Synthesis and leishmanicidal activity of quinoline-triclosan and quinoline-eugenol hybrids

Article abstract of DOI:10.1007/s00044-011-9886-8

In this study, hybrids 7-12 and 19-24 were synthesized via Williamson reaction of O-Quinaldine alkyl bromide plus eugenol and O-triclosan alkyl bromide plus 8-hydroxyquinaldine, respectively. Structures of the products were elucidated by spectroscopic ana

Full text of DOI:10.1007/s00044-011-9886-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3445–3454
DOI 10.1007/s00044-011-9886-8
ORIGINAL RESEARCH
Synthesis and leishmanicidal activity of quinoline–triclosan
and quinoline–eugenol hybrids
•
Victor Arango Jorge J. Domınguez Wilson Cardona
•
•
´
•
•
•
Sara M. Robledo Diana L. Mun˜oz Bruno Figadere
´
Jairo Saez
Received: 23 July 2011 / Accepted: 5 November 2011 / Published online: 19 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, hybrids 7–12 and 19–24 were
synthesized via Williamson reaction of O-Quinaldine alkyl
bromide plus eugenol and O-triclosan alkyl bromide plus
8-hydroxyquinaldine, respectively. Structures of the prod-
ucts were elucidated by spectroscopic analysis. The com-
pounds synthesized were evaluated for antileishmanial
activity against L. panamensis amastigotes and cytotoxic
activity against U-937 cells. The compounds 19, 20, and 21
that are 8-hydroxyquinaldine linked to triclosan by 3, 4,
and 5-carbon space, respectively, were more active against
axenic amastigotes (EC50 = 23.6, 9.7, and 4.1 lg/ml,
respectively). Compounds 19 and 21 were also active
against intracellular amastigotes with EC50 vales of 6.4 and
2.4 lg/ml, respectively, making these compounds promis-
ing for the development of new antileishmanial drugs.
Introduction
The Leishmaniases are a group of diseases caused by dif-
ferent species of the intracellular protozoan parasite of the
genus Leishmania, which affect more than 12 million
people worldwide with 2 million new cases diagnosed
every year (http://www.who.int/leishmananiasis). Current
therapies for the disease are still inadequate. The recom-
mended standard drugs for treatment are still the pentava-
lent antimonial drugs: Pestostam and Glucantime. These
drugs have variable efficacies and toxicities, they are
associated with moderate and severe side effects (Desjeux,
2004; Ouellette et al., 2004), prone to induce resistance
(Croft and Coombs, 2003; Faraut-Gambarelli et al., 1997;
Antoine et al., 1989), and require parenteral administration
during long-term periods (Olliaro and Bryceson, 1993).
The second-line drugs, such as Amphotericin B and its
lipid formulations, are either more toxic or too expensive
for routine use in developing countries. For these reasons,
it is necessary to develop new, effective, non-toxic, and
less expensive drugs for use in the treatment of Leish-
maniasis disease.
Keywords Leishmania ꢀ Antiprotozoal ꢀ Quinoline ꢀ
Hybrids ꢀ Triclosan ꢀ Amastigotes ꢀ Eugenol
The quinolinic core is a structural feature of many
bioactive compounds (Narsinh and Anamik, 2001; Kidwai
et al., 2000; Doube et al., 1998; Vennerstrom et al., 1998;
Dillard et al., 1973). Some of these compounds showed
antiprotozoal activity (Akendengue et al., 1999) including
leishmanicidal activity (Nakayama et al., 2005; Tempone
et al., 2005; Dietze et al., 2001; Mohammed et al., 2003;
Dade et al., 2001; Fournet et al., 1996). Triclosan, a
common antibacterial agent, is an uncompetitive inhibitor
of purified enoyl-acyl carrier protein reductase (ENR)
which has demonstrated inhibitory activity in vitro against
Plasmodium falciparum (Kapoor et al., 2004; Perozzo
et al., 2002; Surolia and Surolia, 2001; McLeod et al.,
´
´
V. Arango ꢀ J. J. Domınguez ꢀ W. Cardona (&) ꢀ J. Saez
´
Instituto de Quımica, Quımica de Plantas Colombianas,
Universidad de Antioquia, A. A 1226, Medellın, Colombia
e-mail: wcardona@matematicas.udea.edu.co;
jaisav@matematicas.udea.edu.co
´
´
S. M. Robledo ꢀ D. L. Mun˜oz
Programa de Estudio y Control de Enfermedades Tropicales
´
(PECET), Universidad de Antioquia, A. A 1226, Medellın,
Colombia
B. Figadere
Laboratoire de Pharmacognosie, Associe0 au CNRS (BIOCIS),
Universite0 de Paris-Sud, 92296 Chatenay-Malabry, France
123

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Med Chem Res (2012) 21:3445–3454
2001). Triclosan analogs that are non-toxic to mammalian
cells (McLeod et al., 2001; Bhargava and Leonard, 1996)
and lack of ENR homologues, have been recently explored
as potential antimalarial therapy (Freundlich et al., 2007;
Freundlich et al., 2006; Chhibber et al., 2006; Freundlich
et al., 2005; Perozzo et al., 2002). On the other hand, the
eugenol-rich essential oil of Ocimun gratissimum pro-
gressively inhibits the Leishmania amazonensis growth and
shows no cytototoxic effects against mammalian cells
(Nakamura et al., 2006).
bromide and 1,x-dihaloalkane (Palit et al., 2009; Chau-
dhuri et al., 2007), and the mixture was stirred continu-
ously. In all reactions, we obtained the bis-alkylquinoline
and the corresponding monoethers (Scheme 1). Then, the
monoethers obtained previously were allowed to react with
eugenol using acetone as solvent and potassium carbonate
as base (Chaudhuri et al., 2007). Bis-alkylquinolines are
not of interest in this study because several of them were
synthesized, and their leishmanicidal activity has already
been reported against L. donovani (Palit et al., 2009).
A small set of quinoline–triclosan hybrids were obtained
in the following way: initially, triclosan was treated with an
equivalent amount of potassium carbonate, heated up to
melting point, and then 1,x-dibromoalkane derivative in
acetone was added. The mixture was stirred under reflux to
complete the reaction. Following purification, we obtained
the bromoalkyltriclosan. The corresponding bromoalkyl-
triclosan derivative obtained previously was added to a
mixture of appropriate amounts of 8-hydroxyquinaldine
with an equivalent amount of potassium carbonate in ace-
tone; the entire mixture was subject to reflux. Following
purification by column chromatography, a total of six
compounds were obtained (Scheme 2). Remarkably, low
yields were obtained when the bromoalkylquinaldine were
used as a reaction intermediate.
The combination of two pharmacological agents into a
single molecule, called hybrid molecules, is an emerging
strategy in medicinal chemistry and drug discovery
research (Keith et al., 2005; Roth et al., 2004). These
hybrid molecules may display dual activity but not neces-
sarily acting on the same biological target (Musonda et al.,
2009; Meunier, 2008; Bollini et al., 2008; Opsenica et al.,
2008; Walsh et al., 2007; Ayad et al., 2001). In the search
of new therapeutic alternatives for the treatment of
Leishmaniasis exhibiting more potent activity against
Leishmania parasites with fewer side effects and combin-
ing the pharmacological properties of quinolines, triclosan,
and eugenol, several, quinoline–triclosan hybrids (A) and
quinoline–eugenol hybrids (B) (Fig. 1) were synthesized
and their cytotoxic and leishmanicidal activities were
determined.
Williamson etherification reactions appealed to us for
the preparation of the desired product. We employed
8-hydroxyquinaldine, eugenol, and triclosan as model
substrates and 1,x-dihalo derivatives of propane, butane,
pentane, octane, nonane, and dodecane as reactants, to
examine whether the length of the spacer between quino-
line moieties has an effect on activity.
Antileishmanial activity
The leishmanicidal activity and cytotoxicity of the com-
pounds synthesized as well as glucantime and Amphoter-
icin B, which were used as control drugs, were evaluated
following the method previously reported in the literature
(Varela et al., 2009; Robledo et al., 2005; Weninger et al.,
2001; Robledo et al., 1999). The results were expressed as
EC50 and LC50 values of compounds and are shown in the
Tables 1 and 2.
Results and discussion
According to the results shown in the Table 1, com-
pounds 7, 9, 20, 21, triclosan, and 8-hydroxy quinoline
are very active against axenic amastigotes of L. panam-
ensis exhibiting an EC50 \ 20.0 lg/ml. Compounds 8, 10,
11, and 19 showed a moderate leishmanicidal activity
with an EC50 ranging between 21.0 and 39.0 lg/ml
(Table 1). No leishmancidal activity was observed for the
compounds 12, 22, 23, and 24 with an EC50 [ 100 lg/ml
(Table 1). Regarding the toxic activity against macro-
phages, the results showed a high toxicity level for the
compounds 7–12, 21, triclosan, and the 8-hydroxy quin-
oline with a LC50 \ 100 lg/ml (Table 1). No apparent
toxicity was observed for the compounds 19, 20, 22, 23,
and 24. The LC50 for these compounds was [200 lg/ml
(Table 1). The best selectivity index was observed for
compounds 20 and 21 with values of [20.6 and 14.4,
respectively.
Chemistry
Appropriate amounts of 8-hydroxyquinaldine were dis-
solved in dichloromethane, aqueous NaOH solution was
added to the solution followed by tetrabutyl ammonium
Cl
Cl
N
O
N
O
O
O
O
O
n
n
Cl
n = 1, 2, 3, 6, 7,10
A
B
Fig. 1 Chemical structures of quinoline–triclosan and quinoline–
eugenol hybrids
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Med Chem Res (2012) 21:3445–3454
3447
Scheme 1 General synthetic
pathway to the quinoline–
eugenol hybrids
NaOH, CH2Cl2
TBAB
+
+
Br
Br
n
N
N
N
OH
O
O
n = 1, 2,3,6,7,10
n
n
Br
O
Compounds 1-6
N
OCH3
OH
K2CO3, Acetone
Reflux, 48 h
7, n = 1
8, n = 2
9, n = 3
10, n = 6
11, n = 7
12, n = 10
3
4
H3CO
N
,
,
3"
5"
2"
1
,
O
5
3"
O
,
n
1"
6
7
6"
,
3"
Cl
,
5"
OH
Cl
3
,
N
6"
3"
4"
O
1) K2CO3, 60-70 oC,10'
1) K2CO3 acetone.
4
N
OH
,
Cl
1
2) acetone, Br
n
Br
O
O
Cl
O
5
Cl
O
reflux, 48 h
n
6
6"
7
reflux, 24 h
Br
Cl
n
19, n = 1
20, n = 2
21, n = 3
22, n = 6
23, n = 7
24, n = 10
Compounds 13-18
n = 1, 2,3,6,7,10
Scheme 2 Synthetic pathway to quinoline–triclosan hybrids
Table 1 In vitro leishmanicidal
Compound
Cytotoxicity
U937 cells
LC50 (lg/ml)
Leishmanicidal
activity
EC50 (lg/ml)
SI
Comment
activity against axenic
amatigotes of L. panamensis
and toxicity of quinoline–
eugenol and quinoline–triclosan
hybrids
7
6.8 ? 0.3
31.3 ? 0.1
10.5 ? 2.1
14.7 ? 1.8
14.1 ? 2.8
82.9 ? 5.4
[200.0
6.9 ? 0.5
38.9 ? 5.8
9.5 ? 0.7
29.6 ? 1.3
21.1 ? 2.2
[100
1.0
Cytotoxic, active
8
0.5
1.0
0.5
0.7
Cytotoxic, moderately active
Cytotoxic, active
9
10
11
12
19
20
21
Cytotoxic, moderately active
Cytotoxic, moderately active
Cytotoxic, no active
[0.8
[8.5
23.6 ? 1.0
9.7 ? 0.9
4.1 ? 0.5
[100.0
No cytotoxic, moderately active
No cytotoxic, active
Cytotoxic, active
[200.0
[20.6
14.4
[2.0
[2.0
[2.0
0.4
59.2 ? 9.8
[200.0
22
No cytotoxic, no active
No cytotoxic, no active
No cytotoxic, no active
Cytotoxic, active
LC50 Lethal Concentration 50;
EC50 Effective Concentration
23
[200.0
[100.0
50; SI selectivity index: LC50
EC50; Cytotoxicity
/
24
[200.0
[100
Triclosan
4.8 ? 0.4
14.2 ? 1.6
7.6 ? 0.2
38.8 ? 2.2
[1,000.0
11.3 ? 1.3
21.4 ? 4.1
2.6 ? 0.3
0.05 ? 0.01
[1,000.0
LC50 \ 100 lg/ml; No
cytoxicity LC50 [ 200 lg/ml;
Active EC50 \ 20 lg/ml;
Moderately active
ELC50 \ 100 lg/ml; No active
EC50 [ 100 lg/ml
Eugenol
0.7
Cytotoxic, moderately active
Cytotoxic, active
8-hydroxy quinaldine
Amphotericin B
Glucantime
2.9
776
Cytotoxic, active
[1.0
No cytotoxic, no active
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Med Chem Res (2012) 21:3445–3454
was observed for compounds 19 and 21 with values of
[31.25 and 24.6, respectively. Overall, compounds 8, 10,
19, and 21 were apparently the most active compounds
showing the highest activities against both axenic and
intracellular amastigotes of L. panamensis, while com-
pounds 7, 9, 11 and 20 showed activities only on the axenic
form of this Leishamania species. Compound 12 showed
activity only against intracellular amastigotes of L. panam-
ensis and was less toxic than the other compounds.
Table 2 In vitro activity of quinoline–eugenol and quinoline–triclosan
hybrids against intracellular amatigotes of L. panamensis
Compound
Leishmanicidal
activity
SI
Comment
EC50 (lg/ml)
8
[31.3
\1.0
Moderately active
Active
10
3.9 ? 1.3
17.4 ? 1.0
6.4 ? 1.4
[100
3.8
4.7
12
Active
19
[31.25
[2.0
24.6
Moderately active
No active
Active
When the leishmanicidal activity was compared to the
cytotoxicity (Fig. 2), we observed that compounds 7, 9,
21, triclosan, 8-hydroxy quinaldine, and amphotericine B
were both cytotoxic on U937 cells and active against
axenic amastigotes of L. panamensis (lower left corner on
the Fig. 2a). Eugenol and the compounds 8 and 10 were
toxic on U937 cells and moderately active against
Leishmania parasites (lower right corner, Fig. 2a); com-
pound 20 was non-toxic but active on Leishmania para-
sites (upper left corner, Fig. 2a), and compounds 11 and
19 showed no cytotoxicity and no activity (upper right
corner, Fig. 2a).
20
21
2.4 ? 0.5
0.04 ? 0.01
6.8 ? 0.5
Amphotericin B
Glucantime
970.0
[147.05
Active
Active
The leishmanicidal activity against the intracellular forms
of L. panamensis was also determined for compounds that
showed high activity (compounds 20 and 21) or moderate
activity (compounds 8, 10, and 19) against axenic amastig-
otes of L. panamensis. Given that compound 12 showed an
apparent low toxicity against the U937 cells and the fact that
for some compounds the biological activity depends on the
metabolism that the molecule suffers inside the host cell,
the leishmanicidal activity against intracellular amastigotes
was also determined for compound 12. Although triclosan,
eugenol, and 8-hydroxyquinaldine showed activities against
axenic amastigotes of L. panamensis, their leishmanicidal
activities against the intracellular form of L. panamensis
were not evaluated because of the high toxicity levels of
these compounds against U937 cells. The activities of these
compounds are summarized in Table 2. Thus, the most
active compounds were 10, 12, 19, and 21 with EC50 of 3.9,
17.4, 6.4, and 2.4 lg/ml, respectively. Compound 8 showed
moderate leishmanicidal activity (EC50 [ 31.3). Compound
20 showed no activity against intracellular amastigotes of
L. panamensis (EC50 [ 100 lg/ml) (Table 2). The best SI
On the other hand, the compounds 10, 12, and 21
showed to be toxic and active on U937 cells and intracel-
lular amastigotes of L. panamensis (lower left corner,
Fig. 2b); the compound 8 was toxic on U937 cells and
moderately active against Leishmania parasites (lower
right corner, Fig. 2b), while compound 19 was non-toxic
but active (upper left corner, Fig. 2b). Finally, the com-
pound 20 showed no cytotoxicity and no activity (upper
right corner, Fig. 2b).
In general, the quinoline–triclosan hybrids are the most
active and least cytotoxic compounds. The quinoline–
eugenol hybrids showed a good activity and low selectivity
index. The relationship between the leishmanicidal activity
and structural facts such as the chain lenght showed that
short alkyl chains increase the biological activity in the
Fig. 2 Comparison between toxicity and leishmanicidal activity of
quinoline–eugenol and quinoline–triclosan hybrids. a Axenic ama-
tigotes of L. panamensis. Filled square 7, filled triangle 8, filled
inverted triangle 9, filled diamond 10, filled circle 11, open square 12,
open triangle 19, open inverted triangle 20, open diamond 21, open
circle 22, times 23, plus 24, asterisk triclosan, shaded square eugenol,
shaded triangle 8-hydroxy quinaldine, shaded inverted triangle
amphothericine B, shaded diamond meglumine antimoniate. b. Intra-
cellular amatigotes of L. panamensis. filled square 8, filled triangle
10, filled inverted triangle 12, filled diamond 19, filled circle 20, open
square 21, open triangle amphothericin B, open inverted triangle
meglumine antimoniate
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Med Chem Res (2012) 21:3445–3454
3449
quinoline–triclosan hybrids, and the quinoline–eugenol
hybrids were active when the alkyl chain is less than nine
carbon atoms.
and evaporated to dryness under reduced pressure. Column
chromatography on silica gel (hexane–ethyl acetate, dif-
ferent ratios) to afford the bis-alkylquinoline in yields
between 15 and 22% and the corresponding monoethers in
yields between 25 and 47%.
Conclusion
A solution of eugenol (1.0 eq) and potassium carbonate
(1.5 eq) in acetone (20 ml) was stirred for 1 h at room
temperature, the corresponding bromoalkylquinaldine
compound obtained previously (1.0 eq) was added, and the
mixture was refluxed for 48 h. Thereafter, the organic layer
was washed with water to remove alkali, using a separating
funnel, isolated, dried over anhydrous magnesium sulfate,
and evaporated to dryness under reduced pressure. Column
chromatography on silica gel (hexane–ethyl acetate, dif-
ferent ratios) to afford the quinoline–eugenol hybrids in
yields between 31 and 53%.
The design, synthesis, and antileishmanial screening of
twelve quinoline derivatives are reported. The results
shown here are promising since several of the compounds
have some potential as leishmanicidal drugs as determined
by both the leishmanicidal activity and the cytotoxicity.
Owing to the high leishmanicidal activity and the low
cytotoxicity observed in vitro for compounds 20 and 21,
other studies on the animal model for leishmaniasis disease
are needed to validate their potential as antileishmanial
drugs. On the other hand, compounds 7, 9, 10, 21, triclosan,
and 8-hydroxy quinoline that were active against Leish-
mania parasite but toxic for mammalian cells, still have
potential to be considered as candidates for antileishmanial
drug development. However, more studies on toxicity
using other cell lines are needed to discriminate whether
the toxicity shown by these compounds is against tumor or
non-tumor cells. Understanding the mechanism of action of
these molecules needs to be carried out as a future objec-
tive to this project.
8-((3-(4-allyl-2-methoxyphenoxy)propyl)oxy)-2-methylquin-
oline (7) Yield 0.44 g (34%); yellow oil; IR tmax: 3055
(=C–H), 1672 (C=C), 1599 (C=N), 1508 (C=C aromatic
1
ring), 1257 (C–O–C), 747 (C–H aromatic ring) cm-1. H
NMR (CDCl3, 300 MHz): d 2.76 (s, CH3), 7.30 (d, H-3,
J = 8.3 Hz), 7.98 (d, H-4, J = 8.4 Hz), 7.25 (d, H-5,
J = 8.3 Hz), 7.31 (t, H-6, J = 8.0 Hz), 7.03 (dd, H-7,
J = 6.9, 1.3 Hz), 4.45 (t, H-10, J = 6.4 Hz), 2.49 (m,
H-20), 4.29 (t, H-30, J = 6.1 Hz), 6.68 (d, H-300,
J = 1.5 Hz), 6.70 (dd, H-500, J = 8.1, 1.5 Hz), 6.90 (d,
H-600, J = 8.1 Hz), 3.83 (s, OCH3), 3.31(d, H-1000,
J = 6.6 Hz), 5.95 (m, H-2000), 5.01(d, H-3000, J = 1.0 Hz),
5.08 (d, H-3000, J = 8.7 Hz). 13C NMR (CDCl3, 75 MHz):
d 25.5 (CH3), 154.0 (C-2), 122.3 (C-3), 135.9 (C-4), 127.5
(C-4a),119.4 (C-5), 125.5 (C-6), 109.3 (C-7), 157.8 (C-8),
139.7 (C-8a), 66.1 0(0 C-10), 28.9 (C-20), 65.8 (C-30), 146.5
(C-100), 149.3 (C-2 ), 112.3 (C-300), 132.9 (C-400), 120.4
(C-500), 113.7 (C-600), 39.6 (C-1000), 137.5 (C-2000), 115.4
(C-3000), 55.8 (OCH3). APCIMS m/z 364 [M ? H]?.
Experimental procedures
Chemistry
IR spectra were recorded on a Perkin–Elmer Spectrum RX
1
I FT-IR system in a KBr disk. H NMR and 13C NMR
spectra were recorded on Bruker 300 MHz spectrometer
using CDCl3 as solvent and TMS as an internal standard.
The chemical shifts are expressed in d ppm. APCIMS and
HRTOFESIMS were run on a Waters Micromass LCT
mass spectrometer. Silica gel 60 (Merck 0.063–0.200
mesh) was used for column chromatography, and precoated
silica gel plates (Merck 60 F254 0.2 mm) were used for
TLC.
8-((4-(4-allyl-2-methoxyphenoxy)butyl)oxy)-2-methylquino-
line (8) Yield 0.40 g (31%); yellow pale oil; IR tmax
:
3055 (=C–H), 1670 (C=C), 1594 (C=N), 1508 (C=C aro-
matic ring), 1261 (C–O–C), 745 (C–H aromatic ring)
1
cm-1. H NMR (CDCl3, 300 MHz): d 2.75 (s, CH3), 7.30
(d, H-3, J = 8.3 Hz), 7.98 (d, H-4, J = 8.4 Hz), 7.27 (d,
H-5, J = 8.4 Hz), 7.35 (t, H-6, J = 8.1 Hz), 7.04 (dd, H-7,
J = 6.9, 1.3 Hz), 4.31 (t, H-10, J = 6.5 Hz), 2.08 (m,
H-20), 2.21 (m, H-30), 4.12 (t, H-40, J = 6.5 Hz), 6.68 (d,
H-300, J = 1.5 Hz), 6.70 (dd, H-500, J = 8.1, 1.5 Hz), 6.83
(d, H-600, J = 8.0 Hz), 3.82 (s, OCH3), 3.31(d, H-1000,
J = 6.4 Hz), 5.94 (m, H-2000), 5.02 (d, H-3000, J = 1.2 Hz);
5.06 (d, H-3000, J = 8.9 Hz). 13C NMR (CDCl3, 75 MHz):
d 25.6 (CH3), 154.1 (C-2), 122.3 (C-3), 135.9 (C-4), 127.6
(C-4a),119.3 (C-5), 125.5 (C-6), 109.0 (C-7), 157.9 (C-8),
137.6 (C-8a), 68.6 (C-1’), 25.5 (C-20), 26.0 (C-30), 68.7
Synthesis of quinoline–eugenol hybrids (7–12)
To a solution of 2-methyl-8-hydroxyquinolina (1.60 g,
10 mmol) in dichloromethane (40 ml) was added aqueous
NaOH solution (10%, 50 ml) followed by the addition of
1 mmol of tetrabutyl ammonium bromide and 10 mmol of
1,x-dibromoalkane derivative. The mixture was stirred at
room temperature for 24–48 h, then the organic layer was
isolated using a separating funnel, washed with water to
remove alkali, dried over anhydrous magnesium sulfate,
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Med Chem Res (2012) 21:3445–3454
(C-40), 146.7 (C-100), 149.3 (C-200), 112.2 (C-300), 132.6
(C-400), 120.3 (C-500), 113.2 (C-600), 39.7 (C-1000), 137.6 (C-2000),
115.4 (C-3000), 55.8 (OCH3). APCIMS m/z 378 [M ? H]?.
(d, H-3, J = 7.4 Hz), 7.97 (d, H-4, J = 8.0 Hz), 7.27 (d,
H-5, J = 7.6 Hz), 7.36 (t, H-6, J = 8.0 Hz), 7.02 (dd, H-7,
J = 7.3, 1.4 Hz), 4.21 (t, H-10, J = 7.2 Hz), 2.02 (m,
H-20), 1.51 (m, H-30), 1.37 (m, H-40), 1.37 (m, H-50), 1.37
(m, H-60), 1.51 (m, H-70), 1.82 (m, H-80), 3.97 (t, H-90,
J = 7.0 Hz), 6.70 (d, H-300, J = 1.5 Hz), 6.69 (dd, H-500,
J = 8.1, 1.5 Hz), 6.80 (d, H-6, J = 8.6 Hz), 3.84 (s,
OCH3), 3.32 (d, H-1000, J = 6.6 Hz), 5.95 (m, H-2000), 5.08
(dd, H-3000, J = 8.6, 1.5 Hz), 5.04 (d, H-3000, J = 1.5 Hz).
13C NMR (CDCl3, 75 MHz): d 25.6 (CH3), 157.8 (C-2),
122.3 (C-3), 136.0 (C-4), 127.6 (C-4a), 120.3 (C-5), 125.5
(C-6), 108.9 (C-7), 154.2 (C-8), 139.8 (C-8a), 69.0 (C-10),
29.2 (C-20), 25.6 (C-30), 28.7 (C-40), 29.2 (C-50), 28.7
(C-60), 25.8 (C-70), 29.3 (C-80), 69.0 (C-90), 146.8 (C-100),
149.3 (C-200), 112.3 (C-300), 135.8 (C-400), 120.3 (C-500),
113.0 (C-600), 39.7 (C-1000), 137.6 (C-2000), 115.4 (C-3000),
55.8 (OCH3). APCIMS m/z 448 [M ? H]?.
8-((5-(4-allyl-2-methoxyphenoxy)pentyl)oxy)-2-methylquin-
oline (9) Yield 0.55 g (43%); yellow pale oil; IR tmax
:
3055 (=C–H), 1672 (C=C), 1599 (C=N), 1508 (C=C aro-
matic ring), 1257 (C–O–C), 747 (C–H aromatic ring)
1
cm-1. H NMR (CDCl3, 300 MHz): d 2.76 (s, CH3), 7.31
(d, H-3, J = 8.3 Hz), 7.98 (d, H-4, J = 8.4 Hz), 7.27 (d,
H-5, J = 8.5 Hz), 7.36 (t, H-6, J = 8.1 Hz), 7.02 (dd, H-7,
J = 7.1, 1.5 Hz), 4.24 (t, H-10, J = 7.0 Hz), 2.10 (m,
H-20), 1.71 (m, H-30), 1.95 (m, H-40), 4.03 (t, H-50,
J = 7.0 Hz), 6.70 (d, H-300, J = 1.5 Hz), 6.69 (dd, H-500,
J = 8.1, 1.5 Hz), 6.81 (d, H-600, J = 8.4 Hz), 3.83 (s,
OCH3), 3.32 (d, H-1000, J = 6.6 Hz), 5.95 (m, H-2000), 5.08
(dd, H-3000, J = 8.6, 1.5 Hz), 5.03 (d, H-3000, J = 1.5 Hz).
13C NMR (CDCl3, 75 MHz): d 25.6 (CH3), 154.1 (C-2),
122.3 (C-3), 135.9 (C-4), 127.6 (C-4a),120.3 (C-5), 125.5
(C-6), 108.9 (C-7), 157.9 (C-8), 139.8 (C-8a), 68.9 (C-10),
28.5 (C-20), 22.5 (C-30), 29.0 (C-40), 68.7 (C-50), 146.7
(C-100), 149.3 (C-200), 119.2 (C-300), 132.6 (C-400), 112.3
(C-500), 113.2 (C-600), 39.7 (C-1000), 137.6 (C-2000), 115.4
(C-3000), 55.8 (OCH3). APCIMS m/z 392 [M ? H]?.
8-((12-(4-allyl-2-methoxyphenoxy) dodecyl)oxy)-2-methyl-
quinoline (12) Yield 0.54 g (45%); yellow oil; IR tmax
:
3050 (=C–H), 1670 (C=C), 1560 (C=N), 1508 (C=C aro-
matic ring), 1257 (C–O–C), 754 (C–H aromatic ring)
1
cm-1. H NMR (CDCl3, 300 MHz): d 2.77 (s, CH3), 7.31
(d, H-3, J = 8.3 Hz), 7.98 (d, H-4, J = 8.4 Hz), 7.27 (d,
H-5, J = 8.5 Hz), 7.34 (t, H-6, J = 8.1 Hz), 7.02 (dd, H-7,
J = 7.3, 1.4 Hz), 4.21 (t, H-10, J = 7.2 Hz), 2.02 (m,
H-20), 1.49 (m, H-30), 1.49 (m, H-40), 1.28 (m, H-50), 1.49
(m, H-60), 1.49 (m, H-70), 1.28 (m, H-80), 1.49 (m, H-90),
1.49 (m, H-100), 1.81 (m, H-110), 3.97 (t, H-120,
J = 7.0 Hz), 6.68 (d, H-300, J = 1.5 Hz), 6.69 (dd, H-500,
J = 8.1, 1.5 Hz), 6.78 (d, H-600, J = 8.6 Hz), 3.83
(s, OCH3), 3.31 (d, H-1000, J = 6.6, 1.5 Hz), 5.94 (m,
H-2000), 5.09 (dd, H-3000, J = 8.6, 1.5 Hz), 5.03 (d, H-3000,
J = 1.5 Hz). 13C NMR (CDCl3, 75 MHz): d 25.6 (CH3),
154.2 (C-2), 122.3 (C-3), 135.9 (C-4), 127.6 (C-4a), 119.1
(C-5), 125.6 (C-6), 108.9 (C-7), 157.9 (C-8), 139.8 (C-8a),
69.0 (C-10), 29.1 (C-20), 25.9 (C-30), 28.7 (C-40), 29.3
(C-50), 29.5 (C-60), 29.5 (C-70), 29.3 (C-80), 28.7 (C-90),
25.9 (C-100), 29.4 (C-110), 69.1 (C-120),146.8 (C-100), 149.3
(C-200), 112.3 (C-300), 132.5 (C-400), 120.3 (C-500), 113.1
(C-600), 39.1 (C-1000), 137.6 (C-2000), 115.4 (C-3000), 55.7
(OCH3). APCIMS m/z 491 [M ? H]?.
8-((8-(4-allyl-2-methoxyphenoxy)octyl)oxy)-2-methylquinoline
(10) Yield 0.61 g (49%); yellow pale oil; IR tmax: 3055
(=C–H), 1672 (C=C), 1594 (C=N), 1510 (C=C aromatic
1
ring), 1258 (C–O–C), 748 (C–H aromatic ring) cm-1. H
NMR (CDCl3, 300 MHz): d 2.76 (s, CH3), 7.31 (d, H-3,
J = 8.2 Hz), 7.97 (d, H-4, J = 8.4 Hz), 7.26 (d,
H-5, = 8.3 Hz), 7.35 (t, H-6, J = 8.1 Hz), 7.02 (dd, H-7,
J = 7.3, 1.4 Hz), 4.21 (t, H-10, J = 7.2 Hz), 2.03 (m,
H-20), 1.42 (m, H-30), 1.42 (m, H-40), 1.42 (m, H-50), 1.42
(m, H-60), 1.82 (m, H-70), 3.97 (t, H-80, J = 7.0 Hz), 6.70
(d, H-300, J = 1.5 Hz), 6.68 (dd, H-500, J = 8.1, 1.5 Hz),
6.80 (d, H-600, J = 8.6 Hz), 3.83 (s, OCH3), 3.31 (d, H-1000,
J = 7.0 Hz), 5.95 (m, H-2000), 5.06 (dd, H-3000, J = 8.4,
1.4 Hz); 5.02 (dd, H-3000, J = 1.4 Hz). 13C NMR (CDCl3,
75 MHz): d 25.6 (CH3), 154.2 (C-2), 122.3 (C-3), 135.9
(C-4), 127.5 (C-4a), 119.1 (C-5), 125.5 (C-6), 108. (C-7),
157.8 (C-8), 139.7 (C-8a), 69.0 (C-10), 28.7 (C-20), 25.8
(C-30), 29.2 (C-40), 29.2 (C-50), 25.8 (C-60), 29.1 (C-70),
68.9 (C-80), 146.8 (C-100), 149.2 (C-200), 112.2 (C-300), 132.5
(C-400), 120.3 (C-500), 113.0 (C-600), 39.7 (C-1000), 137.6
(C-2000), 115.4 (C-3000), 55.8 (OCH3). APCIMS m/z 434
[M ? H]?.
Synthesis of quinoline–triclosan hybrids (19–24)
Triclosan (1.0 g, 3.45 mmol) was mixed with potassium
carbonate (4.0 mmol), heated to 60–70°C and stirred
for 30 min, after cooling the mixture, a solution of
1,x-dibromoalkane derivative (3.45 mmol) in acetone was
added, and the mixture was exposed to reflux for a period
of 24–36 h. The organic layer was washed with water to
remove alkali, using a separating funnel, isolated, dried
over anhydrous magnesium sulfate, and evaporated to
8-((9-(4-allyl-2-methoxyphenoxy)nonyl)oxy)-2-methylquino-
line (11) Yield 0.65 g (53%); yellow pale oil; IR tmax
:
3055 (=C–H), 1672 (C=C), 1600 (C=N), 1506 (C=C aro-
matic ring), 1260 (C–O–C), 745 (C–H aromatic ring)
1
cm-1. H NMR (CDCl3, 300 MHz): d 2.77 (s, CH3), 7.30
123

Med Chem Res (2012) 21:3445–3454
3451
dryness under reduced pressure. Column chromatography
on silica gel (hexane–ethyl acetate, different ratios) to
afford the bromoalkyltriclosan in yield between 78 and
97%. Thereafter, 8-hydroxyquinaldine (1.0 eq) and potas-
sium carbonate (1.2 eq) were mixed in acetone. The mix-
ture was stirred for 30 min and the corresponding
bromoalkyltriclosan derivative obtained previously was
added and placed under reflux for 48 h. Then, to complete
the reaction, the mixture was transferred to a separatory
funnel, and the organic layer was washed with water,
separated, dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure. The residue was
chromatographed over silica gel (hexane–ethyl acetate,
different ratios) to afford the quinoline–triclosan hybrids in
yield between 41 and 75%.
(C-10), 26.1 (C-20), 25.7 (CH3), 25.3 (C-30), ESI–MS (m/z)
503 (M ? H)?.
8-((5-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)pentyl)oxy)-
2-methylquinoline (21) Yield 0.83 g (55%); white solid,
m.p. = 139-142; IR tmax: 1598 (C=N), 1470 (C=C), 1230
1
(C–O–C), 798 (C–H aromatic ring), 740 (C–Cl) cm-1. H
NMR (CDCl3, 300 MHz): d 2.72 (s, CH3), 7.25 (d, H-3,
J = 8.4 Hz), 7.95 (d, H-4, J = 8.4 Hz), 7.31–7.39 (m, H-5
and H-6), 6.94–7.04 (m, H-7, H-600 and H-6000), 4.10
(t, H-10, J = 7.0 Hz), 1.93 (m, H-20), 1.38 (m, H-30), 1.71
(m, H-40), 3.91 (t, H-50, J = 6.3 Hz), 6.57 (d, H-300,
J = 8.4 Hz), 6.87 (dd, H-5000, J = 8.4, 2.5 Hz), 7.37
(d, H-3000, J = 2.5 Hz), 7.01 (dd, H-400, J = 8.7, 2.5), 13C
NMR (CDCl3, 75 MHz): d 158.0 (C-2), 154.2 (C-8),152.6
(C-1000), 150.9 (C-100), 142.8 (C-200), 139.8 (C-8a), 135.9 (C-
4), 130.6 (C-4000), 130.0 (C-3000), 127.6 (C-4a), 127.5 (C-500),
125.6 (C-6),127.5 (C-5000), 124.2 (C-2000), 122.4 (C-3),
122.2 (C-400), 120.8 (C-6000), 119.3 (C-300),117.6 (C-5),
114.6 (C-600), 109.0 (C-7), 68.7 (C-10), 68.7 (C-50), 28.6
(C-40), 28.3 (C-20), 25.7 (CH3), 22.2 (C-30), ESI–MS (m/z)
516 (M ? H)?.
8-((3-(5-(chloro-2-(2,4-dichlorophenoxy)phenoxy)propyl)oxy)-
2-methylquinoline (19) Yield 1.04 g (72%); white solid,
m.p = 112–113; IR tmax : 1596 (C=N), 1474 (C=C), 1229
1
(C–O–C), 797 (C–H aromatic ring), 738 (C–Cl) cm-1. H
NMR (CDCl3, 300 MHz): d 2.75 (s, CH3), 7.27 (d, H-3,
J = 8.5 Hz), 8.0 (d, H-4, J = 8.6 Hz), 7.24-7.41 (m, H-5
and H-6) 4.24 (t, H-10, J = 6.2 Hz), 2.33 (m, H-20), 4.09 (t,
H-30, J = 5.5 Hz), 6.53 (d, H-300, J = 8.8 Hz), 6.86 (dd,
H-5000, J = 7.6, 1.4 Hz), 6.83–7.05 (m, H-7, H-400, H-600
and H-6000). 13C NMR (CDCl3, 75 MHz): d 157.9 (C-2),
154.0 (C-8),152.5 (C-1000), 150.8 (C-100), 142.4 (C-200),
139.7 (C-8a), 135.90 (C-4), 00130.7 (C-4000), 130.0
(C-3000),127.5 (C-4a), 127.5 (C-5 ), 127.5 (C-400), 125.6
(C-6), 123.9 (C-2000), 122.3 (C-6000), 122.2 (C-3), 120.9
(C-5000),119.1 (C-5), 117.2 (C-300),114.6 (C-600), 109.3
(C-7), 65.6 (C-30), 65.0 (C-10), 28.7 (C-20), 25.6 (CH3),
ESI–MS (m/z) 490 (M ? H)?.
8-((8-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)octyl)oxy)-
2-methylquinoline (22) Yield 1.21 g (75%); white solid,
m.p. = 82–83; IR tmax: 1600 (C=N), 1474 (C=C), 1230
1
(C–O–C), 795 (C–H aromatic ring), 740 (C–Cl) cm-1. H
NMR (CDCl3, 300 MHz): d 2.76 (s, CH3), 7.27 (d, H-3,
J = 8.4 Hz), 7.98 (d, H-4, J = 8.5 Hz), 7.31–7.38 (m, H-5
and H-6), 6.92–7.03 (m, H-7, H-600 and H-6000), 4.21 (t,
H-10, J = 7.3 Hz), 2.02 (m, H-20), 1.48 (m, H-30), 1.24 (m,
H-40), 1.24 (m, H-50), 1.24 (m, H-60), 1.60 (m, H-70), 3.87
(t, H-80, J = 6.4 Hz), 6.60 (d, H-300, J = 8.5 Hz), 7.05 (dd,
H-400, J = 8.6, 2.5 Hz), 7.40 (d, H-3000, J = 2.4 Hz), 6.89
(dd, H-5000, J = 8.4, 2.0 Hz), 13C NMR (CDCl3, 75 MHz):
d 157.9 (C-2), 154.2 (C-8),152.6 (C-1000), 151.0 (C-100),
142.7 (C-200), 139.8 (C-8a), 135.9 (C-4), 130.5 (C-4000),
130.0 (C-3000), 127.6 (C-500), 127.5 (C-4a), 127.4 (C-5000),
125.6 (C-6), 124.1 (C-2000), 122.3 (C-3), 122.2 (C-400),
120.7 (C-6000), 119.2 (C-5), 117.5 (C-300),114.5 (C-600),108.9
(C-7), 68.9 (C-10), 68.9 (C-80), 29.2 (C-20), 29.0 (C-70),28.8
(C-30), 28.7 (C-60), 25.8 (C-40), 25.5 (C-50), 25.6 (CH3),
ESI–MS (m/z) 560 (M ? H)?.
8-((4-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)butyl)oxy)-
2-methylquinoline (20) Yield 076 g (45%); white solid,
m.p. = 96-97; IR tmax: 1600 (C=N), 1473 (C=C), 1230
1
(C–O–C), 798 (C–H aromatic ring), 738 (C–Cl) cm-1. H
NMR (CDCl3,300 MHz): d 2.75 (s, CH3), 7.28 (d, H-3,
J = 8.4 Hz), 8.0 (d, H-4, J = 8.4 Hz), 7.33 (d, H-5,
J = 7.2 Hz), 7.33 (t, H-6, J = 6.0 Hz), 6.98 (dd, H-7,
J =0 6.2, 2.0 Hz), 4.21 (t, H-10, J = 5.8 Hz), 1.95 (m,
H-2 ), 1.95 (m, H-30), 4.10 (t, H-40, J = 5.8 Hz), 6.63 (d,
H-300, J = 8.8 Hz), 7.03 (dd, H-400, J = 8.8, 2.4 Hz), 6.98
(d, H-600, J = 2.4 Hz), 7.38 (d, H-3000, J = 2.0 Hz), 6.89
(dd, H-5000, J = 8.4, 2.0 Hz), 6.92 (d, H-6000, J = 8.4 Hz).
13C NMR (CDCl3, 75 MHz): d 158.0 (C-2), 154.1
(C-8),152.5 (C-1000), 151.0 (C-100), 143.0 (C-200), 139.9
(C-8a), 136.0 (C-4), 130.6 (C-4000), 130.1 (C-3000), 127.7 (C-
4a), 127.7 (C-500), 127.6 (C-400), 125.6 (C-6), 124.4 (C-200),
122.4 (C-3), 122.0 (C-6000), 120.9 (C-5000), 119.6 (C-5),
117.9 (C-300),114.9 (C-600), 109.1 (C-7), 69.0 (C-40), 68.5
8-((9-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)nonyl)oxy)-
2-methylquinoline (23) Yield 1.09 g (69%); white solid,
m.p. = 71–73; IR tmax: 1598 (C=N), 1471 (C=C), 1230
1
(C–O–C), 796 (C–H aromatic ring), 738 (C–Cl) cm-1. H
NMR (CDCl3,300 MHz): d 2.76 (s, CH3), 7.27 (d, H-3,
J = 8.6 Hz), 7.98 (d, H-4, J = 8.5 Hz), 7.31–7.39 (m, H-5
and H-6), 6.92–7.02 (m, H-7, H-600 and H-6000), 4.21 (t,
H-10, J = 7.0 Hz), 2.0 (H-20), 1.50 (H-30), 1.23 (H-40), 1.23
123

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Med Chem Res (2012) 21:3445–3454
(H-50), 1.23 (H-60), 1.35 (H-70), 1.58 (H-80), 3.87 (t, H-90,
J = 6.2 Hz), 6.60 (d, H-300, J = 8.8 Hz), 7.05 (dd, H-400,
J = 8.8, 2.4 Hz), 7.41 (d, H-3000, J = 2.4 Hz), 6.89 (dd,
H-5000, J = 8.4, 2.2 Hz), 13C NMR (CDCl3, 75 MHz): d
157.9 (C-2), 154.2 (C-8), 152.6 (C-1000), 151.0 (C-100),
142.8 (C-200), 139.8 (C-8a), 135.9 (C-4), 130.6 (C-4000),
130.0 (C-3000), 127.6 (C-500), 127.5 (C-4a), 127.4 (C-5000),
125.6 (C-6), 124.1 (C-2000), 122.3 (C-3), 122.2 (C-400),
120.7 (C-6000), 119.1 (C-5), 117.5 (C-300), 114.5 (C-600),
108.8 (C-7), 69.0 (C-10), 68.9 (C-90), 29.3 (C-50), 29.0
(C-20), 29.3 (C-80), 28.8 (C-40), 28.7 (C-60), 25.9 (C-70),
25.7 (CH3), 25.6 (C-30), ESI–MS (m/z) 574 (M ? H)?.
corresponding concentrations of the compounds, starting at
200 lg/ml in duplicate. The cells were incubated at 37°C
with 5% CO2 for 72 h in the presence of the compounds, and
then the effect was determined using MTT assay, incubating
at 37°C for 3 h. After 72 h of incubation, the effect of
compounds was determined by measuring the activity of the
mitochondrial dehydrogenase by adding 10 ll/well of MTT
solution (0.5 mg/ml) and incubating at 37°C for 3 h. The
reaction was stopped by adding a 50% isopropanol solution
with 10% sodium dodecyl sulfate for 30 min. Cell viability
was determined based on the quantity of formazan produced,
which was measured by means of a Bio-Rad ELISA reader
set at 570 nm. As a viability test, cultured cells in the absence
of extracts were used; as cytotoxicity controls, Amphotericin
B and meglumine antimoniate were used. The results are
expressed as the Lethal Concentration 50 (LC50) calculated
by the Probit method (Finney, 1971).
8-((12-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)dodecyl)oxy)-
2-methylquinoline (24) Yield 0.67 g (41%); yellow pale
solid, m.p. = 78-80; IR tmax: 1598 (C=N), 1475 (C=C),
1229 (C–O–C), 796 (C–H aromatic ring), 737 (C–Cl)
cm-1. 1H NMR (CDCl3, 300 MHz): d 2.77 (m, CH3), 7.27
(d, H-3, J = 8.7 Hz), 7.98 d, H-4, J = 8.4 Hz), 7.28–7.37
(m, H-5 and H-6), 6.92–7.03 (m, H-7, H-600 and H-6000),
4.23 (t, H-10, J = 7.2 Hz), 2.02 (m, H-20), 1.51 (m, H-30),
1.36 (m, H-40), 1.1 (m, H-50), 1.1 (m, H-60), 0.91 (m, H-70),
1.1 (m, H-80), 1.1 (m, H-90), 1.38 (m, C-100), 1.58 (m,
C-110), 3.87 (t, C-120, J = 6.3 Hz), 6.60 (d, H-300,
J = 8.8 Hz), 7.06 (dd, H-400, J = 8.8, 2.4 Hz), 7.40 (d,
H-3000, J = 2.5 Hz), 6.89 (dd, H-5000, J = 8.4, 2.1 Hz), 13C
NMR (CDCl3, 75 MHz): d 157.9 (C-2), 154.2 (C-8), 152.6
(C-1000), 151.0 (C-100), 142.7 (C-200), 139.8 (C-8a),135.9
(C-4), 130.5 (C-4000), 130.0 (C-3000), 127.6 (C-5000), 127.4
(C-4a), 127.4 (C-5000), 125.6 (C-6), 124.1 (C-2000), 122.3
(C-3), 122.2 (C-400), 120.6 (C-600), 119.1 (C-5), 117.5
(C-300), 114.5 (C-600), 108.8 (C-7), 69.0 (C-120), 68.9 (C-10),
29.5 (C-110), 29.4 (C-70), 29.4 (C-60), 29.1 (C-20), 28.8
(C-40), 28.8 (C-90), 28.7 (C-30), 28.7 (C-100), 25.9
(C-50), 25.6 (C-80), 25.6 (CH3), ESI–MS (m/z) 616
(M ? H)?.
In vitro leishmanicidal activity on axenic
and intracellular amastigotes
Axenic and intracellular amastigotes of GFP-transfected
L. (V.) panamensis strain (MHOM/CO/87/UA140epir
GFP) were used for the in vitro testing of leishmanicidal
activity of the bis-alkylquinolines and quinoline–triclosan
and quinoline–eugenol hybrids.
Activity against axenic amastigotes
The respective ability of the bis-alkylquinolines and quino-
line–triclosan and quinoline–eugenol hybrids to kill axenic
amastigotes of L. (V.) panamensis was determined based
on the viability of the parasites evaluated by the MTT (3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
method as described previously (Taylor et al., 2010). In
short, parasites were cultivated in Schneider’s medium pH
5.4 supplemented with 20% heat inactivated FBS for 3 days
at 32°C. Afterward, they were harvested, washed, and re-
suspended at 2 9 106 axenic amastigotes/ml in fresh med-
ium. Each well of a 96-well plate was seeded with 100 ll of
each parasite suspension (in duplicate), and 100 ll of each
concentration of the test compound was added, starting at
100 lg/ml. Plates were incubated at 32°C. After 72 h of
incubation, the effect of drugs was determined by adding
10 ll/well of MTT and incubating at 32°C for 3 h. The
reaction was stopped, and the quantity of formazan produced
was measured with a Bio-Rad ELISA reader set at 570 nm.
Parasites cultivated in the absence of the compound but
maintained under the same conditions were used as controls
for growth and viability. Parasites cultivated in the presence
of anphotericin B and meglumine antimoniate were used as
controls for leishmanicidal activity.
Biological activity assays
The compounds were subjected to in vitro leishmanicidal
activity on amastigotes and promastigotes of L. panamensis
and cytotoxic activity on mammalian cells.
In vitro cytotoxic activity in mammalian cells
The cytotoxic activity of the compounds was assessed based on
the viability of the human promonocytic cell line U937 (ATCC
CRL-1593.2TM) evaluated by the MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as
describedby Robledoet al. (2005). In brief, cells were grown in
96-well cell-culture dishes at a concentration of 100,000
cells/ml in RPMI-1640 supplemented with 10% FBS and the
123

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Activity against intracellular amastigotes
The effects of the bis-alkylquinolines and quinoline–
triclosan and quinoline–eugenol hybrids against intracel-
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flow cytometry using the methodology described by Varela
et al. (2009). In brief, U937 cells were dispensed in 24-well
plates at a concentration of 300,000 cells/well, which were
treated with 1 lM of Phorbol Myristate Acetate (PMA) for
48 h at 37°C, after which they were infected with prom-
astigotes of L. (V.) panamensis in stationary growth phase
(day 5) in modified NNN medium, a proportion of 1:25
cell/parasite, after 3 h of incubation at 34°C in 5% CO2
non-internalized parasites were washed, and incubated
again at 34°C and 5% CO2 to allow differentiation to
amastigotes form. After 24 h of incubation, the compounds
with the appropriate dilution, not exceeding the LC50, were
added. Infected and treated cells were maintained at 34°C
and 5% CO2 for 72 h. The leishmanicidal effect was
measured in a flow cytometer at 488 nm of excitation
and 525 nm of emission, and determined as described by
Varela etal. (2009). The results are expressed as theEffective
Concentration 50 (EC50) calculated by the Probit statistical
method. The data are the averages of three independent
experiments conducted in duplicate. Infected but untreated
cells were used as control of viability. In addition, infected
cells exposed to Amphotericin B and meglumine antimoni-
ate were used as leishmanicidal activity. The Selectivity
Index (SI) was calculated by dividing the cytotoxic activity
between the leishmanicidal activity (SI = LC50/EC50).
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´
´
Acknowledgment The authors thank Blandine Seon-Meniel for the
help during NMR measurements. This research was supported finan-
cially by the Universidad de Antioquia (Programa de Sostenibilidad).
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