WO2015059337A1 - Polyamine compounds and metal complexes comprising same for the use thereof as antiparasitic agents - Google Patents

Polyamine compounds and metal complexes comprising same for the use thereof as antiparasitic agents Download PDF

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WO2015059337A1
WO2015059337A1 PCT/ES2014/070800 ES2014070800W WO2015059337A1 WO 2015059337 A1 WO2015059337 A1 WO 2015059337A1 ES 2014070800 W ES2014070800 W ES 2014070800W WO 2015059337 A1 WO2015059337 A1 WO 2015059337A1
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use according
hydrogen
compound
same
represent
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PCT/ES2014/070800
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Spanish (es)
French (fr)
Inventor
Xavi RIBAS SALAMAÑA
Miquel Costas Salgueiro
Olaf CUSSÓ FOREST
Anna Company Casadevall
Julio LLORET FILLOL
Manuel SÁNCHEZ MORENO
Clotilde MARÍN SÁNCHEZ
María José Rosales Lombardo
Francisco OLMO ARÉVALO
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Universidad De Granada
Universitat De Girona
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Priority claimed from ES201331558A external-priority patent/ES2439397B1/en
Priority claimed from ES201331559A external-priority patent/ES2440896B1/en
Application filed by Universidad De Granada, Universitat De Girona filed Critical Universidad De Granada
Publication of WO2015059337A1 publication Critical patent/WO2015059337A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/68—One oxygen atom attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of polyamine compounds and metal complexes comprising said polyamine compounds for the treatment of diseases of parasitic origin such as American trypanosomiasis, also known as Chagas disease, or Leishmaniasis.
  • the present invention is framed in the field pharmaceutical and veterinary, as pharmaceutical composition for the treatment and / or prevention of human and animal parasitic diseases
  • Neglected diseases are diseases that affect thousands of people around the world, but do not have effective or adequate treatments. They are mostly infectious tropical diseases that fundamentally affect the poorest population, such as Leishmaniasis and Chagas disease, which have a devastating impact on civilization. These infectious diseases are caused by protozoan parasites that especially affect less developed countries, representing a serious public health problem in the world. It is estimated that almost 50% of the world's population is exposed to this risk of contracting these infections, and that approximately 500 million people suffer from pathologies related to this type of disease every year.
  • Chagas disease is caused by the hemo-flagellated protozoan Trypanosoma cruzi that is transmitted naturally through the bugs of a bed bug hematophagous triatomine (Order Hemiptera). Other routes of transmission are by transfusion of infected blood, organ transplantation and even from mothers to children through pregnancy and lactation, or orally through contaminated food.
  • the acute phase in which the parasite actively multiplies within the host cells and high levels of blood parasitemia are detected; and the chronic phase, which develops 30% of those infected, in which the level of parasitemia is reduced to subpatent blood levels but the parasites form pseudocysts in different organs such as heart, esophagus and colon, causing digestive heart disease or megasyndromes.
  • nifurtimox nitrofuran
  • benznidazole nitroimidazole derivative
  • Leishmaniasis caused by the infection of different species of protozoa of the genus Leishmania is another common disease in tropical and subtropical regions, as well as in temperate areas so it is a cosmopolitan disease. Around 20 million people suffer from this disease, which is transmitted through the bite of Diptera of the genus Phlebotomus or Lutzomyia. Leishmaniasis can occur in different clinical manifestations: visceral, cutaneous or mucocutaneous, with visceral being the most severe form of the disease.
  • leishmaniasis The treatment of leishmaniasis is complicated and in addition, this disease has a substantial morbidity, so expedited therapies are often required.
  • pentavalent derivatives of antimony such as: Sodium stibogluconate (Pentostan) or Meglumina antimoniato (Glucantime).
  • Other medications used are: Amphotericin B (AmBiosome®) that is administered for up to 10 days and does not present 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 of use for pregnant women and children; Pentamidine and Ketoconazole. This problem is exacerbated since repeated population infections or ineffective treatments have caused the resistance of parasites to these therapies.
  • the present invention provides compounds and their metal complexes useful as antiparasitic agents, as well as significantly less toxic than the active principles currently used for diseases of this type and are also active both against the acute phases and against the chronic phases of infections caused by Trypanosoma cruzi or Leishmania spp.
  • the present invention relates to the use of polyamine compounds and metal complexes of said polyamine compounds as antiparasitic agents, in particular for the treatment of leishmaniasis and Chagas disease.
  • alkyl refers in the present invention to aliphatic, linear or branched chains, having 1 to 5 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ferc- butyl, sec-butyl, n-pentyl.
  • the alkyl group has between 1 and 3 carbon atoms. More preferably they are methyl.
  • the alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, aryl carboxylic acid, hydroxyl, amino, amido, ester, carboxylic, ether, thiol, acylamino or carboxamide, which in turn may optionally be substituted.
  • substituents such as halogen, hydroxyl, azide, aryl carboxylic acid, hydroxyl, amino, amido, ester, carboxylic, ether, thiol, acylamino or carboxamide, which in turn may optionally be substituted.
  • the alkyl group is not substituted.
  • alkoxy refers in the present invention to a group of the formula -OR a in which R a is a (C1-C5) alkyl as described above.
  • R a is a (C1-C5) alkyl as described above.
  • R a is a (C1-C5) alkyl as described above.
  • methoxy ethoxy or propoxy.
  • it is a methoxy.
  • halogen is meant a molecule of Cl, Br, I or F. Preferably it is Cl or F.
  • heteroaryl refers, in the present invention, to aromatic heterocyclic rings (mono- or bicyclic) having between 5 and 10 members, including carbon atoms and at least 1 to 3 heteroatoms selected from N, O and S.
  • the heteroaryl group has between 5 and 6 members.
  • Heteroaryl radicals may be optionally substituted by one or more substituents such as alkyl, halogen, hydroxy, alkoxide, thiol, nitro, amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester or combinations of these groups.
  • substituents such as alkyl, halogen, hydroxy, alkoxide, thiol, nitro, amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester or combinations of these groups.
  • they are pyridine rings, which in the complexes are attached to the metals by the N atom.
  • any metal cation is understood, preferably of transition, and more preferably of the first transition series, although metals of the second and third transition series of groups 7-12 of the periodic table may be used, and even more preferably selects between Mn, Fe, Co, Ni, Cu and Zn.
  • the oxidation state of the metal is preferably +2, that is, it is a divalent metal, therefore, when the counter-anion is monovalent, two counter-anions are required to compensate for the load positive.
  • “Parasitic disease” means an infectious disease caused by protozoa, vermes (cestodes, trematodes, nematodes) or arthropods.
  • the parasites are protozoa belonging to the genera Trypanosoma and Leishmania, therefore, it can be said that the compounds are antiprotozoal.
  • Species thereof may be, but not limited to, Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis and Leishmania donovani, among others known to a person skilled in the art.
  • leishmaniasis is a disease caused by a protozoan of the genus 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.
  • Trypanosomiasis are diseases caused in humans or vertebrate animals that are caused by protozoan parasites of the genus Trypanosoma, among them you can find African human trypanosomiasis, also known as sleeping sickness, American trypanosomiasis or Chagas disease or trypanosomiasis in Animals or Nagana
  • the present invention relates to the use of a polyamine compound of general formula (I):
  • Ri, R 2 and R 3 are the same or different and represent a group selected from the list comprising hydrogen, (C 1 -C 5) alkyl, -NR'R ", halogen, (C 1 -C 5) alkoxy and - COOR '"; or
  • Ri and R 3 represent a pinene or quinoline group fused to the pyridine ring;
  • R ', R "and R'" are the same or different and represent a hydrogen or an alkyl group and N-R-N represents the groups:
  • Compounds of formula (I) include both the S, S 'and R, R' conformations, and this is represented by a serpentine bond.
  • the compound of formula (I) is the compound of general formula (I I):
  • R 1 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 .
  • R 2 and R 3 are the same or different and they represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same.
  • Ri is -N (CH 3 ) 2 and R 2 and R 3 are hydrogen; or R1 is methoxy and R 2 and R 3 methyl.
  • the compound of formula (II) is 2,2'- (2S, 2'S) -2,2'-bipyrrolidine-1,1'-diylbis (methylene) bis (N, N-dimethylpyridine-4-amine ): also called (S, S ') - (Me 2 N-Pyr) 2 -bpbp or X15:
  • the compound of formula (I) is the compound of (III):
  • Ri represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 .
  • R 2 and R 3 are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same.
  • RR 2 and R 3 are hydrogen; or R 1 is methoxy and R 2 and R 3 methyl.
  • the compound of formula (I II) is the racemic compound DL-N1, N2-dimethyl-N1, N2-bis (pyridin-2-ylmethyl) cyclohexane-1,2-diamine, also called bpmcn-racemic
  • Another aspect of the present invention relates to the use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I), preferably of formula (II) or (III), as described above, for the Preparation of a medicine for the treatment and / or prevention of parasitic diseases.
  • a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I), preferably of formula (II) or (III), as described above, for the Preparation of a medicine for the treatment and / or prevention of parasitic diseases.
  • the metal complex is of general formula (IV), preferably (V), (VI):
  • R, R 1 5 R 2 and R 3 have been defined above; M is a divalent metal; and X is a monovalent counter-anion.
  • the counter-anion (X) is any pharmaceutically acceptable anion known to a person skilled in the art and can preferably be selected from the list comprising CF 3 S0 3 ⁇ , Cl ⁇ , Br ⁇ , CI0 4 ⁇ , PF 6 " and BF 4 ⁇ , more preferably the counter-anion is
  • R 1 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , CI, -OCH 3 and -COOCH 2 CH 3 , more preferably, R 2 and R 3 , are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same. Even more preferably the compound of formula (II), R is -N (CH 3 ) 2 and R 2 and R 3 are hydrogen; or R is methoxy and R 2 and R 3 methyl.
  • the complexes are of formula (V) and both S, S 'and R, R' conformations are included.
  • the complex comprises as ligand the compounds 2,2'- (2R, 2'R) -2,2 , -bipyrrolidine-1,1 , -diylbis (methylene) bis (N, N-dimethylpyridine -4- amine): also called (R, R ') - (Me 2 N-Pyr) 2 -bpbp, or (2S, 2'S) -1, 1' -bis ((4-methoxy-3,5-dimethylpyridine -2-yl) methyl) -2,2'-bipyrrolidine, also called S, S '- (DMM-Py) 2 (bpbp):
  • the metal is divalent and the complex is of formula (V), more preferably the metal is Mn and even more preferably the complex is X8 'or X12:
  • ( ⁇ 8 ') is of the formula [ ⁇ 2,2' - (2R, 2'R) -2,2'-bipyrrolidine-1, 1'-diylbis (methylene) bis (N, N-dimethylpyridine-4-amine) ⁇ Mn "] (CF 3 S0 3 ) 2 or also called [ ⁇ R, R '- (Me 2 N-Py) 2 (bpbp) ⁇ Mn"] (CF 3 S0 3 ) 2; Y
  • (X12) is of the formula [ ⁇ 2S, 2'S) -1, 1'-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -2,2'-bipyrrolidine ⁇ Mn "] ( CF 3 S0 3 ) 2 or also called [ ⁇ S, S '- (DMM-Py) 2 (bpbp) ⁇ Mn "] (CF 3 S0 3 ) 2 .
  • R represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 , more preferably, R 2 and R 3 , are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same. Yet more preferably the compound of formula (III), Ri, R 2 and R 3 are hydrogen; or Ri is methoxy and R 2 and R 3 methyl. In these compounds of formula (III) both S, S 'and R, R' conformations are included.
  • the complex comprises as ligand the compound (1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2-dimethylcyclohexane -1, 2-diamine, or also called R, R '- (DMM-Py) 2 (bpmcn).
  • the metal is divalent and the complex is of formula (VI), more preferably the metal is Mn and even more preferably the complex is X13:
  • (X13) is of the formula [ ⁇ (1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2-dimethylcyclohexane-1,2-diamine ⁇ Mn "] (CF 3 S0 3 ) 2 or also called [ ⁇ R, R '- (DMM-Py) 2 (bpmcn) ⁇ Mn"] (CF 3 S0 3 ) 2 .
  • the present invention relates to the use of a polyamine compound of the general formula (1-2):
  • R 4 and R 5 are the same or different, and represent a hydrogen, an alkyl group (CrC 5 ) or a -CH 2 -Cy group;
  • R 6 represents a hydrogen, a halogen or an alkyl group (CrC 5 );
  • R 7 and R 9 are the same or different, and represent a hydrogen or an alkyl group (C
  • R 8 represents a group selected from the list comprising hydrogen, -NR'R ", halogen, alkoxy (CC 5 ), -N0 2 and -COOR '";
  • R ', R "and R'" are the same or different and represent a hydrogen or an alkyl group
  • Cy represents a heteroaryl
  • R 4 and R 5 are the same or different, and represent a hydrogen, a methyl or a -CH 2 -Cy group; where Cy is pyridine, more preferably R 4 and R 5 are the same.
  • R 6 represents hydrogen, Cl, F or methyl.
  • R 7 and R 9 are the same or different, and represent hydrogen or methyl; more preferably R 7 and R 9 are the same.
  • R 8 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 , N0 2 and -COOCH 2 CH 3 .
  • R 4 and R 5 are the same and represent a methyl or a -CH 2 -pyridine group; and R 6 , R 7 , R 8 and R 9 are hydrogen.
  • the compound of formula (I-2) is selected from 1,4-dimethyl-7- ⁇ pyridin-2-ylmethyl) -1, 4,7-triazonan, also called PyTACN (X26); and 1, 4,7-tris (pyridin-2-ylmethyl) -1, 4,7-triazonan, also called Py3TACN (X27):
  • Another aspect of the present invention relates to the use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I-2), as described above, for the preparation of a medicament for treatment and / or the prevention of parasitic diseases.
  • the metal complex is of general formula (II-2):
  • R 4 , R 5 , R 6 , R 7 , R 8 and R 9 have been defined above; M is a divalent metal; and X is a monovalent counter-anion.
  • the counter-anion (X) is any pharmaceutically acceptable anion known to a person skilled in the art and can preferably be selected from the list comprising CF 3 S0 3 " , CI “ , Br “ , CI0 4 " , PF 6 “ and BF 4 " , more preferably the counter-anion is CF3SO3-.
  • R 4 and R 5 are the same or different, and represent a hydrogen, a methyl or a group -CH 2 - Cy; where Cy is pyridine, more preferably R 4 and R 5 are the same.
  • R 6 represents hydrogen, Cl, F or methyl.
  • R 7 and Rg are the same or different, and represent hydrogen or methyl; more preferably R 7 and R 9 are the same.
  • R 8 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 , N0 2 and -COOCH 2 CH 3 .
  • R 4 and R 5 are the same and represent a methyl or a -CH 2 -pyridine group; and R 6 , R 7 , R 8 and R 9 are hydrogen.
  • the compound of formula (I-2) is selected from 1,4-dimethyl-7- ⁇ pyridin-2-ylmethyl) -1, 4,7-triazonan, also called PyTACN and 1,4, 7-tris (pyridin-2-ylmethyl) -1, 4,7-triazonan, also called Py3TACN (X27), more preferably is the PyTACN compound.
  • the metal is divalent and the complex is of formula (II-2), more preferably the metal is Mn, Fe or Cu and even more preferably the complex is X1, X6 or X9:
  • X1 is of the formula [ ⁇ 1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano ⁇ Fe] (CF 3 S0 3 ) 2 or also called [ ⁇ PyTACN ⁇ Fe "] ( CF 3 S0 3 ) 2;
  • X6 is of the formula [ ⁇ 1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano ⁇ Mn "] (CF 3 S0 3 ) 2 or also called [ ⁇ PyTACN ⁇ Mn"] (CF 3 S0 3 ) 2 ; Y
  • X9 is of the formula [ ⁇ 1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano ⁇ Cu "] (CF 3 S0 3 ) 2 or also called [ ⁇ PyTACN ⁇ Cu”] (CF 3 S0 3 ) 2 .
  • compositions comprising at least one compound of general formula (I), preferably (II) or (III), or its complexes of general formula (IV), preferably (V) or ( VI), described above, together with at least one pharmaceutically acceptable carrier, optionally this composition may include another active ingredient, for example another antiparasitic compound.
  • a further aspect of the present invention relates to the use of at least one compound of general formula (I), preferably (II) or (III), and its complexes of general formula (IV), preferably (V) or (VI) , described above, for the preparation of a medicament.
  • compositions comprising at least one compound of general formula (I-2) or its complexes of general formula (II-2), described above, together with at least one pharmaceutically acceptable carrier, optionally this composition may include another active ingredient, for example another antiparasitic compound.
  • a further aspect of the present invention relates to the use of at least one compound of general formula (I-2) or its complexes of general formula (II-2), described above, for the preparation of a medicament.
  • Another aspect of the present invention relates to the complex (X13) [ ⁇ (1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2- dimethylcyclohexane-1, 2- diamine ⁇ Mn "] (CF 3 S0 3 ) 2.
  • a further aspect of the present invention relates to the complex (X9) [ ⁇ 1,4-dimethyl-7- (pyridin-2-ylmethyl) -1,4,7-triazonano ⁇ Cu "] (CF 3 S0 3 ) 2 .
  • the compounds and / or complexes described in the present invention are used for the treatment and / or prevention or for the preparation of a medicament for the treatment and / or prevention of protozoan parasitic diseases, more preferably parasites. They belong to the Trypanosomatidae family.
  • the inoculum of parasites to start the culture was 5 x 10 4 cells / ml in 5 ml of medium in 25 cm 2 Falcon® plastic bottles, and kept in an oven at 28 e C.
  • J774.2 macrophages were recloned from J774.2 (European collection o ⁇ cell cultures (ECACC) number 9105151 1) originals of a mouse female tumor of the Balb / c strain, keeping the cultures between 3-9 x 10 5 cells / ml at 37 e C and 5% C0 2 in MEM + culture medium supplemented with 20% SBF-I.
  • the protocol to work with the cells in culture was taking them off the culture bottle where they were attached by cold support and dry blows. The cells were decanted and centrifuged for 5 minutes at 800 rpm.
  • the cells were resuspended in fresh culture medium for counting, by staining with trypan blue (1: 1 dilution) and in a hemocytometric chamber of Neubauer, for subsequent studies (Sánchez-Moreno M, et al., J Antimicrob Chemother 2012; 67: 387-397). These tests were performed by flow cytometry.
  • the macrophages of the J774.2 line were deposited in a sterling (Universal Tube®) and centrifuged at 800 rpm for 5 min, the supernatant was discarded and the cells were resuspended in MEM + Glutamine + 20% SBF-I medium.
  • results were analyzed in the cytometer taking into account that the cells with the intact plasma membrane have a green fluorescence, while the 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.
  • Promastigote forms of L. infantum grown in the manner described above were collected in their exponential phase of growth by centrifugation at 1500 rpm for 10 min. They were resuspended in fresh medium. The concentration of parasites was counted in a hemocytometric chamber of Neubauer and seeded in a 24-well plate at a concentration of 5 x 10 4 parasites / well. The compounds to be tested were dissolved with pure DMSO at a DMSO concentration of 0.01% (v / v), at which this solvent is not toxic nor has any effect on the growth of parasites.
  • the compounds were added to the culture medium in the 24-well plate at a final concentration of: 100, 50, 25, 10 and 1 ⁇ , and at a final volume per well of 500 ⁇ .
  • IN VITRO ACTIVITY TESTS INTRACELLULAR FORMS. Test on amastigote forms
  • the macrophages took off from the culture bottle where they were attached by cold support and dry blows. For this, the culture medium was removed, then the cell surface was covered with EDTA-trypsin and incubated cold for 5 minutes according to the methodology set forth above. 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 resuspending them in MEM + Glutamine with 20% SBF-I. They are counted in a Neubauer chamber and grown in 24-well plates at a rate of 1 x 10 4 cells, in which we had previously introduced a 12mm round coverslip glass into each well. For adhesion, we let the cells grow 24 h at 37 e C in 5% C0 2 .
  • the values obtained are compared with those corresponding to the reference drug, Glucatim®, commonly used in the treatment of leishmaniasis.
  • the analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite used, with IC 50 values up to four higher than the values obtained for the reference compound.
  • IC 50 values against macrophages between 10 and 25 times higher than the reference compound have been obtained, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage at the time of its use as therapeutic agents. Therefore, and from these values, selectivity indices with values between 15 and 150 have been obtained for a group of the compounds studied.
  • c Selectivity index IC 50 of macrophages / IC 50 of extracellular and intracellular forms of parasites. In parentheses: Number of times the SI of the compounds is greater than the SI of the reference drug.
  • the values obtained are compared with those corresponding to the reference drug, Glucatim®, commonly used in the treatment of leishmaniasis.
  • the analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite, with IC 50 values lower than the values obtained for the reference compound, mainly compounds X6 and X27.
  • the values of IC 50 against macrophages exceed 7 to 10 times that of the reference compound, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage to time of its use as therapeutic agents. Therefore, and from these values, the selectivity indices of compounds X6 and X27 exceed that of glucantime by 20 and 164 times for the amastigote forms of both Leishmania species.
  • the analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite used, with IC 50 values up to four higher than the values obtained for the reference compound.
  • IC 50 values against macrophages between 10 and 25 times higher than the reference compound have been obtained, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage at the time of its use as therapeutic agents. Therefore, and from these values, selectivity indices with values between 15 and 150 have been obtained for a group of the compounds studied.
  • Trypanosoma cruzi strain type I was used (IRHOD / CO / 2008 / SN3) (Tellez-Meneses et al., Acta Trop 2008; 108: 26-34) whose geographical origin is Guajira (Colombia) and its biological origin f ⁇ hodnius prolixus in the forms of development: epimastigotes, trypomastigotes and amastigotes.
  • the culture of the epimastigote forms of T was used (IRHOD / CO / 2008 / SN3) (Tellez-Meneses et al., Acta Trop 2008; 108: 26-34) whose geographical origin is Guajira (Colombia) and its biological origin f ⁇ hodnius prolixus in the forms of development: epimastigotes, trypomastigotes and amastigotes.
  • the culture of the epimastigote forms of T was used (IRHOD / CO / 2008 / SN3) (Telle
  • cruzi was performed in vitro in sterility in a single-phase MTL culture medium (Medium Trypanosomes Liquid, Ruiz-Pérez et al., 1986) enriched with 10% (v / v) Fetal Bovine Serum (SBF-I) inactivated at 56 e C / 30 minutes.
  • the inoculum of parasites to start the culture was 5 x 10 4 cells / ml in 5 ml of medium in 25 cm 2 Falcon® plastic bottles and kept in an oven at 28 e C.
  • the cultures were carried out in a manner routine achieving the exponential growth of flagellates until the necessary cell mass is obtained for subsequent studies. (Téllez-Meneses J, et al. Acta Trop 2008; 108: 26-34).
  • Vero cells were established from the kidney of an adult African Green monkey, keeping the cultures at a density of 1 x 10 4 cells / ml at 37 e C and 5% C0 2 .
  • RPMI supplemented with 10% of inactivated fetal bovine serum (SBF-I) was used as culture medium (Sánchez-Moreno M, et al. J Antimicrob Chemother 2012; 67: 387-397). Vero cells were deposited in a sterlin and centrifuged at 800 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI medium. 1 x 10 4 cells / ml were deposited in each well of a 24-well titration plate, incubated for 24 h at 37 e C in a humid atmosphere enriched with 5% C0 2 . This was done to fix the cells.
  • SBF-I inactivated fetal bovine serum
  • 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 ⁇ .
  • the samples were prepared for reading on the flow cytometer. The method followed is that described by (Ormerod, 1994), starting from the cells and the medium present in the wells, to which 100 ⁇ of propidium iodide solution (Pl, 100 ⁇ g / ml) was added (Sigma Chemical Co), incubating at 28 e C in the dark for about 10 minutes, subsequently, 100 ⁇ of fluorescein diacetate (FDA) (Sigma Chemical Co) in solution (100ng / ml) was added and re-incubated at 28 e C in the dark about 10 minutes, and after centrifugation at 1500 rpm for 10 minutes, the precipitate was washed with PBS.
  • Pl propidium iodide solution
  • FDA fluorescein diacetate
  • Metacyclogenesis was induced at a 5-day culture (stationary phase), after being centrifuged at 7000g for 10 min at 10 e C.
  • the parasites were incubated 2 h at 28 e C at a density of 5 x 10 8 parasites / ml in medium TAU (190 mM NaCI, 17 mM KCI, 2 mM MgCI 2 , 2 mM CaCI 2 , 8 mM phosphate buffer, pH 6.0).
  • TAU3AAG medium TAU supplemented with 10 mM L-proline, 50 mM sodium L-glutamate, 2 mM L-sodium aspartate and 10 mM D-glucose ) in 25 ml culture jars in a horizontal position preventing the medium from exceeding 1 cm deep.
  • Epimastigote forms of T. cruzi cultured 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 5 x 4 4 parasites / ml in each well.
  • the compounds to be tested were dissolved in DMSO at a concentration of 0.01% (v / v) 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 ⁇ .
  • the effect of each compound on the growth of epimastigote forms, at the different concentrations tested, was evaluated at 72 h, using a Neubauer hemocytometric chamber and the trypanocidal effect was expressed as IC 50 (concentration required to give an inhibition of 50%, calculated by the analysis of the linear regression of the K c at the concentrations tested) (Sánchez-Moreno M, et al. J Med Chem. 201 1; 54: 970-9).
  • the activity is compared with the control after performing the methodology described in (Junior CO, et al. Biomed Pharmacother 2010; 64: 624-6) with some modifications made in the laboratory.
  • the test is carried out using infected Balb / c mouse blood that has been obtained during the days of maximum parasitemia (day 7 approx. Post infection).
  • the infected blood was diluted with uninfected blood to obtain a concentration of 1 -4 x 10 6 trypomastigotes / ml, then diluted 1: 2 with RPMI 1640 medium-GIBCO, invitrogen®.
  • Stock solutions of the compounds to be tested are prepared at the same time in DMSO.
  • a sample of infected blood and the drug are added to a 96-well plate to a final volume of 200 ⁇ and compound concentrations of 1 to 100 ⁇ , in order to calculate the IC 50 for each.
  • the plates were incubated at 4 e C for 24 h. The experiments were repeated three times. Each sample was examined microscopically (OLYMPUS CX41) for the parasite count using the Neubauer chamber.
  • 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 according to the methodology set forth above. After that, it was transferred to a conical bottom flask of 25 ml capacity (Steriling) to centrifuge them at 800 rpm for 5 minutes, removing the supernatant and counted in Neubauer chamber. Resuspend the cells at a concentration of 1 x 10 4 cells / well in RPMI medium for Vero cells, growing in 24-well plates, in which we had previously introduced a 12 mm round glass cover glass in each well. For adhesion, we let the cells grow 24 h at 37 e C in 5% C0 2 .
  • Steriling 25 ml capacity
  • the Vero cells were infected "in vitro" with 1 x 10 5 cells / well of trypomastigote forms of T. cruzi, obtained according to the methodology cited above.
  • the infection was maintained for 24 hours for the parasite to enter the cell.
  • the culture medium was removed and fresh medium was added with the products to be tested, at the concentrations necessary to remove the IC 50 (100 to 1 ⁇ ).
  • 72 hours after incubation the crystals were removed. Once the crystals were removed, they were 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.
  • Table 5 In vitro activity, toxicity and selectivity index of polyamine compounds and complexes with transition metals evaluated in extracellular and intracellular forms of Trypanosoma cruzi.
  • IC 50 concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at the concentrations used (1, 10, 25, 50 and 100 ⁇ ). 13 Against Vero cells after 72 h of incubation.
  • Table 6 Selectivity index of polyamine compounds and transition metal complexes evaluated in extracellular and intracellular forms of Trypanosoma cruzi.
  • IC 50 concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at the concentrations used (1, 10, 25, 50 and 100 ⁇ ). 13 Against Vero cells after 72 h of incubation.
  • Table 8. Selectivity index of polyamine compounds and transition metal complexes evaluated in extracellular and intracellular forms of Trypanosoma cruzi.

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Abstract

The invention relates to the use of polyamine-based compounds or the metal complexes thereof for the prevention and/or treatment of diseases of parasitic origin such as American trypanosomiasis also known as Chagas disease, or Leishmaniasis.

Description

COMPUESTOS POLIAMÍNICOS Y COMPLEJOS METÁLICOS QUE LOS COMPRENDEN PARA SU USO COMO AGENTES ANTIPARASITARIOS  POLYAMINE COMPOUNDS AND METAL COMPLEXES THAT INCLUDE THEM FOR USE AS ANTIPARASITARY AGENTS
DESCRIPCIÓN DESCRIPTION
La presente invención se refiere al uso de compuestos poliamínicos y complejos metálicos que comprenden dichos compuestos poliamínicos para el tratamiento de enfermedades de origen parasitario como son la Tripanosomiasis americana, también conocida como enfermedad de Chagas, o la Leishmaniasis La presente invención se enmarca en el campo farmacéutico y veterinario, como composición farmacéutica para el tratamiento y/o la prevención de las enfermedades parasitarias humanas y animales The present invention relates to the use of polyamine compounds and metal complexes comprising said polyamine compounds for the treatment of diseases of parasitic origin such as American trypanosomiasis, also known as Chagas disease, or Leishmaniasis. The present invention is framed in the field pharmaceutical and veterinary, as pharmaceutical composition for the treatment and / or prevention of human and animal parasitic diseases
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
Las enfermedades olvidadas (Neglected diseases) son enfermedades que afectan a millares de personas en todo el mundo, pero que no disponen de tratamientos eficaces o adecuados. En su mayoría se trata de enfermedades tropicales infecciosas que afectan fundamentalmente a la población más pobre, como por ejemplo la Leishmaniasis y la enfermedad de Chagas, que ocasionan un impacto devastador en la humanidad. Estas enfermedades infecciosas están causadas por parásitos protozoarios que afectan especialmente a los países menos desarrollados, representando un problema grave de salud pública en el mundo. Se estima que casi el 50% de la población mundial está expuesta a este riesgo de contraer estas infecciones, y que aproximadamente 500 millones de personas sufren cada año patologías relacionadas con este tipo de enfermedades. Neglected diseases are diseases that affect thousands of people around the world, but do not have effective or adequate treatments. They are mostly infectious tropical diseases that fundamentally affect the poorest population, such as Leishmaniasis and Chagas disease, which have a devastating impact on humanity. These infectious diseases are caused by protozoan parasites that especially affect less developed countries, representing a serious public health problem in the world. It is estimated that almost 50% of the world's population is exposed to this risk of contracting these infections, and that approximately 500 million people suffer from pathologies related to this type of disease every year.
En la actualidad no existe tratamiento eficaz frente a estas enfermedades olvidadas, ya que, son de larga duración y algunos son de obligada administración parenteral o intravenosa, además de tener una elevada toxicidad (pancreatitis y toxicidad cardiovascular) con lo que pueden conllevar un efecto letal. Todo esto acentúa el problema sanitario al no haber terapias efectivas además de la aparición de resistencias a los pocos fármacos accesibles existentes. At present there is no effective treatment against these neglected diseases, since they are long-lasting and some are mandatory parenteral or intravenous administration, in addition to having a high toxicity (pancreatitis and cardiovascular toxicity) which can lead to a lethal effect . All this accentuates the health problem as there are no effective therapies in addition to the emergence of resistance to the few existing accessible drugs.
La enfermedad de Chagas está causada por el protozoo hemoflagelado Trypanosoma cruzi que se transmite de forma natural a través de las deyecciones de una chinche triatomina hematófaga (Orden Hemiptera). Otras vías de transmisión son por transfusión de sangre infectada, el trasplante de órganos e incluso de madres a hijos mediante el embarazo y la lactancia, o por vía oral a través de alimentos contaminados. En esta enfermedad se distinguen dos fases, la fase aguda en la que el parásito se multiplica activamente dentro de las células del hospedador y se detectan altos niveles de parasitemia en sangre; y la fase crónica, que desarrollan el 30% de los infectados, en la que el nivel de parasitemia se reduce a niveles subpatentes en sangre pero los parásitos forman pseudoquistes en diferentes órganos como corazón, esófago y colón, ocasionando cardiopatías o megasíndromes digestivos. Se trata de una enfermedad endémica en la mayor parte de América Latina y se ha clasificado por la WHO como la tercera enfermedad tropical más extendida después de la malaria y la esquistosomiasis. Se ha estimado que alrededor de 100 millones de personas están en peligro de ser infectadas y que hay entre 15 y 20 millones de personas infectadas. 50,000 personas mueren cada año como resultado de la infección de este parásito. Chagas disease is caused by the hemo-flagellated protozoan Trypanosoma cruzi that is transmitted naturally through the bugs of a bed bug hematophagous triatomine (Order Hemiptera). Other routes of transmission are by transfusion of infected blood, organ transplantation and even from mothers to children through pregnancy and lactation, or orally through contaminated food. In this disease two phases are distinguished, the acute phase in which the parasite actively multiplies within the host cells and high levels of blood parasitemia are detected; and the chronic phase, which develops 30% of those infected, in which the level of parasitemia is reduced to subpatent blood levels but the parasites form pseudocysts in different organs such as heart, esophagus and colon, causing digestive heart disease or megasyndromes. It is an endemic disease in most of Latin America and has been classified by the WHO as the third most widespread tropical disease after malaria and schistosomiasis. It has been estimated that around 100 million people are in danger of being infected and that there are between 15 and 20 million people infected. 50,000 people die each year as a result of the infection of this parasite.
Desafortunadamente, el tratamiento para la enfermedad de Chagas utilizado en la actualidad es muy limitado y no se ha desarrollado una vacuna efectiva. Los agentes terapéuticos desarrollados hasta la actualidad se basan en estructuras nitro- heterocíclicas como el nitrofurano (nifurtimox) o el derivado del nitroimidazol (benznidazol). Tanto el nifurtimox como el benznidazol sólo son efectivos frente a la fase aguda de la enfermedad, demostrando tener una eficacia muy limitada en la fase crónica. Además son muy tóxicos y causan efectos secundarios severos como pancreatitis y toxicidad cardiaca. Unfortunately, the treatment for Chagas disease used today is very limited and an effective vaccine has not been developed. The therapeutic agents developed to date are based on nitro-heterocyclic structures such as nitrofuran (nifurtimox) or the nitroimidazole derivative (benznidazole). Both nifurtimox and benznidazole are only effective against the acute phase of the disease, proving to have very limited efficacy in the chronic phase. They are also very toxic and cause severe side effects such as pancreatitis and cardiac toxicity.
La leishmaniasis, causada por la infección de diferentes especies de protozoos del género Leishmania es otra enfermedad común en las regiones tropicales y subtropicales, así como en zonas templadas por lo que se trata de una enfermedad cosmopolita. Alrededor de 20 millones de personas padecen esta enfermedad, la cual se transmite a través de la picadura de dípteros del género Phlebotomus o Lutzomyia. La leishmaniasis se puede presentar en diferentes manifestaciones clínicas: visceral, cutánea o mucocutánea, siendo la visceral la forma más severa de la enfermedad. Leishmaniasis, caused by the infection of different species of protozoa of the genus Leishmania is another common disease in tropical and subtropical regions, as well as in temperate areas so it is a cosmopolitan disease. Around 20 million people suffer from this disease, which is transmitted through the bite of Diptera of the genus Phlebotomus or Lutzomyia. Leishmaniasis can occur in different clinical manifestations: visceral, cutaneous or mucocutaneous, with visceral being the most severe form of the disease.
El tratamiento de la leishmaniasis es complicado y además, esta enfermedad presenta una morbilidad sustancial por lo que a menudo se requieren terapias expeditivas. Para el tratamiento de la leishmaniasis se utilizan desde hace más de 70 años derivados pentavalentes del antimonio como: Estibogluconato sódico (Pentostan) o la Meglumina antimoniato (Glucantime). Otros medicamentos utilizados son: Anfotericina B (AmBiosome®) que se administra durante un máximo de 10 días y no presentan toxicidad pero es sumamente caro (1 ,500 a 2,400$ por tratamiento); la miltefosina que es de administración oral pero el tratamiento dura 4 semanas y tiene restricciones de uso para gestantes y niños; la pentamidina y el Ketoconazol. Este problema se agrava dado que las infecciones repetidas de la población o los tratamientos inefectivos han ocasionado la resistencia de los parásitos a estas terapias. The treatment of leishmaniasis is complicated and in addition, this disease has a substantial morbidity, so expedited therapies are often required. For The treatment of leishmaniasis has been used for more than 70 years pentavalent derivatives of antimony such as: Sodium stibogluconate (Pentostan) or Meglumina antimoniato (Glucantime). Other medications used are: Amphotericin B (AmBiosome®) that is administered for up to 10 days and does not present 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 of use for pregnant women and children; Pentamidine and Ketoconazole. This problem is exacerbated since repeated population infections or ineffective treatments have caused the resistance of parasites to these therapies.
Por lo tanto, resulta necesario obtener nuevos agentes terapéuticos más efectivos tanto en las fases agudas como crónicas de las enfermedades mencionadas, y que además sean activos frente a cepas resistentes a los tratamientos habituales. Además es importante que presenten una menor toxicidad con el objetivo de disminuir los efectos secundarios adversos.  Therefore, it is necessary to obtain new more effective therapeutic agents in both the acute and chronic phases of the mentioned diseases, and which are also active against strains resistant to the usual treatments. It is also important that they have a lower toxicity in order to reduce adverse side effects.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención proporciona unos compuestos y sus complejos metálicos útiles como agentes antiparasitarios, así como significativamente menos tóxicos que los principios activos utilizados en la actualidad para enfermedades de este tipo y además son activos tanto frente a las fases agudas como frente a las fases crónicas de la infecciones causadas por Trypanosoma cruzi o por Leishmania spp. The present invention provides compounds and their metal complexes useful as antiparasitic agents, as well as significantly less toxic than the active principles currently used for diseases of this type and are also active both against the acute phases and against the chronic phases of infections caused by Trypanosoma cruzi or Leishmania spp.
Por tanto, la presente invención se refiere al uso de compuestos poliamínicos y complejos metálicos de dichos compuestos poliamínicos como agentes antiparasitarios, en particular para el tratamiento de la leishmaniasis y de la enfermedad de Chagas. Therefore, the present invention relates to the use of polyamine compounds and metal complexes of said polyamine compounds as antiparasitic agents, in particular for the treatment of leishmaniasis and Chagas disease.
Definiciones Definitions
El término "alquilo" se refiere en la presente invención a cadenas alifáticas, lineales o ramificadas, que tienen de 1 a 5 átomos de carbono, por ejemplo, metilo, etilo, n- propilo, /-propilo, n-butilo, ferc-butilo, sec-butilo, n-pentilo. Preferiblemente el grupo alquilo tiene entre 1 y 3 átomos de carbono. Más preferiblemente son metilo. Los grupos alquilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como halógeno, hidroxilo, azida, ácido carboxílico arilo, hidroxilo, amino, amido, éster, carboxílico, éter, tiol, acilamino o carboxamido, que a su vez pueden opcionalmente estar sustituidos. Preferiblemente el grupo alquilo no está sustituido. The term "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 5 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ferc- butyl, sec-butyl, n-pentyl. Preferably the alkyl group has between 1 and 3 carbon atoms. More preferably they are methyl. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, aryl carboxylic acid, hydroxyl, amino, amido, ester, carboxylic, ether, thiol, acylamino or carboxamide, which in turn may optionally be substituted. Preferably the alkyl group is not substituted.
El término "alcoxilo" se refiere en la presente invención a un grupo de fórmula -ORa en la que Ra es un alquilo (C1 -C5) como el descrito anteriormente. Por ejemplo, pero sin limitarse a metoxilo, etoxilo o propoxilo. Preferiblemente es un metoxilo. The term "alkoxy" refers in the present invention to a group of the formula -OR a in which R a is a (C1-C5) alkyl as described above. For example, but not limited to methoxy, ethoxy or propoxy. Preferably it is a methoxy.
Por "halógeno" se entiende por una molécula de Cl, Br, I o F. Preferiblemente es Cl o F. By "halogen" is meant a molecule of Cl, Br, I or F. Preferably it is Cl or F.
Por "heteroarilo" se refiere, en la presente invención, a anillos heterocíclicos aromáticos (mono- o bicíclicos) que tienen entre 5 y 10 miembros, entre ellos átomos de carbono y al menos entre 1 a 3 heteroátomos seleccionados de entre N, O y S. Preferiblemente el grupo heteroarilo tiene entre 5 y 6 miembros. Los radicales heteroarilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como grupos alquilo, halógeno, hidroxilo, alcóxido, tiol, nitro, amino, acilamino, ciano, carboxilato, carboxamida, carboxiéster o combinaciones de estos grupos. Preferiblemente son anillos piridina, que en los complejos están unidos a los metales por el átomo de N. By "heteroaryl" refers, in the present invention, to aromatic heterocyclic rings (mono- or bicyclic) having between 5 and 10 members, including carbon atoms and at least 1 to 3 heteroatoms selected from N, O and S. Preferably the heteroaryl group has between 5 and 6 members. Heteroaryl radicals may be optionally substituted by one or more substituents such as alkyl, halogen, hydroxy, alkoxide, thiol, nitro, amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester or combinations of these groups. Preferably they are pyridine rings, which in the complexes are attached to the metals by the N atom.
Por "meta!' se entiende a cualquier catión metálico, preferiblemente de transición, y más preferiblemente de la primera serie de transición, aunque se pueden usar metales de la segunda y tercera serie de transición de los grupos 7-12 de la tabla periódica, y aún más preferiblemente se selecciona entre Mn, Fe, Co, Ni, Cu y Zn. El estado de oxidación del metal es preferiblemente +2, es decir, es un metal divalente, por tanto, cuando el contraanión es monovalente se precisan de dos contraaniones para compensar la carga positiva. For 'goal!' any metal cation is understood, preferably of transition, and more preferably of the first transition series, although metals of the second and third transition series of groups 7-12 of the periodic table may be used, and even more preferably selects between Mn, Fe, Co, Ni, Cu and Zn. The oxidation state of the metal is preferably +2, that is, it is a divalent metal, therefore, when the counter-anion is monovalent, two counter-anions are required to compensate for the load positive.
Por "enfermedad parasitaria", se entiende una enfermedad infecciosa causada por protozoos, vermes (cestodos, tremátodos, nematodos) o artrópodos. En particular, en la presente invención, los parásitos son protozoos pertenecientes a los géneros Trypanosoma y Leishmania, por tanto, se puede decir que los compuestos son antiprotozoarios. Especies de las mismas, pueden ser, pero sin limitarse, Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis y Leishmania donovani, entre otras conocidas por un experto en la materia. "Parasitic disease" means an infectious disease caused by protozoa, vermes (cestodes, trematodes, nematodes) or arthropods. In particular, in the present invention, the parasites are protozoa belonging to the genera Trypanosoma and Leishmania, therefore, it can be said that the compounds are antiprotozoal. Species thereof, may be, but not limited to, Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis and Leishmania donovani, among others known to a person skilled in the art.
Las enfermedades parasitarias a tratar podrían ser leishmaniasis o tripanosomiasis. La "Leishmaniasis" es una enfermedad causada por un protozoo del género Leishmania y transmitido, principalmente por dípteros conocidos como "sand fly", moscas de la arena o jejenes. Esta enfermedad se produce en humanos y animales vertebrados, como pueden ser marsupiales, cánidos, roedores y primates. Las " Tripanosomiasis" son enfermedades producidas en humanos o animales vertebrados que son causadas por parásitos protozoarios del género Trypanosoma, entre ellas se puede encontrar la tripanosomiasis humana africana, también conocida como enfermedad del sueño, la tripanosomiasis americana o enfermedad de Chagas o la tripanosomiasis en animales o Nagana. The parasitic diseases to be treated could be leishmaniasis or trypanosomiasis. "Leishmaniasis" is a disease caused by a protozoan of the genus 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. "Trypanosomiasis" are diseases caused in humans or vertebrate animals that are caused by protozoan parasites of the genus Trypanosoma, among them you can find African human trypanosomiasis, also known as sleeping sickness, American trypanosomiasis or Chagas disease or trypanosomiasis in Animals or Nagana
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. 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.
Compuestos de fórmula general (I) Compounds of general formula (I)
En un primer aspecto, la presente invención se refiere al uso de un compuesto poliamínico de fórmula general (I): In a first aspect, the present invention relates to the use of a polyamine compound of general formula (I):
Figure imgf000006_0001
Figure imgf000006_0001
(I) donde: Ri, R2 y R3 son iguales o diferentes y representan un grupo seleccionado de la lista que comprende hidrógeno, alquilo (C1 -C5) , -NR'R", halógeno, alcoxilo (C1 -C5) y -COOR'"; o (I) where: Ri, R 2 and R 3 are the same or different and represent a group selected from the list comprising hydrogen, (C 1 -C 5) alkyl, -NR'R ", halogen, (C 1 -C 5) alkoxy and - COOR '"; or
Ri y R3 representan un grupo pineno o quinolina fusionado al anillo de piridina; Ri and R 3 represent a pinene or quinoline group fused to the pyridine ring;
R', R" y R'", son iguales o diferentes y representan un hidrógeno o un grupo alquilo y N-R-N representa los grupos:  R ', R "and R'", are the same or different and represent a hydrogen or an alkyl group and N-R-N represents the groups:
Figure imgf000007_0001
cualquiera de sus isómeros y/o sales farmacéuticamente aceptables, para el tratamiento y/o la prevención de enfermedades parasitarias o para la elaboración de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias.
Figure imgf000007_0001
any of its isomers and / or pharmaceutically acceptable salts, for the treatment and / or prevention of parasitic diseases or for the preparation of a medicament for the treatment and / or prevention of parasitic diseases.
En los compuestos de fórmula (I) se incluyen tanto las conformaciones S,S' como R,R', y ello se representa con un enlace serpenteado. Compounds of formula (I) include both the S, S 'and R, R' conformations, and this is represented by a serpentine bond.
En una realización preferida el compuesto de fórmula (I) es el compuesto de fórmula general (I I): In a preferred embodiment the compound of formula (I) is the compound of general formula (I I):
Figure imgf000007_0002
Figure imgf000007_0002
En una realización más preferida del compuesto de fórmula (I I) R1 representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3 y - COOCH2CH3. En una realización más preferida, R2 y R3, son iguales o diferentes y representan un hidrógeno o un grupo metilo, aún más preferiblemente R2 y R3 son iguales. In a more preferred embodiment of the compound of formula (II) R 1 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 . In a more preferred embodiment, R 2 and R 3 , are the same or different and they represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same.
En otra realización preferida del compuesto de fórmula (II), Ri es -N(CH3)2 y R2 y R3 son hidrógeno; o R1 es metoxilo y R2 y R3 metilo. In another preferred embodiment of the compound of formula (II), Ri is -N (CH 3 ) 2 and R 2 and R 3 are hydrogen; or R1 is methoxy and R 2 and R 3 methyl.
En estos compuestos de fórmula (II) se incluyen tanto las conformaciones S,S' como R,R'. In these compounds of formula (II) both S, S 'and R, R' conformations are included.
En una realización preferida el compuesto de fórmula (II) es 2,2'-(2S,2'S)-2,2'- bipirrolidina-1 ,1 '-diilbis(metileno)bis(N,N-dimetilpiridina-4-amina): también denominado (S,S')-(Me2N-Pyr)2-bpbp ó X15: In a preferred embodiment the compound of formula (II) is 2,2'- (2S, 2'S) -2,2'-bipyrrolidine-1,1'-diylbis (methylene) bis (N, N-dimethylpyridine-4-amine ): also called (S, S ') - (Me 2 N-Pyr) 2 -bpbp or X15:
Figure imgf000008_0001
Figure imgf000008_0001
(X15)  (X15)
En otra realización preferida, el compuesto de fórmula (I) es el compuesto de (III): In another preferred embodiment, the compound of formula (I) is the compound of (III):
Figure imgf000008_0002
Figure imgf000008_0002
(III) En una realización más preferida del compuesto de fórmula (II I) Ri representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3 y - COOCH2CH3. En una realización más preferida, R2 y R3, son iguales o diferentes y representan un hidrógeno o un grupo metilo, aún más preferiblemente R2 y R3 son iguales. (III) In a more preferred embodiment of the compound of formula (II I) Ri represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 . In a more preferred embodiment, R 2 and R 3 are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same.
En una realización más preferida del compuesto es de fórmula (II I), R R2 y R3 son hidrógeno; o R1 es metoxilo y R2 y R3 metilo. In a more preferred embodiment of the compound is of formula (II I), RR 2 and R 3 are hydrogen; or R 1 is methoxy and R 2 and R 3 methyl.
En estos compuestos de fórmula (II I) se incluyen tanto las conformaciones S,S' como R,R'. In these compounds of formula (II I) both S, S 'and R, R' conformations are included.
En otra realización preferida el compuesto de fórmula (I II) es el compuesto racémico DL-N1 ,N2-dimetil-N1 ,N2-bis(piridin-2-ilmetil)ciclohexano-1 ,2-diamina, también denominado bpmcn-racémic In another preferred embodiment the compound of formula (I II) is the racemic compound DL-N1, N2-dimethyl-N1, N2-bis (pyridin-2-ylmethyl) cyclohexane-1,2-diamine, also called bpmcn-racemic
Figure imgf000009_0001
Figure imgf000009_0001
Otro aspecto de la presente invención se refiere al uso de un complejo metálico que comprende un ligando tetradentado que consiste en un compuesto de fórmula general (I), preferiblemente de fórmula (I I) o (I I I), según se han descrito anteriormente, para la preparación de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias. Another aspect of the present invention relates to the use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I), preferably of formula (II) or (III), as described above, for the Preparation of a medicine for the treatment and / or prevention of parasitic diseases.
En una realización preferida el complejo metálico es de fórmula general (IV), preferiblemente (V), (VI): In a preferred embodiment the metal complex is of general formula (IV), preferably (V), (VI):
Figure imgf000010_0001
Figure imgf000010_0001
(IV) (V) (VI) (IV) (V) (VI)
Donde: R, R1 5 R2 y R3 se han definido anteriormente; M es un metal divalente; y X es un contraanión monovalente. Where: R, R 1 5 R 2 and R 3 have been defined above; M is a divalent metal; and X is a monovalent counter-anion.
El contraanión (X) es cualquier anión farmacéuticamente aceptable conocido por un experto en la materia y preferiblemente se puede seleccionar de la lista que comprende CF3S03 ~, Cl~, Br~, CI04 ~, PF6 " y BF4 ~, más preferiblemente el contraanión es The counter-anion (X) is any pharmaceutically acceptable anion known to a person skilled in the art and can preferably be selected from the list comprising CF 3 S0 3 ~ , Cl ~ , Br ~ , CI0 4 ~ , PF 6 " and BF 4 ~ , more preferably the counter-anion is
En una realización preferida de los complejos que comprenden el ligando tetradentado de fórmula (II) como se ha definido anteriormente, R1 representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, CI, -OCH3 y -COOCH2CH3, más preferiblemente, R2 y R3, son iguales o diferentes y representan un hidrógeno o un grupo metilo, aún más preferiblemente R2 y R3 son iguales. Aún más preferiblemente el compuesto de fórmula (II), R es -N(CH3)2 y R2 y R3 son hidrógeno; o R es metoxilo y R2 y R3 metilo. En una realización más preferida el complejos es de fórmula (V) y se incluyen tanto las conformaciones S,S' como R,R'. In a preferred embodiment of the complexes comprising the tetradentate ligand of formula (II) as defined above, R 1 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , CI, -OCH 3 and -COOCH 2 CH 3 , more preferably, R 2 and R 3 , are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same. Even more preferably the compound of formula (II), R is -N (CH 3 ) 2 and R 2 and R 3 are hydrogen; or R is methoxy and R 2 and R 3 methyl. In a more preferred embodiment the complexes are of formula (V) and both S, S 'and R, R' conformations are included.
En una realización preferida más preferida, el complejo comprende como ligando los compuesto 2,2'-(2R,2'R)-2,2,-bipirrolidina-1 ,1 ,-diilbis(metileno)bis(N,N-dimetilpiridina-4- amina): también denominado (R,R')-(Me2N-Pyr)2-bpbp, o (2S,2'S)-1 ,1 '-bis((4-metoxi- 3,5-dimetilpiridin-2-il)metil)-2,2'-bipirrolidina, también denominado S,S'-(DMM- Py)2(bpbp): In a more preferred preferred embodiment, the complex comprises as ligand the compounds 2,2'- (2R, 2'R) -2,2 , -bipyrrolidine-1,1 , -diylbis (methylene) bis (N, N-dimethylpyridine -4- amine): also called (R, R ') - (Me 2 N-Pyr) 2 -bpbp, or (2S, 2'S) -1, 1' -bis ((4-methoxy-3,5-dimethylpyridine -2-yl) methyl) -2,2'-bipyrrolidine, also called S, S '- (DMM-Py) 2 (bpbp):
Figure imgf000011_0001
Figure imgf000011_0001
En una realización más preferida el metal es divalente y el complejo es de fórmula (V), más preferiblemente el metal es Mn y aún más preferiblemente el complejo es X8' o X12: In a more preferred embodiment the metal is divalent and the complex is of formula (V), more preferably the metal is Mn and even more preferably the complex is X8 'or X12:
Figure imgf000011_0002
Figure imgf000011_0002
(Χ8') (X12)  (Χ8 ') (X12)
(Χ8') es de fórmula [{2,2'-(2R,2'R)-2,2'-bipirrolidina-1 ,1 '-diilbis(metileno)bis(N,N- dimetilpiridina-4-amina)}Mn"](CF3S03)2 o también llamado [{R,R'-(Me2N- Py)2(bpbp)}Mn"](CF3S03)2; y (Χ8 ') is of the formula [{2,2' - (2R, 2'R) -2,2'-bipyrrolidine-1, 1'-diylbis (methylene) bis (N, N-dimethylpyridine-4-amine) } Mn "] (CF 3 S0 3 ) 2 or also called [{R, R '- (Me 2 N-Py) 2 (bpbp)} Mn"] (CF 3 S0 3 ) 2; Y
(X12) es de fórmula [{{2S,2'S)-1 ,1 '-bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)-2,2'- bipirrolidina}Mn"](CF3S03)2 o también llamado [{S,S'-(DMM-Py)2(bpbp)}Mn"](CF3S03)2. (X12) is of the formula [{{2S, 2'S) -1, 1'-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -2,2'-bipyrrolidine} Mn "] ( CF 3 S0 3 ) 2 or also called [{S, S '- (DMM-Py) 2 (bpbp)} Mn "] (CF 3 S0 3 ) 2 .
En otra realización preferida de los complejos que comprenden el ligando tetradentado de fórmula (III) como se ha definido anteriormente, R representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3 y -COOCH2CH3, más preferiblemente, R2 y R3, son iguales o diferentes y representan un hidrógeno o un grupo metilo, aún más preferiblemente R2 y R3 son iguales. Aún más preferiblemente el compuesto de fórmula (III), Ri , R2 y R3 son hidrógeno; o Ri es metoxilo y R2 y R3 metilo. En estos compuestos de fórmula (III) se incluyen tanto las conformaciones S,S' como R,R'. In another preferred embodiment of the complexes comprising the tetradentate ligand of formula (III) as defined above, R represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 , more preferably, R 2 and R 3 , are the same or different and represent a hydrogen or a methyl group, even more preferably R 2 and R 3 are the same. Yet more preferably the compound of formula (III), Ri, R 2 and R 3 are hydrogen; or Ri is methoxy and R 2 and R 3 methyl. In these compounds of formula (III) both S, S 'and R, R' conformations are included.
En una realización preferida más preferida, el complejo comprende como ligando el compuesto (1 R,2R)-N1 ,N2-bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)-N1 ,N2- dimetilciclohexano-1 ,2-diamina, o también denominado R,R'-(DMM-Py)2(bpmcn). In a more preferred preferred embodiment, the complex comprises as ligand the compound (1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2-dimethylcyclohexane -1, 2-diamine, or also called R, R '- (DMM-Py) 2 (bpmcn).
En una realización más preferida el metal es divalente y el complejo es de fórmula (VI), más preferiblemente el metal es Mn y aún más preferiblemente el complejo es X13: In a more preferred embodiment the metal is divalent and the complex is of formula (VI), more preferably the metal is Mn and even more preferably the complex is X13:
Figure imgf000012_0001
Figure imgf000012_0001
(X13)  (X13)
(X13) es de fórmula [{(1 R,2R)-N1 ,N2-bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)-N1 ,N2- dimetilciclohexano-1 ,2-diamina}Mn"](CF3S03)2 o también llamada [{R,R'-(DMM- Py)2(bpmcn)}Mn"](CF3S03)2. (X13) is of the formula [{(1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2-dimethylcyclohexane-1,2-diamine } Mn "] (CF 3 S0 3 ) 2 or also called [{R, R '- (DMM-Py) 2 (bpmcn)} Mn"] (CF 3 S0 3 ) 2 .
Uso de compuestos de Fórmula general (1-2) Use of compounds of General Formula (1-2)
En otro aspecto, la presente invención se refiere al uso de un compuesto poliamínico de fórmula general (1-2): In another aspect, the present invention relates to the use of a polyamine compound of the general formula (1-2):
Figure imgf000013_0001
Figure imgf000013_0001
(I-2) donde: R4 y R5 son iguales o diferentes, y representan un hidrógeno, un grupo alquilo (CrC5) o un grupo -CH2-Cy; (I-2) where: R 4 and R 5 are the same or different, and represent a hydrogen, an alkyl group (CrC 5 ) or a -CH 2 -Cy group;
R6 representa un hidrógeno, un halógeno o un grupo alquilo (CrC5) ; R 6 represents a hydrogen, a halogen or an alkyl group (CrC 5 );
R7 y R9 son iguales o diferentes, y representan un hidrógeno o un grupo alquilo (CR 7 and R 9 are the same or different, and represent a hydrogen or an alkyl group (C
C5); C 5 );
R8 representa un grupo seleccionado de la lista que comprende hidrógeno, -NR'R", halógeno, alcoxilo (C C5), -N02 y -COOR'"; R 8 represents a group selected from the list comprising hydrogen, -NR'R ", halogen, alkoxy (CC 5 ), -N0 2 and -COOR '";
R', R" y R'", son iguales o diferentes y representan un hidrógeno o un grupo alquilo  R ', R "and R'", are the same or different and represent a hydrogen or an alkyl group
Cy representa un heteroarilo, Cy represents a heteroaryl,
o cualquiera de sus isómeros y/o sales farmacéuticamente aceptables, para el tratamiento y/o la prevención de enfermedades parasitarias o para la elaboración de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias. or any of its isomers and / or pharmaceutically acceptable salts, for the treatment and / or prevention of parasitic diseases or for the preparation of a medicament for the treatment and / or prevention of parasitic diseases.
En una realización preferida del compuesto de fórmula (I-2), R4 y R5 son iguales o diferentes, y representan un hidrógeno, un metilo o un grupo -CH2-Cy; donde Cy es piridina, más preferiblemente R4 y R5 son iguales. En una realización más preferida R6 representa hidrógeno, Cl, F o metilo. En una realización aún más preferida, R7 y R9 son iguales o diferentes, y representan hidrógeno o metilo; más preferiblemente R7 y R9 son iguales. En una realización más preferida, R8 representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3, N02 y -COOCH2CH3. In a preferred embodiment of the compound of formula (I-2), R 4 and R 5 are the same or different, and represent a hydrogen, a methyl or a -CH 2 -Cy group; where Cy is pyridine, more preferably R 4 and R 5 are the same. In a more preferred embodiment R 6 represents hydrogen, Cl, F or methyl. In an even more preferred embodiment, R 7 and R 9 are the same or different, and represent hydrogen or methyl; more preferably R 7 and R 9 are the same. In a more preferred embodiment, R 8 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 , N0 2 and -COOCH 2 CH 3 .
En otra realización preferida del compuesto de fórmula (I-2), R4 y R5 son iguales y representan un metilo o un grupo -CH2-piridina; y R6, R7, R8 y R9 son hidrógeno. En otra realización más preferida el compuesto de fórmula (I-2) se selecciona de entre 1 ,4-dimetil-7-{piridin-2-ilmetil)-1 ,4,7-triazonano, también denominado PyTACN (X26); y 1 ,4,7-tris(piridin-2-ilmetil)-1 ,4,7-triazonano, también denominado Py3TACN (X27): In another preferred embodiment of the compound of formula (I-2), R 4 and R 5 are the same and represent a methyl or a -CH 2 -pyridine group; and R 6 , R 7 , R 8 and R 9 are hydrogen. In another more preferred embodiment the compound of formula (I-2) is selected from 1,4-dimethyl-7- {pyridin-2-ylmethyl) -1, 4,7-triazonan, also called PyTACN (X26); and 1, 4,7-tris (pyridin-2-ylmethyl) -1, 4,7-triazonan, also called Py3TACN (X27):
Figure imgf000014_0001
Figure imgf000014_0001
(X26) (X27)  (X26) (X27)
Otro aspecto de la presente invención se refiere al uso de un complejo metálico que comprende un ligando tetradentado que consiste en un compuesto de fórmula general (I-2), según se han descrito anteriormente, para la preparación de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias. Another aspect of the present invention relates to the use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I-2), as described above, for the preparation of a medicament for treatment and / or the prevention of parasitic diseases.
En una realización preferida el complejo metálico es de fórmula general (II-2): In a preferred embodiment the metal complex is of general formula (II-2):
Figure imgf000014_0002
Figure imgf000014_0002
(II-2)  (II-2)
Donde: R4, R5, R6, R7, R8 y R9 se han definido anteriormente; M es un metal divalente; y X es un contraanión monovalente. Where: R 4 , R 5 , R 6 , R 7 , R 8 and R 9 have been defined above; M is a divalent metal; and X is a monovalent counter-anion.
El contraanión (X) es cualquier anión farmacéuticamente aceptable conocido por un experto en la materia y preferiblemente se puede seleccionar de la lista que comprende CF3S03 ", CI", Br", CI04 ", PF6 " y BF4 ", más preferiblemente el contraanión es CF3SO3-. En otra realización preferida de los complejos que comprenden el ligando tetradentado de fórmula (I-2) como se ha definido anteriormente, preferiblemente R4 y R5 son iguales o diferentes, y representan un hidrógeno, un metilo o un grupo -CH2-Cy; donde Cy es piridina, más preferiblemente R4 y R5 son iguales. En una realización más preferida R6 representa hidrógeno, Cl, F o metilo. En una realización aún más preferida, donde R7 y Rg son iguales o diferentes, y representan hidrógeno o metilo; más preferiblemente R7 y R9 son iguales. En una realización más preferida, R8 representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, - OCH3, N02 y -COOCH2CH3. En otra realización preferida del compuesto de fórmula (I-2), R4 y R5 son iguales y representan un metilo o un grupo -CH2-piridina; y R6, R7, R8 y R9 son hidrógeno. En otra realización más preferida el compuesto de fórmula (I- 2) se selecciona de entre 1 ,4-dimetil-7-{piridin-2-ilmetil)-1 ,4,7-triazonano, también denominado PyTACN y 1 ,4, 7-tris(piridin-2-ilmetil)-1 ,4,7-triazonano, también denominado Py3TACN (X27), más preferiblemente es el compuesto PyTACN. The counter-anion (X) is any pharmaceutically acceptable anion known to a person skilled in the art and can preferably be selected from the list comprising CF 3 S0 3 " , CI " , Br " , CI0 4 " , PF 6 " and BF 4 " , more preferably the counter-anion is CF3SO3-. In another preferred embodiment of the complexes comprising the tetradentate ligand of formula (I-2) as defined above, preferably R 4 and R 5 are the same or different, and represent a hydrogen, a methyl or a group -CH 2 - Cy; where Cy is pyridine, more preferably R 4 and R 5 are the same. In a more preferred embodiment R 6 represents hydrogen, Cl, F or methyl. In an even more preferred embodiment, where R 7 and Rg are the same or different, and represent hydrogen or methyl; more preferably R 7 and R 9 are the same. In a more preferred embodiment, R 8 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 , N0 2 and -COOCH 2 CH 3 . In another preferred embodiment of the compound of formula (I-2), R 4 and R 5 are the same and represent a methyl or a -CH 2 -pyridine group; and R 6 , R 7 , R 8 and R 9 are hydrogen. In another more preferred embodiment the compound of formula (I-2) is selected from 1,4-dimethyl-7- {pyridin-2-ylmethyl) -1, 4,7-triazonan, also called PyTACN and 1,4, 7-tris (pyridin-2-ylmethyl) -1, 4,7-triazonan, also called Py3TACN (X27), more preferably is the PyTACN compound.
En una realización más preferida el metal es divalente y el complejo es de fórmula (II- 2), más preferiblemente el metal es Mn, Fe o Cu y aún más preferiblemente el complejo es X1 , X6 o X9: In a more preferred embodiment the metal is divalent and the complex is of formula (II-2), more preferably the metal is Mn, Fe or Cu and even more preferably the complex is X1, X6 or X9:
Figure imgf000015_0001
Figure imgf000015_0001
(X1 ) (X6) (X9)  (X1) (X6) (X9)
X1 es de fórmula [{1 ,4-dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano}Fe ](CF3S03)2 o también denominados [{PyTACN} Fe"](CF3S03)2; X1 is of the formula [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano} Fe] (CF 3 S0 3 ) 2 or also called [{PyTACN} Fe "] ( CF 3 S0 3 ) 2;
X6 es de fórmula [{1 ,4-dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano}Mn"](CF3S03)2 o también denominado [{PyTACN}Mn"](CF3S03)2; y X6 is of the formula [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano} Mn "] (CF 3 S0 3 ) 2 or also called [{PyTACN} Mn"] (CF 3 S0 3 ) 2 ; Y
X9 es de fórmula [{1 ,4-dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano}Cu"](CF3S03)2 o también denominado [{PyTACN}Cu"](CF3S03)2. Composiciones farmacéuticas y medicamentos X9 is of the formula [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano} Cu "] (CF 3 S0 3 ) 2 or also called [{PyTACN} Cu"] (CF 3 S0 3 ) 2 . Pharmaceutical compositions and medications
Otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos un compuesto de fórmula general (I), preferiblemente (II) o (III), o sus complejos de fórmula general (IV), preferiblemente (V) o (VI), descritos anteriormente, junto con al menos un vehículo farmacéuticamente aceptable, opcionalmente esta composición puede incluir otro principio activo, por ejemplo otro compuesto antiparasitario. Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (I), preferably (II) or (III), or its complexes of general formula (IV), preferably (V) or ( VI), described above, together with at least one pharmaceutically acceptable carrier, optionally this composition may include another active ingredient, for example another antiparasitic compound.
Un aspecto más de la presente invención se refiere al uso de al menos un compuesto de fórmula general (I), preferiblemente (II) o (III), y sus complejos de fórmula general (IV), preferiblemente (V) o (VI), descritos anteriormente, para la elaboración de un medicamento. A further aspect of the present invention relates to the use of at least one compound of general formula (I), preferably (II) or (III), and its complexes of general formula (IV), preferably (V) or (VI) , described above, for the preparation of a medicament.
Otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos un compuesto de fórmula general (I-2) o sus complejos de fórmula general (II-2), descritos anteriormente, junto con al menos un vehículo farmacéuticamente aceptable, opcionalmente esta composición puede incluir otro principio activo, por ejemplo otro compuesto antiparasitario. Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (I-2) or its complexes of general formula (II-2), described above, together with at least one pharmaceutically acceptable carrier, optionally this composition may include another active ingredient, for example another antiparasitic compound.
Un aspecto más de la presente invención se refiere al uso de al menos un compuesto de fórmula general (I-2) o sus complejos de fórmula general (II-2), descritos anteriormente, para la elaboración de un medicamento. A further aspect of the present invention relates to the use of at least one compound of general formula (I-2) or its complexes of general formula (II-2), described above, for the preparation of a medicament.
Complejos objeto de la invención Complexes object of the invention
Otro aspecto de la presente invención se refiere al complejo (X13) [{(1 R,2R)-N1 ,N2- bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)-N1 ,N2-dimetilciclohexano-1 ,2- diamina}Mn"](CF3S03)2. Another aspect of the present invention relates to the complex (X13) [{(1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) -N1, N2- dimethylcyclohexane-1, 2- diamine} Mn "] (CF 3 S0 3 ) 2.
Otro aspecto más de la presente invención se refiere al complejo (X9) [{1 ,4-dimetil-7- (piridin-2-ilmetil)-1 ,4,7-triazonano}Cu"](CF3S03)2. Uso preferido de los compuestos v/o complejos descritos A further aspect of the present invention relates to the complex (X9) [{1,4-dimethyl-7- (pyridin-2-ylmethyl) -1,4,7-triazonano} Cu "] (CF 3 S0 3 ) 2 . Preferred use of the compounds v / o complexes described
En una realización preferida los compuestos y/o complejos descritos en la presente invención, se usan para el tratamiento y/o la prevención o para la elaboración de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias protozoarias, más preferiblemente los parásitos pertenecen a la familia Trypanosomatidae.  In a preferred embodiment the compounds and / or complexes described in the present invention are used for the treatment and / or prevention or for the preparation of a medicament for the treatment and / or prevention of protozoan parasitic diseases, more preferably parasites. They belong to the Trypanosomatidae family.
EJEMPLOS EXAMPLES
Los siguientes ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto la efectividad del producto de la invención. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention. The invention will be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
Síntesis de los compuestos poliamínicos v complejos metálicos (I), (II), (III), (IV), (V) v (VI) Synthesis of polyamine compounds and metal complexes (I), (II), (III), (IV), (V) v (VI)
La síntesis de los compuestos poliamínicos y de sus complejos metálicos (familias (I), (II), (III), (IV), (V), (VI)) ha sido descrita anteriormente en distintos artículos científicos como por ejemplo (cf.J. Lloret Fillol et al., Nat. Chem. 201 1 , 3, 807-813; Gómez et al., J. Org. Chem. 2013, 78, 1421 -1433; Gómez et al., Angew. Chem. Int. Ed. 2009, 48, 5720 -5723; I. García- Bosch et al., Adv. Synth. Catal. 2012, 354, 65 - 70; I.Prat et al., Chem. Eur. J. 2013, 19, 1908 - 1913).  The synthesis of polyamine compounds and their metal complexes (families (I), (II), (III), (IV), (V), (VI)) has been described previously in different scientific articles such as (cf. J Lloret Fillol et al., Nat. Chem. 201 1, 3, 807-813; Gómez et al., J. Org. Chem. 2013, 78, 1421-1433; Gómez et al., Angew. Chem. Int. Ed. 2009, 48, 5720-5723; I. García- Bosch et al., Adv. Synth. Catal. 2012, 354, 65-70; I.Prat et al., Chem. Eur. J. 2013, 19, 1908-1913).
Síntesis de los compuestos poliamínicos y complejos metálicos (1-2) y (11-2) Synthesis of polyamine compounds and metal complexes (1-2) and (11-2)
La síntesis de los compuestos poliamínicos y de sus complejos metálicos (familias (I- 2) y (11-2)) ha sido descrita anteriormente en distintos artículos científicos como por ejemplo (cf. I. Prat et al., Chem. Eur. J. 2013, 19, 6724-6738; I. Prat et al., Adv. Synth. Catal. 2013, 355, 947-956; A. Company et al., Chem. Eur. J. 2008, 14, 5727 - 5731 ; Gómez et al., Angew. Chem. Int. Ed. 2009, 48, 5720 -5723; Flassbeck et al., Z. Anorg. Allg. Chem. 1992, 608, 60-68; V. Stavila et al., New J. Chem., 2008, 32, 428^135) ENSAYOS BIOLOGICOS The synthesis of polyamine compounds and their metal complexes (families (I-2) and (11-2)) has been described previously in different scientific articles such as for example (cf. I. Prat et al., Chem. Eur. J. 2013, 19, 6724-6738; I. Prat et al., Adv. Synth. Catal. 2013, 355, 947-956; A. Company et al., Chem. Eur. J. 2008, 14, 5727 - 5731; Gómez et al., Angew. Chem. Int. Ed. 2009, 48, 5720-5723; Flassbeck et al., Z. Anorg. Allg. Chem. 1992, 608, 60-68; V. Stavila et al. , New J. Chem., 2008, 32, 428 ^ 135) BIOLOGICAL TESTS
CULTIVO PARASITARIO (PARA Leishmania spp) PARASITARY CULTURE (FOR Leishmania spp)
Formas promastigotas de las especies Leishmania infantum (MCAN/ES/2001/UCM- 10) y Leishmania braziliensis (MHOM/BR/1975/ M2904), fueron cultivadas in vitro en esterilidad, a 28e C en medio de cultivo monofásico MTL (Médium Trypanosomes Liquid) enriquecido con el 10 % (v/v) de suero bovino fetal inactivado frente al complemento (SBF-I) a 56eC/30 minutos. El inoculo de parásitos para iniciar el cultivo fue de 5 x 104 células/ml en 5 mi de medio en frascos de plástico Falcon® de 25 cm2, y mantenidos en una estufa a 28eC. (González P, et al., Int J Antimicro Agents 2005, 25, 136-141 ). Los cultivos se realizaron de manera rutinaria, y renovando el medio cada dos días consiguiendo el crecimiento exponencial de los flagelados hasta conseguir la masa celular necesaria para los posteriores estudios. Promastigote forms of the species Leishmania infantum (MCAN / ES / 2001 / UCM-10) and Leishmania braziliensis (MHOM / BR / 1975 / M2904), were cultured in vitro in sterility, at 28 e C in MTL single phase culture medium (Medium Trypanosomes Liquid) enriched with 10% (v / v) of inactivated fetal bovine serum against complement (SBF-I) at 56 e C / 30 minutes. The inoculum of parasites to start the culture was 5 x 10 4 cells / ml in 5 ml of medium in 25 cm 2 Falcon® plastic bottles, and kept in an oven at 28 e C. (González P, et al. , Int J Antimicro Agents 2005, 25, 136-141). The cultures were performed routinely, and the medium was renewed every two days, achieving the exponential growth of the flagellate until the necessary cell mass was obtained for subsequent studies.
CULTIVOS CELULARES Y ENSAYO DE CITOTOXICIDAD. CELL CULTURES AND CYTOTOXICITY TEST.
Los macrófagos J774,2 fueron reclonados desde J774,2 (European collection oí cell cultures (ECACC) número 9105151 1 ) originales de un tumor de hembra de ratón de la cepa Balb/c, manteniendo los cultivos entre 3-9 x 105 células/ml a 37eC y a 5% de C02 en medio de cultivo MEM + Glutamina suplementado con un 20 % de SBF-I. El protocolo para trabajar con las células en cultivo fue despegándolas del frasco de cultivo donde se encontraban adheridas mediante soporte frío y golpes secos. Se decantaron las células y se centrifugaron durante 5 minutos a 800 rpm. Se resuspendieron las células en medio de cultivo fresco para proceder a su contaje, mediante tinción con azul de tripán (dilución 1 :1 ) y en cámara hemocitométrica de Neubauer, para los estudios posteriores (Sánchez-Moreno M, et al., J Antimicrob Chemother 2012; 67:387-397). Estos ensayos se realizaron mediante citometría de flujo. Los macrófagos de la línea J774,2, se depositaron en un steríling (Universal Tube®) y se centrifugaron a 800 rpm durante 5 min, el sobrenadante se descartó y las células se resuspendieron en medio MEM + Glutamina + 20 % SBF-I. Se depositaron 1 x 104 células/en cada pocilio con un volumen final de 500 μΙ de medio por pocilio en una placa de titulación de 24 pocilios, se incubaron durante 24 h a 37eC en atmósfera húmeda enriquecida con 5% C02. Esto se realizó para que se fijasen las células. Transcurrido este tiempo el medio de cultivo se retiró y se adicionó medio fresco con los productos a ensayar, a las concentraciones de 50, 25, 10 y 1 μΜ. A las 72 horas de la incubación, se procedió a la preparación de las muestras para su lectura en el citómetro de flujo. The J774.2 macrophages were recloned from J774.2 (European collection oí cell cultures (ECACC) number 9105151 1) originals of a mouse female tumor of the Balb / c strain, keeping the cultures between 3-9 x 10 5 cells / ml at 37 e C and 5% C0 2 in MEM + culture medium supplemented with 20% SBF-I. The protocol to work with the cells in culture was taking them off the culture bottle where they were attached by cold support and dry blows. The cells were decanted and centrifuged for 5 minutes at 800 rpm. The cells were resuspended in fresh culture medium for counting, by staining with trypan blue (1: 1 dilution) and in a hemocytometric chamber of Neubauer, for subsequent studies (Sánchez-Moreno M, et al., J Antimicrob Chemother 2012; 67: 387-397). These tests were performed by flow cytometry. The macrophages of the J774.2 line were deposited in a sterling (Universal Tube®) and centrifuged at 800 rpm for 5 min, the supernatant was discarded and the cells were resuspended in MEM + Glutamine + 20% SBF-I medium. 1 x 10 4 cells / were placed in each well with a final volume of 500 μΙ of medium per well in a 24-well titration plate, incubated for 24 h at 37 e C in a humid atmosphere enriched with 5% C0 2 . 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 50, 25, 10 and 1 μΜ. At 72 hours After incubation, the samples were prepared for reading on the flow cytometer.
El método seguido para la preparación de muestras es el descrito por Ormerod, 1994, partiendo de las células y el medio presente en los pocilios, a las cuales se le adicionó 100 μΙ de solución de ioduro de propidio (Pl, 100 g/ml) (Sigma Chemical Co), incubándose a 28eC en oscuridad unos 10 minutos, posteriormente, se añadió 100 μΙ de diacetato de fluoresceína (FDA) (Sigma Chemical Co) en solución (100ng/ml) volviéndose a incubar a 28eC en oscuridad unos 10 minutos, y previa centrifugación a 1500 rpm durante 10 minutos, se procedió a lavar el precipitado con PBS. Finalmente, se analizaron los resultados en el citómetro teniendo en cuenta que las células con la membrana plasmática intacta tienen una fluorescencia color verde, mientras que las células dañadas o muertas presentan una fluorescencia roja. Se calculó el porcentaje de viabilidad. El número de células muertas se determinó por comparación con los cultivos controles. The method followed for the preparation of samples is that described by Ormerod, 1994, starting from the cells and the medium present in the wells, to which 100 μΙ of propidium iodide solution (Pl, 100 g / ml) was added. (Sigma Chemical Co), incubating at 28 e C in the dark for about 10 minutes, subsequently, 100 μΙ of fluorescein diacetate (FDA) (Sigma Chemical Co) in solution (100ng / ml) was added and re-incubated at 28 e C in darkness about 10 minutes, and after centrifugation at 1500 rpm for 10 minutes, the precipitate was washed with PBS. Finally, the results were analyzed in the cytometer taking into account that the cells with the intact plasma membrane have a green fluorescence, while the 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.
ENSAYOS DE ACTIVIDAD IN VÍTRO: FORMAS EXTRACELULARES Ensayo sobre formas promastigotas IN VÍTRO ACTIVITY TESTS: EXTRACELLULAR FORMS Essay on promastigote forms
Las formas promastigotas de L. infantum cultivadas de la forma anteriormente descrita, fueron recolectadas en su fase exponencial de crecimiento mediante centrifugación a 1500 rpm durante 10 min. Se resuspendieron en medio fresco. La concentración de parásitos fue contada en una cámara hemocitométrica de Neubauer y sembrados en una placa de 24 pocilios a razón de una concentración de 5 x 104 parásitos/ pocilio. Los compuestos a ensayar se disolvieron con DMSO puro a una concentración de DMSO de 0,01 % (v/v), a la cual, este disolvente no es tóxico ni tiene ningún efecto sobre el crecimiento de los parásitos. Los compuestos fueron añadidos al medio de cultivo en la placa de 24 pocilios a una concentración final de: 100, 50, 25, 10 y 1 μΜ, y a un volumen final por pocilio de 500 μΙ. El efecto de cada producto sobre el crecimiento de las formas promastigotas, a las diferentes concentraciones ensayadas, se evaluó a las 72 horas, usando una cámara hemocitométrica de Neubauer, y el efecto leishmanicida se expresó como la IC50 (concentración requerida para dar una inhibición del 50%, calculado por el análisis de la regresión lineal de la Kc a las concentraciones ensayadas) (González P, et al. Int J Antimicro Agents 2005; 25:136-141 ). ENSAYOS DE ACTIVIDAD IN VITRO: FORMAS INTRACELULARES. Ensayo sobre formas amastigotas Promastigote forms of L. infantum grown in the manner described above, were collected in their exponential phase of growth by centrifugation at 1500 rpm for 10 min. They were resuspended in fresh medium. The concentration of parasites was counted in a hemocytometric chamber of Neubauer and seeded in a 24-well plate at a concentration of 5 x 10 4 parasites / well. The compounds to be tested were dissolved with pure DMSO at a DMSO concentration of 0.01% (v / v), at which this solvent is not toxic nor has any effect on the growth of parasites. The compounds were added to the culture medium in the 24-well plate at a final concentration of: 100, 50, 25, 10 and 1 μΜ, and at a final volume per well of 500 μΙ. The effect of each product on the growth of promastigote forms, at the different concentrations tested, was evaluated at 72 hours, using a Neubauer hemocytometric chamber, and the leishmanicidal effect was expressed as the IC 50 (concentration required to give an inhibition 50%, calculated by the analysis of the linear regression of the Kc at the concentrations tested) (González P, et al. Int J Antimicro Agents 2005; 25: 136-141). IN VITRO ACTIVITY TESTS: INTRACELLULAR FORMS. Test on amastigote forms
Los macrófagos se despegaron del frasco de cultivo donde se encontraban adheridos mediante soporte frío y golpes secos. Para ello, se eliminó el medio de cultivo, seguidamente se cubrió la superficie celular con EDTA-tripsina y se incubó en frío durante 5 minutos según la metodología expuesta anteriormente. Tras ello, se pasó a un frasco de fondo cónico de 25 mi de capacidad (Steriling) para centrifugarlos a 800 rpm durante 5 minutos, retirándose el sobrenadante y resuspendiéndolos en MEM + Glutamina con 20% SBF-I. Se cuentan en cámara de Neubauer y se cultivan en placas de 24 pocilios a razón de 1 x 104 células, en las que previamente habíamos introducido un cristal cubreobjetos redondo de 12 mm en cada pocilio. Para su adherencia, dejamos crecer las células 24 h a 37eC en 5% C02. The macrophages took off from the culture bottle where they were attached by cold support and dry blows. For this, the culture medium was removed, then the cell surface was covered with EDTA-trypsin and incubated cold for 5 minutes according to the methodology set forth above. 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 resuspending them in MEM + Glutamine with 20% SBF-I. They are counted in a Neubauer chamber and grown in 24-well plates at a rate of 1 x 10 4 cells, in which we had previously introduced a 12mm round coverslip glass into each well. For adhesion, we let the cells grow 24 h at 37 e C in 5% C0 2 .
Una vez adheridas las células, se infectaron "in vitro": Macrófagos J774,2 con 1 x 105 parásitos/pocilio de formas promastigotas metacíclicas en fase estacionaria de crecimiento de las especies de Leishmanias utilizadas (L infantum y L braziliensis) La infección se mantuvo durante 24 horas para que el parásito entrase en la célula. Transcurrido este tiempo el medio de cultivo se retiró y se adicionó medio fresco con los productos a ensayar, a las concentraciones necesarias para sacar la IC50 (100, 50, 25, 10 y 1 μΜ). A las 72 horas de la incubación, se procedió a la retirada de los cristales. Once the cells were adhered, they were infected "in vitro": J774.2 macrophages with 1 x 10 5 parasites / well of stationary promastigote forms in growth phase of the Leishmanias species used (L infantum and L braziliensis) The infection was infected. held 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 remove the IC 50 (100, 50, 25, 10 and 1 μΜ). 72 hours after incubation, the crystals were removed.
Una vez sacados los cristales se colocaron en un portaobjetos. Se fijaron con metanol y se dejaron secar. Una vez fijados y secos, se les añadió DPX (Panreac®), medio de montaje para microscopía. Se tiñeron con Giemsa, para ello, inmediatamente antes de su empleo y en tubo de ensayo, se diluyó 0,2 mi de Azur-Eosina-Azul de Metileno solución según Giemsa DC (Código 251338) con 2 mi de tampón Sorensen (dilución 1 :10), se mezcló bien y se cubrió la preparación dejando colorear durante 20 minutos. Se lavó con agua destilada. Se dejó escurrir y secar en posición vertical. Por último se examinó con el objetivo de inmersión y se contó el número de formas amastigotas intracelulares en un total de 200 células (González P, et al. Int J Antimicro Agents 2005, 25, 136-141 ). Resultados de los Ensayos Once the crystals were removed, they were 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, 0.2 ml of Azur-Eosin-Methylene Blue solution was diluted according to Giemsa DC (Code 251338) with 2 ml of Sorensen buffer (dilution 1 : 10), mixed well and covered the preparation leaving coloring 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 (González P, et al. Int J Antimicro Agents 2005, 25, 136-141). Test Results
Los resultados de los ensayos realizados con los compuestos y complejos de las familias (I), (II), (III), (IV), (V) y (VI) se muestran en las Tablas 1 y 2.  The results of the tests carried out with the compounds and complexes of the families (I), (II), (III), (IV), (V) and (VI) are shown in Tables 1 and 2.
Tabla 1 .- Actividad y toxicidad encontradas para los compuestos poliamínicos y de los complejos con metales de transición estudiados sobre formas extracelulares e intracelulares de Leishmania spp. Table 1 .- Activity and toxicity found for polyamine compounds and transition metal complexes studied on extracellular and intracellular forms of Leishmania spp.
Figure imgf000021_0001
Figure imgf000021_0001
Resultado de tres experimentos independientes. a IC50 = Concentración necesaria para obtener el 50 % de Inhibición, calculada por análisis linear de regresión del valor Kc a las concentraciones empleadas de (1 , 10, 25, 50 y 100 μΜ). b Frente a Macrófagos J774,2 después de 72 h de cultivo. Result of three independent experiments. at IC 50 = Concentration necessary to obtain 50% Inhibition, calculated by linear regression analysis of the Kc value at the concentrations used of (1, 10, 25, 50 and 100 μΜ). b In front of J774.2 macrophages after 72 hours of cultivation.
Tabla 2.- índice de Selectividad encontrados los compuestos poliamínicos y de los complejos con metales de transición estudiados sobre formas extracelulares e intracelulares de Leishmania spp. Table 2.- Selectivity index found polyamine compounds and transition metal complexes studied on extracellular and intracellular forms of Leishmania spp.
ISC IS C
Compuesto  Compound
L. infantum L. braziliensis  L. infantum L. braziliensis
Promastigote Amastigote Promastigote Amastigote  Promastigote Amastigote Promastigote Amastigote
Glucatim® 0,8 0,6 0,6 0,5  Glucatim® 0.8 0.6 0.6 0.5
X8 2,2 (3) 2,4 (4) 2,3 (4) 3,1 (6)  X8 2.2 (3) 2.4 (4) 2.3 (4) 3.1 (6)
X12 73,6 (92) 97 (162) 27,8 (46) 1 1 ,0 (22)  X12 73.6 (92) 97 (162) 27.8 (46) 1 1, 0 (22)
X13 18,8 (24) 9,6 (16) 137,6 (229) 10,0 (20)  X13 18.8 (24) 9.6 (16) 137.6 (229) 10.0 (20)
X15 1 1 ,6 (15) 19,1 (32) 14,7 (24) 20,2 (41 )  X15 1 1, 6 (15) 19.1 (32) 14.7 (24) 20.2 (41)
X25 3,2 (4) 1 1 ,9 (20) 3,6 (6) 12,4 (25) c índice de Selectividad.: IC50 de los macrófagos/IC50 de las formas extracelulares e intracelulares de los parásitos. Entre paréntesis: Numero de veces que la IS de los compuestos es superior a la IS de la droga de referencia. X25 3.2 (4) 1 1, 9 (20) 3.6 (6) 12.4 (25) c Selectivity index .: IC 50 of macrophages / IC 50 of extracellular and intracellular forms of parasites. In parentheses: Number of times that the SI of the compounds is greater than the SI of the reference drug.
Los compuestos estudiados se han evaluado frente a formas extracelulares e intracelulares de Leishmania spp. (L infantum y L. braziliensis), obteniéndose los índices IC50 como indicadores de la actividad de los compuestos frente al protozoo, los índices de toxicidad IC50 frente a Macrofagos y el índice de selectividad obtenido como IS= IC50 frente a macrofagos /IC50 frente formas extracelulares e intracelulares de los parásitos. En este estudio los valores obtenidos se comparan con los correspondientes a la droga referencia, Glucatim®, utilizada comúnmente en el tratamiento de la leishmaniasis. The compounds studied have been evaluated against extracellular and intracellular forms of Leishmania spp. (L infantum and L. braziliensis), obtaining the IC 50 indices as indicators of the activity of the compounds against the protozoan, the toxicity indices IC 50 against Macrophages and the selectivity index obtained as IS = IC 50 against macrophages / IC 50 versus extracellular and intracellular forms of parasites. In this study, the values obtained are compared with those corresponding to the reference drug, Glucatim®, commonly used in the treatment of leishmaniasis.
El análisis de los resultados obtenidos indica que en general los compuestos estudiados son activos frente a las formas intracelulares y extracelulares del parásito utilizadas, con valores de IC50 hasta cuatro superiores a los valores obtenidos para el compuesto referencia. Además en este caso se han obtenido valores de IC50 frente a macrofagos entre 10 y 25 veces superiores al del compuesto referencia, lo cual indica que los nuevos compuestos presentan una baja toxicidad frente a los macrofagos, un hecho que constituye una ventaja a la hora de su uso como agentes terapéuticos. Por lo tanto y a partir de dichos valores se han obtenido índices de selectividad con valores entre 15 y 150 para un grupo de los compuestos estudiados. The analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite used, with IC 50 values up to four higher than the values obtained for the reference compound. In this case, IC 50 values against macrophages between 10 and 25 times higher than the reference compound have been obtained, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage at the time of its use as therapeutic agents. Therefore, and from these values, selectivity indices with values between 15 and 150 have been obtained for a group of the compounds studied.
Los resultados de estos ensayos para los compuestos y complejos de las familias (I-2) y (II-2) se muestran las Tablas 3 y 4. The results of these tests for the compounds and complexes of the families (I-2) and (II-2) are shown in Tables 3 and 4.
Tabla 3.- Actividad y toxicidad encontradas para los compuestos poliamínicos y de los complejos con metales de transición estudiados sobre formas extracelulares e intracelulares de Leishmania spp. Table 3.- Activity and toxicity found for polyamine compounds and transition metal complexes studied on extracellular and intracellular forms of Leishmania spp.
Compuesto IC50 (μ )3 Compound IC 50 (μ) 3
Toxicidad IC50MacrofagosToxicity IC 50 Macrophages
L. infantum L. braziliensis L. infantum L. braziliensis
(MM)b Promastigote Amastigote Promastigote Amastigote ( M M) b Promastigote Amastigote Promastigote Amastigote
Glucatim® 18,0+3,1 24,2+2,6 25,6+1 ,6 30,4±6,1 15,20+1 ,3Glucatim® 18.0 + 3.1 24.2 + 2.6 25.6 + 1, 6 30.4 ± 6.1 15.20 + 1, 3
X1 18,5+1 ,6 20,5+1 ,8 22,3±0,6 21 ,3±0,9 37,9+4,2X1 18.5 + 1, 6 20.5 + 1, 8 22.3 ± 0.6 21, 3 ± 0.9 37.9 + 4.2
X6 8,5±0,4 < 1 5,3±0,3 8,7±0,6 98,5±6,3X6 8.5 ± 0.4 <1 5.3 ± 0.3 8.7 ± 0.6 98.5 ± 6.3
X9 45,7±2,8 37,9±5,4 46,7±4,9 19,7±2,5 147,0±6,9X9 45.7 ± 2.8 37.9 ± 5.4 46.7 ± 4.9 19.7 ± 2.5 147.0 ± 6.9
X26 71 ,7±1 0,0 33,4±3,2 53,2±3,6 30,7±2,5 100,5±4,8X26 71, 7 ± 1 0.0 33.4 ± 3.2 53.2 ± 3.6 30.7 ± 2.5 100.5 ± 4.8
X27 2,56±0,7 10,4±0,1 5,5±0,8 1 1 ,9±0,8 126,1 ±7,1 X27 2.56 ± 0.7 10.4 ± 0.1 5.5 ± 0.8 1 1, 9 ± 0.8 126.1 ± 7.1
Resultados de tres experimentos independientes. a IC50 = Concentración necesaria para obtener el 50 % de Inhibición, calculada por análisis linear de regresión del valor Kc a las concentraciones empleadas de (1 , 10, 25, 50 y 100 μΜ). b Frente a Macrófagos J774,2 después de 72 h de cultivo. Results of three independent experiments. at IC 50 = Concentration necessary to obtain 50% Inhibition, calculated by linear regression analysis of the Kc value at the concentrations used of (1, 10, 25, 50 and 100 μΜ). b In front of J774.2 macrophages after 72 hours of cultivation.
Tabla 4.- índice de Selectividad encontrados los compuestos poliamínicos y de los complejos con metales de transición estudiados sobre formas extracelulares e intracelulares de Leishmania spp. Table 4.- Selectivity index found polyamine compounds and transition metal complexes studied on extracellular and intracellular forms of Leishmania spp.
Figure imgf000023_0001
c índice de Selectividad.: IC50 de los macrófagos/IC50 de las formas extracelulares e intracelulares de los parásitos. Entre paréntesis: Número de veces que la IS de los compuestos es superior a la IS de la droga de referencia.
Figure imgf000023_0001
c Selectivity index .: IC 50 of macrophages / IC 50 of extracellular and intracellular forms of parasites. In parentheses: Number of times the SI of the compounds is greater than the SI of the reference drug.
Los compuestos estudiados se han evaluado frente a formas extracelulares e intracelulares de Leishmania spp. (L. infantum y L braziliensis), obteniéndose los índices IC50 como indicadores de la actividad de los compuestos frente al protozoo, los índices de toxicidad IC50 frente a Macrófagos y el índice de selectividad obtenido como IS= IC50 frente a macrófagos /IC50 frente formas extracelulares e intracelulares de los parásitos. En este estudio los valores obtenidos se comparan con los correspondientes a la droga referencia, Glucatim®, utilizada comúnmente en el tratamiento de la leishmaniasis. The compounds studied have been evaluated against extracellular and intracellular forms of Leishmania spp. (L. infantum and L braziliensis), obtaining the IC 50 indices as indicators of the activity of the compounds against the protozoan, the toxicity indices IC 50 against Macrophages and the selectivity index obtained as IS = IC 50 against macrophages / IC 50 versus extracellular and intracellular forms of parasites. In this study the values obtained are compared with those corresponding to the reference drug, Glucatim®, commonly used in the treatment of leishmaniasis.
El análisis de los resultados obtenidos indica que en general los compuestos estudiados son activos frente a las formas intracelulares y extracelulares del parásito, con valores de IC50 menores a los valores obtenidos para el compuesto referencia, principalmente los compuestos X6 y X27. Además, en todos los compuestos estudiados los valores de IC50 frente a macrófagos superan de 7 a 10 veces al del compuesto referencia, lo cual indica que los nuevos compuestos presentan una baja toxicidad frente a los macrófagos, un hecho que constituye una ventaja a la hora de su uso como agentes terapéuticos. Por lo tanto y a partir de dichos valores los índices de selectividad de los compuestos X6 y X27 superan al del glucantime en 20 y 164 veces para las formas amastigotas de ambas especies de Leishmania. The analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite, with IC 50 values lower than the values obtained for the reference compound, mainly compounds X6 and X27. In addition, in all the compounds studied the values of IC 50 against macrophages exceed 7 to 10 times that of the reference compound, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage to time of its use as therapeutic agents. Therefore, and from these values, the selectivity indices of compounds X6 and X27 exceed that of glucantime by 20 and 164 times for the amastigote forms of both Leishmania species.
El análisis de los resultados obtenidos indica que en general los compuestos estudiados son activos frente a las formas intracelulares y extracelulares del parásito utilizadas, con valores de IC50 hasta cuatro superiores a los valores obtenidos para el compuesto referencia. Además en este caso se han obtenido valores de IC50 frente a macrófagos entre 10 y 25 veces superiores al del compuesto referencia, lo cual indica que los nuevos compuestos presentan una baja toxicidad frente a los macrófagos, un hecho que constituye una ventaja a la hora de su uso como agentes terapéuticos. Por lo tanto y a partir de dichos valores se han obtenido índices de selectividad con valores entre 15 y 150 para un grupo de los compuestos estudiados. The analysis of the results obtained indicates that in general the compounds studied are active against the intracellular and extracellular forms of the parasite used, with IC 50 values up to four higher than the values obtained for the reference compound. In this case, IC 50 values against macrophages between 10 and 25 times higher than the reference compound have been obtained, which indicates that the new compounds have a low toxicity against macrophages, a fact that constitutes an advantage at the time of its use as therapeutic agents. Therefore, and from these values, selectivity indices with values between 15 and 150 have been obtained for a group of the compounds studied.
CULTIVO PARASITARIO (para Trypanosoma cruzi) PARASITARY CULTURE (for Trypanosoma cruzi)
Se utilizó Trypanosoma cruzi cepa tipo I (IRHOD/CO/2008/SN3) (Tellez- Meneses et al., Acta Trop 2008; 108:26-34) cuyo origen geográfico es Guajira (Colombia) y su origen biológico fíhodnius prolixus en las formas de desarrollo: epimastigotas, tripomastigotas y amastigotas. El cultivo de las formas epimastigotas de T. cruzi se realizó in vitro en esterilidad en medio de cultivo monofásico MTL (Médium Trypanosomes Liquid, Ruiz-Pérez y col., 1986) enriquecido con el 10% (v/v) de Suero Bovino Fetal (SBF-I) inactivado a 56eC/ 30 minutos. El inoculo de parásitos para iniciar el cultivo fue de 5 x 104 células/ml en 5 mi de medio en frascos de plástico Falcon® de 25 cm2 y se mantuvieron en una estufa a 28eC. Los cultivos se realizaron de manera rutinaria consiguiendo el crecimiento exponencial de los flagelados hasta conseguir la masa celular necesaria para los posteriores estudios. (Téllez-Meneses J, y col. Acta Trop 2008; 108:26-34). Trypanosoma cruzi strain type I was used (IRHOD / CO / 2008 / SN3) (Tellez-Meneses et al., Acta Trop 2008; 108: 26-34) whose geographical origin is Guajira (Colombia) and its biological origin fíhodnius prolixus in the forms of development: epimastigotes, trypomastigotes and amastigotes. The culture of the epimastigote forms of T. cruzi was performed in vitro in sterility in a single-phase MTL culture medium (Medium Trypanosomes Liquid, Ruiz-Pérez et al., 1986) enriched with 10% (v / v) Fetal Bovine Serum (SBF-I) inactivated at 56 e C / 30 minutes. The inoculum of parasites to start the culture was 5 x 10 4 cells / ml in 5 ml of medium in 25 cm 2 Falcon® plastic bottles and kept in an oven at 28 e C. The cultures were carried out in a manner routine achieving the exponential growth of flagellates until the necessary cell mass is obtained for subsequent studies. (Téllez-Meneses J, et al. Acta Trop 2008; 108: 26-34).
CULTIVOS CELULARES Y ENSAYO DE CITOTOXICIDAD. CELL CULTURES AND CYTOTOXICITY TEST.
Las células Vero fueron establecidas desde el riñon de un mono Verde Africano adulto, manteniendo los cultivos a una densidad 1 x 104 células/ml a 37eC y 5% de C02. El procedimiento para trabajar con las células en cultivo fue tripsinizar las células adheridas mediante lavado con PBS y añadiendo la cantidad suficiente de tripsina/EDTA (200ml de PBS + 0,1 g de EDTA y 200 mi de PBS + 0,5 g de tripsina. Se mezclan las dos soluciones. pH= 7,2-7,4 y se filtra). Se incubaron durante 5-10 minutos las células. Se decantaron las células y se centrifugaron durante 5 minutos a 800 rpm. Se resuspendieron las células en un medio de cultivo nuevo. Como medio de cultivo se utilizó RPMI suplementado con un 10 % de suero bovino fetal inactivado (SBF-I) (Sánchez-Moreno M, et al. J Antimicrob Chemother 2012; 67:387-397). Las células Vero, se depositaron en un esterlín y se centrifugaron a 800 rpm durante 5 minutos, el sobrenadante se descartó y las células se resuspendieron en medio RPMI. Se depositaron 1 x104 células/ml en cada pocilio de una placa de titulación de 24 pocilios, se incubaron durante 24 h a 37eC en atmósfera húmeda enriquecida con 5% C02. Esto se realizó para que se fijasen las células. Transcurrido este tiempo el medio de cultivo se retiró y se adicionó medio fresco con los productos a ensayar, a las concentraciones de 100, 50, 25, 10 y 1 μΜ. A las 72 horas de la incubación, se procedió a la preparación de las muestras para su lectura en el citómetro de flujo. El método seguido es el descrito por (Ormerod, 1994), partiendo de las células y el medio presente en los pocilios, a las cuales se le adicionó 100 μΙ de solución de ioduro de propidio (Pl, 100 μg/ml) (Sigma Chemical Co), incubándose a 28eC en oscuridad unos 10 minutos, posteriormente, se añadió 100 μΙ de diacetato de fluoresceína (FDA) (Sigma Chemical Co) en solución (100ng/ml) volviéndose a incubar a 28eC en oscuridad unos 10 minutos, y previa centrifugación a 1500 rpm durante 10 minutos, se procedió a lavar el precipitado con PBS. Finalmente, se analizaron los resultados teniendo en cuenta que las células con la membrana plasmática intacta tienen una fluorescencia color verde, mientras que las células dañadas o muertas presentan una fluorescencia roja. Se calculó el porcentaje de viabilidad. El número de células muertas se determinó por comparación con los cultivos controles (Marín C, et al. J Nat Prod 201 1 ; 74:744-750). ENSAYOS DE ACTIVIDAD IN VITRO: FORMAS EXTRACELULARES. Vero cells were established from the kidney of an adult African Green monkey, keeping the cultures at a density of 1 x 10 4 cells / ml at 37 e C and 5% C0 2 . The procedure for working with the cells in culture was trypsinizing the adhered cells by washing with PBS and 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 are mixed, pH = 7.2-7.4 and filtered). The cells were incubated for 5-10 minutes. The cells were decanted and centrifuged for 5 minutes at 800 rpm. The cells were resuspended in a new culture medium. RPMI supplemented with 10% of inactivated fetal bovine serum (SBF-I) was used as culture medium (Sánchez-Moreno M, et al. J Antimicrob Chemother 2012; 67: 387-397). Vero cells were deposited in a sterlin and centrifuged at 800 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI medium. 1 x 10 4 cells / ml were deposited in each well of a 24-well titration plate, incubated for 24 h at 37 e C in a humid atmosphere enriched with 5% C0 2 . 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 μΜ. At 72 hours after incubation, the samples were prepared for reading on the flow cytometer. The method followed is that described by (Ormerod, 1994), starting from the cells and the medium present in the wells, to which 100 μΙ of propidium iodide solution (Pl, 100 μg / ml) was added (Sigma Chemical Co), incubating at 28 e C in the dark for about 10 minutes, subsequently, 100 μΙ of fluorescein diacetate (FDA) (Sigma Chemical Co) in solution (100ng / ml) was added and re-incubated at 28 e C in the dark about 10 minutes, and after centrifugation at 1500 rpm for 10 minutes, the precipitate was washed with PBS. Finally, the results were analyzed taking into account that the cells with the intact plasma membrane have a green fluorescence, while the 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 (Marín C, et al. J Nat Prod 201 1; 74: 744-750). IN VITRO ACTIVITY TESTS: EXTRACELLULAR FORMS.
Transformación de formas epimastiqotas a formas tripomastiqotas metacíclicas Transformation of epimastiqotas forms to metacyclic tripomastiqotas forms
La metaciclogénesis fue inducida a un cultivo de 5 días (fase estacionaria), tras ser centrifugado a 7000g durante 10 min a 10eC. Los parásitos fueron incubados 2h a 28eC a una densidad de 5 x 108 parásitos/ml en medio TAU (190 mM NaCI, 17 mM KCI, 2 mM MgCI2, 2 mM CaCI2, 8 mM phosphate buffer, pH 6,0). Después, los parásitos se incubaban a la dilución 1 :100 durante 96h a 28eC en medio TAU3AAG (TAU suplementado con 10 mM L-prolina, 50 mM L-glutamato sódico, 2 mM L- aspartato sódico y 10 mM D-glucosa) en frascos de cultivo de 25 mi en posición horizontal impidiendo que supere 1 cm de profundidad el medio. (Cardoso J, et al. Mem Inst Oswaldo Cruz 2010; 5:1026-32). Metacyclogenesis was induced at a 5-day culture (stationary phase), after being centrifuged at 7000g for 10 min at 10 e C. The parasites were incubated 2 h at 28 e C at a density of 5 x 10 8 parasites / ml in medium TAU (190 mM NaCI, 17 mM KCI, 2 mM MgCI 2 , 2 mM CaCI 2 , 8 mM phosphate buffer, pH 6.0). The parasites were then incubated at 1: 100 dilution for 96h at 28 e C in TAU3AAG medium (TAU supplemented with 10 mM L-proline, 50 mM sodium L-glutamate, 2 mM L-sodium aspartate and 10 mM D-glucose ) in 25 ml culture jars in a horizontal position preventing the medium from exceeding 1 cm deep. (Cardoso J, et al. Mem Inst Oswaldo Cruz 2010; 5: 1026-32).
Formas epimastiqotas Epimastiqotas forms
Las formas epimastigotas de T. cruzi cultivadas de la forma anteriormente descrita fueron recolectadas en su fase exponencial de crecimiento mediante centrifugación a 1500 rpm durante 10 min. El número de parásitos fue contado en una cámara hemocitométrica de Neubauer y sembrados en una placa de 24 pocilios a razón de una concentración de 5 x104 parásitos/ml en cada pocilio. Epimastigote forms of T. cruzi cultured 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 5 x 4 4 parasites / ml in each well.
Los compuestos a ensayar se disolvieron en DMSO a una concentración de 0,01 % (v/v) concentración a la cual este disolvente no es tóxico ni tiene ningún efecto sobre el crecimiento de los parásitos. Los compuestos fueron añadidos al medio de cultivo a una concentración final de: 100, 50, 25, 10 y 1 μΜ. El efecto de cada compuesto sobre el crecimiento de las formas epimastigotas, a las diferentes concentraciones ensayadas, se evaluó a las 72 h, usando una cámara hemocitométrica de Neubauer y el efecto tripanocida se expresó como la IC50 (concentración requerida para dar una inhibición del 50 %, calculado por el análisis de la regresión lineal de la Kc a las concentraciones ensayadas) (Sánchez-Moreno M, y col. J Med Chem. 201 1 ; 54:970- 9). The compounds to be tested were dissolved in DMSO at a concentration of 0.01% (v / v) 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 μΜ. The effect of each compound on the growth of epimastigote forms, at the different concentrations tested, was evaluated at 72 h, using a Neubauer hemocytometric chamber and the trypanocidal effect was expressed as IC 50 (concentration required to give an inhibition of 50%, calculated by the analysis of the linear regression of the K c at the concentrations tested) (Sánchez-Moreno M, et al. J Med Chem. 201 1; 54: 970-9).
Formas Tripomastiqotas Tripomastiqotas forms
La actividad (porcentaje de reducción de parásitos) es comparado con el control después de realizar la metodología descrita en (Júnior CO, y col. Biomed Pharmacother 2010; 64:624-6) con algunas modificaciones realizadas en el laboratorio. El ensayo se lleva a cabo usando sangre infectada de ratón Balb/c que ha sido obtenida durante los días de máxima parasitemia (día 7 aprox. post infección). La sangre infectada se diluía con sangre no infectada para obtener una concentración de 1 -4 x 106 tripomastigotas/ml, entonces se diluía 1 :2 con RPMI 1640 medium-GIBCO, invitrogen®. Soluciones stock de los compuestos a ensayar se preparan al mismo tiempo en DMSO. Una muestra de sangre infectada y la droga se añaden a una placa de 96 pocilios hasta un volumen final de 200 μΙ y las concentraciones de compuesto de 1 a 100 μΜ, a fin de calcular la IC50 para cada uno. Para reproducir las condiciones de banco de sangre, las placas se incubaron a 4 eC durante 24 h. Los experimentos se repitieron tres veces. Cada muestra se examinó microscópicamente (OLYMPUS CX41 ) para el recuento de parásitos utilizando la cámara de Neubauer. The activity (percentage of parasite reduction) is compared with the control after performing the methodology described in (Junior CO, et al. Biomed Pharmacother 2010; 64: 624-6) with some modifications made in the laboratory. The test is carried out using infected Balb / c mouse blood that has been obtained during the days of maximum parasitemia (day 7 approx. Post infection). The infected blood was diluted with uninfected blood to obtain a concentration of 1 -4 x 10 6 trypomastigotes / ml, then diluted 1: 2 with RPMI 1640 medium-GIBCO, invitrogen®. Stock solutions of the compounds to be tested are prepared at the same time in DMSO. A sample of infected blood and the drug are added to a 96-well plate to a final volume of 200 μΙ and compound concentrations of 1 to 100 μΜ, in order to calculate the IC 50 for each. To reproduce blood bank conditions, the plates were incubated at 4 e C for 24 h. The experiments were repeated three times. Each sample was examined microscopically (OLYMPUS CX41) for the parasite count using the Neubauer chamber.
ENSAYOS DE ACTIVIDAD IN VÍTRO: FORMAS INTRACELULARES. IN VÍTRO ACTIVITY TESTS: INTRACELLULAR FORMS.
Ensayo de amastiqotas Amastiqotas test
Las células Vero se despegaron del frasco de cultivo donde se encontraban adheridos mediante tripsinización. Para ello, se eliminó el medio de cultivo, seguidamente se cubrió la superficie celular con EDTA-tripsina y se incubó en frío durante 5 minutos según la metodología expuesta anteriormente. Tras ello, se pasó a un frasco de fondo cónico de 25 mi de capacidad (Steriling) para centrifugarlos a 800 rpm durante 5 minutos, retirándose el sobrenadante y se cuentan en cámara de Neubauer. Resuspendiendo las células a una concentración de 1 x104 células/ pocilio en medio RPMI para las células Vero, cultivándose en placas de 24 pocilios, en las que previamente habíamos introducido un cristal cubreobjetos redondo de 12 mm en cada pocilio. Para su adherencia, dejamos crecer las células 24 h a 37eC en 5% C02. 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 according to the methodology set forth above. After that, it was transferred to a conical bottom flask of 25 ml capacity (Steriling) to centrifuge them at 800 rpm for 5 minutes, removing the supernatant and counted in Neubauer chamber. Resuspend the cells at a concentration of 1 x 10 4 cells / well in RPMI medium for Vero cells, growing in 24-well plates, in which we had previously introduced a 12 mm round glass cover glass in each well. For adhesion, we let the cells grow 24 h at 37 e C in 5% C0 2 .
Una vez adheridas las células, se infectaron "in vitro" las células Vero con 1 x 105 células/ pocilio de formas tripomastigotas de T. cruzi, obtenidos según la metodología citada anteriormente. La infección se mantuvo durante 24 horas para que el parásito entre a la célula. Transcurrido este tiempo el medio de cultivo se retiró y se adicionó medio fresco con los productos a ensayar, a las concentraciones necesarias para sacar la IC50 (100 a 1 μΜ). A las 72 horas de la incubación, se procedió a la retirada de los cristales. Una vez sacados los cristales se colocaron en un portaobjetos. Se fijaron con metanol y se dejaron secar. Una vez fijados y secos, se les añadió DPX (Panreac®), medio de montaje para microscopía. Se tiñeron con Giemsa, para ello, inmediatamente antes de su empleo y en tubo de ensayo, se diluyó 0,2 mi de Azur-Eosina-Azul de Metileno solución según Giemsa DC (Código 251338) con 2 mi de tampón Sórensen (dilución 1 :10), se mezcló bien y se cubrió la preparación dejando colorear durante 20 minutos. Se lavó con agua destilada. Se dejó escurrir y secar en posición vertical. Por último se examinó con el objetivo de inmersión y se contó el número de amastigotas intracelulares en un total de 200 células. Once the cells were adhered, the Vero cells were infected "in vitro" with 1 x 10 5 cells / well of trypomastigote forms of T. cruzi, obtained according to the methodology cited above. 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 remove the IC 50 (100 to 1 μΜ). 72 hours after incubation, the crystals were removed. Once the crystals were removed, they were 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, 0.2 ml of Azur-Eosin-Methylene Blue solution was diluted according to Giemsa DC (Code 251338) with 2 ml of Sórensen buffer (dilution 1 : 10), mixed well and covered the preparation leaving coloring 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 amastigotes in a total of 200 cells was counted.
Los datos obtenidos en los ensayos anteriores realizados con los compuestos y complejos de las familias (I), (II), (III), (IV), (V) y (VI) se muestran en la Tabla 5. The data obtained in the previous tests carried out with the compounds and complexes of the families (I), (II), (III), (IV), (V) and (VI) are shown in Table 5.
Tabla 5.- Actividad in vitro, toxicidad e índice de selectividad de los compuestos poliamínicos y de los complejos con metales de transición evaluados en formas extracelulares e intracelulares de Trypanosoma cruzi. Table 5.- In vitro activity, toxicity and selectivity index of polyamine compounds and complexes with transition metals evaluated in extracellular and intracellular forms of Trypanosoma cruzi.
Figure imgf000028_0001
Figure imgf000028_0001
Los resultados representan la media obtenida en cuatro experimentos separados. a. IC50 = concentración requerida para dar el 50% de inhibición, calculada por análisis de regresión lineal a partir de los valores Kc en las concentraciones utilizadas (1 , 10, 25, 50 y 100 μΜ).13 Frente a células Vero después de 72 h de incubación. The results represent the average obtained in four separate experiments. a . IC 50 = concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at the concentrations used (1, 10, 25, 50 and 100 μΜ). 13 Against Vero cells after 72 h of incubation.
Tabla 6.- índice de selectividad de los compuestos poliamínicos y de los complejos con metales de transición evaluados en formas extracelulares e intracelulares de Trypanosoma cruzi.
Figure imgf000029_0001
Table 6.- Selectivity index of polyamine compounds and transition metal complexes evaluated in extracellular and intracellular forms of Trypanosoma cruzi.
Figure imgf000029_0001
Los resultados representan la media obtenida en cuatro experimentos separados.3 índice de selectividad =IC50 Células Vero/IC50formas extracelulares e intracelularesdel parásito. Entre paréntesis: número de veces que el compuesto excede el índice de selectividad de la droga referencia (para formas extracelulares e intracelulares del Trypanosoma cruzi). The results represent the average obtained in four separate experiments. 3 selectivity index = IC 50 Vero cells / IC 50 extracellular and intracellular forms of the parasite. In parentheses: number of times the compound exceeds the selectivity index of the reference drug (for extracellular and intracellular forms of Trypanosoma cruzi).
Estos valores se han comparado con los valores obtenidos para el Benznidazol ya que se trata del fármaco comúnmente utilizado para el tratamiento de esta enfermedad. Los resultados obtenidos indican que en general los compuestos estudiados son menos tóxicos que el Benznidazol frente a células Vero con valores de IC50 entre 10 y 30 veces superiores que el IC50 del compuesto referencia. En algunos casos como para los compuestos X1 2 y X1 3 su actividad es mucho mayor que el compuesto referencia ya que presentan valores de IC50 menores tanto en formas extracelulares como intracelulares. De acuerdo con estos resultados estos compuestos tienen índices de selectividad elevados con valores comprendidos entre 15 y 80. Todo ello indica que dichos compuestos presentan una actividad adecuada para su uso como agentes anti T. cruzi. These values have been compared with the values obtained for Benznidazole since it is the drug commonly used for the treatment of this disease. The results obtained indicate that in general the compounds studied are less toxic than Benznidazole against Vero cells with IC 50 values between 10 and 30 times higher than the IC 50 of the reference compound. In some cases, as for compounds X1 2 and X1 3, their activity is much greater than the reference compound since they have lower IC 50 values in both extracellular and intracellular forms. According to these results, these compounds have high selectivity indices with values between 15 and 80. All this indicates that said compounds have an activity suitable for use as anti T. cruzi agents.
Los datos obtenidos en los ensayos anteriores realizados con los compuestos y complejos de las familias (I-2) y (I I-2) se muestran en la Tabla 7. The data obtained in the previous tests carried out with the compounds and complexes of the families (I-2) and (I I-2) are shown in Table 7.
Tabla 7.- Actividad in vitro, toxicidad e índice de selectividad de los compuestos poliamínicos y de los complejos con metales de transición evaluados en formas extracelulares e intracelulares de Trypanosoma cruzi. IC50 (M )a Toxicidad Table 7.- In vitro activity, toxicity and selectivity index of polyamine compounds and complexes with transition metals evaluated in extracellular and intracellular forms of Trypanosoma cruzi. IC 50 (M) to Toxicity
IC50 IC 50
Compuesto Epimastigote Amastigote Trypomastigote Cel. Vero Compound Epimastigote Amastigote Trypomastigote Cel. Vero
intracelular (MM)b intracellular ( M M) b
Benznidazol 15,8±1 ,1 23,3±4,6 22,4±1 ,9 13,6±0,9 Benznidazole 15.8 ± 1, 1 23.3 ± 4.6 22.4 ± 1, 9 13.6 ± 0.9
X1 40,7±2,1 20,8±1 ,7 19,0±0,8 17,9±3,1  X1 40.7 ± 2.1 20.8 ± 1, 7 19.0 ± 0.8 17.9 ± 3.1
X6 52,5±8,5 21 ,5±2,6 30,0±1 ,4 80,0±7,7  X6 52.5 ± 8.5 21, 5 ± 2.6 30.0 ± 1, 4 80.0 ± 7.7
X9 51 ,6±3,6 41 ,5±8,5 33,2±2,9 1 17,7±6,2  X9 51, 6 ± 3.6 41, 5 ± 8.5 33.2 ± 2.9 1 17.7 ± 6.2
X26 93,8±9,1 34,9±2,3 23,8±0,5 80,2±9,0  X26 93.8 ± 9.1 34.9 ± 2.3 23.8 ± 0.5 80.2 ± 9.0
X27 7,3±0,8 7,0±0,9 5,7±0,8 71 ,6±6,3  X27 7.3 ± 0.8 7.0 ± 0.9 5.7 ± 0.8 71, 6 ± 6.3
Los resultados representan la media obtenida en cuatro experimentos separados. a. IC50 = concentración requerida para dar el 50% de inhibición, calculada por análisis de regresión lineal a partir de los valores Kc en las concentraciones utilizadas (1 , 10, 25, 50 y 100 μΜ).13 Frente a células Vero después de 72 h de incubación. The results represent the average obtained in four separate experiments. a . IC 50 = concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at the concentrations used (1, 10, 25, 50 and 100 μΜ). 13 Against Vero cells after 72 h of incubation.
Tabla 8.- índice de selectividad de los compuestos poliamínicos y de los complejos con metales de transición evaluados en formas extracelulares e intracelulares de Trypanosoma cruzi. Table 8.- Selectivity index of polyamine compounds and transition metal complexes evaluated in extracellular and intracellular forms of Trypanosoma cruzi.
Figure imgf000030_0001
Figure imgf000030_0001
Los resultados representan la media obtenida en cuatro experimentos separados.3 índice de selectividad =IC50 Células Vero/IC50formas extracelulares e intracelularesdel parásito. Entre paréntesis: número de veces que el compuesto excede el índice de selectividad de la droga referencia (para formas extracelulares e intracelulares del Trypanosoma cruzi). The results represent the average obtained in four separate experiments. 3 selectivity index = IC 50 Vero cells / IC 50 extracellular and intracellular forms of the parasite. In parentheses: number of times the compound exceeds the selectivity index of the reference drug (for extracellular and intracellular forms of Trypanosoma cruzi).
Estos valores se han comparado con los valores obtenidos para el Benznidazol ya que se trata del fármaco comúnmente utilizado para el tratamiento de esta enfermedad. Los resultados obtenidos indican que en general los compuestos estudiados son menos tóxicos que el Benznidazol frente a células Vero con valores de IC50 entre 6-9 veces superiores que el IC50 del compuesto referencia. En algunos casos, como por ejemplo el X27 su actividad es mucho mayor que la del compuesto de referencia ya que presentan valores de IC50 menores tanto en formas extracelulares como intracelulares. De acuerdo con estos resultados algunos de estos compuestos como el X27 tienen índices de selectividad elevados con valores comprendidos entre 17 y 21 . Todo ello indica que dichos compuestos presentan una actividad adecuada para su uso como agentes anti T. cruzi. These values have been compared with the values obtained for Benznidazole since it is the drug commonly used for the treatment of this disease. The results obtained indicate that in general the compounds studied are less toxic than Benznidazole against Vero cells with IC 50 values between 6-9 times higher than the IC 50 of the reference compound. In some cases, as per For example, the X27 activity is much higher than that of the reference compound since they have lower IC 50 values in both extracellular and intracellular forms. According to these results some of these compounds such as X27 have high selectivity indices with values between 17 and 21. All this indicates that said compounds have an activity suitable for use as anti T. cruzi agents.

Claims

REIVINDICACIONES
1 . Uso de un compuesto poliamínico de fórmula general (I) one . Use of a polyamine compound of general formula (I)
Figure imgf000032_0001
Figure imgf000032_0001
(I)  (I)
donde: R R2 y R3 son iguales o diferentes y representan un grupo seleccionado de la lista que comprende hidrógeno, alquilo (C1 -C5) , -NR'R", halógeno, alcoxilo (C1 -C5) y -COOR'"; o Ri y R3 representan un grupo pineno o quinolina fusionado al anillo de piridina; R', R" y R"\ son iguales o diferentes y representan un hidrógeno o un grupo alquilo (C1 -C5) ; y donde N-R-N representa los grupos: where: RR 2 and R 3 are the same or different and represent a group selected from the list comprising hydrogen, (C 1 -C5) alkyl, -NR'R ", halogen, (C 1 -C5) alkoxy and -COOR '"; or Ri and R 3 represent a pinene or quinoline group fused to the pyridine ring; R ', R "and R" \ are the same or different and represent a hydrogen or a (C 1 -C 5) alkyl group; and where NRN represents the groups:
Figure imgf000032_0002
o cualquiera de sus isómeros y/o sales farmacéuticamente aceptables, para la elaboración de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias.
Figure imgf000032_0002
or any of its isomers and / or pharmaceutically acceptable salts, for the preparation of a medicament for the treatment and / or prevention of parasitic diseases.
2. Uso según la reivindicación 1 , donde el compuesto es de fórmula general (II): 2. Use according to claim 1, wherein the compound is of general formula (II):
Figure imgf000033_0001
donde R1 5 R2 y R3 se han definido en la reivindicación 1 . 3. Uso según la reivindicación 1 , donde el compuesto es de fórmula (III)
Figure imgf000033_0001
wherein R 1 5 R 2 and R3 have been defined in claim 1. 3. Use according to claim 1, wherein the compound is of formula (III)
Figure imgf000033_0002
donde R1 5 R2 y R3 se han definido en la reivindicación 1 .
Figure imgf000033_0002
wherein R 1 5 R 2 and R 3 have been defined in claim 1.
Uso según cualquiera de las reivindicaciones anteriores, donde R representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3 y - COOCH2CH3. Use according to any of the preceding claims, wherein R represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 and - COOCH 2 CH 3 .
Uso según cualquiera de las reivindicaciones anteriores, donde R2 y R3, son iguales o diferentes y representan un hidrógeno o un grupo metilo. Use according to any of the preceding claims, wherein R 2 and R 3 , are the same or different and represent a hydrogen or a methyl group.
Uso según cualquiera de las reivindicaciones anteriores, donde R2 y R3 son iguales. Use according to any of the preceding claims, wherein R 2 and R 3 are the same.
7. Uso según cualquiera de las reivindicaciones 2 a 6 donde el compuesto es de fórmula (II), Ri es -N(CH3)2 y R2 y R3 son hidrógeno; o R1 es -OCH3 y R2 y R3 metilo. 7. Use according to any of claims 2 to 6 wherein the compound is of formula (II), Ri is -N (CH 3 ) 2 and R 2 and R 3 are hydrogen; or R 1 is -OCH 3 and R 2 and R 3 methyl.
8. Uso según cualquiera de las reivindicaciones 3 a 6, donde el compuesto es de fórmula (III) y R R2 y R3 son hidrógeno. 8. Use according to any of claims 3 to 6, wherein the compound is of formula (III) and RR 2 and R3 are hydrogen.
9. Uso según la reivindicación 1 , donde el compuesto se selecciona entre 2,2'- (2S,2'S)-2,2,-bipirrolidina-1 ,1 '-diilbis(metileno)bis(N,N-dimetilpiridina-4-amina); DL- N1 ,N2-dimetil-N1 ,N2-bis(piridin-2-ilmetil)ciclohexano-1 ,2-diamina. 9. Use according to claim 1, wherein the compound is selected from 2,2'- (2S, 2'S) -2,2 , -bipyrrolidine-1, 1'-diylbis (methylene) bis (N, N-dimethylpyridine-4 -amine); DL-N1, N2-dimethyl-N1, N2-bis (pyridin-2-ylmethyl) cyclohexane-1,2-diamine.
10. Uso de un complejo metálico que comprende un ligando tetradentado que consiste en un compuesto de fórmula general (I) según se describe en cualquiera de las reivindicaciones 1 a 9, para la preparación de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias. 10. Use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I) as described in any one of claims 1 to 9, for the preparation of a medicament for the treatment and / or prevention of Parasitic diseases.
1 1 . Uso según la reivindicación 10, donde los metales se seleccionan de la lista que comprende Mn, Fe, Co, Ni, Cu y Zn. eleven . Use according to claim 10, wherein the metals are selected from the list comprising Mn, Fe, Co, Ni, Cu and Zn.
12. Uso según cualquiera de las reivindicaciones 10 u 1 1 , donde los metales son metales divalentes. 12. Use according to any of claims 10 or 1, wherein the metals are divalent metals.
13. Uso según la reivindicación anterior, donde los complejos son de fórmula general13. Use according to the preceding claim, wherein the complexes are of general formula
(V) o (VI): (V) or (VI):
Figure imgf000034_0001
Figure imgf000034_0001
(V) (VI) donde: Ri a R3 se han definido en cualquiera de las reivindicaciones 1 a 9; M es un metal divalente según se describe en la reivindicación 12; y X es un contraanión. (V) (VI) wherein: Ri to R 3 have been defined in any one of claims 1 to 9; M is a divalent metal as described in claim 12; and X is a counter-anion.
14. Uso según la reivindicación anterior, donde el contraanión se selecciona de la lista que comprende CF3S03 ~, Cl", Br", CI04 ", PF6 " y BF4 ". 14. Use according to the preceding claim, wherein the counteranion is selected from the list comprising CF 3 S0 3 ~ , Cl " , Br " , CI0 4 " , PF 6 " and BF 4 " .
15. Uso según la reivindicación anterior donde el contraanión CF3S03 ~. 15. Use according to the preceding claim wherein the CF 3 S0 3 ~ counter-anion.
16. Uso según la reivindicación 10, donde los complejos se seleccionan de entre: 16. Use according to claim 10, wherein the complexes are selected from:
[{2,2'-(2R,2,R)-2,2,-bipirrolidina-1 ,1 '-diilbis(metileno)bis(N,N-dimetilpiridina-4- amina)}Mn"](CF3S03)2; [{(2S,2'S)-1 ,1 '-bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)- 2,2'-bipirrolidina}Mn"](CF3S03)2; [{(1 R,2R)-N1 ,N2-bis((4-metoxi-3,5-dimetilpiridin-2- il)metil)-N1 ,N2-dimetilciclohexano-1 ,2-diamina}Mn"](CF3S03)2.. [{2,2 '- (2R, 2 , R) -2,2 , -bipyrrolidine-1,1'-diylbis (methylene) bis (N, N-dimethylpyridine-4-amine)} Mn "] (CF 3 S0 3 ) 2 ; [{(2S, 2'S) -1, 1'-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) - 2,2'-bipyrrolidine} Mn "] (CF 3 S0 3 ) 2 ; [{(1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2- yl) methyl) -N1, N2-dimethylcyclohexane-1,2-diamine} Mn "] (CF 3 S0 3 ) 2 ..
17. Uso según cualquiera de las reivindicaciones anteriores, donde la enfermedad parasitaria es una enfermedad parasitaria protozoaria. 17. Use according to any of the preceding claims, wherein the parasitic disease is a protozoan parasitic disease.
18. Uso según cualquiera de las reivindicaciones anteriores, donde los parásitos pertenecen a la familia Trypanosomatidae. 18. Use according to any of the preceding claims, wherein the parasites belong to the Trypanosomatidae family.
19. Uso según la reivindicación anterior, donde los parásitos pertenecen a la especie Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis o Leishmania donovani. 19. Use according to the preceding claim, wherein the parasites belong to the species Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis or Leishmania donovani.
20. Uso según cualquiera de las reivindicaciones anteriores, donde la enfermedad es leishmaniasis o tripanosomiasis. 20. Use according to any of the preceding claims, wherein the disease is leishmaniasis or trypanosomiasis.
21 . Uso según la reivindicación anterior, donde la tripanosomiasis es la enfermedad de Chagas. twenty-one . Use according to the preceding claim, where trypanosomiasis is Chagas disease.
22. Uso de los compuestos de fórmula general (I) descritos en cualquiera de las reivindicaciones 1 a 9, o de los complejos descritos en cualquiera de las reivindicaciones 10 a 16, para la elaboración de un medicamento. 22. Use of the compounds of general formula (I) described in any of claims 1 to 9, or of the complexes described in any of claims 10 to 16, for the preparation of a medicament.
23. Composición farmacéutica que comprende al menos un compuesto descrito según cualquiera de las reivindicaciones 1 a 9 o un complejo según cualquiera de las reivindicaciones 10 a 16, junto con al menos un vehículo farmacéuticamente aceptable. 23. Pharmaceutical composition comprising at least one compound described according to any one of claims 1 to 9 or a complex according to any of claims 10 to 16, together with at least one pharmaceutically acceptable carrier.
24. Complejo de fórmula [{(1 R,2R)-N1 ,N2-bis((4-metoxi-3,5-dimetilpiridin-2-il)metil)- N1 ,N2-dimetilciclohexano-1 ,2-diamina}Mn"](CF3S03)2- 24. Complex of formula [{(1 R, 2R) -N1, N2-bis ((4-methoxy-3,5-dimethylpyridin-2-yl) methyl) - N1, N2-dimethylcyclohexane-1,2-diamine} Mn "] (CF 3 S0 3 ) 2-
25. Uso de un compuesto de fórmula general (I-2): 25. Use of a compound of general formula (I-2):
Figure imgf000036_0001
Figure imgf000036_0001
donde: R4 y R5 son iguales o diferentes, y representan un hidrógeno, un grupo alquilo (C1 -C5) o un grupo -CH2-Cy; R6 representa un hidrógeno, un halógeno o un grupo alquilo (CrC5) ; R7 y R9 son iguales o diferentes, y representan un hidrógeno o un grupo alquilo (CrC5) ; R8 representa un grupo seleccionado de la lista que comprende hidrógeno, -NR'R", halógeno, alcoxilo (CrC5) , -N02 y -COOR'" ; R', R" y R'", son iguales o diferentes y representan un hidrógeno o un grupo alquilo (C C5) ; y Cy representa un heteroarilo, where: R 4 and R 5 are the same or different, and represent a hydrogen, a (C1-C5) alkyl group or a -CH 2 -Cy group; R 6 represents a hydrogen, a halogen or an alkyl group (CrC 5 ); R 7 and R 9 are the same or different, and represent a hydrogen or an alkyl group (CrC 5 ); R 8 represents a group selected from the list comprising hydrogen, -NR'R ", halogen, alkoxy (CrC 5 ), -N0 2 and -COOR '"; R ', R "and R'", are the same or different and represent a hydrogen or an alkyl group (CC 5 ); and Cy represents a heteroaryl,
o cualquiera de sus isómeros y/o sales farmacéuticamente aceptables para la elaboración de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias.  or any of its isomers and / or pharmaceutically acceptable salts for the preparation of a medicament for the treatment and / or prevention of parasitic diseases.
26. Uso según la reivindicación 25, donde R4 y R5 son iguales o diferentes, y representan un hidrógeno, un metilo o un grupo -CH2-Cy; donde Cy es piridina. 26. Use according to claim 25, wherein R 4 and R 5 are the same or different, and represent a hydrogen, a methyl or a -CH 2 -Cy group; where Cy is pyridine.
27. Uso según cualquiera de las reivindicaciones 25 o 26, donde R4 y R5 son iguales. 27. Use according to any of claims 25 or 26, wherein R 4 and R5 are the same.
28. Uso según cualquiera de las reivindicaciones 25 a 27, donde R6 representa hidrógeno, Cl, F o metilo. 28. Use according to any of claims 25 to 27, wherein R 6 represents hydrogen, Cl, F or methyl.
29. Uso según cualquiera de las reivindicaciones 25 a 28, donde R7 y R9 son iguales o diferentes, y representan hidrógeno o metilo. 29. Use according to any of claims 25 to 28, wherein R 7 and R 9 are the same or different, and represent hydrogen or methyl.
30. Uso según cualquiera de las reivindicaciones 25 a 29, donde R7 y Rg son iguales. 30. Use according to any of claims 25 to 29, wherein R 7 and Rg are the same.
31 . Uso según cualquiera de las reivindicaciones 25 a 30, donde R8 representa un grupo seleccionado de la lista que comprende hidrógeno, -N(CH3)2, Cl, -OCH3, N02 y -COOCH2CH3. 31. Use according to any of claims 25 to 30, wherein R 8 represents a group selected from the list comprising hydrogen, -N (CH 3 ) 2 , Cl, -OCH 3 , N0 2 and -COOCH 2 CH 3 .
32. Uso según cualquiera de las reivindicaciones 25 a 31 , donde R4 y R5 son iguales y representan un metilo o un grupo -CH2-piridina; y R6, R7, R8 y R9 son hidrógeno. 32. Use according to any of claims 25 to 31, wherein R 4 and R 5 are the same and represent a methyl or a -CH 2 -pyridine group; and R 6 , R 7 , R 8 and R 9 are hydrogen.
33. Uso según la reivindicación 25, donde el compuesto se selecciona entre 1 ,4- dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano; y 1 ,4,7-tris(piridin-2-ilmetil)-1 ,4,7- triazonano. 33. Use according to claim 25, wherein the compound is selected from 1,4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonan; and 1, 4,7-tris (pyridin-2-ylmethyl) -1, 4,7-triazonan.
34. Uso de un complejo metálico que comprende un ligando tetradentado que consiste en un compuesto de fórmula general (I-2) según se describe en cualquiera de las reivindicaciones 25 a 33 para la preparación de un medicamento para el tratamiento y/o la prevención de enfermedades parasitarias. 34. Use of a metal complex comprising a tetradentate ligand consisting of a compound of general formula (I-2) as described in any of claims 25 to 33 for the preparation of a medicament for treatment and / or prevention of parasitic diseases.
35. Uso según la reivindicación 34, donde los metales se seleccionan de la lista que comprende Mn, Fe, Co, Ni, Cu y Zn. 35. Use according to claim 34, wherein the metals are selected from the list comprising Mn, Fe, Co, Ni, Cu and Zn.
36. Uso según cualquiera de las reivindicaciones 34 o 35, donde los metales son metales divalentes. 36. Use according to any of claims 34 or 35, wherein the metals are divalent metals.
37. Uso según la reivindicación anterior, donde los complejos son de fórmula general (II-2): 37. Use according to the preceding claim, wherein the complexes are of the general formula (II-2):
Figure imgf000038_0001
Figure imgf000038_0001
( 11-2)  (11-2)
Donde: R4 a R9 se han definido en cualquiera de las reivindicaciones 1 a 9; M es un metal divalente según se describe en la reivindicación 12; y X es un contraanión. Where: R 4 to R 9 have been defined in any one of claims 1 to 9; M is a divalent metal as described in claim 12; and X is a counter-anion.
38. Uso según la reivindicación anterior, donde el contraanión se selecciona de la lista que comprende CF3S03 ~, Cl", Br", CI04 ", PF6 " y BF4 ". 38. Use according to the preceding claim, wherein the counteranion is selected from the list comprising CF 3 S0 3 ~ , Cl " , Br " , CI0 4 " , PF 6 " and BF 4 " .
39. Uso según la reivindicación anterior donde el contraanión CF3S03 ~. 39. Use according to the preceding claim wherein the CF 3 S0 3 ~ counter-anion.
40. Uso según la reivindicación 34, donde los complejos se seleccionan de entre: 40. Use according to claim 34, wherein the complexes are selected from:
[{1 ,4-dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano}Fe"](CF3S03)2; [{1 ,4-dimetil-7- (piridin-2-ilmetil)-1 ,4,7-triazonano}Mn"](CF3S03)2; y [{1 ,4-dimetil-7-(piridin-2-ilmetil)- 1 ,4,7-triazonano}Cu"](CF3S03)2. [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano} Fe "] (CF 3 S0 3 ) 2 ; [{1, 4-dimethyl-7- (pyridin- 2-ylmethyl) -1, 4,7-triazonan} Mn "] (CF 3 S0 3 ) 2 ; and [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1,4,7-triazonano} Cu "] (CF 3 S0 3 ) 2 .
41 . Uso según cualquiera de las reivindicaciones 25 a 40, donde la enfermedad parasitaria es una enfermedad parasitaria protozoaria. 41. Use according to any of claims 25 to 40, wherein the parasitic disease is a protozoan parasitic disease.
42. Uso según cualquiera de las reivindicaciones 25 a 41 , donde los parásitos pertenecen a la familia Trypanosomatidae. 42. Use according to any of claims 25 to 41, wherein the parasites belong to the Trypanosomatidae family.
43. Uso según la reivindicación anterior, donde los parásitos pertenecen a la especie Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis o Leishmania donovani. 43. Use according to the preceding claim, wherein the parasites belong to the species Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum, Leishmania brazilensis or Leishmania donovani.
44. Uso según cualquiera de las reivindicaciones 25 a 43, donde la enfermedad es leishmaniasis o tripanosomiasis. 44. Use according to any of claims 25 to 43, wherein the disease is leishmaniasis or trypanosomiasis.
45. Uso según cualquiera de las reivindicaciones anteriores, donde la tripanosomiasis es la enfermedad de Chagas. 45. Use according to any of the preceding claims, wherein trypanosomiasis is Chagas disease.
46. Uso de los compuestos de fórmula general (1-2) descritos en cualquiera de las reivindicaciones 25 a 34, o de los complejos descritos en cualquiera de las reivindicaciones 35 a 41 , para la elaboración de un medicamento. 46. Use of the compounds of general formula (1-2) described in any of claims 25 to 34, or of the complexes described in any of claims 35 to 41, for the preparation of a medicament.
47. Composición farmacéutica que comprende al menos un compuesto descrito según cualquiera de las reivindicaciones 1 a 9 o un complejo según cualquiera de las reivindicaciones 10 a 16, junto con al menos un vehículo farmacéuticamente aceptable. 47. Pharmaceutical composition comprising at least one compound described according to any one of claims 1 to 9 or a complex according to any one of claims 10 to 16, together with at least one pharmaceutically acceptable carrier.
48. Complejo [{1 ,4-dimetil-7-(piridin-2-ilmetil)-1 ,4,7-triazonano}Cu"](CF3S03)2. 48. Complex [{1, 4-dimethyl-7- (pyridin-2-ylmethyl) -1, 4,7-triazonano} Cu "] (CF 3 S0 3 ) 2.
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FR2868320A1 (en) * 2004-03-31 2005-10-07 Centre Nat Rech Scient CONTRAST AGENT FOR MAGNETIC RESONANCE IMAGING

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FR2868320A1 (en) * 2004-03-31 2005-10-07 Centre Nat Rech Scient CONTRAST AGENT FOR MAGNETIC RESONANCE IMAGING

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