ES2414291B2 - MACROCYCLIC COMPOUNDS OF SCORPING TYPE AND ITS USE AS ANTIPARASITARIES. - Google Patents

Abstract

Scorpiondo macrocyclic compounds and their use as antiparasitic. The present invention refers to macrocyclic compounds of the scorpion type, which acquire said nomenclature due to the particular shape of their chemical structure formed by a macrocyclic body and a hanging arm where different radicals can be substituted. The present invention further comprises the use of said compounds as medicaments, particularly in the treatment of diseases caused by parasites, preferably by Trypanosoma cruzi or Leishmania spp parasites, such as Chagas disease or Leishmaniasis.

Description

MACROCYCLIC COMPOUNDS OF SCORPING TYPE AND ITS USE AS ANTIPARASITARIES

FIELD OF THE INVENTION

The present invention refers to macrocyclic compounds of the scorpion type, which acquire said nomenclature due to their particular chemical structure formed by a macrocyclic body and a hanging arm where different radicals can be substituted. The present invention further comprises the use of said compounds as medicaments, particularly in the treatment of diseases caused by parasites, preferably by Trypanosoma cruzi or Leishmania spp. Parasites, such as Chagas disease or Leishmaniasis.

STATE OF THE TECHNIQUE

Parasitism is the biological association in which an organism (“the parasite”) lives on (ectoparasite) or inside (endoparasite) of the body of another organism (“host” or “host”) from which it obtains its nutrients. Thus, parasitism is a process by which a species improves its ability to survive using as a host individuals of other species to cover their basic and vital needs. The result of this association is mainly beneficial for the parasite, which translates into the improvement of its reproductive fitness (in English, reproductive fitness) and, generally, is detrimental to the reproductive fitness of the host.

As seen above, parasites can be classified into different groups based on their location in the host and the influence they exert on it. Thus, the parasites that live inside the host are called endoparasites and, those that live outside, are called ectoparasites. On the other hand, parasitoid parasites are characterized by their ability to cause the death of the host organism.

It has been shown that the parasite's survival is closely linked to the ability of its enzyme superoxide dismutase (FeSOD) to evade damage caused by toxic host radicals [S. Ghosh S. Goswami, S. Adhya, Biochem.

J. 2003, 369, 447]. Thus, the FeSOD of the parasite plays an important role as part of the antioxidant defense in parasites that develop diseases such as Chagas disease (Trypanosoma cruzi) or Leishmaniasis (Leishmania spp.) [N. Le Trang, S. R. Meshnick, K. Kitchener, J. W. Eaton, J. Biol. Chem., 1983, 258, 125].

Specifically, Leishmaniasis is a disease caused by different species of protozoa of the genus Leishmania. The clinical manifestations of the disease range from skin ulcers that heal spontaneously, to fatal forms in which severe inflammation of the liver and spleen occurs. The disease by its zoonotic nature, affects both dogs and humans. However, wild animals such as opossums, coatis and jurumíes among others, are asymptomatic carriers of the parasite, so they are considered as reservoir animals. The cutaneous form of the disease (cutaneous leishmaniasis) in humans is characterized by the appearance of painless skin ulcers in the area of the bite which can heal spontaneously or remain chronically for years. Visceral leishmaniasis is the clinical form that takes more lives worldwide. This presentation can be fatal if it is not treated on time. It is characterized by inflammation of the liver and spleen, accompanied by severe abdominal distension, loss of body condition, malnutrition and anemia.

The current treatment of Leishmaniasis is based on the intramuscular application of antimony-based drugs such as meglumine antimoniate (Glucantime®) for a period of 20 to 30 days. However, as demonstrated in the examples of the present invention, these drugs show high toxicity and have not proven effective in the chronic stage of the disease.

Leishmania specimens show two morphologies during their life cycle: Chagas disease is a tropical parasitic disease, usually chronic, caused by Trypanosoma cruzi. The natural reservoir is made up of armadillos, marsupials, rodents, bats and wild primates, as well as certain domestic animals such as dogs, cats, even rats and guinea pigs. It is commonly transmitted to man by hematophagous triatomines, such as the Triatoma infestans, which transmits the parasite when it defecates on the bite that it has made to feed itself. It can also be transmitted by transfusion of contaminated blood, by the intake of food contaminated by the parasite or vertically from the infected mother to the fetus. The insect that transmits this disease can become infected if it bites a person who has the infection, and thus acquire the ability to continue spreading this parasite.

!

Promastigote: elongated shape  with flagellum  previous. Is present  in  he

intestine and salivary glands of the invertebrate (vector).

!

Amastigote: spherical shape and with a very short scourge, which does not protrude from the

flagellar bag, so that it is only noticeable in the electron microscope.

It reproduces within macrophages and cells of the reticuloendothelial system of the

vertebrate host. Infections occur in the skin (skin), skin

and mucous membranes (mucocutaneous) or in the organs (visceral).

It is estimated that 15 to 17 million people are infected by Chagas disease every year, of which about 50,000 die. The acute childhood stage is characterized by fever, lymphadenopathy, enlarged liver and spleen and, occasionally, myocarditis or meningoencephalitis with a severe prognosis. In the chronic stage, which reaches between 30% and 40% of all chagasic patients, there is usually severe diffuse cardiomyopathy, or pathological dilation (megasyndromes) of the esophagus and colon, megaesophagus and megacolon, respectively.

The most commonly used medication for the treatment of Chagas disease is Benzinidazole (BZN) ®. However, as in the case of commercial drugs for the treatment of Leishmaniasis, and it is evident in the examples of the present invention, these drugs are not very effective, mainly in the chronic phase (more than 30 days of treatment) of the disease and have a very high toxicity.

Trypanosoma cruzi has three different forms:

!

Amastigote: spherical or oval, is the reproductive form inside mammalian cells.

!

Epimastigote: elongated and with the cinetoplast located before the nucleus, it is the reproductive form in the digestive tract of invertebrates and in culture media.

!

Trypomastigote: also elongated, but with the cinetoplast located subsequently to the nucleus. It is found in the blood of mammals and is the infective form of them. This form is not divided.

Thus, although Leishmaniasis and Chagas disease are diseases caused by parasites that affect millions of people around the world, there are currently very limited therapeutic remedies of doubtful efficacy. In addition, as explained, those that are used entail serious problems, since their effectiveness is variable, being long and expensive treatments, and, in addition, they are associated with serious toxic effects.

Therefore, given the incidence and relevance of the diseases mentioned above, it seems necessary to use antiparasitic compounds capable of treating such diseases effectively at all stages of the process, including the chronic stage, and which, in addition, does not are toxic to patients, or at least have a reduced toxicity with respect to the reference compounds currently used.

DESCRIPTION OF THE INVENTION

The present invention solves the technical problem explained above through the use of macrocyclic compounds of the scorpion type for the treatment of diseases caused by parasites, preferably by the parasites Trypanosoma cruzi or Leishmania spp, such as Chagas disease or Leishmaniasis.

The compounds used in the present invention as antiparasitic are compounds of formula (I):

(I) where R can be H or a hydrocarbon chain comprising the substituents R1 and / or R2, where R1 is an amino group substituted by R2 or unsubstituted, R2 comprises

10 carbocycles and / or heterocycles that may be substituted by R3 or unsubstituted, and R3 comprises formula (I) where R is a hydrocarbon chain.

As shown in the examples, the present invention makes a comparison of

the compounds of general formula (I) against Benzinidazol® (BZN) and Glucantime® 15 which are reference drugs used in the treatment of diseases

caused by Trypanosoma cruzi and Leishmania spp respectively. How I know

evidence in the examples, the compounds used in this invention are

structurally different from said reference drugs, they have a toxicity

about ten times lower and show results of antiparasitic activity 20 in both the acute and chronic phases. In a preferred aspect the carbocycle is the

Benzene and the heterocycle is pyridine.

In a preferred aspect, the compounds used in the present invention as

Antiparasitic compounds are compounds of formula (I), where R is a radical selected from the group consisting of H, - (C1-C20) -NHR1, where the hydrocarbon chain is linear, branched, saturated or unsaturated and - (C1-C20 ) -R2, where the hydrocarbon chain is linear, branched, saturated or unsaturated; where R1 is a radical selected from the group consisting of -H and - (C1-C20) where the hydrocarbon chain is linear, branched, saturated or unsaturated and where the terminal carbon of said chain has a substituent comprising 1-7 rings, the rings being saturated, partially unsaturated or aromatic, isolated or condensed, where the rings have 3-8 elements, each of these elements independently selected from the group consisting of C, CH, CH2, N, S, NH, NH +, C = O, O, C-R3; where R2 is equal to R1 except -H; where R3 is - (C1-C10) -R4; and where R4 is:

In an even more preferred embodiment, the compounds used in the present invention as antiparasitic are the following:

(II)

(III)

(IV)

(V)

(SAW)

(VII)

(VIII)

(IX)

(X)

(XI)

(XII)

(XIII)

(XIV)

(XV)

(XVI)

(XVII)

(XVIII)

The use of the compounds of formula (I) as antiparasitic agents is preferably carried out against Chagas disease (caused by Trypanosoma cruzi) or Leishmaniasis (caused by Leishmania spp.). Since the parasite's survival is closely linked to the ability of its enzymes (FeSOD) to avoid damage caused by the toxic radicals of its host and, therefore, the parasite's FeSOD plays a relevant role as part of the defense. Antioxidant in the parasites that develop said diseases, in the present invention compounds of formula (I) capable of inhibiting the FeSOD of the parasite have been used, limiting their antioxidant defense against toxic host radicals.

As shown in the examples, the present invention compares the compounds of general formula (I) against Benzinidazol® (BZN) and Glucantime® which are reference drugs used in the treatment of diseases caused by Trypanosoma cruzi and Leishmania spp respectively. As evidenced in the examples, the compounds used in this invention are structurally different from said reference drugs, have a toxicity about ten times lower and show antiparasitic activity results in both the acute and chronic phases.

Although, as demonstrated in Example 15, Tables 1-3, all the compounds that share the formula (I) are effective in the treatment of diseases caused by parasites, while maintaining a low level of toxicity (substantially less than the reference compounds), the particularly preferred compounds of the present invention for use as an antiparasitic are the compounds of formula (II), (IV) and (V) which have an even lower toxicity level than the rest of Scorpio-type macrocyclic compounds studied in the present invention, obtaining for the compound (V) the highest percentage reduction in the number of parasites.

Thus, the first aspect of the present invention refers to the use of a compound of formula (I), where R can be H or a hydrocarbon chain comprising the

R1 and / or R2 substituents, where R1 is an amino group substituted by R2 or unsubstituted, R2 comprises carbocycles and / or heterocycles that may be substituted by R3 or unsubstituted, and R3 comprises formula (I) where R is a chain hydrocarbon, preferably using a compound of formula (I), where R is a radical selected from the group consisting of H, - (C1-C20) -NHR1, where the hydrocarbon chain is linear, branched, saturated or unsaturated and - (C1-C20) -R2, where the hydrocarbon chain is linear, branched, saturated or unsaturated; where R1 is a radical selected from the group consisting of -H and - (C1-C20) where the hydrocarbon chain is linear, branched, saturated or unsaturated and where the terminal carbon of said chain has a substituent comprising 1-7 rings , the rings being saturated, partially unsaturated or aromatic, isolated or condensed, where the rings have 3-8 elements, each of these elements independently selected from the group consisting of C, CH, CH2, N, S, NH, NH +, C = O, O, C-R3; where R2 is equal to R1 except -H; where R3 is - (C1-C10) -R4; Y

15 where R4 is:

, and even more preferably to the use of a compound selected from the compounds of formula (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X ), (XI),

20 (XII), (XIII), (XIV), (XV), (XVI), (XVII) and (XVIII), for the preparation of a pharmaceutical or veterinary composition for the treatment of diseases caused by parasites. Likewise, this first claimable aspect comprises any of the above compounds for use in the treatment of diseases caused by parasites, that is, as antiparasitic.

The compounds of formula (I) described in the present invention can be used as active ingredients in human patients or animals and can also be prepared in pharmaceutical compositions, or formulations, and administered, according to the knowledge in the state of the art of development. galenic, in different ways such as: topically, by intradermal injection or orally by capsules, dragees or tablets. Similarly, solid forms are made in the presence of the necessary excipients selected from the group comprising but not limited to: mannitol, polyvinyl pyrrolidone, microcrystalline cellulose, silica gel, talcum, magnesium stearate, titanium oxide, dyes and antioxidants. Therefore, the present invention also covers the pharmaceutical compositions themselves

or veterinarians comprising said compounds of formula (I) and, optionally, pharmaceutically acceptable excipients.

The compounds studied in the present invention overcome the problems cited in the state of the art by being effective in the chronic phase of the disease (see Example 16) and having a decreased toxicity with respect to the compounds used as reference (see Example 15). Thus, the compounds studied in this invention have lower IC50 values than the reference compounds, which has important implications because the lower said value, the smaller the amount of compound necessary to cause the desired antiparasitic effect. Therefore, the use of a smaller amount of compound to achieve this effect will significantly reduce its side effects, including toxicity.

In a preferred aspect the compounds used in accordance with the first aspect of the invention form a complex with Mn (II).

In another preferred aspect, the diseases treated by the compounds of the first aspect of the invention are Chagas disease or Leishmaniasis, which means that the compounds of the invention are effective against the parasites causing said diseases, namely Trypanosoma cruzi or Leishmania spp.

Another aspect of the invention refers to a method of treating diseases caused by parasites, such as Chagas disease or Leishmaniasis, which comprises administering to the patient a therapeutically effective amount of at least one of the compounds mentioned in the First aspect of the invention. In the present invention, therapeutically effective amount is understood as that which causes the treated disease to reverse or improve its symptoms.

The third aspect of the invention refers to a compound of formula (I), selected from the compounds of formula (X), (XI), (XIV) and (XV) and, in a preferred aspect, the use thereof To prepare a medicine. Alternatively, this preferred aspect also covers said compounds of formula (X), (XI), (XIV) and (XV) for use as a medicament. The last aspect of the invention refers to a pharmaceutical or veterinary composition comprising at least one compound of formula (X), (XI), (XIV) and

(XV) and pharmaceutically acceptable excipients mentioned above.

Brief description of the figures

Figure 1. This figure shows the percentage reduction in vitro in the number of trypomastigotes (infectious form of the Trypanosoma cruzi parasite) (ordinate axis) according to the different compounds of the invention tested at different concentrations (abscissa axis). It is observed that for the P3py compound (formula V), at a concentration of 50μM, the highest percentage reduction of the number of trypomastigotes is obtained.

Figure 2. In this figure, parasitemia (number of parasites in blood) in the acute phase of Chagas disease in mice is studied. The presence of trypomastigotes in blood is shown in mice (ordinate axis) as a function of the treatment time with the compounds of the invention in days (abscissa axis). The dose used of the compounds of the invention and the reference drug (BZN) is 5 mg / kg. It is observed that in mice where compound P (formula II), P2py (formula IV) and P3py (formula V) has been injected, the presence of parasites is quite minor, particularly with the compound P3py (formula V). Control (- ▲ -) and dose of 5 mg / Kg received from: BZN (-Δ-), P (- □ -), P2py (-∀-) and P3py (- ● -).

On the other hand, this figure shows the results obtained from the parasitemia curve, which shows that the compounds of formula II (P), formula IV (P2py) and formula V (P3py) are the most effective at the beginning of the chronic phase (at 30 days preferably at 60 days post infection) since they significantly reduce the number of parasites in the blood.

Figure 3. This figure shows a study of the activity of compounds P (formula II), P2py (formula IV) and P3py (formula V) in the capacity of infestation and the growth of T. cruzi parasites (axis of ordinates) depending on the treatment time in days (axis of abscissa). Control (- ▲ -), BZN (-Δ-), P (- □ -), P2py (- ♦ -) and P3py (- ● -). Measured at a concentration of IC25:

A) Infestation capacity.

B) Number of amastigotes (reproductive form of the parasite) per Vero cell

infected.

C) Number of trypomastigotes (infectious form of the parasite) in the middle of

culture.

In these graphs it can be seen that the compounds of the invention P (formula II), P2py (formula IV) and P3py (formula V), show a percentage of parasite growth inhibition greater than that of the reference drug, particularly in the case of P3py compound (formula V), and also decrease the infestation capacity.

Figure 4. This figure shows a study of inhibition of the different superoxide dismutase enzymes (Cu-ZnSOD, MnSOD and FeSOD) since the survival of the parasite is closely linked to the ability of the enzymes to avoid damage caused by toxic radicals of your host

A) In vitro inhibition (%) of the CuZn-SOD enzyme from human erythrocytes (activity 23.36 ± 4.21 U / mg). B) In vitro inhibition (%) of the Escherichia coli Mn-SOD enzyme (activity 18.12 ± 5.32 U / mg). C) In vitro inhibition (%) of the FeSOD enzyme of T. cruzi (epimastigotes) (activity 20.77 ± 3.18 U / mg).

It can be seen that the compounds of the invention are capable of inhibiting the parasite FeSOD enzyme with an IC50 value (concentration required to achieve 50% inhibition) lower than in the rest of the enzymes. Thus, it can be said that the compounds of the invention carry out a selective or specific action in the inhibition of the FeSOD enzyme of the parasite.

Figure 5. Images of the ultrastructural alterations taken by transmission electron microscope (TEM) in epimastigotes of Trypanosoma cruzi treated with the different compounds.

(one)

Control parasite of T. cruzi, shows organelles with their characteristic appearance, such as reservosomes (R), mitochondria (M), glycosomes (G), microtubules (MT) and flagellum (F). (Bar: 0.583 μ).

(2)

 Epimastigotes of T. cruzi treated with compound P (formula II) that appeared broken (see arrow) and with little electrodense cytoplasm of granular appearance (see arrow). (Bar: 1.00 μ).

(3)

 Epimastigotes of T. cruzi treated with P2py (formula IV) show swollen mitochondria (M) and cytoplasmic membrane scalloping (see arrow). (Bar: 1.00 μ).

(4)

 Epimastigotes of T. cruzi treated with P3py (formula V) show abundant acidocalcisomas (A), huge strongly electrodesos glycosomes (G) and large vacuoles (V). (Bar: 0.583 μ)

The conclusions obtained from the ultrastructural study indicate damage to the parasite's cellular structure as a result of the effect of the compounds studied in the present invention. The damage at the level of the organelles is key in the energetic metabolism of the parasite (mitochondria and glycosomes) which will cause cell infeasibility.

Figure 6. Representation of the 1H-NMR spectrum in T. cruzi epimastigotes treated with the compounds tested in the invention (IC25 concentration): (A) Control (without treatment); (B) Compound of formula (II) (P); (C) Compound of formula (IV) (P2py); (D) Compound of formula (V) (P3py). Lac (L-lactate), Ala (L-alanine); Ac (acetate); S (succinate); Eth (ethanol) and Gly (glycerol). A decrease in metabolites excreted by the parasite is observed, as a consequence of an alteration in its metabolism by the action of antiparasitic compounds.

Figure 7. This figure shows the effectiveness of the compounds P (formula II), P2py (formula IV) and P3py (formula V) in chronic phase. Parasitic antibody levels are shown that show the efficacy of the compounds of the invention, in the chronic phase of the disease (in vivo model in chronic phase). The decrease in parasitic antibody levels is indicative of the effectiveness of the compounds used in the present invention.

Detailed description of the invention

The examples set forth below are intended to illustrate the invention without limiting the scope thereof.

EXAMPLES

TESTS CARRIED OUT IN T. CRUZI

Example 1. Cultivation of parasites.

T. cruzi SN3 strain was isolated from IRHOD / CO / 2008 / SN3. This nomenclature refers to the host from which the parasite was isolated: I (insect), RHOD (rhodnius), the country (Colombia) and the year of isolation (2008). Specifically the

The parasite was isolated from Rhodnius prolixus from Guajira (Colombia) [Biological characterization of Trypanosoma cruzi stocks from domestic and sylvatic vectors in Sierra Nevada of Santa Marta, Colombia. Jair Téllez-Meneses, Ana María Mejía-Jaramillo, Omar Triana-Chávez Acta Tropica 108 (2008) 26–34, 2008)].

Epimastigote forms were grown in axenic medium (Grace's Insect Medium Gibco®) supplemented with 10% inactive fetal bovine serum (FBS) at 28 ° C in tissue culture bottles. To obtain the parasite suspension, the epimastigote culture (in exponential growth phase) was concentrated by centrifugation at 400 g for 10 minutes and the number of flagella was counted in a hemocytometer.

Example 2. Transformation of epimastigotes to metacyclic forms.

The passage to metacyclic trypomastigote (form found in the vector in the last section of the intestine and which are actually infective forms for the mammalian host) was induced by culturing the parasites at 28 ° C in modified medium (Grace's medium Gibco® ) for 12 days [Osuna, A .; Adroher, F. J .; Lupiañez, J. A. Cell. Differ Dev. 1990, 30, 89–95]. After 12 days of culture at 28 ° C, the metacyclic forms were counted in a Neubauer hemocytometer. The proportion of metacyclic forms was around 40% at this stage.

Example 3. Preparation of the cell culture where the compounds are tested.

Vero cells were grown in RPMI medium (Rowell Park Memorial Institute Medium) (Gibco®) supplemented with 10% fetal bovine serum, in 95% humidified air, atmosphere with 5% CO2 at 37 ° C for two days. For the evaluation of cytotoxicity, the cells were placed in 25ml bottles (Sterling) and centrifuged at 100g for 5 minutes. The culture medium was removed, adding fresh medium to a final concentration of 1x105 cells / ml. This cell suspension was distributed in the culture dish with a ratio of 100 µl / well and incubated for two days at 37 ° C in a humidified atmosphere enriched with 5% CO2. The medium was removed, and

fresh medium was added together with each compound tested (at concentrations of 100, 50, 25, 10 and 1 μM). After 72 hours of treatment, cell viability was determined by flow cytometry [Clotilde Marín, Inmaculada Ramírez-Macías, Angeles López-Céspedes, Francisco Olmo, Noelia Villegas, Jesús G. Díaz, María José Rosales, Ramón Gutierrez-Sánchez, and Manuel Sánchez-Moreno J. Nat. Prod. 2011, 74, 744-750].

Example 4. In vitro activity test: extracellular forms.

Test of the compounds in epimastigotes Epimastigote forms were collected in exponential growth phase and distributed in culture dishes (with 24 wells) at a final concentration of 5x104 parasites / well. The compounds of the invention listed in Table 1 of Example 15 and Benzinidazol® were dissolved in MTL liquid medium (Mediun Trypanosome Liquid), and tested at the following concentrations: 100, 50, 25, 10 and 1 μM. The effects of each compound against epimastigote forms were visualized at 72 hours using a Neubauer hemocytometer. The effect of the compounds on the parasites is expressed in IC50 values, that is, the concentration required to achieve a 50% inhibition calculated by a linear regression analysis from the Kc values of the concentrations used (Kc represents the concentrations which are tested of the products and used in linear regression to calculate the IC50).

The results are shown in Table 1 of Example 15.

Test of the compounds of the invention in trypomastigotes The compounds used in the invention were evaluated in the trypomastigote form of T. cruzi. BALB / c mice infected with T. cruzi were used after 7 days of infection. Blood was obtained by cardiac puncture using 3.8% sodium citrate as an anticoagulant in a blood / anticoagulant ratio of

7: 3 Parasitemia in infected mice was 1x105 parasites / ml. The

Test compounds were diluted in PBS medium to give a final concentration of 10, 25 and 50 μM for each product. Aliquots (20 μL) of each solution in the culture dishes (96 wells) were mixed with 55 μL of infected blood containing the parasites at a concentration close to 1x106 parasites / ml. Infected blood was used as control in PBS medium. The culture dishes were stirred for 10 minutes at room temperature and kept at 4 ° C for 24 h. Each solution was examined with a microscope (OLYMPUS CX41) in order to count the parasites using the Neubauer hemocytometer. The activity (percentage reduction of parasites) was compared with the control [Boiani, M .; Boiani, L .; Denicola, A .; Torres de Ortiz, S .; Serna, E .; Vera from Bilbao, N .; Sanabria, L .; Yaluff, G .; H. Nakayama, H .; Rojas de Arias, A .; Vega, C .; Rolan, M .; Gómez-Barrio, A .; Cerecetto, H .; González, M. 2H-Benzimidazole 1,3-dioxide derivatives: a new family of water-soluble antitrypanosomatidagents. J. Med. Chem. 2006, 49, 3215-3224] [Gerpe, A .; Aguirre, G .; Boiani, L .; Cerecetto, H .; González, M .; Olea-Azar, C .; Rigol, C .; Maya, J. D .; Morello, A .; Piro, O. E .; Aran, V. J .; Roof, A .; López de Ceráin, A. L .; Monge, A .; Rojas, M. A .; Yaluff, G. IndazoleN-oxide derivatives as antiprotozoalagents: synthesis, biologicalevaluation and mechanism of actionstudies. Bioorg Med. Chem. 2006, 14, 3467-3480].

Example 5. In vitro activity test: intracellular forms.

Cultivation of axenic amastigotes Axenic amastigotes are an artificial form achieved by transformation into a special culture for in vitro study, since the natural intracellular form can only be studied when it is internalized in macrophage cells.

The axenic amastigotes of T. cruzi were cultivated following the methodology described in [David Moreno, Daniel Plano, Ylenia Baquedano, Antonio Jiménez-Ruiz, Juan Antonio Palop, Carmen Sanmartín Parasitol Res (2011) 108: 233-239]. Therefore, the transformation of epimastigotes to amastigotes was achieved after three days of culture in M199 medium (Invitrogen, Leiden, Holland) supplemented with 10% FCS (Foetal calf serum) heat inactivated, 1 g / L β- Alanine, 100 mg / LL

asparagine, 200 mg / L sucrose, 50 mg / L sodium pyruvate, 320 mg / L malic acid, 40 mg / L fumaric acid, 70 mg / L succinic acid, 200 mg / L α-ketoglutarate acid, 300 mg / L citric acid, 1.1 g / L sodium bicarbonate, 5 g / L MONTH (4morpholineethanesulfonic acid), 0.4 mg / L hemin, 10 mg / L gentamicin pH 5.4 a 37 ° C The effect of each compound against axenic amastigotes was tested at 48 hours using a Neubauer hemocytometer. The effect is expressed in IC50 values, it is that is, the concentration required to achieve 50% inhibition calculated by means of a linear regression analysis based on the Kc values of the concentrations used.

The results are shown in Table 1 of Example 15. Test of compounds in intracellular amastigotes Vero cells were grown in RPMI medium in 95% humidified environment, 5% CO2 atmosphere and 37 ° C. These cells were cultured for two days at a density of 1x104 cells / well in 24-well microplates (Nunc). Subsequently, Vero cells were infected in vitro with the metacyclic forms of

T. cruzi, at a ratio of 10: 1 and were maintained for 24 hours at 37 ° C in an atmosphere of 5% CO2. Extracellular parasites were removed by washing, and infected cultures were incubated with the compounds of the invention at the following concentrations: 1, 10, 25, 50 and 100 μM and subsequently cultured for 72 hours in RPMI medium and 10% of inactivated bovine fetal serum. The activity of the compounds was determined from the reduction of the percentage of amastigotes in the cultures. The values are the average of four individual determinations [González P, Marín C, Rodríguez-González I, Hitos AB, Rosales MJ, Reina M, DÃaz JG, Gonzalez-Coloma A, Sanchez-Moreno M. In vitro activity of C20-diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum. Int J Antimicrob Agents 2005; 25: 136–41]. The effect is shown, as in the previous cases, expressed as IC50.

The results are shown in Table 1 of Example 15.

Example 6. Test of the compounds in the capacity of infestation and growth of parasites.

Vero cells were cultured in RPMI medium as described above. Subsequently, the cells were infected in vitro with the metacyclic forms of T. cruzi, with a 10: 1 ratio. The compounds of the invention (IC25 concentrations) were added immediately after infection and incubated for 12 hours at 37 ° C under 5% CO2. Extracellular parasites and compounds tested were removed by washing, and infected cultures were grown for 10 days in fresh medium. The fresh medium was added every 48 hours. The activity of each compound of the invention tested was determined from the percentage of infected cells and the number of amastigotes per infected cell in treated and untreated cultures, in samples fixed with methanol and stained with Giemsa. The percentage of infected cells and the average number of amastigotes per infected cell was determined by analyzing more than 100 host cells distributed in randomly chosen microscopic fields. Values are expressed as the average of four individual determinations. The number of trypomastigotes in the medium was determined as described previously [Osuna, A .; Adroher, F. J .; Lupiañez, J. A. Cell. Differ Dev. 1990, 30, 89–95].

The results are illustrated in Figure 3 where it can be seen that the compounds of the invention P (formula II), P2py (formula IV) and P3py (formula V) show a percentage of parasite growth inhibition greater than that of the reference drug , particularly in the case of the P3py compound (formula V), and, in addition, decrease the infestation capacity.

Example 7. Test of the compounds in the inhibition of the SOD of the parasite.

Cultivated parasites as described above were subjected to centrifugation. The pellet was suspended in 3 ml of STE buffer (0.25 M sucrose, 25 mM Tris-HCl, 1 M EDTA, pH 7.8) and was subjected to 3 sonication cycles, 30 seconds at 60 V. The sonication homogenate was centrifuged at 1500g for 5 minutes at 4 ° C, and the pellet was washed 3 times with ice-cold STE buffer. This fraction was centrifuged (2500 g for 10 minutes at 4 ° C) and subsequently collected in supernatant. Protein concentrations were determined by the Bradford method [Bradford MM. A refined and sensitive method for the quantification of microquantities of protein-dye binding. Anal Biochem 1976; 72: 248].

A) The activity of the superoxide dismutase enzyme FeSOD was determined according to the method described in [Beyer WF and Fridovich I. 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Bicohem 161: 559-566] which estimates the reduction of NTB (nitro blue tetrazolium) by superoxide ions. Each cuvette, located in the Stock solution (50mM phosphate buffer, pH 7.8, 54 ml, L-Methionine 3 ml, NBT 2 ml, Triton-X-100 1.5 ml.), Shows compounds at different concentrations (0.1, 0.5, 1 , 5, 12.5, 25 μM) and riboflavin. The absorbance was determined at 560 nm in a spectrophotometer. After 10 minutes under light and under agitation conditions, the absorbance was determined again. Human CuZn-SOD and Mn-SOD enzymes, coenzymes and substrates used in these assays were obtained from Sigma Chemical Co, Germany. The data obtained were analyzed with the Newman-Keuls test.

The results are illustrated in Figure 4 where it can be seen that the compounds used in the invention are capable of inhibiting the parasite FeSOD enzyme with an IC50 value (concentration required to achieve 50% inhibition) lower than in the rest of the enzymes

Example 8. Secretion of metabolites.

Epimastigote cultures of T. cruzi (initial concentration of 5 × 105 cells / ml) received IC25 concentrations of the compounds except in the case of control cultures. After incubation for 96 hours at 28 ° C, the cells were centrifuged at 400g for 10 minutes. The supernatant was collected to determine the metabolites secreted through 1 H-NMR, and the results were expressed in parts per million (ppm), using sodium 2,2-dimethyl-2-silapentane-5-sulphonate as the reference signal [ Fernández-Becerra C, Sánchez-Moreno M, Osuna A, Opperdoes FR. Comparative aspects of energy metabolism in plant trypanosomatids. J Eukaryotic Microbiol 1997; 44: 523-9]. See Figure 6.

Example 9. Ultrastructural alterations.

The parasites were grown at a density of 5 × 105 cells / ml in the corresponding medium, each containing the compounds of the invention at IC25 concentration. After 96 hours, the cultures were centrifuged at 400 g for 10 minutes and the pellets produced were washed in PBS medium and mixed with 2% (v / v) p-formaldehyde / glutaraldehyde in 0.05M of cacodylate buffer (pH 7.4 ) for 4 hours at 4 ° C. After this, the pellets were prepared to be observed by transmission electron microscopy using the technique described in [González P, Marín C, Rodríguez-González I, Hitos AB, Rosales MJ, Reina M, DÃaz JG, Gonzalez-Coloma A, Sanchez-Moreno M. In vitro activity of C20-diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum. Int J Antimicrob Agents 2005; 25: 136–41].

The results are shown in Figure 5 where the effect of the compounds of the invention on parasites is illustrated.

Example 10. Test of trypanocidal activity in vivo.

A group of three BALB / c female mice (aged 6 to 8 weeks and 20-25g in weight) maintained under standard conditions, were infected with a 1x105 concentration of metacyclic forms of T. cruzi through the intraperitoneal route.

The animals were divided into the following groups:

! Group 1: uninfected and untreated.

! Group 2: infected with T. cruzi but not treated.

! Group 3: not infected but treated with 1 mg / kg weight / day, for five

consecutive days (7 to 12 days post-infection) by intraperitoneal route

[Díaz, J. G .; Carmona, A. J .; Perez de Paz, P .; Werner, H. Phytochem.

Letters 2008, 1, 125-129].

! Group 4: infected and treated for five consecutive days (7 to 12 days

post-infection) with the compounds of the invention and Benzinidazol®. The treatments began seven days after the infection of the animal. The compounds of the invention were administered in a manner similar to that explained above and at the same concentrations. A blood sample (5 μl) was taken from the mandibular vein of each treated mouse and diluted in 1:15 ratio (50 μl of citrate buffer: 0.1 M citric acid, 0.1 M sodium citrate and 20 μL of lysis buffer at pH 7.2: 2M Tris-Cl, MgCl2).

The number of metacyclic forms of T. cruzi in the bloodstream was collected every three days from days 7 to 60 after infection. The number of metacyclic forms was observed at 200 microscopic fields.

Circulating anti-T antibodies. Cruzi, on days 60 and 90 of post-infection, were quantitatively evaluated by immunoassay. The blood, diluted in a 1:50 ratio in PBS, was reacted with an antigen composed of the FeSOD enzyme secreted from the T. cruzi epimastigotes. The results are expressed as the absorbance ratio for each sample at 490 nm versus the reference value. The reference value of each reaction is the average of the values determined in the negative controls plus three times the standard deviation [Longoni SS, Marín C, Sauri-Arceo CH, López-Cespedes A, Rodríguez-Vivas RI, Villegas N, Escobedo -Ortegón J, Barrera-Pérez MA, Bolio-Gonzalez ME, Sánchez-Moreno M. An Iron-Superoxide Dismutase Antigen-Based Serological Screening of Dogs Indicates Their Potential Role in the Transmission of Cutaneous Leishmaniasis and Trypanosomiasis in Yucatan, Mexico. Vector Borne Zoonotic Dis. 2011 Feb 16].

TESTS CARRIED OUT IN LEISHMANIA SPP.

Example 11. In vitro culture of macrophages.

J774.2 macrophages were recloned from the original J774.2 of a tumor, maintaining the cultures at a concentration between 3-9 x 105 cells / ml at 37 ° C and 5% CO2. The procedure for working with the cells in culture was trypsinizing the adhered cells by washing with PBS, adding sufficient amount of trypsin / EDTA (200 ml of PBS + 0.1 g of EDTA and 200 ml of PBS + 0.5 g of trypsin ). The two solutions were mixed at pH 7.2-7.4 and filtered. The cells were incubated for 5-10 minutes. Subsequently, said cells were decanted and centrifuged for 5 minutes at 800 rpm. Finally they were resuspended in a new culture medium. MEM + Glutamine supplemented with 20% inactivated fetal bovine serum (SBF-I) was used as culture medium.

Example 12. In vitro culture of Leishmania spp.

Promastigote forms of two species of the genus Leishmania were used:

-

L. Viannia braziliensis (MHOM / BR / 75 / M2904).

-

L. Leishmania infantum (MCAN / ES / 2001 / UCM-10).

Promastigote forms of the two Leishmania species were obtained from the culture at 28 ° C in 10% MTL medium of SBF-I. Starting the culture with an inoculum of 5 x 104 cells / ml in 5 ml of medium in 25 cm2 Falcon® plastic bottles, always working in sterility.

Example 13. Biological tests in vitro.

Essays on promastigote forms of Leishmania spp. Promastigote forms of Leishmania spp, grown in the manner described above, were collected in their exponential phase of growth by centrifugation at 1500 rpm for 10 min. The number of parasites was counted in a hemocytometric chamber of Neubauer and seeded in a 24-well plate at a concentration of 5x104 parasites in each well.

The compounds to be tested were dissolved in DMSO at a concentration of 0.01% (v / v), a concentration at which this solvent is not toxic or has any effect on the growth of parasites. The compounds were added to the culture medium at a final concentration of: 100, 50, 25, 10 and 1 #M. The effect of each compound on the growth of promastigote forms, at the different concentrations tested, was evaluated at 72 h, using a Neubauer hemocytometric chamber and the leishmanicidal effect was expressed as the IC50 (concentration required to give an inhibition of 50 %, calculated by the analysis of the linear regression of the Kc at the concentrations tested).

Essays on amastigote forms of Leishmania spp. The tests on amastigote forms were carried out by carrying out the following methodology. J774.2 macrophages were detached from the culture flask where they were attached by trypsinization and by dry blows. For this, the culture medium was removed, then the cell surface was covered with EDTAtripsin and incubated for 5 minutes. After that, it was transferred to a 25 ml conical bottom flask (Steriling) to centrifuge them at 800 rpm for 5 minutes, removing the supernatant, and the cells were counted in Neubauer chamber. The cells were cultured in 24-well plates, in which a 12mm round glass cover glass had previously been introduced into each well at a concentration of 1x104 cells in each well. For adhesion, cells were grown 24-48 h at 37 ° C in 5% CO2.

Once the cells were adhered, they were infected in vitro with 1x105 cells of amastigote forms in the stationary phase of Leishmania spp. They were incubated for 24 hours at 37 ° C in 5% CO2. After that time, the parasites that were free in the culture medium, were removed by several washes with fresh medium. The medium was then changed to cell cultures (MEM + 20% SBFI Glut) and the test compound was added at a concentration of 1, 10, 25, 50 and 100 µM. It was allowed to incubate for 72 hours at 37 ° C in 5% CO2.

After 72 hours, the crystals were removed and placed on a slide. They were fixed with methanol and allowed to dry. Once fixed and dry, DPX (Panreac®), a microscopy mounting medium, was added. They were stained with Giemsa, for this, immediately before use and in a test tube, 1 ml of AzurEosine-Methylene Blue solution was diluted according to Giemsa DC (Code 251338) with 10 ml of pH 7.2 buffer solution (Code 252164), the preparation was mixed and covered leaving coloring for 20 minutes. It was washed with pH 7.2 buffer solution (Code 252164). It was allowed to drain and dry in an upright position. Finally, it was examined with the objective of immersion and the number of intracellular amastigote forms in a total of 200 cells of each crystal was counted.

The antiparasitic activity of the products was determined with the number of intracellular amastigote present at the different concentrations and in the control. And it was expressed as the IC50 (concentration required to give a 50% inhibition, calculated by the linear regression analysis of the Kc at the concentrations tested).

Example 14. Cytotoxicity tests.

These tests were performed by flow cytometry. Macrophages of the J774.2 line, were deposited in a tube (steriling) and centrifuged at 800rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in MEM + Glutamine medium with 20% SBF.

1x104 cells were deposited in each well of a 24-well titration plate, incubated for 24-48 h at 37 ° C in a humid atmosphere enriched with 5% CO2. This was done to fix the cells. After this time the culture medium was removed and fresh medium was added with the products to be tested, at

concentrations of 100, 50, 25, 10 and 1 #M. At 72 hours after incubation, the samples were prepared for reading on the flow cytometer.

The method followed was that described by [Ormerod MG. 1994. Flow Cytometry. A practical Approach. 2nd Edition. IRL Press (Oxford University Press)], starting from the cells and the medium present in the wells, to which 100 μl of propidium iodide solution (PI, 100 μg / ml) (Sigma Chemical Co) was added, incubating at 28 ° C in darkness about 10 minutes. Subsequently, 100 μl of fluorescein diacetate (FDA) (Sigma Chemical Co) in solution (100 ng / ml) was added and re-incubated at 28 ° C in the dark for about 10 minutes, and after centrifugation at 1500 rpm for 10 minutes, the procedure was to wash the precipitate with PBS. Finally, the results were analyzed taking into account that the cells with the intact plasma membrane emit green color when the esterases act on the FDA, while the cells that have lost the integrity of the membrane and are not viable, emit in the range of 580 nm when penetrating the PI by passive diffusion and specifically binding to the nucleic acids thereof. Flow cytometry analysis was carried out with a FACS Vantage flow cytometer (Becton Dickinson).

The percentage of viability was calculated. The number of dead cells was determined by comparison with the control cultures. The IC50 was calculated using the linear regression analysis of the Kc at the concentrations tested.

RESULTS

Example 15. Results of the comparative study of the compounds of the invention against Benzinidazol® and Glucantime®.

Initially the study of the compounds of the invention and their complexes with Mn (II) was carried out in comparison with Benzinidazole (BZN) (reference drug for Trypanosoma cruzi) in which toxicity, in vitro activity was measured and the selectivity index (SI) in the epimastigote and amastigote forms of Trypanosoma cruzi (Table 1).

Table 1

Compound

Activity a Toxicity b Sic

Ep.

Am. Axén. Am. Intrac.  Ep. Am. Axén. Am. Intrac.

BZN

15.9 ± 1.1 18.9 ± 1.5 23.3 ± 4.6 13.6 ± 0.9 0.8 0.7 0.6

II

6.5 ± 0.8 4.0 ± 0.4 7.8 ± 0.7 269.5 ± 16.1 41.4 (52) 66.6 (95) 34.5 (57)

II *

12.9 ± 1.2 14.9 ± 2.0 15.7 ± 1.5 234.9 ± 20.6 18.1 (23) 15.7 (22) 14.9 (25)

IV

8.7 ± 2.0 10.2 ± 0.8 10.0 ± 1.3 366.2 ± 14.7 42.2 (53) 35.9 (51) 36.4 (61)

IV *

14.9 ± 2.4 12.8 ± 1.6 14.2 ± 2.3 2614 ± 21.1 17.6 (22) 20.5 (29) 18.4 (31)

III

10.4 ± 1.2 12.8 ± 1.5 15.7 ± 2.0 324.9 ± 23.7 31.2 (39) 25.3 (36) 20.7 (34)

III *

15.1 ± 1.6 18.2 ± 2.3 19.5 ± 2.2 271.3 ± 23.1 18.0 (22) 14.9 (21) 13.9 (23)

V

8.1 ± 1.3 9.3 ± 1.0 9.5 ± 0.6 425.2 ± 29.6 52.7 (66) 45.5 (65) 44.8 (75)

V *

20.9 ± 1.7 21.2 ± 2.3 21.5 ± 1.1 271.9 ± 22.3 13.0 (16) 12.8 (18) 12.6 (21)

SAW

20.9 ± 1.9 19.2 ± 2.4 20.2 ± 1.8 167.3 ± 22.6 8.0 (10) 8.7 (12) 8.3 (14)

SAW*

21.3 ± 1.9 26.5 ± 2.7 20.2 ± 2.4 214.3 ± 31.0 10.1 (13) 8.1 (12) 10.6 (18)

a Activity: expressed as IC50 (μM): concentration required to achieve 50% of inhibition. b Toxicity: expressed as IC50 (μM) in Vero cells after 72 hours of culture.

5 c SI: selectivity index = IC50 in Vero cells / intracellular and extracellular IC50 of the parasite. The most active compounds against intracellular forms of the parasite are those whose SI value is greater than 50 times with respect to the reference compound. (): number of times the SI value of the compound of the invention exceeds the SI value of the reference compound.

10 * Compound in the form of a complex with Mn (II). Ep .: epimastigote form. Am. Axén .: amastigote form. Am. Intrac .: intracellular amastigote form.

Subsequently, the study of the compounds of the invention and their

complexes with Mn (II) compared to Glucantime® (reference drug for

the Leishmania spp). In Leishmania infantum toxicity, activity in

vitro and the selectivity index (SI) of compounds in promastigote forms and

amastigote (Table 2).

Table 2

Compound

Activity a Toxicity b Sic

Pro. Extrac.

Am. Axén. Am. Intrac.  Pro. Extrac. Am. Axén. Am. Intrac.

Glucantime®

18.0 31.0 30.8 15.2 0.8 0.5 0.5

II

18.9 29.2 25.6 231.6 12.2 (15) 8.0 (16) 9.0 (18)

II *

40.7 40.4 30.7 218.6 5.4 (7) 5.4 (11) 7.1 (14)

IV

15.5 25.4 19.6 254.0 16.4 (20) 10.0 (20) 13.0 (26)

IV *

21.2 77.8 50.3 275.7 13.0 (16) 3.5 (7) 5.5 (11)

III

21.7 28.1 17.4 365.3 18.8 (23) 13.0 (26) 21.0 (42)

III *

38.2 45.0 60.1 387.9 10.2 (13) 8.6 (17) 6.5 (13)

V

18.8 15.9 6.3 467.8 24.9 (31) 29.4 (59) 74.3 (149)

V *

40.8 33.7 50.1 223.9 5.5 (7) 6.6 (13) 4.5 (9)

SAW

24.2 29.8 44.9 220.4 9.1 (11) 7.4 (15) 4.9 (10)

SAW*

27.0 33.6 61.6 258.1 9.6 (12) 7.7 (15.4) 4.2 (8)

a Activity: expressed as IC50 (μM): concentration required to achieve 50% of inhibition. b Toxicity: expressed as IC50 (μM) in J774.2 macrophages after 72 hours of culture.

5 c SI: selectivity index = IC50 in macrophages / intracellular and extracellular IC50 of the parasite. The most active compounds against intracellular forms of the parasite are those whose SI value is greater than 20 times with respect to the reference compound. (): number of times the SI value of the compound of the invention exceeds the SI value of the reference compound.

10 * Compound in the form of a complex with Mn (II). Pro. Extrac .: Promastigote extracellular form. Am. Axén .: axenic amastigote form. Am. Intrac .: intracellular amastigote form.

In addition, the study of the compounds of the invention and their complexes was carried out.

with Mn (II) compared to Glucantime® (reference drug for

Leishmania spp) in Leishmania braziliensis. Toxicity, activity in was measured

vitro and the selectivity index (SI) of compounds in amastigote forms and

promastigote (Table 3).

Table 3

Compound

Activity a Toxicity b Sic

Pro. Extrac.

Am. Axén. Am. Intrac.  Pro. Extrac. Am. Axén. Am. Intrac.

Glucantime®

25.6 30.9 19.7 15.20 0.6 0.5 0.8

II

11.8 16.0 10.0 231.6 19.6 (30) 14.5 (29) 23.2 (29)

II *

32.8 54.3 30.4 218.6 6.8 (11) 4.0 (8) 7.3 (9)

IV

11.3 16.0 10.4 254.0 22.5 (37) 15.9 (32) 24.4 (30)

IV *

12.4 26.7 16.4 275.7 22.2 (37) 10.3 (20) 16.8 (21)

III

12.3 24.9 16.6 365.3 29.7 (49) 14.7 (29) 22.0 (27)

III *

29.5 59.2 28.9 387.9 13.1 (22) 6.6 (13) 13.4 (17)

V

23.0 23.9 10.3 467.8 20.3 (34) 19.6 (39) 45.4 (57)

V *

21.2 41.6 47.0 223.9 10.6 (18) 5.4 (11) 4.8 (6)

SAW

15.5 31.4 19.1 220.4 14.2 (24) 7.0 (14) 11.5 (14)

SAW*

25.5 42.6 77.8 258.1 10.1 (17) 6.1 (12) 3.3 (4)

a Activity: expressed as IC50 (μM): concentration required to achieve 50% of inhibition. b Toxicity: expressed as IC50 (μM) in J774.2 macrophages after 72 hours of culture.

5 c SI: selectivity index = IC50 in macrophages / intracellular and extracellular IC50 of the parasite. The most active compounds against intracellular forms of the parasite are those whose SI value is greater than 20 times with respect to the reference compound. (): number of times the SI value of the compound of the invention exceeds the SI value of the reference compound.

10 * Compound in the form of a complex with Mn (II). Pro. Extrac .: Promastigote extracellular form. Am. Axén .: axenic amastigote form. Am. Intrac .: intracellular amastigote form.

15 In view of the results obtained from this example, it is shown that

Compounds of the invention, characterized by formula (I), are effective in the

treatment of diseases caused by parasites maintaining a low level of

toxicity, particularly the three least toxic compounds: Pytren (P) (formula II),

Pytren2py (P2py) (formula IV) and Pytren3py (P3py) (formula V).

Example 16. Effectiveness of the compounds of the invention in chronic phase.

A group of Balb / c strain mice was taken 140 days post infection with the idea of reproducing the chronic phase of treatment. From the seventh day of post infection they were administered the treatment consisting of 5 doses of 100 μl to finally gather a concentration of 15 mg / kg weight. Subsequently, blood samples were taken to monitor the state of parasitemia in the mouse by fresh counting, as well as to assess the immune status by means of the ELISA test.

On day 140 the mice were immunocompromised by two cycles of cyclophosphamide (50 mg / kg day weight, 4 days treatment with 3 rest) and subsequently a final sample was obtained where organs (heart and liver) were included after necropsy to be evaluated by pathological studies.

After examination of fresh parasitemia, a sharp decrease in the number of blood parasites was observed. In addition, the absence of significant variations between immunosuppressed and non-immunosuppressed animals that had been treated with the product was observed, which is indicative of a trypanocidal effect by the product at the time of treatment. It is interesting to note that there are no useful drugs for the chronic phase that is produced after 30 days when the parasite moves to organs such as liver, esophagus and heart. The products tested constitute valid drugs for the treatment of the chronic phase of parasitemia. Figure 7 shows the levels of parasitic antibodies that show the efficacy of the compounds of the invention in the chronic phase of the disease (in vivo model in chronic phase).

Figure 2 shows the results obtained from the parasitemia curve in which it can be seen that the compounds of formula (II) (P), formula (IV) (P2py) and formula

(V) (P3py) are the most effective at the beginning of the chronic phase (day 60) since they significantly reduce the number of parasites in the blood.

Example 17. Synthesis of the compounds used in the present invention.

The compound of formula (II) was reacted with the aldehyde group of a compound of formula R-COH, where R comprises the substituents R1 and / or R2, where R1 is an amino group substituted by R2 or unsubstituted, R2 comprises carbocycles and / or heterocycles that may be substituted by R3 or unsubstituted, and R3 comprises the formula (I) where R is a hydrocarbon chain, the two compounds being preferably dissolved in ethanol or other alcohol, and subsequently sodium borohydride or other was subsequently added reducer for the reduction of the imine formed. The mixture was conveniently treated to extract the reaction product, from which the ammonium salt is prepared by the addition of hydrochloric acid. The salts of the different compounds are solid and purified through successive washes with ethanol. The compounds are perfectly characterized by NMR (Nuclear Magnetic Resonance) spectroscopy, mass spectrometry, crystallographic analysis and through elemental analysis.

In addition, the compounds used in the present invention can be presented as complexes with Mn (II) for which the ligand and the corresponding metal (in this case Mn (II)) are mixed in an aqueous solution in molar ratio 1: 1 (L: M), the pH of the solution is adjusted to the formation pH of the species and allowed to evaporate slowly.

Synthesis of the compound of formula (XI)

The synthesis of this compound was achieved following a modification of the Richman-Atkins procedure [B. Verdejo, A. Ferrer, S. Blasco, C. E. Castillo, J. González, J. Latorre, M. A. Mañez, M. G. Basallote, C. Soriano and E. García-Spain, Inorg. Chem., 2007, 46, 5707] by reacting the pertosylated train polyamine with 2,6-bis (bromomethyl) pyridine in a 1: 1 molar ratio, preferably using K2CO3 as a base, and refluxing in CH3CN. Detosylation was carried out with HBr / HAc to give Intermediate 1, as indicated in Scheme 1.

The compound of formula (XI) was obtained by the reaction of this intermediate detosylated, in its free amine form, with anthracene-9-carbaldehyde in dry ethanol, followed by an in situ reduction with sodium borohydride. The product precipitated in Hydrochloride salt form (Scheme 1). Scheme 1:

6- [4- (1-Pyrene) -3-azabutyl] -3,6,9-triaza-1- (2,6) -pyridinecyclodecaphane (XI). The compound of formula (II) was dissolved in a dry solvent (preferably ethanol) and pyrene-1-carbaldehyde dissolved in the same solvent was added dropwise. It stirred

10 the reaction for 2 hours. A reducer (in this case NaBH4) was added and the resulting solution was stirred for 1 hour at room temperature. The solvent was removed under reduced pressure. The resulting residue was treated with water and dichloromethane. The organic phase was separated and removed under reduced pressure. The resulting residue was dissolved in ethanol and precipitated as hydrochloride salt (XI).

15 1H NMR (D2O, 300MHz): δH 8.10 (d, J = 8Hz, 1H), 8.06 (d, J = 8Hz, 1H), 7.95 (d, J = 4Hz, 1H), 7.92 (t, J = 6Hz , 1H), 7.90 (t, J = 6Hz, 1H), 7.86 (m, 1H), 7.81 (t, J = 6Hz, 2H), 7.79 (d, J = 5Hz, 1H), 7.70 (d, J = 10Hz, 1H), 7.30 (d, J = 8Hz, 2H), 4.62 (s, 2H), 4.46 (s, 4H), 3.22 (m, 2H), 3.05 (m, 4H), 2.89 (t, J = 8Hz, 2H), 2.69 (t, J = 5Hz, 4H).

Synthesis of the compounds of formula (XIV) and (XV)

5 By reacting the pertosylated form of the Intermediate 1 product with N- (3bromopropyl) phthalimide in dry CH3CN at reflux, a white product was obtained which was deprotected with hydrazine and detosylated with HBr / HAc to obtain Intermediate 2. As in the previous case, by reacting this detosylated intermediate in

In the form of free amine with the corresponding aldehyde in dry ethanol, followed by the reaction in situ with sodium borohydride and precipitating the product in the form of hydrochloride, these two compounds (XIV and XV) were synthesized, as shown in the Scheme 2.

Scheme 2

OR

N

N OR Br N TsN NTs K2CO3 N2H4

TsN

NTs

CH3CN EtOH Reflux

H2NN N

NHTs

HBr / HAc / PhOH

N

HN NH

CHO R

N

N NHCl HCl (g) EtOH

HN

NaBH4

NH

Ts

N

Reflux

NH · nHBr H2N

N

H

H

N

N

R

Intermediate 2

6- [8- (2-Pyridyl) -3,7-diazaoctyl] -3,6,9-triaza-1- (2,6) -pyridinecyclodecaphane (XIV). The compound of formula (IX) was dissolved in dry ethanol and pyridine-2-carbaldehyde dissolved in dry ethanol was added dropwise. The reaction was stirred for 2 hours. NaBH4 was added and the resulting solution was stirred for 1 hour at room temperature. Ethanol was removed under reduced pressure. The resulting residue was treated with water and dichloromethane. The organic phase was separated and removed under reduced pressure. The resulting residue was dissolved in ethanol and precipitated as hydrochloride salt (XIV). 1H NMR (D2O, 300MHz): δH 8.83 (d, J = 5Hz, 1H), 8.51 (t, J = 6Hz, 1H), 8.05 (m, 3H), 8.00 (m, 3H), 7.96 (m, 3H ), 7.48 (d, J = 9Hz, 2H), 4.69 (s, 6H), 3.37 (m, 5H),

3.32 (m, 5H), 3.10 (t, J = 6Hz, 2H), 2.98 (m, 4H), 2.28 (m, 2H). 13C NMR (D2O, 75.43MHz): δC 149.2, 146.4, 145.5, 140.1, 127.5, 122.6, 51.3, 50.8, 49.9, 48.9, 46.3, 45.3, 44.0, 23.1. Anal. Calc. For C22H35N7 · 5HCl: C, 42.9; H, 7.2; N, 15.9. Experimental: C, 42.4; H, 7.5; N, 15.4.

6- [8- (4-pyridyl) -3,7-diazaoctyl] -3,6,9-triaza-1- (2,6) -pyridinecyclodecaphane (XV). The compound of formula (IX) was dissolved in dry ethanol and pyridine-4-carbaldehyde dissolved in dry ethanol was added dropwise. The reaction was stirred for 2 hours. NaBH4 was added and the resulting solution was stirred for 1 hour at room temperature. Ethanol was removed under reduced pressure. The resulting residue was treated with water and dichloromethane. The organic phase was separated and removed under reduced pressure. The resulting residue was dissolved in ethanol and precipitated as hydrochloride salt (XV). 1H NMR (D2O, 300MHz): δH 8.92 (d, J = 8Hz, 2H), 8.25 (d, J = 8Hz, 2H), 8.00 (t, J = 8Hz, 1H), 7.51 (d, J = 9Hz, 2H), 4.70 (s, 2H), 4.68 (s, 4H), 3.39 (m, 5H), 3.33 (m, 5H), 3.13 (t, J = 6Hz, 2H), 2.96 (t, J = 6Hz, 4H), 2.30 (m, 2H). 13C NMR (D2O, 75.43MHz): δC 151.7, 149.2, 142.4, 140.1, 127.9, 122.6, 51.3, 50.8, 49.9, 48.8, 46.3, 45.6, 45.3, 44.1, 23.1. Anal. Calc. For C22H35N7 · 5HCl · 5H2O: C, 39.4; H, 7.5; N, 14.6. Experimental: C, 39.6; H, 7.1; N, 14.3.

Synthesis of the compound of formula (X)

The 5- (2-Aminoethyl) -2,5,8-triaza [12] -2,6-pyridinophane and 4-carboxaldehyde-2,9-phenanthroline were dissolved in dry ethanol and allowed to stir at room temperature for 2 hours. The imine formed was preferably reduced with NaBH4 and left another 2 hours stirring at room temperature. The solvent was removed in the rotary evaporator. The solid obtained was extracted with H2O and CH2Cl2. The organic phase was separated and removed under reduced pressure. The resulting residue was dissolved in ethanol and

5 was precipitated as hydrochloride salt, as shown in Scheme 3. Yield 60%.

1H NMR (500 MHz, D2O): δ (ppm) = 8.69 (d, J = 5 Hz, 1H), 8.67 (d, J = 4 Hz, 1H),

8.12 (d, J = 8 Hz, 1H), 7.86 (t, J = 8 Hz, 1H), 7.67 (d, J = 4 Hz, 1H), 7.58 (dd, J1 = 8

10 Hz, J2 = 5 Hz, 1H), 7.52 (d, J = 8 Hz, 1H), 7.35 (d, J = 8 Hz, 2H), 7.33 (d, J = 8 Hz, 1H), 4.62 (s , 2H), 4.55 (s, 4H), 3.44 (t, J = 7 Hz, 2H), 3.26-3.18 (m, 4H), 3.09 (t, J = 7 Hz, 2H), 2.89 (t, J = 5 Hz, 4H). 13C NMR (125 MHz, D2O): δ (ppm) = 149.35, 148.95, 147.59, 141.41, 140.31, 139.89, 139.65, 128.01, 126.03, 124.28, 122.06, 121.56, 51.01, 50.56, 49.57, 46.82,

15 45.94, 44.18.

Scheme 3

Claims (10)

1. Use of a compound of formula (I): (I) where R can be H or a hydrocarbon chain comprising the R1 and / or R2 substituents, where R1 is an amino group substituted by R2 or unsubstituted, R2 comprises carbocycles and / or heterocycles that may be substituted by R3 or unsubstituted, and R3 comprises formula (I) where R is a hydrocarbon chain; for the preparation of a pharmaceutical or veterinary composition intended for the treatment of diseases caused by parasites. 2. Use according to claim 1, wherein in the compound of formula (I) the carbocycle is benzene and the heterocycle is pyridine. 3. Use according to claims 1 or 2, wherein the compound is selected from the following: (II) (III) (IV) (V) (SAW) (VII) (VIII)

(IX)

(X)

(XI)

(XII) (XIII) (XIV) (XV) (XVI) (XVII) (XVIII) 4. Use of a compound of formula (II), (III), (IV), (V), (VI), (VII), (VIII), 5 (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) or (XVIII), where the compound forms a complex with Mn ( II), for the preparation of a pharmaceutical or veterinary composition intended for the treatment of diseases caused by parasites. Use according to any one of claims 1 to 4, characterized in that the diseases treated are Chagas disease caused by Trypanosoma cruzi or leishmaniasis caused by Leishmania spp. 6. Use according to claim 5, characterized in that the diseases are treated in the chronic phase thereof. 7. Compound selected from the group comprising: (X)

(XI)

(XIV)

(XV)

8.

Compound according to claim 7, characterized by forming complexes with Mn (II).

9.

Use of any of the compounds of claims 7-8 to prepare a medicament.

10.

Pharmaceutical or veterinary composition comprising at least one compound of any of claims 7-8 and pharmaceutically acceptable excipients.

FIGURE 1 FIGURE 2 FIGURE 3A % Inhibition ΔBZN 39% □ P 73% ♦ P2py 77% ● P3py 86% FIGURE 3B % Inhibition ΔBZN 12% □ P 46%

♦

P2py 60%

●

P3py 69%

FIGURE 3C FIGURE 4A FIGURE 4B FIGURE 4C FIGURE 5 FIGURE 6 A FIGURE 6 B FIGURE 6 C FIGURE 6 D FIGURE 7 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201132035 SPAIN Date of submission of the application: 16.12.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

WO 2011033163 A2 (UNIVERSITAT DE VALENCIA) 24.03.2011, page 4, lines 23-31; pages 5-6, compounds 1-3; page 6, lines 19-23; page 7, lines 1-6,23-28. 1-10

TO

GONZÁLEZ, J. et al. "Tritopic phenanthroline and pyridine tail-tied aza-scorpiands." Organic and Biomolecular Chemistry 2010, Volume 8, pages 2367-2376. [Available online on 03/24/2010). See page 2367, summary and introduction; page 2368, table 1; page 2369, column 2, paragraph 2; figure 4. 1-10

TO

BLASCO, S. et al. "Hydrogen and Copper ion Induced Molecular Reorganizations in Two New Scorpiand-Like Ligands Appended with Pyridine Rings." Inorganic Chemistry 2010, Volume 49, Number 15, pages 7016-7027. [Available online on 07.08.2010). See page 7016, summary; page 7017, table 1; page 7018, column 1, paragraph 3. 1-10

TO

SÁNCHEZ-MORENO, M. et al. "In vitro leishmanicidal activity of imidazole-or pyrazole-based benzo [g] phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species". Journal of Antimicrobial Chemotherapy 2012, Volume 67, pages 387-397. [Advance Access] [Available online 29.11.2011]. See page 1, summary; page 3, figure 3. 1-10

TO

GHOSH, S. et al. "Role of superoxide dismutase in survival of Leishmania within macrophage." Biochemical Journal 2003, Volume 369, pages 447-452. See page 447, summary. 1-10

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 27.03.2013

Examiner G. Esteban García Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201132035 CLASSIFICATION OBJECT OF THE APPLICATION C07D471 / 08 (2006.01) A61K31 / 439 (2006.01) A61P33 / 02 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C07D, A61K, A61P Electronic databases consulted during the search (name of the database and, if possible, terms of search used) EPODOC, WPI, REGISTRY, HCAPLUS, MEDLINE, BIOSIS, XPESP, NPL, EMBASE, CHEMSPIDER, PUBMED State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201132035 Date of Completion of Written Opinion: 03.23.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-10 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-10 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201132035 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 2011033163 A2 03-24-2011

D02

GONZÁLEZ, J. et al. Organic and Biomolecular Chemistry 2010, Vol. 8, pp. 2367-2376. 03-24-2010

D03

BLASCO, S. et al. Inorganic Chemistry 2010, Vol. 49, No. 15, pp. 7016-7027. 07.08.2010

D04

SÁNCHEZ-MORENO, M. et al. Journal of Antimicrobial Chemotherapy 2012, Vol. 67, pp. 387-397. 11/29/2011

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention is the use of a compound of general formula (I) for the preparation of a pharmaceutical or veterinary composition for the treatment of diseases caused by parasites; a compound selected from the group comprising the compounds (X), (XI), (XIV) and (XV); the use of said compounds to prepare a medicament; and a pharmaceutical or veterinary composition comprising at least one of these compounds. Document D01 discloses compounds (1) and (2), polyazapyridinophane macrocycles with a quinoline heterocycle attached to the central backbone of the molecule (see page 4, lines 23-31), which correspond to compounds (VII) and ( VIII) of the invention, not claimed (see pages 5 and 6). These compounds, obtained from the compound (3), which has the unsubstituted ethylamine chain (compound II of the invention), form coordination complexes with a mimetic manganese (II) atom of the enzyme superoxide dismutase or SOD (see page 6, lines 19-23). These complexes can be used in therapy for the treatment of diseases (in humans or animals) whose etiology is based on abnormalities of the activity or lack of endogenous SOD, acting primarily as anti-inflammatory, analgesic and antioxidant antioxidant protective antioxidants (see page 7, lines 1-6, 23-28). Document D02 discloses two receivers derived from azascorpiands of formulas L1 (compound XVII of the invention) and L2 (compound XVIII of the invention), in addition to the precursors L3 and L4 (compounds II and IV of the invention, respectively) (see page 2367, summary; page 2368, table 1). These compounds have the ability to coordinate to metal ions, such as Zn (II) (see page 2369, column 2, paragraph 2; figure 4), in addition to potential applications in pharmaceutical chemistry (see page 2367, introduction). None of these compounds are included in the claims of the application object of this report. Document D03 discloses ligands consisting of a tris (2-aminoethyl) amine structure linked to the 2.6 positions of a pyridine that acts as a spacer through functionalized methylene groups (see page 7016, summary), such as L1 compounds (compound IV of the application), L2 (compound V of the application), L4 (compound III of the application), in addition to the precursor L3 (compounds II of the application) (see page 7017, table 1). These compounds, which are not claimed in the present application, form coordination complexes with metal ions, such as Cu (II) (see page 7018, column 1, paragraph 3), as opposed to the compounds of the invention that form complexes of Mn (II). Document D04 discloses a series of compounds derived from benzo [g] phthalazine which possess imidazole or pyrazole radicals (see page 1, summary), in which the benzo [g] phthalazine group acts as a spacer between the two imidazole or pyrazole moieties, through triethylene chains functionalized with an amino group (see page 3, figure 3, compounds 1, 3, 5). These compounds have antiparasitic activity, in particular, they were active in vitro against two Leishmania species, being less toxic to mammalian cells than the reference drugs. The cited documents show only the state of the art of the field to which the invention belongs. None of them, taken alone or in combination with the others, disclose or contain any suggestion that could direct the person skilled in the art towards the use of a compound of general formula (I) for the preparation of a pharmaceutical or veterinary composition intended for treatment. of diseases caused by parasites; nor towards the concrete compounds (X), (XI), (XIV) and (XV); and, therefore, neither towards the use of these to prepare a medicament nor towards a pharmaceutical or veterinary composition comprising them. Therefore, it is considered that the object of the application meets, regarding the state of the art, the requirements of novelty and inventive activity set forth in Articles 6.1 and 8.1 of Patent Law 11/86. State of the Art Report Page 4/4