WO2019150161A1 - Specific inhibitors of akt-like kinase of leishmania spp and trypanosoma cruzi with leishmanicidal and trypanocidal activity - Google Patents

Abstract

The present invention relates to compounds and drugs for the prophylaxis and treatment of leishmaniasis and Chagas disease, diseases caused by the parasites Leishmania panamensis and Trypanosoma cruzi. Two specific chemical inhibitors against the pleckstrin homology (PH) domain of the ΑKT-like protein (PKB) of L. panamensis and T. cruzi were identified: the inhibitor UBMC1 and the inhibitor UBMC4. Also, the anti-parasitic activity of the in vitro intracellular compounds (IC50 ) and the cytotoxic effects thereof (LC50) in different human cells were determined. After conducting a series of experiments, it was concluded that the compounds UBMC1 and UBMC4 were compounds that showed efficiency against L. panamensis and T. cruzi and acted against the AKT-like kinase (PKB), leading to the damaging of the parasite's DNA and, subsequently, its death. The compounds UBMC1 and UBMC4 displayed low toxicity for human macrophages and other human cells.

Description

 SPECIFIC INHIBITORS OF THE QUINASA AKT-LIKE DE LEISHMANIA SPP AND TRYPANOSOMA CRUZI WITH LEISHMANICIDE AND TRIPANOCID ACTIVITY

 1. FIELD OF THE INVENTION

The present invention relates to the industrial pharmaceutical sector, specifically to obtaining and developing compounds and medicaments for the prophylaxis and treatment of leishmaniasis and chagas disease, diseases caused by parasites Leishmania spp. and Trypanosoma cruzi. Such compounds and medicaments comprise specific inhibitors of the AKT-like enzyme kinase of the Leishmania spp. and Trypanosoma cruzi.

2. BACKGROUND OF THE INVENTION

Leishmaniasis is a disease caused by parasites belonging to the genus Leishmania, and Chagas disease is caused by the parasite Trypanosoma cruzi in the americas. These parasites as a whole belong to the family called Trypanosomatidae, that is, they are evolutionarily close and therefore share homologous proteins that can be a common therapeutic target among them.

Between the two diseases, an estimated 82,000 deaths occur each year with high morbidity rates that generate high costs for the health system and families. Given these characteristics and the complex host-parasite interactions, the disease becomes difficult to treat, given the small number of medications available for treatment, with the aggravating understanding of its mechanisms of action.

Currently, there is no vaccine against Chagas disease or leishmaniasis, or effective prophylactic drugs, so the control of the latter disease is based mainly on antimony treatment pentavalent; However, the findings and reports of resistance to this line of treatment show the need to find new therapeutic alternatives.

The medications currently marketed for the treatment of leishmaniasis are: Glucantime, Amphotericin B, Pentamidine and Miltefosine, which are medications with variable biological activity, have high costs, and some of them, such as Glucantime and Amphotericin B do not liposomal, they are highly toxic to the patient causing little adherence to the treatment and even the death of the patient. In addition, many of the strains circulating in Leishmania spp. in several endemic areas of the world they present different mechanisms of resistance to Glucantime, the most used medicine. In turn, the medications currently marketed to treat Chagas disease are benznidazole, and nifurtimox. These medications have the great limitation of not being very effective in the chronic phase of the disease, phase in which most patients are diagnosed. Likewise, resistance to benznidazole has been reported.

Some documents have reported medications that comprise AKT protein kinase inhibitors in order to treat different diseases and conditions. One such case is US2010292244 which describes a new compound that inhibits AKT protein kinases. The compound has utility as rheumatoid arthritis, osteoarthritis, eye diseases, endometriosis, atherosclerosis, asthma, obesity, Alzheimer's, COPD, multiple sclerosis, infections, particularly bacterial, viral, retroviral or parasitic infections (by increasing apoptosis), lung disease, Parkinson's, among others.

Another is the case of document US5663155 which describes pharmaceutical compositions useful for the prevention and treatment of parasitic infections by eukaryotic organisms. Said compositions contain an adenosine derivative comprising a 5-membered pentose ring coupled to a purine. The mechanism of action of the adenosine derivative is aimed at the disruption of processes Parasite enzymes that are related to purine metabolism, purine derivatives and purine type chemical structures. Purines and related compounds are involved in biosynthetic and enzymatic pathways such as nucleotide biosynthesis comprising the synthesis of ATP and GTP, nucleic acid and protein biosynthesis, intracellular and extracellular cell signaling, mitosis, meiosis, DNA replication, transcription of the RNA, folate metabolism, activities of protein kinases A and C, the purine cycle, excretion, absorption and secretion, and other processes considered essential for life. Parasites are very sensitive to the interference of these processes, and therefore, compositions that are specifically directed to these reactions can be used to selectively eliminate an infection or improve pathogenic symptoms associated with a parasitic infection by reducing parasitemia. without unnecessary damage to the host.

Likewise, US2004254094 discloses cyclin-dependent kinase inhibitors to block the replication of any virus, bacterium or parasite dependent on CDK activity for proliferation. The use of such inhibitors blocks DNA replication of the pathogen. The candidate substance may be a small molecule inhibitor, a protein or fragment thereof, or even a nucleic acid molecule or portions thereof, for example, nucleoside analogs.

For its part, document WOO 134201 discloses a method to inhibit, prevent or treat the clinical manifestations of neurofibromatosis type 1 (NF1), for example, in cells that are heterozygous or homozygous by administering an agent that restores normal intracellular signaling. as well as growth control. In this, a method for identifying agents that inhibit the activity of different proteins such as Raf, MEK, Rae, PAK1, PAK2, ERK2, ERK1, P13K, among others, is particularly known. Another is the case of document US2011059934 which discloses a class of new compounds and pharmaceutical compositions comprising a kinase inhibitor for treating or preventing diseases or disorders associated with a parasite. In particular, such compounds can be used to treat malaria, leishmaniasis and / or Chagas disease.

Compositions and methods for eliminating, inhibiting growth and / or inhibiting the reproduction of a kinetoplast or apicomplex protozoan are also disclosed in US2010311742. Such compositions comprise an enzyme tyrosine kinase inhibitor, which contributes to cell proliferation, cell differentiation and carcinogenesis.

For its part, US2010055201 discloses methods and compositions for the treatment and prevention of parasitic infections, using diindolylmethane-related indones, which selectively promote apoptosis of parasite-infected host cells. The direct pro-apoptotic activity and the interaction of diindolylmethane-related indones with other anti-parasitic and pro-apoptotic agents allow to be more effective in chemotherapy and to be complementary to the co-administration of anti-protozoal vaccines.

Other documents are specifically aimed at the treatment of leishmaniasis and trypanosomiasis, as does WO / 2008/022422, which is related to pharmaceutical compositions that inhibit signaling pathways that make possible the integration of Trypanosoma cruzi cDNA in the host genome.

Finally, document US9101573 is related to obtaining new anti-inflammatory, immuno-regulator and ethical anti-diabetic drugs that comprise component C lantionin synthetase (LANCL) proteins as therapeutic targets. In order to find and discover new medications against inflammatory diseases and disorders several were performed experiments, and compounds, new drugs and readapted drugs that bind to LANCL2 / PPAR were obtained. However, it should be noted that unlike the present invention, these novel medications are not directed to the treatment of leishmaniasis and Chagas disease.

Notwithstanding the foregoing, a drug that inhibits the enzyme kinase AKT-like (PKB) of the parasites Leishmania spp and Trypanosoma cruzi for the treatment of leishmaniasis and Chagas disease is not disclosed in the literature. Therefore, the technical problem that solves the present invention consists in the finding of new compounds that result in a new therapeutic alternative for leishmaniasis and Chagas disease, which are obtained by a process of searching for compounds that act on protein kinases specific.

3. BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the obtaining and development of compounds and medicaments for the prophylaxis and treatment of leishmaniasis and chagas disease, diseases caused by parasites Leishmania spp. and Trypanosoma cruzi.

AKT - // 7ce kinase in trypanosomatids, is a homologous protein of human serine / threonine protein kinase (PKB) or AKT that could be involved in the survival and anti-stress cellular response of these parasites. Through bioinformatics methodologies, two specific chemical inhibitors directed against the pleckstrin (PH) domain of the AKT - // ke (PKB) protein were identified, common domain to all Leishmania spp and T. cruzi species: the UBMC1 inhibitor had an energy interaction-free of -10.4 kcal / mol for AKT - // ke of L. panamensis and -7.9 for AKT- like of T. cruzi and the UBMC4 inhibitor that has an interaction-free energy of - 8.2 kcal / mol for the AKT - // ke of L. panamensis and of -6.3 for the AKT - // ke of T. cruzi. 4. BRIEF DESCRIPTION OF THE FIGURES

F1GURE í. Transmission Electron Microscopy of promastigotes of

Leishmania panamensis incubated with UBMC4.

F1GURA 2. Transmission Electron Microscopy of Epimastigotes of

Trypanosoma cruzi incubated with UBMC4.

F1GURA 3. Evaluation of the% hypodiploidia of the compounds UBMC1 and UBMC4 in promastigotes of Leishmania panamensis.

F1GURE 4. Evaluation of mitochondrial membrane potential in promastigotes of Leishmania panamensis treated with UBMC1 and UBMC4.

F1GURE 5. Genotoxicity of promishigotes of Leishmania panamensis incubated with UBMC1.

F1GURE 6. Genotoxicity of promastigotes of Leishmania panamensis incubated with UBMC4.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the obtaining and development of specific inhibitors of the enzyme kinase AKT-like parasites Leishmania spp and Trypanosoma cruzi, for the prophylaxis and treatment of leishmaniasis and Chagas disease, diseases caused by these parasites.

In order to obtain these specific compounds, a search strategy was developed through computational tools and the study of molecular signaling pathways that allowed the identification of two chemical compounds present in a database containing millions of chemical compounds

(hilpy / z-no.dock-ng.org /).

The search method consists in first identifying a therapeutic target, that is, selecting a white protein from hundreds of proteins that a parasite can have. For this, the latest advances in cancer therapeutics disclosed in Wu DW et al, 2016 and Politz O, et al, 2017 are used and are extrapolated to the field of parasitology.

To obtain optimal compounds and / or medications for the treatment of leishmaniasis and Chagas disease, the promising targets studied by the pharmaceutical industry that are related to the specific inhibition of protein kinases are selected. Taking into account the above, the search for the appropriate kinase is performed to be inhibited in Leishmania spp. and Trypanosoma cruzi together. The process consists of a computational search that aims to identify the AKT-like kinase of the parasites from their sequenced genome, then a 3D structure of the kinase is generated by computational methods (l-tasser), as it does not have a structure crystalline to work in the dockig-molecular, using the AutoDock Vina program. Once the structure is available, a specific place of interaction between the PH domain of the kinase and the chemical compound is identified. This strategy has as a theoretical basis the doctoral thesis work of Varela, R.E., 2013 of the University of Salamanca-Spain, which is described in more detail below.

According to previous studies by Varela, R.E., 2013, it was shown that a kinase inhibitor used in cancer also has an effect on the parasites studied and therefore, it is concluded that homologous proteins may exist between signaling pathways.

According to the findings found, the signaling route to be studied is the Pl3K / AKT / mTOR route, where the main kinase is the AKT protein, a protein involved in the survival and cellular response anti stress of these parasites for being an anti-apoptotic kinase. Additionally, not only is the AKT-like kinase identified, but it is also possible to identify a specific kinase site that could be inhibited without affecting other kinases. This specific site is known as the pleckstrin (PH) domain.

Using a bioinformatic approach that includes the modeling of the 3D structure (l-Tasser) of the AKT - // 7ce protein (RAC) and molecular docking (AutoDock Vina program) against a sub library of 600,000 compounds (ZINC ¹² database) they look for specific chemical inhibitors against the pleckstrin (PH) domain of the AKT - // 7ce (RAC) protein of L. panamensis and T. cruzi.

The best 10 complexes obtained from the PH-ligand domain are subjected to a Molecular Dynamics (MD) analysis and docking resulting in the UBMC1 inhibitor with an interaction-free energy of -10.4 kcal / mol for the AKT - // 7ce of L panamensis and -7.9 for the AKT - // 7ce deT. Cruzi Subsequently, a structural homolog of the UBMC1 inhibitor is sought, giving rise to the UBMC4 inhibitor that has an interaction-free energy of -8.2 kcal / mol for the AKT - // 7ce of L. panamensis and -6.3 for the AKT - // 7ce of T. cruzi.

Bioinformatics Methodology

Sequence analysis and 3D modeling of the AKT-like of Leishmania panamensis using bioinformatics tools

From the previous cloning and sequencing of the AKT-like protein of Leishmania panamensis, it is compared with the homologous protein of L. braziliensis, L. major, L. infantum. For this purpose multiple sequence alignment is performed through the Omega Clustal program (Sievers et al, 2011). In parallel, alignment is performed using BLAST (Basic Local Alignment Search Tool) against the AKT protein from other organisms including the human. Percentages of sequence identity are calculated and identification of conserved motifs of the protein in the different alignments is performed.

Based on the annotated sequence of the Leishmania panamensis AKT-like, (GenBank KP258183) the 3D structure of the protein is modeled, using the I-TASSER web server (Roy et al, 2010). This modeling approach is selected due to the lack of unique templates with adequate identity percentages (> 60%), which limits modeling based on protein homology. Through this template-based modeling strategy through l-TASSER, a protein structure model can be established, which exposes its plecsktrin (PH) domain. The quality of the resulting model is analyzed using the Ramachandran point diagram and the energy stability curves on the SwissModel server (Biasianini et al, 2014) before and after subjecting the protein to the minimization protocol with UCSF Chimera version 1.10 (Petersen et al, 2004).

Virtual screening of potential AKT inhibitors - // ke of Leishmania panamensis

A set of approximately 600,000 compounds is evaluated using a virtual screening protocol against the structure of the AKT - // ke of Leishmania panamensis. The sub-library of compounds is selected from the Zinc database (lrwin and Shoichet, 2005) and a filter of all the compounds evaluated is made based on the rule of five of Lipinski (Lipinski CA, 2004). With respect to the molecular docking site, a particular region of the protein around the pleckstrin (PH) domain is selected as a potential allosteric site within the kinase. Prior to the start of the simulations, protein and ligand are parameterized with the AutoDock Tools program (Morris et al, 2009) by adding hydrogen bridges to the polar side of the chain and partial estimation of the charges using Gasteiger's methodology. Additionally, the ligands are configured in a flexible conformation based on the active twisting of the bridges present in the 3D structure. After the parameterization of the inputs, the conjugates are they submit to the molecular docking protocol using the AutoDock Vina program (Trott et al, 2010) using an exhaustivity of 20. The results of the virtual screening are initially filtered using the binding-free energy score between the protein and the ligand, in the Docking scale, where 0 is poor interaction and - 14 is the best interaction. From the interactions performed, the compounds with the best interaction energy score are selected and subsequently sent for chemical synthesis. Through a virtual screening against a sub library of compounds of the Zinc database, molecular docking is done against the pleckstrin domain of the AKT - // ke of L. panamensis, where the first 14 compounds that had the best molecular docking score, on a scale of 0 to -14, where 0 is a poor interaction between the compound and the protein and -14 is the best interaction.

Compounds for the prophylaxis and treatment of leishmaniasis and Chagas disease

Taking into account the previous methodology, compounds with different scores in molecular docking were obtained and those that presented the best interaction between the compound and the protein were selected.

Thus, in one embodiment of the invention, compound UBMC1 is a compound of formula (1):

(I) en donde

(I) where

 X is a heteroatom selected from the group O, N or S;

 Y is a heteroatom selected from the group O, N or S;

 Z is a heteroatom selected from the group O, N or S; Y

A is selected from the group of sulfhydryl, alkylsulifda, sulfonyl or sulfoxide.

In another embodiment of the invention, compound UBMC1 is a compound of formula

(I):

en donde

where

 X, Y, Z are N, where N can form a primary amine, a secondary amine or a tertiary amine; Y

A is a sulfonyl group.

In a new embodiment of the invention, the UBMC4 compound is a compound of formula (11):

(II)

where

 X is a heteroatom selected from the group O, N or S;

 Y is a heteroatom selected from the group O, N or S;

 Z is selected from a group of H, hydroxy, alkoxy, alkoxide ester, or alkylcarboxyl; and A is a heteroatom selected from the group of O, N or S.

In a further embodiment of the invention, the UBMC4 compound is a compound of formula (11):

en donde

where

 X, Y, are N, where N can form a primary amine, a secondary amine or a tertiary amine;

 Z is a hydroxy; Y

A is an S.

In another embodiment of the invention, the compound compound UBMC1 is a compound of formula (111):

A new embodiment of the invention, the UBMC1 compound is the compound 3- [4- (5,5-dioxidedibenzo [b, d] thien-2-yl) -5-phenyl-lH-imidazol-2-yl] -3a , 7a-dihydro-lH-indole.

In another embodiment of the invention, the compound compound UBMC4 is a compound of formula (IV):

(IV)

In a further embodiment of the invention, compound UBMC4 is compound 4-

(5-phenyl-4- {8-thiatriccyclo [7.4.0.0 ² , ⁷ ] trideca-l (9), 2 (7), 3,5,10,12-hexaen-4-yl} -lH- imidazole- 2-yl) phenol.

A preferred embodiment of the invention includes a pharmaceutical composition comprising the compound of formula (111), the compound of formula (IV) and excipients.

So far there is not a single compound or drug in the world that is used against parasites using the described methodology, and no AKT-like kinase inhibitor of parasites has been identified.

The main advantage in the development of specific inhibitors for the AKT-like protein of Leishmania panamensis and Trypanosoma cruzi is that these new compounds are targeted treatments, that is, knowing the therapeutic target, structural modifications can be made directly that improve the affinity of the compound with the cell target in the parasite, which is novel since it has not been possible with current medications. Its production can be economical, less than USD $ 2422/100 mg of UBMC1 and USD $ 295 / 100mg of UBMC4, with the advantage that the UBMC4 compound has better pharmacokinetic properties than UBMC1 and that they are suitable for use in patients, because they have low toxicity and comply with the Lipinski rule (Lipinski, CA, 2004) that is, compounds that due to their chemical characteristics can be supplied in a composition oral. This is very important for the control of leishmaniasis and Chagas disease because it reduces treatment costs and increases adherence to treatment.

Antiparasitic activity assays of intracellular compounds in vitro IC50 and their cytotoxic effects LC50 in different human cells indicate that this drug has great potential to be used in patients. In addition to the above, these compounds could also function as temporary prophylactics for tourists visiting tropical areas. On the other hand, these inhibitors could be used in combination therapies to improve their effectiveness and avoid the emergence of resistance, which is currently presented in medications used for the treatment of leishmaniasis and Chagas disease.

The two chemical compounds identified have similarities and differences in their chemical structure. In the similarities of the compound it is understood that the pharmacophore of the compound is present, that is, its antiparasitic part.

6. EXAMPLES

Example 1. In vitro evaluation of the leishmanicidal activity of inhibitors in intracellular amastigotes of L. panamensis

The activity of the compounds was evaluated in intracellular amastigotes of Leishmania panamensis transfected with the green fluorescent protein (GFP) gene (strain MHOM / CO / 87 / UA140-pIR-eGFP). For this, the U937 cells were adjusted to a concentration of 300,000 cells / mL maintained in RPM1 1640 at 10% SFB medium and treated with 0.1 pg / mL forbol myristate acetate (PMA); they were deposited in each well of a dish for cell culture of 24 wells. After 72 hours of incubation at 37 ° C, 5% C02 to favor adhesion and differentiation to macrophages of U-937 cells, the cells were infected with promastigotes of L. panamensis in stationary phase of growth in a 15: 1 ratio parasite: cell. The cells and the parasites were incubated for three hours at 34 ° C, 5% C02. Non-internalized parasites were removed by washing with PBS. New medium was added and the infected cells were incubated again at 34 ° C, 5% C02.

After 24 hours, the medium was removed and new medium was added containing each concentration of the compound to be evaluated. The compounds were evaluated at a concentration of 50 pg / mL and the percent inhibition was determined. It is important to clarify that those compounds that showed cytotoxic activity at a CE <20 pg / mL, were evaluated at 5 pg / mL. After 72 hours of incubation in the presence of the compounds, their effect on the viability of intracellular amastigotes was determined by flow cytometry. For this, the infected and treated cells were detached from the bottom of each well and the suspended cells were read in the flow cytometer (Cytomics FC 500MPL) at 488 nm excitation and 525 nm emission counting at least 10,000 events (59) .

Subsequently, the compounds that showed% inhibition> 50% at the initial concentration evaluated of 20 pM / mL were evaluated at four base concentrations 1 in 4. The tests were carried out at least twice with three replicates for each concentration evaluated.

Example 2. In vitro evaluation of the cytotoxic activity of inhibitors

Cytotoxic activity was evaluated for compounds UBMC1 and UBMC4 since there were no data of this nature for these compounds in the cell lines of interest. In the cytotoxicity assays the following cell lines were used: Human liver cells (HepG2) and human rectal colon epithelial carcinoma cells (CaCo2). In addition, primary cultures of macrophages derived from human monocytes (huMDM) obtained from healthy donors were used, after signing the informed consent. Cell lines were maintained under standard culture conditions at 37 ° C with 5% CO2. HepG2 and CaCo2 cells were cultured in DMEN medium with 5% fetal bovine serum (SFB) and antibiotics. All cell types were kept in incubation at 37 ° C and 5% C02. The huMDMs were obtained from peripheral blood monocytes from a healthy volunteer donor. To this end, 50mL of defibrinated whole blood was mixed in a 1: 1 ratio with saline phosphate buffered (Dulbecco's phosphate saline-DPBS) free of calcium and magnesium; This mixture was centrifuged in a Ficoll-Hypaque 1077 density gradient in a 3: 1 ratio (blood-ficoll) for mononuclear separation, centrifuging at 200 rpm for 20 minutes at 22 ° C. The phase corresponding to mononuclear was taken, the cells were washed twice with DPBS solution, centrifuging each time at 1300rpm for 10 minutes. After the last wash, the cells were resuspended in RPM1 1640 medium with 10% autologous serum at a concentration of 0.3xl0 ⁶ cells per mL. In each well of a 24-well cell culture dish, one mL of the cell suspension was deposited and the dish was incubated at 37 ° C and 5% C02 for 72 hours to allow differentiation of monocytes to macrophages. The cytotoxic activity of the compounds was evaluated according to the ability to cause death of mammalian cells HepG2, CaCo2 and on primary cultures of huMDM using the MTT method 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazole, Sigma ), following the procedure described by other authors (Robledo et al, 2005).

For the MTT method, HepG2, CaCo2 and huMDM cells were adjusted to a concentration of 0.1 x 10 ⁶ cells / mL for suspended cells, 0.25 x 10 ⁶ cells / mL for adherent cell lines and 0.5 x 10 ⁶ cells / mL for huMDM, in RPM1 1640 medium supplemented with 10% fetal bovine serum (SFB) and the corresponding concentration of the compound. For each of the compounds, six double serial concentrations from 40 pg / rnL were evaluated. In each well of a 96-well cell culture dish, 100 pL of cells and 100 pL of each compound were dispensed at the respective concentration. The dish was incubated at 37 ° C, 5% C02 for 72 hours. Then 20 pL of MTT (5 mg / mL) was added to each well and the dish was incubated again at 37 ° C for three hours. The amount of formazan produced by viable cells was quantified according to the optical densities (OD) obtained in an EL1SA reader (Bio-Rad), with readings at 570 nm. As control of Cytotoxicity cells exposed to Amphotericin B®, Chloroquine® and Doxorubicin were used. As viability control cells grown in medium alone were used. Each concentration was evaluated in triplicate in two different trials. Each concentration of the compound, as well as the blank, was evaluated in triplicate, in two different experiments.

Example 3. Determination of lethal concentrations 50 (CLso), inhibition percentages and effective concentrations 50 (CEso) of the compounds.

The cytotoxicity was determined according to the percentages of viability and mortality obtained for the experimental condition (compounds, positive control drugs, medium only) and the results were expressed as Lethal concentration 50 (LC50), that is, the concentration at which the 50% cell death, calculated by Probit analysis, a parametric linear regression method that allows the analysis of the dose-response relationship (JD, 1978). The viability percentage was calculated using the following formula, where the optical densities (D.O) of the control well correspond to 100% viability:

D.0 exposed cells

 D.0 control cells

In turn, the percentage of mortality corresponds to 100 -% viability. The lethal concentration 50 (LC50) was then calculated for each compound by the Probit method (JD, 1978) using as data the% viability and mortality of the cells obtained for each concentration evaluated in each cell type (CaCo2 and HepG2).

For leishmanicidal activity, the percentage of infected cells was first calculated based on the number of positive events green fluorescence (parasites) and cells by point diagram analysis, as well as the parasitic load according to the analysis of the average fluorescence intensity (1FM) for the green fluorescence channel. Then, the percentage of infection inhibition obtained for each compound at the concentration of 20mM was calculated using the following formulas:

Infected cells in the presence of compound

 % infection = - x 100

 Infected cells in the absence of compound (negative control)

In turn, the percentage of inhibition corresponds to 100% infection. The Effective Concentration 50 (EC50) was then calculated for each compound by the Probit method (JD, 1978) using as data the% infection and infection inhibition obtained for each concentration evaluated for L. panamensis.

Example 4. Analysis of the phosphorylation of AKT- / zfce in promastigotes of L. panamensis subjected to cellular stress.

To obtain proteins, 1.5xl0 ⁷ promastigotes were lysed with 180pL lysis buffer (150mm NaCl, 10mm HEPES, 1% CHAPS and O.lmM sodium orthovanadate), supplemented with protease inhibitor. Between 40-60pg of total protein extract was run on a 12% SDS-PAGE polyacrylamide gel. The proteins separated in the gel were transferred to a PVDF membrane (Thermo Scientific 0.45pm 26.5cm x 3.7cm), blocked with 5% skimmed milk powder (% w / v) in TBST IX buffer (50mM TR1S, 150mM NaCl and 0.1% (% v / v) of Tween 20, pH 7.6) overnight at 4 ^Q C under constant agitation and incubated at 4 ^Q C for 12 hours with the following antibodies: total anti-AKT that recognizes a sequence of 15 amino acids and anti-AKTp, which recognizes phosphorylated threonine in a sequence of 15 amino acids, 1: 1000 dilution in TBST IX with 5% milk. The reactivity of the antibodies was checked with an anti-rabbit lgG antibody conjugated to horseradish peroxidase (HRP) (Sigma Aldrich), using chemiluminescence (Super signal west peak chemiluminescent substrate, Thermo Scientific) using auto-radiography films (CL-XPOSURETM Film Thermo Scientific 18cm X 24cm, clear blue X-Ray Film) to visualize the immunoreactivity of the bands.

Example 5. In vitro evaluation of AKT- / zfce inhibitors computationally identified under conditions of cellular stress in promastigotes of L. panamensis

Of the compounds chemically synthesized and evaluated in promastigotes and intracellular amastigotes of L. panamensis subjected to cellular stress, only two showed good activity; in their order, were the compounds encoded as UBMC1 and UBMC4, which at IOmM of final concentration and in just 6 hours showed inhibitory effect on the parasites in culture. The analysis of cell cycle and mitochondrial membrane potential, showed a normal control (+ SFB); with 7% of hypodiploid parasites, a control without SFB (-SFB) hypodiploidy of 3%, among the non-serum deprived parasites and treated with the UBMC1 inhibitor, 29% hypodiploidy was evident and in those depleted serum (stressed) and treated with this inhibitor, 64.3% hypodiploidy was observed. With respect to the UBMC4 inhibitor in the case of non-private parasites of SFB and treated hypodiploidy of 34.3% was observed, while those deprived parasites of SFB (stressed) and treated showed hypodiploidy of 52.7% as illustrated in Figure 3.

Regarding the evaluation of the mitochondrial membrane potential, it should be mentioned that the normal promastigote control showed D10C6 positive cells equal to 5%, while the private control of SFB showed 44.6% positive cells. In the non-depleted parasites of SFB and treated with UBMC1 25.8% were positive for D10C6, while those depleted of SFB (stressed) and treated only 5.5% were positive for D10C6. On the other hand, those non-depleted parasites of SFB and treated with UBMC4, 19.3% were positive for D10C6 while in the group of deprived of SFB (stressed) and treated with UBMC4 15.3% of positive cells were obtained in the mareaje with D10C6, as illustrated in Figure 4. Example 6. In vitro evaluation of compounds UBMC1 and UBMC4 in Leishmania panamensis and Trypanosoma cruzi

The two inhibitors were evaluated in vitro under different conditions of cellular stress: Nutritional (serum deprivation) and thermal (temperature rise to 37 ° C) and compound activity was determined by flow cytometry for cell cycle and mitochondrial membrane potential (IP and DIOC6).

It was observed that these compounds induce in vitro in the parasites loss of mitochondrial membrane potential> 90%, changes in morphology and high percentages of cells with hypodiploidy.

The antiparasitic activity of the intracellular compounds in vitro was also determined, evaluating the IC ₅₀ inhibitory concentration in intracellular amastigotes in U-937 macrophages infected with promastigotes that are subsequently transformed into amastigotes (Table 1) (Robledo S., et al., 2005 ).

Table 1. Result of the leishmanicidal activity of the UBMC1 and UBMC4 inhibitors in intracellular amastigotes of L. panamensis (IC50).

a IC ₅₀ Inhibitory concentration in amastigotes of L. panamensis.

Table 1 shows that the UBMC4 compound inhibits 50% of the parasite population at a concentration less than 5 micromolar, being more active against T. cruzi. For the compound identified as UBMC1, a larger dose close to double is needed to obtain the same result as the UBMC4 compound. However, greater antiparasitic activity against T. cruzi is also observed. On the other hand, a virtual prediction of the chemical properties of compound UBMC1 and UBMC4 was made (Table 2).

Table 2. Virtual Prediction of the chemical properties of the compounds

 PM <500: Molecular weight less than 500 Dalton

 logDs <5: Distribution coefficient under physiological conditions

 logP <5: Water distribution coefficient

 N + 0 <10: Accept Hydrogen

 Donor H <5: Hydrogen donor

 USD / 100 mg: Price in dollars for each lOOmg

Table 2 shows some of the chemical characteristics of the compounds when compared to each other. The UBMC4 molecule has a lower molecular weight because it has a smaller number of heterocyclic groups in its structure. This difference is also evident in the LogP and LogD partition coefficient between the molecules, where it is evident that the UBMC4 compound is less hydrophobic by losing these chemical groups. Additionally, the cost of compound UBMC4 is eight times lower because part of the structure of compound 1 that made its synthesis more expensive, is no longer essential to maintain the biological activity of the compound.

Additionally, a comparison was made between the toxicity observed between the compounds and the different exposed cell lines. Being these intracellular parasites it can be seen that the compounds also have an action in the intracellular form.

Table 3. Cytotoxicity result (LC50) of the UBMC1 and UBMC4 inhibitors in hepatocellular carcinoma (HepG2), colorectal carcinoma (CaCo2) cell lines and primary macrophage derived cultures of human monocytes (hMDM).

 to IC50 Inhibitory concentration in amastigotes of L. panamensis

 b LC50 Lethal concentration.

Table 3 shows the values of the intracellular activity of chemical compounds in cells that parasites normally infect during their life cycle. The intracellular form of the parasite is called amastigote, it is housed and divided into the parasitophous vacuole of macrophages to cause disease. The UBMC4 compound showed greater intracellular activity than the UBMC1 compound, with approximately half the dose being needed to obtain the same antiparasitic effect as the UBMC1 compound. Additionally, the cytotoxicity of these compounds in different human cell lines was evaluated to predict the possible toxic effect or not of the compounds in cells of different organs. Caco2 cells that are located in colon epithelium and HepG2 cells of liver epithelium were evaluated and had no toxicity associated with either compound. In the case of the UBMC1 compound, this showed a lower selectivity index in a human macrophage line compared to the UBMC4 compound, but in any case the parasitic activity of the compounds is less than that necessary to be toxic in the evaluated cells. Consequently, the chemical compounds showed low toxicity to human macrophages and other human cells (Table 3).

Experimentally, a strong biological activity of the compound was observed against intracellular forms of the parasites (Table 1), and as mentioned, its low toxicity in human cells was demonstrated in macrophages derived from human-hMDM monocytes (Table 3). Likewise, it was proved that the compounds do not inhibit the Human AKT enzyme when found constitutively expressed for example in tumors in hepatocellular carcinoma-HepG2 and colorectal epithelial adenocarcinoma-Caco-2 (Table 3). Something that should be highlighted is the index of selectivity of the UBMC4 molecule which can be> 8.7, which demonstrates the good properties of this molecule to be used in humans. It was also shown that the computational model is capable of predicting compounds with biological activity, and that the AKT-like kinase exists in parasites, particularly in L. panamensis and T. cruzi.

In this way, the pharmacophore is reached with the structural differences of the chemical compounds. It was observed that the mechanism of death is through mitochondrial damage and this generates an apoptosis-like death. Finally, it was also shown that the location of the protein is cytoplasmic.

Example 7. Transmission electron microscopy

Transmission electron microscopy allowed to observe the ultra-structure of the treated parasites (promastigotes of L. panamensis) and the changes within them; for example, Figure 1A shows promastigotes cultured in RPM1 medium supplemented with 10% SFB (unstressed parasites) and normal morphology of the promastigote nucleus and cytoplasm (1A); however, in the Figures IB, 1C and ID, the changes that the parasites treated with the UBMC4 IOmM inhibitor are shown, where it could be shown that with respect to the control the treated parasites suffer cytoplasmic shortening; that is, they change their elongated shape and take an oval morphology, nuclear morphology is lost in just one hour after treatment (Figure IB) to the point where the very vacuolated cytoplasm is observed where practically the nucleus is indistinguishable and has The scourge was completely lost two hours after treatment (Figure ID). Subjecting parasites to nutritional stress; that is, no longer cultured in RPM1 medium supplemented with fetal bovine serum; but in PBS buffer, the same previous test was performed. On the other hand, in Figure 1A control parasites are observed, on the contrary showing a round nucleus (N) with peripheral chromatin, compact Kinetoplast DNA (K) and some cytoplasmic lipid vacuoles (L) and the endoplasmic reticulum (ER) .

Contrary to what was observed in Figure 1A, in Figure IB parasites treated for 1 hour with UBMC4 are observed. There appear more electrodense lipid drops (L) and there is a slightly enlarged mitochondria (M), along with cellular vocalization.

In Figures 1C and ID, parasites treated for 2 hours with UBMC4 are observed. It shows drops of electrodense lipids (L) and enlarged mitochondria (M). There are also some parasites that have empty cytoplasms (D) with several vacuoles.

Figure 2 compiles the observations made in this regard in Epimastigotes of Trypanosoma cruzi incubated with UBMC4; where, for example, a control Epimastigote (2A) is observed. B C D. Epimastigotes treated with IOmM of UBMC4 per lh. E, F are epimastigotes treated with IOmM of UBMC4 for 2 hours, show fragmentation of kinetoplast and core DNA. The nuclear chromatin quite altered, condensed on the periphery of the nucleus / chromatin compacted and fragmented into pieces.

Compounds UBMC1 and UBMC4 are compounds effective against L. panamensis and T. cruzi that act against the AKT - // 7ce kinase which would be an important new therapeutic target.

Example 8. Comet test

Taking into account nuclear fragmentation and what was observed in chromatin and kinetoplast in the photographs of transmission electron microscopy, the test Comet or genotoxicity test performed on promastigotes of L. panamensis stressed, unstressed and treated with compounds UBMC1 and UBMC4, similar results were obtained. In the case of UBMC1, Figure 5 shows the average percentage of tail DNA in Leishmania panamensis cells analyzed by alkaline comet DNA assay. It was possible to show that although in the treatment group that corresponds to the normal control of cells the DNA in the tail is 10% and in the control -SFB (Stressed without treatment) it is 20%, in the group + SFB to IOmM of UBMC1 at two hours after treatment the DNA in the tail is almost 40%, while in the -SFB group treated with 10mM of UBMC1 at two hours after treatment as mentioned above it was not possible to determine the percentage of DNA in the comet's tail , because the damage generated was so great that it was not possible to quantify due to the lack of heads of nuclei and tails of the comet, so for this group this parameter could not be determined.

In the case of UBMC4, Figure 6 shows the average percentage of tail DNA in Leishmania panamensis cells analyzed by alkaline comet DNA assay. It was evidenced that although in the treatment group that corresponds to the normal control of cells the DNA in the tail is 20% and in the control -SFB (Stressed without treatment) it is less than 60%, in the group + SFB at 10mM of UBMC1 at two hours post treatment the DNA in tail is almost 60%, while in the private group of SFB and treated with 10mM of UBMC4 at two hours post treatment it was not possible to determine the percentage of DNA in tail of the comet , because the damage generated in the DNA was so great that it was not possible to quantify.

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Claims

1. A compound of formula (II):

where  X is a heteroatom selected from the group 0, N or S;  Y is a heteroatom selected from group 0, N or S;  Z is selected from a group of H, hydroxy, alkoxy, alkoxide ester, or alkylcarboxyl; and A is a heteroatom selected from the group of 0, N or S. 2. The compound according to claim 1, wherein:

(P) where  X, Y, are N, where N can form a primary amine, a secondary amine or a tertiary amine;  Z is a hydroxy; Y A is an S. 3. The compound according to the preceding claims, wherein said compound of formula (II) is 4- (5-phenyl-4- {8-thiacyclo [7.4.0.0 ² , ⁷ ] tridecal (9), 2 (7), 3,5,10,12-hexaen-4-yl} -lH-imidazol-2-yl) phenol. 4. A compound of formula (I):

where  X is a heteroatom selected from the group 0, N or S;  Y is a heteroatom selected from group 0, N or S;  Z is a heteroatom selected from the group 0, N or S; Y A is selected from the group of sulfhydryl, alkylsulifda, sulfonyl or sulfoxide. 5. The compound according to claim 4, wherein:  X, Y, Z are N, where N can form a primary amine, a secondary amine or a tertiary amine; Y A is a sulfonyl group. 6. The compound according to claims 4 and 5, wherein said compound of formula (I) is 3- [4- (5,5-dioxidedibenzo [b, d] thien-2-yl) -5-phenyl- lH- imidazol-2-yl] -3a, 7a-dihydro-lH-indole. 7. A pharmaceutical composition comprising the compound of formula (I), the compound of formula (II) and excipients. 8. The pharmaceutical composition according to claim 7, comprising 4- (5-phenyl-4- {8-thiacyclo [7.4.0.0 ² , ⁷ ] trideca-l (9), 2 (7), 3.5 , 10,12-hexaen-4- and l} -lH-imidazol-2-yl) phenol; 3- [4- (5,5-Dioxidedibenzo [b, d] thien-2-yl) -5-phenyl-l-imidazol-2-yl] -3a, 7a-dihydro-lH-indole and excipients.