Trypanocidal structure-activity relationship for cis- and trans-methylpluviatolide

Article abstract of DOI:10.1016/j.phytochem.2008.04.002

The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi gGAPDH. The cytotoxicity of the compounds w

Full text of DOI:10.1016/j.phytochem.2008.04.002

Phytochemistry 69 (2008) 1890–1894
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Trypanocidal structure–activity relationship for cis- and trans-methylpluviatolide
R. da Silva a, J. Saraiva b, S. de Albuquerque b, C. Curti c, P.M. Donate d, T.N.C. Bianco a,
J.K. Bastos c, M.L.A. Silva a,
*
a Núcleo de Ciências Exatas e Tecnológicas, Universidade de Franca, Avenida Dr. Armando Salles de Oliveira, 2001, 14404-600 Franca, SP, Brazil
b Departamento de Análises Clínicas, Toxicológicas e Bromatológicas da Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
Avenida do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil
c Departamento de Ciências Farmacêuticas da Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
Avenida do Café, s/n, 14040-903 Ribeirão Preto, SP, Brazil
d Departamento de Química da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo,
Avenida Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro
against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi
gGAPDH. The cytotoxicity of the compounds was assessed by the MTT method using LLC-MK2 cells. The
effect of the compounds on peroxide and NO production were also investigated. The mixture of the trans
stereoisomers displayed trypanocidal activity (IC50 ꢀ89.3 lM). Therefore, it was separated by chiral HPLC,
furnishing the (+) and (–)-enantiomers. Only the (–)-enantiomer was active against the parasite (IC50
ꢀ18.7 lM). Despite being inactive, the (+)-enantiomer acted as an antagonistic competitor. Trans-meth-
ylpluviatolide displayed low toxicity for LLC-MK2 cells, with an IC50 of 6.53 mM. Furthermore, methyl-
pluviatolide neither inhibited gGAPDH activity nor hindered peroxide and NO production at the
evaluated concentrations.
Received 25 October 2007
Received in revised form 8 February 2008
Available online 12 May 2008
Keywords:
Lignans
Methylpluviatolide
Structure–activity relationship
Trypanocidal activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
mada, 1996; Umezawa et al., 1997a; Xu et al., 2001). Third, the
predominant enantiomers of furofuran, furan, and dibenzylbutane
The lignan class of compounds presents a conspicuous chemical
diversity, which varies substantially with respect to their enantio-
meric composition. Natural lignans usually occur as either pre-
dominantly one enantiomer or as enantiomeric mixtures of
various enantiomeric compounds (% e.e’. values) (Umezawa et al,
1997a).
lignans vary among plant species, and they differ even within the
organs of a single plant species (Arctium lappa) (Umezawa et al.,
1991; Umezawa et al., 1992; Umezawa et al., 1997b). Fourth, the
absolute configurations of the predominant enantiomers of various
lignans isolated from a single plant specimen are sometimes
different.
In the last decade, the search for antiprotozoal compounds from
natural sources has intensified (Rocha et al., 2005; Da Silva Filho
et al., 2004; Caniato and Puricelli, 2003). This search has been
mainly in plants, which continue to be a major source of biologi-
cally active metabolites that may provide lead compounds for
the development of new drugs.
The enantiomeric properties of lignans can be summarized into
four statements. First, dibenzylbutyrolactone lignans can be opti-
cally pure (>99% e.e.), while furofuran and furan lignans can be
found as a mixture of both enantiomers of varying enantiomeric
composition (Umezawa et al., 1997a). Secondly, most dibenzylbu-
tyrolactone lignans are levorotatory, except for the lignans present
in Thymelaeaceae plants and Selaginella doederleinii, which are dex-
trorotatory (Chen et al., 2001; Lin et al., 1994; Umezawa and Shi-
In nature, it is possible to find two enantiomeric forms of the
same lignan, but these forms bear different stereochemistry,
depending on the plant species in which it occurs. In the mean-
time, only one of the lactone lignan enantiomers, (+)- or (–), has
been found to occur in the same plant. Therefore, biological assays
carried out with either plant extracts containing lignan lactones or
its isolated lignans generally allow the evaluation of only one
enantiomer, making it impossible to determine whether the other
enantiomer displays activity.
Although lignan lactones are present in numerous plants used
in popular medicine (Bianchi et al., 1968; Broomhead and Dewick,
1990; Cho et al., 1999), their trypanocidal activity against Trypan-
osoma cruzi has only recently been discovered by Bastos et al.
(1999), who has investigated the trypanocidal activity of the Zanth-
oxyllum naranjillo lignans, and discovered one of the most powerful
compounds regarding trypanocidal activity, (-)-methylpluviatolide
(1) (Bastos et al., 1999; Silva et al., 2003), (Fig. 1).
* Corresponding author. Tel.: +55 16 3711 8871; fax: +55 16 3711 8878.
E-mail address: mlasilva@unifran.br (M.L.A. Silva).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.04.002

R. da Silva et al. / Phytochemistry 69 (2008) 1890–1894
1891
32.0 lM (39.2 1.0), 128.0 lM (55.0 5.6) and 256 lM
(67.9 0.0), with an IC50 (lM mLꢁ1) of 89.3.
O
O
OCH3
OCH3
OCH3
OCH3
O
O
Although the concentrations used by Bastos et al. (1999) dif-
fered from ours, the fact that the racemic mixture of the trans ste-
reoisomer evaluated this time displayed lower activity than the
pure compound evaluated before might indicate that the (+)-trans
enantiomer should compete for the active sites in the parasite and
might act as an antagonist in relation to the (–)-trans enantiomer.
On the basis of these results, a separation of the trans-stereoiso-
mer from the racemic mixture was undertaken by chiral HPLC
using an analytical Chiracel OJ (4.6 ꢂ 250 mm) column, aiming to
evaluate the trypanocidal activity of each enantiomer separately.
The chromatogram gave a well resolved peak separation, allowing
the isolation of both enantiomers. Therefore, it was possible to
compare the trypanocidal activity of both isomers, since in our pre-
vious work only the (–) isomer from Zanthoxylum narangillo was
isolated (Bastos et al., 1999). The results showed that the (+) –
trans-methylpluviatolide (1) displayed the following percentage
of lysis: 8.0 lM (5.3 2.5), 32.0 lM (7.6 3.4) and 128.0 lM
(9.9 2.5), with IC50 (lM mLꢁ1) of 1.3 ꢂ 106. The (–) – trans- meth-
ylpluviatolide (1) displayed the following percentage of lysis:
8.0 lM (40.6 3.6), 32.0 lM (52.3 4.3) and 128.0 lM
(79.7 0.0), with IC50 (lM mLꢁ1) of 18.7.
The results show that the (+)-enantiomer is completely inactive,
whereas the (–)-enantiomer displayed good activity, with an IC50
(lM) = 18.7 and 79.7% lyses, at concentrations lower than those
used by Bastos et al. (1999), which corresponded to 50 lg/mL,
IC50 = 1.3 lg and 100% lyses. These results indicate that, despite
being completely inactive, the (+)-enantiomer of (1) blocks the ac-
tion of the (–)-enantiomer when they are present in a racemic mix-
ture. It should be taken into consideration that the (+)-enantiomer
might bind to the active sites as a competitive antagonist, which
may be confirmed by comparison of the IC50 value (lM) of the race-
mic mixture with that of the (–)-enantiomer itself.
O
O
O
O
(1)
(2)
Fig. 1. Structural formulas of trans-methylpluviatolide (1) and cis-methylpluviato-
lide (2).
Lignans, one of the longest known classes of natural products,
have attracted much interest over the years on account of their
broad range of biological activities, such as antileishmanial (Barata
et al., 2000), antimalarial (Zhang et al., 2001), antitumor (Bastos
et al., 1996), and anti-inflammatory (Souza et al., 2004) activities,
among others. Recently, our group reported the significant trypan-
ocidal and antileishmanial activities of dibenzylbutyrolactone lign-
ans (Souza et al., 2005; Royo et al., 2003; Bastos et al., 1999). Such
results aroused the interest within our research group to study the
effect of the stereochemistry and the absolute configuration of
methylpluviatolide on its trypanocidal activity. To this end, meth-
ylpluviatolide was synthesized in its trans (1) and cis (2) racemic
forms (Fig. 1), allowing us to evaluate the trypanocidal activity
not only of a mixture of these two stereoisomers, but also of the
pure enantiomers, which were separated by chiral HPLC.
2. Results and discussion
The results obtained against T. cruzi show that the racemic cis-
stereoisomer (2) is inactive, while the racemic trans-stereoisomer
(1) displays trypanocidal activity, with an IC50 ꢀ89.3 lM. Further-
more, the latter stereoisomer (1) presents low toxicity for LLC-MK2
cells, with an IC50 ꢀ6.53 mM. (Fig. 2).
Some metabolic routes were evaluated, such as the methylpluv-
iatolide mechanism of action. In this regard, trypanosomatids are
highly dependent on glycolysis for ATP production (Opperdoes,
1987; Opperdoes and Michels, 2001). The enzyme glycosomal glyc-
eraldehyde-3-phosphate dehydrogenase (gGAPDH) catalyzes the
Our result is different from that obtained for the pure (–)-trans-
methylpluviatolide (1) by Bastos et al. (1999), who reported 100%
activity at a concentration of 50 lg and an IC50 value of 1.3 lg
mLꢁ1. This result indicates that the trans isomer of this compound
should display higher trypanocidal activity. However, it should be
pointed out that the obtained results were undertaken for the race-
mic mixture of cis and trans-methylpluviatolide isomers. The (+/–)-
cis (1) displayed the following percentage of lysis: 8.0 lM
(1.4 1.2), 32.0 lM (8.7 2.7), 128.0 lM (16.8 3.9) and 256 lM
(32.2 4.7), with an IC50 (lM mLꢁ1) of 641.0. The (+/–)-trans (2)
displayed the following percentage of lysis: 8.0 lM (24.6 1.9),
oxidative phosphorylation of D-glyceraldehyde-3-phosphate into
1,3-diphosphoglycerate in the presence of NAD+ and inorganic
phosphate, and can be one of the preferred steps for inhibition
(Bakker et al., 1999; Bakker et al., 2000). However, the obtained re-
sults demonstrated that methylpluviatolide does not exert inhibi-
tory activity on this enzyme, displaying 0% inhibition at 100 lM.
The effect of methylpluviatolide (1) on nitric oxide and H2O2
production is shown in Figs. 3 and 4, respectively. The trypanocidal
activity of several types of natural and synthetic compounds has
been partly attributed to reactive oxygen species generated by
the reduction of drugs (Uchiyama et al., 2005). Regarding the ob-
tained results, the production of NO by cells was significantly low-
er when treated with methylpluviatolide (Figs. 3 and 4). Likewise,
the literature reports that benzonidazole, a drug used for the treat-
ment of Chagas disease, also inhibits the production of NO by mod-
ifying the balance between pro and anti-inflammatory mediators,
with important consequences for the development of the disease
(Piaggio et al., 2001; Stoppani, 1999). Fig. 4 shows that methyl-
pluviatolide displays a weak effect in enhancing the peroxide pro-
duction by T. cruzi. Some drugs, such as nifurtimox and gentian
violet, act in the parasite by increasing the production of free rad-
icals (Raether and Hanel, 2003).
100
80
60
EC50=6.53mM
40
20
Moreover, T. cruzi is more sensitive to free radicals, as it has a
deficient antioxidant mechanism in comparison with host cells.
This is especially the case for hydrogen peroxide, as T. cruzi does
not produce catalase, an antioxidant enzyme responsible for the
conversion of hydrogen peroxide into water and molecular oxygen
-1.25 -1.00 -0.75 -0.50 -0.25
Log dose (mM)
0.00
0.25
0.50
Fig. 2. Cytotoxic potential of (–)-methylpluviatolide (1) for LLC-MK2 cells.

1892
R. da Silva et al. / Phytochemistry 69 (2008) 1890–1894
4. Experimental
4.1. Reagents, solvents and compound identification procedures
Sulfanilamide, L-glutamine, tetrazolium salt, Triton x, NaHCO3,
20,70-dichlorodihydrofluorescein diacetate (H2DCFDA), t-butyl
hydroperoxide and Hepes-KOH were purchased from Sigma–Al-
drich Co., St. Louis, MO, USA. Dimethyl sulfoxide (DMSO) isopropa-
nol and sucrose were purchased from Merck Co., whereas RPMI-
1640 and fetal calf serum were obtained from GIBCO. Penicillin
and streptomycin were acquired from Invitrogen. Optical rotations
were measured at k = 589 nm on a Schmidt–Haensch polartronic
HH8 polarimeter (Berlin, Germany) using a 1.0 cell. A high-perfor-
mance liquid chromatography (HPLC) apparatus equipped with an
LC-10ADVP pump, an SPD-M10AVP diode array detector and a
DGU-14A degasser (Shimadzu, Kyoto, Japan) was used for both iso-
lation and purity determination of the methylpluviatolide isomers,
using an analytical Chiracel OJ (4.6 ꢂ 250 mm) column at a mobile
phase flow rate (EtoH n-hexane: 65:35, v/v) of 0.9 mL/min and
sample volume injection of 20 lL. The solvents used were of chro-
matographic grade.
Fig. 3. Nitric oxide production of mouse peritoneal macrophages treated with m-
ethylpluviatolide (1). (C: cells control without T. cruzi infection; Ci: infected cells
control).
4.2. Preparation of compounds
T-BOOH
500
400
300
200
100
0
control
Cis and trans-methylpluviatolide were prepared from interme-
diate 3, which was obtained by a procedure described by Landais
et al. (1991) and Charlton and Chee (1997). Hydrogenolysis of 3
under the conditions described in Scheme 1 furnished the trans-
steroisomer (1) in 80% yield. Acetylation of 3 followed by an elim-
ination reaction with DBU produced a benzylidenebenzyl lignan,
which furnished the cis-steroisomer (2) in 78% yield after hydroge-
nation (scheme 1).
methylpluviatolide
Coupling constants of 15.2 Hz and 3.8 Hz between protons 8
and 80 confirmed the trans- and cis-geometries for stereoisomers
1 and 2, respectively.
The trans-methylpluviatolide racemic mixture was separated by
chiral HPLC, and the fraction corresponding to each peak was col-
lected and analyzed in the polarimeter for determination of the
specific rotation of each enantiomer. The peak with a retention
time of 36.20 min was identified as (+)-methylpluviatolide (1)
Y
Bolívia
Strain
Fig. 4. Effects of methylpluviatolide (1) on H2O2 produced by T. cruzi. (RFU: relative
fluorescence unit; T-BOOH: t-butyl hydroperoxide).
25
½aꢃD +20.8 (c 1.51, CHCl3), and the peak with a retention time of
25
43.70 min was identified as (–)-methylpluviatolide (1) ½aꢃD -20.8
(Turrens and Mccord, 2006). Therefore, we could conclude that the
trypanocidal mechanism of action of this class of compounds
might not be related to the increase in the production of peroxides.
Recently, we have described the racemic 70-oxi-methylpluviato-
lide, racemic 70-hydroxi-methylpluviatolide and racemic 70-oxi-6-
nitro-methylpluviatolide as potential respiratory chain complex I
inhibitors, a finding of possible relevance due to either the toxico-
logical or therapeutical importance of these compounds (Saraiva
et al., 2005).
(c 1.51, CHCl3).
4.3. Parasites and culture procedure
Trypomastigote forms of the Y strain of T. cruzi were used for
the trypanocidal assay and were cultivated in the LLMCK2 cell
lineage (Norval, 1979). The cells were cultivated in RPMI-1640
medium (GIBCO), supplemented with 2 mM L-glutamine, 10 mM
NaHCO3, 100 U/mL penicillin, 100 lg/mL streptomycin, and 5%
inactivated fetal calf serum. The culture was kept at 37 °C in an
atmosphere of 5% CO2 and 95% humidity. Blood samples contain-
ing trypomastigote forms were added to the culture cell at a ratio
of 3:1, the supernatant was removed after 24 h, and the medium
was replaced. After 7 days under the same culture condition, the
supernatant was removed and centrifuged, furnishing free trypo-
mastigote forms of the parasite for the bioassay. The free trypo-
mastigote forms were transferred to a 96-well microtitre plate,
each well was filled with 1 ꢂ 106 trypomastigote forms of the
parasite, and each test compound was added to the wells. After
24-h incubation, activity was evaluated by using a colorimetric
technique at 595 nm with the addition of a tetrazolium salt
[MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide], as described by Muelas-Serrano et al. (2000). Negative
(solvent plus medium) and positive (gentian violet at 250 lg/
Therefore, other metabolic routes should be evaluated to eluci-
date the mechanisms of action of (–)-methylpluviatolide.
3. Conclusion
In conclusion, it is possible to state that the trans-stereochemis-
try is the one responsible for the trypanocidal activity of methyl-
pluviatolide (1), probably because it allows the correct binding of
this compound to the active sites on the parasite, which is not pos-
sible with the cis-stereoisomer. As for the absolute configuration of
the trans-stereoisomers, the inactive (+)-enantiomer probably acts
as an antagonist, hindering the binding of the (–)-enantiomer to
the active sites on the parasite, therefore decreasing the trypanoci-
dal activity of methylpluviatolide when it is present as a racemic
mixture.

R. da Silva et al. / Phytochemistry 69 (2008) 1890–1894
1893
O
O
O
OH
8
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
a
O
O
b,c,d
O
8'
O
O
O
O
O
O
(2)
(3)
(1)
Reaction condition: (a) H2, 4 atm, Pd/C, EtOH, HClO4, 60 h, rt; (b) THF, Ac2O, Et3N,
DMAP, 2h, rt; (c) DBU, CH2Cl2, 5h, rt; (d) H2, 4 atm, Pd/C, EtOH, 4h, rt.
Scheme 1.
mL) controls were also run in parallel. All the assays were under-
taken in triplicate.
a concentration of 32 lM, followed by addition of 5 lM H2DCFDA,
and the system was incubated at room temperature for 35 min.
The parasites were then removed by centrifugation (8000g, 4 °C,
10 min), and the fluorescence of the supernatant was determined
at 485 nm as described by Fang and Beattie (2003). t-Butyl hydro-
peroxide and a parasite-containing solution of 10% DMSO were
used as positive and negative controls, respectively. The assays
were performed in triplicate.
4.4. Cytotoxicity assay
The cytotoxic action of the compound was evaluated by the
MTT method as described by Sieuwerts et al. (1995). In a 96-well
microtitre plate, LLC-MK2 cells (5 ꢂ 105 cells/well) were cultivated
in RPMI-1640 medium supplemented as described above. The
compound was evaluated at concentrations of 64, 256, 512, and
2560 lM for 24 h, at 37 °C, under a 5% CO2 and 95% humidity atmo-
sphere. After this period, the cells were incubated with MTT (5 mg/
mL) for 240 min, and the reaction was interrupted by addition of
acidified isopropanol. A microtitre plate reader (TECAN-SUNRISE)
was used to obtain the results (570 nm). The negative (cells and
solvent) and positive (cells treated with Triton X-100 20% solution)
cytotoxicity controls were run in parallel. All assays were carried
out in triplicate.
The formula % cytotoxicity = [1 – (Y – Md)/(N – M)] ꢂ 100 was
used to calculate the cytotoxicity percentage, where Y is the mean
optical density of wells with cells and different concentrations of
compound; Md is the mean optical density of wells with medium
and different concentrations of compound; M is the mean optical
density of wells with medium only; and N is the mean optical den-
sity of wells with cells and no compound (negative control). The le-
vel of 50% cellular cytotoxicity (CC50) was obtained by a sigmoidal
dose-response curve. Stock solutions were prepared by dissolving
the compound in pure dimethylsulfoxide (DMSO), and aliquots of
the stock solution were added to cultures to give a final concentra-
tion of DMSO lower than 0.2%.
4.7. Nitric oxide measurement
The effects on the NO production by mouse peripheral macro-
phages were evaluated using the method previously reported by
Giorgio et al. (1998). Briefly, peritoneal exudate cells were col-
lected from the peritoneal cavities of male balb/c mice, previously
injected intraperitoneally with 3% thioglycolate medium 96 h be-
fore, and washed with 6 mL of ice-cold phosphate-buffered saline
(PBS). Cells were cultured in 96-well-flat-bottomed plates at
5 ꢂ 105 cells/well. The cultures were infected with T. cruzi at a
10:1 parasite:cell ratio. The methylpluviatolide was added to the
medium at a concentration of 8, 32, and 128 lM. Cultures were
incubated at 37 °C in 5% CO2 for 24 h. Nitric oxide was measured
in the supernatants by using the Griess reaction (Green et al.,
1982). The Griess reagent was prepared by mixing equal volumes
of 1% sulfanilamide in 5% H2PO4 and 0.1% naphthylethylene dia-
mine dihydrochloride. Equal amounts of culture supernatant and
Griess reagent were combined, and incubated for 10 min at room
temperature. Absorbance measured at 540 nm was compared to
a sodium nitrite standard curve and data were expressed in lM.
Acknowledgements
4.5. Enzymatic inhibition studies
The authors are thankful to Dr. Mônica Tallarico Pupo from Fac-
uldade de Ciências Farmacêuticas de Ribeirão Preto-USP for her
collaboration with the gGAPDH studies, as well as Fundacßão de
Amparo à Pesquisa do Estado de São Paulo (FAPESP Process 04/
13368-7, 03/01475-0, 98/14956-7 and 05/01550-8), Coordenado-
ria de Aperfeicßoamento de Pessoal do Ensino Superior (CAPES),
and Conselho Nacional de Desenvolvimento Científico e Tecnológ-
ico (CNPq) for financial support.
The enzymatic activities of the T. cruzi gGAPDH enzyme have
been determined according to a previously reported procedure
by Vieira et al. (2001), using the forward reaction. The percentage
of remaining activity was calculated by comparison with an inhib-
itor-free control experiment.
4.6. Production of H2O2 and other peroxides
This activity was evaluated by quantification of the oxidation
product of H2DCFDA, a dichlorofluorescein compound (Carthcart
et al., 1983). To this end, trypomastigote forms of Y and Bolivian
strain of T. cruzi were cultured in the LLC-MK2 cell line (Hull
et al., 1962) and suspended (1 ꢂ 106 parasites/ml) in a medium
containing 125 mM sucrose, 65 mM KCl, and 10 mM Hepes-KOH,
pH 7.2. Then, methypluviatolide (1) was added to the medium at
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