanalysis from Liljefors et al., which suggests
that the C-40 position is highly relevant for the biologi-
cal activity. On the other hand, the antileishmanial
activity and the cytotoxicity remained unaffected by
replacement of the hydroxy or acetoxy groups at C-20
by an O-tetra-acetyl-b-d-glucosyl group (8 and 9 vs 10).
169.9 s 168.5 s 168.2 s
20.8 c
169.3 s
Ac-20
Ac-60
20.8 cf
168.8 sg
21.4 c 20.8cf
168.5 s 168.8 sg
21.5 c 20.4 c
169.6 s 169.5 s
To compare the differences in compound susceptibility
among parasite species, we assayed compounds 1–11 on
L. tropica and L. infantum (Table 3). Among the three
parasite species, L. braziliensis proved to be the most
susceptible line to the compounds assayed, with 7 exhi-
biting the highest relative activity. Compounds 6 and 7
were the most potent against L. infantum (IC50 9.11 mg/
mL) and showed low toxicity (94 and 90% viability at
5.0 and 2.4 mg/mL, respectively), while 4 was the most
interesting compound against L. tropica with an IC50 of
6.69 mg/mL and 91% viability at 10.0 mg/mL.
aValues are based on DEPT, HSQC and HMBC experiments.
bꢀgOverlapping signals.
compounds with CH3COCH2CH2Rand RCOCH 2CH3
moieties, respectively, and a series of synthetic com-
pounds with a benzocyclopentanone system (24–27) and
different substituents on the aromatic ring.
In comparison to ketoconazol as reference drug, com-
pounds 1–11, which are all C6–C3–C6, revealed pro-
nounced effects against promastigotes of L. braziliensis
(Table 3), while compounds 12–27 were inactive
(IC50>100 mg/mL). Analysis of the in vitro anti-
leishmanial activity of 1–7 indicates that the B-ring
substitution did not play a major role for the anti-
leishmanial activity, since compounds 1 and 2 showed
similar activity, although the toxicity on macrophages is
5-fold higher for 2. Substitution of hydroxyl groups for
acetate groups not only increased the antileishmanial
activity but also decreased the cytotoxicity on macro-
phages. Thus, activity of the monoacetylated, diacety-
lated, and triacetylated derivatives, 5, 6 and 7, are
around 2-, 7- and 8-fold, respectively, higher than that
Regarding molecular structure and function, these in
vitro studies showed that the antileishmanial activity of
the potentially active compounds (1–11) appears to have
structural features in common since all contain a C6–
C3–C6 system, which seems to be a requirement for
antileishmanial activity. In addition, these results, in
accordance with modelling studies and the effects of
chalcones on promastigotes of Leishmania major repor-
ted by Christensen et al.,15 support the fact that the
pharmacophore is the two aromatic rings, and the pro-
panone chain just functions as a spacer, and that the
ability to inhibit parasite growth apparently depends on
the presence and ratio of lipophilic/hydrophilic sub-
stituents at both aromatic rings, as concluded by Kayser

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A. Hermoso et al. / Bioorg. Med. Chem. 11 (2003) 3975–3980
Table 3. Antileishmanial activity of natural compounds 1 and 2, derivatives 3–7, and synthetic related compounds 8–11
Compd
IC50 (mg/mL)
Cytotoxicitya
L. braziliensis
L. tropica
L. infantum
(mg/mL)
% Viability
1
2
3
4
5
6
7
8
27.04
28.47
13.97
11.89
12.53
4.08
2.98
0.66
0.44
0.79
21.29
3.82
12.10
6.69
13.67
11.17
9.29
9.30
8.02
15.30
6.35
9.13
20.36
10.03
9.11
9.11
9.11
12.62
14.10
11.60
21.32
20.0
20.0
15.0
10.0
10.0
5.0
2.4
1.0
0.5
0.8
40
8
84
91
35
94
90
65
58
50
91
9
10
9.30
11.60
41.17
11
Ketoconazol
3.65
34.89
3.6
aCytotoxicity to murine macrophages J774.
et al.7 On the other hand, the relevance of the acetate
groups to the antileishmanial activity of dihy-
drochalcones, which markedly altered activity but also
reduced the inherent toxicity, represents an advance in
the search for novel antiprotozoal agents from natural
sources.
Bioassays
Parasite strain. L. braziliensis strain MHOM/PE195/
LQ7 was originally isolated at La Convencion Province,
Cusco, Peru, and identified by isoenzyme analysis. L.
tropica (MON 58/LEM 2578) and L. infantum (MON
183/LEM 2592) strains were also used in this study.
Earlier work and the results that we report in this paper
reinforce the interest for C6–C3–C6 compounds, chal-
cones, dihydrochalcones, and related compounds as
potential drugs against the leishmaniasis diseases at a
time when there is an urgent need for leads on new
innovative drugs. This study also provides a rational
explanation for the use of P. elongatum in antiparasitic
ethnomedicine.
In vitro effect on the promastigote forms of Leishmania
spp. For the in vitro studies samples were dissolved in
dimethyl sulfoxide (DMSO) and further dilutions were
made with RPMI 1640 medium. Promastigotes were
adapted for growth at 22 ꢁC in RPMI 1640 modified
medium (Gibco) and supplemented with 20% heat-
inactivated fetal bovine serum.16 Logarithmic phase
cultures were used for experimental purposes, and the in
vitro susceptibility assay was performed in sterilized 24-
well microtiter plates (CorningTM). To these wells were
added 2.5ꢂ104/well parasites, and the drug concen-
tration to be tested. The final volume was 500 mL in
each well.
Experimental
General
Optical rotations were measured on a Perkin-Elmer 241
automatic polarimeter and the [a]D are given in 10ꢀ1
Growth of promastigotes was monitored after 48 h by
counting the number of motile promastigotes micro-
scopically in a Neubauer camera. Percentage of inhibi-
tion and 50% inhibitory concentrations (IC50) were
calculated by linear regression analysis with 95% con-
fidence limits. Tests were performed at least in triplicate on
three different days in order to verify the results. Ketoco-
nazol was used as positive control (IC50 34.8 mg/mL).
deg cm2 gꢀ1. IRspectra were recorded in CHCl on a
Bruker IFS 55 spectrophotometer and UV spectra were
3
1
collected in absolute EtOH on a Jasco V-560. H and
13C NMRspectra were recorded on a Bruker Avance
400 NMRspectrometer at 400 MHz and 100 MHz,
respectively. EI–MS and HR-EI–MS were recorded on
a Micromass Autospec spectrometer. TLC 1500/LS 25
Schleicher and Schuell foils were used for thin-layer
chromatography. Silica gel (particle size 40–63 mM,
Merck) and Sephadex LH-20 (Pharmacia), were used
for column chromatography.
Cytotoxic assays. Murine macrophages J-774 (ATCC
TIB-67) cell line were grown in RPMI 1640 (GIBCO
BRL) medium supplemented with 10% SBFI, and
maintained at 37 ꢁC in 5% CO2 and 90% humidity.
Macrophages were resuspended in growth medium at a
final concentration of 1ꢂ166 cells/mL, placed in a 96-
well culture microtiter plate (100 mL/well), and incu-
bated with the test drug for 48 h. Cytotoxicity was
assessed using the colorimetric MTT [3-(3,4-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduc-
tion assay,17 and cytotoxic effects were expressed as a
percentage in cell viability at a concentration of the
Plant material
P. elongatum was collected in Valle de Urubamba,
Microcuenca de Cusichaca, Cusco, Peru, in February
1992.
A voucher specimen (no. CUZ-028801 A.
Tupayachi 2103) is deposited in the Vargas Herbarium,
Faculty of Biology, Universidad Nacional de San
Antonio Abad del Cusco, Peru.

A. Hermoso et al. / Bioorg. Med. Chem. 11 (2003) 3975–3980
3979
1
compound tested close to the IC50 value obtained
against L. braziliensis.
834; H NMR d: see Table 1; 13C NMR d: see Table 2;
EI–MS m/z%: 414 (M+, 23), 396 (19), 372 (23), 354
(37), 330 (45), 312 (57), 288 (44), 270 (64), 209 (17), 167
(100), 140 (20), 120 (44), 107 (37); HR-EI–MS: m/z
414.13223 (calcd for C22H22O8 414.13147).
Extraction and isolation
The aerial part of P. elongatum (460 g) was extracted
with ethanol in a Soxhlet apparatus, yielding 88 g of
residue which was chromatographed by dry flash chro-
matography on Si gel, using n-hexane–EtOAc mixtures
of increasing polarity to afford 35ꢂ100 mL frs, which
were reduced to 5 frs by TLC: A (0–5%, n-hexane–
EtOAc), B (5–15%), C (15–25%), D (25–45%), and E (45–
100%). The n-hexane–EtOAc (1:1) eluting fraction was
then chromatographed on Sephadex LH-20 (n-hexane–
CHCl3–MeOH, 2:1:1) and silica gel (n-hexane–1,4-dioxan,
3:2) to yield compounds 1 (38.0 mg) and 2 (16.4 mg).14
Synthetic compounds
Compounds 12–27 were purchased from Sigma and
used without further purification. The derivatives 8–11
were synthesized as follows, purified and authenticated
by analytical and spectroscopic methods.
Compounds 8 and 9. Compound 14 (23.0 mg) was trea-
ted under the conditions already described for the
synthesis of compounds 3 and 4, to give derivatives 8
(2.7 mg) and 9 (17.0 mg).
Acetylation of 1. Acetic anhydride (10 drops) was added
to compound 1 (24.0 mg) dissolved in pyridine (five
drops), and the mixture left at room temperature for 16
h. EtOH (3ꢂ2 mL) was added and carried almost to
dryness in a rotavapor, and this process was repeated
with CHCl3 (3ꢂ2.0 mL), and purified by preparative
TLC with a mixture of n-hexane–AcOEt (1:1), to give
derivatives 3 (8.5 mg) and 4 (12.7 mg).
Compound 10. Acetic anhydride (8 drops) and dimethy-
laminepyridine (2.0 mg) was added to compound 12 (6.0
mg) dissolved in pyridine (15 drops), and the mixture
was treated under the same conditions described above
for the acetylation of 1, to give derivative 10 (4.1 mg).
Compound 11. Compound 13 (8.0 mg) was treated
under the conditions already described for the synthesis
of 10, to give derivative 11 (6.8 mg).
Compound 3. Lacquer; UV lmax nm: 340, 276; IR nmax
cmꢀ1: 3545, 2928, 2853, 1769, 1735, 1610, 1369, 1211,
1
1154, 837; H NMR d: see Table 1; 13C NMR d: see
Table 2; EI–MS m/z%: 314 (M+, 25), 272 (22), 255 (14),
209 (3), 167 (100), 140 (28), 91 (10); HR-EI–MS: m/z
314.11505 (calcd for C18H18O5 314.11542).
Acknowledgements
This work has been supported by the DGES Projects,
BQU2000-0870-CO2-01 (I.A. and I.L.) and PPQ2000-
1655-CO2-01 (A.G.), and Project 238-56/99 from Uni-
versidad de La Laguna (A.H., J.E. and B.V.). We thank
Prof. Alfredo Tupayachi for kindly providing the plant
material.
Compound 4. Lacquer; UV lmax nm: 340, 269; IR nmax
cmꢀ1: 2923, 2853, 1774, 1686, 1617, 1459, 1367, 1182,
1
1038, 877; H NMR d: see Table 1; 13C NMR d: see
Table 2; EI–MS m/z%: 356 (M+, 6), 314 (21), 296 (12),
272 (50), 255 (27), 209 (29), 167 (100), 140 (56), 91 (28);
HR-EI/MS: m/z 356.12668 (calcd for C20H20O6
356.12599).
References and Notes
Acetylation of 2. Compound 2 (9.0 mg) was treated
under the same conditions as described above, to give
derivatives 5 (3.5 mg), 6 (1.9 mg) and 7 (1.5 mg).
1. Hirst, S. L.; Staplet, L. A. Parasitol. Today 2000, 16, 1.
2. Thakur, C. P.; Sinha, P. K.; Singh, R. K.; Hassan, S. M.;
Narain, S. Trans. R. Soc. Trop. Med. Hyg. 2000, 94, 696.
3. Faraut-Gambarelli, F.; Piarroux, R.; Deniau, M.; Giu-
siano, B.; Marty, P.; Michel, G.; Faugere, B.; Dumon, H.
Antimicrob. Agents Chemother. 1997, 41, 827.
4. Berman, J. D. Clin. Infect. Dis. 1997, 24, 684.
5. Mcgregor, A. Lancet 1998, 351, 575.
6. Nielsen, S. F.; Christensen, S. B.; Cruciani, G.; Kharazmi,
A.; Liljefors, T. J. Med. Chem. 1998, 41, 4819.
Compound 5. Lacquer; UV lmax nm: 338, 285; IR nmax
cmꢀ1: 3370, 3310, 2920, 2851, 1754, 1720, 1626, 1593,
1428, 1366, 1216, 1191, 836, 807; 1H NMR d: see Table 1;
13C NMR d: see Table 2; EI–MS m/z%: 330 (M+, 5),
285 (50), 242 (8), 209 (4), 167 (100), 150 (14), 140 (2),
135 (11); HR-EI–MS: m/z 330.10697 (calcd for
C18H18O6 330.11034).
7. Kayser, O.; Kiderlen, A. Phytother. Res. 2001, 15, 148.
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Compound 6. Amorphous solid; UV lmax nm: 339, 277;
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1187, 910, 832; 1H NMR d: see Table 1; 13C NMR d: see
Table 2; EI/MS m/z%: 372 (M+, 5), 330 (13), 312 (5),
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HR-EI–MS: m/z 372.11883 (calcd for C20H20O7
372.12090).
Compound 7. Lacquer; UV lmax nm: 339, 279; IR nmax
cmꢀ1: 2924, 2853, 1759, 1620, 1507, 1369, 1189, 886,
´
indıgena en la cuenca del rıo Madre de Dios, Peru´; Lacase, D.,
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