Human African trypanosomiasis (HAT, sleeping sickness) and American trypanosomiasis (Chagas disease) are endemic zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Trypanosoma cruzi, respectively). Just a few old drugs are available for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. Only fexinidazole has been recently incorporated into the arsenal for the treatment of HAT. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these pathogens by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: pyridine-2-thiolato-1-oxide ligand (mpo) and polypyridyl ligands (NN). Three [Ru(mpo)(dppf)(NN)](PF6) compounds and their derivatives with chloride as a counterion were synthesized and fully characterized in solid state and solution. They showed in vitro activity on bloodstream T. brucei (EC50 = 31-160 nM) and on T. cruzi trypomastigotes (EC50 = 190-410 nM). Compounds showed the lowest EC50 values on T. brucei when compared to the whole set of metal-based compounds previously developed by us. In addition, several of the Ru compounds showed good selectivity toward the parasites, particularly against the highly proliferative bloodstream form of T. brucei. Interaction with DNA and generation of reactive oxygen species (ROS) were ruled out as potential targets and modes of action of the Ru compounds. Biochemical assays and in silico analysis led to the insight that they are able to inhibit the NADH-dependent fumarate reductase from T. cruzi. One representative hit induced a mild oxidation of low molecular weight thiols in T. brucei. The compounds were stable for at least 72 h in two different media and more lipophilic than both bioactive ligands, mpo and NN. An initial assessment of the therapeutic efficacy of one of the most potent and selective candidates, [Ru(mpo)(dppf)(bipy)]Cl, was performed using a murine infection model of acute African trypanosomiasis. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described, but was unable to control parasite proliferation in vivo, probably because of its rapid clearance or low biodistribution in the extracellular fluids. Future studies should investigate the pharmacokinetics of this compound in vivo and involve further research to gain deeper insight into the mechanism of action of the compounds.

Human African trypanosomiasis (sleeping sickness) and leishmaniasis are prevalent zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Leishmania spp). Additionally, both are co-endemic in certain regions of the world. Only a small number of old drugs exist for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these trypanosomatid parasites by including in a single molecule 1,1 '-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate li-gands: 8-hydroxyquinoline derivatives (8HQs) and polypyridyl ligands (NN). Three [Ru(8HQs)(dppf)(NN)](PF6) compounds were synthesized and fully characterized. They showed in vitro activity on bloodstream Trypanosoma brucei (IC50 140-310 nM) and on Leishmania infantum promastigotes (IC50 3.0-4.8 mu M). The compounds showed good selectivity towards T. brucei in respect to J774 murine macrophages as mammalian cell model (SI 15-38). Changing hexafluorophosphate counterion by chloride led to a three-fold increase in activity on both parasites and to a two to three-fold increase in selectivity towards the pathogens. The compounds affect in vitro at least the targets of the individual bioactive moieties included in the new chemical entities: DNA and generation of ROS. The compounds are stable in solution and are more lipophilic than the free bioactive ligands. No clear correlation between lipophilicity, interaction with DNA or generation of ROS and activity was detected, which agrees with their overall similar anti-trypanosoma potency and selectivity. These compounds are promising candidates for further drug development.

In the search for a more effective chemotherapy for the treatment of Human African Trypanosomiasis, a disease caused by the parasite Trypanosoma brucei, the development of ferrocenyl compounds has arisen as a promising strategy. In this work, five new Pd-Fe heterobimetallic [PdII(L)(dppf)](PF6) compounds, including 8-hydroxyquinolyl derivatives HL1-HL5 as bioactive ligands and dppf = 1,1'-bis(diphenylphosphino)ferrocene as the organometallic co-ligand, were synthesized and fully characterized in the solid state and in solution. Molecular structures of three compounds were solved by single crystal X-ray diffraction methods. The compounds displayed submicromolar or micromolar IC50 values against bloodstream T. brucei (IC50: 0.33-1.2 μM), and good selectivity towards the pathogen (SI: 4-102) with respect to mammalian macrophages (cell line J774). The new Pd complexes proved to be 2-fold to 45-fold more potent than the drug nifurtimox but most of them are less active than their Pt analogues. Potential molecular targets were studied. The complexes interact with DNA but they do not alter the intracellular thiol-redox homeostasis of the parasite. In order to understand and predict the main structural determinants on the anti-T. brucei activity, a search of quantitative structure-activity relationships (QSAR) was performed including all the [M(L)(dppf)](PF6) complexes, where M = Pd(ii) or Pt(ii), currently and previously developed by us. The correlation obtained shows the relevance of the electronic effects, the lipophilicity and the type of metal. According to the QSAR study, compounds with electron-withdrawing ligands, higher lipophilicity and harboring Pt would result in higher T. brucei cytotoxicity. From the whole series of [M(L)(dppf)](PF6) compounds developed, where M = Pt(ii) or Pd(ii) and HL = 8-hydroxyquinolyl derivatives, Pt-dppf-L4 (IC50 = 0.14 μM, SI = 48) was selected to perform an exploratory pre-clinical study in infected mice. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described and exerts an anti-proliferative effect on parasites, which extends animal survival but is not curative.

Searching for a more effective chemotherapy for the treatment of Human African trypanosomiasis, the disease caused by the parasite Trypanosoma brucei, and cancer, in the current work five new [Pt-II(L)(dppf)](PF6) compounds, with HL = 8-hydroxyquinoline derivatives and dppf = 1,1'-bis(diphenylphosphino)ferrocene, were synthesized and fully characterized. Crystal structures of three compounds were solved by XRD. The compounds displayed fairly good activity against bloodstream T. brucei, with IC50 values in the submicromolar range (IC50: 0.14-0.93 mu M), and good selectivity towards the pathogen (SI: 11- 48) with respect to mammalian macrophages (cell line J774). Coordination to the {Pt-dppf} moiety led, in most cases, to an enhancement of the activity in respect to the bioactive ligands (11 to 41 fold). Cytotoxicity was assessed against wildtype (A2780) and cisplatinresistance (A2780cisR) ovarian cancer cell lines. Four [Pt-II(L)(dppOl(PF6) compounds were more active (IC50: 1.2-4.4 mu M) than cisplatin (IC50: 26.0 mu M) on A2780 cells and showed far superior activity than the reference drug against A2780cisR cells. Platinum levels in A2780 cells showed poor correlation between cellular uptake and the cytotoxic activity. All the complexes interacted with DNA and the most active ones induced reactive oxygen species (ROS) formation which suggested that the mechanism of action for these complexes may be mediated by oxidative stress and interaction with DNA that could act as a potential molecular target for this type of complexes. Some complexes of this series could be considered new hits for the development of prospective agents against trypanosomatid parasites and ovarian cancer.

Searching for prospective agents against infectious diseases, four new ferrocenyl derivatives, [M(L)(dppf)4] (PF6), with M = Pd(II) or Pt(II), dppf = 1,1'-bis(diphenylphosphino) ferrocene and HL = tropolone (HTrop) or hinokitiol (HHino), were synthesized and characterized. Complexes and ligands were evaluated against the bloodstream form of T. brucei, L. infantum amastigotes, M. tuberculosis (MTB) sensitive strain and MTB clinical isolates. Complexes showed a significant increase of the anti-T. brucei activity with respect to the free ligands ( > 28- and > 46-fold for Trop and 6- and 22-fold for Hino coordinated to Pt-dppf and Pd-dppf, respectively), yielding IC50 values < 5 mu M. The complexes proved to be more potent than the antitrypanosomal drug Nifurtimox. The new ferrocenyl derivatives were more selective towards the parasite than the free ligands. The Pt compounds were less toxic on J774 murine macrophages (mammalian cell model), than the Pd ones, showing selectivity index values (SI = IC50 murine macrophage/IC50 T. brucei) up to 23. Generation of the {M-dppf} compounds lead to a slightly positive impact on the anti-leishmanial potency. Although the ferrocenyl derivatives were more active on sensitive MTB than the free ligands (MIC90 = 9.88-14.73 mu M), they showed low selectivity towards the pathogen. Related to the mechanism of action, the antiparasitic effect cannot be ascribed to an interference of the compounds with the thiol-redox homeostasis of the pathogen. Fluorescence measurements pointed at DNA as a probable target of the new compounds. (Pt(Trop)(dppOl(PF6) and [Pt(Hino)(dppf)] (PF6) could be considered prospective anti-T. brucei agents that deserve further research.

Introducao: Em 2015 o agente etiologico Mycobacterium tuberculosis (MTB), causador da tuberculose, alcancou 10,4 milhoes de pessoas no mundo, ficando em primeiro lugar entre as doencas infecto-contagiosas que mais matam pessoas. O seu dificil combate e devido principalmente ao surgimento das cepas resistentes, por isso o descobrimento de novas moleculas se faz necessario. Neste trabalho determinou-se a concentracao inibitoria minima (CIM90), que e a concentracao capaz de inibir 90% do crescimento bacteriano dos ligantes hinokitiol (hino) e tropolona (trop) e seus complexos com Pt (platina) e Pd (paladio contra MTB H37Rv (ATCC 27294) e quatro cepas clinicas resistentes a farmacos. Determinou-se tambem o IC50, que e a concentracao capaz de manter 50% da viabilidade celular, atraves do ensaio de citotoxicidade em celulas MRC-5 (fibroblasto pulmonar humano). Determinou-se o indice de seletividade (IS) que representa quanto um composto e mais seletivo para a morte do microrganismo sem causar dano celular. Objetivo: Avaliacao do potencial anti-MTB dos ligantes hino e trop, seus complexos com Pd e Pt e seus precursores. Metodologia: Tanto o REMA, para determinar a CIM90, quanto o ensaio de citotoxicidade para determinar o IC50, sao metodos de microdiluicao em placas de 96 pocos que utilizam a resazurina como revelador. O inoculo bacteriano utilizado foi de aproximadamente 105 UFC/mL e o intervalo de concentracoes foi 25 - 0,098μg/mL. Para o ensaio de citotoxicidade o intervalo de concentracoes utilizado foi de 100 - 0,39μg/mL. Para demostrar o IS, calculou-se a divisao dos valores obtidos de IC50/MIC. Resultados e discussao: Com os resultados do REMA nos observamos que a atividade anti-MTB de hino (35.46μM) e trop (29.02μM) sozinhos foram menos ativos que quando complexados com Pd e Pt. Encontramos entre Pd-hino (13.14μM), Pd-trop (9.01μM), Pt-hino (14.73μM), Pt-trop (17.60μM), que Pd-trop e o mais ativo. Ja os melhores valores de IS foram para trop (7,02) e hino (7,03), pois os ligantes sozinhos sao menos toxicos para as celulas. Nos isolados clinicos nos observamos que a atividade foi menor que contra a cepa sensivel (H37Rv), mas ainda observamos atividade para trop and Pd-trop confirmando o seu potencial anti-MTB, pois, trop apresentou atividade contra 3 cepas (18,30-99,44μM) e Pd-trop contra 2 (9,05-25,64μM) das 4 cepas testadas. Conclusao: Esses dados demonstram que os ligantes hino e trop apresentam atividade anti-MTB, mas a complexacao com Pd e Pt melhora esta atividade. No entanto, a complexacao com os metais levou a um aumento na citotoxicidade, apenas hino e trop, quando nao complexados, apresentaram IS superiores a 7 enquanto os complexos apresentaram IS menor que 2. A pesquisa de novas moleculas e uma necessidade, visto o aumento da resistencia aos antibioticos disponiveis.