Use of proton-ionizable pyrazole-derived esters and their corresponding salts for the treatment of c

专利摘要:
Use of proton-ionizable pyrazole-derived esters and their corresponding salts for the treatment of chagas disease and leishmaniasis. Use of esters derived from pyrazole, preferably protonation-ionizable derivatives in neutral form or in the form of salts (pyrazolates), as medicaments and more particularly for the prevention and/or treatment of diseases of parasitic origin such as chagas disease or leishmaniasis (Machine-translation by Google Translate, not legally binding)
公开号:ES2566228A1
申请号:ES201431309
申请日:2014-09-11
公开日:2016-04-11
发明作者:Felipe REVIRIEGO PICON；Pilar Navarro Torres；Vicente J. ARAN REDO；Manuel SANCHEZ MORENO；Clotilde MARIN SANCHEZ；Francisco OLMO AREVALO；Inmaculada RAMIREZ MACIAS；Enrique GARCIA-ESPAÑA MONSONIS；Maria Teresa ALBELDA GIMENO
申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad de Granada；Universitat de Valencia；
IPC主号:

专利说明:

DESCRIPTION

Use of proton-ionizable pyrazole-derived esters and their corresponding salts for the treatment of Chagas disease and leishmaniasis.
 5
The present invention relates to the use of proton-ionizable pyrazole derived esters in neutral form or in the form of salts (pyrazolates), as medicaments and more particularly for the prevention and / or treatment of diseases of parasitic origin such as the disease of Chagas or leishmaniasis.
 10 STATE OF THE TECHNIQUE

Chagas disease (American trypanosomiasis) is an infectious tropical disease cataloged in the group of so-called "neglected deseases", which affects thousands of people in Latin America in areas of population 15 with low economic resources, and therefore represents a serious health problem.

It is a parasitic disease, usually chronic, caused by the hemophlagelated protozoan parasite Trypanosoma cruzi (T. cruzi). The natural reservoir 20 consists of armadillos, marsupials, rodents, bats and wild primates, in addition to certain domestic animals (dogs and cats). It is transmitted to man by hematophagous triatomines such as Triatoma infestans, Rhodius prolixus or Triatoma dimidiata, which defecate on the bites that they themselves have made to feed. The disease can also be transmitted by non-vector mechanisms such as transfusion of contaminated blood or organ donation, by ingestion of contaminated food or vertically from an infected mother to the fetus.

Initially, the acute infection phase is normally asymptomatic, and most patients do not realize that they have been infected; but this acute phase 30 subsequently evolves into a chronic state, which reaches between 30% and 40% of all chagasic patients, in which severe complications are manifested 10-30 years after infection. Severe diffuse cardiomyopathy, peripheral nervous system disorders, pathological dilation of the esophagus and colon, megaesophagus, and megacolon, respectively, occur. Many 35 of them suffer heart failure and sudden death. Due to the large increase in trips
international and immigration, Chagas disease has spread, not only throughout Latin America, but also to the United States, Canada, Spain, Italy and other countries.

There is currently no adequate therapy or effective vaccine. The two 5 drugs primarily used to treat Chagas disease are two nitroaromatic heterocycles discovered more than three decades ago: nifurtimox (Nfx, 3-methyl-4- (5-nitrofurfurylidenamino) tetrahydro-4H-1,4-thiazine-1 , 1-dioxide, Lampit®, which has recently been discontinued by Bayer), and benznidazole (Bzn, N-benzyl-2- (2-nitroimidazol-1-yl) acetamide, Roche's Rochagan®, now manufactured by 10 LAFEPE in Brazil). The use of such medications in the acute phase is widely accepted, but their efficacy in the chronic phase is quite controversial, and it is estimated that parasitological cure in this phase is only obtained in 10-20% of patients [Maya, J.D. et al., Comp. Biochem Physiol Part A 2007, 146, 601-620]. In addition, they cause severe side effects such as pancreatitis and cardiac toxicity. The most commonly used clinically is benznidazole, although in adults it generates a considerable number of adverse side effects, such as digestive, hematological, dermatological and neurological disorders [Castro, J.A. et al., Hum. Exp. Toxicol. 2006, 25, 471-479].
 twenty
Another worrying aspect of Chagas disease is the high reactivation capacity of parasitemia in immunosuppressed individuals. It has been proven that cured patients who have subsequently undergone kidney or liver transplantation, diagnosed with AIDS, or treated with anticancer chemotherapy, underwent reactivation of Chagas disease with a very aggressive clinical course leading to meningoencephalitis and / or acute myocarditis . In fact, when patients with chronic chagasic heart disease undergo cardiac transplantation, reactivation of parasitemia occurs and benznidazole treatment only leads to temporary remission, but T. cruzi infection persists [Campos, S.V. et al., J. Heart Lung Transp. 2008, 27, 597-602]. 30

Considering these facts, it can be concluded that there is an urgent need to find new drugs that are less toxic to patients and more effective in the chronic phase of Chagas disease than benznidazole, currently used, and that also have the ability to reduce the reactivation of parasitemia in cases of immunodeficiency.

Another important disease caused by kinetoplastic protozoan parasites is leishmaniasis, caused by infection with different species of protozoa of the genus Leishmania, which is transmitted through the bite of diptera of the genera Phlebotomus and Lutzomyia. Currently, leishmaniasis is endemic in 98 5 countries, mainly in America, but also in Europe and Asia, with more than 350 million people at risk, more than 2 million new cases each year, and an annual mortality exceeding 60,000 patients The World Health Organization has ranked it 9th among the most severe infectious diseases. 10

Leishmaniasis can occur with different clinical manifestations: (I) visceral, which is the most severe of all; (II) the skin, which causes nodules and ulcers that may persist for years; and (III), the mucocutaneous, which causes permanent lesions in the mouth, nose or genital mucosa. Visceral leishmaniasis is the 15 clinical form that claims the most lives worldwide. It can be fatal if not treated in time and is characterized by inflammation of the liver and spleen, accompanied by severe abdominal distension, loss of body condition, malnutrition and anemia.

Leishmania infantum (L. infantum) is considered to be the main etiologic agent of visceral leishmaniasis in southeastern Europe. It uses dogs as a reservoir and mainly affects children, although co-infection with HIV and the increasing use of chemotherapy for immunosuppression in transplants have led to a considerable increase in the percentage of cases in adults. Another significant species is Leishmania braziliensis (L. braziliensis) that mainly affects the Andean countries, and the Amazon basin, and causes cutaneous and mucocutaneous leishmaniasis.

The treatment of leishmaniasis is complicated. There are no effective vaccines and diagnostic tests are not specific due to poor control measures of the vector [Barrett, M.P. et al., Curr. Top. Med. Chem. 2002, 2, 471-482]. Chemotherapy is the main weapon to combat the clinical manifestations of most forms of leishmaniasis. The medications that have been commonly used so far are sodium stibogluconate (Pentostan®) and meglumine antimony agent (Glucantime®). But they are very ineffective and cause a multitude of toxic side effects such as nausea, vomiting, diarrhea, skin rashes,
dizziness, cardiac arrhythmia, hypotension, hepatitis and pancreatitis. The current treatment with Glucantime, is based on intramuscular application for a period of 20 to 30 days. However, this drug shows a high toxicity and is also not effective [Palumbo, E., Am. J. Ther. 2009, 16, 178-182].
 5
Other medications used are: amphotericin B (AmBiosome®), which is administered for a maximum of 10 days and has no toxicity, but is extremely expensive ($ 1,500 to $ 2,400 per treatment); Miltefosine that is orally administered but the treatment lasts 4 weeks and has restrictions on use for pregnant women and children Pentamidine and Ketoconazole. On the other hand, repeated infections or the 10 ineffective treatments have caused resistance of the parasites to these therapies [Singh, N. et al., Indian J. Med. Res. 2006, 123, 411-422]. Therefore, it is necessary to obtain new antileishmanial agents of lower cost, greater effectiveness and with less adverse side effects.
 fifteen
The search for new drugs for the treatment of Chagas disease and leishmaniasis has focused mainly on its potential action on essential and exclusive components of trypanosomatids. Trypanosomatid-specific enzymes include iron superoxide dismutase (FeSOD), which plays a fundamental role in the survival of parasites such as T. cruzi and Leishmania spp., For their ability to avoid damage caused by toxic radicals of the host. Since FeSOD is not present in humans, it can be considered an attractive target for the search for new drugs for the treatment of Chagas disease and leishmaniasis. Since prosthetic groups are essential in all enzymatic processes, changes in the active center of the enzyme, either by dissociation of the metal or by changes in coordination geometry, could be an effective way to deactivate its antioxidant action, and possibly affect both the growth and survival of the parasite cells.
 30
It has been previously described that alkylamino derivatives of benzo [g] phthalazine or phthalazine with antiparasitic activity in vitro and / or in vivo against T. cruzi and Leishmania spp., Showed a potent capacity to inhibit the FeSOD of the respective parasites [Sánchez -Moreno, M. et al., J. Med. Chem. 2012, 55, 9900-9913]. Similarly, macrocyclic and macrobicyclic polyamines containing pyrazole rings, which inhibit FeSOD from T. cruzi, showed trypanosomicidal activity in the acute phases.
and chronic Chagas disease [Sánchez-Moreno, M. et al., J. Med. Chem. 2012, 55, 4231-4243]. However, the use of simple acyclic esters derived from proton-ionizable pyrazole and their corresponding salts (pyrazolates), to which this invention refers, is not described so far, neither as FeSOD inhibitors nor as antiparasitic agents for Treatment of 5 Chagas disease and leishmaniasis.
DESCRIPTION OF THE INVENTION

The present invention provides the use of low toxicity and low cost compounds, which possess a potent antiparasitic activity against the Kinetoplastea class and in particular against the Trypanosomatidae family, in particular trypanosomicidal activity in Chagas disease, more particularly, in cases of immunodeficiency in which reactivation of parasitemia frequently occurs, in addition to activity against leishmaniasis. In general, said compounds are characterized by a potent efficacy to inhibit the enzyme iron superoxide dismutase (FeSOD). Since the parasite's survival is closely linked to the ability of its enzymes (FeSOD) to prevent damage caused by the toxic radicals of its host and, therefore, FeSOD plays a relevant role as part of the antioxidant defense in the parasites that cause said diseases, in the present invention compounds of formula (I) and (II) capable of inhibiting the FeSOD of Trypanosoma and Leishmania have been used, limiting their antioxidant defense against toxic host radicals.
 25
As evidenced in the examples, the compounds of the invention are structurally different from known drugs and have a much lower toxicity. Particularly significant are the results of antiparasitic activity and low toxicity obtained in vivo against T. cruzi, in the acute and chronic phase of Chagas disease, as well as in conditions of immunodeficiency in which reactivation processes occur. parasitemia

A first aspect of the present invention relates to the use of a compound of general formula (I) or (II) (hereafter compounds of the invention):
 35


where:
R1 and R2 may be the same or different and represent a (C1-C10) alkyl group;
R3 is selected from hydrogen or a (C1-C10) alkyl group; and 5
X + is a pharmaceutically acceptable monovalent cation;
For the preparation of a medicament, preferably the medicament is an antiparasitic agent.

Therefore, another aspect of the present invention relates to the compounds of general formula (I) or (II), described in the present invention, for use as a medicament.

The term "alkyl" refers in the present invention to linear or branched aliphatic chains, having 1 to 10 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert. butyl. Preferably the alkyl group has 1 to 4 carbon atoms.

In a preferred embodiment the compounds of the invention R3 is hydrogen.
 twenty
In another preferred embodiment, R1 and / or R2 is a (C1-C4) alkyl group, more preferably R1 and R2 are the same.

In a preferred embodiment, X + is an alkali cation, more preferably it is selected from Li +, Na + or K +, even more preferably it is Na +. 25

In a preferred embodiment the compound of the invention is selected from:
Dimethyl 1H-pyrazol-3,5-dicarboxylate;
3,5-bis (methoxycarbonyl) sodium pyrazolate;
Diethyl 1H-pyrazol-3,5-dicarboxylate; 30
3,5-bis (ethoxycarbonyl) sodium pyrazolate;
Dipropyl 1-pyrazol-3,5-dicarboxylate;
Sodium 3,5-bis (propoxycarbonyl) pyrazolate;
Diisopropyl 1-pyrazol-3,5-dicarboxylate; Y
1 H -pyrazol-3,5-dibutyl dicarboxylate.
 5
In a more preferred embodiment, the compounds of the invention are diethyl 1H-pyrazol-3,5-dicarboxylate or sodium 3,5-bis (ethoxycarbonyl) pyrazolate, even more preferably the compound is 1H-pyrazole-3,5- diethyl dicarboxylate.

Another aspect of the present invention relates to the use of a compound of general formula (I) or (II) as previously described, for the preparation of a medicament for the prevention and / or treatment of a parasitic disease, which preferably it is based on the inhibition of the parasitic FeSOD enzyme, more preferably for the prevention and / or treatment of diseases caused by a parasite of the Kinetoplastea class, more preferably of the Trypanosomatidae family, more preferably the parasites are of the Trypanosoma or Leishmania genus . Species thereof, may be, but are not limited to Trypanosoma cruzi, Leishmania infantum, Leishmania brazilensis, Leishmania donovani, Leishmania tropica or Leishmania chagasi, among others, known to a person skilled in the art.
 twenty
The parasitic diseases to be treated could be leishmaniasis or trypanosomiasis. "Leishmaniasis" is a disease caused by a protozoan of gender Leishmania and transmitted, mainly by diptera known as "sand fly", sand flies or jejenes. This disease occurs in humans and vertebrate animals, such as marsupials, canids, rodents and Primates The "trypanosomiasis" are diseases caused in humans or vertebrate animals that are caused by protozoan parasites of the genus Trypanosoma, including African human trypanosomiasis, also known as sleeping sickness, American trypanosomiasis or Chagas disease, or trypanosomiasis in animals or Nagana Preferably, trypanosomiasis is Chagas disease, preferably in its acute phase or in its chronic phase, particularly in immunocompromised individuals in which reactivation of parasitemia occurs.

Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (I) or (II) as described.
previously, together with a pharmaceutically acceptable carrier, said composition may optionally comprise another active ingredient, preferably the active ingredient is an antiparasitic. Furthermore, the use of said composition will be in a therapeutically effective amount.
 5
The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
 10
The pyrazole esters of the present invention and their pharmaceutically acceptable salts, as well as the pharmaceutical compositions containing them, can be used together with other drugs, or additional active ingredients, preferably antiparasitic, to provide a combination therapy. Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or not administration to a compound of general formula (I) or (II) or to the pharmaceutical composition that understands them.

Examples of an additional active ingredient include, but are not limited to: 20 meglumine antimoniate, sodium stibogluconate, amphotericin B, ketoconazole, miltefosine, paromomycin, pentamidine, allopurinol, itraconazole, gamma interferon, bleomycin, levamisole, mebendazole, metronidazole, metronidazole, metronidazole Miconazole, minomycin, methyluracil, nifurtimox, diminazeno, cyclologuanyl palmoate, emetine, furazolidone, rifampicin, isoniazid, interleukin 2, trimethoprim-sulfamethoxazole, suramin, 25 melarsoprol, eflornithine, chloroquine, quinine, tinine , pharmaceutically acceptable solvates, stereoisomers or prodrugs administered simultaneously or sequentially to at least one of the compounds of formula (I) or (II) described in the present invention.
 30
Therefore, another aspect of the present invention relates to a combined preparation for use separately, simultaneously or sequentially comprising at least one compound of general formula (I) or (II) as described above and another active ingredient, preferably an antiparasitic. More preferably the use of that combined preparation for the manufacture of a medicament for the treatment and / or prevention of a disease caused by a parasite, preferably of the
Kinetoplastea class, more preferably of the Trypanosomatidae family, more preferably the parasites are of the genus Trypanosoma or Leishmania. Species thereof, may be, but are not limited to Trypanosoma cruzi, Leishmania infantum, Leishmania brazilensis, Leishmania donovani, Leishmania tropica or Leishmania chagasi, among others, known to a person skilled in the art. 5

In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, for the characteristics of the compounds, as well as the age, condition of the patient, the severity of the alteration or disorder, and the route and frequency of administration.

Said therapeutic composition can be prepared in solid form or in suspension, in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention may be administered by any appropriate route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.

A further aspect of the present invention relates to a compound selected from: 3,5-bis (methoxycarbonyl) sodium pyrazolate; Dipropyl 1-pyrazol-3,5-dicarboxylate; Sodium 3,5-bis (propoxycarbonyl) pyrazolate; and 1 H -pyrazol-3,5-dibutyl dicarboxylate.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. 30
BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Evaluation of compounds 3 and 4 on the rate of infection and division of intracellular forms of T. cruzi in culture of infected Vero cells. (A)% of 35 infection. (B) number of amastigotes per infected cell. (C) number of
trypomastigotes in the culture medium. The values are results of four independent experiments; the legend indicates the value of the reduction of the corresponding parameter in relation to the control for each of the tested compounds.
 5
FIG. 2.- (A) In vitro inhibition (%) of CuZnSOD of human erythrocytes with compounds 3 and 4. (B) In vitro inhibition (%) of FeSOD of epimastigote forms of T. cruzi with compounds 3 and 4. Concentrations tested: 1-100 µM; the legend indicates the IC50 values of the corresponding SOD obtained for each of the compounds tested. IC50 values for erythrocytes were obtained by mathematical extrapolation.

FIG. 3.- Evaluation of compounds 3 and 4 on the rate of infection and division of intracellular forms of L. infantum in culture of J774.2 macrophages infected with L. infantum. (A)% of infection. (B) number of amastigotes per infected cell. 15 Measured at an IC25 concentration; the legend indicates the value of the reduction of the corresponding parameter in relation to the control for each of the tested compounds.

FIG. 4.- Evaluation of compounds 3 and 4 on the rate of infection and division 20 of intracellular forms of L. braziliensis in J774.2 macrophage culture infected with L. braziliensis. (A)% of infection. (B) number of amastigotes per infected cell. Measured at a concentration of IC25; the legend indicates the value of the reduction of the corresponding parameter in relation to the control for each of the tested compounds. 25

FIG. 5.- (A) In vitro inhibition (%) of CuZnSOD from human erythrocytes by compounds 3 and 4. (B) In vitro inhibition (%) of FeSOD from promastigote forms of L. infantum by compounds 3 and 4. ( C) In vitro inhibition (%) of FeSOD of promastigote forms of L. braziliensis by compounds 3 and 4. Concentrations tested: 1-100 µM; the legend indicates the IC50 values of the corresponding SOD obtained for each of the compounds tested. IC50 values for erythrocytes were obtained by mathematical extrapolation.

FIG. 6.- Parasitemia in the murine model during the acute phase of 35 Chagas disease. Dose administered: 25 mg / kg; the value of the reduction is indicated in the legend
of the number of trypomastigotes / mL in relation to the control (untreated animals) for each of the compounds tested.

FIG. 7.- Percentage of reactivation of parasitemia after the administration of an immunosuppressive treatment cycle for both the group of control mice (untreated animals) and those treated.

FIG. 8.- Total levels of anti-T Ig-G. cruzi expressed in absorbance units (OD, optical density) for groups of control mice (untreated animals) and treated at different days after infection (p.i.). For the day 120 p.i. The groups were divided into two subgroups: immunosuppressed (IS) and not immunosuppressed.

FIG. 9.- Result of the PCR amplification for post mortem mouse organs (day 120 p.i.), through the use of specific primers for the parasite superoxide dismutase gene (sod-b). Streets: (M) Base pair marker, 15 (1) Positive PCR control, (2) Negative PCR control, (3) Heart of the infection control group, (4) Heart of the group infected and treated with the compound 4, (5) Heart of the infected group and treated with compound 3, (6) Heart of the immunosuppressed infection control group, (7) Heart of the infected group, treated with compound 4 and immunosuppressed, (8) Heart of the infected group, treated with compound 3 and immunosuppressed.
EXAMPLES

The invention will now be illustrated by studies carried out by the 25 inventors, which show the specificity and effectiveness of the compounds of the invention.

Example 1
Preparation of the compounds of general formula (I) and (II) 30
The compounds of the general formula (I) were prepared by esterification of the 1H-pyrazol-3,5-dicarboxylic acid with the corresponding alcohol and the compounds of the general formula (II) were prepared by treating the esters (I) with sodium hydroxide.

General procedure for the synthesis of compounds of formula (I):
when R1 and R2 are equal and are represented as R and R3 is H


Methodology: To a solution of 1 H -pyrazol-3,5-dicarboxylic acid monohydrate (5.00 g; 28.7 mmol) in 100 mL of the appropriate alcohol (ROH), a stream of hydrogen chloride gas was passed to saturation, keeping stirring for 24 5 h. After that time, the solvent was removed to dryness and a 10% aqueous solution of NaCO3H was added until a basic pH was reached. The reaction mixture was extracted with chloroform and the organic phase was dried with MgSO4. Solvent removal leads to the desired product in the form of a chromatographically pure oil that solidifies shortly. 10

1 H -pyrazol-3,5-dimethyl dicarboxylate (1)

Reagents: 1H-pyrazol-3,5-dicarboxylic acid monohydrate, methanol. Yield: 4.86 g (92%) (lit. 86%; Schenck, T.G. et al., Inorg. Chem. 1985, 24, 2334-2337). P. f. 154-15 155 oC. Elemental analysis (C7H8N2O4): Theoretical% C 45.66,% H 4.38,% N 15.21; Found% C 45.77,% H 4.30,% N 15.07. max (KBr) / cm-1 3369 (NH), 1729, 1709 (CO). 1 H NMR (500 MHz; DMSO-d6) : 14.71 (1 H, s), 7.21 (1 H, s), 3.84 (3H, s). 13C NMR (125 MHz; DMSO-d6) : 160.7, 143.3, 135.9, 111.3, 52.5. FAB-MS (m / z): 185 (MH +).
 twenty
Diethyl 1H-pyrazol-3,5-dicarboxylate (3)

Reagents: 1H-pyrazol-3,5-dicarboxylic acid monohydrate, ethanol. Yield: 5.48g (90%). P. f. 54-55 oC (hexane) (lit., m.p. 53-54 oC; Iturrino, L. et al., Eur. J. Med. Chem. 1987, 22, 445-451). Elemental analysis (C9H12N2O4): Theoretical% C 50.94,% H 5.70,% N 25 13.20; Found% C 50.80,% H 5.51,% N 13.34. max (KBr) / cm-1 3260 (NH), 1730 (CO). 1 H NMR (300 MHz; DMSO-d6) : 14.62 (1 H, s), 7.18 (1 H, s), 4.31 (4H, c), 1.31 (6H, t).
13C NMR (75 MHz; DMSO-d6) : 160.9, 159.0, 143.6, 134.7, 110.8, 60.9, 14.1. FAB-MS (m / z): 213 (MH +).

Dipropyl 1-pyrazol-3,5-dicarboxylate (5)
 5
Reagents: 1H-pyrazol-3,5-dicarboxylic acid monohydrate, propanol. Yield: 6.35 g (92%). P. f. 53-55 oC. Elemental analysis (C11H16N2O4): Theoretical% C 54.99,% H 6.71,% N 11.66; Found% C 54.91,% H 6.65,% N 11.71. max (KBr) / cm-1 3443 (NH), 1732 (CO). 1 H NMR (500 MHz; DMSO-d6) : 14.64 (1 H, s), 7.17 (1 H, s), 4.19 (4H, t), 1.67 (4H, m), 0.92 (6H, t). 13C NMR (125 MHz; DMSO-d6) 161: 161.2, 158.6, 143.8, 10 134.7, 110.8, 66.3, 21.54, 10.2. FAB-MS (m / z): 241 (MH +).

Diisopropyl 1-pyrazol-3,5-dicarboxylate (7)

Reagents: 1H-pyrazol-3,5-dicarboxylic acid monohydrate, isopropanol. Yield: 6.21 g (90%). P. f. 50-52 oC. Elemental analysis (C11H16N2O4): Theoretical% C 54.99,% H 6.71,% N 11.66; Found% C 55.07,% H 7.08,% N 11.62. max (KBr) / cm-1 3432 (NH), 1724 (CO). 1 H NMR (500 MHz; DMSO-d6) : 14.58 (1 H, s), 7.11 (1 H, s), 5.09 (4H, m), 1.28 (6H, s), 1.26 (6H, s). 13C NMR (125 MHz; DMSO-d6) 161: 161.0, 158.5, 144.4, 135.4, 111.1, 69.0, 22.0. FAB-MS (m / z): 241 (MH +). twenty
1 H -pyrazol-3,5-dibutyl dicarboxylate (8)

Reagents: 1H-pyrazol-3,5-dicarboxylic acid monohydrate, butanol. Yield: 6.78 g (88%). P. f. 46-47 oC. Elemental analysis (C13H20N2O4): Theoretical% C 58.19,% H 7.51,% N 10.44; Found% C 58.17,% H 7.42,% N 10.40. max (KBr) / cm-1 3437 (NH), 1727 25 (CO). 1 H NMR (500 MHz; DMSO-d6) : 14.66 (1 H, s), 7.16 (1 H, s), 4.25 (4H, t), 1.65
(4H, m), 1.37 (4H, m), 0.89 (6H, t). 13C NMR (125 MHz; DMSO-d6) : 160.3, 143.8, 135.4, 111.2, 64.9, 30.6, 19.1, 14.0. ESMS + (m / z): 269 (MH +).

General procedure for the synthesis of sodium salts of formula (II):
when R1 and R2 are equal and are represented as R 5


Methodology: To a solution of the 1H-pyrazol-3,5-dicarboxylic acid ester (1.00 g) in 30 mL of the corresponding alcohol (ROH), an equimolecular amount of sodium hydroxide dissolved in 10 mL of the same alcohol was slowly added . The reaction mixture 10 was maintained with stirring and at room temperature. After the time indicated in each case, the solvent was concentrated and the solid obtained was filtered.

3,5-bis (methoxycarbonyl) sodium pyrazolate (2) 15

Reagents: 1H-pyrazol-3,5-dicarboxylic acid methyl ester, methanol. Reaction conditions: stirring at room temperature 48 h. Yield: 1.06 g (95%). P. f .:> 215 oC (decomp.). Elemental analysis (C7H7N2O4Na):% C 40.79,% H 3.42,% N 13.59; Found% C 40.52,% H 3.55,% N 13.42. max (KBr) / cm-1 1704 (CO). 1H NMR 20 (500 MHz; DMSO-d6) : 6.97 (1H, s), 3.68 (6H, s). 13C NMR (125 MHz; DMSO-d6) : 163.3, 142.5, 111.0, 50.3. FAB-MS (m / z): 207 (MH +).

3,5-bis (ethoxycarbonyl) sodium pyrazolate (4)
 25
Reagents: 1H-pyrazol-3,5-dicarboxylic acid ethyl ester, ethanol. Reaction conditions: stirring at room temperature 1 h. Yield: 1.07 g (97%). P. f .: 213-
214 oC (lit.3, mp 212-214 oC) (Reviriego, F. et al., J. Am. Chem. Soc. 2006, 128, 16458-16459. Elemental analysis (C9H11N2O4Na):% C 46.15, % H 4.70,% N 11.96; Found% C 46.03,% H 4.68,% N 12.10 max (KBr) / cm-1 1670 (CO). 1 H NMR (500 MHz ; DMSO-d6) : 6.69 (1H, s), 4.16 (4H, c), 1.25 (6H, t). 13C NMR (125 MHz; DMSO-d6) : 163.6, 142.4, 111.1, 58.5, 14.4 EM-FAB (m / z): 235 (MH +).

3,5-bis (propoxycarbonyl) sodium pyrazolate (6)

Reagents: 1H-pyrazol-3,5-dicarboxylic acid propyl ester, propanol. Reaction conditions: stirring at room temperature 48 h. Yield: 1.06 g (97%). P. f .: 10> 300 oC. Elemental analysis (C11H15N2O4Na):% C 50.38,% H 5.77,% N 10.68; Found% C 50.72,% H 5.70,% N 10.43. max (KBr) / cm-1 1724, 1696 (CO). 1 H NMR (500 MHz; DMSO-d6) : 6.93 (1H, s), 4.05 (4H, t), 1.64 (4H, m), 0.92 (6H, t). 13C NMR (125 MHz; DMSO-d6) : 163.9, 142.5, 110.9, 63.9, 21.7, 10.3. FAB-MS (m / z): 263 (MH +).
 fifteen
In vitro activity assays against T. cruzi and Leishmania spp.
As demonstrated in Examples 2 (Table 1) and 3 (Tables 2 and 3), the compounds that share the general formula (I) (compounds 1, 3, 5, 7 and 8) and the general formula (II ) (compounds 2, 4, and 6) are effective in vitro in the treatment of diseases caused by the T. cruzi and Leishmania spp. parasites, while maintaining a low toxicity level (substantially lower than the reference compounds ). In general, all of them selectively inhibit the FeSOD of the parasites T. cruzi and Leishmania spp. in relation to CuZnSOD.

Example 2 25
Procedure for evaluation of in vitro activity and toxicity of compounds 1-8 against epimastigote (extracellular) and amastigote (intracellular) forms of T. cruzi, toxicity against Vero cells and selectivity indices (IS)
Methodology: For these studies, T. cruzi type I was used (strain SN3, isolated in 30 Guajira in northern Colombia in humans, 2006) [Téllez-Meneses, J. et al., Acta Trop. 2008, 108, 26-34]. The culture of the epimastigote forms of T. cruzi was performed in vitro.
in sterility in single phase MTL (Medium Trypanosomes Liquid) culture medium enriched with 10% (v / v) fetal bovine serum (SBF-I) inactivated at 56 ° C for 30 minutes. The inoculum of parasites to start the culture was 5 x 104 cells / mL in 5 mL of medium in 25 cm2 Falcon® plastic bottles and kept in an oven at 28 ° C. Cultures were performed routinely achieving exponential growth of flagellate until the necessary cell mass was obtained for subsequent studies. For the performance of the trials both epimastigote and trypomastigote and amastigote forms were used.

Epimastigote forms of T. cruzi cultured in the manner described above 10 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 5 x104 parasites / well.
 fifteen
The compounds to be tested were dissolved in dimethylsulfoxide (DMSO), a solvent that at a final concentration in the test medium of 0.01% (v / v) does not show toxicity or have any effect on the growth of parasites. The compounds were added to the culture medium at final concentrations of 100, 50, 25, 10 and 1 µM. The effect of each compound on the growth of epimastigote forms, at the 20 different concentrations tested, was evaluated at 72 h, using a Neubauer hemocytometric chamber and the trypanocidal effect was expressed as the IC50 (concentration required to produce an inhibition of the 50%, calculated by the analysis of the linear regression of the Kc at the concentrations tested) [Sánchez-Moreno, M. et al., J. Med. Chem. 2011, 54, 970-979]. 25

For the study of the in vitro effect on intracellular forms of T. cruzi, the experimental model designed by the authors of the present invention was used [Ramírez-Macías, I. et al., Parasitol. Int. 2012, 61, 405-413]. Vero cells were detached from the culture flask where they were attached by trypsinization. For this, the culture medium was removed, then the cell surface was covered with EDTA-trypsin and incubated cold for 5 minutes. After that, the mixture was passed to a conical bottom flask of 25 mL capacity (steriline), centrifuged at 800 rpm for 5 minutes, the supernatant was removed and the cells were counted in Neubauer chamber. Vero cells were resuspended at a concentration of 1 x 104 cells / well in RPMI culture medium, cultured in 24-well plates, in each of the
which had previously introduced a 12mm round glass coverslip. For adhesion, the cells were left 24 h at 37 ° C in 5% CO2.

Once the cells were adhered, they were infected in vitro with 1 x 105 cells / well of trypomastigote forms of T. cruzi [Ramírez-Macías, I. et al., Parasitol. Int. 2012, 61, 5 405-413]. The infection was maintained for 24 hours for the parasite to enter the cell. After this time the culture medium was removed and fresh medium was added with the products to be tested, at the concentrations necessary to obtain the IC50 (100 to 1 μM). 72 hours after incubation, the crystals were removed. 10

Once the crystals were removed, they were placed on a slide. They were fixed with methanol and allowed to dry. Once fixed and dried, DPX (Panreac®), microscopy mounting medium was added and stained with Giemsa; for this, immediately before use and in a test tube, Azur-Eosin-Methylene Blue 15 solution was diluted according to Giemsa DC (Code 251338) with Sörensen buffer (dilution 1:10), mixed well and the preparation was covered Letting color for 20 minutes. It was washed with distilled water. 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 was counted. twenty

For the study of non-specific toxicity against Vero cells, the experimental model designed by the authors of the present invention was used [Marín, C. et al., J. Nat. Prod. 2011, 74, 744-750]. Vero cells were established from the kidney of an adult African green monkey, keeping the cultures at a density of 1 x 104 25 cells / mL at 37 ° C and 5% CO2. The cells were deposited in a sterile and centrifuged at 800 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI medium. 1 x 104 cells / well were deposited in 24-well titration plates, incubated for 24 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 the concentrations of 100, 50, 25, 10 and 1 μM. At 72 hours after incubation, the samples were prepared for reading in the flow cytometer following a previously described procedure. The cells and the medium present in the wells were split, 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 cells with intact plasma membrane 5 have a green fluorescence, while damaged or dead cells have a red fluorescence. The percentage of viability was calculated. The number of dead cells was determined by comparison with the control cultures.
 10
Table 1.- Activity, toxicity and selectivity indices found for compounds 1-8 against extracellular and intracellular forms of T. cruzi. Results of four independent experiments. aIC50 = concentration required to obtain 50% inhibition, calculated by linear regression analysis of the Kc value at the concentrations used (1, 10, 25, 50 and 100 µM). bConcentration needed to obtain 50% inhibition against Vero cells after 72 h of culture. Selectivity index = IC50 of Vero cells / IC50 of extracellular and intracellular forms of T. cruzi. In parentheses: number of times the SI of the compounds is greater than the IS of the reference drug.
 twenty
 Compounds  IC50 µMa Toxicity IC50 cel. Vero (µM) b ISc
 Epimastigote forms  Intracellular amastigote forms Epimastigote forms Intracellular amastigote forms
 Benznidazole  15.8 ± 1.1 23.3 ± 4.6 13.6 ± 0.9 0.9 0.6
 one  40.5 ± 6.3 16.9 ± 2.8 186.5 ± 9.3 4.6 (5) 11.0 (18)
 2  27.5 ± 3.2 27.1 ± 1.1 139.1 ± 9.3 5.0 (6) 5.1 (9)
 3  17.2 ± 2.4 10.8 ± 0.7 385.4 ± 11.5 22.4 (25) 35.7 (59)
 4  16.5 ± 1.6 9.3 ± 5.8 404.1 ± 13.3 24.5 (27) 43.4 (72)
 5  38.7 ± 7.7 23.6 ± 1.4 154.5 ± 12.5 4.0 (4) 6.5 (11)
 6  34.5 ± 2.2 34.5 ± 1.7 170.2 ± 13.8 4.9 (6) 4.9 (8)
 7  31.2 ± 3.8 30.9 ± 4.7 179.4 ± 13.1 5.7 (6) 5.8 (10)
 8  35.8 ± 4.1 23.7 ± 3.3 242.1 ± 10.6 6.8 (7) 10.2 (17)


Particularly preferred compounds of the present invention for use as an antiparasitic against T. cruzi are the compounds of formula 3 and 4, which have a level of non-specific toxicity against Vero cells much lower than that of benznidazole (Bzn) and even less than the rest of the esters and pyrazolates 5 studied in the present invention.

Example 3

Procedure for evaluating the in vitro activity of compounds 1-8 10 against promastigote (extracellular) and amastigote (intracellular) forms of L. infantum and L. braziliensis, macrophage toxicity and selectivity indices
Methodology: For this study, promastigote and amastigote forms of two species of the genus Leishmania were used: 15
- L. braziliensis (MHOM / BR / 1975 / M2904).
- L. infantum (MCAN / ES / 2001 / UCM-10).
The Leishmania species have been maintained in our laboratory for several years by means of cultures at 28 ° C in MTL medium enriched with 10% inactive fetal bovine serum (FCSI) and so that they do not lose their virulence, 20 periodic passes are made in vivo in mice strain Balb / c [Téllez-Meneses, J. et al., Acta Trop. 2008, 108, 26-34].

Once the exponential phase of development was reached (106 promastigotes / mL), the culture was centrifuged at 1000 g for 10 minutes in order to concentrate the flagellated forms in a button. The amastigote forms were obtained in vitro after infestation of macrophages of the J774.2 line (ECACC 85011428) according to the method developed by the authors of the present invention [González, P. et al., J. Antimicrob. Agents 2005, 25, 136-141].
 30
For in vitro assays of promastigote forms, promastigote forms were started in exponential growth phase, grown at 28 ° C in MTL medium supplemented with 10% FCSI of the two Leishmania strains. The promastigote forms test is performed in a similar way to that described for the epimastigote forms of T. cruzi. 35

For studies on amastigote forms, similar to that described for T. cruzi, only J774.2 macrophages with promastigote forms in stationary phase of growth of the two Leishmania strains are used, according to the methodology described above .
 5
Throughout all the experiments, studies were conducted aimed at knowing the differences in the sensitivity of the parasites against drugs of activity known as benznidazole in the case of T. cruzi and Glucantime for the two Leishmania strains, which allowed us Compare your behavior with the results obtained with our products. 10

Table 2.- Activity, toxicity and selectivity indices found for compounds 1-8 against extracellular and intracellular forms of Leishmania infantum. Results of four independent experiments. aIC50 = concentration necessary to obtain 50% inhibition, calculated by linear regression analysis of the Kc value at the concentrations used (1, 10, 25, 50 and 100 µM). bConcentration needed to obtain 50% inhibition against macrophages J 774.2 after 72 h of culture. Selectivity index = IC50 of macrophages / IC50 of extracellular and intracellular forms of L. infantum. In parentheses: number of times the SI of the compounds is greater than the IS of the reference drug 20

 Compounds  IC50 µMa Toxicity IC50 macrophages (µM) b ISc
 Promastigote forms  Intracellular amastigote forms Promastigote forms Intracellular amastigote forms
 Glucantime  18.0 ± 3.1 30.0 ± 2.7 15.2 ± 1.3 0.8 0.5
 one  23.5 ± 4.2 18.4 ± 2.0 166.2 ± 11.4 7.1 (9) 9.0 (18)
 2  17.5 ± 3.2 22.4 ± 1.5 222.4 ± 21.2 12.7 (16) 9.9 (20)
 3  24.4 ± 1.5 16.3 ± 0.9 233.5 ± 15.6 9.6 (12) 14.3 (29)
 4  5.9 ± 0.2 6.7 ± 1.0 288.6 ± 23.1 48.9 (61) 43.1 (86)
 5  15.6 ± 0.8 7.6 ± 0.6 170.2 ± 13.5 10.9 (14) 22.4 (45)
 6  17.7 ± 2.5 21.3 ± 1.0 264.3 ± 16.9 14.9 (19) 12.4 (25)
 7  19.7 ± 0.8 2.8 ± 0.7 158.1 ± 7.4 8.0 (10) 56.1 (113)
 8  24.8 ± 2.6 19.1 ± 1.1 271.1 ± 16.1 10.9 (14) 14.2 (28)


Table 3.- Activity, toxicity and selectivity indices found for compounds 1-8 against extracellular and intracellular forms of Leishmania 25 braziliensis. Results of four independent experiments. aIC50 = concentration necessary to obtain 50% inhibition, calculated by linear regression analysis
of the Kc value at the concentrations used (1, 10, 25, 50 and 100 µM). bConcentration needed to obtain 50% inhibition against macrophages J 774.2 after 72 h of culture. Selectivity index = IC50 of macrophages / IC50 of extracellular and intracellular forms of L. braziliensis. In parentheses: number of times the SI of the compounds is greater than the IS of the reference drug. 5
 Compounds  IC50 µMa Toxicity IC50 macrophages (µM) b ISc
 Promastigote forms  Intracellular amastigote forms Promastigote forms Intracellular amastigote forms
 Glucantime  25.6 ± 1.6 31.1 ± 3.0 15.2 ± 1.3 0.6 0.5
 one  21.4 ± 1.7 18.2 ± 0.9 166.2 ± 11.4 7.8 (13) 9.1 (18)
 2  15.9 ± 0.8 23.2 ± 3.5 222.4 ± 21.2 14.0 (23) 9.6 (19)
 3  18.8 ± 1.8 9.3 ± 0.8 233.5 ± 15.6 12.4 (21) 25.1 (50)
 4  23.8 ± 3.2 6.8 ± 0.7 288.6 ± 23.1 12.1 (20) 42.4 (85)
 5  16.0 ± 1.4 17.9 ± 2.2 170.2 ± 13.5 10.6 (18) 9.5 (19)
 6  16.6 ± 1.4 14.7 ± 0.5 264.3 ± 16.9 15.9 (26) 18.0 (36)
 7  32.1 ± 2.9 21.5 ± 1.5 158.1 ± 7.4 4.9 (8) 7.35 (15)
 8  36.2 ± 3.6 20.8 ± 1.4 271.1 ± 16.1 7.5 (13) 13.0 (26)

Particularly preferred compounds for use as an antiparasitic against Leishmania spp. are those of formula 3, 4 and 7. Specifically, compounds 3 and 4 are much more active against extracellular (promastigote) and intracellular (amastigote) forms of L. infantum and L. braziliensis than the reference medicine, Glucantime , and much less toxic about macrophages.

Example 4
Procedure for the evaluation of compounds 3 and 4 on the rate of infection and the growth of parasites in culture media of Vero 15 cells infected with T. cruzi
Methodology: Vero cells were cultured under the same conditions outlined above for 2 days. The cells were then infected in vitro with metacyclic forms of T. cruzi, in a 10: 1 ratio. Compounds 3 and 4 (at IC25 concentration) were added immediately after infestation and incubated for 12 h at 37 ° C in 5% CO2. Non-phagocytic parasites and products were removed by washing and infected cultures were grown for 10 days in fresh medium, adding fresh medium every 48 hours. The activity of the compounds was determined by the% of infected cells, number of amastigotes per infected cell and number of trypomastigotes in the medium. Treated and untreated cultures were fixed and stained with methanol and Giemsa. The% of infected cells and the average number of amastigotes per infected cell was determined by analysis of
200 Vero cells under a microscope every 48 hours. The number of trypomastigotes in the medium was determined using a Neubauer chamber.

In parasite growth assays in culture media of Vero cells infected with T. cruzi, compounds 3 and 4 effectively reduce the rate of infection, the number of amastigotes per infected cell, and the number of trypomastigotes in the culture medium in relation to control (see FIG. 1). Typical benznidazole values in these trials were 23% (FIG. 1A), 13% (FIG. 1B) and 46% (FIG. 1C) [Sánchez-Moreno, M. et al., J. Med. Chem. 2012 , 55, 4231-4243].
 10
Example 5
Procedure for assessing the ability of compounds 3 and 4 to inhibit the FeSOD of epimastigotes of T. cruzi in relation to the CuZnSOD of human erythrocytes
Methodology: The previously selected compounds were evaluated as 15 inhibitors of the FeSOD enzyme exclusive to parasitic protozoa of the Kinetoplastea class, and therefore absent in the mammalian host, in order to validate this new and attractive target for the design of new drugs with Antiparasitic activity due to its ability to inactivate the action of the FeSOD of the parasite by capturing its metal ion through competitive complexation mechanisms. In parallel, FeSOD (parasite) / CuZnSOD (human) selectivity tests were performed. The inhibition of the new compounds on FeSOD / CuZnSOD activity was quantified in a spectrophotometer at 560 nm according to a technique described previously [Beyer, W.F. & Fridovich, I., Anal Biochem. 1987, 161, 559-66], based on the reduction of tetrazolium nitro blue (NBT) by superoxide ions and that the authors of the present invention have routinely tested for trypanosomatids [Sánchez-Moreno, M. et al., J. Med. Chem. 2011, 54, 970-979; Ramírez-Macías, I. et al., J. Antimicrob. Chemother 2011, 66, 913-919].
 30
In these assays, FeSOD has been shown to effectively and selectively inhibit epimastigote forms of T. cruzi in relation to the CuZnSOD of human red blood cells (see FIG. 2).

 35

Procedure for assessing the effect of compounds 3 and 4 on the rate of infection and the growth of parasites in culture media of macrophages infected with L. infantum and L. braziliensis
Methodology: J774.2 macrophages, preserved in the laboratory by cryopreservation in liquid nitrogen and successive passes in culture with essential medium 5 (MEM) + glutamine + 20% FCSI, were deposited in a steriline and centrifuged at 100 g for 5 min, the supernatant was discarded and the cells resuspended in the same medium at a final concentration of 1 x 106 cells / mL.

Then, 10 µl of the cell suspension was deposited in each well of a 24-well 10 culture plate (each with a round 10 mm diameter glass cover glass) in a total volume of 500 µl, and incubated for 24 h at 37 ºC in humid atmosphere enriched with 5% CO2.

The cells were infected in vitro with promastigote forms of the two species of Leishmania, in a 10: 1 ratio. Compounds 3 and 4 (at an IC25 concentration) were added immediately after infestation and incubated for 12 h at 37 ° C in 5% CO2. The non-phagocytic parasites and the products were removed by washing and the infected cells were cultured for 10 days in fresh medium, which was added every 48 hours. The activity of the compounds was determined by the% of infected cells and the number of amastigotes per infected cell [Sánchez-Moreno, M. et al., J. Med. Chem. 2011, 54, 970-979]. Treated and untreated cultures were fixed and stained with methanol and Giemsa. The% of infected cells and the average number of amastigotes per infected cell was determined by the analysis of 200 cells under a microscope every 48 hours. 25

In parasite growth assays in J774.2 macrophage culture media infected with L. infantum and L. braziliensis, compounds 3 and 4 also effectively reduce the rate of infection and division of intracellular forms relative to control (see FIG 3 and 4). The values of these tests are results of 30 four independent experiments. Typical glucantime values in these trials were 24% (FIG. 3A), 30% (FIG. 3B), 22% (FIG. 4A) and 37% (FIG. 4B) [Marín, C. et al., Eur J. Med. Chem. 2013, 62, 466-477].

 35

Example 7
Procedure for assessing the ability of compounds 3 and 4 to inhibit the FeSOD of promastigotes of L. infantum and L. braziliensis in relation to the CuZnSOD of human erythrocytes.
Methodology: The procedure used to evaluate the capacity of inhibition of the FeSOD enzyme of the promastigote forms of L. infantum and L. braziliensis in relation to the CuZnSOD of human erythrocytes was the same as described previously in the case of T. cruzi.

Parallel to what happens in the T. cruzi assays, the two selected compounds (3 and 4) effectively and selectively inhibit the FeSOD of promastigotes of L. infantum and L. braziliensis in relation to the CuZnSOD of human erythrocytes (see FIG. 5).

Example 8 15
Procedure for assessing the toxicity of compounds 3 and 4 in vivo
Methodology and results: As a model, albino mice of the strain Balb / c, lot of 5 animals of the same age (6-8 weeks) and weight (25-30 g) and female sex were used, which were administered an initial dose of 100 mg / kg of each compound on alternative days in order to find out the lethal dose 50 (LD50). In view of the fact that after administering 500 mg / kg weight no apparent toxic effect or death of any specimen was achieved, doses of 500 mg / kg of each compound were administered on alternative days until reaching 6 g / kg, where it was decided to cease the experiment considering that these levels of administration of the compound showed their "non-toxicity". A routine biochemical parameter analysis was performed one week after the end of the treatment and no significant alteration was observed. The only appreciation outside the norm was that in the immediate 15-20 minutes after administration of the compounds the mice showed some hyperactivity after their transfer to the cage, which ceased after this period and left no sequelae or change in appreciable behavior. 30

In vivo toxicity tests in the murine model (albino mice of the strain Balb / c) have made it clear that at doses between 500 mg / kg and 6 g / kg, the two selected compounds (3 and 4) do not produce toxic effects Apparent neither the death of any animal occurs. 35


Example 9
Procedure for in vivo evaluation of the activity of compounds 3 and 4 in the acute phase of Chagas disease
Methodology: Albino mice of the strain Balb / c, a batch of 6 5 animals of the same age (6-8 weeks) and weight (25-30 g), and female, were used as a model. Initially the lethal dose (LD50) of the selected compounds was sought (as seen in a previous example), as well as their permanence and serum stability. As inoculum, metacyclic trypomastigotes were used. Each animal was inoculated with 5 x 105 metacyclic trypomastigotes, intraperitoneally. Experimental animals were treated under the norms and principles of the international guide for biomedical research with experimental animals and with the authorization of the Bioethics Committee of the University of Granada. The lots were distributed as follows: a) a lot for the study of the development of the disease [acute phase and chronic phase (Example 10)], positive control group 15 composed of infected animals without any treatment, and b) a second batch group: animals infected and treated with different concentrations of the compounds under study (one sublot per product and concentration to be tested).

Parasitemia, number of circulating parasites, was quantified through chamber 20 of blood cell count. Peripheral blood, obtained by puncture of the maxillary vein, was used in phosphate buffer (PBS). This count was performed every 3 days, for 30 days (end of the acute phase). The administration of the products was carried out on the fifth day of the infection and, once the parasitization was confirmed, different concentrations of the products were tested, according to the data of the LD50 and the dosage 25 at the dose established for each product and during 5 days. Similar groups of mice were treated with benznidazole (5 mg / kg of weight / day for 5 days), under the same conditions as described above.

Both compounds 3 and 4 showed a high efficacy of parasitemia 30 inhibition in the acute phase (see FIG. 6).

Example 10
In vivo evaluation procedure of compounds 3 and 4 on the percentages of reactivation of parasitemia after immunosuppression in the chronic phase of Chagas disease
Methodology: After day 60, the animals enter the chronic phase; at day 120 of the experiment an immunosuppression situation with cyclophosphamide monohydrate is induced as described [Cencig, S. et al., PLoS Negl. Trop Dis. 2011, 5, e1216]; Finally, the slaughter of the animals takes place for the extraction of tissues and organs that will be subjected to evaluation of the effect of the compounds at the level of the parasitemia after the treatment, by counting (acute and chronic phase), ELISA (comparison of the acute phase and chronic) and tissue PCR (chronic phase).

Once day 120 (advanced chronic phase) was reached, the mice were divided into two subgroups, one that remained under the same conditions and another that underwent immunosuppression therapy (induced by cyclophosphamide monohydrate). This added variable allowed to determine the degree of reactivation of parasitemia and therefore the parasitological cure or not of the specimens (see FIG. 7, 8 and 9). In FIG. 7, specifically, the percentage of reactivation of parasitemia is shown, by fresh counting of the specimens that were immunosuppressed 15 (both treated and controls); the percentage of reactivation of parasitemia, which in the control is 60%, falls to 5% in the presence of compound 4, and to 24% in the presence of compound 3. In FIG. 8 the variations in antibody concentrations against T. cruzi, signal of the immunological state of the mouse are observed; it shows how the chronic phase has been reached in a normal situation, treated 20 or not, an immunological equilibrium is reached (chronicity of the disease); this equilibrium state is artificially broken with immunosuppression, so that in the control situation there is a considerable increase in immunoglobulin levels as a result of the presence of the parasite in blood, while in those treated with compounds 3 and 4 levels remain virtually constant, indicators of lower parasitemia. The results of the post mortem analysis shown in FIG. 9, which compares results of elimination of parasitemia in heart tissues of non-immunosuppressed and immunosuppressed animals analyzed by the PCR technique. In the absence of immunosuppression, both by treatment with compound 3 and with 4, no band is detected (signal of decreased parasitemia). But in tissues of the heart from previously immunocompromised animals, in the case of treatment with compound 4 no band is observed, while in the case of treatment with compound 3 a fainter band is observed, signal of a lower parasitemia with Regarding control.

权利要求:
Claims (19)
[1]


1. Use of a compound of general formula (I) or (II)
 5

where:
R1 and R2 may be the same or different and represent a (C1-C10) alkyl group;
R3 is selected from hydrogen or a (C1-C10) alkyl group; and 10
X + is a pharmaceutically acceptable monovalent cation;
for the preparation of a medicine.

[2]
2. Use according to claim 1, wherein R3 is hydrogen.
 fifteen
[3]
3. Use according to any of claims 1 or 2, wherein R1 and / or R2 is a (C1-C4) alkyl group.

[4]
4. Use according to any of claims 1 to 3, wherein R1 and R2 are the same.
 twenty
[5]
5. Use according to any of claims 1 to 4, wherein X + is an alkali cation.

[6]
6. Use according to any of claims 1 to 5, wherein X + is selected from Li +, Na + or K +, preferably Na +. 25

[7]
7. Use according to claim 1, wherein the compound is selected from:
Dimethyl 1H-pyrazol-3,5-dicarboxylate;
3,5-bis (methoxycarbonyl) sodium pyrazolate;
Diethyl 1H-pyrazol-3,5-dicarboxylate; 30
3,5-bis (ethoxycarbonyl) sodium pyrazolate;
Dipropyl 1-pyrazol-3,5-dicarboxylate;
Sodium 3,5-bis (propoxycarbonyl) pyrazolate;
Diisopropyl 1-pyrazol-3,5-dicarboxylate; Y
1 H -pyrazol-3,5-dibutyl dicarboxylate.

[8]
8. Use according to the preceding claim, wherein the compound is 1 H -pyrazol-3,5-5 diethyl dicarboxylate or sodium 3,5-bis (ethoxycarbonyl) pyrazolate.

[9]
9. Use of a compound of general formula (I) or (II) as described in any of claims 1 to 8, for the preparation of a medicament for the prevention and / or treatment of parasitic diseases through Parasitic FeSOD enzyme inhibition.

[10]
10. Use according to the preceding claim, wherein the diseases are diseases caused by a parasite of the Kinetoplastea class.
 fifteen
[11]
11. Use according to claim 10, wherein the parasite is from the Trypanosomatidae family.

[12]
12. Use according to claim 11, wherein the parasites are of the genera Leishmania or Trypanosoma. twenty

[13]
13. Use according to claim 12, wherein the parasites are of the species Trypanosoma cruzi, Leishmania infantum Leishmania braziliensis, Leishmania donovani, Leishmania tropica or Leishmania chagasi.
 25
[14]
14. Use according to any of claims 9 to 13, wherein the disease is trypanosomiasis.

[15]
15. Use according to claim 14, wherein the disease is Chagas disease. 30

[16]
16. Use according to any of claims 9 to 13, wherein the disease is leishmaniasis in humans or animals.

[17]
17. Pharmaceutical composition comprising at least one compound of general formula (I) or (II) as described in any one of claims 1 to 8, together with a pharmaceutically acceptable carrier.

[18]
18. Pharmaceutical composition according to claim 17, further comprising at least one other antiparasitic active ingredient.

[19]
19. Compound selected from:
3,5-bis (methoxycarbonyl) sodium pyrazolate; 5
Dipropyl 1-pyrazol-3,5-dicarboxylate;
Sodium 3,5-bis (propoxycarbonyl) pyrazolate; Y
1 H -pyrazol-3,5-dibutyl dicarboxylate.
 10

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同族专利:

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公开号 | 公开日

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ES2566228B1|2017-02-08|

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WO2016038238A1|2016-03-17|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2022008784A1|2020-07-10|2022-01-13|Universidad De Granada|Use of simple acyclic polyamines for the treatment of diseases caused by parasites of the genus leishmania|US7863301B2|2003-08-21|2011-01-04|Wisconsin Alumni Research Foundation|Potentiators of insulin secretion|

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BR112015002779A2|2012-08-17|2019-10-15|Hoffmann La Roche|process for the preparation of pyrazole carboxylic acid derivatives|

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申请号 | 申请日 | 专利标题

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ES201431309A|ES2566228B1|2014-09-11|2014-09-11|Use of proton-ionizable pyrazole-derived esters and their corresponding salts for the treatment of Chagas disease and leishmaniasis|ES201431309A| ES2566228B1|2014-09-11|2014-09-11|Use of proton-ionizable pyrazole-derived esters and their corresponding salts for the treatment of Chagas disease and leishmaniasis|

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PCT/ES2015/070658| WO2016038238A1|2014-09-11|2015-09-10|Use of ester derivatives of pyrazole proton-ionizable compounds and the corresponding salts thereof for the treatment of chagas disease and leishmaniasis|