Synthesis and antileishmanial activities of novel 3-substituted quinolines

Article abstract of DOI:10.1128/AAC.49.3.1076-1080.2005

The antileishmanial efficacy of four novel quinoline derivatives was determined in vitro against Leishmania chagasi, using extracellular and intracellular parasite models. When tested against L. chagasi-infected macrophages, compound 3b demonstrated 8.3-f

Full text of DOI:10.1128/AAC.49.3.1076-1080.2005

Synthesis and Antileishmanial Activities of
Novel 3-Substituted Quinolines
André Gustavo Tempone, Ana Cláudia Melo Pompeu da
Silva, Carlos Alberto Brandt, Fernanda Scalzaretto
Martinez, Samanta Etel Treiger Borborema, Maria Amélia
Barata da Silveira and Heitor Franco de Andrade Jr.
Antimicrob. Agents Chemother. 2005, 49(3):1076. DOI:
10.1128/AAC.49.3.1076-1080.2005.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2005, p. 1076–1080
0066-4804/05/$08.00ϩ0 doi:10.1128/AAC.49.3.1076–1080.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 3
Synthesis and Antileishmanial Activities of Novel
3-Substituted Quinolines
Andr´e Gustavo Tempone,1,2* Ana Cl´audia Melo Pompeu da Silva,3 Carlos Alberto Brandt,4
Fernanda Scalzaretto Martinez,1 Samanta Etel Treiger Borborema,1
Maria Am´elia Barata da Silveira,3 and Heitor Franco de Andrade, Jr.1
Laboratorio de Parasitologia, Divisao de Biologia Medica, Instituto Adolfo Lutz,2 Laboratorio de Protozoologia,
Instituto de Medicina Tropical,1 Departamento de Farma´cia, Faculdade de Ciˆencias Farmacˆeuticas,
Universidade de Sao Paulo,3 and Instituto Butantan,4 Sao Paulo, Brazil
Received 16 July 2004/Returned for modification 25 August 2004/Accepted 12 November 2004
The antileishmanial efficacy of four novel quinoline derivatives was determined in vitro against Leishmania
chagasi, using extracellular and intracellular parasite models. When tested against L. chagasi-infected mac-
rophages, compound 3b demonstrated 8.3-fold greater activity than did the standard pentavalent antimony. No
significant activity was found for compounds 3a, 4a, and 4b. The antilesihmanial effect of compound 3b was
independent of host cell activation, as demonstrated by nitric oxide production. Ultrastructural studies of
promastigotes treated with compound 3b showed mainly enlarged mitochondria, with matrix swelling and
reduction in the number of cristae. Synthetic analogues based on the quinoline ring structure, already an
established template for antiparasitic drugs, could provide further useful compounds.
Protozoan parasites affect 3 billion people, with malaria and
trypanosomatid parasites causing the greatest morbidity (10,
28). Leishmaniasis is estimated to afflict 12 million people
worldwide, causing disease ranging from skin lesions in cuta-
neous leishmaniasis to a progressive and fatal hepatospleno-
megaly in visceral leishmaniasis (9).
most active compound were investigated through ultrastruc-
tural studies and the potential activation of macrophages.
MATERIALS AND METHODS
Materials. Sodium dodecyl sulfate was purchased from Merck. Lipopoly-
sacharide (LPS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(Thiazol blue; MTT), M-199 and RPMI-PRϪ 1640 medium (without phenol red)
were purchased from Sigma. Pentavalent antimony (Aventis-Pharma) and pen-
tamidine (Eurofarma) were used as the standard drugs. Sodium nitrite, sulfanil-
amide, and N-1-naphthylethylenediamine dihydrochloride (NED) were pur-
chased from Merck.
Treatment of leishmaniasis suffers from problems of drug
resistance and severe toxicity (9) and requires parenteral ad-
ministration (14). Despite the great advances in many scientific
fields, the 92-year-old antimonials (29) are still the main treat-
ment in the majority of developing countries. In addition,
problems have occurred with the manufacture and supply of
antimonials in developing countries (20) and high levels of
arsenic contaminants have also been described (22). Second-
line drugs, such as pentamidine and amphotericin B, are im-
portant in combined therapy or in cases of antimony treatment
failures (3). Experimental studies have identified the antican-
cer drug miltefosine as an effective antileishmanial agent (8).
However, clinical trials in India have identified gastrointestinal
toxicity and teratogenicity in association with this drug (9).
Therefore, the development of new antiprotozoal compounds
with improved pharmacological properties is imperative.
Several drugs based on the quinoline structure have im-
proved the therapy of protozoal diseases, especially malaria
(5). These have also shown considerable in vivo efficacy against
Leishmania parasites; this is exemplified by the drug WR 6026,
which is currently undergoing clinical trials for its effectiveness
in treating visceral leishmaniasis (11). In this paper, we de-
scribe the synthesis of four novel 3-substituted quinolines with
allyl and cinnamyl motifs covalently attached to the quinoline
entity. In vitro parasite models are used to evaluate their an-
tileishmanial activity. Possible mechanisms of action for the
Animals and parasites. Animals were supplied by the Animal Breeding Fa-
cility at the Faculty of Medicine of S˜ao Paulo University. They were maintained
in sterilized cages under a controlled environment, receiving water and food ad
libitum. Animal procedures were performed under the approval of the Research
Ethics Commission, according to the Guide for the Care and Use of Laboratory
Animals from the National Academy of Sciences (http://www.nap.edu). Leish-
mania chagasi (MHOM/BR/1972/LD) was maintained in golden hamsters. Ap-
proximately 60 to 70 days postinfection, amastigotes were obtained from the
hamster spleen by differential centrifugation and the parasite burden was deter-
mined using the Stauber equation (24). Promastigotes were maintained in M-199
medium supplemented with 10% calf serum and 0.25% hemin at 24°C.
Determination of the 50% inhibitory concentration (IC50). The antileishma-
nial activity against promastigotes was determined as described elsewhere (27),
using pentamidine as the standard drug. Parasite viability was determined using
the MTT assay (25). The antileishmanial activity against intracellular amastigotes
was determined with infected macrophages (26), using pentavalent antimony as
the standard drug. The parasite burden was defined as the number of infected
macrophages in a total of 400 cells. Each assay was performed in triplicate.
Cytotoxicity assay. RAW 264.7 cells (ATCC TIB-71) were seeded (4 ϫ 104/
well) in 96-wells microplates and incubated with different drug concentrations for
48 h at 37°C in a humidified mixture of 5% CO2 and 95% air in an incubator. The
cell viability was determined using the MTT assay. The selectivity index (S.I.) was
calculated using the following equation: S.I. ϭ IC50 (RAW 264.7 cells)/IC50
(Leishmania amastigotes).
Ultrastructural studies. L. chagasi promastigotes were incubated with com-
pound 3b at 10 ␮g mlϪ1 for both 30 and 60 min at 24°C in 24-well plates. They
were then processed (12) and observed under a transmission electron microscope
(JEOL).
Nitric oxide production. The nitric oxide production by macrophages was
measured in the presence of compound 3b by the Griess reaction (19). Peritoneal
macrophages were incubated for 24 h at 37°C with compound 3b (10 ␮g mlϪ1).
LPS (10 ␮g mlϪ1) was used to induce NO up-regulation and was incubated alone
with compound 3b (10 ␮g mLϪ1) under the same conditions. The absorbance
* Corresponding author. Mailing address: Lab. Parasitologia, Div.
Biologia Medica Medical, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355,
8° andar CEP, 01246-000 Sao Paulo, Brazil. Phone: 55 3066-7010. Fax:
55 3088-5237. E-mail: atempone@ial.sp.gov.br.
1076

VOL. 49, 2005
Test agent
NEW QUINOLINE DERIVATIVES AGAINST L. CHAGASI
TABLE 1. Effect of novel quinoline compounds on L. chagasi and mammalian cytotoxicitya
1077
S.I.
IC50 (␮g mlϪ1) (95% C.I.)b for:
Cytotoxicity; IC50 (␮g mlϪ1
)
(95% C.I.)
Promastigotes Amastigotes
3a
0.79 (0.45–1.37)
0.091 (0.06–0.13)
18.78 (8.02–44.00)
1.79 (0.73–4.23)
Ͼ15
3.55 (3.34–3.78)
31.60 (19.82–50.38)
26.89 (20.47–35.34)
13.77 (10.75–17.62)
22.00 (16.24–29.80)
Ͼ100
3b
7.57
4a
Ͼ15
Ͼ15
4b
Sb Vc
29.55 (28.09–31.09)
Ͼ3.38
Pentamidine
2.02 (1.75–2.35)
2.57 (2.23–2.96)
a Promastigotes were incubated with compounds and standard drugs for 24 h at 24°C. RAW 264.7 cells were incubated for 48 h at 37°C, and the viability of both
parasite and RAW cells was determined using the MTT assay.
b C.I., confidence interval.
c Sb V, pentavalent antimony.
was determined at 540 nm using a Multiskan MS (Uniscience) microplate
reader.
Drug synthesis. Ethyl-2-acetylpent-4-enoate (compound 1a) and ethyl-(4E)-2-
concentrations, in a dose-dependent manner. A strong anti-
acetyl-5-phenylpent-4-enoate (compound 1b) were obtained by a previously de-
determined against L. chagasi promastigotes. Table 1 shows
that three of these compounds killed promastigotes at low
parasitic effect was observed for compounds 3b and 3a (IC50 Ͻ
0.8 ␮g mlϪ1) compared to the standard drug, pentamidine,
which had an IC50 of 2.02 ␮g mlϪ1. All compounds killed 100%
of parasites at the maximal concentration of 50 ␮g mlϪ1; how-
ever, compound 4a was the least potent (IC50 ϭ 18.78 ␮g
mlϪ1) and the most toxic against mammalian cells. Compound
4b showed a significant antiparasitic activity with an IC50 of
1.79 ␮g mlϪ1, being at least 8.5-fold less toxic for mammalian
cells than the standard drug pentamidine.
scribed method (1, 2). Ethyl-(2Z)-2-(1-anilinoethylidene)pent-4-enoate (compound
2a) and ethyl-(2Z,4E)-2-(1-anilinoethylidene)-5-phenylpent-4-enoate (compound
2b) were obtained from ketoester 1a or 1b and excess of aniline in the presence
of molecular sieves: 5 Å (0.1 g/mmol) at 60°C for 24 h (16). 3-Substituted
4-hydroxyquinolines (compounds 3a and 3b) and 3-substituted 4-chloroquino-
lines (compounds 4a and 4b) were obtained by a previously described method (6,
7, 15).
(i) 3-Allyl-2-methylquinolin-4-ol (compound 3a). The yield was 1.494 g (75%)
of a pale yellow solid with a melting point of 244°C to 245°C. 1H nuclear
magnetic resonance (NMR) (DMSO-d6): 2.25 (s, 3H), 3.18 (d, 2H, J ϭ 6.0 Hz),
4.81 (d, 1H, J ϭ 9.3 Hz), 4.86 (d, 1H, J ϭ 15.2 Hz), 5.73 (ddt, 1H, J ϭ 15.2, J ϭ
9.3, and J ϭ 6.0 Hz), 7.16 (t, 1H, J ϭ 7.8 Hz), 7.37 (d, 1H, J ϭ 7.8 Hz), 7.48 (t,
1H, J ϭ 7.8 Hz), 7.95 (d, 1H, J ϭ 7.8 Hz), 11.37 (br s, 1H). 13C NMR (DMSO-d6):
17.3, 28.7, 114.0, 116.1, 117.5, 122.4, 123.3, 125.1, 131.0, 136.4, 139.1, 146.7, 175.1.
MS (m/z): 199 [M]ϩ, 184, 92, 77. Analysis calculated for C13H13NO (199.25): C,
78.37; H, 6.58; N, 7.03. Found: C, 78.17; H, 6.39; N, 7.11.
(ii) 2-Methyl-3-[(2E)-3-phenylprop-2-enyl]quinolin-4-ol (compound 3b). The
yield was 1.542 g (56 %) of a yellow solid with a melting point of 238°C to 240°C.
1H NMR (DMSO-d6): 2.33 (s, 3H), 3.35 (d, 2H, J ϭ 5.6 Hz), 6.18 (dt, 1H, J ϭ
16.0 and J ϭ 5.7 Hz), 6.29 (d, 1H, J ϭ 16.0 Hz), 7.07 (t, 1H, J ϭ 7.1 Hz), 7.10 to
7.15 (m, 4H), 7.25 (d, 1H, J ϭ 7.2 Hz), 7.40 (d, 1H, J ϭ 8.0 Hz), 7.50 (t, 1H, J ϭ
7.5 Hz), 7.99 (d, 1H, J ϭ 7.9 Hz), 11.43 (br s, 1H). 13C NMR (DMSO-d6): 17.5,
27.8, 116.3, 117.5, 122.4, 123.4, 125.1, 125.4, 125.7, 126.7, 128.4, 128.9, 131.0,
137.2, 139.1, 146.8, 175.2. MS (m/z): 275 [M]ϩ, 184, 92, 77. Analysis calculated for
(ii) Intracellular amastigotes. Peritoneal macrophages were
infected with L. chagasi amastigotes and treated with the test
compounds over the concentration range of 15 to 0.117 ␮g
mlϪ1 (twofold dilution) for 120 h. Only compound 3b showed
a significant reduction in the number of intracellular amasti-
gotes, with an IC50 of 3.55 ␮g mlϪ1. The sigmoid dose-response
curve showed 100% parasite inhibition for macrophages
treated with a drug concentration of less than 8.0 ␮g mlϪ1 (Fig.
1). Despite the susceptibility of mammalian cells (RAW 264.7)
to the quinoline compounds in a range of 13 to 32 ␮g mlϪ1
,
the S.I. of compound 3b was 7.5 (Table 1). Compound 3b
C
19H17NO (275.35): C, 82.88; H, 6.22; N, 5.09. Found: C, 82.57; H, 6.29; N, 5.01.
(iii) 3-Allyl-4-chloro-2-methylquinoline (compound 4a). The yield was 2.068 g
(95%) as a brown solid with a melting point of 140°C. 1H NMR (CDCl3): 3.23 (s,
3H), 3.86 (d, 2H, J ϭ 7.0 Hz), 4.98 (d, 1H, J ϭ 17.1 Hz), 5.23 (d, 1H, J ϭ 10.2 Hz),
5.93 (m, 1H), 7.89 (t, 1H, J ϭ 7.4 Hz), 8.02 (t, 1H, J ϭ 8.4 Hz), 8.39 (d, 1H, J ϭ
8.5 Hz), 9.06 (d, 1H, J ϭ 8.5 Hz). 13C NMR (CDCl3): 19.2, 33.6, 118.2, 122.2,
125.0, 125.9, 130.3, 130.8, 131.6, 134.1, 137.7, 150.6, 157.0. MS (m/z): 219, 217
[M]ϩ, 184, 182, 169, 167, 77. Analysis calculated for C13H13NCl (217.69): C,
71.73; H, 5.55; N, 6.43, Cl, 16.29. Found: C, 71.28; H, 5.39; N, 6.61, Cl, 16.59.
(iv) 4-Chloro-2-methyl-3-[(2E)-3-phenylprop-2-enyl]quinoline (compound
4b). The yield was 2.203 g (75%) as a brown solid with a melting point of 108°C.
1H NMR (CDCl3): 2.84 (s, 3H), 3.93 (d, 2H, J ϭ 3.4 Hz), 6.26 to 6.39 (m, 1H),
6.30 (d, 1H, J ϭ 15.9 Hz), 7.19 to 7.29 (m, 5H), 7.61 (t, 1H, J ϭ 8.3 Hz), 7.74 (t,
1H, J ϭ 8.3 Hz), 8.11 (d, 1H, J ϭ 8.3 Hz), 8.23 (d, 1H, J ϭ 8.3 Hz). 13C NMR
(CDCl3): 23.6, 33.63, 124.4, 124.7, 125.6, 125.9, 126.1, 127.3, 127.5, 128.0, 129.8,
130.2, 131.7, 136.7, 145.8, 158.7. MS (m/z): 295, 293 [M]ϩ, 260, 258, 182, 180, 77.
Analysis calculated for C19H16NCl (293.799): C, 77.68; H, 5.49; N, 4.77; Cl, 12.06.
Found: C, 77.38; H, 5.31; N, 4.62; Cl, 11.59.
Statistical analysis. Data represent the mean and standard deviation of du-
plicate or triplicate samples from two or three independent assays. The IC50s
were calculated using sigmoid dose-response curves in Graph Pad Prism 3.0
software, and the 95% confidence intervals are included in parentheses.
FIG. 1. Determination of the IC50 of compound 3b against L. cha-
gasi-infected macrophages. Pentavalent antimony was used as a stan-
dard drug. Macrophages were treated for 120 h at 37°C with drugs, and
the number of infected macrophages in Giemsa-stained glass cover-
slips was determined by light microscopy. Dose-response curves were
obtained in GraphPad Prism 3.0 software. Data are mean and standard
deviation.
RESULTS
Determination of the IC50. (i) Promastigotes. The antileish-
manial activity of the four quinoline compounds was initially

1078
TEMPONE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Ultrastructural effects of compound 3b on Leishmania promastigotes. (B and C) Promastigotes were incubated with compound 3b at
10 ␮g mlϪ1 for 30 (B) and 60 min (C) at 24°C and subsequently observed under a transmission electron microscope. (A) Untreated cells were used
as parasite controls. m, mitochondria; n, nucleus; g, granules; v, vacuoles.
was not shown to be toxic to macrophages by light micros-
copy, as confirmed by normal morphology and attachment
to plates.
Nitric oxide production. Peritoneal macrophages, both LPS
activated and nonactivated, were incubated for 24 h in the
presence of compound 3b. The nitrite content was evaluated by
the Griess reaction. Our results showed that compound 3b did
not produce significantly more nitrite than untreated controls
and consequently did not up-regulate NO production by mac-
rophages at 10 ␮g mlϪ1 (data not shown).
Ultrastructural studies. To evaluate the intracellular dam-
age caused in Leishmania promastigotes by compound 3b, the
ultrastructural modifications were investigated by transmission
electron microscopy after a short-term incubation (Fig. 2). The
images clearly demonstrate time-dependent damage, with
most significant changes at 60 min of incubation. After 60 min
of incubation (Fig. 2C), compound 3b induced an enlargement
of the kinetoplast and a considerable enhancement in the num-
ber of lipid granules and vacuoles. After 30 min of incubation
(Fig. 2B), initial changes in promastigotes were observed as an
increase in the number of vacuoles and lipid granules, which
were much more pronounced at 60 min incubation.
Drug synthesis. The synthesis of chloroquinolines (com-
pounds 4a and 4b) and quinoline (compounds 3a and 3b)
derivatives is shown in Fig. 3. Among the methods for gener-
ating ␤-enaminones, condensation of amines and ␤-dicarbonyl
compounds is a classical method, in which the azeotropic re-
moval of water is achieved by refluxing in an aromatic solvent.
In this paper, ␤-enamine esters (compounds 2a and 2b) were
prepared by the reaction of the dicarbonyl compounds with
aniline in the presence of the molecular sieve (5 Å) and the
products were easily separated from the remaining aniline. The
resulting derivatives were then cyclized using phenyl ether to
yield 4-hydroxyquinolines (compounds 3a and 3b), which then
gave the 4-chloroquinolines derivatives (compounds 4a and
4b) after treatment with phosphorus oxychloride.
DISCUSSION
Previous studies using quinoline compounds have shown
potent in vitro and in vivo antileishmanial activity (23), and
recent clinical trials have also been undertaken using these
compounds (30). In this work, we have demonstrated the effi-
cacy of novel quinoline derivatives against L. chagasi parasites.

VOL. 49, 2005
NEW QUINOLINE DERIVATIVES AGAINST L. CHAGASI
1079
FIG. 3. Synthesis of quinolines 3a through 4b.
The synthesis of these quinoline compounds, in addition to
being relatively simple, resulted in large amounts of pure ma-
terials, as confirmed by physical and spectroscopic data. Our
results showed antileishmanial activity for all compounds
tested against promastigotes, with most IC50s being lower than
for the standard drug pentamidine. The chemical structure of
quinolines and their biological activity relationship showed
that the substitution of the ClϪ (compound 4b) by OHϪ (com-
pound 3b) in the quinoline ring caused a 19-fold increase in the
antiparasitic effect. Likewise, the same substitution for com-
pounds 4a and 3a showed similar improvements in anti-
parasitic effect, suggesting that the OHϪ at position 4 might be
essential for the antileishmanial activity. Based on these re-
sults, we suggest that the substitution of allyl by cinnamyl
groups in the quinoline ring might have contributed to a better
antileishmanial effect. Furthermore, the four quinoline com-
pounds had less toxicity than pentamidine when incubated with
mammalian cells. The introduction of the OHϪ group resulted
in a decrease in cytotoxicity, which could be an improvement if
we also consider the improvement of its antiparasitic effect.
Compound 3b had the lowest IC50 of any compounds tested
against L. chagasi promastigotes, with an IC50 of 0.091 ␮g
mlϪ1. It was at least 22-fold more effective than the standard
drug, pentamidine. However, antileishmanial activity against
promastigotes does not guarantee activity against intracellular
amastigotes, the clinically more relevant form of the parasite.
The intracellular amastigotes have also been shown to express
different enzymatic patterns, which could improve the parasite
defense (21). Incubation of L. chagasi-infected macrophages
with quinoline compounds demonstrated no significant anti-
leishmanial activity for compounds 3a, 4a, and 4b at the highest
concentration. Only compound 3b showed efficacy, with an
IC50 of 3.55 ␮g/ml, which was 8.3-fold more active than the
standard pentavalent antimony (IC50 ϭ 29.55 ␮g/ml). Further-
more, this in vitro activity was similar to that of other anti-
leishmanial quinolines, such as WR 6026, which had an IC50 of
1.6 ␮g/ml against Leishmania tropica-infected macrophages
(6). The relationship between mammalian toxicity and anti-
parasitic effect, given by the S.I., demonstrated that compound
3b was at least 7.6-fold more harmful to intracellular amasti-
gotes than to mammalian cells (RAW 264.7). These results
also suggest that the introduction of OHϪ into the quinoline
ring might have contributed to the improved antileishmanial
activity, compared to compound 4b, which had a ClϪ instead of
OHϪ in position 4. Furthermore, the potential in vivo anti-
leishmanial activity of novel 2-substituted quinolines, originally
isolated from medicinal plants, has been described previously
(13).
The inefficacy of the other quinoline compounds against
intracellular amastigotes might have been due to one of the
following: poor drug uptake by macrophages and subsequently
low drug concentration reaching the parasitophorous vacuole;
inactivation of the quinoline compounds inside macrophages;
or metabolic differences of amastigotes, such as the enzymatic
antioxidant system (catalase or superoxide dismutase) (27).
Compound 3b was chosen for further investigations since it
had the lowest IC50 against promastigotes, the lowest deviation
(95% confidence interval), and efficacy against intracellular
amastigotes.
Macrophages, the target cells in therapy of leishmaniasis,
play an important role in the immunological control of intra-
cellular parasites through the production of cytokines and
oxygen metabolites (4). One of the main mechanisms is the
up-regulation of nitric oxide inside the cell, which is an effec-
tive mediator of amastigote killing (18). We have investigated
the possible activation of macrophages induced by compound
3b by incubation of this compound with both LPS-activated
and nonactivated macrophages. No up-regulation of nitric ox-
ide was seen when compound 3b was incubated in the presence
of nonactivated macrophages. These results indicate that com-
pound 3b might have a specific antiparasitic activity rather than
causing activation of NO production by macrophages. In ad-
dition, efficacy against extracellular promastigotes supports the
hypothesis of a specific antiparasitic activity for compound 3b.
Ultrastructural studies of promastigotes treated with com-
pound 3b (10 ␮g mlϪ1) suggested damage to Leishmania mi-
tochondria, showing an enlargement of the matrix and a re-
duction in the number of cristae clearly observed after 1 h of

1080
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ANTIMICROB. AGENTS CHEMOTHER.
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administered 8-aminoquinoline, in the treatment of visceral leishmaniasis
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Fuentes, H. Nakayama, A. Schinini, and R. Hocquemiller. 1996. In vivo
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incubation. Furthermore, an increase in the numbers of elec-
tron-dense granules and vacuoles within the parasite cytoplasm
was observed, suggesting a time-dependent damage to intra-
cellular organelles. The naphthoquinones have previously been
shown to alter the mitochondrial membrane potential in Leish-
mania spp. (17), causing extensive and irreversible damage,
which dramatically affected parasite survival.
In conclusion, compound 3b had the most potent antileish-
manial activity and was not toxic to mammalian cells at con-
centrations required to kill parasites. Experimental in vivo
studies are under investigation in our laboratory, using both
free drug and drug entrapped in phosphatidylserine-rich lipo-
somes. Liposomes have been described as an effective drug-
targeting tool in leishmaniasis (26). Further investigations of
antiparasitics using quinolines as the lead structure for the
design and synthesis of novel pharmacological compounds rep-
resent an important and cost-effective strategy for addressing
neglected diseases as leishmaniasis.
17. Luque-Ortega J. R., O. M. Rivero-Lezcano, S. L. Croft, and L. Rivas. 2001.
In vivo monitoring of intracellular ATP levels in Leishmania donovani pro-
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ACKNOWLEDGMENTS
We thank Tracy Garnier (London School of Hygiene and Tropical
Medicine) for manuscript revision and Cleuza Takakura for micros-
copy assistance.
This work was supported by grants from Funda¸c˜ao de Amparo `a
Pesquisa do Estado de S˜ao Paulo (FAPESP 99/08491-4), LIM-49
HCFMUSP and CNPq.
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