Ruthenium complexes for the treatment of protozoan diseases of medical and veterinary importance

The auxotrophic parasite Toxoplasma gondii is a cause of toxoplasmosis. It affects all warm-blooded species, and around one third of the world human population is affected. The Current medication consists in combination treatments based on pyrimethamine with sulfadiazine. Drugs clinically used to treat toxoplasmosis are not specific, being mainly developed for other diseases, have important side effects, and only effective during the acute stage of infection but they do not eradicate bradyzoite tissue cysts. In consequence, the development of new effective anti-Toxoplasma medications, exhibiting also improved tolerability, is of great importance. Trithiolato-bridged dinuclear ruthenium(II)-arene compounds demonstrated interesting antiparasitic efficacy against Toxoplasma gondii, Neospora caninum and Trypanosoma brucei. Very little is known about their cellular targets and potential mode of action. It was hypothesized that improved in vivo properties and a better knowledge of their exact cellular targets and mode of action can be obtained using the 'conjugate approach' consisting in anchoring judiciously chosen bioactive molecules onto the diruthenium core. This thesis was focused on the synthesis and biological activity evaluation of various hybrid molecules in which fluorophores, metabolites and drugs were appended on the trithiolato-bridged dinuclear ruthenium(II)-arene moiety. The aims of this approach were: (i) to investigate the cellular targets/mode of action by tracing fluorophore-tagged compounds inside cells/parasites, (ii) to improve the cellular uptake of the complexes by conjugating metabolites necessary for the parasites growth, exploiting the parasite auxotrophies and metabolic peculiarities in Trojan horse strategy, and (iii) to obtain compounds with increased antiparasitic efficacy by coupling with a drug relevant to the treatment of toxoplasmosis. Five libraries of dyads based on the diruthenium moiety were synthesized and fully characterized. Where applicable (conjugates with fluorophores), photophysical properties were also measured. The new conjugates and representative intermediates were investigated to assess the impact of compound exposure upon T. gondii β-gal tachyzoites grown in HFF (human foreskin fibroblasts) and noninfected HFF. For each series the influence of the nature of the anchored organic fragment, the type and length of the linking unit were evaluated. The most active and selective compounds were also submitted to dose-response studies. Seven compounds with IC50 values in nanomolar range displayed promising activity and selectivity. For some compounds TEM (transmission electron microscopy) and confocal microscopy tests were also performed. The TEM images suggest that the parasite’s mitochondrion is the preferred targets of the compounds and, importantly, that the host’s mitochondrion (HFF) is not affected. Overall a library of 93 trithiolato-bridged dinuclear ruthenium(II)-arene complexes decorated with different organic substituents were developed. 7 compounds with interesting cytotoxicity/selectivity profile were identified and can be submitted for further in vivo studies

Synthesis and Antiparasitic Activity of New Conjugates—Organic Drugs Tethered to Trithiolato-Bridged Dinuclear Ruthenium(II)–Arene Complexes

Tethering known drugs to a metalorganic moiety is an efficient approach for modulating the anticancer, antibacterial, and antiparasitic activity of organometallic complexes. This study focused on the synthesis and evaluation of new dinuclear ruthenium(II)–arene compounds linked to several antimicrobial compounds such as dapsone, sulfamethoxazole, sulfadiazine, sulfadoxine, triclosan, metronidazole, ciprofloxacin, as well as menadione (a 1,4‐naphtoquinone derivative). In a primary screen, 30 compounds (17 hybrid molecules, diruthenium intermediates, and antimicrobials) were assessed for in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β‐galactosidase (T. gondii β‐gal) at 0.1 and 1 μM. In parallel, the cytotoxicity in noninfected host cells (human foreskin fibroblasts, HFF) was determined by an alamarBlue assay. When assessed at 1 μM, five compounds strongly impaired parasite proliferation by >90%, and HFF viability was retained at 50% or more, and they were further subjected to T. gondii β‐gal dose‐response studies. Two compounds, notably 11 and 13, amide and ester conjugates with sulfadoxine and metronidazole, exhibited low IC50 (half‐maximal inhibitory concentration) values 0.063 and 0.152 μM, and low or intermediate impairment of HFF viability at 2.5 μM (83 and 64%). The nature of the anchored drug as well as that of the linking unit impacted the biological activity

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii β-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model

In vitro antibacterial activity of dinuclear thiolato-bridged ruthenium(II)-arene compounds.

The antibacterial activity of 22 thiolato-bridged dinuclear ruthenium(II)-arene compounds was assessed in vitro against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. None of the compounds efficiently inhibited the growth of the three E. coli strains tested, and only compound 5 exhibited a medium activity against this bacterium [MIC (minimum inhibitory concentration) of 25 µM]. However, a significant antibacterial activity was observed against S. pneumoniae, with MIC values ranging from 1.3 to 2.6 µM for compounds 1-3, 5, and 6. Similarly, compounds 2, 5-7, and 20-22 had MIC values ranging from 2.5 to 5 µM against S. aureus. The tested diruthenium compounds have a bactericidal effect significantly faster than that of penicillin. Fluorescence microscopy assays performed on S. aureus using the BODIPY-tagged diruthenium complex 15 showed that this type of metal compound enters the bacteria and does not accumulate in the cell wall of gram-positive bacteria. Cellular internalization was further confirmed by inductively coupled plasma mass spectrometry experiments. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appears to significantly influence the antibacterial activity. Thus, if overall a decrease of the bactericidal effect with the increase of compounds' molecular weight is observed, however, the complexes bearing larger benzo-fused lactam substituents had low MIC values. This first antibacterial activity screening demonstrated that the thiolato-diruthenium compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies. IMPORTANCE The in vitro assessment of diruthenium(II)-arene compounds against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus showed a significant antibacterial activity of some compounds against S. pneumoniae, with minimum inhibitory concentration (MIC) values ranging from 1.3 to 2.6 µM, and a medium activity against E. coli, with MIC of 25 µM. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appear to significantly influence the antibacterial activity. Fluorescence microscopy showed that these ruthenium compounds enter the bacteria and do not accumulate in the cell wall of gram-positive bacteria. These diruthenium(II)-arene compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies, especially the compounds bearing larger benzo-fused lactam substituents

In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii.

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 μM but had an impact at 2 μM, and induced zebrafish embryotoxicity at 20 μM, but not at 2 or 0.2 μM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue

The Quest of the Best – A SAR Study of Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds Presenting Antiparasitic Properties

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula [(η6-arene)2Ru2(μ2-SR)3]+, symmetric and [(η6-arene)2Ru2(μ2-SR1)2(μ2-SR2)]+, mixed, respectively) is reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by introducing chemical modifications at the level of bridge thiols, and they were grouped into eight families according to their structural features. The selected fittings were guided by previous results and focused on a fine-tuning of the physico-chemical and steric properties. Newly synthesized complexes were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-crystal X-ray structures were obtained. The in vitro biological assessment of the compounds was realized by applying a three-step screening cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing β-galactosidase (T. gondii-β-gal) grown in human foreskin fibroblast monolayers (HFF) and assessment of toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii) studies on the effects in murine splenocytes. A primary screening was performed at 1 and 0.1 μM, and resulted in the selection of 39 compounds that inhibited parasite proliferation at 1 μM by more than 95% and reduced the viability of HFF by less than 49%. In the secondary screening, dose-response assays showed that the selected compounds exhibited half maximal inhibitory concentration (IC50) values for T. gondii-β-gal between 0.01 μM and 0.45 μM, with 30 compounds displaying an IC50 lower than 0.1 μM. When applied to non-infected HFF monolayers at 2.5 μM, 8 compounds caused more than 90% and 31 compounds more than 30% viability impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher than 50% at 2.5 μM. These derivatives were assessed for potential immunosuppressive activities. First, splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysaccharide (LPS) with compounds applied at 0.1 and 0.5 μM. Subsequently, the 5 compounds exhibiting the lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation. Overall, compound 55 [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-o-CF3)2(μ2-SC6H4-p-OH)]Cl exhibited the most favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic efficacy and drug-like propertie

Synthesis, Photophysical Properties and Biological Evaluation of New Conjugates BODIPY - Dinuclear Trithiolato-Bridged Ruthenium(II)-Arene Complexes.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii β-gal (RH strain tachyzoites expressing β-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza- s -indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs ester), as well as that of the length and nature (alkyl vs aryl) of the spacer between the dye and the diruthenium(II) complex moiety, upon the fluorescence and the biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of these series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes to be used for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro