Synthesis and biological evaluation of new quinoline derivatives as antileishmanial and antitrypanosomal agents

Article abstract of DOI:10.1016/j.bioorg.2018.10.053

As a part of our project aimed at developing new safe chemotherapeutic agents against tropical diseases, a series of aryl derivatives of 2- and 3-aminoquinoline, some of them new compounds, was designed, synthesized, and evaluated as antiproliferative age

Full text of DOI:10.1016/j.bioorg.2018.10.053

Accepted Manuscript
Synthesis and biological evaluation of new quinoline derivatives as antileish-
manial and antitrypanosomal agents
Santiago N. Chanquia, Facundo Larregui, Vanesa Puente, Carlos Labriola, Elisa
Lombardo, Guadalupe García Liñares
PII:
S0045-2068(18)31072-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.053
YBIOO 2587
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
24 September 2018
25 October 2018
26 October 2018
Please cite this article as: S.N. Chanquia, F. Larregui, V. Puente, C. Labriola, E. Lombardo, G. García Liñares,
Synthesis and biological evaluation of new quinoline derivatives as antileishmanial and antitrypanosomal agents,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.053
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Synthesis and biological evaluation of new quinoline derivatives as
antileishmanial and antitrypanosomal agents
Santiago N. Chanquia,a Facundo Larregui,a Vanesa Puente,b Carlos Labriola,c Elisa
Lombardo,b* and Guadalupe García Liñaresa*
aLaboratorio de Biocatálisis. Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y
Naturales,Universidad de Buenos Aires, Ciudad Universitaria,Pabellón 2, piso 3, C1428EGA Buenos Aires, Argentina.
Centro de Investigaciones sobre Porfirias y Porfirinas (CIPYP, UBA-CONICET), Hospital de Clínicas José de San Martín, Avenida
Córdoba 2351, 1120 Buenos Aires, Argentina.
b
C Instituto de Investigaciones Bioquímicas, Av. Patricias Argentinas 435C1405BWE Buenos Aires, Argentina
Abstract
As a part of our project aimed at developing new safe chemotherapeutic agents
against tropical diseases, a series of aryl derivatives of 2- and 3-aminoquinoline, some of
them new compounds, was designed, synthesized, and evaluated as antiproliferative agents
against Trypanosoma cruzi, the parasite responsible for American trypanosomiasis
(Chagas’ disease), and Leishmania mexicana, the etiological agent of Leishmaniasis. Some
of them showed a remarkable activity as parasite growth inhibitors. Fluorine-containing
derivatives 11b and 11c were more than twice more potent than geneticin against
intracellular promastigote form of Leishmania mexicana exhibiting both IC50 values of 41.9
M. The IC50 values corresponding to fluorine and chlorine derivatives 11b-d were in the
same order than benznidazole against epimastigote form. These drugs are interesting
examples of effective antiparasitic agents with outstanding potential not only as lead drugs
but also to be used for further in vivo studies. In addition, the obtained compounds showed
no toxicity in Vero cells, which makes them good candidates to control tropical diseases.
Regarding the probable mode of action, assayed quinoline derivatives interacted with
hemin, inhibiting its degradation and generating oxidative stress that is not counteracted by
the antioxidant defense system of the parasite.
Keywords: quinoline derivatives; leishmaniasis; Chagas’ disease; hemin interaction;
oxidative damage
Corresponding author. e-mail: linares@qo.fcen.uba.ar; elombardo@qb.fcen.uba.ar

1. Introduction
American trypanosomiasis (Chagas’ disease) and leishmaniasis are among the most
prevalent parasitic diseases worldwide [1]. It has been estimated that around 20 million
people are infected and over 40 million individuals are at risk of infection by the
hemoflagellated protozoan Trypanosoma cruzi, the responsible agent of Chagas’ disease
[2]. T. cruzi has a complex life cycle possessing four main morphological forms [3]. It
multiplies within the insect gut as an epimastigote form and is disseminated as a non-
dividing metacyclic trypomastigote. In the mammalian host, T. cruzi proliferates as the
intracellular amastigote form, which is afterwards released into the bloodstream as a non-
dividing highly infective trypomastigote [3, 4]. The mechanism of transmission in
developed countries, where Chagas’ disease is not endemic, is via the placenta, by blood
transfusion or migration of individuals [5]. T. cruzi can also be orally trasnmitted by food
ingestion [6].
Leishmaniasis is also considered as one of the most important parasitic diseases and,
taking into account the number of infected, it is found second, after malaria [7, 8].
Leishmaniasis are a group of clinical diseases produced by at least 17 species and some
mutants of the protozoan Leishmania and transmitted to the humans by the bite of the
female phlebotomine sandfly [3, 9]. The disease comprises three major syndromes:
cutaneous, mucocutaneous and visceral leishmaniasis. Leishmania mexicana is one of the
species that produce cutaneous leishmaniasis [10]. It is estimated that there are almost 1.3
million new cases of leishmaniasis and around 30,000 deaths annually [8, 11, 12]. In
addition, the incidence of leishmaniasis and HIV co-infection has been increased due to
appearance of visceral leishmaniasis in urban areas [13, 14].
The existing chemotherapy either for Chagas’ disease or leishmaniasis remains
deficient. In the first case, the chemotherapy is based on two drugs empirically discovered,
nifurtimox (1) and benznidazole (Bnz) (2) (Fig 1). Although both of these compounds are
able to cure at least 50% of recent infections, they present severe side effects [15, 16].
The chemotherapy for leishmaniasis has been based for over 60 years on the use of
pentavalent antimonial drugs being meglumine antimoniate (Glucantime) (3) or sodium
stibogluconate (Pentostam) (4) the first-choice drugs (Fig 1) [17]. However, these drugs are
not satisfactory due to their toxic effects and their growing rates of resistance in several

parts of the world [18, 19]. Second line drugs include pentamidine (5) and amphotericin B
(6), which are mainly employed in cases where the antimonials are not effective [20]. There
are disadvantages associated with the use of these drugs such as their high cost, the need for
long-term treatment, the loss of effectiveness due to parasite resistance and severe
undesired effects [19, 21, 22]. Recently, miltefosine (7) [23, 24] was approved for the
treatment of human visceral leishmaniasis infections, but it is believed that it is teratogenic
[25, 26]. Two interesting drugs under clinical trials are sitamaquine (8) [27, 28] and the
antibiotic paromomycin (9) (Fig 1) [3, 29]. Extensive studies of new and diverse potential
antileishmanial drugs have been made in the last few years.
Figure 1. Chemotherapy of Chagas’disease and leishmaniasis. Chemical structure of
current drugs clinically employed for the treatment of these diseases.
However, the problems of drug resistance and the side effects of the chemotherapies used at
present have not yet been solved. Consequently, the development of novel, safe and
affordable compounds with potent anti-parasitic activity is urgently needed [21, 30].
Quinoline derivatives, mainly reported as antimalarial drugs, are also known to have
activity against Leishmania spp, trypanosomes, bacteria and tumors [31]. Chloroquine
(CQ), one of the most successful antimalarial drugs [32]. showed antimicrobial, anti-
inflammatory activities and also it is used for the treatment of autoimmune diseases [33-

36]. Some 2-substituted quinolines were shown to be effective in the treatment of
cutaneous leishmaniasis and in mice infected with T. cruzi [37, 38]. Tipifarnib, another
quinoline derivative with anticancer activity, and other analogs have also shown
antiparasitic activity [39-41].
Although the mechanism of action of CQ and other aminoquinolines used as antimalarial
drugs is not yet fully understood, it is thought to involve an interaction with heme,
inhibiting its polymerization to hemozoin [42-46]. The latter is a nontoxic form of heme,
which avoids the pro-oxidant properties of the free heme that would lead to the parasite
death [31].
In the present study, we report the synthesis of a series of 2- and 3-arylaminoquinoline
derivatives 10a-f and 11a-f (Fig 2), some of them new compounds, which were biologically
evaluated against T. cruzi and L. mexicana. Finally, the participation of heme in the
mechanism of action of these compounds was also investigated.
Figure 2. Aryl 2- and 3-aminoquinoline derivatives. Chemical structure of new synthesized
compounds.
2. Results and Discussions
2.1. Synthesis of arylaminoquinolines
It is known that a large number of biologically active compounds have aromatic
heterocyclic rings in their structure [47-53]. In particular, quinoline derivatives have shown
antibacterial, anti-inflammatory and antiparasitic activity [54-58]. In the course of our
program aimed at searching new biologically active nitrogen drugs, we have previously
reported the synthesis and biological properties of different compounds [47, 59-61]. In this

case, we have decided to prepare new compounds containing different substituents at
quinoline ring.
A series of twelve 2- and 3-arylaminoquinoline derivatives were designed and synthesized
considering principles of medicinal chemistry such as ring transformation or inclusion of
suitable groups. For example, substitution in the aryl ring by the introduction of halogen
atoms, an acid group or an unsaturated side chain could increase potency and improve
metabolic stability. Taking this into account we synthesized the compounds 10-11 (Fig 2).
Thus, the aryl amino quinolines were straight forwardly prepared via a coupling reaction
that involves an aryl boronic acid and an appropriate aromatic amine (Fig 3). Boronic acids
are known reagents to form heteroatom-carbon bonds. Likewise, phenylboronic acids are
efficient arylating agents. This N-arylation methodology may be performed under mild
conditions at room temperature in the presence of pyridine and cupric acetate as catalysts. It
was observed that the yield of the reaction is remarkably dependent on the nature of the
substrate and the substitution on the boronic acid [62-64].
Figure 3. Preparation of 2- and 3-arylaminoquinoline derivatives. Synthetic strategy for
obtention of desired compounds.
Starting from commercially available 2- (12) and 3-aminoquinoline (13), the reaction
involved their coupling with various substituted phenylboronic acids (14a-f) in dry
dichloromethane. Aryl 2- and 3-amino quinolines derivatives, 10a-f and 11a-f respectively,
were obtained from good to very good yield (Table 1).

Table 1. Yields in the synthesis of new aryl amino quinoline derivatives (10a-f and 11a-f).
R1
2-Arylaminoquinoline (10) 3-Arylaminoquinoline (11)
Yield (%)
Yield (%)
2-F
3-F
66
70
75
72
45
68
70
80
85
82
50
75
a
b
c
d
e
f
4-F
4-Cl
4-CO2H
4-CH=CH2
The reaction conditions correspond to those described in Materials and Methods.
As it is observed, the reaction yields were influenced by the substituent at the phenyl group
and its position in the quinoline ring. The reaction yields with 3-aminoquinoline (11a-f)
were higher than those with the 2-amino isomer (10a-f). Higher yields were obtained with
halogen substituents, mainly at the 4 position of the phenyl ring. All compounds were fully
characterized through their 1H and 13C NMR, HRMS and IR spectra.
2.2 Anti-Leishmania mexicana activity
The effect of different concentrations of the 2- and 3-arylaminoquinolines (10a-f and 11a-f)
on promastigote form of Leishmania mexicana growth was studied during 10 days.
Geneticin, a well-known antiparasitic agent, was used as a standard inhibitor at a
concentration of 50 g/mL. A remarkable inhibitory effect on L. mexicana growth was
observed in the case of 10b, 11b, 11c and 11d. The rest of the tested compounds were
active but at a concentration higher than 50 g/mL. For the lowest dosage of 10 g/mL,
50% inhibition was detected after 6 days of incubation of 11b and 11c. With 25 g/mL, 25
and 37.5% inhibition respectively was observed after 2 days of exposure and a maximum of
100% was reached after 10 days for both compounds. Figure 4 shows the growth curves of
promastigote L. mexicana in the presence of compounds 10b, 11b, 11c and 11d.

Figure 4. Effect of 10b, 11b-d concentration on Leishmania mexicana growth rate.
Promastigotes were grown at 28 °C for 10 days in the presence of different concentrations
of 10b and 11b-d compounds (10-100 µg/ml). Growth inhibition was estimated by
counting the parasites, every 2 days, using a Neubauer chamber. p values  0.05 (*) and 
0.01 (**) were considered significant.

These four compounds were the most potent against L. mexicana with IC50 values of 75.6,
41.9, 41.9 and 98.1M (day 6 of growth) for 10b, 11b, 11c and 11d respectively, showing
better activity than the standard inhibitor geneticin (IC50 100 M) (Table 2).
Table 2. Biological activity of quinoline derivatives against T. cruzi (epi-, trypo- and
amastigote forms) and L. mexicana (promastigote form).
Comp
IC50 (M)
Tc trypo
Cytotoxicity
Lm pro
> 200
Tc epi
>15
Tc amas
>> 50
CC50 (M)
>> 500
811.02 ±
>> 500
>> 500
>> 500
>> 500
>> 500
419.95 ±
616.02 ±
>> 500
>> 500
>> 500
10a
10b
75.6± 1.0
> 200
> 15
58.4±0.8
>> 50
> 100
> 100
185
> 15
10c
> 200
> 15
>> 50
10d
> 200
>> 15
>> 50
ND
10e
> 200
>> 15
>> 50
ND
10f
> 200
> 15
>> 50
ND
11a
41.9± 1.0
41.9± 0.8
13.51 ± 1.20
11.29 ± 0.63
22.8± 0.9
10.9 ± 1.2
25.8 ± 3.0
>> 50
> 100
>> 100
> 100
ND
11b
11c
98.1 ± 1.6 12.01 ± 0.18
11d
> 200
> 200
-
>> 15
>> 15
7.56 ± 0.5
-
11e
>> 50
ND
11f
38.43 ± 4.2 4.88 ± 0.60
-
85
Bnz
Geneticin
100
-
IC50 values were estimated by non-linear regression from the growth inhibition percentage values and log of
drug concentration. CC50 on Vero cells was calculated by non-linear regression from the percentage of viable
cells and the log of drug concentration.

The fluorinated products 11b, 11c and 10b are the most potent, indicating that the presence
of the fluorine atom increased the inhibitory activity. Besides, the best compound was the
3-aminoquinoline derivative containing fluorine at 4-position (11c). The presence of a
fluorine atom could increase the logP value improving cell membrane penetration by a
better water/octanol distribution coefficient, resulting in a higher biological activity [65].
Substitution with chlorine at 4-position of the phenyl group (11d) afforded a compound
which showed similar activity to geneticin.
2.3. Anti-Trypanosoma cruzi activity
In a first screening, all synthesized compounds were evaluated as growth inhibitors against
the epimastigote form of Trypanosoma cruzi at concentrations ranging from 2.5 µM to 15
µM in three independent assays. Benznidazole (Bnz), the drug currently used for treatment
of Chagas’ disease, was used as a positive control. Halogen containing 3-aminoquinoline
derivatives (11b-d) were again the most potent compounds showing an inhibitory effect in
the same order of benznidazole. The rest of the tested compounds were inactive until
concentration of 15 Figure 5 shows the growth curves that allowed to obtain IC50
values of 13.5, 11.3 and 12.0 M for 11b, 11c and 11d, respectively (Table 2).
Figure 5. Effect of 11b, 11c and 11d concentration on the proliferation of Trypanosoma
cruzi epimastigotes. The parasites were grown at 28 °C for 4 days with different

concentrations (2.5 -15 µM) of tested compounds. Growth rate was evaluated by counting
the parasites using a Neubauer chamber. p values  0.05 (*) and  0.01 (**) were
considered significant.
The promising finding of the antiparasitic activity of compounds 11b, 11c and 11d
against T. cruzi epimastigotes prompted us to study their activity against bloodstream stage
(trypomastigote form) and intracellular stage (amastigote form) (Table 2). Compounds
11b, 11c and 11d were also those that showed better trypanocidal activity on trypo- and
amastigote forms. From Table 2, we conclude that compounds 11b-d show the highest
antiparasitic activity on both L. mexicana and T. cruzi. In addition, it is important to note
that compound 11c showed IC50 values in the same order of Bzn.
2.4. Cytotoxic activity
The results of cytotoxic activity of compounds 11b, 11c and 11d on Vero cells are
also shown in Table 2. For concentrations of these compounds as high as 100 µM no
cytotoxic effect was observed, while for Bnz was of 85 ± 5 µM.
2.5. Hemin interaction
Trypanosomatids protozoa show a strict requirement of heme-compounds such as
hemoglobin, hematin or hemin during their growth [30]. This nutritional characteristic is
due to its inability to synthesize heme. Since high levels of hemin and related compounds
have an important cytotoxic effect mediated by ROS generation, trypanosomatids present
different pathways to counteract the oxidative stress that an excess of heme would generate.
In Plasmodium spp. this detoxification is carried out by the conversion of heme into
hemozoin, a non-toxic polymerized form of heme. In contrast, in Leishmania spp. and
Trypanosoma cruzi, the cytotoxic effect of heme is avoided by its conversion into
biliverdin, in which heme oxygenase enzyme would be involved [31].
It is known that quinoline drugs develop their antimalarial activity through the
formation of quinoline-heme complexes, which inhibit the heme polymerization into
hemozoin, leading to the death of the parasite. According to their structures, our quinoline
derivatives could exercise their antiparasitic action by the formation of heme-quinoline

complexes which could inhibit heme detoxification. To prove this possible mode of action,
we evaluate the binding of quinolines 11b-d to heme (Fig 6). These compounds have
effectively manifested an interaction with hemin similar than that shown by chloroquine.
Figure 6. Interaction of quinoline derivatives 11b-d with hemin. After interacting the
hemin (7.5 µM) with different concentrations of 11b-d quinolines or chloroquine (22.5-90
µM), the absorption spectra of hemin was recorded. The absorbance ratio at 430 and 400
nm (A430/A400) was used to quantify modifications in the Soret band. p values  0.05 (*)
were considered significant.
2.6. Intracellular redox state
Assuming that quinoline derivatives inhibit heme degradation, its presence in the
parasite would generate oxidative stress. Given that the amount of reduced low molecular
mass thiols is indicative of the redox state of the cell, we expect that the treatment with the
compounds 11b, 11c and 11d affects SH-group levels, because of the formation of oxidant
species. For the evaluation of the oxidative stress induced by these quinolines, T. cruzi
epimastigotes were cultured in the presence of compounds 11b-d (35 μM) during 3, 6 and 9
h. Our results (Fig 7A) showed that SH-content remains constant and equal to the untreated
parasites (about 52.31 nmol/mg protein), for treatment with the three quinolines for 3 and 6
hours, diminishing between 43%, 35% and 39% for 11b, 11c and 11d compounds,

respectively after 9 h of treatment. The level of SH-groups decrease to counteract oxidative
injury induced by treatment with quinolines, confirming the proposed mechanism of action.
Together with these determinations, the intracellular oxidative state was assessed by
flow cytometry. As we see in the Figure 7B the behavior of the three quinolines was
similar, an increase of about 4 times in the fluorescence intensity of the H2DCFDA-loaded
epimastigotes was observed after 9 h of treatment. This evidences an oxidative stress that
persists despite the decrease in the level of thiols, and that would be responsible for the
death of the parasite treated with the different quinolines.
Figure 7. Thiol groups content and intracellular oxidative stress during the treatment with
11b-d derivatives. T. cruzi epimastigotes, which had been cultured for 4 days, were
incubated with 35 µM 11b-d compounds during 3, 6 or 9 h. (A) The thiol groups were
measured spectrophotometrically after reacting with DTNB. (B) Intracellular oxidative
status was evaluated by flow cytometry using probe H2DCFDA. Gmt/Gmc ratios were
calculated as indicated in Materials and Methods.

3. Materials and methods
3.1. General
Chemicals and solvents used in the synthesis were purchased from Merck Argentina and
Sigma-Aldrich Argentina and used without further purification. Standard solutions of
synthezised compounds were prepared in dimethyl sulphoxide (DMSO) at a final
concentration that never exceeded 0.5% DMSO. Hemin, CPRG and H2DCFDA were
obtained from Sigma Chem. Co. (Saint Louis, MO, USA). Benznidazole (Bnz) was kindly
provided by Roche (Argentina). All other chemicals were of the highest purity
commercially available.
3.2. General procedure for the synthesis of arylaminoquinolines
A flask is loaded with 2-amino or 3-aminoquinoline (1 mmol), cupric acetate (1 mmol), the
corresponding arylboronic acid (2 mmol) and 4Å molecular sieves. The reaction mixture is
diluted with dichloromethane (5.0 mL) and pyridine (2 mmoL) is added. After stirring the
heterogeneous reaction mixture for 18 h at 25 °C under nitrogen atmosphere, the resulting
slurry is filtered and the product is isolated from the organic filtrate by column
chromatography (silica gel) employing mixtures of hexane-EtOAc as eluent (7:3 – 2:3). To
monitor the reaction progress aliquots were withdrawn and analyzed by TLC performed on
commercial 0.2 mm aluminum-coated silica gel plates (F254), using EtOAc:hexane 3:2 as
developing solvent and visualized by 254 nm UV or immersion in an aqueous solution of
1
(NH4)6Mo7O24.4H2O (0.04 M), Ce(SO4)2 (0.003 M) in concentrated H2SO4 (10%). H
NMR and 13C NMR spectra were recorded at room temperature in CDCl3 as solvent using a
1
Bruker AM-500 NMR instrument operating at 500.14 MHz and 125.76 MHz for H and
1
13C respectively. The H NMR spectra are referenced with respect to the residual CHCl3
proton of the solvent CDCl3 at  = 7.26 ppm. Coupling constants are reported in Hertz
(Hz). 13C NMR spectra were proton decoupled and are referenced to the middle peak of the
solvent CDCl3 at  = 77.0 ppm. Splitting patterns are designated as: s, singlet; d, doublet; t,
triplet; q, quadruplet; qn, quintet; dd, double doublet, etc. High Resolution Mass
Spectrometry was recorded with Thermo Scientific EM/DSQ II – DIP. The results were
within ±0.02% of the theoretical values.

1. 2-(2-Fluor-phenylamino) quinoline (10a). Yield 66% of pure compound as a yellow
oil. 1H NMR (CDCl3) 6.60 (m, 2H, H-14, H-16), 6.97 (d, J = 8.5 Hz, 1H, H-3), 7.16 (m, 2
H, H-13, H-15), 7.33 (dt, J1 = 1.2 Hz, J2 = 8.0 Hz, 1 H, H-6), 7.61 (dt, J1 = 1.1 Hz, J2 = 8.2
Hz, 1H, H-7), 7.67 (dd, J1 = 1.1 Hz, J2 = 8.0 Hz, 1 H, H-8), 7.83 (dd, J1 = 1.1 Hz, J2 = 8.0
Hz, 1 H, H-5), 7.97 (d, J = 8.4 Hz, 1H, H-4); 13C NMR (CDCl3) 111.5 (C-3), 115.6 (d, J
= 23.4 Hz, C-13), 122.3 (d, J = 9.6 Hz, C-14, C-16), 123.9 (C-8), 124.5 (C-10), 126.8 (C-
15), 127.7 (C-5, C-6), 130.4 (C-7), 130.5 (d, J = 9.6 Hz, C-11), 138.5 (C-4), 147.3 (C-9),
153.9 (C-2), 164.7 (d, J = 244.3 Hz, C-12); HRMS: [M + Na]+ Calcd. C15H11FN2Na
261.0804. Found: C15H11FN2Na 261.0812.
2. 2-(3-Fluor-phenylamino) quinoline (10b). Yield 70% of pure compound as a light
1
yellow oil. H NMR (CDCl3) 6.77 (ddd, J1 = 2.3 Hz, J2 = 8.3Hz, 1H, H-14), 6.98 (d, J =
8.6 Hz, 1H, H-3), 7.28 (m, 2 H, H-15, H-16), 7.35 (dt, J1 = 1.1 Hz, J2 = 8.0 Hz, 1 H, H-6),
7.62 (m, 2 H, H-7, H-12), 7.67 (dd, J1 = 1.3 Hz, J2 = 7.9 Hz, 1 H, H-8), 7.84 (dd, J1 = 1.2
Hz, J2 = 8.0 Hz, 1 H, H-5), 7.98 (d, J = 8.6 Hz, 1H, H-4); 13C NMR (CDCl3) (d, J =
24.4 Hz, C-12), 109.5 (d, J = 22.4 Hz, C-14), 111.2 (C-3), 111.9 (C-16), 115.4 (d, J = 9.5
Hz, C-15), 123.6 (C-8, C-10), 127.5 (C-5, C-6), 130.1 (C-7), 130.4 (d, J = 9.6 Hz, C-11),
138.2 (C-4), 147.1 (C-9), 153.6 (C-2), 162.4 (d, J = 244.3 Hz, C-13); HRMS: [M + Na]+
Calcd. C15H11FN2Na 261.0804. Found: C15H11FN2Na 261.0799.
3. 2-(4-Fluor-phenylamino) quinoline (10c). Yield 75% of pure compound as a yellow
1
oil. H NMR (CDCl3) 6.89 (d, J = 8.2 Hz, 1H, H-3), 7.06 (d, J = 8.6 Hz, 2 H, H-13, H-
15), 7.31 (dt, J1 = 1.2 Hz, J2 = 7.6 Hz, 1 H, H-6), 7.55 (d, J = 8.7 Hz, 2 H, H-12, H-16),
7.60 (dt, J1 = 1.5 Hz, J2 = 7.5 Hz, 1 H, H-7), 7.64 (dd, J1 = 1.0 Hz, J2 = 7.8 Hz, 1 H, H-8),
13
7.77 (dd, J1 = 1.0 Hz, J2 = 7.6 Hz, 1 H, H-5), 7.92 (d, J = 8.0 Hz, 1H, H-4); C NMR
(CDCl3) 111.4 (C-3), 115.8 (d, J = 22.5 Hz, C-13, C-15), 122.6 (d, J = 7.8 Hz, C-12, C-
16), 122.7 (C-8), 123.2 (C-10), 126.6 (C-6), 127.4 (C-5), 129.9 (C-7), 136.1 (C-11), 137.9
(C-4), 147.5 (C-9), 154.4 (C-2), 160.0 (d, J = 242.6 Hz, C-14); HRMS: [M + Na]+ Calcd.
C15H11FN2Na 261.0804. Found: C15H11FN2Na 261.0810.

4. 2-(4-Chloro-phenylamino) quinoline (10d). Yield 72% of pure compound as a light
1
yellow oil. H NMR (CDCl3) 6.94 (d, J = 9.0 Hz, 1H, H-3), 7.32-7.33 (m, 3 H, H-6, H-
13, H-15), 7.52 (d, J = 7.8 Hz, 2 H, H-12, H-16), 7.61 (dt, J1 = 1.5 Hz, J2 = 7.1 Hz, 1 H, H-
7), 7.66 (dd, J1 = 1.5 Hz, J2 = 8.1 Hz, 1 H, H-8), 7.79 (dd, J1 = 1.3 Hz, J2 = 8.4 Hz, 1 H, H-
13
5), 7.95 (d, J = 8.9 Hz, 1H, H-4); C NMR (CDCl3) 111.6 (C-3), 116.7 (C-12, C-16),
121.8 (C-8), 123.5 (C-6), 124.1 (C-10), 126.4 (C-14), 127.5 (C-5), 129.5 (C-12, C-16),
130.1 (C-7), 136.7 (C-11), 138.2 (C-4), 149.1 (C-9), 154.6 (C-2); HRMS: [M + Na]+ Calcd.
C15H11ClN2Na 277.0508. Found: C15H11ClN2Na 277.0512.
5. 2-(4-Carboxy-phenylamino) quinoline (10e). Yield 45% of pure compound as a light
yellow oil. 1H NMR (CDCl3) 6.88 (d, J = 8.0 Hz, 1H, H-3), 7.30 (dt, J1 = 1.2 Hz, J2 = 7.6
Hz, 1 H, H-6), 7.59 (dt, J1 = 1.4 Hz, J2 = 7.6 Hz, 1 H, H-7), 7.64 (dd, J1 = 1.2 Hz, J2 = 7.7
Hz, 1 H, H-8), 7.70 (d, J = 8.6 Hz, 2 H, H-12, H-16), 7.77 (dd, J1 = 1.1 Hz, J2 = 7.6 Hz, 1
13
H, H-5), 7.92 (d, J = 7.9 Hz, 1H, H-4), 8.00 (d, J = 8.6 Hz, 2 H, H-13, H-15); C NMR
(CDCl3) 112.2 (C-3), 116.7 (C-12, C-16), 123.5 (C-8, C-14), 124.0 (C-10), 127.4 (C-6),
128.2 (C-5), 130.7 (C-7), 132.9 (C-13, C-15), 138.7 (C-4); 141.7 (C-11); 148.3 (C-9);
155.2 (C-2), 165.2 (-CO2H); HRMS: [M + Na]+ Calcd. C16H12N2NaO2 287.0796. Found:
C16H12N2NaO2 287.0790.
6. 2-(4-Vinyl-phenylamino) quinoline (10f). Yield 68% of pure compound as a light
orange oil. 1H NMR (CDCl3) 5.16 (dd, J1 = 0.9 Hz, J2 = 10.9 Hz, 1H, H-18a), 5.66 (dd, J1
= 0.9 Hz, J2 = 17.5 Hz, 1H, H-18b), 6.67 (dd, J1 = 10.9 Hz, J2 = 17.5 Hz, 1H, H-17), 6.91
(d, J = 8.9 Hz, 1H, H-3), 7.30 (m, 3 H, H-6, H-13, H-15), 7.50 (d, J = 7.9 Hz, 2 H, H-12, H-
16), 7.58 (dt, J1 = 1.6 Hz, J2 = 7.0 Hz, 1 H, H-7), 7.63 (dd, J1 = 1.5 Hz, 2 = 8.0 Hz, 1 H, H-
8), 7.77 (dd, J1 = 1.2 Hz, J2 = 8.5 Hz, 1 H, H-5), 7.93 (d, J = 9.0 Hz, 1H, H-4); 13C NMR
(CDCl3) 110.0 (C-3), 112.7 (=CH2), 116.7 (C-12, C-16), 120.21 (C-8), 121.9 (C-6),
122.5 (C-10), 124.8 (C-14), 125.9 (C-5), 127.9 (C-13, C-15), 128.5 (C-7), 135.1 (C-11),
136.0 (CH=), 136.6 (C-4), 147.5 (C-9), 153.0 (C-2); HRMS: [M + Na]+ Calcd. C17H14N2Na
269.1055. Found: C17H14N2Na 269.1053.

7. 3-(2-Fluor-phenylamino) quinoline (11a). Yield 70% of pure compound as a yellow
1
oil. H NMR (CDCl3) .05 (m, 4H, H-13, H-14, H-15, H-16), 7.39 (dt, J1 = 1.3 Hz, J2 =
7.9 Hz, 1 H, H-7), 7.49 (dt, J1 = 1.1 Hz, J2 = 8.0 Hz, 1H, H-6), 7.54 (dd, J1 = 1.1 Hz, J2 =
8.0 Hz, 1 H, H-4), 7.60 (dd, J1 = 1.1 Hz, J2 = 8.0 Hz, 1 H, H-5), 7.98 (d, J = 8.4 Hz, 1H, H-
8), 8.64 (s, 1H, H-2); 13C NMR (CDCl3) 114.3 (C-4), 114.9 (d, J = 23.4 Hz, C-13), 122.7
(d, J = 8.5 Hz, C-16), 123.9 (d, J = 8.3 Hz, C-14), 124.9 (C-5), 125.0 (C-6), 125.4 (d, J =
2.5 Hz, C-15), 125.8 (C-7), 127.4 (C-10), 127.6 (C-8), 129.5 (d, J = 26 Hz, C-11), 136.4
(C-9), 142.1 (C-3), 143.0 (C-2), 157.4 (d, J = 244.0 Hz, C-12); HRMS: [M + Na]+ Calcd.
C15H11FN2Na 261.0804. Found: C15H11FN2Na 261.0809.
8. 3-(3-Fluor-phenylamino) quinoline (11b). Yield 80% of pure compound as a yellow
oil. 1H NMR (CDCl3) 7.07 (ddd, J1 = 2.1 Hz, J2 = 8.2 Hz, 1H, H-14), 7.16 (m, 2 H, H-15,
H-16), 7.37 (dt, J1 = 1.0 Hz, J2 = 8.2 Hz, 1 H, H-12), 7.47 (dt, J1 = 1.2 Hz, J2 = 8.0 Hz, 1 H,
H-7), 7.50 (dt, J1 = 1.1 Hz, J2 = 8.1 Hz, 1H, H-6), 7.54 (dd, J1 = 1.2 Hz, J2 = 8.1 Hz, 1 H,
H-4), 7.61 (dd, J1 = 1.0 Hz, J2 = 8.0 Hz, 1 H, H-5), 7.99 (d, J = 8.3 Hz, 1H, H-8), 8.65 (s,
1H, H-2); 13C NMR (CDCl3) (d, J = 24.2 Hz, C-12), 111.5 (d, J = 22.7 Hz, C-14),
111.2 (C-3), 113.7 (d, J = 2.5 Hz, C-16), 115.7 (C-4), 117.2 (d, J = 8.2 Hz, C-15), 126.4
(C-5, C-6), 127.2 (C-7), 128.8 (C-10), 129.0 (C-8), 130.8 (d, J = 8.0 Hz, C-11), 137.8 (C-
9), 143.4 (C-3), 144.4 (C-2), 158.8 (d, J = 242.0 Hz, C-13); HRMS: [M + Na]+ Calcd.
C15H11FN2Na 261.0804. Found: C15H11FN2Na 261.0810.
9. 3-(4-Fluor-phenylamino) quinoline (11c). Yield 85% of pure compound as light yellow
1
colorless oil. H NMR (CDCl3) 5.89 (ws, 1H, NH), 7.07 (t, J = 8.2 Hz, 2H, H-13, H-15),
7.16 (dd, J1 = 4.7 Hz, J2 = 8.4 Hz, 2 H, H-12, H-16), 7.45 (dt, J1 = 1.3 Hz, J2 = 7.8 Hz, 1 H,
H-7), 7.51 (dt, J1 = 1.4 Hz, J2 = 6.7 Hz, 1 H, H-6), 7.57 (d, J = 2.6 Hz, 1 H, H-4), 7.61 (dd,
13
J1 = 1.5 Hz, J2 = 8.0 Hz, 1 H, H-5), 7.99 (d, J = 8.2 Hz, 1H, H-8), 8.65 (s, 1H, H-2); C
NMR (CDCl3) 115.7 (C-4), 116.3 (d, J = 22.7 Hz, C-13, C-15), 121.5 (d, J = 7.8 Hz, C-
12, C-16), 126.3 (C-5), 126.4 (C-6), 127.2 (C-7), 128.8 (C-10), 129.0 (C-78), 137.5 (d, J =
2.6 Hz, C-11), 137.8 (C-9), 143.5 (C-3), 144.4 (C-2), 158.7 (d, J = 242.0 Hz, C-14);
HRMS: [M + Na]+ Calcd. C15H11FN2Na 261.0804. Found: C15H11FN2Na 261.0808.

10. 3-(4-Chloro-phenylamino) quinoline (11d). Yield 82% of pure compound as a light
1
yellow oil. H NMR (CDCl3) 6.02 (ws, 1H, NH), 7.11 (d, J = 8.7 Hz, 2H, H-13, H-15),
7.30 (d, J = 8.7 Hz, 2 H, H-12, H-16), 7.49 (dt, J1 = 1.6 Hz, J2 = 7.0 Hz, 1 H, H-7), 7.51 (dt,
J1 = 1.6 Hz, J2 = 6.9 Hz, 1 H, H-6), 7.66 (dd, J1 = 1.8 Hz, J2 = 8.2 Hz, 1 H, H-5), 7.69 (d, J
= 2.7 Hz, 1 H, H-4), 8.01 (d, J = 8.3 Hz, 1H, H-8), 8.69 (d, J = 2.7 Hz, 1H, H-2); 13C NMR
(CDCl3) 117.6 (C-4), 119.7 (C-12, C-16), 126.5 (C-5), 126.9 (C-6), 127.1 (C-14), 127.3
(C-7), 128.7 (C-10), 128.8 (C-8), 129.6 (C-13, C-15), 136.6 (C-11), 140.5 (C-9), 143.6 (C-
3), 144.8 (C-2); HRMS: [M + Na]+ Calcd. C15H11ClN2Na 277.0508. Found: C15H11ClN2Na
277.0505.
11. 3-(4-Carboxy-phenylamino) quinoline (11e). Yield 50% of pure compound as a
1
yellow oil. H NMR (CDCl3) 6.00 (ws, 1H, NH), 7.47 (dt, J1 = 1.6 Hz, J2 = 7.0 Hz, 1 H,
H-7), 7.49 (dt, J1 = 1.6 Hz, J2 = 6.9 Hz, 1 H, H-6), 7.63 (dd, J1 = 1.8 Hz, J2 = 8.2 Hz, 1 H,
H-5), 7.67 (d, J = 2.7 Hz, 1 H, H-4), 7.75 (d, J = 8.7 Hz, 2H, H-13, H-15), 7.91 (d, J = 8.7
13
Hz, 2 H, H-12, H-16), 8.00 (d, J = 8.3 Hz, 1H, H-8), 8.67 (d, J = 2.7 Hz, 1H, H-2); C
NMR (CDCl3) 116.9 (C-12, C-16), 117.8 (C-4), 125.2 (C-14), 126.6 (C-5), 127.0 (C-6),
127.5 (C-7), 128.9 (C-10), 129.0 (C-8), 130.4 (C-13, C-15), 140.4 (C-11), 140.6 (C-9),
143.7 (C-3), 145.0 (C-2), 167.8 (-CO2H); HRMS: [M + Na]+ Calcd. C16H12N2NaO2
287.0796. Found: C16H12N2NaO2 287.0801.
12. 3-(4-Vinyl-phenylamino) quinoline (11f). Yield 75% of pure compound as a dark
yellow oil. 1H NMR (CDCl3) 5.16 (dd, J1 = 0.8 Hz, J2 = 10.9 Hz, 1H, H-18a), 5.66 (dd, J1
= 0.8 Hz, J2 = 17.5 Hz, 1H, H-18b), 6.04 (ws, 1H, NH), 6.67 (dd, J1 = 10.9 Hz, J2 = 17.6
Hz, 1H, H-17), 7.12 (d, J = 8.5 Hz, 2 H, H-6, H-12, H-16), 7.38 (d, J = 8.5 Hz, 2 H, H-13,
H-15), 7.46 (dt, J1 = 1.6 Hz, J2 = 6.8 Hz, 1 H, H-7), 7.51 (dt, J1 = 1.6 Hz, J2 = 6.8 Hz, 1 H,
H-6), 7.62 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1 H, H-5), 7.72 (d, J = 2.8 Hz, 1H, H-4), 7.99 (dd,
13
J1 = 1.2 Hz, J2 = 8.6 Hz, 1 H, H-8), 8.68 (d, J = 2.7 Hz, 1H, H-2); C NMR (CDCl3)
112.1 (=CH2), 117.5 (C-4), 118.3 (C-12, C-16), 125.0 (C-14), 126.5 (C-5), 126.7 (C-6),
127.2 (C-7), 127.5 (C-13, C-15), 128.9 (C-8), 131.9 (C-10), 136.1 (CH=), 136.7 (C-11),
141.4 (C-9), 143.5 (C-3), 144.9 (C-2); HRMS: [M + Na]+ Calcd. C17H14N2Na 269.1055.
Found: C17H14N2Na 269.1061.

3.3. Parasites
Leishmania mexicana promastigotes were grown in a culture medium containing
3.3% brain-heart infusion (Difco); 0.3% tryptose (Difco); 0.3% disodium phosphate; 0.04%
potassium chloride; 0.03% dextrose. pH was about 7.5, without the need for adjustment.
After sterilization penicillin (100 IU mL-1), streptomycin (100 g mL-1), hemin (20 g mL-
1) and 20% v/v heat-inactivated fetal calf serum was added. Cultures were performed at 28
°C in 15 mL cylindrical flasks with screw caps, containing 2 mL of medium. Shaking was
performed manually, twice a day. All cultures were started with inocula taken from
exponential growth parasite in the same medium.
Trypanosoma cruzi epimastigotes (Tulahuén strain) were grown, in the same liquid medium
as the composition mentioned for L. mexicana added with hemin (20 g mL-1), under the
conditions described previously [66]. At the time of inoculating the parasite this medium was
supplemented with penicillin (100 IU mL-1), streptomycin (100 g mL-1) and 10% v/v heat-
inactivated fetal calf. Cultures were routinely maintained in the exponential phase by weekly
passages at 28 °C. Bloodstream trypomastigotes were obtained from infected CF1 mice by
cardiac puncture, at the peak of parasitemia on day 15 post-infection. Trypomastigotes were
routinely maintained by infecting 21 days-old CF1 mice. Inbred CF1 male mice were nursed
at Facultad de Medicina, Universidad de Buenos Aires. Animals were handled in accordance
with guidelines established by the Animal Care and Use Committee of the Argentine
Association of Specialists in Laboratory Animals (AADEALC).
3.4. In vitro assays for anti-leishmanial activity
The susceptibilities of the promastigote form of Leishmania to synthetic compounds were
tested by culturing them in cell-free medium at 28 °C. Growth experiments with
promastigotes of L. mexicana were initiated with 5 x 106 parasites/mL, and the inhibitors
were added at 10, 25, 50 and 100 g/mL from concentrated stock solutions in DMSO at
this moment.
A solution of 50 g/mL of geneticin was used as a positive control. Parasite grow was
evaluated by reading parasites in a Neubauer chamber every two day for ten days. DMSO
concentration in the cultures (0.1%) did not interfere with parasite growth or morphology.

The IC50 (50 % inhibitory concentration) was determined by non-linear regression analysis
of the data (Sigma Plot 12 Software).
3.5. In vitro assays for anti-trypanocidal activity
To evaluate the growth inhibition of epimastigotes, 0.75 - 1.25 x 107 parasites/ml were
cultured in the presence of 2.5 to 15 µM of the compounds or Bnz (used as positive control)
for 4 days. Cells growth was assessed by counting the number of cells per ml of culture
using a Neubauer chamber and was expressed as cellular density (CD). The percentage of
inhibition (%I) was calculated as: %I = {1-[(CD4t-CD0)/(CD4c-CD0)]} x 100, where the
different CDs represent the cellular density of: CD4t, treated parasites on day 4; CD0,
parasites on day 0; and CD4c, untreated parasites (control) on day 4.
The trypanocidal effects of different quinolines and Bnz were also tested on bloodstream
trypomastigotes according to a standard WHO protocol slightly modified [67]. Briefly,
mouse blood containing trypomastigotes (1.5 x 106 parasites/ml) were seeded (150 µl/well)
in duplicate in a 96-well plate and treated with different concentrations of each compound
(0.45 to 450 µM) or Bnz (0.38 to 380 µM). Plates were incubated for 24 h, and surviving
parasites were counted in a Neubauer chamber. The results were expressed as the
percentage of lysed parasites (%L) relative to the number of parasites in the control: %L =
[1 - (CDt/(CDc)] x 100, where CDt and CDc represents the cellular density of treated and
untreated parasites, respectively.
For analysis of amastigotes, J774 macrophages line was infected with transfected
bloodstream trypomastigotes, which expressing the ??-galactosidase gene, using a parasite:
cell ratio of 10:1. Then 24 h, the cultures were washed and different concentration of each
compound (2 to 50 M) was added in fresh RPMI medium without phenol red. After 7
days, the galactosidase activity was measured employing CPRG as substrate and recording
absorbance at 570 nm in a Microplate Reader (Bio-Rad Laboratories). Blanks employing
uninfected cell were performed. The percentage of inhibition was calculated as %??
=[1]×100, where ?? represents the mean ??570 value recorded for ??it, treated infected
cells; ??nit, treated noninfected cells; ??ic, untreated infected cells; and ??nic, untreated
noninfected cells. To calculate the IC50 (50% inhibitory concentration) for each parasite
form, I% or %L values were plotted against the logarithm (log) of drug concentration (μM)

and fitted to a sigmoidal curve determined by a non-linear regression (Sigma Plot 12
software).
3.6. Cytotoxicity assay
Cytotoxic activity was evaluated in vitro, using Vero cells. The cells were cultured
in RPMI medium at 37 °C 5% CO2. Cells, 9 x 105 cell/mL were seeded in a 24-well plate
and then 24 h different concentrations of compounds (5 - 100 µM) or Bnz (5 - 200 µM)
were added. After 48 h of incubation, cells were washed twice with PBS and 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was added at a final
concentration of 0.5 mg/ml. Plates were incubated for 1h at 37 °C. Finally, blue precipitates
were dissolved in 0.5 mL DMSO and were read on a plate reader (Spectra CountTM BS
10001) at a wavelength of 570 nm. Values from blank wells containing only medium and
reagents were subtracted from the values of the samples. All MTT assays were repeated
two times by using three samples per assay. The CC50 (50% cytotoxic concentration) was
calculated by non-linear regression analysis (sigmoidal curve) from the percentage of
viable cells and the decimal logarithm (log) of drug concentration.
3.7. Hemin binding assay
The interaction of the quinolines with the hemin (which is in non-polymerization
conditions) was evaluated spectrophotometrically by monitoring the hemin Soret band. The
assay system contained 0.23 M sodium phosphate buffer pH 7.4, 1% SDS, 7,5 µM hemin
and different concentrations of the compounds to be tested (22,5 to 90 µM). The absorption
spectra of hemin was measured using a Hewlett-Packard 8452-Diode Array
spectrophotometer. The absorbance ratio at 430 and 400 nm (A430/A400) was used to
quantify modifications in the Soret band. Chloroquine was used as positive control.
3.8. Reduced thiol groups assay
T. cruzi epimastigotes growing in logarithmic phase were incubated with 11b-d (35
μM) during 3, 6 or 9 h. Treated-parasites were harvested by centrifugation at 12,000 xg for
10 min, washed once, and resuspended in 50 mM sodium phosphate buffer pH 7.4. Cells in
suspension were disrupted by sonication in an MSE Soniprep 150 ultrasonic disintegrator

for 45 s. The resulting homogenate (H) was employing for measuring the level of SH-
groups and protein content. To quantify thiols, the H homogenate was treated with 50%
trichloroacetic acid to a final concentration of 5%, the precipitated protein was discarded by
centrifugation and reduced thiols measured in the supernatant using the chromogenic
compound 5,5’-dithiobis-2-nitrobenzoate (DTNB), as already described [46]. Protein
concentration was determined according to the method described by Lowry et al [68], and
these values were considered to express the content of SH-groups per mg of protein.
3.9. Intracellular oxidative activity assay
The induction of intracellular oxidative stress was assessed using the oxidant-
sensitive fluorescent probe H2DCFDA. T. cruzi epimastigotes growing in logarithmic phase
were incubated with 11b-d (35 μM) during 3, 6 or 9 h. Treated-parasites were harvested
and stained for 45 min in the dark with 10 M H2DCFDA at 37 ºC. As positive control,
parasites were treated with 0.2 mM H2O2. The fluorescence intensity of dichlorofluorescein
(DCF) in cells was then analyzed in a Becton Dickinson FACScalibur flow cytometer with
an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Results were
expressed by the ratio Gmt/Gmc, where Gmt and Gmc correspond to the geometric mean of
histograms obtained for treated and untreated (control) cells respectively. Flow cytometry
data were analysed employing WinMDI 2.9 software.
3.10. Statistical analysis
Results are representative of three to four separate experiments, performed in
duplicate or triplicate. Data are expressed as means ± standard errors of the mean (SEMs).
The significance of differences was evaluated using Student´s t test, or One-way ANOVA;
p values  0.05 (*) and  0.01 (**) were considered significant.
4. Conclusions
This work describes the synthesis and biological evaluation of arylaminoquinoline
derivatives, some of them new compounds, which were fully characterized by
spectroscopic methods. They were obtained in good yield with a simple reaction with
affordable starting materials. All compounds were evaluated against promastigote

Leishmania mexicana and epimastigote, trypomastigote and amastigote forms of
Trypanosoma cruzi. Fluorine and chlorine derivatives of 3-aminoquinoline (11b-c) showed
to be very effective as antiparasitic agents on L. mexicana and T. cruzi exhibiting an
efficacy comparable to that of presented by with IC50 values of the same order than used
control compounds. These compounds were capable to inhibit the degradation of heme,
inducing intracellular oxidative damage, which is not countered by the antioxidative
defense system of the parasite. In addition, they did not showed citotoxicity, therefore these
compounds offer excellent perspective as potential drugs for chemotherapy of neglected
diseases as trypanosomiasis and leishmaniasis.
Supporting Information: Spectral data for compounds 10-11, associated with this article
are supplied (for reviewers' information only).
Acknowledgments
We thank UBA (UBACYT 20020130100212), CONICET (PIP11220110100653) and
Bunge y Born Foundation for partial financial support. G.G.L, E.L., C.L. and A.B. are
Research Members of CONICET (Argentina).
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Graphical Abstract
Neglected
Tropical
Diseases
Trypanosomiasis
Leishmaniasis

Highlights:
 New 2- and 3-arylaminoquinolines were prepared and totally spectroscopy
characterized.

Fluorine derivatives showed a remarkable activity against L. mexicana and T. cruzi.
 Heme polymerization inhibition is the action mechanism proposed.
 All of synthesized derivatives did not showed citotoxicity.