BR102020019121B1 - METALOAZASTEROL COMPOUND, DELTA 24-STEROL METHYLTRANSFERASE INHIBITOR, PHARMACEUTICAL COMPOSITION AND USE OF THE COMPOUND - Google Patents
METALOAZASTEROL COMPOUND, DELTA 24-STEROL METHYLTRANSFERASE INHIBITOR, PHARMACEUTICAL COMPOSITION AND USE OF THE COMPOUND Download PDFInfo
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- BR102020019121B1 BR102020019121B1 BR102020019121-7A BR102020019121A BR102020019121B1 BR 102020019121 B1 BR102020019121 B1 BR 102020019121B1 BR 102020019121 A BR102020019121 A BR 102020019121A BR 102020019121 B1 BR102020019121 B1 BR 102020019121B1
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- sterol
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Abstract
COMPOSTO METALOAZASTEROL INIBIDOR DA ENZIMA DELTA 24-ESTEROL METILTRANSFERASE, COMPOSIÇÃO FARMACÊUTICA E USO DO COMPOSTO. A presente invenção se refere a um novo composto, diclorobis(22- hidrazona-imidazolina-2-il-col-5-en-3β-ol)zinco(II), que consiste em um metaloazasterol resultante da complexação de zinco com duas moléculas de 22-hidrazona-imidazolina-2-il-col-5-en-3β-ol (H3) que atua inibindo seletivamente a enzima delta 24-esterol metiltransferase (24-EMT) essencial para síntese de ergosterol e outros esteróis 24-alquilados de membrana em parasitas e fungos, sendo, portanto, particularmente útil no tratamento de doenças parasitárias, tais como leishmaniose e doença de Chagas, e doenças fúngicas. Outros aspectos da presente invenção consistem em uma composição farmacêutica que compreende o novo composto, bem como o uso desteMETALOAZASTEROL COMPOUND, DELTA 24-STEROL METHYLTRANSFERASE INHIBITOR, PHARMACEUTICAL COMPOSITION AND USE OF THE COMPOUND. The present invention relates to a new compound, dichlorobis(22-hydrazone-imidazoline-2-yl-col-5-en-3β-ol)zinc(II), which consists of a metalloazasterol resulting from the complexation of zinc with two molecules of 22-hydrazone-imidazoline-2-yl-col-5-en-3β-ol (H3) that acts by selectively inhibiting the enzyme delta 24-sterol methyltransferase (24-EMT) essential for the synthesis of ergosterol and other 24-alkylated sterols membrane in parasites and fungi, and is therefore particularly useful in the treatment of parasitic diseases, such as leishmaniasis and Chagas disease, and fungal diseases. Other aspects of the present invention consist of a pharmaceutical composition comprising the new compound, as well as the use of this
Description
[0001] A presente invenção se refere a um novo composto de metaloazasterol, diclorobis(22-hidrazona-imidazolina-2-il-col-5-en-3β-ol)zinco(II), resultante da complexação de zinco com duas moléculas de 22-hidrazona-imidazolina-2-il-col-5-en- 3β-ol (H3) que atua inibindo seletivamente a enzima Δ24-esterol metiltransferase (24EMT) essencial para síntese de ergosterol e outros esteróis 24-alquilados de membrana de parasitas e fungos, sendo particularmente útil no tratamento de doenças parasitárias, tais como leishmaniose e doença de Chagas, e doenças fúngicas. Outros aspectos da presente invenção consistem em uma composição farmacêutica que compreende o novo composto, bem como o uso deste.[0001] The present invention relates to a new metalloazasterol compound, dichlorobis(22-hydrazone-imidazolin-2-yl-col-5-en-3β-ol)zinc(II), resulting from the complexation of zinc with two molecules of 22-hydrazone-imidazoline-2-yl-col-5-en-3β-ol (H3) that acts by selectively inhibiting the enzyme Δ24-sterol methyltransferase (24EMT) essential for the synthesis of ergosterol and other 24-alkylated membrane sterols of parasites and fungi, being particularly useful in the treatment of parasitic diseases, such as leishmaniasis and Chagas disease, and fungal diseases. Other aspects of the present invention consist of a pharmaceutical composition comprising the new compound, as well as the use thereof.
[0002] Doenças parasitárias e fúngicas constituem um problema de saúde pública mundial. Os tratamentos mais tradicionais utilizados no combate dessas patologias foram desenvolvidos há décadas e apresentam alta toxicidade associada a graves efeitos colaterais, além de custos frequentemente elevados e difícil administração, sobretudo nas circunstâncias em que devem ocorrer em longo prazo. Essa situação revela uma demanda urgente pelo desenvolvimento de novos compostos, mais eficazes e ativos contra cepas já resistentes, mas que possuam níveis de toxicidade inferiores aos compostos conhecidos.[0002] Parasitic and fungal diseases are a global public health problem. The more traditional treatments used to combat these pathologies were developed decades ago and present high toxicity associated with serious side effects, in addition to often high costs and difficult administration, especially in circumstances where they must occur in the long term. This situation reveals an urgent demand for the development of new compounds, more effective and active against strains that are already resistant, but which have lower toxicity levels than the known compounds.
[0003] Os compostos azólicos, tradicionalmente utilizados no combate de infecções causadas por fungos e protozoários, atuam na rota de biossíntese de esteróis, em enzimas comuns tanto à formação de esteróis de membrana desses parasitas (ergosterol) como de mamíferos (colesterol), o que resulta em toxicidade elevada e, consequentemente, efeitos colaterais indesejados.[0003] The azole compounds, traditionally used to combat infections caused by fungi and protozoa, act in the sterol biosynthesis route, in enzymes common to both the formation of membrane sterols of these parasites (ergosterol) and of mammals (cholesterol), the which results in high toxicity and, consequently, unwanted side effects.
[0004] Nesse sentido, fármacos que atuam inibindo seletivamente a rota biossintética do ergosterol se mostram uma opção terapêutica menos tóxica e mais eficiente, uma vez que esse esterol é constituinte apenas de membranas de plantas, protozoários e fungos, não estando presente em células de mamíferos.[0004] In this sense, drugs that act by selectively inhibiting the biosynthetic route of ergosterol are shown to be a less toxic and more efficient therapeutic option, since this sterol is a constituent only of membranes of plants, protozoa and fungi, not being present in cells of mammals.
[0005] Compostos inibidores seletivos da biossíntese dos esteróis de membrana desses microrganismos podem ter como um dos alvos a enzima Δ24-esterol metiltransferase (24-EMT ou 24-SMT em inglês), uma enzima da biossíntese de ergosterol restrita a plantas, protozoários e fungos.[0005] Selective inhibitor compounds of the membrane sterol biosynthesis of these microorganisms may have as one of the targets the enzyme Δ24-sterol methyltransferase (24-EMT or 24-SMT), an ergosterol biosynthesis enzyme restricted to plants, protozoa and fungi.
[0006] Dentre as moléculas conhecidas por atuarem como inibidores da 24-EMT, estão os azasteróis, que atuam inibindo seletivamente a enzima Δ24-esterol metiltransferase, apresentando, portanto, menor toxicidade para mamíferos. Compostos dessa classe mostraram efeitos antimicrobianos contra Trypanosoma cruzi, Leishmania spp. (J. Med. Chem. 2003, 46, 22, 4714-4727), Pneumocystis spp. (Antimicrob. Agents Chemother. 1997; 41:1428-1432), Paracoccidioides brasiliensis (Antimicrob. Agents Chemother. 2003, 47:2966-2970), Sporothrix spp (Front Microbiol. 2016 Mar 11; 7:311), entre outros agentes patogênicos.[0006] Among the molecules known to act as inhibitors of 24-EMT, are the azasterols, which act by selectively inhibiting the enzyme Δ24-sterol methyltransferase, presenting, therefore, less toxicity to mammals. Compounds of this class showed antimicrobial effects against Trypanosoma cruzi, Leishmania spp. (J. Med. Chem. 2003, 46, 22, 4714-4727 ), Pneumocystis spp. (Antimicrob. Agents Chemother. 1997; 41:1428-1432), Paracoccidioides brasiliensis (Antimicrob. Agents Chemother. 2003, 47:2966-2970), Sporothrix spp (Front Microbiol. 2016 Mar 11; 7:311), among other agents pathogens.
[0007] Magaraci, F. et al. em J. Med. Chem. 2003, 46, 22, 4714-4727, avaliaram a atividade de azasteróis sobre T. cruzi e Leishmania spp. mostrando que alguns, por inibição da 24-EMT, atuam alterando a composição do esterol de membrana e possivelmente a proliferação desses parasitas. Há ainda diversos outros estudos que discutem o mecanismo dos azasteróis como inibidores da 24-EMT sobre diferentes espécies de Trypanossoma spp., Leishmania spp. e Plasmodium spp.[0007] Magaraci, F. et al. in J Med. Chem. 2003, 46, 22, 4714-4727, evaluated the activity of azasterols on T. cruzi and Leishmania spp. showing that some, by inhibiting 24-EMT, act by altering the membrane sterol composition and possibly the proliferation of these parasites. There are also several other studies that discuss the mechanism of azasterols as 24-EMT inhibitors on different species of Trypanosoma spp., Leishmania spp. and Plasmodium spp.
[0008] Dentre os azasteróis, a molécula 22-hidrazona-imidazolina-2-il-col-5-en- 3β-ol, denominada H3, foi desenhada para ser um inibidor específico e reversível da 24EMT. Visbal, G. et al. demonstraram a ação antiproliferativa de H3 contra o fungo dimórfico Paracoccidioides brasiliensis (J. Steroids. 2011,76, 1069-1081 e H. Curr. Drug Targets: Infect Disorders. 2005, 5, 211-226) e Vivas, J. et al. sobre o fungo Cryptococcus neoformans (E. Invest. Clin. 2011, 52, 312-322). Mais precisamente, foi verificado que o composto afeta a biossíntese do ergosterol, inibindo seletivamente a enzima Δ24-esterol metiltransferase no fungo P. brasiliensis. O processo de síntese de H3 já é conhecido do estado da técnica.
[0009] Navarro et al. em Transition Met. Chem. 40, 707-713 (2015) propuseram modificações estruturais em hidrazonas, sendo uma delas a molécula de H3, (nomeada no artigo como Him2) por meio da coordenação com o átomo de platina (Pt). O intuito foi o de verificar sua atividade contra T. cruzi e Leishmania mexicana, uma vez que estudos prévios realizados por Visbal et al. em J Inorg Biochem 102:547-554 (2008) já haviam demonstrado que complexos de hidrazonas combinadas ao átomo de platina poderiam ter efeito sobre L. mexicana.[0009] Navarro et al. at Transition Met. Chem. 40, 707-713 (2015) proposed structural modifications in hydrazones, one of which is the H3 molecule, (named in the article as Him2) through coordination with the platinum atom (Pt). The aim was to verify its activity against T. cruzi and Leishmania mexicana, since previous studies carried out by Visbal et al. in J Inorg Biochem 102:547-554 (2008) had already demonstrated that hydrazone complexes combined with the platinum atom could have an effect on L. mexicana.
[0010] A abordagem com o uso da platina foi de realizar a síntese com uma relação estequiométrica 2:1 por analogia aos complexos de cisplatina reportados como agentes antineoplásicos. Na literatura, há uma ampla variedade de complexos de platina (II) com essa relação estequiométrica que apresentam uma geometria planar quadrada. É conhecido ainda que relações estequiométricas de 3:1 ou 4:1 são mais difíceis de serem obtidas devido a efeitos estéricos na geometria planar quadrada, que é a preferencial da platina (II).
[0011] Foram, então, realizados estudos da atividade biológica em promastigotas da L. mexicana e epimastigotas de T cruzi, agentes causadores, respectivamente, de Leishmaniose e Mal de Chagas, doenças classificadas como negligenciadas. Foi demonstrado que as formas promastigota de L. mexicana e epimastigota de T. cruzi foram susceptíveis ao H3 e seu complexo metálico de platina trans-[Pt(H3)2(Cl)2].[0011] Studies were then carried out on the biological activity in promastigotes of L. mexicana and epimastigotes of T cruzi, causative agents, respectively, of Leishmaniasis and Chagas' disease, diseases classified as neglected. The promastigote forms of L. mexicana and epimastigote of T. cruzi were shown to be susceptible to H3 and its platinum metal complex trans-[Pt(H3)2(Cl)2].
[0012] Ambos os compostos mostraram uma concentração inibitória mínima (CIM) de 10 μM para os modelos biológicos utilizados. Porém, a uma concentração mais baixa, de 5 μM, apenas o H3 manteve uma inibição de 100%, enquanto o complexo metálico de platina trans-[Pt(H3)2(Cl)2] mostrou uma inibição da proliferação celular de 50% sobre o parasita T. cruzi. Com isso verificou-se que o átomo de platina não teve o efeito esperado de aumentar a atividade de H3, uma vez que o complexo metálico não apresentou diferença significativa em comparação à atividade de H3. Assim, foi concluído que não necessariamente um metaloazasterol apresentaria atividade superior ao azasterol que lhe deu origem.[0012] Both compounds showed a minimum inhibitory concentration (MIC) of 10 μM for the biological models used. However, at a lower concentration of 5 μM, only H3 maintained a 100% inhibition, while the platinum metal complex trans-[Pt(H3)2(Cl)2] showed a 50% inhibition of cell proliferation. on the T. cruzi parasite. Thus, it was verified that the platinum atom did not have the expected effect of increasing the activity of H3, since the metallic complex did not present a significant difference in comparison to the activity of H3. Thus, it was concluded that not necessarily a metalloazasterol would present superior activity to the azasterol that gave rise to it.
[0013] Por outro lado, estudos de citotoxicidade sobre células linfocitárias humanas mostraram que ambos compostos, a uma concentração final de 10 μM, apresentaram pouco ou nenhum efeito sobre as células linfocitárias humanas.[0013] On the other hand, cytotoxicity studies on human lymphocyte cells showed that both compounds, at a final concentration of 10 μM, had little or no effect on human lymphocyte cells.
[0014] Embora não haja estudos na literatura que correlacionem seguramente os metais de transição com a atividade biológica, são conhecidos alguns estudos que indicam que metais como platina, ouro e cobre têm preferência por diferentes grupos funcionais. Por exemplo, o ouro tende a se coordenar ao átomo de enxofre, de modo que um de seus alvos é a enzima tioredoxina redutase (TrxR), enquanto a platina tem preferência por átomos de nitrogênio, sendo amplamente reconhecido que os compostos de platina interagem com o DNA pelas suas bases nitrogenadas.[0014] Although there are no studies in the literature that securely correlate transition metals with biological activity, some studies are known that indicate that metals such as platinum, gold and copper have a preference for different functional groups. For example, gold tends to coordinate with the sulfur atom, so one of its targets is the enzyme thioredoxin reductase (TrxR), while platinum has a preference for nitrogen atoms, and it is widely recognized that platinum compounds interact with DNA by its nitrogenous bases.
[0015] Com efeito, as principais evidências para esta interação incluem os seguintes aspectos: (a) indução do crescimento de filamentos em bactérias; (b) indução de lise em bactérias lisogênicas; (c) inibição preferencial da síntese de DNA em relação a síntese de RNA e proteínas em culturas de células; e (d) mutagênese.[0015] Indeed, the main evidences for this interaction include the following aspects: (a) induction of filament growth in bacteria; (b) inducing lysis in lysogenic bacteria; (c) preferential inhibition of DNA synthesis over RNA and protein synthesis in cell cultures; and (d) mutagenesis.
[0016] No artigo “Metal-azasterol complexes: Synthesis, characterization, interaction studies with DNA And TrxR and Biological Evaluation”, (J. Mex. Chem. Soc, México , v. 61, n. 2, p. 146-157, 2017) é apresentado o novo composto azasterol 22- hidrazina-imidazolina-2-il-col-5-en-3β-ol (AH3), análogo a H3, e seus derivados resultantes da complexação com ouro (Au) e cobre (Cu), sob as formas de [Au(AH3)Cl] e [Cu(AH3)2(H2O)](NO3)2, respectivamente. O estudo averiguou a ação de AH3 e de seus complexos metálicos em dois alvos moleculares, o DNA e a TrxR.[0016] In the article “Metal-azasterol complexes: Synthesis, characterization, interaction studies with DNA And TrxR and Biological Evaluation”, (J. Mex. Chem. Soc, Mexico , v. 61, n. 2, p. 146-157 , 2017) the new azasterol compound 22-hydrazine-imidazoline-2-yl-col-5-en-3β-ol (AH3), analogous to H3, and its derivatives resulting from complexation with gold (Au) and copper ( Cu), in the forms of [Au(AH3)Cl] and [Cu(AH3)2(H2O)](NO3)2, respectively. The study investigated the action of AH3 and its metal complexes on two molecular targets, DNA and TrxR.
[0017] Os resultados encontrados neste estudo demonstraram que ambos complexos metaloazasteróis foram capazes de interagir com o DNA, provavelmente, por ligações covalentes, e que o complexo com Au foi também capaz de inibir fortemente a TrxR.[0017] The results found in this study demonstrated that both metalloazasterol complexes were able to interact with DNA, probably by covalent bonds, and that the Au complex was also able to strongly inhibit TrxR.
[0018] Além disso, foi pesquisada a atividade dos três compostos contra o fungo Sporothrix spp, causador da principal micose subcutânea na América Latina. Os resultados obtidos mostraram que, dentre os azasteróis analisados, o metaloazasterol [Au(AH3)Cl] teve um melhor efeito antiproliferativo sobre o fungo, tendo a menor concentração inibitória mínima (CIM) na faixa de 0,16 - 0,3 (μM), com efeito superior, inclusive, ao itraconazol, fármaco de referência no tratamento da esporotricose.[0018] In addition, the activity of the three compounds against the fungus Sporothrix spp, which causes the main subcutaneous mycosis in Latin America, was investigated. The results obtained showed that, among the analyzed azasterols, metalloazasterol [Au(AH3)Cl] had a better antiproliferative effect on the fungus, having the lowest minimum inhibitory concentration (MIC) in the range of 0.16 - 0.3 (μM ), with a superior effect, even to itraconazole, the reference drug in the treatment of sporotrichosis.
[0019] Contudo, os resultados acima demonstrados não foram melhores que os já obtidos em 2016 por Borba-Santos et al. em Front. Microbiol., 7:311, p. 1-13, nos quais a molécula isolada de H3 mostrou uma concentração inibitória mínima (CIM) na faixa de 0,15 - 0,1 (μM) quando testada com o mesmo fungo, indicando uma atividade biológica superior de H3 em relação à AH3 e seus derivados metálicos, o que possivelmente ocorre em razão da geometria de coordenação dos compostos metálicos e seus estados de oxidação. Neste mesmo estudo também foi demonstrado que H3 inibe a 24-EMT afetando a homeostase do ergosterol.[0019] However, the results shown above were no better than those already obtained in 2016 by Borba-Santos et al. in Front. Microbiol., 7:311, p. 1-13, in which the isolated H3 molecule showed a minimum inhibitory concentration (MIC) in the range of 0.15 - 0.1 (μM) when tested with the same fungus, indicating a superior biological activity of H3 in relation to AH3 and their metallic derivatives, which possibly occurs due to the coordination geometry of metallic compounds and their oxidation states. In this same study it was also shown that H3 inhibits 24-EMT affecting ergosterol homeostasis.
[0020] Assim, são conhecidos no estado da técnica outros fatores, por exemplo, fisiológicos, bioquímicos e de reconhecimento molecular, que afetam as atividades biológicas de cada complexo metálico de forma diferenciada. Com efeito, questões estéricas (como a seleção de ligantes estericamente compatíveis) e diferentes geometrias de coordenação de cada complexo metálico são particularmente importantes nesta análise, ao passo que ainda diante da interação com a célula, sobretudo no caso de organismos vivos, há variações na atuação da molécula.[0020] Thus, other factors are known in the state of the art, for example, physiological, biochemical and molecular recognition, which affect the biological activities of each metal complex differently. Indeed, steric issues (such as the selection of sterically compatible ligands) and different coordination geometries of each metal complex are particularly important in this analysis, while even in the face of the interaction with the cell, especially in the case of living organisms, there are variations in the molecule action.
[0021] Existe ainda na literatura extenso material acerca dos efeitos do zinco sobre a atividade biológica. De acordo com o artigo “The Biochemical Basis of Zinc Physiology” de B.L.Vallee et al (Physiological Reviews, v. 73, no. 1, Janeiro 1993), o zinco é definido como o único elemento pré, pós e de transição reconhecido como não- tóxico, sendo essencial para a preservação e perpetuação das espécies a longo prazo por meio de seu papel fundamental na síntese, transcrição e tradução da mensagem genética e no curto prazo, como pré-requisito para o anabolismo e catabolismo de todos os alimentos essenciais e seus intermediários, participando como um componente funcionalmente obrigatório na ação de enzimas metabólicas relevantes.[0021] There is still extensive material in the literature about the effects of zinc on biological activity. According to the article “The Biochemical Basis of Zinc Physiology” by B.L.Vallee et al (Physiological Reviews, v. 73, no. 1, January 1993), zinc is defined as the only pre, post and transition element recognized as non-toxic, being essential for the preservation and perpetuation of the species in the long term through its fundamental role in the synthesis, transcription and translation of the genetic message and in the short term, as a prerequisite for the anabolism and catabolism of all essential foods and their intermediates, participating as a functionally obligatory component in the action of relevant metabolic enzymes.
[0022] No que se refere à homeostase, o estudo “Zinc essentiality and toxicity. Biophysical aspects” de Y.Harmaza et al. (Biophysics, 2014, Vol. 59, No. 2, pp. 264-275.) estabelece que a concentração admissível (fisiológica) de zinco no soro humano é de 215 μM, enquanto o nível de Zn2+ intracelular livre na maioria das células é extremamente baixo (<1 nM), ao passo que a célula possui um sistema regulatório sofisticado para manter baixo o zinco intracelular mesmo com elevados níveis extracelulares. Porém, é feita referência a um vasto material sobre os efeitos negativos das concentrações elevadas de zinco nos biossistemas, tanto em condições fisiológicas quanto patológicas, lançando dúvidas sobre o efeito positivo das chamadas concentrações “farmacológicas” de zinco.[0022] With regard to homeostasis, the study “Zinc essentiality and toxicity. Biophysical aspects” by Y.Harmaza et al. (Biophysics, 2014, Vol. 59, No. 2, pp. 264-275.) establishes that the permissible (physiological) concentration of zinc in human serum is 215 μM, while the level of free intracellular Zn2+ in most cells is extremely low (<1 nM), whereas the cell has a sophisticated regulatory system to keep intracellular zinc low even at high extracellular levels. However, reference is made to extensive material on the negative effects of high concentrations of zinc on biosystems, both under physiological and pathological conditions, casting doubt on the positive effect of so-called “pharmacological” concentrations of zinc.
[0023] Já na área alimentícia, em artigo intitulado “Antifungal properties of Zinc- compounds against toxigenic fungi and mycotoxin” de Geovana D. Savi et al. (Int. J. Food Science and Technology 2013, 48, 1834-1840) há uma análise sobre as propriedades antifúngicas e anti-micotoxinas do zinco.[0023] In the food field, in an article entitled “Antifungal properties of Zinc- compounds against toxigenic fungi and mycotoxin” by Geovana D. Savi et al. (Int. J. Food Science and Technology 2013, 48, 1834-1840) there is a review of the antifungal and antimycotoxin properties of zinc.
[0024] De acordo com a conclusão desse estudo, os compostos de zinco testados podem apresentar forte atividade antifúngica mesmo em baixas concentrações, em que os tratamentos interferiram no metabolismo celular dos fungos. É destacada ainda a necessidade de mais estudos do zinco como um agente fungicida eficaz para aplicações agrícolas e de segurança alimentar, cabendo observar o limite superior tolerável para consumo, e eventuais implicações de sua incorporação em filmes e outros materiais de embalagem, bem como sua aplicação na agricultura.[0024] According to the conclusion of this study, the zinc compounds tested may have strong antifungal activity even at low concentrations, in which the treatments interfered with the cellular metabolism of fungi. It is also highlighted the need for further studies of zinc as an effective fungicidal agent for agricultural and food safety applications, and it is important to observe the tolerable upper limit for consumption, and possible implications of its incorporation in films and other packaging materials, as well as its application. in the farming.
[0025] Com relação ao uso do zinco como agente antiparasitário, o texto “Anthelmintic effects of zinc oxide and iron oxide nanoparticles against Toxocara vitulorum” de Ruhollah Dorostkar et al. (Int Nano Lett (2017) 7:157-164) avalia nanopartículas de óxido de zinco (ZnO) e óxido de ferro (FeO) quanto aos seus possíveis efeitos anti-helmínticos in vitro contra Toxocara vitulorum, concluindo que estas moléculas, de fato, exercem efeitos anti-helmínticos por meio da indução de estresse oxidativo / nitrosativo. Porém, é feita referência também ao mecanismo de “indução de apoptose” subjacente ao efeito antiparasitário do ZnO, sendo relatado que após o tratamento de promastigotas de Leishmania major com nanopartículas de ZnO, surgiram efeitos necróticos e apoptóticos no parasita. A este respeito, é bem documentado que nanopartículas de óxido metálico liberam espécies reativa de oxigênio (ROS) em grande quantidade e, assim, exercem seus efeitos citotóxicos.[0025] Regarding the use of zinc as an antiparasitic agent, the text “Anthelmintic effects of zinc oxide and iron oxide nanoparticles against Toxocara vitulorum” by Ruhollah Dorostkar et al. (Int Nano Lett (2017) 7:157-164) evaluates zinc oxide (ZnO) and iron oxide (FeO) nanoparticles for their possible in vitro anthelmintic effects against Toxocara vitulorum, concluding that these molecules, in fact, , exert anthelmintic effects through the induction of oxidative/nitrosative stress. However, reference is also made to the “apoptosis induction” mechanism underlying the antiparasitic effect of ZnO, and it is reported that after treatment of Leishmania major promastigotes with ZnO nanoparticles, necrotic and apoptotic effects appeared in the parasite. In this regard, it is well documented that metal oxide nanoparticles release reactive oxygen species (ROS) in large amounts and thus exert their cytotoxic effects.
[0026] Desta forma, embora a literatura técnica disponível e discutida acima aponte para o zinco como um potencial agente microbiano, que é relativamente bem aceito pela baixa toxicidade in vivo, ainda há uma carência de evidências acerca de como seria a atividade biológica deste elemento coordenado com H3 sobre os alvos dos agentes patogênicos, especificamente se forem considerados os resultados não animadores da complexação de H3 com platina, que apesar de apresentar um efeito biológico desejável, nos testes in vitro não apresentou efeito superior ao H3 isolado.[0026] Thus, although the technical literature available and discussed above points to zinc as a potential microbial agent, which is relatively well accepted due to its low toxicity in vivo, there is still a lack of evidence about what the biological activity of this element would be. coordinated with H3 on the targets of pathogens, specifically if one considers the non-exciting results of the complexation of H3 with platinum, which, despite having a desirable biological effect, in in vitro tests did not show a superior effect to H3 alone.
[0027] Diante da referida carência de evidências, por meio da presente invenção, chegou-se à conclusão de que o composto resultante da coordenação de 2 moléculas de H3 ao zinco, sob a forma de diclorobis(22-hidrazona-imidazolina-2-il-col-5-en-3β- ol)zinco(II), também referido pela notação Zn(H3)2 daqui em diante, consiste em uma alternativa menos tóxica e biologicamente mais eficaz para o tratamento de doenças parasitárias e fúngicas, uma vez que apresenta toxicidade inferior e efeito biológico surpreendentemente superior quando comparada à molécula de H3.[0027] In view of the aforementioned lack of evidence, through the present invention, it was concluded that the compound resulting from the coordination of 2 molecules of H3 to zinc, in the form of dichlorobis(22-hydrazone-imidazoline-2- yl-col-5-en-3β-ol)zinc(II), also referred to by the notation Zn(H3)2 hereinafter, is a less toxic and biologically more effective alternative for the treatment of parasitic and fungal diseases, a since it has lower toxicity and surprisingly superior biological effect when compared to the H3 molecule.
[0028] A presente invenção tem por objetivo prover um composto metaloazasterol particularmente útil como agente terapêutico no tratamento de infecções fúngicas e parasitárias, como a causada, por exemplo, por Leishmania amazonensis, devido à sua ação como inibidor seletivo da enzima Δ24-esterol metiltransferase (24-EMT) presente nesses organismos e essencial para síntese de ergosterol e outros esteróis 24-alquilados de membrana, atuando desta forma com significativa redução da toxicidade.[0028] The present invention aims to provide a metalloazasterol compound particularly useful as a therapeutic agent in the treatment of fungal and parasitic infections, such as that caused, for example, by Leishmania amazonensis, due to its action as a selective inhibitor of the enzyme Δ24-sterol methyltransferase (24-EMT) present in these organisms and essential for the synthesis of ergosterol and other 24-alkylated membrane sterols, thus acting with a significant reduction in toxicity.
[0029] Portanto, o composto da presente invenção pertence ao campo dos compostos de azasteróis, mais especificamente, metaloazasteróis, com atividade no tratamento de doenças parasitárias, tais como leishmaniose e doença de Chagas, bem como outras doenças fúngicas, com a característica adicional de ser complexada com zinco, com a produção de efeitos inesperados e imprevisíveis diante do estado da técnica.[0029] Therefore, the compound of the present invention belongs to the field of azasterol compounds, more specifically, metalloazasterols, with activity in the treatment of parasitic diseases such as leishmaniasis and Chagas disease, as well as other fungal diseases, with the additional feature of be complexed with zinc, with the production of unexpected and unpredictable effects given the state of the art.
[0030] Este objetivo é alcançado por meio da complexação de zinco (Zn) com duas moléculas de 22-hidrazona-imidazolina-2-il-col-5-en-3β-ol (H3), que resulta em uma molécula de diclorobis(22-hidrazona-imidazolina-2-il-col-5-en-3β-ol)zinco(II), aqui nomeada como Zn(H3)2, com atividade biológica superior e toxicidade inferior à molécula de H3 isolada.[0030] This objective is achieved through the complexation of zinc (Zn) with two molecules of 22-hydrazone-imidazoline-2-yl-col-5-en-3β-ol (H3), which results in a molecule of dichlorobis (22-hydrazone-imidazoline-2-yl-col-5-en-3β-ol)zinc(II), here named as Zn(H3)2, with superior biological activity and inferior toxicity to the isolated H3 molecule.
[0031] Outro objetivo da presente invenção consiste em prover uma composição farmacêutica que compreende um veículo farmaceuticamente aceitável, bem como o composto voltado para o tratamento de doenças parasitárias e fúngicas, sendo particularmente útil como agente terapêutico no tratamento de infecções causadas Leishmania amazonensis.[0031] Another objective of the present invention is to provide a pharmaceutical composition that comprises a pharmaceutically acceptable carrier, as well as the compound aimed at the treatment of parasitic and fungal diseases, being particularly useful as a therapeutic agent in the treatment of infections caused by Leishmania amazonensis.
[0032] A Figura 1 mostra as alterações na membrana plasmática de formas promastigotas de L. amazonensis observadas por microscopia eletrônica de varredura tratadas com H3 e Zn(H3)2 a uma concentração de 3 μM comparada a uma amostra de controle.[0032] Figure 1 shows the changes in the plasma membrane of promastigote forms of L. amazonensis observed by scanning electron microscopy treated with H3 and Zn(H3)2 at a concentration of 3 μM compared to a control sample.
[0033] A Figura 2 mostra imagens de microscopia eletrônica de transmissão que revelam os efeitos obtidos nas mitocôndrias de formas promastigotas de L. amazonensis, tratadas com H3 e Zn(H3)2 na concentração de 3 μM.[0033] Figure 2 shows transmission electron microscopy images that reveal the effects obtained in the mitochondria of promastigote forms of L. amazonensis, treated with H3 and Zn(H3)2 at a concentration of 3 μM.
[0034] A Figura 3 é um gráfico que representa o efeito de Zn(H3)2 no percentual de infecção de macrófagos por formas amastigostas de L. amazonensis após 48 horas de tratamento, usando diferentes concentrações. Por meio desses dados foram calculadas a IC50 (concentração inibitória média) e a CIM (concentração inibitória mínima) do composto.[0034] Figure 3 is a graph representing the effect of Zn(H3)2 on the percentage of infection of macrophages by amastigotes forms of L. amazonensis after 48 hours of treatment, using different concentrations. Through these data, the IC50 (mean inhibitory concentration) and MIC (minimum inhibitory concentration) of the compound were calculated.
[0035] De acordo com um aspecto da presente invenção, é provido um composto com atividade no tratamento de doenças parasitárias, tais como leishmaniose e doença de Chagas, e doenças fúngicas.[0035] According to one aspect of the present invention, there is provided a compound having activity in the treatment of parasitic diseases, such as leishmaniasis and Chagas disease, and fungal diseases.
[0036] O composto pertence ao grupo dos azasteróis é representado pela fórmula a seguir:
[0037] Em uma modalidade alternativa, R1 e R2 são ligantes estericamente estáveis e com geometrias distintas de coordenação para cada complexo metálico (podendo ser iônicos ou neutros), sendo selecionados, preferencialmente, dentre fosfinas, nitratos, acetatos, sulfato, fluoreto, brometo, iodeto, grupos alquílicos, hidroxilas e água.[0037] In an alternative embodiment, R1 and R2 are sterically stable ligands and with distinct coordination geometries for each metal complex (can be ionic or neutral), being selected, preferably, among phosphines, nitrates, acetates, sulfate, fluoride, bromide , iodide, alkyl groups, hydroxyl groups and water.
[0038] Nos testes realizados acerca de atividade biológica sobre as formas promastigota e amastigota de L. amazonensis foram obtidos os resultados apresentados nas Tabelas 1 e 2 a seguir, mais precisamente referentes às concentrações inibitórias média e mínima inibitórias para as duas formas do parasita. Tabela 1 - Concentração inibitória média (IC50) e Concentração inibitória mínima (CIM) sobre formas promastigotas de L. amazonensis.
[0039] Para os fins da presente invenção, IC50 se refere à concentração do composto necessária para reduzir a 50% o crescimento populacional dos parasitas, in vitro, sendo, portanto, uma medida da eficácia da substância testada. Em outras palavras, neste caso, a IC50 foi utilizada para determinar a dose necessária para reduzir em 50% a quantidade das formas promastigotas e amastigotas do parasita quando comparados às amostras controle.[0039] For the purposes of the present invention, IC50 refers to the concentration of the compound necessary to reduce to 50% the population growth of the parasites, in vitro, being, therefore, a measure of the effectiveness of the substance tested. In other words, in this case, the IC50 was used to determine the dose needed to reduce the amount of promastigote and amastigote forms of the parasite by 50% when compared to control samples.
[0040] Já a CIM, concentração inibitória mínima (CIM), corresponde à mais baixa concentração de uma substância capaz de inibir o crescimento do parasita.[0040] Already the MIC, minimum inhibitory concentration (MIC), corresponds to the lowest concentration of a substance capable of inhibiting the growth of the parasite.
[0041] Os dados referentes à IC50 e a CIM do composto Zn(H3)2 foram extraídos da Figura 3.[0041] Data referring to IC50 and MIC of the compound Zn(H3)2 were extracted from Figure 3.
[0042] Os resultados obtidos revelam que o átomo de zinco (Zn) afeta positivamente a atividade de H3, sobre as formas promastigotas e, especialmente, sobre as formas amastigotas intracelulares, que são as formas de maior interesse clínico, por serem formas infectantes do parasita.[0042] The results obtained reveal that the zinc atom (Zn) positively affects the activity of H3, on the promastigote forms and, especially, on the intracellular amastigote forms, which are the forms of greatest clinical interest, as they are infective forms of the parasite.
[0043] Mais particularmente, em uma análise comparativa entre os compostos H3 e complexo Zn(H3)2, as Tabelas 1 e 2 revelam que a IC50 do complexo Zn(H3)2 para formas amastigotas em 48 horas foi aproximadamente 17 vezes menor que a de H3, demonstrando, portanto, que Zn(H3)2 é cerca de 17 vezes mais potente que H3 em amostras tratadas após 48 horas. Além disso, a concentração inibitória mínima (CIM) em 48 horas do complexo Zn(H3)2, necessária para inibir o crescimento do parasita, foi 5 vezes menor quando comparada à CIM de H3 no mesmo intervalo de tempo. Esses dados comprovam a eficácia superior do complexo Zn(H3)2 quando comparado ao H3.[0043] More particularly, in a comparative analysis between compounds H3 and the Zn(H3)2 complex, Tables 1 and 2 reveal that the IC50 of the Zn(H3)2 complex for amastigote forms at 48 hours was approximately 17-fold lower than that of H3, thus demonstrating that Zn(H3)2 is about 17 times more potent than H3 in samples treated after 48 hours. Furthermore, the 48-hour minimum inhibitory concentration (MIC) of the Zn(H3)2 complex, required to inhibit the growth of the parasite, was 5-fold lower when compared to the MIC of H3 at the same time interval. These data prove the superior efficacy of the Zn(H3)2 complex when compared to H3.
[0044] Ainda estudos em macrófagos murinos foram realizados para determinação da CC50 em 48 horas (concentração de citotoxicidade necessária para reduzir 50% das células viáveis), em que foi demonstrado que foi necessária uma concentração mínima de aproximadamente 5 μM de Zn(H3)2 para que o composto fosse capaz de reduzir a viabilidade das células murinas tratadas com Zn(H3)2 em 50% em 48 horas. Uma concentração, portanto, muito superior à dose de 32nM necessária para inibir em 50% as formas amastigotas, clinicamente infectantes, do parasita.[0044] Further studies on murine macrophages were performed to determine the CC50 in 48 hours (concentration of cytotoxicity necessary to reduce 50% of viable cells), in which it was shown that a minimum concentration of approximately 5 μM of Zn(H3) was required. 2 so that the compound was able to reduce the viability of murine cells treated with Zn(H3)2 by 50% in 48 hours. A concentration, therefore, much higher than the dose of 32nM necessary to inhibit by 50% the clinically infective amastigote forms of the parasite.
[0045] Foi realizada a separação e identificação dos esteróis livres pela técnica analítica de cromatografia gasosa acoplada ao espectrômetro de massas, onde foram calculados seus porcentuais ponderados em relação ao total de seus conteúdos. Os esteróis na forma livre, tais como utilizados nesta análise, são importantes porque integram a arquitetura da membrana plasmática e outras organelas internas da célula.[0045] The separation and identification of free sterols was performed by the analytical technique of gas chromatography coupled to the mass spectrometer, where their weighted percentages were calculated in relation to the total of their contents. Free sterols, as used in this analysis, are important because they integrate the architecture of the plasma membrane and other internal organelles of the cell.
[0046] As análises da composição de esteróis livres das formas promastigotas de L. amazonensis nas amostras controle e tratadas (Tabelas 3 e 4) revelaram como principais esteróis das amostras controle (não tratados) ergosta-5,7,24 (24')-trien-3β-ol (5-deidroepisterol) e ergosta-7,24(24')-dien-3β-ol (episterol), representando aproximadamente 80% do total de esteróis. Outros esteróis minoritários, como zimosterol, ergosta-5,7,9(11)-22-tetraen-3β-ol, ergosta-7,22,24(24')-trien-3β-ol, ergosta-5,7,22,24(24')-teraen-3β-ol, ergosta-5,8,22-trien-3β-ol, ergosta-5,24(24')-dien- 3β-ol 14α-metil-ergosta-8,24(24')-dien-3β-ol, neoepisterol, ergosta-5,7,9(10)-24(24')- tetraen-3β-ol, lanosterol, estigmasta-5,7,22-trien-3β-ol, estigmasta-7,22-trien-3β-ol foram detectados, e a soma deles representou cerca de 13% do total de esteróis (Tabelas 3 e 4). Além disso, o colesterol que é tomado passivamente do meio de crescimento alcançou aproximadamente 7%.[0046] The analysis of the composition of free sterols of the promastigote forms of L. amazonensis in the control and treated samples (Tables 3 and 4) revealed that the main sterols of the control samples (untreated) were ergot-5,7,24 (24') -trien-3β-ol (5-dehydroepisterol) and ergost-7,24(24')-dien-3β-ol (episterol), representing approximately 80% of the total sterols. Other minor sterols such as zymosterol, ergost-5,7,9(11)-22-tetraen-3β-ol, ergost-7,22,24(24')-trien-3β-ol, ergost-5,7, 22,24(24')-teraen-3β-ol, ergost-5,8,22-trien-3β-ol, ergost-5,24(24')-dien-3β-ol 14α-methyl-ergosta-8 ,24(24')-dien-3β-ol, neoepisterol, ergost-5,7,9(10)-24(24')-tetraen-3β-ol, lanosterol, stigmasta-5,7,22-trien- 3β-ol, stigmasta-7,22-trien-3β-ol were detected, and their sum represented about 13% of the total sterols (Tables 3 and 4). Furthermore, the cholesterol that is passively taken from the growth medium reached approximately 7%.
[0047] Parasitas cultivados na presença de concentrações crescentes de H3 (Tabela 3) exibiram uma redução significativa da porcentagem dos esteróis endógenos. Ao mesmo tempo, o efeito do H3 na concentração mais baixa de 3 μM causou um aumento dos intermediários metabólicos colesta-5,7,24-trien-3β-ol e colesta-7,24- trien-3β-ol em 69% e 12%, respectivamente, atingindo um total de 81%.[0047] Parasites cultured in the presence of increasing concentrations of H3 (Table 3) exhibited a significant reduction in the percentage of endogenous sterols. At the same time, the effect of H3 at the lowest concentration of 3 μM caused an increase in the metabolic intermediates cholesta-5,7,24-trien-3β-ol and cholesta-7,24-trien-3β-ol by 69% and 12%, respectively, reaching a total of 81%.
[0048] Com efeito, os estudos de análise de esteróis na forma livre nos parasitas de L. amazonensis em controles e tratados, mostraram que, com diferentes concentrações inibitórias de H3, os esteróis principais do parasita (5-desidro-episterol e episterol) são substituídos por esteróis do tipo colesta como colesta-5,7,24-trien-3β-ol e colesta-7,24-dien-3β-ol. Estas evidências mostram claramente que há inibição da enzima 24-EMT.[0048] Indeed, studies of analysis of sterols in the free form in the parasites of L. amazonensis in controls and treated, showed that, with different inhibitory concentrations of H3, the main sterols of the parasite (5-dehydro-episterol and episterol) are replaced by colesta-type sterols such as colesta-5,7,24-trien-3β-ol and colesta-7,24-dien-3β-ol. These evidences clearly show that there is inhibition of the 24-EMT enzyme.
[0049] Do mesmo modo, foi realizada a avaliação com o composto Zn(H3)2 sobre as formas amastigotas da L. amazonensis (tabela 4). O resultado obtido foi similar ao de H3, com a diferença de que a menor concentração usada de Zn(H3)2 foi de 2 μM, e o acúmulo dos esteróis colesta-5,7,24-trien-3β-ol e colesta-7,24-trien-3β-ol foi de 54% e 21%, respectivamente, atingindo um total de 75%.[0049] In the same way, the evaluation was carried out with the compound Zn(H3)2 on the amastigote forms of L. amazonensis (Table 4). The result obtained was similar to that of H3, with the difference that the lowest concentration of Zn(H3)2 used was 2 μM, and the accumulation of cholesta-5,7,24-trien-3β-ol and cholesta- 7,24-trien-3β-ol was 54% and 21%, respectively, reaching a total of 75%.
[0050] Outro fator de destaque é que em ambos casos (H3 e Zn(H3)2), o percentual de colesterol aumenta de forma dose-dependente. No caso da H3, a uma concentração de 7 μM, o colesterol atinge o valor de 14%. Entretanto, Zn(H3)2 à mesma concentração causa um acúmulo do colesterol de 17%, um valor, aproximadamente, 20% superior em relação ao efeito do H3.[0050] Another important factor is that in both cases (H3 and Zn(H3)2), the percentage of cholesterol increases in a dose-dependent manner. In the case of H3, at a concentration of 7 μM, cholesterol reaches a value of 14%. However, Zn(H3)2 at the same concentration causes a cholesterol accumulation of 17%, a value approximately 20% higher than the effect of H3.
[0051] O aumento do colesterol com o uso de ambos compostos indica o esgotamento dos esteróis endógenos pela inibição da enzima Δ24-esterol metiltransferase, o que tem como consequência a perda da viabilidade celular.[0051] The increase in cholesterol with the use of both compounds indicates the depletion of endogenous sterols by the inhibition of the enzyme Δ24-sterol methyltransferase, which results in the loss of cell viability.
[0052] As Tabelas 3 e 4, a seguir, se referem ao tempo de retenção e ao percentual de esteróis livres presentes no crescimento de L. amazonensis, tal como já discutido, na ausência e na presença, respectivamente, de H3 e Zn(H3)2 em diferentes concentrações, quando analisados pela técnica de cromatografia gasosa acoplada com espectrômetro de massas. Tabela 3 - Tempo de retenção (min) e percentual dos esteróis livres presentes no crescimento de L. amazonensis na ausência e presença de H3.
[0053] A análise da morfologia das formas promastigotas do parasita L. amazonensis por microscopia eletrônica de varredura, de acordo com Figura 1, mostra diferenças significativas entre as amostras tratadas com H3 e Zn(H3)2 quando comparadas às amostras controle, evidenciadas por alterações na morfologia do corpo celular, na ultraestrutura de membrana plasmática e superfície celular, sob a forma de enrugamento de membrana, vistas de forma mais intensa nas amostras tratadas com Zn(H3)2.[0053] The analysis of the morphology of the promastigote forms of the parasite L. amazonensis by scanning electron microscopy, according to Figure 1, shows significant differences between the samples treated with H3 and Zn(H3)2 when compared to the control samples, evidenced by changes in the morphology of the cell body, in the ultrastructure of the plasma membrane and cell surface, in the form of membrane wrinkling, seen more intensely in the samples treated with Zn(H3)2.
[0054] Nas imagens da Figura 2, obtidas por microscopia eletrônica de transmissão, verifica-se a ocorrência de lesão mitocondrial, a qual é típica de células tratadas com inibidores da 24-EMT. Porém, foi verificado que as alterações mitocondriais foram mais intensas com o complexo Zn(H3)2 da presente invenção. Outro aspecto observado foi a compactação do DNA nuclear que teve significativa alteração com o complexo Zn(H3)2 da invenção, bem como a presença de grandes autofagossomas.[0054] In the images of Figure 2, obtained by transmission electron microscopy, there is the occurrence of mitochondrial damage, which is typical of cells treated with 24-EMT inhibitors. However, it was found that the mitochondrial changes were more intense with the Zn(H3)2 complex of the present invention. Another aspect observed was the compaction of the nuclear DNA, which was significantly altered with the Zn(H3)2 complex of the invention, as well as the presence of large autophagosomes.
[0055] Para a obtenção do complexo Zn(H3)2 da presente invenção (cujo esquema de síntese é apresentado abaixo), a solução do esterol hidrazona H3 em metanol é adicionada gota a gota, sob agitação, a uma solução do cloreto de zinco, também em metanol. Em seguida, a solução é submetida a refluxo por quatro horas, e após arrefecimento, o precipitado é separado por filtração e lavado com água e éter dietílico. Finalmente, o sólido branco obtido, é seco.[0055] To obtain the Zn(H3)2 complex of the present invention (whose synthesis scheme is presented below), the solution of the sterol hydrazone H3 in methanol is added dropwise, under stirring, to a solution of the zinc chloride , also in methanol. Then, the solution is refluxed for four hours, and after cooling, the precipitate is filtered off and washed with water and diethyl ether. Finally, the white solid obtained is dried.
[0056] Em uma modalidade preferida do processo para a obtenção do composto da presente invenção, são empregadas as seguintes quantidades no processo de obtenção: - solução do esterol hidrazona H3 (0,24 mmol) em metanol (30 mL); - solução em agitação do cloreto de zinco (0,12 mmol) em metanol (15 mL); - água para lavagem (3 x 20 mL); - éter dietílico (3 X 20 mL).
[0057] Nas tabelas seguintes de 1H-RMN e 13C-RMN, pela técnica de espectroscopia de ressonância magnética nuclear, são indicados os deslocamentos químicos mais significativos (ppm) dos sinais dos prótons e carbonos -13 do ligando H3 e do complexo Zn(H3)2Cb. Análise elementar foi calculada para Zn(H3hCb^3H2O [C, 59.1; H, 8.5; N, 11.0] e encontrados C, 59.4; H, 8.3; N, 11.3. Tabela 5 - 1H-RMN. Deslocamentos químicos em ppm dos sinais dos prótons do ligando H3 e complexo Zn(H3)2CL
[0058] A presente invenção pode se apresentar ainda na forma de uma composição farmacêutica contendo o composto
[0059] Por fim, é provido ainda o uso do composto
Claims (8)
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