In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of New Arylamine Mannich Base-Type Derivatives

Article abstract of DOI:10.1021/acs.jmedchem.6b00784

Chagas disease is a neglected tropical disease with 6-7 million people infected worldwide, and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity, and a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model results show that compounds 3, 4, 7, and 10 induced a remarkable decrease in parasitemia levels in acute phase and the parasitemia reactivation following immunosuppression, and curative rates were higher than with benznidazole. These high antiparasitic activities encourage us to propose these compounds as promising molecules for developing an easy to synthesize anti-Chagas agent.

Full text of DOI:10.1021/acs.jmedchem.6b00784

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Article
In vitro and in vivo anti-Trypanosoma cruzi activity
of new arylamine Mannich base-type derivatives
Elsa Moreno-Viguri, Carmen María Jiménez Montes, Rubén Martín Escolano, Mery Jhenny Santivañez
Veliz, Álvaro Martín-Montes, Amaya Azqueta, Marina Jimenez Lopez, Salvador Zamora-Ledesma, Nuria
Cirauqui Díaz, Adela Lopez de Cerain, Clotilde Marín, Manuel Sánchez-Moreno, and Silvia Pérez-Silanes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00784 • Publication Date (Web): 23 Nov 2016
Downloaded from http://pubs.acs.org on November 23, 2016
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Journal of Medicinal Chemistry
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In vitro and in vivo antiꢀTrypanosoma cruzi activity of
new arylamine Mannich baseꢀtype derivatives.
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Elsa MorenoꢀViguri, Carmen JiménezꢀMontes Rubén MartínꢀEscolano, Mery SantivañezꢀVeliz,
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¥, ǂ
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Alvaro MartinꢀMontes, Amaya Azqueta, Marina JimenezꢀLopez, Salvador Zamora Ledesma, Nuria
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¥,ǂ
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§,*
†,*
Cirauqui, Adela López de Ceráin, Clotilde Marín, Manuel SánchezꢀMoreno, Silvia PérezꢀSilanes
†
Department of Organic and Pharmaceutical Chemistry, Institute of Tropical Health, Universidad de
¥
Navarra, Pamplona, Spain. Department of Pharmacology and Toxicology, Universidad de Navarra,
ǂ
Pamplona, Spain. IdiSNA, Navarra Institute for Health Research, Recinto de Complejo Hospitalario de
§
Navarra, Pamplona, Spain. Departamento de Parasitología, Instituto de Investigación Biosanitaria
Ɨ
(
ibs.GRANADA), Hospitales Universitarios De Granada/Universidad de Granada, Granada, Spain.
Department of Pharmaceutical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
ABSTRACT. Chagas disease is a neglected tropical disease with 6ꢀ7 million people infected worldwide
and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to
discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of
identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity and
with a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of
high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model
results show that compounds 3, 4, 7 and 10 induced a remarkable decrease in parasitemia levels in acute
phase and the parasitemia reactivation following immunosuppression, and curative rates were higher
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than with benznidazole. These high antiꢀparasitic activities encourage us to propose these compounds as
promising molecules for developing an easy to synthesize antiꢀChagas agent.
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INTRODUCTION
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Chagas disease (CD), is caused by the protozoa Trypanosoma cruzi (T. cruzi) which is naturally
transmitted by hematophagous insects, presents risk factors strongly linked to low socioeconomic
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, 2
factors and it is classified as a neglected tropical disease according to WHO. CD is one of the most
important medical problems in rural areas of the 21 endemic Latin America countries. Moreover, in the
past decades migration and travelling have extended the spread of CD to other continents including
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North America, Europe and parts of the Western Pacific, where there is now a significant number of
individuals with this disease. The worldwide incidence of CD has decreased thanks to the control
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programs; however, an estimated 6ꢀ7 million persons are infected worldwide. Approximately 20% of
Latin American countries are endemic for this disease and up to 30ꢀ40% of patients will suffer chronic
form of the disease in their lifetimes–i.e. important cardiac or digestive problems.
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Currently, the two drugs recommended for CD are benznidazole (BZN) and nifurtimox (NFX) In
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general terms, BZN is the first line drug in most endemic countries because it is tolerated better but
some of its secondary effects include dermatitis or neuropathy. Both drugs reduce parasitism during the
acute and early CD stage, but drug efficacy during the chronic phase is limited. In addition, the efficacy
of BNZ and NFX could change according to the geographical area, as consequence of a different
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susceptibility of the drugs by the different strains of T. cruzi. This background, including side effects
and therapeutic deficiencies of these drugs, justifies the urgent need to continue researching in order to
discover novel therapeutic alternatives. The ideal drug defined by its Target Product Profile (TPP) is
outlined in Table 1. Briefly, it should present an efficacy at least equal to BZN and with a better safety
profile. An oral drug in onceꢀaꢀday inꢀhouse treatment for less than 30 days is desired and the new drug
11ꢀ13
should be active against most parasite species.
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Journal of Medicinal Chemistry
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Table 1. Proposed TPP for Chagas disease modified from DNDi.
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Drug repositioning has emerged as one of the most fruitful strategies for improving and accelerating
the drug development process. This strategy has become one of the most powerful tools for identifying
compounds for the treatment of a disease different from the one they were originally designed for, used
not only in the academic field but also in pharmaceutical companies. Different methods are considered
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under this strategy but 28 out of the 50 small molecules approved by the FDA between 1999 and 2008
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were discovered by phenotypic based drug design.
In this context we decided to screen our inꢀhouse
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chemical laboratory library with the purpose of searching drugs against of CD.
We found some
arylamine Mannich base derivatives tested before for their in vitro affinity at the 5ꢀHT transporter and
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HT receptors or 5ꢀHT receptors as potential antidepressants and against Plasmodium falciparum
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as a potential antimalarials and in every case they were not active. However risk of potential offꢀtarget
activities derived from a possible CSN activity is unlikely.
Herein we present the synthesis of arylaminoketoneꢀtype compounds and their in vitro evaluation
against T. cruzi. Furthermore we show that the preliminary genotoxicity screening test revealed that this
family of compounds is not genotoxic with or without metabolic activation, thereby providing the ideal
chemical space for the development of new, potent and safe agents for the treatment of CD. Highꢀ
selectivity indexes and the genotoxicity screening have been the established cutꢀoff to move into in vivo
assays in BALB/c mice, where fresh blood examination was used to quantify the parasitemia. Also a
cure was assayed by PCR, as was the reactivating of parasitemia in blood following
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immunosuppression. The mechanism of action has been explored at metabolic levels by H Magnetic
Nuclear Resonance, and the study has been completed by testing their activity as potential Feꢀ
Superoxide dismutase inhibitors. Using computational approaches, we suggest a binding mode for the
compounds to the enzyme.
RESULTS AND DISCUSSION
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Chemistry. It is essential to identify the purpose of the synthesis of new small molecules. During the
drug discovery process, mainly when facing NTD, the synthetic routes should allow the preparation of
an important amount of different small molecules with a variety of substituents in order to explore and
modify polarity, solubility, volume, rigidity and some other parameters that could influence activity and
toxicity. Cost and commercial availability of chemicals should also be taken into consideration as well
as the solvent and waste volumes. With these ideas in mind, the retrosynthetic analysis of the target
compounds (1-20) suggested methylketones and arylamines as the appropriate building blocks (Table
2). Compounds could be prepared in one step synthetic route with exception of compounds 10 and 13.
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In this case, both arylamines had to be prepared as previously reported.
The general method shown
in Figure 1 was used to prepare the compounds presented in this work. The desired compounds were
obtained condensing the adequate methylketone (commercially available) with the appropriate
arylamine via Mannich reaction in acidic medium and using 1,3ꢀdioxolane as the solvent and source of
formaldehyde.
Table 2. Relationship of synthesized compounds.
Figure 1. General method of synthesis of new arylaminoketone derivatives.
The results of the in silico study of some relevant molecular properties related to the drugꢀlikeness of
the compounds are shown in Table 3. It can be observed that none of them have a molecular weight
higher than 500 g/mol, or an estimated partition coefficient (logP) higher than 5. Compounds 3, 6, 10,
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5 and 19 present a number of hydrogenꢀbond acceptors higher than 5 but lower than 10, while the
number of donors is lower than 5 for all compounds. Therefore, all compounds fulfill the Lipinski “rule
of five”. Finally, together with the acceptable partition coefficient values, the Polar Surface Area (PSA)
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of the compounds is lower than 140 Å , and the aqueous solubility (logS) is similar or higher than ꢀ4,
which anticipates good oral bioavailability.
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Table 3. Molecular properties of the new compounds calculated with the DataWarrior program.
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Journal of Medicinal Chemistry
In vitro Trypanocidal Evaluation. Information was gathered by the in vitro evaluation of activity of
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compounds 1-20 with respect to intraꢀ and extracellular forms of T. cruzi SN3, Arequipa and Tulahuen
strains obtained as detailed in the experimental section. Assays have been carried out in strains from
different locations, hosts and tropisms, in order to determine if there are any differences in drug
performance.
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Extracellular form (epimastigote) is the most commonly used owing to its easy culture in laboratory,
but tests obtained on intracellular amastigotes (responsible for the chronic phase of CD) and blood
trypomastigotes are more indicative due to these form of the parasite is the developed form in vertebrate
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hosts . According to some authors, for deeming a compound as potential antichagasic agent, it has to
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meet certain criteria . In the first place, the IC value has to be around 10 µM and the in second place
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the SI has to be 50 times higher than that of the reference drug. IC values for the 3 studied strains in
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both their extracellular and intracellular forms are shown in Table 4.
Table 4 shows the IC values found at 72 h exposure using concentrations of 1ꢀ100 µM on assaying
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compounds 1ꢀ20 vs. 3 strains studied and the 3 forms of parasite cited above. The IC values for the
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drug BZN (reference) served for comparisons. Ten compounds (3, 4, 7, 10, 11, 13-15, 18 and 19) were
shown to fulfill this first requisite. It was shown that the compounds meet this first requirement. Those
compounds are the most active of the tested compounds for the three parasite forms assayed with an
IC value lower than the one corresponding to the reference drug.
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Table 4. In vitro activity and toxicity for the 20 arylaminoketone derivatives on extraꢀ and
intracellular forms of Trypanosoma cruzi strains.
On the other hand, the cytotoxicity of compounds 1-20 was tested against Vero cells (Table 5) and a
lesser cytotoxicity than the one of the reference drug was observed. Interestingly, toxicity data against
Vero cells after 72 h of culture showed that all derivatives assayed were considerably less cytotoxic than
drug reference BZN. Cytotoxicity IC values ranged from 30.1 µM to 2846.3 µM in sharp contrast with
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that obtained for BZN (13.6 µM). Only compounds 11 and 18 were more cytotoxic than BZN.
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Table 5. Selectivity index and in vitro toxicity for the 20 derivatives of arylaminoketone intra and
extracellular of T .cruzi strains.
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In accordance with the above in vitro data, Table 5 presents the most notable selectivity index (SI)
values, indicating in brackets how often the SI of each compound surpassed the SI of BNZ. This Table
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shows that excellent results were found for compounds 3, 4, 7 and 10 in all the parasite forms tested.
Those drugs were chosen for further in vitro and in vivo assays and the possible mechanisms of action.
In this case experiments were carried out only in the Arequipa strain as there are no significant
differences between the performances of the compounds on the other studied strains.
From a structural point of view, it can be said that the benzothiophene derivatives are less cytotoxic
than the rest of the derivatives. All of the benzothiophene derivatives (1-10) present IC in Vero cells
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higher than 70 µM; moreover, the selected compounds (3, 4, 7, 10) present IC higher than 700 µM in
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contrast with BZN (13.6 µM).
In vitro genotoxicity screening test. The potential genotoxicity of the compounds (1-5, 7, 10, 14, 16-
18) was screened using the SOS/umu test. The concentrations tested for each compounds were
previously determined in a toxicity study in the test system. The SOS/umu screening test revealed that
none of the tested compounds were genotoxic with or without metabolic activation. Meanwhile, both
NFX and BZN were also tested and both of them were genotoxic in the tested conditions. The main
purpose of including the SOS/umu test in the preliminary assays is to study the genotoxicity capacity of
the most active compounds in vitro so that only compounds that are not genotoxic will move on to
further studies. This test is convenient as a screening assay because it is a fast, simple and efficient
method for testing the genotoxicity of a large number of samples. For these reasons, compounds 3, 4
and 7, that had met the criteria for being moved on to in vivo models, were included in the SOS/umu
test. Moreover, compounds 1, 2, 5, 10, 14 and 16-18 had been previously assayed in the genotoxicity
screening test. These compounds came from our inꢀhouse chemical library and their in vitro
trypanocidal activity had been studied, showing interesting results. Therefore and before synthesizing
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Journal of Medicinal Chemistry
new related derivatives, we decided to explore their genotoxicity capacity to be sure that these new
derivatives could be promising not only because of their potency but also because of their safeness.
Both the trypanocidal activity and genotoxicity screening test mentioned above supported the idea of
carrying out more detailed in vitro and in vivo assays with benzothiophenes 3, 4, 7 and 10 in order to get
more information about their mode of action and their apparent advantages with respect to drug
reference BZN.
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Infectivity assay. For fuller knowledge concerning the trypanocidal action of four compounds (3, 4, 7
and 10), a subsequent determination was made of their impact on the intracellular replication and
infectivity of amastigotes of T. cruzi (Figure 2). A sharp decline in the infection rate of the host cell
occurred when the benzothiophene derivatives 3, 4, 7 and 10 were added to the infected Vero cells.
Also, the four compounds proved to have greater effectiveness than did the reference drug, this reducing
the infective capacity by 22% on day 10. (Figure 2A). Measuring of the mean number of amastigotes
per infected Vero cell (Figure 2B) led to similar results: at the end of the treatment, the benzothiophene
derivatives had decreased the number of amastigotes present in the cells by 45 to 74%, whereas BZN
allowed only a poor reduction of 30%. With regard to the number of trypomastigote forms released in
the medium (Figure 2C), on the whole, inhibition values were less significant. Even so, all the
compounds showed a reduction by 47 to 68% and almost doubled the inhibitory effect of BZN (20%),
which clearly remained less effective. Ultimately, infectivity tests confirmed the in vitro antiparasitic
activity shown in Table 4 and 5 for the four compounds, and also their higher efficacy with respect to
the reference drug.
Figure 2. Reduction of the infection of Trypanosoma cruzi in Vero cells treated with (ꢀ■ꢀ) BZN, (ꢀꢁꢀ)
3, (ꢀ□ꢀ) 4, (ꢀ●ꢀ) 7 and (ꢀ▲ꢀ) 10. (A) Rate of infection; (B) mean number of amastigotes per infected
Vero cells; (C) number of trypomastigotes in the culture medium. Measured at IC .Values are the
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means of the three separate experiments.
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In Vivo Anti-T. cruzi Activity in BALB/c Albino Female Mice. Since compounds 3, 4, 7 and 10
exhibited notable in vitro SI values in comparison to BZN (the reference drug) and the screening test
showed no genotoxicity, those compounds were employed in more in vivo studies using BALB/c mice.
Their antꢀtrypanosomal action prior to the acute CD phase to 40 days postꢀinfection (pi) was studied
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first. Infection was imposed by intraperitoneal inoculation of metacyclic blood trypomastigotes (5x10 )
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using Arequipa T. cruzi which results in lower mortality than do other approaches , and compound
doses of 50 and 100 mg/kg body weight were tested by oral administration on alternating days (7−15
days pi). The parasites were counted in 5 µL blood samples taken from the mandibular vein. Notably,
no mice died after receiving compounds 3, 4, 7 and 10. Figure 3 shows the effect caused by the
benzothiophene derivatives on the parasitemia levels in blood during the acute phase of CD (day 0ꢀ40
pi). (In Figure 3 only the results for the experiments performed with 100 mg/kg body weight dose are
shown; not showing results for experiments with 50 mg/kg body weight dose, due to the evident
majority effect of this first dose). A reduction of parasitemia in mice treated with the tested compounds
was evident as soon as the very beginning of the treatment (day 7 pi), and it was maintained until the
end of the acute phase (day 40 pi), and not only until the end of period of treatment (day 15 pi). In
Figure 3 shows that mice treated with compounds 4 and 7, parasitemia was undetected in day 25 pi.
This appears to indicate that parasites were eliminated, or their number depleted to nonꢀdetectable levels
with a 100 dilution factor for counting in optic microscope, or maybe they migrated to organs. In the
three detailed cases, they could no longer be detected in blood.
Figure 3. Parasitemia in mice with acute CD. In (ꢀ♦ꢀ) control, (ꢀ▲ꢀ) BZN, (ꢀ●ꢀ) compound 3, (ꢀ■ꢀ)
compound 4, (ꢀꢁꢀ) compound 7 and (ꢀ□ꢀ) compound 10. In all cases, compounds were orally
administered using, for each compound, 100 mg/kg of body mass. Values constitute means of six mice
±
standard deviation. Treatment days are represented in grey.
Performance of drugs during the chronic phase (where currently used drugs are not as effective as
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they should) was tested.
The parasitemia remaining in blood at day 90 pi is practically undetectable
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due to the parasite, in the amastigote form, is nested inside target organs. This involves that
immunocompromised individuals cured with apparent success show high reactivation ability of the
parasitemia. Furthermore, apparently cured patients submitted to liver or kidney transplantation, treated
with anticancer chemotherapy or diagnosed with AIDS, showed clinically highly aggressive CD
reactivation. As a means of providing information on this question, mice that were treated as indicated
above, at 90 days pi received three successive immunosuppression cycles using cyclophosphamide
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28
monohydrate, as described above. The aim was to reactivate the parasitemia, previously under the
control of the immunological system of the mice. The tridimensional graph of Figure 4 shows the
reactivation caused by compounds due to this reactivation is proportional to the survival rate of the
parasites. Parasitemia reactivation reached 65% with respect to the acute phase in the control group of
infected and untreated mice, whereas it decreased to 0% in the treated mice with 100 mg/kg body
weight group revealing a significant effect on the chronic course of infection. Moreover, it can be
observed that parasitemia reactivation decreased to 0% in mice treated with compound 7 not only with
100 mg/kg but also with 50 mg/kg.
Figure 4. Immunosuppression in vivo assay for mice untreated and treated with 100 mg/kg of body
mass of BZN, 3, 4, 7 and 10. Figure shows the reactivation of blood parasitemia after the
immunosuppression cycles by fresh blood containing parasitemia to the peak day during acute phase.
ELISA (enzymeꢀlinked immunosorbent assay) was employed to detect the total IgꢀG levels using Feꢀ
SOD isolated by us as antigen source. An assessment of the immune status of the mice was made
possible by the detection of total IgG, since this reflects the protection level that should be ascribed to
the compounds tested, in combination with the animal’s natural protection. Results from the
experiments using ELISA were verified using the parasitemia assay conducted as described above
(Figure 5). The ELISA test indicated that the mice receiving compounds 3, 4, 7, and 10 maintained
steady total IgG levels, as chronically infected mice also did; levels proved lower than in control after
immunosuppression, whereas control levels rose following parasitemia reactivation.
Figure 5. Shows differences in the IgG levels measured by ELISA at different day postꢀinfection.
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Finally the curative effect of the compounds in the extracted target organs was evaluated. Figure 6
only shows the PCR results of organs in the different groups of mice treated with 100 mg/kg body
weight, because this dosage had better results. The controls showed the ubiquitous presence of the
parasite in the 8 target organs. In addition, the figure shows the group of mice treated with BZN, in
which 6 out of 8 target organs were infected. In contrast, the group of mice treated with the compounds
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, 4, 7 and 10 showed less infected target organs than reference drug BZN, especially compounds 4 and
. Every organ of mice treated with drug 4 was not infected. In the case of compound 7, target organs
were also not infected, while the two remaining target organs presented 83% less parasites than control.
Cure assessment in chronic T. cruzi infection is controversial, mainly because of the lack of reliable
29
tests to ensure parasite elimination. We should mention that identifying by PCR whether experimental
animals have been cured is a critical question. This technique is able to detect a single parasite in 5 ml
of blood and the main utility is to confirm failure to cure, because even consistently negative results
3
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cannot prove that tissue parasites have been completely eliminated.
Bioluminiscence imaging
models techniques are being used currently to determinate if experimental animals have been cured.
However, we can state that there are many evidences of cure (or, at least, a considerable reduction of the
number of parasites), so the negative results of 5 independent samples by PCR and the absence of
reactivation in blood after immunosuppression (double checking for demonstrating cure).
Immunosupresion is the formula to demonstrate curation in all animal testing models, either
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bioluminescenceꢀbased tests or the ones that employ PCR as double checking ones.
The detection methodology has been improved significantly by specific PCR of genes and sequences
with the development of the technology TaqMan, in the use of fluorogenic probes and the measurement
of realꢀtime amplification reaction used in the detection of sequences of constant regions and variables
of minicircles of kinetoplast DNA. These results are quite useful to measure the parasitic load in chronic
patients earlier and later after the treatment, and for the future establishment of reliable criteria of
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patient healing submitted to therapy .
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Figure 6. Polymerase chain reaction (PCR) analysis of the 8 target organs on day 120 after infection.
Lanes: M, base pair marker; 1: PCR negative control; 2: PCR positive control; 3: PCR adipose tissue; 4:
PCR brain tissue; 5: PCR heart tissue; 6: PCR esophagus tissue; 7: PCR stomach tissue; 8: PCR bone
marrow tissue; 9: PCR muscle tissue; 10: PCR lung tissue. * This means that 1/6 of the corresponding
organ PCR products showed 300 bp band on electrophoresis.
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Finally, Table 6 shows weight percentage of spleens from different groups of mice in both doses of
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0 and 100 mg/kg body weight. Control group showed more weight percentage when compared with
both groups of mice treated with 50 and 100 mg/kg body weight, which means that all compounds
reduced splenomegaly and, therefore, infection rates. Moreover, groups of mice treated with 100 mg/kg
body weight showed less weight percentage compared with the same groups of mice treated with a
dosage of 50 mg/kg body weight. Compounds 3 and 7 showed lower splenomegaly.
Table 6. Weight percent of spleens of different groups of mice.
Inhibition of the T. cruzi Fe-SOD Enzyme. The most studied therapeutic targets are enzymes, one
of them is the trypanosomatid FeꢀSOD, which presents structural and biochemical differences with
respect to their human homologues. This enzyme plays a crucial role allowing the cell to avoid damage
34, 35
caused by the oxidative burst.
Taking this in account, we have given importance to iron superoxide
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dismutase (FeꢀSOD), a trypanosomatid exclusive enzyme that is absent in other eukaryotic cells.
Alternatively, trypanosomatids have a shared redox system in which trypanothione and glutathione team
up to protect the parasite from reactive oxygen species (ROS). Trypanothione [bis(glutathionyl)
spermidine] is synthesized from spermidine, the major polyamine in these parasites. Therefore,
alterations in the biosynthesis of this amine can interfere in the defense mechanisms of the parasites
against oxidative stress. Also noteworthy is the trypanothione synthase enzymes (trys) and
trypanothione reductase (tryr), involved in the biosynthetic process trypanothione, which is the main
defense system against oxidative trypanosomatids damage. The enzyme glutathione peroxidase
3
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constitutes the third block of main antioxidant enzymes of the parasite.
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As mentioned above, the antiparasitic activity of other benzothiophene derivatives partly accounted
for by their inhibition of parasitic SOD. This could view as a possible target for designing new
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molecules capable of acting on metal ion carried within their structure.
As a means of gaining more evidence in this regard, the effects exerted on T. cruzi Arequipa strain Feꢀ
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SOD by compounds 3, 4, 7 and 10 were assessed at concentrations of between 1 µM to 100 µM.
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Figure 7A shows the data on inhibition, and the related IC values appear in brackets. Figure 7B, for
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comparative purposes, presents the action of these same compounds regarding CuZnꢀSOD from human
erythrocytes. Reflecting that the four compounds acted against FeꢀSOD. Nevertheless Figure 7A
indicates that the most trypanocidal compounds 3, 7 and 10 highly inhibited the enzyme activity, while
the inhibitory activity of compound 4 was clearly lower. The graph shows that benzothiophene 3
reached 100% inhibition of FeꢀSOD at a 50 µM concentration and with an IC of 7.8 µM and that
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compounds 7 and 10 almost reached 100% inhibition at a slightly lower concentration, with IC values
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of 16.8 and 71.1 µM respectively. As the assay against FeSOD shows, compound 4 is the least efficient,
as shown in its in vivo effects. However, it had curative effect in chronic phase. Some other mechanisms
of action could be considered and the chance of a multi target compound should not be discarded.
Figure 7. A) Inhibition in vitro (%) of FeꢀSOD from epimastigotes of Trypanosoma cruzi for
compounds (activity 25.4 ± 3.1 U/mg). B) Inhibition in vitro (%) of CuZnꢀSOD from human
erythrocytes for compounds (activity 23.36± 2.5 U/mg). Activity differences in the control homogenate
vs. the sample incubated using compounds were identified by the NewmanꢀKeuls test. Values are the
average of three separate rate determinations.
Interestingly, the effect of compound 3 on human CuZnꢀSOD was substantially similar to the case of
FeꢀSOD (Figure 7A), since inhibitory activity scarcely reached 100% at the 50 µM concentration. The
other three compounds (4, 7 and 10) did not show inhibition to CuZnꢀSOD, not even at 100 µM. These
compounds are remarkable selective inhibitors of FeꢀSOD.
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Metabolite excretion. Trypanosomatids proved incapable of completely degrade glucose to CO , thus
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excreting into the medium part of their hexose skeleton as partly oxidized fragments. The percentage
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and nature of this excretion depended on the pathway that was used in glucose metabolism. In T.
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cruzi, the catabolism product are mainly acetate, succinate, Lꢀalanine and Dꢀlactate.
In order to obtain some information about the effects of 3, 4, 7 and 10 on the glucose metabolism of
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the parasite, we registered the H NMR spectrum of T. cruzi epimastigotes treated with the test
compounds (Supl information); the final excretion products were qualitatively and quantitatively
identified. Figure 8 shows the results obtained and the comparison with control epimastigotes.
Excretion of some metabolites, were disturbed in the treatment with the compounds, with the succinate
being the most affected, and showing an increase of 199% for compound 10, 142% for compound 3, 22
% for 4 and an increase of 109% for compound 7; lesser alterations were observed in the other
metabolites.
All of these data could be interpreted on the basis of a change in the succinate pathways occurring in
the presence of the compounds under investigation. Otherwise, it is interesting to note that the increase
in succinate with these compounds indicates catabolic changes that could be related to mitochondria
malfunction, due to the redox stress produced by inhibition of the mitochondrionꢀresident FeꢀSOD
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enzyme.
Figure 8. Percentages of variation among peaks of catabolites excreted by epimastigotes of
Trypanosoma cruzi exposed to compounds 3, 4, 7 and 10 at their IC25 in comparison to a control
incubated 96 h.
Mutagenicity studies. Ames test
Taking into account the great potential of compounds 4 and 7, we decided to include them in the
Ames test to explore their mutagenicity capacity before considering them for further preclinical
development. The results obtained from the Ames test for compound 4 and 7 are shown in Tables 7 and
8, respectively. The evaluated doses of the compound were determined in a previous solubility study on
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the test system and 2.25 µg/plate and 0.75 µg/plate were established as the highest concentrations to be
tested in the experimental conditions for compounds 4 and 7, respectively. No signs of cytotoxicity or
precipitation were observed during the experiment. Both compounds were not mutagenic in any of the
two tested conditions, as no evidence of doseꢀrelated or more than twoꢀfold increase in the mean
number or revertants was observed with or without metabolic activation. Therefore, compounds 4 and 7
could be pointed as interesting candidates for the development of an antiꢀChagas agent
Table 7. Results of the Ames test of compound 4.
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Table 8. Results of the Ames test of compound 7.
Docking study of benzothiophenes 3, 4, 7 and 10 in the T. cruzi Fe-SOD and the human CuZn-
SOD enzymes.
In order to gain insight into the binding mode of the new benzothiophene derivatives in the FeꢀSOD
enzyme, docking studies were carried out using the crystal structure of both mitochondrial (PDB id
4
DVH) and cytosolic (PDB id 2GPC) FeꢀSOD trypanothione cruzi proteins. The results showed that the
compounds bind in a deep funnel at the top of the dimer interface, which gives access to the active site
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(
metal site) (Figure 9a). This cavity has already been suggested as the inhibitor binding site. Even
when the binding mode of the compounds in both enzymes was similar, the affinity was higher for the
mitochondrial protein. However, we believe that this is merely an artifact produced by the different
orientation of Lys36 (Lys35 in the mitochondrial enzyme), which lays inside the active site in the PDB
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GPC and outside of it in PDB 4DVH. Based on our studies, we suggest the mitochondrial protein with
PDB code 4DVH as best suited for docking studies on FeꢀSOD trypanothione cruzi enzyme. The
compounds bind in an aromatic pocket formed by aminoacids Try32, His28, Trp161 and Phe119. This
hydrophobic pocket is flanked by polar residues at both sides: Glu162, Arg173, Val116 and Asn117 at
one side, and Lys35 and Gln68 at the other. Just like the fluoro substituents of compounds 4 and 7, the
nitro group of compounds 3 and 10 perform electrostatic interactions with Lys35, Gln68 and Asn117.
As expected, the ligand does not perform interactions with the metal, which binds well inside the active
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Journal of Medicinal Chemistry
site. However, it could act by inhibiting substrate access to the active site, or even by disrupting
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dimerization, as observed for other FeꢀSOD inhibitors.
Docking studies of the four compounds on the human CuZnꢀSOD were also carried out in order to
understand the reason for compound 3 presenting no selectivity, contrary to the other three compounds.
Previous crystal structures show that organic fragments bind to this enzyme in different regions of its
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surface.
Our blind docking results coincide with this observation, no single binding site was found.
However, for all four compounds, one of the two best scored poses presented the inhibitor bound on the
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dimer interface. This interface has already been suggested as the inhibitors binding site.
Therefore,
we decided to perform another docking run defining this one as binding region. The results suggest a
binding score around 10 times higher for compound 3 when compared with the other three compounds,
coinciding with our experimental findings. Our docking results show compound 3 performing
electrostatic interactions between the sulfur atom and Asp53, and between the nitro group and Lys9.
Both interactions are absent in the other compounds (Figure 9b).
Figure 9. Docking results for the binding of benzothiophenes 3 (upꢀleft), 4 (downꢀleft), 7 (upꢀright) and
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0 (downꢀright) to the Trypanosoma cruzi FeꢀSOD (a) and the human CuZnꢀSOD (b) enzymes. In the
center, an image of the entire enzyme with the compounds binding at the top of the dimer interface is
shown. Residues forming the inhibitor binding site are shown. Color scheme: oxygen (red), nitrogen
(blue), sulfur (yellow), carbon (pale pink for compound 3, orange for compound 4, blue for compound 7
and magenta for compound 10. For the proteins, one monomer is shown in green and the other in
yellow). The ions are shown as spheres. Figures were created with The PyMOL Molecular Graphics
System, Version 1.8 Schrödinger, LLC.
CONCLUSIONS
In conclusion, of the 20 arylaminoketone derivatives tested, in vitro and in vivo experiments allowed
us to identify 4 compounds (3, 4, 7 and 10) which showed improved trypanocidal properties and less
toxicity. In addition, they present higher activity for treating Chagas disease during acute phase, higher
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curative rates and larger spectrum of action than the reference drug benznidazole. Moreover, none of the
compounds included in the toxicological studies were genotoxic or mutagenic. Therefore, they are
promising molecules for the development of an easy to synthesize antiꢀChagas agent, and this allows us
to think that these compounds could be immediately implemented into a step further within the
preclinical phase. Finally, it merits mention that due to their different mechanisms of action, combined
therapies should be considered for obtaining improved efficiency.
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EXPERIMENTAL SECTION
CHEMISTRY
General methods. All solvents and chemicals were used as purchased without further purification.
Thin layer chromatography (TLC) using silica Alugram SIL G/UV254 (layer: 0.2 mm) (Machereyꢀ
Nagel GmbH&Co. KG., Düren, Germany) and detection with UV light was used to monitor reactions.
Conventional flash column chromatography was carried out with silica gel 60 (0.040_0.063 mm,
Merck). Flash column chromatography was developed on a CombiFlash®R (TELEDYNE ISCO,
f
Lincoln, USA) instrument with Silica RediSep®R columns. All of the synthesized compounds were
f
1
chemically characterized by infrared (IR), proton nuclear magnetic resonance ( H NMR), carbon
1
3
nuclear magnetic resonance ( C NMR) and elemental microanalyses (CHN). IR spectra were recorded
on a Nicolet Nexus FTIR (Thermo, Madison, WI, U.S.) in KBr pellets. Data are reported as vibrational
ꢀ1
frequency (cm ) and intensity is defined as strong (s), medium (m) and weak (w). In some cases, 2D
NMR experiments (COSY, HMBC, HMQC) were carried out for the appropriate peaks assignment.
NMR spectra were recorded in DMSOꢀd or CDCl solutions on a Bruker 400 Ultrashield instrument.
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3
The data are reported as follows: chemical shift in ppm from internal tetramethylsilane (TMS) standard
on the δ scale, multiplicity (bs = broad singlet, s = singlet, d = doublet, dd = double doublet, ddd =
double double doublet, t = triplet, m = multiplet) and coupling constants (J) values given in hertz (Hz).
(See supporting information) All compounds were purified to ≥96% purity as determined by elemental
microanalysis results obtained on a CHNꢀ900 elemental analyzer (Leco, Tres Cantos, Spain) from
vacuumꢀdried samples. The analytical results for C, H, and N were within ±0.4 of the theoretical values.
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Melting points (mp) were determined with a Mettler FP82 + FP80 apparatus (Greifensee, Switzerland)
and are not corrected.
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Synthesis. Compounds 2, 3, 6, 10, 12 and 13 were previously reported.
General methodology for the preparation of arylamine Mannich base derivatives (1, 4, 5, 7-9,
1, 14-20). A solution of the substituted methylketone (1.0 eq), the corresponding arylamine (1.0 eq),
,3ꢀdioxolane (5 mL) and concentrated HCl (1 mL) was heated at reflux. The reaction was followed by
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TLC until conversion (30 min ꢀ 2 h). NaOH 2 M was added to basic pH and the product was extracted
with CH Cl . The organic extracts were combined, dried over anhydrous Na SO , filtered and
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concentrated under reduced pressure. The residue was purified by column chromatography using
CH Cl /methanol 95:5 (v/v) as mobile phase or Automated Flash Chromatography eluting in gradient
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with CH Cl /methanol 99:1 (v/v). The desired compounds were precipitated with diethyl ether.
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-(benzo[b]thiophen-3-yl)-3-(4-(4-trifluoromethylphenyl)piperazin-1-yl)propan-1-one
ꢀ1
hydrochloride (1). Yield: 14%. mp: 209ꢀ210 ºC. IR (KBr) ν cm : 2552 (s, νHCl); 2458 (s, νHCl); 1660 (s,
1
νC=O); 1335 (s, ν ); 1166 (s, ν ); 1113 (s, ν ); 1073 (s, ν ). H NMR (DMSOꢀd , 400 MHz) δ ppm:
CꢀF
CꢀF
CꢀF
CꢀF
6
1
7
4
1.45ꢀ11.26 (bs, 1H, HCl); 9.15(s, 1H, H ); 8.61 (d, 1H, H , J =7.9 Hz); 8.12 (d, 1H, H , J =7.8 Hz);
2 4 4ꢀ5 7 7ꢀ6
.57 (d, 2H, H +H , J =8.7 Hz); 7.54ꢀ7.45 (m, 2H, H +H ); 7.18 (d, 2H, H +H , J2’ꢀ3’=8.7 Hz);
3’
5’
3’ꢀ2’
5
6
2’
6’
.10ꢀ4.00 (m, 2H, 2H ); 3.77 (t, 2H, COꢀCH , J=6.9 Hz); 3.72ꢀ3.62 (m, 2H, 2H ); 3.55 (t, 2H, CH ꢀN,
p
2
p
2
13
J=6.8 Hz); 3.34ꢀ3.19 (m, 4H, 2H +2H ). C NMR (DMSOꢀd , 100 MHz) δ ppm: 191.91; 151.97;
2
p
6p
6
3
140.51; 139.36; 135.99; 133.42; 125.89; 125.47; 124.51: 123.00; 114.91 (2C); 126.30 (2C, q, J =4.0
1
2
Hz); 124.78 (q, J =268.0 Hz); 119.04 (q, J =31.5 Hz); 50.52 (3C); 44.30 (2C); 34.01. Anal. calcd for
C H F N OS.HCl: C 58.08%, H 4.87%, N 6.16%. Found: C 57.81%, H 4.80%, N 6.32%.
22
21
3
2
1-(5-fluorobenzo[b]thiophen-3-yl)-3-(4-(4-trifluoromethylphenyl)piperazin-1-yl)propan-1-one
ꢀ1
(
4). Yield: 26%. mp: 143ꢀ144 ºC. IR (KBr) ν cm : 1664 (s, νC=O); 1331 (s, ν ); 1243 (s, ν ); 1107 (s,
CꢀF
CꢀF
1
ν ). H NMR (CDCl , 400 MHz) δ ppm: 8.48 (dd, 1H, H , J =10.3 Hz, J =2.3 Hz); 8.40 (s, 1H,
CꢀF
3
4
4ꢀF
4ꢀ6
H ); 7.80 (dd, 1H, H , J =8.8 Hz, J =4.8 Hz); 7.48 (d, 2H, H +H , J3’ꢀ2’=8.6 Hz); 7.20 (ddd, 1H, H6,
2
7
7ꢀ6
7ꢀF
3’
5’
J =8.7 Hz, J =8.7 Hz, J =2.4 Hz); 6.92 (d, 2H, H +H , J2’ꢀ3’=8.6 Hz); 3.30ꢀ3.26 (m, 4H,
6
ꢀF
6ꢀ7
6ꢀ4
2’
6’
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H +2H ); 3.23 (t, 2H, COꢀCH , J=7.2 Hz); 2.94 (t, 2H, CH ꢀN, J=7.2 Hz); 2.72ꢀ2.68 (m, 4H,
3p 5p 2 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
1
2
H +2H ). C NMR (CDCl , 100 MHz) δ ppm: 193.73; 161.82 (d, J =243.1 Hz); 153.20; 138.56;
2
p
6p
3
3
3
1
137.91 (d, J =10.2 Hz); 134.87; 134.84; 126.37 (2C, q, J =4.0 Hz); 124.72 (q, J =270.1 Hz); 123.27 (d,
3
2
2
2
J =9.4 Hz); 120.55 (q, J =32.6 Hz); 114.56 (d, J =25.7 Hz); 114.52 (2C); 111.62 (d, J =25.2 Hz); 53.13;
5
6
2.98 (2C); 47.98 (2C); 37.81. Anal. calcd for C H F N OS: C 60.54%, H 4.62%, N 6.42%. Found: C
22 20 4 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.37%, H 4.30%, N 6.39%.
-(5-fluorobenzo[b]thiophen-3-yl)-3-(4-(4-chlorophenyl)piperazin-1-yl)propan-1-one (5). Yield:
1
ꢀ1
1
33%. mp: 156ꢀ157 ºC. IR (KBr) ν cm : 1660 (s, ν ); 1246 (s, ν ). H NMR (DMSOꢀd , 400 MHz) δ
C=O
CꢀF
6
ppm: 9.17 (s, 1H, H ); 8.32 (dd, 1H, H , J =10.6 Hz, J =2.6 Hz); 8.15 (dd, 1H, H , J =8.8 Hz, J
7ꢀ
2
4
4ꢀF
4ꢀ6
7
7ꢀ6
F=5.2 Hz); 7.88 (ddd, 1H, H , J =9.0 Hz, J =8.9 Hz, J =2.8 Hz); 7.21 (d, 2H, H +H , J3’ꢀ2’=9.2
6
6ꢀF
6ꢀ7
6ꢀ4
3’
5’
Hz); 6.93 (d, 2H, H +H , J =9.2 Hz); 3.27 (t, 2H, COꢀCH , J=7.0 Hz); 3.14ꢀ3.07 (m, 4H,
2
’
6’
2’ꢀ3’
2
13
2
H +2H ); 2.80 (t, 2H, CH ꢀN, J=7.2 Hz); 2.62ꢀ2.54 (m, 4H, 2H +2H ). C NMR (DMSOꢀd , 100
3p 5p 2 2p 6p 6
1
3
MHz) δ ppm: 212.89; 160.96 (d, J =240.8 Hz); 149.56; 142.13; 137.43 (d, J =10.5 Hz); 135.06 (d,
4
4
3
J =1.3 Hz); 133.64 (d, J =4.5 Hz); 128.46 (2C); 124.57 (d, J =9.7 Hz); 122.24; 116.70 (2C); 113.90 (d,
2
2
J =25.2 Hz); 109.96 (d, J =24.9 Hz); 52.64; 52.30 (2C); 47.72 (2C); 36.91. Anal. Calcd. for
C H ClFN OS: C 62.60%, H 5.00%, N 6.95%. Found: C 62.23%, H 4.63%, N 6.87%.
21 20
2
1
-(5-fluorobenzo[b]thiophen-3-yl)-3-(4-(4-fluorophenyl)piperazin-1-yl)propan-1-one (7). Yield:
ꢀ1
1
1
8%. mp: 156ꢀ157 ºC. IR (KBr) ν cm : 1661 (s, νC=O); 1175 (s, ν ); 1131 (s, ν ). H NMR (CDCl ,
CꢀF CꢀF 3
400 MHz) δ ppm: 8.48 (dd, 1H, H , J =10.4 Hz, J = 2.6 Hz); 8.39 (s, 1H, H ); 7.79 (dd, 1H, H , J
7ꢀ
4
4ꢀF
4ꢀ6
2
7
6=
8.8 Hz, J =4.8 Hz); 7.19 (ddd, 1H, H , J =8.6 Hz, J =8.6 Hz, J =2.5 Hz); 6.99ꢀ6.93 (m, 2H,
7ꢀF 6 6ꢀF 6ꢀ7 6ꢀ4
H +H ); 6.89ꢀ6.84 (m, 2H, H +H ); 3.22 (t, 2H, COꢀCH , J=7.3 Hz); 3.14ꢀ3.11 (m, 4H, 2H +2H );
3
’
5’
2’
6’
2
3p
5p
13
2.94 (t, 2H, CH ꢀN, J=7.3 Hz); 2.71ꢀ2.68 (m, 4H, 2H +2H ). C NMR (CDCl , 100 MHz) δ ppm:
2
2p
6p
3
1
1
4
1
93.82; 161.78 (d, J =243.8 Hz); 157.15 (d, J =239.9 Hz); 147.86 (d, J =2.2 Hz); 138.56; 137.89 (d,
3
4
4
3
3
J =10.7 Hz); 135.11 (d, J =1.6 Hz); 134.84 (d, J =4.5 Hz); 123.23 (d, J =9.5 Hz); 117.80 (2C, d, J =7.6
2
2
2
Hz); 115.48 (2C, d, J =22.0 Hz); 114.51 (d, J =25.6 Hz); 111.60 (d, J =25.2 Hz); 53.26 (2C); 53.15;
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Journal of Medicinal Chemistry
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0.14 (2C); 37.81. Anal. Calcd. for C H F N OS: C 65.27%, H 5.22%, N 7.25%. Found: C 64.94%, H
21 20 2 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
.18%, N 6.88%.
1-(benzo[b]thiophen-3-yl)-3-(4-benzylpiperazin-1-yl)propan-1-one hydrochloride (8). Yield:
ꢀ1
1
1
2%. mp: 202ꢀ203 ºC. IR (KBr) ν cm : 2515 (m, νHCl); 2406 (m, νHCl); 1664 (s, νC=O). H NMR
(DMSOꢀd , 400 MHz) δ ppm: 11.93 (s, 2H, HCl); 9.04 (s, 1H, H ); 8.60 (d, 1H, H , J =7.9 Hz); 7.11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6 2 4 4ꢀ5
(d, 1H, H , J =7.6 Hz); 7.71ꢀ7.57 (m, 2H, H +H ); 7.56ꢀ7.36 (m, 5H, H -H ); 4.36 (s, 2H, CH2ꢀbenzyl);
7 7ꢀ6 5 6 2’ 6’
13
3
.84ꢀ3.66 (m, 6H, COꢀCH +2H +2H ); 3.66ꢀ3.19 (m, 6H, CH ꢀN+2H +2H ). C NMR (DMSOꢀd ,
2 3p 5p 2 2p 6p 6
100 MHz) δ ppm:. 181.92; 161.21; 150.86; 140.51; 139.36; 135.99; 128.75; 125.87 (2C); 125.47 (2C);
1
24.98; 124.52 (2C); 123.00; 73.22; 57.70 (3C); 47.33 (2C); 36.99. Anal. Calcd. for C H N OS.2HCl:
22 24 2
C 60.41%, H 5.99%, N 6.40%. Found: C 60.02%, H 6.08%, N 6.27%.
1-(benzo[b]thiophen-3-yl)-3-(4-(3-fluorobenzyl)piperazin-1-yl)propan-1-one hydrochloride (9).
ꢀ1
1
Yield: 12%. mp: 214ꢀ215 ºC. IR (KBr) ν cm : 2525 (m, νHCl); 2449 (m, νHCl); 1668 (s, νC=O). H NMR
(DMSOꢀd , 400 MHz) δ ppm: 11.45ꢀ11.26 (bs, 2H, HCl); 9.05(s, 1H, H ); 8.61 (d, 1H, H , J =7.7
6
2
4
4ꢀ5
Hz); 8.12 (d, 1H, H , J =7.8 Hz); 7.66ꢀ62 (m, 2H, H +H ); 7.57ꢀ7.52 (m, 4H, H +H +H +H ); 4.41
7
7ꢀ6
5
6
2’
4’
5’
6’
(s, 2H, CH2ꢀbenzyl); 3.85ꢀ3.65 (m, 12H, 8H +COCH +CH N). Anal. Calcd. for C H FN OS.2HCl: C
p
2
2
22 23
2
5
8.02%, H 5.53%, N 6.15%. Found: C 58.40%, H 5.71%, N 6.00%.
-(thiophen-3-yl)-3-(4-(3-trifluoromethylphenyl)piperazin-1-yl)propan-1-one
1
hydrochloride
ꢀ1
(11). Yield: 18%. mp: 177ꢀ178 ºC. IR (KBr) ν cm : 2459 (m, νHCl); 1682 (s, νC=O); 1166 (s, ν ); 1120
CꢀF
1
(s, ν ); 1076 (s, ν ). H NMR (DMSOꢀd , 400 MHz) δ ppm: 11.45ꢀ11.28 (bs, 1H, HCl); 8.61 (s, 1H,
CꢀF
CꢀF
6
H ); 7.68 (dd, 1H, H , J =4.6 Hz, J =2.5 Hz); 7.55 (d, 1H, H , J =5.0 Hz); 7.47 (t, 1H, H , J =7.9
2
5
5ꢀ4
5ꢀ2
4
4ꢀ5
5’ 5ꢀ4’
Hz); 7.37ꢀ7.24 (m, 2H, H +H ); 7.16 (d, 1H, H , J =7.3 Hz); 4.04ꢀ3.93 (m, 2H, COꢀCH ); 3.70ꢀ
2
’
6’
4’
4’ꢀ5’
2
1
3
3.57 (m, 4H, 2H +2H ); 3.52ꢀ3.43 (m, 2H, CH ꢀN); 3.28ꢀ3.14 (m, 4H, 2H +2H ). C NMR
3
p
5p
2
2p
6p
2
(DMSOꢀd , 100 MHz) δ ppm: 190.90; 149.84; 141.07; 134.29; 130.10; 129.92 (q, J =30.7 Hz); 127.76;
6
1
3
3
1
5
5
26.32; 124.28 (q, J =272.0 Hz); 119.22; 115.65 (q, J =3.6 Hz); 111.03 (q, J =3.5 Hz); 50.57 (2C);
0.35; 44.86 (2C); 33.73. Anal. Calcd. for C H F N OS.HCl: C 53.40%, H 4.98%, N 6.92%. Found: C
1
8
19
3
2
3.02%, H 5.02%, N 6.85%.
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1
-(naphthalen-2-yl)-3-(4-(4-trifluoromethylphenyl)piperazin-1-yl)-propan-1-one (14). Yield:
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ1
1
6
6%. mp: 150ꢀ151 ºC. IR (KBr) ν cm : 1677 (s, ν ); 1148 (s, ν ); 1098 (s, ν ); 1065 (s, ν ). H
C=O CꢀF CꢀF CꢀF
NMR (DMSOꢀd , 400 MHz) δ ppm: 8.72 (s, 1H, H ); 8.14 (d, 1H, H , J =7.7 Hz); 8.03ꢀ7.97 (m, 3H,
6
1
8
8ꢀ7
H -H ); 7.69ꢀ7.59 (m, 2H, H +H ); 7.48 (d, 2H, H +H , J =8.8 Hz); 7.04 (d, 2H, H +H , J2’ꢀ3’=8.8
3
5
6
7
3’
5’ 3’ꢀ2’
2’
6’
Hz); 3.37 (t, 2H, COꢀCH , J=7.2 Hz); 3.27ꢀ3.21 (m, 4H, 2H +2H ); 2.79 (t, 2H, CH ꢀN, J=7.2 Hz);
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3p
5p
2
13
2
1
.61ꢀ2.55 (m, 4H, 2H +2H ). C NMR (DMSOꢀd , 100 MHz) δ ppm: 199.19; 153.24; 135.03;
2p 6p 6
3
34.00; 132.23; 129.95; 129.60; 128.61; 128.26; 127.64; 126.91; 126.13 (2C, q, J =3.8 Hz); 124.99 (q,
1
2
J =270.1 Hz); 123.55; 117.78 (q, J =32.0 Hz); 114.12 (2C); 52.92; 52.41 (2C); 46.96 (2C); 35.81. Anal.
Calcd. for C H F N O: C 69.89%, H 5.62%, N 6.79%. Found: C 69.82%, H 5.72%, N 6.76%.
2
4
23
3
2
1
-(naphthalen-2-yl)-3-(4-(4-nitrophenyl)piperazin-1-yl)-propan-1-one (15). Yield: 86%. mp: 190ꢀ
ꢀ1
1
191 ºC. IR (KBr) ν cm : 2806 (s, νCꢀH); 1674 (s, νC=O). H NMR (DMSOꢀd , 400 MHz) δ ppm: 8.74 (s,
6
1
H, H ); 8.14 (d, 1H, H , J =7.8 Hz); 8.04 (m, d, 2H, H +H , J3’ꢀ2’=9.4 Hz); 8.02ꢀ7.98 (m, 3H, H3-
1 8 8ꢀ7 3’ 5’
H ); 7.70ꢀ7.60 (m, 2H, H +H ); 7.03 (d, 2H, H +H , J =9.4 Hz); 3.48ꢀ3.42 (m, 4H, 2H +2H );
5
6
7
2’
6’
2’ꢀ3’
3p
5p
13
3.40 (t, 2H, COCH , J=7.2 Hz); 2.80 (t, 2H, CH ꢀN, J=7.2 Hz); 2.62ꢀ2.56 (m, 4H, 2H +2H ). C
2
2
2p
6p
NMR (DMSOꢀd , 100 MHz) δ ppm: 199.07; 154.68; 136.75; 134.98; 133.92; 132.19; 129.94; 129.56;
6
1
3
1
28.57; 128.20; 127.60; 126.86; 125.66 (2C); 123.50; 112.55 (2C); 52.75; 52.24 (2C); 46.23 (2C);
5.71. Anal. Calcd. for C H N O : C 70.93%, H 5.95%, N 10.79%. Found: C 70.99%, H 5.85%, N
23
23
3
3
0.58%.
3-(4-(4-fluorophenyl)piperazin-1-yl)-1-(naphthalen-2-yl) propan-1-one (16). Yield: 15%. mp: 98ꢀ
ꢀ1
1
9
9 ºC. IR (KBr) ν cm : 2800 (m, νCꢀH); 1676 (s, νC=O); 1177 (s, νarCꢀF); 1134 (s, νarCꢀF). H NMR (CDCl ,
3
4
00 MHz) δ ppm: 8.50 (s, 1H, H ); 8.04 (dd, 1H, H , J =8.6 Hz, J =1.7 Hz); 7.97 (d, 1H, H , J =8.0
1
8
8ꢀ7
8ꢀ6
3
3ꢀ4
Hz); 7.91 (d, 1H, H , J =8.8 Hz); 7.88 (d, 1H, H , J =8.7 Hz); 7.64ꢀ7.54 (m, 2H, H +H ); 6.97 (dd,
5
5ꢀ6
4
4ꢀ3
6
7
2
H, H +H , J3’ꢀ2’=9.0 Hz, J3’ꢀF=8.4 Hz); 6.88 (dd, 2H, H +H , J2’ꢀ3’=9.2 Hz, J2’ꢀF=4.6 Hz); 3.37 (t, 2H,
3’ 5’ 2’ 6’
COꢀCH , J=7.4 Hz); 3.17ꢀ3.12 (m, 4H, 2H +2H ); 2.97 (t, 2H, CH ꢀN, J=7.4 Hz); 2.75ꢀ2.70 (m, 4H,
2
3p
5p
2
13
1
4
2
H +2H ). C NMR (CDCl , 100 MHz) δ ppm: 198.93; 159.17 (d, J =239.1 Hz); 147.89 (d, J = 2.2
2p 6p 3
Hz); 135.60; 134.22; 132.51; 129.71; 129.54; 128.51 (2C); 127.77; 126.81; 123.78; 117.82 (2C, d,
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Journal of Medicinal Chemistry
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2
J =7.9 Hz); 115.48 (2C, d, J =22.1 Hz); 53.28 (2C); 53.25; 50.14 (2C); 36.33. Anal. Calcd. for
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C H FN O: C 76.22%, H 6.40%, N 7.73%. Found: C 76.46%, H 6.57%, N 7.60%.
23
23
2
1-(benzo[d][1,3]dioxol-5-yl)-3-(4-(4-trifluoromethylphenyl)piperazin-1-yl)propan-1-one
(17).
ꢀ1
Yield: 21%. mp: 107ꢀ108 ºC. IR (KBr) ν cm : 1669 (s, ν ); 1158 (s, ν ); 1111 (s, ν ); 1072 (s, ν
C=O
CꢀF
CꢀF
Cꢀ
1
F). H NMR (DMSOꢀd , 400 MHz) δ ppm: 7.65 (1H, d, H , J =8.2 Hz); 7.48 (d, 2H, H +H , J3’ꢀ2’=8.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
6
6ꢀ7
3’
5’
Hz); 7.47 (s, 1H, H ); 7.04 (dd, 3H, H +H +H , J =J =8.7 Hz); 6.13 (s, 2H, 2H ); 3.28ꢀ3.2 (m, 4H,
4
2’
6’
7
2’ꢀ3’ 7ꢀ6
2
2
H +2H ); 3.16 (t, 2H, COꢀCH , J=7.1 Hz); 2.70 (t, 2H, CH ꢀN, J=7.1 Hz); 2.59ꢀ2.52 (m, 4H,
3p 5p 2 2
13
2H +2H ). C NMR (DMSOꢀd , 100 MHz) δ ppm: 197.06; 153.16; 151.28; 147.71; 131.35; 126.01
2
p
6p
6
3
1
2
(
2C, q, J =3.7 Hz); 124.91 (q, J =270.5 Hz); 124.28; 117.66 (q, J =32.0 Hz); 114.01 (2C); 107.92;
1
07.33; 101.89; 52.89; 52.29 (2C); 46.85 (2C); 35.45. Anal. Calcd. for C H F N O : C 62.06%, H
21 21 3 2 3
5.21%, N 6.89%. Found: C 62.06%, H 5.60%, N 7.03%.
1
-(benzo[d][1,3]dioxol-5-yl)-3-(4-(4-chlorophenyl)piperazin-1-yl)propan-1-one (18). Yield: 20%.
ꢀ1
1
mp: 105ꢀ106 ºC. IR (KBr) ν cm : 1675 (s, νC=O); 1241 (s, νCꢀO). H NMR (DMSOꢀd , 400 MHz) δ ppm:
6
7.64 (dd, 1H, H , J =8.2 Hz, J =1.3 Hz); 7.46 (s, 1H, H ); 7.20 (d, 2H, H +H , J3’ꢀ2’=8.9 Hz); 7.02
6
6ꢀ7
6ꢀ4
4
3’
5’
(
d, 1H, H , J =8.2 Hz); 6.92 (d, 2H, H +H , J =8.9 Hz); 6.12 (s, 2H, 2H ); 3.14 (t, 2H, COꢀCH ,
7 7ꢀ6 2’ 6’ 2’ꢀ3’ 2 2
J=7.1 Hz); 3.11ꢀ3.05 (m, 4H, 2H +2H ); 2.69 (t, 2H, CH ꢀN, J=7.1 Hz); 2.57ꢀ2.51 (m, 4H,
3p
5p
2
13
2
H +2H ). C NMR (DMSOꢀd , 100 MHz) δ ppm: 197.20; 151.35; 149.78; 147.78; 131.40; 128.55
2p 6p 6
(2C); 124.38; 122.22; 116.74 (2C); 108.00; 107.38; 101.97; 52.98; 52.49 (2C); 47.95 (2C); 35.52. Anal.
Calcd. for C H ClN O : C 64.43%, H 5.68%, N 7.51%. Found: C 64.61%, H 5.86%, N 7.40%.
2
0
21
2
3
1
-(benzo[d][1,3]dioxol-5-yl)-3-(4-(4-nitrophenyl)piperazin-1-yl)propan-1-one (19). Yield: 50%.
ꢀ1
1
mp: 164ꢀ165 ºC. IR (KBr) ν cm : 1665 (s, νC=O); 1241 (s, νCꢀO). H NMR (DMSOꢀd , 400 MHz) δ ppm:
6
8.04 (d, 2H, H +H , J =9.4 Hz); 7.65 (dd, 1H, H , J =8.2 Hz, J =1.4 Hz); 7.47 (s, 1H, H ); 7.06ꢀ
3
’
5’ 3’ꢀ2’
6
6ꢀ7
6ꢀ4
4
7
.00 (m, 3H, H +H +H ); 6.13 (s, 2H, 2H ); 3.46ꢀ3.40 (m, 4H, 2H +2H ); 3.16 (t, 2H, COꢀCH ,
7 2’ 6’ 2 3p 5p 2
1
3
J=7.0 Hz); 2.70 (t, 2H, CH ꢀN, J=7.1 Hz); 2.57ꢀ2.51 (m, 4H, 2H +2H ). C NMR (DMSOꢀd , 100
2
2p
6p
6
MHz) δ ppm: 197.08; 154.66; 151.33; 147.75; 136.75; 131.35; 125.64 (2C); 124.35; 112.54 (2C);
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1
07.98; 107.38; 101.94; 52.78; 52.17 (2C); 46.21 (2C); 35.42. Anal. Calcd. for C H N O : C 62.65%,
20 21 3 5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H 5.52%, N 10.96%. Found: C 62.29%, H 5.43%, N 10.61%.
1-(benzo[d][1,3]dioxol-5-yl)-3-(4-(4-fluorophenyl)piperazin-1-yl)propan-1-one (20). Yield: 19%.
ꢀ1
1
mp: 96ꢀ97 ºC. IR (KBr) ν cm : 1676 (s, νC=O); 1242 (s, νCꢀO). 1132 (s, ν ); 1109 (s, ν ). H NMR
CꢀF
CꢀF
(DMSOꢀd , 400 MHz) δ ppm: 7.65 (d, 1H, H , J =8.0 Hz); 7.47 (s, 1H, H ); 7.07ꢀ6.99 (m, 3H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6 6 6ꢀ7 4
H +H +H ); 6.93 (dd, 2H, H +H , J2’ꢀ3’=8.9 Hz, J2’ꢀF=4.6 Hz); 6.13 (s, 2H, 2H ); 3.14 (t, 2H, COꢀ
3
’
5’
7
2’
6’
2
CH , J=7.0 Hz); 3.08ꢀ2.99 (m, 4H, 2H +2H ); 2.69 (t, 2H, CH -N, J=7.1 Hz); 2.59ꢀ2.52 (m, 4H,
2
3p
5p
2
13
1
2H +2H ). C NMR (DMSOꢀd , 100 MHz) δ ppm: 197.10; 155.84 (d, J =235.4 Hz); 151.26; 147.82
2
p
6p
6
4
3
2
(d, J =1.9 Hz); 147.71; 131.35; 124.28; 116.92 (2C, d, J =7.5 Hz); 115.10 (2C, d, J =21.8 Hz); 107.92;
1
07.32; 101.89; 52.94; 52.59 (2C); 48.85 (2C); 35.49. Anal. Calcd. for C H FN O : C 67.40%, H
20 21 2 3
5.94%, N 7.86%. Found: C 67.30%, H 6.04%, N 7.75%.
Calculation of molecular properties. The drugꢀlikeness of the compounds was estimated with
2
2
Osiris, using the standꢀalone DataWarrior program. The information about the methods used to
evaluate each property is available on the program web site http://www.openmolecules.org.
BIOLOGY
In vitro Trypanocidal Evaluation
Parasite strain culture. Epimastigote and trypomastigote forms: Epimastigote assays. The
epimastigote forms of T. cruzi SN3 strain (IRHOD/CO/2008/SN3, DTU I) that were isolated from the
5
3
household triatomine Rhodnius prolixus (from Guajira, Colombia). Epimastigotes of T. cruzi Arequipa
strain (MHOM/Pe/2011/ Arequipa, DTU V) isolated from a human and Tulahuen strain
(TINF/CH/1956/Tulahuen, DTU VI) were cultivated in vitro using trypanosome liquid medium (MTL)
containing 10% inactivated fetal bovine serum and maintained at 28°C in an air atmosphere, in Roux
2
54
flasks (Corning, USA) of 75 cm surface area, as described in previous studies. The epimastigote
forms transformed into metacyclic forms by metacyclogenesis triggered by culturing a 5ꢀdayꢀold
epimastigote forms that had been harvested by centrifuging (7000 g) at 10 ºC for 10 min. Next the
8
parasites were incubated at 28 ºC for 2 h (density of 5 x 10 cells/mL) in TAU medium (190 mM NaCl,
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Journal of Medicinal Chemistry
1
7 mM KCl, 2 mM MgCl , 2 mM CaCl , 8 mM phosphate buffer, pH 6.0). The parasites were then
2 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
incubated at a dilution of 1:100 (final concentration of epimastigotes: 5 × 10 cells/mL) at 28 ºC f or 96
h in TAU3AAG medium (TAU supplemented with 10 mM Lꢀproline, 50 mM Lꢀsodium glutamate, 2
mM Lꢀsodium aspartate and 10 mM Dꢀglucose) in 25 mL flasks with a layer culture medium no more
5
5
than 1 cm in deep.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Trypomastigote Forms assays. The activity (parasiteꢀreduction %) was compared to control with the
54
method described above , with certain modifications in our lab. The test of drug action against T. cruzi
used Balb/c albino mouse blood collected during peak parasitemia (day 7). The infected blood was
6
diluted using uninfected mouse blood at a concentration of 4 x 10 trypomastigotes/mL. Then, in RPMI
1
640 mediumꢀGIBCO (2 x 106 trypomastigotes/mL), a dilution was made to 1:2. Stock solutions of the
compounds were prepared in DMSO. The IC values were calculated using, samples of infected blood,
5
0
adding each drug to wells in a 96ꢀmicrowell plate for a final 200 µL volume for drug concentrations of
, 10, 25, 50, and 100 µM., and the plates were then incubated for 24 h at 4ꢀ to 8 ºC. Each experiment
1
was replicated three times (OLYMPUS CX41) and the parasites were counted in the Neubauer chamber.
Cytotoxicity and Cell Culture Tests. Vero cells (EACC number 84113001) originally extracted from
monkey kidney, were cultured for two days in RPMI (Gibco), supplemented with 10% inactivated fetal
bovine serum in 95% humidified air, 5% CO at 37 ºC. The cytotoxicity test was performed for Vero
2
54
cells following a methodology described previously. Flow cytometry was used to determine cell
viability after 72 h of treatment. Propidium iodide solution (100 mg/mL) was added at 100 ꢂL/well and
incubated at 28 °C for 10 min in darkness. Then, fluorescein diacetate (100 ng/mL) was added at 100
ꢁ
L/well and incubated using the same conditions. The cells were collected by centrifuging (400 g for 10
min), and finally the precipitate was washed using PBS (phosphate buffered saline). Flow cytometry
was performed with a FACSVantage flow cytometer (Becton Dickinson), and the percentage of viability
was computed in comparison to control. The IC50 was calculated with linearꢀregression analysis
employing the K values corresponding to the concentrations used (0.1 to 100 ꢂM).
c
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In Vitro Activity. Epimastigote Forms Assays (Extracellular Forms). The compounds obtained and
BZN (the reference drug), dissolved in DMSO (Panreac, Barcelona, Spain) at a 0.01% (v/v)
concentration and assayed, proved nontoxic to the parasites and not inhibitory to their growth, according
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
56
to a procedure reported on a previous work. The compounds, at doses of 100, 50, 25, 10, l, 0.5, 0.25,
and 0.1 ꢂM were added to the culture medium. For each compound, the effects against T. cruzi
epimastigote forms were assayed at 72 h with a hemocytometric chamber (Neubauer). The antiꢀ
trypanosomatid effect is expressed as the IC . That is, the concentration needed for 50% growth
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
inhibition, determined by linear regression with the K values of the concentrations used.
c
Amastigote Assays (Intracellular Forms). Vero cells grown as indicated above in RPMI medium
4
(
Gibco), were inoculated at 1 × 10 cells/well in 24ꢀwell microplates (Nunc) that had rounded coverslips
and cultured 2 days. Then the Vero cells were infected for 24 h in vitro using metacyclic forms at a 10:1
ratio. After the removal of nonꢀphagocytic parasites by washing, the drugs (100, 50, 25, 10, 1, 0.5 0.25
and 0.1 ꢂM) were added. Vero cells were incubated with the drugs at 37°C for 72 h in 5% CO . The
2
activity of the drugs was evaluated based on the number of amastigotes in treated as well as untreated
cultures, in preparations that were methanolꢀfixed and Giemsaꢀstained. The number of amastigotes was
estimated analyzing 200 host cells in randomly taken microscopic fields. The anti trypanosomal effect
was expressed as the IC .
5
0
In vitro genotoxicity screening
Bacteria. S. typhimurium TA1535/pSK1002 was obtained from the German Collection for
Microorganisms and Cell cultures (DSMZ). Chemicals. DMSO, dextrose, NaCl, ampicillin, oꢀ
nitrophenolꢀβꢀDꢀgalactopyranoside (ONPG), Na CO , Na HPO 2H O, NaH PO .H O, MgSO .7H O,
2
3
2
4.
2
2
4
2
4
2
KCl, βꢀmercaptoethanol and sodium dodecyl sulphate were obtained from SigmaꢀAldrich;
Bactotryptone was obtained from BD; and rat S9 mix (Mutazyme) was obtained from Moltox.
Reagents preparation for SOS/umu test. TGA culture medium was prepared dissolving 10 g
bactotryptone, 2 g dextrose and 5 g NaCl in 1 L of water and sterilizing during 30 minutes at 121 ºC. Bꢀ
buffer was prepared dissolving 20.18 g Na HPO .2H O, 5.5 g NaH PO .H O, 0.75 g KCl, 0.25 g
2
4
2
2
4
2
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Journal of Medicinal Chemistry
MgSO .7H O, 1.0 g SDS, 2.7 mL βꢀmercaptoethanol in 1 L water adjusting pH value to 7. Stop reagent
4
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Na CO 1 M) was prepared dissolving 106 g of Na CO in 1 L water. Phosphate buffer (for ONPG
2 3 2 3
dissolution) was prepared dissolving 1.086 g Na HPO .2H O and 0.538 g NaH PO .H O in 100 mL
2
4
2
2
4
2
water and adjusting pH value to 7. ONPG solution was prepared dissolving 45 mg ONPG in 10 mL
phosphate buffer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SOS/umu test. The SOS/umu test was carried out according to the method of Oda et al. (1985) and
57, 58
Reifferscheid et al. (1991), with some modifications.
The test strain S. typhimurium
TA1535/pSK1002 was used. It was preꢀincubated overnight in TGA medium with ampicillin (50 ꢂg/mL)
by shaking in an incubator at 37 °C in order to obtain exponentially growing bacteria. The test was
performed in the absence and the presence of external metabolic activation system (rat S9 mix) in order
to determine the possible genotoxic effects of any metabolite. In each test performed, negative and
positive controls were included. DMSO was used as solvent control, and 4ꢀnitroquinolineꢀNꢀoxide (4ꢀ
NQO) and 2ꢀaminofluorene (2ꢀAF) were used as positive controls with and without of S9 mix.
Test procedure was as follows: firstly, each compound tested was dissolved in DMSO at 40 mg/mL
and 11 serial ½ dilutions were prepared in a 96ꢀwell plate (plate A; final volume in each well was 10
ꢂL). Next, 70 ꢂL of water were added to each well. In another two 96ꢀwell plates (plates B; one for the
test with S9 and the other without S9), 10 ꢂL S9 mix or 10 ꢂL PBS, were added , respectively, followed
by the addition of 25 ꢂL of each previously prepared compound. Finally, 90 ꢂL of exponentially
growing bacteria was added and incubated during 4 hours by shaking at 37 ºC. After the incubation
period, absorbance at 600 nm was measured in order to evaluate toxicity on S. typhimurium
TA1535/pSK1002. Toxicity was calculated as follows:
Survival percentage = (A600 for each concentration tested/media A600 for negative control)*100
Afterwards, for the determination of βꢀgalactosidase activity, in two new 96ꢀwell plates (plates C),
1
50 ꢂL of ONPG solution were added to each well and 30 ꢂL of the contents of each well of plates B
were transferred to these plates C. Both plates were incubated for 30 minutes by shaking at 28 ºC,
avoiding direct light exposure. After the incubation period, 120 ꢂL of stop reagent were added and the
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reaction was stopped. Absorbance (420 nm) was then measured immediately, and βꢀgalactosidase
activity (relative units; RU) was calculated as follows:
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
βꢀgalactosidase enzymatic units = A420 for each concentration tested/A600 for each concentration
tested
Finally, the induction factor (IF) was calculated as follows:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IF = βꢀgalactosidase RU for each concentration tested/media βꢀgalactosidase RU for negative control,
where: media βꢀgalactosidase RU for negative control = media A420 for negative control/media A600
for negative control
In the same way, βꢀgalactosidase relative units were calculated for both positive controls, and the test
was only considered valid if the positive controls reached an induction factor ≥2 under the given test
conditions.
Thus, a compound was consider genotoxic, as it induces the induction of the umu operon, when in any
of the conditions studied (with or without metabolic activation), the induction factor was ≥2 at non
cytotoxic concentrations (bacteria survival percentage ≥80%), presenting a doseꢀresponse relationship.
Wells in which compound precipitation was observed, were discarded from analysis.
Infectivity Assay
Vero cells were cultured under the same conditions for two days. The, cells were then infected in vitro
with T. cruzi metacyclic forms, at a 10:1 ratio. Immediately after infection, the drugs (IC25
concentrations) were added, followed by incubation at 37°C for 12 h in 5% CO . After removal of the
2
drugs and nonphagocytosed parasites by washing, the infected cultures were grown in fresh medium for
4
1
1
0 day. Every 48 h, fresh culture medium was added. The activity of the drugs was determined from
the % of infected cells, the number of amastigotes per infected cell, and the number of trypomastigotes
in the medium, both in treated as well as untreated cultures, and in methanolꢀfixed as well as Giemsaꢀ
stained preparations. A determination was made every 48 h of the mean number of amastigotes per
infected cell and the % of infected cells by examining 200 host cells in randomly taken microscopic
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fields. The trypomastigotes in the medium were counted every 48 h in a hemocytometric chamber
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(Neubauer).
In Vivo Trypanocidal Activity Assay
For the following purposes, in the in vivo stage, experimental tests were performed: i) to assess the
influence that the compounds exerted on the parasitemia levels postꢀ treatment by a Neubauer chamber
count for 40 days as an acuteꢀphase marker; ii) to provide a measure of the parasitemia reactivation
following immunosuppression as a chronicꢀphase marker; iii) to compare the levels of the chronic and
acute phases of IgꢀG using ELISA as an immuneꢀresponse indicator; and iv) for detection of the
parasite’s presence using PCR in target organs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Mice Infection and Treatment. The mice were handled following the international guide for
biomedical research using experimental animals and the experiment was approved by the ethics board of
the University of Granada (Spain). Groups of six female BALB/c albino mice 8 to 10 weeks old and
weighing 25 to 30 g, were kept under standard conditions and given free access to standard chow and
5
water. The mice were infected intraperitoneally using 5 x 10 blood trypomastigotes (T. cruzi Arequipa
strains) from the blood of mice previously infected. These animals were split in the three groups: I,
untreated infected mice constituted the positive control group; II, infected mice treated using the
reference drug made up the BZN group; and III, infected mice treated using the compounds under
investigation comprised the study group. The tested compounds were administered beginning on the day
7 after confirmation of the infection, and dosages of 50 and 100 mg/kg body mass per day were given
orally on 5 alternating days (day 7 to 15 pi). In each mouse, peripheral blood was taken from the
mandibular vein (5 ꢂL samples) and this was diluted in 495 ꢂL PBS solution (1:100). The circulating
parasites were counted in a Neubauer chamber every 3 days for 40 d (acute phase). The number of
bloodstream forms detected was expressed in parasites/mL.
Immunosuppression Induced by Cyclophosphamideꢀ and Cure Assessment. At day 90 pi, the groups in
which the parasitemia levels had significantly fallen, (irrespective of the treatment the mice received)
and could not be detected by microscopic examination of fresh blood, were subjected to 3 cycles of
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immunosuppression using CP (cyclophosphamide monohydrate ISOPAC®) as follows: 4 days
consecutively on which the mice were given 1 intraperitoneal injection/day (dosage 200 mg/kg·w/day),
and 3 days off. The effectiveness of this immunosuppression technique for evaluating cryptic infection
was corroborated by the mortality rate of nearly 100% in chronically untreated mice and/or by the high
parasitemia rate. The parasitemia was assessed within 1 week after the last injection of CP, following
the procedure explained above for the acute phase in order to quantify the trypomastigote forms in blood
as the reactivation rate. The mice were finally bled by heart puncture under gaseous anesthesia (CO2).
Blood was incubated for 2 hours at 37 ºC and then over night at 4º C in order to allow clotting.
Subsequently, the serum was obtained after centrifuging the supernatant twice at 1000 and 2700 g,
consecutively. The serum was used for ELISA and biochemical analysis as explained further on in this
paper. Thanks to our previous in vivo studies, we observed that the tropism of T. cruzi strain (Arequipa)
occurs in the following organs: adipose, brain, heart, esophagus, stomach, bone marrow, muscle and
lung. Therefore these 8 target organs were harvested and immediately flushed free of blood by gentle
infusion of preꢀwarmed PBS for the purpose of avoid contamination of collected tissue with blood
parasites. Next, they were used for DNA extraction, as explained below. In addition, spleens were
weighed to evaluate inflammation of these organs in different groups of mice.
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ELISA Tests. FeꢀSOD excreted from the parasite, cultured and processed as described by Lopezꢀ
59
Céspedes et al. was used as antigen. Circulating antibodies in serum against T. cruzi, from day 40 to
day 120 postꢀinfection (including sera postꢀimmunosuppression), were qualitatively and quantitatively
assessed using ELISA. The serum was centrifuged from whole blood and diluted in PBS to 1:80. The
absorbance was read at 492 nm using a microplate reader (Sunrise, TECAN). All samples, in triplicate,
were analyzed in polystyrene microtiter plates. For calculating the cutoff value, the mean and standard
deviations of the optical density of the negative control sera were used.
PCR and DNA Extraction. The organs to be studied were thawed and then ground using a Potterꢀ
Elvehjem for purification using Wizard® Genomic DNA Purification Kit (Promega). The PCR was
conducted with two primers from our laboratory (unpublished data), based on the published SOD
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Page 29 of 62
Journal of Medicinal Chemistry
(
superoxide dismutase) sequence of T. cruzi CL Brenner (GenBank accession No. XM_808937); this
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amplifies a fragment pertaining to T. cruzi SOD gene b of c. 300 pb. The amplification was performed
in Thermal Cycler TM MyCycler thermocycler (BioRad).
Studies of the action mechanism of
Superoxide Dismutase (SOD) Inhibition Studies. T. cruzi epimastigotes cultured as indicated above
were centrifuged and collected. After the suspension of the pellet in 3 mL of STE buffer (0.25 M
sucrose, 25 mM Tris–HCl, 1 M EDTA, pH 7.8), the cells were broken up by ultrasonication (three
cycles) at 60 W every 30 s. Following 1500ꢀg centrifugation of the homogenate at 4ºC for 5 min and,
the pellet was washed in iceꢀcold STE buffer three times and centrifuged (2500 g at 4ºC for 10 min)
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The supernatant was collected to determine its protein concentration using the Bradford method. The
activities of FeꢀSOD and CuZnꢀSOD (iron and copperꢀzinc superoxide dismutases) (were determined by
61
the method of Beyer and Fridovich, to measure the extent to which nitrobluetetrazolium (NBT) was
reduced by superoxide ions. To each cuvette, the following was added: 1.5 mL of Triton Xꢀ100 1%
−
1
(v/v)], 2 mL of NBT (1.41 mg, 10 mL ), and 845 ꢂL of stock solution [3 mL of Lꢀmethionine (300 mg,
−l
1
0 mL ), as well as 30 ꢂL of the homogenate fraction of the parasite, 10 ꢂL riboflavin (0.44 mg, 10
−
l
mL ), and an equivalent volume of the compound solution. For each compound, 5 concentrations were
used: 1 to 100 ꢂM. For the control experiment, the volume was raised to 1000 ꢂL using 50 mM
potassium phosphate buffer (pH 7.8), while 30 ꢂL of the homogenate fraction of the parasite were added
to the mixtures that contained the compounds. A spectrophotometer was used to measure the absorbance
(A ), at 560 nm. Each cuvette was placed under lamp light for 10 min with constant stirring, and a
0
measurement was made of the absorbance (A ). The substrates, coenzymes, and human CuZnꢀSOD used
1
6
2
in these tests were from Sigma Chemical Co. The data were submitted to the Newman–Keuls test.
Metabolite excretion. Cultures of epimastigotes of T. cruzi Arequipa strain at a starting concentration
5
of 5×10 cells/mL) received the IC dosage of each compound (excepting control cultures). After 72 h
25
of incubation for at 28°C the cells underwent centrifugation (400 g) for 10 min. After supernatant
1
collection, the excreted metabolites were determined by H NMR; using sodium 2,2ꢀdimethylꢀ2ꢀ
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