Anti-leishmanial activity of neolignans from Virola species and synthetic analogues

Article abstract of DOI:10.1016/S0031-9422(00)00240-5

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkage

Full text of DOI:10.1016/S0031-9422(00)00240-5

Phytochemistry 55 (2000) 589±595
www.elsevier.com/locate/phytochem
Anti-leishmanial activity of neolignans from Virola species and
synthetic analogues$
Lauro E. S. Barata a,*, Lourivaldo S. Santos b, Pedro H. Ferri c, J. David Phillipson d,
Angela Paine e, Simon L. Croft e
a
Â
Instituto de Quõmica-Universidade Estadual de Campinas (UNICAMP), CP 6154, (13083-970), Campinas-SP, Brazil
b
Â
Departamento de Quõmica-CCEN, Universidade Federal do Para, CP 8600, (66075-110), Belem-PA, Brazil
Â
Instituto de Quõmica e GeocieÃncias, Universidade Federal de Goias, (74001-970), Goiania-GO, Brazil
Â
c
Â
Â
Ã
dCentre for Pharmacognosy, The School of Pharmacy, University of London, London WC1N 1AK, UK
eDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
Received 11 January 2000
Abstract
Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[20,60-dimethoxy-40-(E)-propenylphenoxy]-
phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether lin-
kages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amasti-
gotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 mM but inactive against intracellular
amastigotes.The natural neolignan from V. pavonis was active against promastigotes at 100 mM. The highest selective activity was
found in those compounds with sulphur bridges.The b-ketosul®de (3,4-dimethoxy)-8-(40-methylthiophenoxy)-propiophenone pro-
duced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for ®ve consecutive
days (P>0.05). # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Virola surinamensis; Virola pavonis; Myristicaceae; Leishmania donovani; b-Ketoethers; b-Ketosul®des; b-Ketoamines; Neolignans; Anti-
leishmanial activity
1. Introduction
and allopurinol are currently on clinical trial, results for
some have been equivocal while others still depend
upon parenteral administration. There remains an
urgent need for new therapeutic agents (Olliaro and
Bryceson, 1993; Berman, 1997). In studies to identify
new anti-leishmanial compounds, a range of plant pro-
ducts, including indole and isoquinoline alkaloids,
chalcones, quinones and terpenes, showed activity
against Leishmania spp. (Wright and Phillipson, 1990;
Iwu et al., 1994); licochalcone A and propylquinolone
derivatives look particularly promising (Chen et al.,
1994; Fournet et al., 1996). Diarylheptanoids, an iso-
¯avonoid (Araujo et al., 1998) and a chalcone were also
active in L. amazonensis (Torres-Santos et al., 1999a,b).
Advances in the ®ght against leishmaniasis using
natural products have been recently reviewed (Akendengue
et al., 1999).
Leishmaniasis is a severe debilitating disease, infecting
an estimated 1,400,000 people annually worldwide
(Ashford et al., 1992), many of whom live in rural areas
without access to treatment. The disease is caused by
haemo¯agellate protozoan parasites that survive and
multiply in macrophages in the mammalian host and are
transmitted by phlebotomine sand¯ies. The ®rst choice of
drugs, the pentavalent antimonials, sodium stibogluconate
and meglumine antimoniate, require parenteral admin-
istration under medical supervision, and side e€ects
frequently occur (Olliaro and Bryceson, 1993; Berman,
1997). Although a few new drugs, such as the lipid
amphotericin B formulations, paromomycin, ketoconazole
As part of a programme to identify novel anti-leish-
manial compounds, natural and synthetic neolignans,
derivatives and analogues, were tested for activity against
L. donovani in culture and animal models. Neolignans,
$
In memorium of Ralph A. Neale.
* Corresponding author. Tel.: +55-19-788-3153; fax: +55-19-788-
3023.
E-mail address: lbarata@iqm.unicamp.br (L. Barata).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00240-5

590
L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
which occur in the Myristicaceae and other primitive
plant families, are usually dimers of the oxidative cou-
pling of allyl and propenyl phenols (Gottlieb, 1978).
They have a wide range of biological e€ects including
antifungal, anti¯ammatory, neuroleptic, antihepatoxic,
anticancer and anti-PAF activities (MacRae and Tow-
ers, 1984; Lima et al., 1987; Barata et al., 1991; Santos,
1991). QSAR studies of synthetic neolignans and related
active compounds against L. donovani have recently
been correlated (Costa et al., 1995, 1999).
anilinium-propiophenone (23), and the b-ketosul®des 8-
(4-chlorophenoxy)-propiophenone (24), 3,4-dimethoxy-
8-(40-methylthiophenoxy)-propiophenone (25), 3,4-dimeth-
oxy-8-(40-chlorothiophenoxy)-propiophenone (26) and
3,4-dimethoxy-8-(thiophenoxy)-propiophenone (27), were
synthesized through Friedel-Crafts reaction of a ben-
zene derivative, bromination of the resultant aromatic
ketone with bromine, condensation in basic medium of
the appropriate a-bromoketone with aniline or a deri-
vative and with thiophenol or a derivative. These
furnished the b-ketoamines and the b-ketosul®des
(respectively) in good yield. Alternatively, the a-bromo-
ketone could be generated by hydro-bromination of
abundant Brazilian natural products such as iso-eugenol
or iso-safrole with NBS:H2O leading to a bromidrine
with posterior oxidation (Santos et al., 1992). All spec-
tral data of synthesized compounds were in agreement
with the expected products. The structure of b-keto-
sul®de 27 was con®rmed through X-ray analysis (Lar-
iucci et al., 1995).
The activity of compounds 1a and 2±27 was assessed
against L. donovani promastigotes and amastigotes in
vitro, (Tables 1 and 2), the activity of 8-phenoxy-aceto-
phenone (7) and compounds 24±27 was assessed in vivo.
Surinamensin 1a, a neolignan isolated from Virola
surinamensis and selected for its anti-schistosomal
activity (Barata et al., 1978), was active in vitro against
L. donovani promastigotes at 50 mM, but showed no
selective toxicity when tested against L. donovani amas-
tigotes in the mouse peritoneal macrophage model. In an
assay using extracellular promastigotes, 3,4-dimethoxy-8
-[30 -methoxy-40 -(E)-propenylphenoxy]-propiophenone
(2), 3,4-methylenedioxy-8-[30-methoxy-40-(E)-propenyl-
phenoxy]-propiophenone (3), 3,4-dimethoxy-8-(40-meth-
oxyphenoxy)-propiophenone (12), 3,4-dimethoxy-8-[30 -
methoxy-40-(E)-allylphenoxy]-propiophenone (16), and
compounds 19, 21 and 24±26 were active in the range of
30 and 100 mM (Table 1). The natural neolignan 20
from V. pavonis was active at 100 mM. The rest of the
compounds were inactive.
2. Results and discussion
The natural neolignans used in this study were iso-
lated from Virola surinamensis (Rol.) Warb and V.
pavonis (A.DC.) A.C. Smith leaves, (Myristicaceae),
collected in the Amazon rainforest, Brazil. Sur-
inamensin 1a and virolin 1b, biologically active 8.O.40
neolignans (Gottlieb, 1978; Oliveira and Sampaio, 1978),
were previously isolated from V. surinamensis (Barata et
al.,1978). The absolute con®guration of 1a was deter-
mined using diastereomeric O-methyl mandelic esters
(Santos and Barata, 1990). The 8.O.40-neolignan 3,4,5-
trimethoxy-8-[30,50-dimethoxy-40-(E)-propenylphenoxy]-
phenylpropane (20), was isolated from V. pavonis as
previously described (Ferri and Barata, 1992).
b-Ketoether compounds (2±18) were obtained as pre-
viously described (Santos and Barata, 1990; Barata et al.,
1991; Santos, 1991). 2-methyl-3-(40-hydroxy-30-methox-
yphenyl)- 1,4-(200-hydroxy)-benzodioxane (19) and 4-
hydroxy-3-methoxy-8-[30, 50-dimethoxy-40-(E)-propenyl-
phenoxy]-phenylpropane (21) were synthesized as
reported (Ferri and Barata, 1992). The b-ketoamines 8-
anilinium-propiophenone (22) and (3,4-dimethoxy)-8-
Table 1
In vitro anti-L.donovani promastigote activity of synthetic neolignan
analogues
Compounda,b
Inhibition at 100 mMc
Inhibition at 30 mM
In tests against amastigotes in macrophages, 1a, 2, 7-
hydroxy-8-phenoxy-phenylethane (4), 8-phenoxy-aceto-
phenone-oxime (6), 3,4-dimethoxy-8-(40-chlorophenoxy)-
propiophenone (15), 8-(30-methylphenoxy-acetophenone
(18), 19, 22 and 23 were active at 100 mM but also toxic
to macrophages, while 3,4-dimethoxy-8-(20-methoxy-
phenoxy)-propiophenone (14) and 15 were selectively
toxic to macrophages. 3, 7-Amine-8-phenoxy-phenyl-
ethane (5), 16, 3,4-dimethoxy-8-[20-( 10, 30-dimethoxy-
phenoxy)]-propiophenone (17) and 27 were active at 100
mM against intracellular amastigotes, while 8-(20-nitro-
phenoxy)-acetophenone (11) showed signi®cant activity
against intracellular amastigotes at 30 mM (Table 2)
although it was not active against promastigotes. Com-
pounds 24, 25 and 26, (all with a sulphur bridge) were
active against both amastigotes and promastigotes
2
++++
++++
++++
++
++++
+++
+++
3
12
16
19
21
24
25
26
+++
++
++
++
+++
++++
++
+++
a
Compounds 4±11, 13±15, 17, 18, 22, 23 and 27 were inactive at
100 mM.
b
Compound 20, a natural neolignan from V.pavonis, produced
+++ inhibition at 100 mM.
c
Key: ++++All promastigotes rounded up and non-motile after
48 h; +++ all promastigotes rounded up and only slight movement
visible after 48 h; ++ Moderate inhibition of promastigote motility
after 48 h; + Slight inhibition of promastigote motility after 48 h.

L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
591
Table 2
In vitro anti-L. donovani amastigote activity of synthetic neolignan analogues
Compound
%Ia at 100 mM Æ
%I at 30 mM Æ
%I at 10 mM Æ
ED50
Toxic to M1s at
S.E.
S.E.
S.E.
3
90.7Æ0.5
90.2Æ0.5
100Æ0
10.6Æ1.4
0Æ0
0Æ0
50.05 (54.26±46.39)
100 mM
300 mM
100 mM
300 mM
300 mM
Not toxic at 300 mM
Not toxic at 300 mM
Not toxic at 300 mM
Not toxic at 300 mM
5
0Æ0
11
16
17
24
25
26
27
91.9Æ0.6
0Æ0
0Æ0
91.9Æ0.3
41Æ6.7
0Æ0
0Æ0
0Æ0
100Æ0
46.3Æ5
70.6Æ3.3
90.2Æ0.9
0Æ0
0Æ0
27.69 (29.66±25.52)
24.32 (26.52±21.73)
100Æ0
0Æ0
100Æ0
0Æ0
83.3Æ1.5
%I at 27 mg
Sbv/mlÆS.E.
81.3Æ1.5
0Æ0
%I at 9 mg
Sbv/mlÆS.E.
77.4Æ5.2
%I at 3 mg
Sbv/mlÆS.E.
23.0 Æ3.8
IC5O
Na-stibogluconate
5.32 (5.8197±4.8182)
a
% I=% Inhibition of amastigotes.
(Tables 1 and 2) and showed the greatest selectivity. 8-
(20-methoxyphenoxy)-acetophenone (8), 8-(40-methox-
yphenoxy)-acetophenone (9), 8-(40-chlorophenoxy)-
acetophenone (10) and 3,4-dimethoxy-8-phenoxy-pro-
piophenone (13) were inactive at 100 mM against pro-
mastigotes and amastigotes. Some of the compounds
which showed in vitro activity were further tested
against L. donovani in the BALB/c mouse model. Only
compound 25 showed signi®cant anti-leishmanial activ-
ity in vivo, reducing the liver amastigote load by 42% (P
> 0.05) at a dose of 100 mg/kg/day for 5 days (Table 3).
load. Compounds 5, 11 and 17 were not active against
promastigotes, although they were active against amas-
tigotes. This may be due to biochemical or metabolic
di€erences between the two stages of the parasite, or to
variations in the intracellular concentration of the drug
by the extracellular promastigotes and the macrophages.
Preliminary mode of action studies on promastigotes
of b-ketosulphides compounds showed that they induce
aberrant non-motile forms, with displaced nuclei and
absent or reduced ¯agella, suggesting microtubule inhi-
bition. Further studies are needed in order to elucidate
the mechanism(s) of action of these compounds.
3. Conclusions
4. Experimental
b-Ketoethers 3, 5, 11, 16 and 17 and b-ketosul®des 24,
25, 26 and 27 were selectively toxic to amastigotes in
vitro, but 24, 25 and 26 showed the greatest selectivity,
suggesting that the C8 sulphur bond may play a part in
the anti-L. donovani activity of these compounds. How-
ever, only compound 25 was signi®cantly active in vivo,
producing the greatest reduction in amastigote liver
4.1. General experimental procedures
Mps. (uncorr.) were taken on a Ko¯er microscopic
apparatus. NMR spectra were determined on a Bruker
AW-80 instruments (80 MHz) and a 300 MHz in CDCl3
solution, using TMS as internal reference. Chemical
shifts are expressed as d values and coupling constants
(J) are expressed in Hertz (Hz). IR spectra were recor-
ded in KBr ®lm and measured with a Perkin-Elmer
model 399B spectrophotometer. EIMS were obtained
by direct probe insertion at 70 eV.
Table 3
In vivo activity of synthetic neolignan analogues and sodium sti-
bogluconate against L. donovani in BALB/c micea
Compound
Dose level (mg/kg/day for 5 days)
%I
7
100
100
100
26
4.2. Plant material
24
25
1
42
P > 0.05
19
Leaves of Virola pavonis (A.DC) A.C. Smith (Myr-
isticaceae) were collected in the Amazon forest by Dr.
Hipolito F. Paulino Filho, near the Guapore river, State
of Rondonia, Brazil, and authenticated by Dr. William
A. Rodrigues (Instituto Nacional de Pesquisa da Ama-
zonia). A voucher specimen is deposited in the herbar-
ium of INPA, Manaus, Brazil. Leaves of Virola
surinamensis (Rol.) Warb were collected in Belem, State
of Para, Brazil. A voucher specimen (No 166061) has
26
27
100
100
17
97
Na-stibogluconate
45 (mg Sbv/kg/day)
P > 0.05
74
P > 0.05
15 (mg Sbvkg/day)
a
P > 0.05 indicates the signi®cant di€erence between number
of parasites in the drug treated mice compared with the untreated
controls.

592
L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
been deposited in the herbarium of Empresa Brasileira
de Pesquisa Agropecuaria (EMBRAPA-Divisao de
Botanica), Belem-PA, Brazil.
(4.5 ml of solvent/mmol of thiophenol) was stirred for
10 min at room temp. After this period, a solution of a-
bromoketone in ethyl methyl ketone (1.5 ml of solvent/
mmol of ketone) was added dropwise and the reaction
mixture was treated until re¯ux began, this being main-
tained for 6±8 h. The cooled solution was ®ltered and
the residue washed with CH2Cl2. The resultant ®ltrate
was conc., diluted with H2O and extracted thoroughly
with CH2Cl2 (Â4). Organic extracts were combined,
washed with 5% NaOH solution, satd. NaCl soln. and
dried (Na2SO4). The solution was ®ltered, concentrated
under reduced pressure, to give after puri®cation, 78-
85% yields of the b-ketosul®des.
4.3. Isolation
Surinamensin 1a was isolated from the leaves of V.
surinamensis as previously described (Barata et al., 1978;
Santos and Barata, 1990). 3,4,5-Trimethoxy-8-[30,50-
dimethoxy-40-(E)-propenylphenoxy]-phenylpropane (20)
was isolated from V. pavonis as previously described
(Ferri and Barata, 1992).
4.4. General procedure for synthesis of ꢀ-ketoamines
4.5.1. 2-(4-Chlorophenoxy)-propiophenone 24
A solution of the a-bromoketone (1 equiv.) (Barata et
at., 1978; Santos and Barata, 1990; Santos, 1991) in
treated butanone (5 ml of solvent/mmol of ketone) was
added to a mixture of the aromatic amine (2.5 equiv.) in
butanone (5 ml of solvent/mmol of amine) and
NaHCO3 (1.1 equiv.). The reaction was stirred at 80ꢀC
for 3±6 h. It was then left to cool, and H2O was added
and the solution was extracted with CH2Cl2. The
organic phase was washed with 2% HCl solution, dried
(Na2SO4), ®ltered and evaporated at reduced pressure.
The crude residue was recrystallized from MeOH to
produce an 80±82% yield of the following b-ketoa-
mines.
The crude residue obtained from 4-chlorothiophenol
(1.40 g, 9.67 mmol) of K2CO3 (2.33 g, 16.99 mmol) and
of 8-bromo-propiophenone (2.0 g, 9.39 mmol) after 7 h
of reaction was recrystallized from MeOH yield 24 (2.02
g) as colorless crystals, mp 70.0±71.5ꢀC (78% yield).
KBr
max
IRꢁ
(cm 1): 3060, 2820, 1675 (CˆO), 1595, 1580; 1H
NMR spectral data (80 MHz, CCl4): 1.44 (3H, d, J=
7.0 Hz, H-9), 4.46 (1H, q, J= 7.0 Hz, H-8), 7.10±8.05
(9H, m, H-Ar); EIMS (probe) 70 eV, mlz (rel. int.): 276
[M]+ (34), 171 [C8H8SCl]+ (52), 143 [C6H5SCl]+ (11),
105 [C7H5O]+ (100).
4.5.2. (3, 4-Dimethoxy)-8-(40-methylthiophenoxy)-
propiophenone 25
4.4.1. 8-Anilinium-propiophenone 22
The crude residue obtained from 4-methylthiophenol,
(1.87 g, 15.08 mmol) K2CO3 (3.64 g, 26.37 mmol) and
3,4-dimethoxy-8-bromo-propiophenone (4.62 g, 14.62
mmol) after 7.0 h of reaction, was recrystallized from
The crude product obtained from 1.50 g (16.13 mmol)
of aniline, 0.60g (7.11 mmol) of NaHCO3 and 1.37 g
(6.46 mmol) of 3,4-dimethoxy-8-bromo-propiophenone
after 3 h of reaction was recrystallized from MeOH
a€ording 1.90 g of 22 as crystalline solid, mp 103.0±
MeOH to give of 25 (3.87g) as colourless crystals, mp
KBr
80.0±81.0ꢀC. IRꢁ
(cm 1): 3040, 2840, 1665 (CˆO),
max
105.0ꢀC (82% yield). H NMR spectral data (80 MHz,
1580, 1570, 1380, 1H NMR spectral data (80 MHz,
CDCl3): d 1.40 (3H, d, J =7.0 Hz, H-9), 2.30 (3H, s,
H-10), 4.36 (1 H, q, J= 7.0 Hz, H-8), 6.60±7.60 (6H, m,
H-Ar); EIMS (probe) 70 eV, m/z (rel. int.): 316 [M]+
(15), 165 [C9H9O3]+ (100), 151 [C9H11S]+ (12), 123
[C7H7S]+ (7).
1
CCl4): d 1.50 (3H, d, J=7.0 Hz, H-9), 4.50±4.80 (1 H,
m, NH), 5.10 (1H, q, J=7.7 Hz, H-8), 6.20±8.20 (8H, m,
H-Ar).
4.4.2. (3,4-Dimethoxy)-8-anilinium-propiophenone 23
The crude product obtained from 1.70 g (18.31 mmol)
of aniline, 0.68 g (8.10 mmol) of NaHCO3 and 2.00 g
(7.30 mmol) of 3,4-dimethoxy-8-bromo-propiophenone
after 6 h of reaction was recrystallized from MeOH
4.5.3. (3, 4-Dimethoxy)-8-(40-chlorothiophenoxy)-
propiophenone 26
The crude residue obtained from 4-chlorothiophenol,
(1.36 g, 9.43 mmol) K2CO3 (2.27 g, 16.48 mmol) and
3,4-dimethoxy-8-bromo-propiophenone (2.50 g, 9.16
mmol) after 8.0 h of reaction, was recrystallized
from MeOH to give 26 (2.52 g) as colorless crystals,
mp 90±94ꢀC (81.7% yield). IR nmKaBxr (cm 1): 3085,
producing 1.68 g of 23 as a crystalline solid, mp 109±
KBr
110ꢀC (80% yield). IRꢁ
(cm 1): 3370 (NH), 1670
max
(CˆO); 1H NMR spectral data (300 MHz, CDCl3:CCI4,
8:2): d 1.45 (3H, d, J=8.8 Hz, H-9), 3.89 (3H, s, OMe-
3), 3.84 (3H, s, OMe-4), 4.48 (IH, m, NH), 5.15(1H, q,
J= 8.8 Hz, H-8) 6.50±7.70 (8H, m, H-Ar).
1
2850, 1670 (CˆO), 1595, 1585, 1480. H NMR spectral
data (80 MHz, CDCl3): d 1.50 (3H, d, J =7.0 Hz, H-9),
3.86 (3H, s, OMe-3) 3.92 (3H, s, OMe-4), 4.59 (1H, q,
J=7.0 Hz, H-8), 6.70±7.80 (7H, m, H-Ar); EIMS
(probe) 70 eV, m/z (rel. int.): 336 [M]+ (10), 165
[C9H9O3]+ (100).
4.5. General procedure for synthesis of ꢀ-Ketosul®des
A solution of 1.03 equiv. of thiophenol or derivative
and 1.8 g of dry K2CO3 in treated ethyl methyl ketone

L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
593
4.5.4. (3, 4-Dimethoxy)-8-(thiophenoxy)-
propiophenone 27
CD 1 mice and maintained in RPMI 1640 medium with
10% heat-inactivated fetal calf serum and 50 mg/ml
1
The crude residue obtained from thiophenol, (2.07 g,
18.86 mmol) K2CO3 (4.55 g, 32.97 mmol) and 3,4-
dimethoxy-8-bromo-propiophenone (5.0 g, 18.31 mmol)
after 6.0 h of reaction was recrystallized from MeOH to
gentamycin at a density of 105 cells ml in eight well
tissue-culture chamber slides (Labtek Products, Miles
Laboratories) at 37ꢀC in a 5% CO2/air mixture. After
24 h, the macrophages were infected with L. donovani
amastigotes isolated from the spleen of an infected
hamster, at a ratio of 10:1.
Test compounds were added to the infected macro-
phages in fresh medium 24 h later, on day 2, and again
on days 4 and 7. Each compound was tested in quad-
ruplicate, initially at 300, 100, 30 and 10 mM. On day
nine, the macrophage monolayers were ®xed with
methanol and stained with Giemsa stain. The percen-
tage of infected cells was determined. Damage to mac-
rophages was assessed in order to determine toxicity.
Where selective anti-leishmanial activity was found, the
compound was re-tested at an appropriate range of
concentrations. ED50 values were calculated by linear
regression analysis with P95 ®ducial limits.
give 27 (4.70 g) as colorless crystals, mp 59.0±61.0ꢀC
KBr
(85% yield). IRꢁ
(cm 1): 3060, 2840, 1670 (CˆO),
max
1590, 1580, 1520, 1470, 1275; 1H NMR spectral data (80
MHz,CDCl3): d 1.52 (3H, d, J =7.0 Hz, H-9), 3.88 (3H,
s, OMe-3), 3.92 (3H, s, OMe-4), 4.60 (1 H, q, J =7.0
Hz, H-8), 6.70±7.80 (8H, m, H-Ar); EIMS (probe) 70
eV, mlz (rel. int.): 302 [M]+ (10), 165 [C9H9O3]+ (100).
4.6. Biological evaluation
The in vitro sensitivity of L. donovani (MHOM/ET/
67/L82) amastigotes to test compounds was determined
in a mouse peritoneal macrophage model (Neal and Croft,
1984). Mouse peritoneal macrophages were isolated from

594
L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
Acknowledgements
4.7. In vitro promastigote assays
Assays were carried out in 96-well microplates using
L. donovani promastigotes grown in Schneider's med-
ium with 10% fetal calf serum. Each compound was
tested in quadruplicate at 100, 30 and 10 mM with a
promastigote suspension at 106/ml per well, and incu-
bated at 24ꢀC for 48 h. Promastigote motility was
assessed by inverted microscopy.
Angela Paine received ®nancial support from the Sir
Halley Stewart Trust. Simon Croft received ®nancial
support from the UNDP/World Bank/WHO Special
Programme for Research and Training in Tropical Dis-
eases. We also acknowledge FAPESP for ®nancial support,
and CAPES and CNPq for fellowships to LSS and PHF.
4.8. In vivo testing against Leishmania donovani
References
Female BALB/c mice (B and K Ltd.), 18±20 g, were
infected by the tail vein with 5Â 106 amastigotes of L.
donovani derived from a hamster spleen. One week after
infection, mice were individually dosed subcutaneously,
once per day for ®ve days, with compounds 7, 24, 25, 26
and 27 suspended in 0.25% cellacol. Three days after
completion of drug treatment, mice were sacri®ced,
livers were removed, weighed, and smears prepared. The
liver amastigote load of untreated and treated mice was
calculated from liver weight (mg)Ânumber of amasti-
gotes per 500 liver cells.
Araujo, C.A.C., Alegro, L.V., Leon, L.L., 1998. Antileishmanial
activity of compounds extracted and characterized from Cen-
trolobium sclerophyllum. Phytochemistry 49, 751±754.
Akendengue, B., Ngou-Milama, E., Laurens, A., Hocquemiller, R.,
1999. Recent advances in the ®ght against leishmaniasis with natural
products. Parasite. J. Soc. Francaise de Parasitologie 6, 3±8.
Ashford, R.W., Desjeux, P., de Raadt, P., 1992. Estimation of popu-
lation at risk of infection and number of cases of leishmaniasis.
Parasitol. Today 8, 104±105.
Barata, L.E.S., Santos, L.S., Fernandes, A.M.A.P., Ferri, P.H., Paulo,
M.Q., Neal, R., Jourdan, M.C., 1991. Natural and synthetic bioac-
tive neolignans. Abstracts of II Brazilianisch-Deustsches Sympo-
sium fur Natursto€chemie, Hannover, pp. 18±21.

L.E.S. Barata et al. / Phytochemistry 55 (2000) 589±595
595
Barata, L.E.S., Baker, P.M., Gottlieb, O.R., Ruveda, E.A., 1978.
Neolignans of Virola surinamensis. Phytochemistry 17, 783±786.
Berman, J.D., 1997. Human leishmaniasis: Clinical, diagnostic and
chemotherapeutic developments in the last 10 years. Clinical Infec-
tious Diseases 24, 684±703.
microbiana de neolignanas 8.O.40 e derivados sinteticos-l. Cienc.
Cult. e Saude 9 (2), 55±57.
MacRae, W.D., Towers, G.H.N., 1984. Biological activities of lignans.
Phytochemistry 23, 1207±1220.
Neal, R.A., Croft, S.L., 1984. An in vitro system for determining the
activity of compounds against the intracellular amastigote form of
L. donovani. J. Antimicrob. Chemother. 14, 463±475.
Oliveira, M.M., Sampaio, M.R.P., 1978. Acao da neolignana virolina e
derivados em leucemia experimental. Ciencia e Cult. (Suppl.), 32, 104.
Olliaro, P.L., Bryceson, A.D.M., 1993. Practical progress and new
drugs for changing patterns of leishmaniasis. Parasitol. Today 9,
323±328.
Chen, M., Christensen, S.B., Theander, T.G., Kharazmi, A., 1994.
Antileishmanial activity of licochalcone A in mice infected with
Leishmania major and in hamsters infected with Leishmania dono-
vani. Antimicrorobial Agents and Chemotherapy 38, 1339±1344.
Costa, M.C.A., Barata, L.E.S., Takahata, Y., 1995. SAR analysis of
synthetic neolignans and related compounds which are antileishma-
niasis active compounds using pattern recognition methods.
Theochem Ð J. Mol. Struct. 340, 185±192.
Santos, L.S., 1991. PhD thesis. Synthesis and biological activities of
8.O.40 neolignans, derivatives and correlated compounds. Campi-
nas, SP, Brazil.
Costa, M.C.A., Barata, L.E.S., Takahata, Y., 1999. Conformation of
Neolignans that binds to the arginine residue in adenosine kinase
from Leishmania donovani. Theochem Ð J. Mol. Struc. 464, 281±287.
Ferri, P.H., Barata, L.E.S., 1992. Neolignans and a new phenylpro-
panoid from Virola pavonis leaves. Phytochemistry 31, 1375±1377.
Fournet, A., Ferreira, M.E., Rojas de Arias, A., Torres de Ortiz, S.,
Fuentes, S., Nakayama, H., Schinini, A., Hocquemiller, R., 1996. In
vivo oral and intralesional administration of 2-substituted quino-
lines in experimental treatment of new world cutaneous leishma-
niasis caused by Leishmania amazonensis. Antimicrobial Agents and
Chemotherapy 40, 2447±2451.
Santos, L.S., Barata, L.E.S., 1990. Determination of absolute con®g-
uration of (+)- surinamensin via diastereomeric o-methyl mandelic
esters. J. Braz. Chem. Soc. 1, 47±49.
Santos, L.S., Ferreira, L.B., Barata, L.E.S., 1992. New synthetic route
to 8.O.40 neolignans using abundant Brazilian natural products. The
5th Brazilian Meeting on Organic Synthesis, Campinas-SP-Brazil,
75.
Torres-Santos, E.C., Moreira, D.L., Kaplan, M.A.C., Meirelles,
M.N.L., Rossi-Bergmann, B., 1999a. The selective e€ect of 20,60-
dihydroxy-40-methoxychalcone isolated from Piper aduncum on
Leishmania amazonensis. Antimicrob. Agents Chemother. 43, 1234±
1241.
Gottlieb, O.R., 1978. Neolignans. Fortschr. Chem. Org. Naturst. 35,
1±72.
Iwu, M.M., Jackson, J.E., Schuster, B.G., 1994. Medicinal plants in
the ®ght against leishmaniasis. Parasitol. Today 10, 65±68.
Lariucci, C., Homar, L.I.B., Ferri, P.H., Santos, L.S., 1995. The crys-
tal structure of 1-(30,40-dimethoxyphenyl)-1-propanone, a Platelet
Activating Factor (PAF-acether) antagonist. Anais Assoc. Bras.
Quõm. 44 (3), 22±27.
Torres-Santos, E.C., Rodrigues-Jr, J.M.R., Moreira, D.L., Kaplan,
M.A.C., Rossi-Bergmann, B., 1999b. Improvement of in vitro and
in vivo antileishmanial activity of 20, 60-dihydroxy-40-methox-
ychalcone by encapsulation in poly (d, l-lactide) nanoparticles.
Antimicrob. Agents Chemother. 43, 1776±1778.
Lima, E.O., Maia, R.F., Barbosa, R.C.S.B.C., Xavier-Filho, L.,
Paulo, M.Q., Santos, L.S., Barata, L.E.S., 1987. Atividade anti-
Wright, C.W., Phillipson, J.D., 1990. Medicinal plants against proto-
zoal diseases. Phytother. Res. 4, 127±139.