In vitro activities of new 2-substituted quinolines against Leishmania donovani

Article abstract of DOI:10.1128/AAC.01299-10

A series of 9 quinolines and 18 styrylquinolines was evaluated for the drugs' in vitro antileishmanial activities and cytotoxicities. The 7-aroylstyrylquinoline scaffold appeared to be the most promising one, with the most interesting compound, no. 35, exhibiting a 50% inhibitory concentration (IC₅₀) of 1.2 μM and a selectivity index value of 121.5. Compound 35 was 10-fold and 8-fold more active than miltefosine and sitamaquine, the reference compounds, with selectivity indexes 607-fold and 60-fold higher, respectively. Copyright

Full text of DOI:10.1128/AAC.01299-10

In Vitro Activities of New 2-Substituted
Quinolines against Leishmania donovani
Philippe M. Loiseau, Suman Gupta, Aditya Verma, Saumya
Srivastava, S. K. Puri, Faten Sliman, Marie Normand-Bayle
and Didier Desmaele
Antimicrob. Agents Chemother. 2011, 55(4):1777. DOI:
10.1128/AAC.01299-10.
Published Ahead of Print 10 January 2011.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2011, p. 1777–1780
0066-4804/11/$12.00 doi:10.1128/AAC.01299-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 4
In Vitro Activities of New 2-Substituted Quinolines against
Leishmania donovaniᰔ
Philippe M. Loiseau,1* Suman Gupta,2 Aditya Verma,2 Saumya Srivastava,2 S. K. Puri,2 Faten Sliman,3
Marie Normand-Bayle,3 and Didier Desmaele3
Groupe Chimioth´erapie Antiparasitaire, UMR 8076 CNRS BioCis, Facult´e de Pharmacie, Univ Paris-Sud,
92290-Chaˆtenay-Malabry, France1; Division of Parasitology, Central Drug Research Institute (CSIR), Lucknow,
India2; and Laboratoire de Synth`ese Organique et Pharmacochimie, UMR 8076 CNRS BioCis,
Facult´e de Pharmacie, Universit´e Paris-Sud, 92290-Chaˆtenay-Malabry, France3
Received 23 September 2010/Returned for modification 8 November 2010/Accepted 2 January 2011
A series of 9 quinolines and 18 styrylquinolines was evaluated for the drugs’ in vitro antileishmanial activities
and cytotoxicities. The 7-aroylstyrylquinoline scaffold appeared to be the most promising one, with the most
interesting compound, no. 35, exhibiting a 50% inhibitory concentration (IC50) of 1.2 ␮M and a selectivity
index value of 121.5. Compound 35 was 10-fold and 8-fold more active than miltefosine and sitamaquine, the
reference compounds, with selectivity indexes 607-fold and 60-fold higher, respectively.
Leishmaniasis is a family of parasitic diseases that affect
about 12 million people in tropical and subtropical areas in the
form of three clinical expressions: visceral leishmaniasis, which
is fatal in the absence of treatment; muco-cutaneous leish-
maniasis; and cutaneous leishmaniasis, which is often self-
curing. Classical drugs such as antimonials (Pentostam and
Glucantime) are toxic, and drug resistance is increasing
dangerously in the field (3). A liposomal amphotericin B
formulation (AmBisome) less toxic than amphotericin B de-
oxycholate is gradually becoming the first-line therapy, espe-
cially in immunocompromised patients, but this drug must be
administered by a parenteral route (11). Miltefosine (Im-
pavido) was the first drug registered against visceral leishman-
iasis in the last decade; however, its toxicity and the appear-
ance of drug resistance justify the search for new chemical
series in order to find an orally safe and active drug (8).
Quinolines substituted at the 2-position have shown in vivo
activities against Leishmania donovani, and many compounds
have been synthesized over the last decade (14). The Drug for
Neglected Diseases Initiative (DNDi) has been considering
this series for evaluation in preclinical development for about
a year and a half. However, although promising, the series still
requires improvements, and here we report the in vitro anti-
leishmanial evaluation of new quinoline derivatives, including
2-[2-aryl(ethenyl)]-substituted quinoline (2-styrylquinolines)
bearing additional aroyl/acyl groups at the C-7 position. In
addition, some compounds within this series were recently
shown to display substantial antiviral activity in HIV-infected
cells (13, 22).
amide compound 17 (13). Similarly, bromination of the C-5
position and protection of the salicylic moiety provided 5-bro-
moquinaldine compound 2, which was engaged in a modified
Suzuki cross-coupling reaction to give 5-arylated derivatives 14
to 16 (19). Styrylquinoline compounds 19 to 27 were prepared
from the corresponding quinaldine compound 3 by Perkin-type
condensation in refluxing acetic anhydride, followed by hydro-
lysis in a pyridine-water mixture (10, 16, 21, 22). Finally, the
7-aroyl-stryryquinoline derivatives 28 to 35 were obtained via
the 7-bromostyrylquinoline compound 5 according to a three-
step sequence involving lithiation followed by condensation
with the required aldehyde, manganese dioxide oxidation, and
deprotection (15) (Fig. 1).
The antileishmanial evaluation of these compounds was then
performed on Leishmania donovani amastigotes by using the
luciferase-transfected Leishmania donovani (strain MHOM/
IN/80/Dd8) promastigotes maintained in the laboratory of the
Division of Parasitology, Central Drug Research Institute,
Lucknow, India, since 2005 as described by Sunduru et al. (20).
In order to assess the activity of compounds against the amas-
tigote stage of the parasite, the mouse macrophage cell line
J-774A.1, infected with promastigotes expressing the luciferase
firefly reporter gene, was used. Cells were seeded in a 96-well
plate at a density of 4 ϫ 104 cells per ml in a final volume of 100
␮l in RPMI 1640 containing 10% fetal calf serum, and the
plates were incubated at 37°C in a CO2 incubator. After 24 h,
the medium was replaced with fresh medium containing sta-
tionary-phase promastigotes (4 ϫ 105/100 ␮l/well). Promasti-
gotes were engulfed by the macrophage and transformed there
into amastigotes. The test compounds were added at 2-fold
dilutions in up to 7 points in fresh complete medium starting
from a 100 ␮M concentration, and the plates were incubated at
37°C in a CO2 incubator for 72 h. After incubation, the drug-
containing medium was decanted and 50 ␮l phosphate-buff-
ered saline (PBS) was added in each well and mixed with an
equal volume of Steady-Glo luciferase assay substrate dis-
solved in Steady-Glo luciferase assay buffer. After gentle shak-
ing for 1 to 2 min, the readings were recorded in a luminometer
(1, 4, 17). The values were expressed as relative luminescence
The synthesis of most of the compounds has been previously
reported. Briefly, Kolbe carbonation of 8-hydroxyquinaldine
afforded the pivotal hydroxyacid compound 1 (9), which was
further elaborated into the 5-iodoquinaldine compound 12 and
* Corresponding author. Mailing address: Groupe Chimiothe´rapie
Antiparasitaire, UMR 8076 CNRS BioCis, Facult´e de Pharmacie, Uni-
versit´e Paris-Sud, 92290-Chˆatenay-Malabry, France. Phone: 33 (0) 1 46
83 55 53. Fax: 33 (0) 1 46 83 55 57. E-mail: philippe.loiseau@u-psud.fr.
ᰔ Published ahead of print on 10 January 2011.
1777

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ANTIMICROB. AGENTS CHEMOTHER.
dium. After incubation, 25 ␮l of MTT reagent (5 mg/ml) in
PBS medium, followed by syringe filtration, was added to each
well and incubated at 37°C for 2 h. At the end of the incubation
period, the supernatants were removed by inverting the plate
completely without disturbing the cell layer, and 150 ␮l of pure
DMSO was added to each well. After 15 min of shaking, the
readings were recorded as absorbance at 544 nm on a micro-
plate reader. The cytotoxic effects were expressed as 50%
lethal dose (i.e., as the concentration of a compound which
provoked a 50% reduction in cell viability compared to cells in
culture medium alone). Fifty percent cytotoxic concentration
(CC50) values were estimated as previously described (5, 12).
The selectivity index (SI) for each compound was calculated as
the ratio between cytotoxicity (CC50) and activity (IC50)
against Leishmania amastigotes.
FIG. 1. General synthetic scheme for the quinoline and styrylquino-
line derivatives evaluated in this study.
Among the simple quinolines (Table 1) and the styrylquino-
line derivatives (Table 2), three compounds exhibited an IC50
for parasites of less than 10 ␮M (compounds 17, 18, and 20).
The most interesting compound was compound 18, with an
IC50 for L. donovani intramacrophage amastigotes of 4.1 ␮M
and a selectivity index of 8.3. A clear-cut structure-activity
relationship showed that the introduction of a carboxyl group
at any position was responsible for both a dramatic decrease in
the antileishmanial activity and a decrease in the cytotoxicity
decrease. This observation was confirmed when two carboxyl
groups were introduced into the same molecule, resulting in no
activity and no cytotoxicity (compound 27). These results could
be ascribed to an excessive hydrophilicity limiting the drug-
parasite membrane interactions or a reaction between the car-
boxyl group with some compounds of the culture medium
preventing the entry of the compound into the parasite.
Among the 7-aroylstyrylquinolines (Table 3), the most in-
teresting compound was compound 35, which exhibited an
IC50 of 1.2 ␮M and a selectivity index of 121.5. Compound 35
was 10-fold and 8-fold more active than miltefosine and sita-
maquine, the reference compounds, with selectivity indexes
607-fold and 60-fold higher, respectively.
units (RLU). Data were transformed into a graphic program
(Excel). The 50% inhibitory concentration (IC50) for antileish-
manial activity was calculated by nonlinear regression analysis
of the concentration-response curve by using the four-param-
eter Hill equations. The in vitro Leishmania donovani intra-
macrophage amastigote system used to evaluate the antileish-
manial activity of the compounds was the most relevant one,
since it takes into account the pharmacokinetics barriers that a
compound has to overcome before entering the parasite.
KB cells were used to evaluate the cytotoxicity of the com-
pounds to mammalian cells, which allowed us to determine an
in vitro selectivity index. The cell viability was determined
with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide (MTT) assay (12). Exponentially growing KB
cells at a density of 1 ϫ 105 cells per ml in a 100-␮l final volume
were incubated in a 96-well plate with test drugs for 72 h. The
test compounds were added at 3-fold dilutions for up to 7
points in complete medium starting from a 400 ␮M concen-
tration and were incubated at 37°C in a humidified mixture of
CO2 and 95% air in an incubator. Podophyllotoxin was used
as a reference drug, and control wells containing dimethyl
sulfoxide (DMSO) without drugs were also included in the
experiment. Stock solutions of compounds were initially dis-
solved in DMSO and further diluted with fresh complete me-
Compound 34 had an IC50 of 2.1 ␮M and a selectivity index
of 27.3. These compounds exhibited the best selectivity indexes
in their series, despite the presence of a nitro group. The
presence of the nitro group at the meta position greatly in-
TABLE 1. In vitro antileishmanial activity and cytotoxicity results for compounds 6 and 10 to 17
SI (CC50
/
Compound
R1
R2
R3
R4
IC50 (␮M)
CC50 (␮M)
IC50 ratio)a
6
H
H
H
I
H
Ph
Br
CO2H
H
CO2H
CHO
CO2H
CO2H
CO2H
OH
CH3
H
16.1
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
5.9
75.4
335
4.6
Ͻ3.3
Ͻ3.0
Ͻ3.3
Ͻ1.8
Ͻ3.2
Ͻ1.6
10
11
12
13
14
15
16
17
OH
CO2H
OH
OH
OH
OH
OH
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
303.5
331.5
181.9
329.9
169.7
Ͼ400
32.9
4-MePh
3-Quinolinyl
H
p-FPhCH2NHCO
5.5
a The selectivity index (SI) is defined as the ratio of CC50 on KB cells to IC50 on L. donovani intramacrophage amastigotes.

VOL. 55, 2011
ANTILEISHMANIAL STYRYLQUINOLINES
TABLE 2. In vitro antileishmanial activity and cytotoxicity results for styrylquinolines 18 to 27
1779
SI (CC50
/
Compound
R1
R2
R3
R4
R5
R6
IC50 (␮M)
CC50 (␮M)
IC50 ratio)a
18
19
20
21
22
23
24
25
26
27
H
H
H
H
H
H
H
H
H
CN
OH
NO2
OH
OAc
OH
OH
OH
OH
OH
OH
H
OH
OAc
H
OAc
NH2
SO2CH3
F
Cl
H
OH
OH
OAc
H
OAc
H
H
F
OH
OH
OH
4.1
21.3
34.5
39.7
8.3
1.8
H
H
H
H
H
6.9
4.0
0.6
H
13.5
9.8
35.6
263.3
193.8
299.8
Ͼ400
Ͼ400
0.7
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
H
Ͼ100
Ͼ100
Ͼ100
79.6
Ͻ0.3
Ͻ2.6
Ͻ1.9
3.7
H
H
H
OH
H
Ͼ100
Ͼ100
CO2H
a The selectivity index (SI) is defined as the ratio of CC50 on KB cells to IC50 on L. donovani intramacrophage amastigotes.
creased the selectivity index, as evidenced by the much lower
selectivity index of the parent compound, no. 28.
leishmanial activity and low toxicity, is the most interesting
compound to emerge from more than 150 derivatives of 2-sub-
stituted quinolines that have now been synthesized and evalu-
ated. It has now been selected as a candidate for evaluation in
vivo with L. donovani mouse or hamster models via the DNDi
pipeline.
Recent work has confirmed the interesting antileishmanial
properties of other quinoline series (2, 6, 18). In addition,
quinolines have recently been found to inhibit leishmanial
GDP-mannose-pyrophosphorylase, an enzyme system produc-
ing a range of mannose-rich glycoconjugates that are essential
for parasite survival and virulence (7). This potential for selec-
tive action against a Leishmania-specific target makes quino-
lines a promising series of antileishmanial drugs. Moreover, we
have tried to select quinoline-resistant L. donovani promasti-
gotes in the lab by in vitro drug pressure and have only ob-
tained a slight decrease in sensitivity since the IC50s were no
more than twice those of the wild-type line (data not shown).
This encouraging result is an additive justification for further
studies of 2-substituted quinolines.
This work was supported by the Drug for Neglected Disease Initia-
tive (DNDi).
The transgenic L. donovani promastigotes were originally procured
from Neena Goyal, Division of Biochemistry, Central Drug Research
Institute, Lucknow, India.
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(␮M)
(␮M)
IC50 ratio)a
28
Ph
2.5
20.0
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58.7
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a The selectivity index (SI) is defined as the ratio of CC50 on KB cells to IC50
on L. donovani intramacrophage amastigotes.

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