Antileishmanial activity of quinazoline derivatives: Synthesis, docking screens, molecular dynamic simulations and electrochemical studies

Article abstract of DOI:10.1016/j.ejmech.2014.12.051

A series of quinazoline-2,4,6-triamine were synthesized and evaluated in vitro against Leishmania mexicana. Among them, N6-(ferrocenmethyl)quinazolin-2,4,6-triamine (H2) showed activity on pro-mastigotes and intracellular amastigotes, as well as low cytot

Full text of DOI:10.1016/j.ejmech.2014.12.051

Accepted Manuscript
Antileishmanial activity of quinazoline derivatives: Synthesis, docking screens,
molecular dynamic simulations and electrochemical studies
Cesar Mendoza-Martínez, Norma Galindo-Sevilla, José Correa-Basurto, Victor
Manuel Ugalde-Saldivar, Rosa Georgina Rodríguez-Delgado, Jessica Hernández-
Pineda, Cecilia Padierna-Mota, Marcos Flores-Alamo, Francisco Hernández-Luis
PII:
S0223-5234(14)01160-X
10.1016/j.ejmech.2014.12.051
EJMECH 7611
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 August 2014
Revised Date: 23 November 2014
Accepted Date: 28 December 2014
Please cite this article as: C. Mendoza-Martínez, N. Galindo-Sevilla, J. Correa-Basurto, V.M. Ugalde-
Saldivar, R.G. Rodríguez-Delgado, J. Hernández-Pineda, C. Padierna-Mota, M. Flores-Alamo, F.
Hernández-Luis, Antileishmanial activity of quinazoline derivatives: Synthesis, docking screens,
molecular dynamic simulations and electrochemical studies, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2014.12.051.
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Antileishmanial activity of quinazoline derivatives: Synthesis, docking
screens, molecular dynamic simulations and electrochemical studies
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Cesar Mendoza-Martínez , Norma Galindo-Sevilla , José Correa-Basurto , Victor Manuel
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Ugalde-Saldivar , Rosa Georgina Rodríguez-Delgado , Jessica Hernández-Pineda , Cecilia
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Padierna-Mota , Marcos Flores-Alamo , Francisco Hernández-Luis
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Programa de Maestría y Doctorado en Ciencias Químicas, Universidad Nacional
Autónoma de México, México, DF 04510, México
Departamento de Farmacia, Departamento de Química Inorgánica y Nuclear, Facultad
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de Química, Universidad Nacional Autónoma de México, México DF 04510, México
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Departamento de Infectología, Instituto Nacional de Perinatología, México DF 11000,
México
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Escuela Superior de Medicina, Instituto Politécnico Nacional, México DF 11340, México
Laboratorio de Especialidades Inmunológicas S.A. de C.V., México DF 07580, México
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*Corresponding author: Tel +525 56225287, E-mail address: franher@unam.mx

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ABSTRACT
A series of quinazoline-2,4,6-triamine were synthesized and evaluated in vitro against
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Leishmania mexicana. Among them, N -(ferrocenmethyl)quinazolin-2,4,6-triamine (H2)
showed activity on promastigotes and intracellular amastigotes, as well as low cytotoxicity
in mammalian cells. Docking and electrochemical studies showed the importance of both
the ferrocene and the heterocyclic nucleus to the observed activity. H2 is readily oxidized
electrochemically, indicating that the mechanism of action probably involves redox
reactions.
Keyboard: Leishmania mexicana; quinazoline; antiprotozoan activity

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1. Introduction
The search for antiparasitic molecules has recently become important because of the
emergence of drug resistant strains, the toxicity of the known molecules, increased poverty
and the percentage of the affected population [1]. Parasitic tropical diseases affect hundreds
of millions of people worldwide, however, it has been neglected to develop drugs against
these diseases because they primarily affect people in poor regions of the world [1].
Leishmaniasis, African trypanosomiasis, and Chagas disease are vector-borne diseases
caused by parasites of the kinetoplastida order [2]. Leishmaniasis is a set of devastating
diseases caused by the obligate intracellular protozoa parasites of the Leishmania genus,
which are transmitted by a group of 50 species and subspecies of phebotomine insects.
About 1.5 million of new cases of cutaneous leishmaniasis and 500 000 new cases of
visceral disease occur each year. Cutaneous leishmaniasis is endemic in more than 70
countries worldwide [3]. A major emerging problem is co-infection of Leishmania with
human immunodeficiency virus, especially because there is no effective treatment for these
patients [3]. Conventional chemotherapy relies on multiple parenteral administrations of
pentavalent antimonials that are considerably toxic and induce resistance. Second-line
drugs, such as amphotericin B and its lipid formulations, are either too toxic or too
expensive for routine use in developing countries [4]. Recently, miltefosine, a
phosphocholine analogue originally developed as an anticancer agent, was introduced as a
drug against visceral leishmaniasis, but its effectiveness has still not been conclusively
determined and there have already been reported cases of resistance [5]. Because
chemotherapy for leishmaniasis is still inefficient, there is an urgent need for the
development of new efficient and safe drugs.

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In order to find new drugs against leishmaniasis, the computational design of new drugs
against parasites has been based on the knowledge and availability of new information
from molecular targets [7,8]. With the goal of following this strategy, we chose
pteridine reductase (PTR) as the target of the study, which is a protein that appears to be
important to the resistance mechanism of Leishmania. This enzyme is NADPH-
dependent with oxide reductase activity, and pterins are its natural substrate [7].
However, under conditions of cellular stress or when folate metabolism is brought
down by molecules that inhibit dihydrofolate reductase (DHFR), PTR may reduce
folate, therefore allowing the production of DNA through the salvage route, causing
parasite resistance [9-11]. New approaches in the design of antifolates should consider
the importance of PTR to discover new molecules capable of inhibiting DHFR and/or
PTR through rational design [7].
The isoform PTR1 of Leishmania major was co-crystallized with quinazoline-2,4,6-
triamine (TAQ) [12]. By analyzing the interactions of TAQ with the active site of
PTR1, the chemical modification of TAQ at position 6 was considered because the
reception site near this position is large and possesses both polar and non-polar regions
(Figure 1). Berman and co-workers, showed the importance of the lipophilic
substituents at position 6 of the quinazoline ring, decreasing the ED50 against L. major.
In addition, they have shown that, by increasing the size of the substituent, the ED50
increased [13]. In this case, the selection of the chemical substituent at position 6 was
based on its potential to function as an anti-protozoal scaffold. With this in mind,
nitrobenzene, ferrocene, benzimidazole, anisole and phenol moieties were selected as
the substituents in H1, H2, H3, H4 and H5, respectively (Table 1). Ferrocene is present
in ferroquine, an anti-malarial drug that is currently under clinical evaluation and has

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got a great potential [14]. A number of studies have shown that the introduction of the
ferrocene core may significantly enhance the molecule’s (desirable) bioactive
properties. The ferrocene unit might act as a hydrophobic spacer and/or
lipophilicity/bioavailability enhancer (enabling an easier way through cell membranes)
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[15]. It is also known that ferrocene Fe /Fe redox chemistry might contribute to the
bioactivity of ferrocene derivatives [16]. Benzimidazole is a privileged structure in
antiparasitic molecules [17]; particularly, in a compound prepared in our group, 5,6-
dicloro-2-(trifluoromethyl)-1H-benzimidazole, denoted in this work as G2 [18].
Nitrobenzene, anisole and phenol groups are present in several anti-parasitic drugs [19,
20].
With the aim of exploring PTR1 binding site surfaces of the protein, we employed
docking studies using TAQ as ligand and attempted to reproduce this ligand is in the
previously reported crystal structure [12]. Next, we performed a molecular coupling of
the proposed molecules (H1–H5) into active site of Leishmania PTR1 and DHFR. The
affinity of all the target compounds to both enzymes was higher than the affinity of
TAQ. Subsequently, the proposed compounds were synthesized and once their structure
was elucidated by x-ray crystallographic, they were tested in vitro against the
promastigote form of L. mexicana. The most active was H2. For this reason, chemical
modifications were carried out to the H2 structure (Figure 2) in order to explore
changes in biological responses that could cause these structural modifications. Finally,
the reduction potential for this group of compounds was carried out using DMSO as
aprotic solvent. The data in aprotic solvent did not only describe the situation in that
way but it also allowed us to obtain biological significance of interpretation.
2. Results and discussion

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2.1.Chemistry
Compounds H4 and H5 were first synthesized and evaluated as antichagasic and
antiplasmodial agents by Davoll, and co-workers [21]. Since they were not evaluated
against Leishmania, these molecules were synthesized in this work in order to explore their
biological activity against L. mexicana.
All the TAQ derivatives were synthesized according to the route outlined in Scheme 1. The
antiprotozoal compound G2 (5,6-dicloro-2-(trifluoromethyl)-1H-benzimidazole) was
prepared in our laboratory using known procedures [18]. As shown in Scheme 1,
nucleophilic substitution of the bromine atom in 2-bromo-1,1-diethoxyethane by G2
afforded 5,6-dichloro-1-(2,2-diethoxyethyl)-2-(trifluoromethyl)-1H-benzimidazole (1).
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Treatment of 1 with boron tribromide (BBr ) followed by hydrolysis gave [5,6-dichloro-2-
(trifluoromethyl)-1H-benzimidazole-1-yl]acetaldehyde (2). Preliminary attempts to obtain
the aldehyde group with hydrochloric acid [22], I /acetone [23] and FeCl [24] were
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unsuccessful. The cyclocondensation of commercially available 5-nitroanthranilonitrile
with guanidine hydrochloride yielded 6-nitroquinazoline-2,4-diamine (3), which was
treated with acetic anhydride followed by hydrogenation under a hydrogen atmosphere
using palladium on charcoal to yield N,N'-(6-nitroquinazoline-2,4-diyl)diacetamide (4).
Subsequent condensation of 4 with the corresponding aldehyde gave the imine derivatives.
The imine group was reduced with sodium borohydride (NaBH ) to yield the proposed
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molecules (H1–H5).
Of all the compounds synthesized only H2 gave crystals adequate to carry out an single-
crystal X-ray analysis. These studies revealed that H2 in ethanol crystallizes in the triclinic
space group P-1. A view of H2 is presented in Figure 3, and selected bond distances and
torsion angles are listed in Table 2. The asymmetric unit of the H2 compound consists of

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N -(ferrocenylmethyl)quinazolin-2,4,6-triamine and three ethanol solvate molecules in
general positions. The quinazolin-2,4,6-triamine shows planarity with a rms deviation of
the fitted atoms of 0.0265. The C2-C1-C19-N1 (106.47°) and C5-C1-C19-N1 (70.20°)
torsion angles show a nearly perpendicular arrangement between the ferrocenyl and
quinazoline groups.
In the crystal lattice, the neutral ferrocenyl complex and the ethanol solvate molecules
participate in multiple hydrogen bonding interactions, such as N-H···O, N-H···N and O-
H···O (Figure 4). The majority of the hydrogen bond distances range from 1.87-2.25 Å
(Table 3). The interaction between the nitrogen donor atom (N4) and the acceptor atom
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(N2) forms the R (8) motif, whereas the N1-H1F···O1C hydrogen bond shows the D (17)
motif. All of these interactions are present in the three crystalline axes and favor three-
dimensional growth.
In order to determine the part of the molecule that provides the anti-Leishmania effect when
quinazoline or ferrocene are used, a series of ferrocene derivatives (HO2, HO4, H2A,
FBC) were synthesized via reductive amination with varying reagents (Scheme 2).
The structures of all the compounds synthesized were characterized by spectroscopic and
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spectrometric data. In the H NMR spectra of the title compounds, the doublet in the range
4.5–3.9 ppm, integrating for two protons, confirmed the presence of methylene linking the
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substituent to quinazoline nucleus; whereas in the C NMR spectra, the same signal of the
secondary carbon of the methylene was seen at δ value of 46 ppm. For the same group of
compounds, the signal of proton of the NH exocyclic at position 6 was recorded as triplet at
δ values in the region 6–5 ppm. For H2, HO4, HO2, H2A and FBC, typical ferrocene
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signals were showed in the region of 4.3– 4.0 ppm in the H NMR spectra, and 60–90 ppm

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in the C NMR spectra. For those compounds having exocyclic amines groups at position
2 and 4 of quinazoline nucleus, the signal derived from two protons was observed at δ
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values 5.6 and 7.0 ppm, respectively. Regarding H NMR spectra of HO2 and HO4, the
exocyclic amine group, at position 4 and 2, presented a signal to 7.73 and 5.91 ppm,
respectively. Finally, in the downfield region of the same spectrum, the signal of one proton
at 10.44 (HO2) and 10.78 (HO4) ppm revealed the presence of endocyclic NH of
quinazoline moiety, suggesting the presence of carbonyl group at position 2 and 4,
respectively. This suggestion was supported by IR spectra of both compounds, which
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exhibited stretch carbonyl characteristic band at 1624 and 1644 cm , respectively.
Additionally, energetic stability studies confirmed that the most stable tautomers (Gibbs
energy of 0 Kcal) for both molecules are those having the carbonyl group. All these data
indicate that in HO2 and HO4 the C-O bond is present as carbonyl group and the most
stable tautomeric form should be as proposed (Figure 5).
2.2.Modeling studies
With the purpose of estimating the interactions of the synthesized compounds with PTR
and DHFR, the affinity to both enzymes was analyzed in a theoretical docking study. The
crystal structure of tcDHFR (Trypanosoma cruzi dihydrofolate reductase ID-PBD: 2H2Q)
was used to construct a homologous enzyme, L. major was used as a model for L.
mexicana. The DHFR sequence in L. mexicana is unknown; however, the three species (T.
cruzi, L. major and L. mexicana) are evolutionarily very close. The molecular dynamics
protocol was applied to relax the built protein, and a snapshot at 10 ns was obtained for the
docking of all the molecules. The root media square deviation (RMSD) and the radius of
gyration (RGyr) indicated that the protein reached convergence (Figure 6). The crystal

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structure of L. major pteridin reductase (1 ID-PBD: 1WOC) was obtained from the Protein
Data Bank, and molecular docking was performed with this protein.
The free energy analysis of the molecular interaction between the TAQ derivatives and the
DHFR and PTR1 enzymes showed that all of the compounds bind more strongly to the
receptor site than TAQ, and the mode of binding was very similar (Figure 7). In the case of
the ferrocene derivatives (HO2, HO4 and H2A), the differences in the interaction energies
were a result of the substituents at positions 2 and 4 (Table 5). For this study, the stability
of the different tautomers of HO2 and HO4 was analyzed (Figure 5) with the intention of
using only the most stable.
2.3. Biological activity
Obtained compounds were tested in vitro against promastigotes form of L. mexicana.
Initially, the compounds were incorporated into the media at 100 µM and their ability to
inhibit growth of the parasite was evaluated in comparison to the control (no drug added to
the media). Glucantime was used as the leishmanicidal of reference; pyrimethamine and
trimetoprim were used as DHFR inhibitors models. Subsequently, the IC50 (50% inhibitory
growth concentration) concentration was determined. With exception of H2 and H4, all
compounds resulted with poor or null activity (Table 6). Compound H2 was the most active
(IC = 0.93 µM); the second place went to H4 (IC = 14.59 µM). The components of H2
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(ferrocene and TAQ) had a poor antileishmanial activity individually. Interestingly, H2
showed antiparasitic activity from the first hour of being in contact with the parasites. This
behavior may be due to the ferrocene moiety contributes with its lipophilicity and redox
properties. On the other hand, with exception of H4, FBC showed slightly higher activity
than the quinazoline derivatives, despite having only one phenyl group attached to

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ferrocene unit. In H2, the TAQ portion was necessary for leishmanicidal activity because
when the 2- and 4-positions were modified by acetylation (H2A) or substitution of the NH
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group (HO2, HO4), the activity was lost (Table 6). In the light of these results, only H2
was selected for the following studies in order to gain an understanding of its biological
activity.
There is no obvious correlation with the solubility, however, the solubility of H2 is greater
than ferrocene alone and less than triaminquinazoline. This, of course, could be a cause of
antileishmanial activity shown because ferrocene is dissolved in greater amounts. However,
H2A is much more soluble than most derivatives (except TAQ, which is extremely soluble)
but this is not enough to improve the activity, so, that interaction of the amines of TAQ are
important.
2.3.1. Activity of H2 against intracellular amastigotes
The study on intracellular amastigotes revealed that H2 maintains strong leishmanicidal
activity. This compound is able to kill intracellular parasites inside the macrophages;
therefore, it may be useful for in vivo treatment of the disease. Note that although the
antileishmanial activity observed is for intracellular amastigotes, H2 eliminates 97% of the
parasites at 12 µM (Figure 8 B). After exposure of the infected macrophages to H2 for 72
h, removal of this compound and reculturing the parasites in fresh media, parasite survival
was observed at 3 and 6 µM, whereas concentrations of 12, 25, and 50 µM effectively
killed the intracellular parasite and survival was not observed (Figure 8 C).
2.3.2. DHFR inhibition
As a second approach, the inhibition of DHFR was explored, as shown by the theoretical
studies (Table 7). For experimental studies, the parasites were exposed to above LC50 of H2

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(a concentration that shows an effect but does not kill all of the parasites) after they had
been previously exposed to folic acid, folinic acid and ferulic acid. Folic acid competes for
the active site of DHFR, as it is the natural substrate. Folinic acid does not need to undergo
metabolism to contribute to DNA synthesis, and ferulic acid has an antioxidant effect.
Although the effect anti-leishmanial of H2 was decreased, differences were observed
among folic acid, folinic acid and ferulic acid. The greatest inhibitor of the H2
antileishmanial effect was folic acid, which showed a 20% increase in parasite survival
compared with the control. Trimethoprim is a drug that inhibits DHFR and was used as a
positive control. Parasite survival increased to 100% when the parasites were treated with
folic acid before trimethoprim, demonstrating that its mechanism of action involved DHFR
inhibition. An excess of folic acid was added in all cases to ensure reversibility of DHFR
inhibition from the tested compounds. This complete reversibility of the antileishmanial
effect with trimethoprim was not observed in the case of H2, in which parasite survival
only reached 20% (Table 7). Therefore, we assumed that the mechanism of action of the
antileishmanial effect of H2 partially occurs via DHFR, but other mechanisms, such as a
redox mechanism, could be occurring simultaneously as result of the presence of ferrocene
in the molecule.
When the activity of H2A (acetylated H2) was evaluated in the docking study, improved
biological activity compared to H2 was expected due to its better affinity for the DHFR and
PTR receptors. However, the results were not consistent with this hypothesis. This
inconsistency can be explained by the alternate mechanism of the antileishmanial effect of
H2 and its derivatives, which is a redox mechanism resulting from ferrocene, that endows
the molecule with remarkable performance against Leishmania, as demonstrated by its

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good biological activity. The compounds TAQ and 4 (TAQ acetylated) behaved in accord
with the theoretical docking prediction.
2.3.3. Effect of H2 on oxygen consumption by the parasite
When the parasite is exposed to H2, it quickly becomes quiescent, it loses its activity as
their shape wraps around, and it is finally lysed. The inhibition of parasite replication due to
DHFR or PTR should take several hours to affect the parasite, but instead, the parasite dies
in the presence of H2 as soon as 20 min after it is added; thus, an alternative mechanism
must be occurring. Because of this result, we investigated the inhibition of oxygen
consumption, which could indicate whether the compound is able to modify the
mitochondrial activity of the parasite. Thus, promastigotes of Leishmania parasites were
treated with different concentrations of H2 during 1 and 2 hours. If the viability of the
parasite is immediately affected, then a decrease in oxygen consumption will be detected by
the increase in fluorescence. The H2 treatment reduced the fluorescence score to a level
similar to that of PBS without the Leishmania controls, indicating that this drug somehow
affects the mitochondria, the organelles that consume oxygen (Figure 9). Whether this
effect is due to the respiratory chain remains to be determined.
Dithionite was used as a positive control for the abolition of oxygen concentration in the
medium. For a mitochondrial damage positive control, a group of parasites was treated with
sodium cyanide during two hours. In the case of H2, the decreased oxygen consumption,
fall the fluorescence. In the case of treatment with cyanide, a similar effect was observed
(Table 8).
Effect of H2 on the Leishmania mitochondria. Mitotracker green is a fluorochrome that
joins the mitochondria independent of their potential. Mitochondria are stained at
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nanomolar concentrations of Mitotracker Green FM . Fluorescence results from the

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accumulation of lipids in the mitochondria and indicates if the cells have active
mitochondria. If no fluorescence is observed, the mitochondria and cells are not viable, i.e.,
the cells are undergoing cell death.
Propidium iodide is a fluorescent molecule that intercalates between DNA and RNA bases
and is commonly used in quantitative tests of DNA content. Propidium iodide cannot
permeate the cell membrane of healthy cells.
L. mexicana promastigotes that were untreated showed intense green fluorescence, as
expected, and practically no parasite red stain was observed, indicating that the cells were
viable and the mitochondria were fully functional.
When the promastigotes were subjected to different concentrations of the H2, the intense
Mitotracker green FM fluorescence was not observed as the H2 concentration increased,
and the propidium iodide fluorescence was observed, indicating parasite death (Figure 10).
2.3.4. Cytotoxic effect of H2 on eukaryotic cells
The cytotoxicity of H2 was analyzed and compared with others antiparasitic compounds
(nifurtimox and pyrimethamine). At a concentration of 100 µM, H2 was slightly cytotoxic
compared with the solution without the compound (Figure 11). The cytotoxicity observed
in the vehicle solution should be due to DMSO. The tested concentration (100 µM) is
higher than the IC50 against L. mexicana. Furthermore, evaluations were conducted to
determine the influence of compounds on the metabolic activation of cells. In Figure 12, it
can be seen that the pyrimethamine not affect the metabolic activity of cells, similar to
control. Compound H2 had an activity of 82%, slightly more toxic than pyrimethamine.
Moreover, cells exposed to nifurtimox presented a 42% metabolic activity, indicating
toxicity of this compound.

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The cytotoxicity was also evaluated in macrophages, using the Alamar Blue indicator. In
this evaluation the cells remained viable after 24 h at all concentrations compared to the
control. This reinforces the hypothesis that the compound H2, is not cytotoxic (Figure 12).
These results hold the interest to continue studying the compound H2 as anti-protozoan.
Electrochemical studies
Electrochemical studies were carried out to evaluate the importance of the redox process on
the biological activity, particularly with compounds containing the ferrocene moiety. We
found that the half-wave potential of ferrocene not changed in the molecules.
It is important mentioning that of all of the compounds, H2 is oxidized most easily. H2 has
an oxidation potential of -0.086 V (IA), and the magnitude of the current intensity observed
in this process is three times higher than those of TAQ and the other compounds. That
could be very important for the biological activity (Figure 13). We propose that the
nitrogen in position 1 of quinazoline nucleus is oxidized because this is the location of the
highest electron density (Figure 14).
The presence of the ferrocene group is important for the biological activity, which is
demonstrated by the parasite growth inhibition caused by ferrocene and FBC. However, for
+
ferrocene derivatives, the oxidation potential of the Fc/Fc couple does not substantially
change (Table 9); therefore, the difference in activity between them results from the
modification of the quinazoline nucleus.
3. Conclusions
Leishmaniasis has become a health issue due to its high incidence, especially in
underdeveloped countries. There is an urgent need to develop molecules that can better
treat the disease because existing molecules have become inefficient. In this study, we have
two new antileishmanial agents (H2 and H4). The first presented the most pronounced

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potency and activity against promastigotes and amastigotes stages of parasite. The results
demonstrated that leishmanicidal activity of H2 is due to multiple mechanisms of action,
which are the most significant advantage of this molecule, because they could reduce the
likelihood of the parasite developing resistance. It is interesting to note that this
antiparasitic effect is largely due to the presence of the ferrocene group. This moiety
affords distinct characteristics to the molecule, primarily as result of the readily oxidized
iron atom. Previous studies have shown that the ferrocene group can produce ROS,
principally hydroxyl radicals, to exert its effect. Additionally, the quinazoline-2,4-diamine
scaffold is required for biological activity, as demonstrated by the poor biological activities
of HO2, HO4, H2A and FBC. In this sense, the exocyclic amino groups must be retained
because they are to contribute to receptor interaction. It is worth mentioning that H2 is
electrochemically oxidized more easily with respect other quinazoline derivatives,
indicating that the mechanism of action involves a type of redox reaction. Regarding the
inhibition of DHFR or PTR is still involved, but it is not the primary mechanism of action
of this compound because the consequences of their enzymatic inhibition would require
several hours for their display; H2 initiates its biological action from the first hour after
being applied to parasites.
Other possibility is that the mitochondria are actively affected during the action of H2. The
decrease in oxygen consumption (similar to that observed with cyanide) clearly indicates
the relationship of the mechanism to inhibition of the respiratory chain, which results in
apoptosis of the parasite. However, more studies are required to identify the extent of this
influence on mitochondrial function. Furthermore, the fact that the promastigotes become
permeable to propidium iodide is an indicator of the cell membrane damage caused by H2.

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We are currently clarifying these hypotheses. A feature last, in cytotoxicity studies, H2 did
not showed severe toxicity in eukaryotic cells.
In regard to H4, which also presented an interesting activity, we could indicate that
likely it has a good interaction with its receptor owing to the substituent lipoid that
constitutes. Its mechanism of action may be different from H2 because it does not
present in its structure the ferrocene entity, and its antiparasitic effect is manifested after
24 h of exposure. We will focus on studying in greater detail the mechanism of activity
and optimization of this compounds in the future.
Finally, we emphasize that there are new opportunities to develop novel drugs that are
more active against intracellular parasites, such as Leishmania, and that present only
low toxicity to the host. The results presented in this study advance the fight against
orphan parasitic diseases, which often cause death.
4. Experimental section
70 3.1.Chemistry
71
72
73
74
75
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77
78
79
80
Melting points were determined in open capillary tubes with a Büchi B-540 melting point
apparatus and are uncorrected. Reactions were monitored by TLC on 0.2 mm precoated
silica gel 60 F254 plates (E. Merck) and were visualized by irradiation with a UV lamp.
Silica gel 60 (70–230 mesh) was used for column chromatography. IR spectra were
1
recorded with a FT Perkin Elmer 16 PC spectrometer on KBr disks. H NMR spectra were
measured with a Varian EM-390 (300 MHz) spectrometer. Chemical shifts are given in
ppm relative to tetramethylsilane (Me Si, δ = 0), and J values are given in Hz. Splitting
4
patterns have been designated as follows: s, singlet; d, doublet; q, quartet; dd, doublet of
doublet; t, triplet; m, multiplet; br. s, broad singlet. MS data were recorded on a JEOL JMS-
SX102A spectrometer by FAB [(FAB(+)]. Elemental analyses (C, H, N) for the new

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compounds were performed on a Fisons EA 1108 instrument and were within ±0.4% of the
theoretical values of the proposed structures. The catalytic hydrogenation reactions were
carried out in a Parr shaker hydrogenation apparatus. The starting materials 5-
nitroanthranilonitrile, guanidine hydrochloride, and aniline are commercially available
(Sigma Aldrich).
The percent purity for compounds H1, H2, H3, H4, H5, H2A, HO2, HO4 and FBC was
calculated by chromatographic analysis. Standard solutions of all of the compounds were
dissolved in methanol grade HPLC, and the volume of injection was 20 µL. The methanol
and acetonitrile used for the mobile phase were of chromatographic grade (J.T. Baker,
®
Phillipsburg, NJ, U.S.A). Water was deionized and osmosed using a Milli-Q system
(Millipore, Bedford, MA, U.S.A). The phosphates were of analytical grade. The HPLC
system consisted of a Waters Alliance e2695 separation module autosampler and a 2489
dual λ absorbance UV/Visible detector coupled with EmpowerTM software (Waters,
®
Milford, MA, U.S.A). The analytical column was an Acentis RP-Amide column (150 mm
x 4.6 mm ID, particle size 5 µm) (Supelco, Sigma-Aldrich, U.S.A) protected by a
compatible guard column. The mobile phase consisted of 10 mM phosphate buffer at pH
3.3 and acetonitrile (60:40 v/v). The assay run time was 8 min with a flow rate of 1.00
mL/min, and it was carried out at 30 °C. Absorbance was measured at 240 and 272 nm.
4.1.1. 5,6-dicloro-1-(2,2-dietoxiethyl)-2-(trifluoromethyl)-1H-benzimidazole (1)
Compound G2 (1.43 g, 3.8 mmol) was reacted with 2-bromo-1,1-diethoxyethane (5.7 mol)
and sodium carbonate (1.19 g, 11.23 mmol) in N,N-dimethylformamide (1 mL) at 110 °C
for 10 h. Then, the reaction mixture was poured in cold water, and the solid was filtered and
2 2
dried. Recrystallization of the powder from CH Cl yielded cream-colored crystals (1)

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(1.93 g, 92%, Mp: 76.8–77.6 °C), TLC (hexane/CHCl /ethyl acetate = 50/35/15) R = 0.65.
3
f
1
IR (KBr): 3437 (C-H), 1133 (C-O), 1121 (C-F), 1078 (C-Cl). H NMR (DMSO-d
J = 7, 6H, CH ), 3.41 (m, J = 9, J = 7, 2H, OCH ), 3.74 (m, J = 9, J
4.33 (d, J = 5, 2H, NCH ), 4.66 (t, J = 5, 1H, OCHO), 7.79 (s, 1H, CH, aromatic), 7.89 (s,
6
): 1.09 (t,
3
1
2
2
1
2 2
= 7, 2H, OCH ),
2
1
3
1H, CH, aromatic); C NMR (DMSO-d ): 15.20, 48.77, 64.65, 101.72, 114.27, 120.20,
6
122.34, 128.21, 129.79, 135.53, 140.11, 142.09. Anal. Calcd for C H N O : C, 46.61; H,
1
2
13
5
2
2.93; N, 27.18. MS (+FAB) m/z = 371 (M+1).
4.1.2. [5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole-1-yl]acetaldehyde
Compound 1 (1 g, 2.7 mmol) was dissolved en CH Cl (4 mL) and reacted with a solution 1
M of BBr in CH Cl (10.77 mL, 10.77 mmol). Then, the reaction mixture was transferred
(2)
2
2
3
2
2
into cold water, and the solution was extracted with ethyl acetate (three portions of 50 mL).
The organic layer was concentrated and yielded a dark oil, which was immediately used in
the next reaction (1.93 g, 99%) (TLC, hexane/CHCl /ethyl acetate = 50/35/15; R = 0.51).
3
f
4.1.3. 6-nitroquinazoline-2,4-diamine (3)
Sodium hydroxide (3.4 g, 85 mmol) was added to a solution of guanidine hydrochloride
(3.65 g, 38 mmol) in ethanol, and the reaction was stirred for 20 min at room temperature.
The solution was then stirred under reflux for 6 h with 5-nitroanthranilonitrile (5 g, 31
0
mmol) in 1-propanol (40 mL). Then, the reaction mixture was cooled to 0 C. The solid was
filtered off, washed with cold water, washed with cold ethanol and dried. An orange solid
product was obtained (5.4 g, 86%, Mp: 360 °C), TLC (2-butanol/acetic acid/water =
1
80/20/5) R = 0.4. IR (KBr): 3464 and 3440 (N-H), 1614 (C-H, aromatic), 1325 (NO ). H
f
2
NMR (DMSO-d ): 6.77 (s, 2H, NH ), 7.22 (d, J = 9, 1H, aromatic), 7.86 (s, 2H, NH ), 8.21
6
2
2
1
3
(dd, J = 3, J = 9, 1H, aromatic), 9.08 (d, J = 3, 1H, aromatic); C NMR (DMSO-d6):
1
2

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108.84, 121.92, 124.87, 126.63, 139.12, 157.28, 163.00, 163.025. Anal. Calcd for
C
12
H
13
N
5
O
2
: C, 46.83; H, 3.44; N, 34.13.
4.1.4. N-[2-(acetylamino)-6-aminoquinazolin-4-yl]acetamide (4)
o
Compound 3 (1 g, 4.87 mmol) and acetic anhydride (1 mL) were heated at 100 C for 12 h.
Then, the reaction mixture was poured into cold water, and the solid was filtered off,
washed with cold water at pH 7 and dried. A yellow solid (3a) was obtained (0.89 g, 65%,
Mp: 278 °C), TLC (CHCl /MeOH = 60/40) R = 0.8. Anal. Calcd for C H N O : C, 49.83;
3
f
12 11
5
4
H, 3.83; N, 24.21. Found: C, 49.99; H, 3.11; N, 23.81.
The catalytic reduction of 3a (0.5 g, 1.73 mmol) with hydrogen and Pd/C (10%) (0.05 g)
was carried out on a Parr assembly at 60 psi at room temperature for 1 h. The catalyst was
then removed by filtration, and the filtrate was concentrated on a rotavapor under reduced
pressure to yield 4 (0.34 g, 76%, Mp = 238 °C), TLC (CHCl /MeOH = 60/40) R = 0.67. IR
3
f
1
(KBr): 3353 and 3230 (N-H). H NMR (DMSO-d ): 2.2 (s, 3H, CH ), 2.34 (s, 3H, CH ),
6
3
3
5.61 (br. s, 2H, NH ), 7.04 (d, J = 3, 1H, aromatic), 7.28 (dd, J = 8, J = 3, 1H, aromatic),
2
1
2
1
3
7.5 (d, J = 8, 1H, aromatic), 10.46 (br. s, 1H, NH amide), 10.22 (br. s, 1H, NH amide); C
NMR (DMSO-d ): 24.51, 102.37, 116.82, 126.02, 127.48, 145.16, 146.55, 150.08, 156.08,
6
169.18, 170.73. Anal. Calcd for C12
H, 4.52; N, 25.20.
13 5 2
H N O : C, 55.59; H, 5.05; N, 27.01. Found:C, 57.0;
4.1.5. 2-amino-6-nitroquinazolin-4(3H)-one (5)
Compound 3 (2 g, 9.7 mmol) was refluxed in 75 mL of HCl (6 N) for 2 weeks. The solution
was then cooled and adjusted to pH 7 with a saturated solution of Na CO . The solid was
2
3
filtered and washed repeatedly with water. A yellow solid was obtained (1.5 g, 75%, Mp:
351.5-352.5 °C), TLC (CHCl /MeOH = 80/20) R = 0.53. IR (KBr): 3572 and 3437 (N-H),
3
f
1
1704 (C=O), 1330 (NO
2
). H NMR (DMSO-d
6
2
): 7.06 (br. s, 2H, NH ), 7.28 (d, J = 9, 1H,

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aromatic), 8.30 (dd, J = 3, J = 9, 1H, aromatic), 8.61 (d, J = 3, 1H, aromatic), 11.74 (br. s,
1
2
1
3
1H, NH); C NMR (DMSO-d
6
): 116.71, 123.13, 125.23, 127.76, 141.05, 154.87, 156.84,
162.04. Anal. Calcd for C12
N, 28.12.
13 5 2
H N O : C, 46.61; H, 2.93;N, 27.18. Found:C, 47.0; H, 2.71;
4.1.6. 4-amino-6-nitroquinazolin-2(1H)-one (6)
Urea and 5-nitroantranilonitrile (2 g) were heated at 160 ° C for 2 hours with agitation. The
mixture was then transferred into cold water. The solid was filtered and washed repeatedly
with water. A yellow compound was obtained (0.34 g, 77%, Mp: 351.7- 354.4 °C), TLC
(CHCl
3
/MeOH = 80/20) R
f
= 0.82. IR (KBr): 3444 and 3333 (N-H), 1703 (C=O), 1317
): 7.24 (d, J = 9, 1H, aromatic), 8.09 (br. s, 2H, NH ), 8.37 (dd,
1
(NO
2
). H NMR (DMSO-d
6
2
1
3
J
1
= 2, J
2
= 9, 1H, aromatic), 9.07 (d, J = 2, 1H, aromatic), 11.29 (br. s, 1H, NHCO). C
NMR (DMSO-d ): 108.22, 116.29, 122.19, 128.96, 131.89, 141.24, 147.67, 156.09, 163.73.
6
Anal. Calcd for C H N O : C, 46.61; H, 2.93; N, 27.18. Found: C, 45.64; H, 2.74; N,
1
2
13
5
2
28.01.
4.1.7. Synthesis of compounds H1, H2, H3, H4 and H5
A solution of the appropriate aldehyde (1.6 mmol) and compound 4 (0.4144 g, 1.6 mmol)
in methanol (80 mL) was stirred at 60 ºC for 2 days. Then, the reaction mixture was cooled
4
to 0 °C, and NaBH (0.09 g, 2.37 mmol) was added. Subsequently, the reaction mixture
was stirred at room temperature for 12 h. Next, the reaction mixture was concentrated on a
rotavapor apparatus under reduced pressure, and cold water was added. The solid was
filtered off, washed with cold water and dried.
6
4.1.8. N -(4-nitrobenzyl)quinazoline-2,4,6-triamine (H1)
Red powder (0.41 g, 82%, Mp: 215-218 °C). TLC (2-butanol/acetic acid/water = 80/20/5)
1
R
f
= 0.54. IR (KBr): 3369 (N-H), 1517 (-NO
2
); H NMR (DMSO-d
6
): 4.45 (d, J = 6, 2H,

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CH ), 5.71 ( br. s, 2H, NH ), 6.25 (t, J = 6, 1H, NH), 7.03 (m, 5H, NH C-H quinazoline),
2
2
2,
1
3
7.67 (d, J = 8, 2H, p-nitrobenzyl), 8.21 (d, J = 8, 2H, p-nitrobenzyl); C NMR (DMSO-d
6
):
46.16 (CH ), 100.66 (CH, quinazoline), 110.93 (C, quinazoline), 123.17 (CH, quinazoline),
2
123.32 (CH, p-nitrobenzyl), 128.44 (CH, p-nitrobenzyl), 142.35 (C-NH, quinazoline),
144.44 (C, quinazoline), 146.36 (C, p-nitrobenzyl), 148.91 (C-NO ), 158.18 (C-NH ,
2
2
quinazoline), 161.435 (C-NH , quinazoline); Anal. Calcd for C H N O : C, 58.06; H,
2
15 14
6
2
4.55; N, 27.08. Found: C, 57.0; H, 4.52; N, 25.20. HPLC purity of 99.9% (retention time =
3.841 min).
6
4.1.9. N -(ferrocenmethyl)quinazolin-2,4,6-triamine (H2)
Dark yellow powder (62%, Mp: 210.6-211 °C). TLC (2-butanol/acetic acid/water =
1
80/20/5) R
f
= 0.53. IR (KBr): 3393 (N-H), 1613 (C-N), 823 (C-H ferrocene). H NMR
(DMSO-d ): 3.98 (t, J = 6, 2H, CH ), 4.12 (s, 2H, ferrocene), 4.20 (s, 5H, ferrocene), 4.32
6
2
(s, 2H, ferrocene), 5.68 (t, J = 6, 2H, NH ), 6.23 (s, 2H, NH ), 7.03 (m, 3H, aromatic
2
2
1
3
quinazoline), 11.24 (br. s, 1H, NH). C NMR (DMSO-d ): 43.01 (CH ), 67.33 (CH,
6
2
ferrocene), 68.40 (CH, ferrocene), 86.10 (C, ferrocene), 100.25 (CH, quinazoline), 110.73
(C, quinazoline), 123.52 (CH, quinazoline), 124.99 (CH, quinazoline), 142.84 (C-NH,
2 2
quinazoline), 145.11 (C-NH, quinazoline), 158.34 (C-NH , quinazoline), 161.48 (C-NH ,
quinazoline). Anal. Calcd for C19 : C, 61.14; H, 5.13; N, 18.76. Found: C, 61.14; H,
H19FeN
5
4.92; N, 18.03. HPLC purity of 99.9% (retention time = 3.947 min). The X-ray structure
was determined.
6
4.1.10. N -{2-[5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazol-1-yl]ethyl}quinazoline-
2,4,6-triamine (H3)
Brown powder (0.62 g, 85%, Mp: 133-136 °C), TLC (2-butanol/acetic acid/water =
1
80/20/5) R
f
= 0.59. IR (KBr): 3350 (N-H), 1273 (C-N), 742 (C-F); H NMR (DMSO-d
6
):

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3.53 (br. s, 2H, CH ), 4.55 (br. s, 2H, CH ), 5.61 (s, 1H, NH), 5.65 (s, 2H, NH ). 6.84 (s,
2
2
2
1H, quinazoline), 7.07 (m, 4H, NH
2
, CH quinazoline), 7.92 (br. s, 1H, benzimidazole), 8.13
): 42.89 (CH ), 44.21 (CH ), 100.21 (CH,
1
3
(s, 1H, benzimidazole); C NMR (DMSO-d
6
2
2
quinazoline), 110.67 (C, quinazoline), 114.29 (CH, benzimidazole), 117.29 (C,
benzimidazole), 121.91 (C-H, benzimidazole), 123.27 (CH, quinazoline), 124.89 (CH,
quinazoline), 126.31 (C-Cl, benzimidazole), 127.88 (C-Cl, benzimidazole), 135.31 (C,
benzimidazole), 139.76 (C-CF ), 140.00 (CF ), 141.80 (C-NH, quinazoline), 144.45 (C,
3
3
quinazoline), 156.66 (C-NH , quinazoline), 161.98 (C-NH , quinazoline). MS (+FAB) m/z
2
2
= 456 (M+1); HRMS (+FAB) calcd for C18
H15Cl
2
F
3
N7: 456.07. Found: 456.07. HPLC
purity of 99.9% (retention time = 3.188 min).
6
4.1.11. N -(4-methoxybenzyl)quinazoline-2,4,6-triamine (H4)
Yellow powder (0.19 g, 40%, Mp: 204.1-205.2 °C). TLC (CHCl /MeOH = 80/20) R =
3
f
0.73. IR (KBr): 3450 and 3328 (N-H), 1639 (C-N), 1469 and 1563 (C=C Ar), 1243 (C-O-
1
C); H NMR (DMSO-d ) ppm: 3.72 (s, 3H, CH ), 4.20 (d., J = 6, 2H, CH ), 5.53 (br. s, 2H,
6
3
2
NH ), 5.81 (t, J = 6, 1H, NH), 6.89 (d, J = 9, 2H, p-methoxybenzyl), 6.96 (br. s, 3H, NH ,
2
2
1
3
CH quinazoline), 7.03 (m, 2H, CH quinazoline), 7.35 (d, J = 9, 2H, p-methoxybenzyl); C
NMR (DMSO-d ): 46.69 (CH ), 55.45 (CH -O), 100.89 (CH, quinazoline), 111.15 (C,
6
2
3
quinazoline), 114.02 (CH, p-methoxybenzyl), 123.91 (CH, quinazoline), 125.35 (CH,
quinazoline), 129.42 (CH, p-methoxybenzyl), 132.31 (C, p-methoxybenzyl), 143.26 (C-
NH, quinazoline), 145.37 (C, quinazoline), 158.57 (C-O, p-methoxybenzyl), 158.70 (C-
NH , quinazoline), 161.85 (C-NH , quinazoline). MS (+FAB) m/z = 296 (M+1); HRMS
2
2
(+FAB) calcd for C H N O: 296.15. Found: 296.15. HPLC purity of 99.9% (retention
1
6
18
5
time = 3.597 min).
4.1.12. 4-{[(2,4-diaminoquinazolin-6-yl)amino]methyl}phenol (H5)

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Yellow powder (0.36 g, 79%, Mp: 192.1-193.7 °C). TLC (CHCl /MeOH = 70/30) R =
3
f
1
0.65. IR (KBr): 3355 (OH), 3206 (NH), 1626 (CN), 1466 and 1568 (C=C Ar); H NMR
(DMSO-d ): 4.14 (d, J = 3, 2H, CH ), 5.51 (br. s, 2H, NH ), 5.70 (t, J = 3, 1H, NH), 6.73
(d, J = 8, 2H, p-hydroxybenzyl), 6.96 (br. s, 3H, NH , CH quinazoline), 7.03 (m, 2H,
6
2
2
2
1
3
quinazoline), 7.22 (d, J = 8, 2H, p-hydroxybenzyl), 9.23 (br. s, 1H, OH); C NMR
(DMSO-d ): 47.01 (CH ), 101.79 (CH, quinazoline), 110.78 (C, quinazoline), 115.45 (CH,
6
2
p-hydroxybenzyl), 124.31 (CH, quinazoline), 126.11 (CH, quinazoline), 129.48 (CH, p-
hydroxybenzyl), 130.10 (C, p-hydroxybenzyl), 144.21 (C-NH, quinazoline), 156.79 (C-O,
p-hydroxybenzyl), 159.71 (C-NH
2
), 162.49 (C-NH
2
, quinazoline); MS (+FAB) m/z = 282
(M+1); HRMS (+FAB) calcd for C15
(retention time = 3.081 min).
16 5
H N
O: 282.13. Found: 282.13. HPLC purity of 92 %
4.1.13. N-{2-(acetylamino)-6-[(ferrocenmethyl)amin]quinazolin-4-yl}acetamide (H2A)
A solution of ferrocencarboxaldehyde (0.5317 g, 2.48 mmol), compound 4 (0.5850 g, 2.25
mmol), and acetic acid (one drop) in N,N-dimethylformamide (1 mL) was stirred at 85 ºC
for 1 h. Then, the reaction mixture was cooled to 0 °C, and NaBH (0.09 g, 2.43 mmol) was
4
added. Subsequently, the reaction mixture was stirred at room temperature for 12 h. Then,
the reaction mixture was concentrated on a rotavapor apparatus under reduced pressure, and
cold water was added. The solid was filtered off, washed with cold water and dried. Yellow
powder (0.495 g, 48%, Mp: 218.3-220.9 °C). TLC (CHCl /MeOH = 80/20) R = 0.76. IR
3
f
1
(KBr): 3369 and 3244 (N-H), 1693 y 1668 (C=O), 825 (C-H ferroceno). H NMR (DMSO-
d ): 2.2 (s, 3H, CH ), 2.49 (s, 3H, CH ), 4.1 (d, J = 6, 2H, CH ), 4.13 (t, J = 2, 2H,
6
3
3
2
ferrocene), 4.22 (s, 5H, ferrocene), 4.34 (t, J = 2, 2H, ferrocene), 6.17 (t, J = 6, 1H, NH),
7.10 (d, J = 2, 1H, quinazoline), 7.4 (dd, J = 9, J = 2.4, 1H, quinazoline), 7.47 (d, J = 9, 1H,
1
3
quinazoline), 10.52 (br. s, 1H, NH amide), 10.2 (br. s, 1H, NH amide). C NMR (DMSO-

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d ): 24.38 and 25.31 (CH ,amide), 42.40 (CH ), 67.43 (CH, ferrocene), 68.43 (CH,
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ferrocene), 85.55 (C, ferrocene), 97.99 (CH, quinazoline), 115.45 (C, quinazoline), 125.86
(CH, quinazoline), 127.13 (CH, quinazoline), 145.11 (C-NH, quinazoline), 146.13 (C,
quinazoline), 149.87 (C-amide, quinazoline), 155.58 (C-NHCO, quinazoline), 169 (C=O,
amide), 171.75 (C=O, amide). EM-[FAB(+)] m/z=457. HRMS (+FAB) calcd for
C H FeN O : 457.1196. Found: 457.1170. HPLC purity of 99.9% (retention time = 3.435
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min).
4.1.14. 2-amino-6-[(4-ferrocenylmethyl)amino]quinazolin-4(3H)-one (HO4)
The catalytic reduction of 4 (1 g, 4.85 mmol) with hydrogen and Pd/C (10%) (0.1 g) was
performed on a Parr assembly at 60 psi at room temperature for 1 h. The catalyst was
filtered off, and the filtrate was concentrated on a rotavapor apparatus under reduced
pressure. A light brown compound (0.6 g) was obtained (4a, yield= 70%), and it rapidly
darkened; therefore, it was used immediately. A solution of ferrocencarboxaldehyde (0.21
g, 0.9813 mmol) and the compound obtained from the above reduction (4a) (0.15 g, 0.8514
mmol) in methanol (80 mL) was stirred at 60 ºC for 2 days. Then, the reaction mixture was
cooled to 0 ºC, and NaBH (0.064 g, 1.69 mmol) was added. Subsequently, the reaction
4
mixture was stirred at room temperature for 12 h. Then, the reaction mixture was
concentrated on a rotavapor apparatus under reduced pressure, and cold water was added.
The solid was filtered off, washed with cold water and dried. Brown powder (0.030 g, 10%,
Mp: 171.2-173.1),TLC (CHCl /MeOH = 80/20) R = 0.6. IR (KBr): 3410 (N-H), 1644
3
f
1
(C=O), 825 (C-H ferrocene). H NMR (DMSO-d ): 3.99 (d, J = 6, 2H, CH ), 4.12 (br. s,
6
2
2H, ferrocene), 4.18 (s, 5H, ferrocene), 4.26 (br. s., 2H, ferrocene), 5.64 (t, J = 6, 1H, NH),
1
3
5.9 (br. s, 2H, NH ), 7.02 (m, 3H, CH quinazoline), 10.78 (br. s, 1H, NHCO). C NMR
2
6 2
(DMSO-d ): 42.77 (CH ), 67.26 (CH, ferrocene), 68.26 (CH, ferrocene), 68.14 (CH,

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ferrocene), 86.28 (C, ferrocene), 104.28 (CH, quinazoline), 117.62 (CH, quinazoline),
122.33 (CH, quinazoline), 133.40 (C, quinazoline), 143.99 (C, quinazoline), 151.62 (C-
2 4
NH ), 166.48 (C=O). EM-[FAB(+)] m/z=375. HRMS (+FAB) calcd for C19H19FeN O:
375.0903. Found: 375.0865. HPLC purity of 98 % (retention time = 4.364 min).
4.1.15. 4-amino-6-[(4-ferrocenylmethyl)amino]quinazolin-2(1H)-one (HO2)
The catalytic reduction of 5 (1 g, 4.85 mmol) with hydrogen and Pd/C (10%) (0.1 g) was
performed on a Parr assembly at 60 psi at room temperature for 1 h. The catalyst was then
filtered, and the filtrate was concentrated on a rotavapor apparatus under reduced pressure.
A light brown compound (0.3 g) was obtained (5a, 35%), and it rapidly darkened;
therefore, it was used immediately. A solution of the appropriate aldehyde (0.21 g, 0.9813
mmol) and the compound obtained from the above reduction (5a) (0.15 g, 0.8514 mmol) in
methanol (80 mL) was stirred at 60 ºC for 2 days. Then, the reaction mixture was cooled to
0 °C, and NaBH (0.09 g, 1.5 eq.) was added. Subsequently, the reaction mixture was
4
stirred at room temperature for 12 h. Then, the reaction mixture was concentrated on a
rotavapor apparatus under reduced pressure, and cold water was added. The solid was
filtered off, washed with cold water and dried. Brown powder (0.040 g, 13%, Mp: 186.3-
187.7 °C), TLC (CHCl
3
/MeOH = 80/20) R
f
= 0.27. IR (KBr): 3366 and 3366 (N-H), 1628
): 3.97 (d, J = 5, 2H, CH ), 4.13 (br. s,
1
(C=O), 826 (C-H ferrocene). H NMR (DMSO-d
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2H, ferrocene), 4.23 (s, 5H, ferrocene), 4.34 (br. s, 2H, ferrocene), 5.47 (t, J = 5, 1H, NH),
6.94 (d, J = 7, 1H, CH, quinazoline), 7.01 (d, J = 7, 1H, CH, quinazoline), 7.07 (s, 1H, CH,
1
3
quinazoline), 7.73 (br. s, 2H, NH ), 10.44 (br. s, 1H, NHCO). C NMR (DMSO-d ): 43.28
2
6
(CH ), 67.85 (CH, ferrocene), 68.88 (CH, ferrocene), 69.66 (CH, ferrocene), 86.37 (C,
2
ferrocene), 103.02 (CH, quinazoline), 116.08 (CH, quinazoline), 123.18 (CH, quinazoline),
2
144.06 (C, quinazoline), 156.37 (C=O), 160.89 (C-NH ). EM-[FAB(+)] m/z=375. HRMS

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(+FAB) calcd for C H FeN O: 375.0903. Found: 375.0865. HPLC purity of 99.9%
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(retention time = 3.604 min).
4.1.16. N-ferrocenylmethylaniline (FBC)
A solution of ferrocencarboxaldehyde (0.15 g, 0.7 mmol) and aniline (0.072 g, 0.77 mmol)
in methanol(30 mL) was stirred at 60 ºC during 2 days. Then, the reaction mixture was
cooled to 0 °C, and NaBH (0.080 g, 2.11 mmol) was added. Subsequently, the reaction
4
mixture was stirred at room temperature for 12 h. Then, the reaction mixture was
concentrated on a rotavapor apparatus under reduced pressure, and cold water was added.
The solid was filtered off, washed with cold water and dried. Brown powder (0.089 g, 40%,
Mp: 84.9-85.3 °C). TLC (hexane/ethyl acetate = 80/20) R
f
= 0.67. IR (KBr): 3369 and 3244
): 3.95 (d, J = 3,
1
(N-H), 1693 and 1668 (C=O), 825 (C-H ferrocene). H NMR (DMSO-d
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2H, CH ), 4.09 (br. s, 2H, ferrocene), 4.18 (s, 5H, ferrocene), 4.26 (br. s, 2H, ferrocene),
2
5.60 (br. s, 1H, NH), 6.51 (t, J = 6, 1H, aromatic), 6.64 (d, J = 9, 2H, aromatic), 7.47 (t, J =
1
3
6, 2H, aromatic). C NMR (DMSO-d ): 42.23 (CH ), 67.24 (CH, ferrocene), 68.40 (CH,
6
2
ferrocene), 86.47 (C, ferrocene), 112.12 (CH, meta-aniline), 115.59 (CH, para-aniline),
128.77 (ortho-aniline), 148.68 (C-NH, aniline). EM-[FAB(+)] m/z=291. HRMS (+FAB)
:
calcd for C17H18FeN 291.0705. Found: 291.0690.
4.2. X-ray crystallographic
The H2 crystal obtained from ethanol was mounted on a glass fiber and was studied with an
Oxford Diffraction Gemini "A" diffractometer with a CCD area detector (λMoKα = 0.71073
Å, monochromator: graphite) source equipped with a sealed tube X-ray source at 130 K.
The unit cell constants were determined with a set of 15/3 narrow frame/run (1° in ω)
scans. A data set consisted of 303 frames of intensity data collected with a frame width of

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1° in ω, a counting time of 25 s/frame, and a crystal-to-detector distance of 55.00 mm. The
double pass method of scanning was used to exclude any noise. The collected frames were
integrated using an orientation matrix determined from the narrow frame scans. The
CrysAlisPro and CrysAlis RED software packages were used for data collection and data
integration [25]. Analysis of the integrated data did not show any decay. The final cell
constants were determined by a global refinement of 6196 reflections (θ < 26.06 °). The
collected data were corrected for absorbance using an analytical numerical absorption
correction, which employs a multifaceted crystal model based on expressions of the Laue
symmetry using equivalent reflections [26]. Structure solution and refinement were
performed with the following programs: the SHELXL97 program for molecular graphics,
the ORTEP-3 program for Windows and the WinGX software for the preparation of
materials for publication [27,28,29].
2
2 2
The full-matrix least-squares refinement was conducted by minimizing (Fo - Fc ) . All of
the non-hydrogen atoms were refined anisotropically. The H atoms of the methanol solvent
(H-O) and the amine group (H-N) were located in a difference map and refined
isotropically with a U (H) value of 1.5 and 1.2 U for (O) and (N), respectively. The H
iso
eq
atoms attached to C atoms were placed in geometrically idealized positions and refined as
riding on their parent atoms, with C—H = 0.95 – 0.99 Å with Uiso (H) = 1.2Ueq(C) for
methylene and aromatic groups, and Uiso (H) = 1.5 Ueq(C) for the methyl group. The crystal
data and experimental details of the structure determinations are listed in Table 5.
4.3. Modeling studies
To construct DHFR Leishmania major, the Trypanosoma cruzi enzyme (ID-PDB: 2H2Q
Key) was used as the template. First, the amino acids were corrected with the Modeler

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program [30]. Classical MD simulations were performed using the NAMD2.6 program
using the CHARMM27 force field, and water molecules were added using the VMD
program [31,32]. The structure was neutralized with 4 sodium ions after being immersed in
a TIP3P water box containing 9249 water molecules. The equilibration protocol began with
1500 minimization steps followed by 30 ps of MD at 310 K with fixed protein atoms. Then,
the entire system was minimized for 1500 steps (at 0 K) and then heated gradually from 10
to 310 K by temperature reassignment during the first 60 ps of 100 ps equilibration
dynamics without restraints. The final step was a 30 ps NTP dynamics using the Nose-
Hoover Langevin piston pressure control 28 at 310 K and 1.01325 bar for density (volume)
fitting. From this point, the simulation was continued in the NTV ensemble during 5 ns.
The periodic boundary conditions and the particle-mesh Ewald method were applied for a
complete electrostatics calculation. The dielectric water constant was used, and the
temperature was maintained at 310 K using Langevin dynamics. The structure was allowed
to converge, which was reached at 10 ns. A snapshot was obtained at this time for the
computational docking.
The docking was also performed for PTR from the structure for this enzyme obtained after
crystallization with triaminoquinazoline (ID-PDB: 1WOC), which also validated the study.
The program AutoDock4 was used [33]. For the docking of both proteins, the water
molecules were removed, and the active site was defined as all of the residues within a grid
of 60 Aº x 60 Aº x 60 Aº centered in the active site, with an initial population of 100
7
randomly placed individuals and a maximum number of 1.0 X 10 energy evaluations. The
compounds for docking were drawn in Gauss view 3.0. Before docking, the compounds
were subjected to energy minimization using the hybrid functional B3LYP with a 6, 31
G(d,p) basis set. The crystallographic structure was taken as the starting point for