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Nov 19, 2011 - Synthesis and leishmanicidal activity of quinoline–triclosan and quinoline–eugenol hybrids. Victor Arango • Jorge J. Domınguez • Wilson ...
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. Domı´nguez • Wilson Cardona Sara M. Robledo • Diana L. Mun˜oz • Bruno Figadere • Jairo Sa´ez



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 products were elucidated by spectroscopic analysis. The compounds 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 promising for the development of new antileishmanial drugs. Keywords Leishmania  Antiprotozoal  Quinoline  Hybrids  Triclosan  Amastigotes  Eugenol

V. Arango  J. J. Domı´nguez  W. Cardona (&)  J. Sa´ez Instituto de Quı´mica, Quı´mica de Plantas Colombianas, Universidad de Antioquia, A. A 1226, Medellı´n, Colombia e-mail: [email protected]; [email protected] S. M. Robledo  D. L. Mun˜oz Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, A. A 1226, Medellı´n, Colombia B. Figadere Laboratoire de Pharmacognosie, Associe0 au CNRS (BIOCIS), Universite0 de Paris-Sud, 92296 Chatenay-Malabry, France

Introduction The Leishmaniases are a group of diseases caused by different 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 recommended standard drugs for treatment are still the pentavalent 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 Leishmaniasis disease. 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.,

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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 progressively inhibits the Leishmania amazonensis growth and shows no cytototoxic effects against mammalian cells (Nakamura et al., 2006). 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 necessarily 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 combining 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 quinoline moieties has an effect on activity.

Results and discussion Chemistry Appropriate amounts of 8-hydroxyquinaldine were dissolved in dichloromethane, aqueous NaOH solution was added to the solution followed by tetrabutyl ammonium

Cl

Cl N

N

O O

O O

O

O n

n Cl

A

n = 1, 2, 3, 6, 7,10

B

Fig. 1 Chemical structures of quinoline–triclosan and quinoline– eugenol hybrids

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bromide and 1,x-dihaloalkane (Palit et al., 2009; Chaudhuri et al., 2007), and the mixture was stirred continuously. 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 bromoalkyltriclosan derivative obtained previously was added to a mixture of appropriate amounts of 8-hydroxyquinaldine with an equivalent amount of potassium carbonate in acetone; 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. Antileishmanial activity The leishmanicidal activity and cytotoxicity of the compounds synthesized as well as glucantime and Amphotericin 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. According to the results shown in the Table 1, compounds 7, 9, 20, 21, triclosan, and 8-hydroxy quinoline are very active against axenic amastigotes of L. panamensis 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 macrophages, the results showed a high toxicity level for the compounds 7–12, 21, triclosan, and the 8-hydroxy quinoline 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.

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Scheme 1 General synthetic pathway to the quinoline– eugenol hybrids

+ N

Br

n

Br

NaOH, CH 2Cl2

+ N

TBAB

OH

N

O

n = 1, 2,3,6,7,10

O n

n

Br

O

Compounds 1-6

N

OCH3 OH K 2 CO3, Acetone Reflux, 48 h

5

, 1 O

6

H3 CO n

7

, 2"

3"

O

3"

, 1" 6"

,

5"

7, n = 1 8, n = 2 9, n = 3 10, n = 6 11, n = 7 12, n = 10

, 3" Cl O

1) K2 CO 3, 60-70

oC,10'

1) K2CO 3 acetone.

Cl Cl

Cl

2) acetone, Br

n Br

reflux, 24 h n = 1, 2,3,6,7,10

O

4

OH

n

O

N

5

reflux, 48 h Cl

, 1 O

6 Br

, 5" , 6"

3

N

O

OH

Cl

Cl

3" n

19, n 20, n 21, n 22, n 23, n 24, n

4"

O 6"

7

Compounds 13-18

Cl

N

Cl

3 4

=1 =2 =3 =6 =7 = 10

Scheme 2 Synthetic pathway to quinoline–triclosan hybrids

Table 1 In vitro leishmanicidal activity against axenic amatigotes of L. panamensis and toxicity of quinoline– eugenol and quinoline–triclosan hybrids

LC50 Lethal Concentration 50; EC50 Effective Concentration 50; SI selectivity index: LC50/ EC50; Cytotoxicity 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

Compound

Cytotoxicity U937 cells LC50 (lg/ml)

Leishmanicidal activity EC50 (lg/ml)

SI

Comment

7

6.8 ? 0.3

6.9 ? 0.5

8

31.3 ? 0.1

38.9 ? 5.8

0.5

Cytotoxic, moderately active

9

10.5 ? 2.1

9.5 ? 0.7

1.0

Cytotoxic, active

10

14.7 ? 1.8

29.6 ? 1.3

0.5

Cytotoxic, moderately active

11

14.1 ? 2.8

21.1 ? 2.2

0.7

Cytotoxic, moderately active

12

82.9 ? 5.4

[100

19 20

[200.0 [200.0

23.6 ? 1.0 9.7 ? 0.9

1.0

[0.8 [8.5 [20.6

Cytotoxic, active

Cytotoxic, no active No cytotoxic, moderately active No cytotoxic, active

21

59.2 ? 9.8

4.1 ? 0.5

22

[200.0

[100.0

[2.0

No cytotoxic, no active

23

[200.0

[100.0

[2.0

No cytotoxic, no active

24

[200.0

[100

[2.0

No cytotoxic, no active

14.4

Cytotoxic, active

Triclosan

4.8 ? 0.4

11.3 ? 1.3

0.4

Eugenol

14.2 ? 1.6

21.4 ? 4.1

0.7

Cytotoxic, moderately active

8-hydroxy quinaldine

7.6 ? 0.2

2.6 ? 0.3

2.9

Cytotoxic, active

776

Cytotoxic, active

Amphotericin B

38.8 ? 2.2

0.05 ? 0.01

Glucantime

[1,000.0

[1,000.0

[1.0

Cytotoxic, active

No cytotoxic, no active

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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 amastigotes 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

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 compounds 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. panamensis and was less toxic than the other compounds. 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); compound 20 was non-toxic but active on Leishmania parasites (upper left corner, Fig. 2a), and compounds 11 and 19 showed no cytotoxicity and no activity (upper right corner, Fig. 2a). On the other hand, the compounds 10, 12, and 21 showed to be toxic and active on U937 cells and intracellular 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 compound 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 amatigotes 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. Intracellular 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

Table 2 In vitro activity of quinoline–eugenol and quinoline–triclosan hybrids against intracellular amatigotes of L. panamensis Compound

Leishmanicidal activity EC50 (lg/ml)

8

[31.3

10

3.9 ? 1.3

12

17.4 ? 1.0

19

6.4 ? 1.4

20

[100

21

2.4 ? 0.5

Amphotericin B

0.04 ? 0.01

Glucantime

6.8 ? 0.5

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SI

Comment

\1.0 3.8 4.7 [31.25 [2.0 24.6

Moderately active Active Active Moderately active No active Active

970.0

Active

[147.05

Active

Med Chem Res (2012) 21:3445–3454

quinoline–triclosan hybrids, and the quinoline–eugenol hybrids were active when the alkyl chain is less than nine carbon atoms.

Conclusion 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 Leishmania 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 objective to this project.

Experimental procedures Chemistry IR spectra were recorded on a Perkin–Elmer Spectrum RX I FT-IR system in a KBr disk. 1H 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. 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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and evaporated to dryness under reduced pressure. Column chromatography on silica gel (hexane–ethyl acetate, different ratios) to afford the bis-alkylquinoline in yields between 15 and 22% and the corresponding monoethers in yields between 25 and 47%. 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, different ratios) to afford the quinoline–eugenol hybrids in yields between 31 and 53%. 8-((3-(4-allyl-2-methoxyphenoxy)propyl)oxy)-2-methylquinoline (7) Yield 0.44 g (34%); yellow oil; IR tmax: 3055 (=C–H), 1672 (C=C), 1599 (C=N), 1508 (C=C aromatic ring), 1257 (C–O–C), 747 (C–H aromatic ring) cm-1. 1H 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 (C-10 ), 28.9 (C-20 ), 65.8 (C-30 ), 146.5 (C-100 ), 149.3 (C-200 ), 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]?. 8-((4-(4-allyl-2-methoxyphenoxy)butyl)oxy)-2-methylquinoline (8) Yield 0.40 g (31%); yellow pale oil; IR tmax: 3055 (=C–H), 1670 (C=C), 1594 (C=N), 1508 (C=C aromatic ring), 1261 (C–O–C), 745 (C–H aromatic ring) cm-1. 1H 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

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(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]?. 8-((5-(4-allyl-2-methoxyphenoxy)pentyl)oxy)-2-methylquinoline (9) Yield 0.55 g (43%); yellow pale oil; IR tmax: 3055 (=C–H), 1672 (C=C), 1599 (C=N), 1508 (C=C aromatic ring), 1257 (C–O–C), 747 (C–H aromatic ring) cm-1. 1H 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). 13 C 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-((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 ring), 1258 (C–O–C), 748 (C–H aromatic ring) cm-1. 1H 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]?. 8-((9-(4-allyl-2-methoxyphenoxy)nonyl)oxy)-2-methylquinoline (11) Yield 0.65 g (53%); yellow pale oil; IR tmax: 3055 (=C–H), 1672 (C=C), 1600 (C=N), 1506 (C=C aromatic ring), 1260 (C–O–C), 745 (C–H aromatic ring) cm-1. 1H NMR (CDCl3, 300 MHz): d 2.77 (s, CH3), 7.30

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(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). 13 C 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-((12-(4-allyl-2-methoxyphenoxy) dodecyl)oxy)-2-methylquinoline (12) Yield 0.54 g (45%); yellow oil; IR tmax: 3050 (=C–H), 1670 (C=C), 1560 (C=N), 1508 (C=C aromatic ring), 1257 (C–O–C), 754 (C–H aromatic ring) cm-1. 1H 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]?. 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

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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 potassium carbonate (1.2 eq) were mixed in acetone. The mixture 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%. 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 (C–O–C), 797 (C–H aromatic ring), 738 (C–Cl) cm-1. 1H 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), 130.7 (C-4000 ), 130.0 (C-3000 ),127.5 (C-4a), 127.5 (C-500 ), 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-((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 (C–O–C), 798 (C–H aromatic ring), 738 (C–Cl) cm-1. 1H 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 = 6.2, 2.0 Hz), 4.21 (t, H-10 , J = 5.8 Hz), 1.95 (m, H-20 ), 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). 13 C 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 (C4a), 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

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(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 (C–O–C), 798 (C–H aromatic ring), 740 (C–Cl) cm-1. 1H 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 (C4), 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-((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 (C–O–C), 795 (C–H aromatic ring), 740 (C–Cl) cm-1. 1H 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-((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 (C–O–C), 796 (C–H aromatic ring), 738 (C–Cl) cm-1. 1H 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

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(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)?. 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)?. 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 TM CRL-1593.2 ) evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as described by Robledo et 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

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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). 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 quinoline–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 resuspended at 2 9 106 axenic amastigotes/ml in fresh medium. 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.

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Activity against intracellular amastigotes The effects of the bis-alkylquinolines and quinoline– triclosan and quinoline–eugenol hybrids against intracellular amastigotes of L. (V.) panamensis were evaluated by 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 promastigotes 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 et al. (2009). The results are expressed as the Effective 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 antimoniate were used as leishmanicidal activity. The Selectivity Index (SI) was calculated by dividing the cytotoxic activity between the leishmanicidal activity (SI = LC50/EC50). Acknowledgment The authors thank Blandine Se´on-Me´niel for the help during NMR measurements. This research was supported financially by the Universidad de Antioquia (Programa de Sostenibilidad).

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