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 fo ‐ cused 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 antimicro ‐ bials) were assessed for in vitro activity against transgenic T. gondii tachyzoites constitutively ex ‐ pressing β‐ galactosidase ( T. gondii β‐ gal) at 0.1 and 1 μ M. In parallel, the cytotoxicity in noninfected host cells (human foreskin fibroblasts, HFF) was determined by an alamarBlue assay. When as ‐ sessed at 1 μ M, 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 stud ‐ ies. Two compounds, notably 11 and 13 , amide and ester conjugates with sulfadoxine and metroni ‐ dazole, exhibited low IC 50 (half ‐ maximal inhibitory concentration) values 0.063 and 0.152 μ M, and low or intermediate impairment of HFF viability at 2.5 μ M (83 and 64%). The nature of the anchored drug as well as that of the linking unit impacted the biological activity. an ‐ timicrobial drugs to trithiolato diruthenium(II)–arene moieties is a promising approach for obtaining new compounds presenting different toxicity profiles than the parent organ ‐ ometallic complexes. The conjugates obtained in this study can be evaluated for other pharmacological applications antiproliferative activity on cancer cells as antibacterials).

Toxoplasmosis is considered one of the most common parasitic diseases affecting approximately one-third of the world's population. In immunocompetent hosts, the infection is usually controlled and asymptomatic, but in immunocompromised persons, such as AIDS patients or persons undergoing immunosuppressive therapy, newly acquired reactivated toxoplasmosis can cause serious complications such as toxoplasmic encephalitis or ocular toxoplasmosis [33], and primary infection during pregnancy, can lead to abortions or fetal malformation. The current therapeutic options are suboptimal, target only the acute disease, and do not eradicate the parasite in chronic infections encysted organisms (bradyzoites) [34,35]. Additionally, adverse side effects are frequently reported [34,36]. Thus, safer, and more effective treatment options are needed.
Cationic trithiolato dinuclear ruthenium(II)-arene complexes represent promising scaffolds. Tethering a functional molecule (e.g., fluorophore, metabolite, drug) to a metal framework is one of the strategies used for tracking, directing, or modulating the biological activity of metal-based complexes [37][38][39]. In numerous cases, conjugation to organometallic moieties led to the enhanced biological activity of the parental drug [4,40]. The trithiolato diruthenium compounds showed high stability and post-functionalization of the bridge thiols proved a useful method to introduce valuable modifications [41][42][43]. This approach proceeds under mild conditions and constitutes an efficient entry to conjugates with biorelevant moieties. The easy access and upscaling of certain mixed trithiolato diruthenium compounds bearing derivatizable groups (e.g., OH, SH, NH2, CO2H) [27,43] (as B, Figure 1) allowed the development and investigation of hybrid structures functionalized with the anticancer drug chlorambucil [41], 7-amino-coumarin fluorophores [43], and peptides [42]. This type of structure is aimed at improving the cytotoxicity against cancer cells [41,42], the water solubility [42] and the selectivity and antiparasitic activity on T. gondii [43]. In this study, a series of structurally diverse antimicrobial agents presenting also relevant anti-Toxoplasma activity have been selected to be conjugated to the trithiolato diruthenium core (Figure 1) [34,36,44,45]. Ruthenium(II)-arene, as well as iridium(III)-and rhodium(III)-cyclopentadienyl are among the favored organometallic units to be tested in intramolecular combination with various drugs.
Another example is a series of organo-ruthenium, rhodium and iridium derivatives of sulfadoxine anchored on N,N′-chelate pyridylimino quinolylimino-bidentate ligands [50] (e.g., D, Figure 2). Screening for in vitro activity against Plasmodium falciparum chloroquine-sensitive and -resistant strains and Mycobacterium tuberculosis showed the activity to be dependent on the organometallic unit, with ruthenium complexes being inactive. The rhodium and iridium compounds inhibited parasite growth with IC50 values in the sub-and low micromolar range, with no significant toxicity towards human embryonic kidney cells (HEK293). Moreover, sulfadoxine was not active in most of the assays, supporting the hypothesis that organometallic conjugates of drugs can beneficially affect bioactivity.
Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol, Figure 1) is an antibacterial agent that was shown to inhibit the in vitro proliferation of T. gondii tachyzoites in the low nanomolar range [52,53]. As triclosan presents poor water solubility and oral bioavailability, various studies focused on derivatives aiming at increased solubility and potency [54][55][56], as well as improved drug delivery and pharmacological properties against T. gondii [57][58][59]. Metronidazole (1-β-hydroxyethyl-2-methyl-5-nitroimidazole, Figure 1) is used to treat antibacterial and antiprotozoal infections [60,61]. A significant reduction of brain cysts was observed in a mouse model of chronic toxoplasmosis after combined treatment with spiramycin and metronidazole [62]. The potential use of ruthenium(II)-benzene metronidazole complex E as a hypoxic cell cytotoxic agent has been assessed, revealing a higher selective toxicity for E compared to the free metronidazole [63].
The antimicrobial drugs, the newly obtained conjugates and the associated intermediates were submitted to a first in vitro screening, assessing the activity against a transgenic T. gondii strain constitutively expressing β-galactosidase (T. gondii β-gal) grown in human foreskin fibroblasts (HFF). In parallel, the cytotoxicity of these compounds was evaluated in noninfected HFF by the alamarBlue assay. The compounds exhibiting interesting antiparasitic activity and low cytotoxicity were subjected to T. gondii IC50 determination.
In the conjugates, the nature of the linker between the diruthenium unit and the drug molecule might be very important for the stability and the biological activity of the hybrid molecule. To extend the purpose of this study, in addition to ester and amide bonds, the use of the triazole ring as a linker was also investigated.

Conjugates with Sulfa-Drugs (Dapsone, Sulfamethoxazole, Sulfadiazine, Sulfadoxine)
Conjugates 8, 9, 10 and 11 were obtained in modest yields (29,24,24 and 35%, respectively) by the amide coupling of the carboxy diruthenium intermediate 2 with commercially available sulfa-drugs dapsone, sulfamethoxazole, sulfadiazine and sulfadoxine in the presence of the coupling agents HOBt and EDCI, in basic conditions (DIPEA) (Scheme 3). The reduced solubility of the starting amines led to poor conversions and yields of isolated pure compounds. Scheme 3. Synthesis of the amide conjugates with the sulfa-drugs dapsone 8, sulfamethoxazole 9, sulfadiazine 10, sulfadoxine 11, and ester conjugates with triclosan 12 and metronidazole 13.

Conjugates with Triclosan and Metronidazole
Ester conjugates with triclosan and metronidazole 12 and 13 were obtained by reacting carboxy complex 2 with the corresponding drugs, both containing free hydroxy groups (Scheme 3). Reactions were performed using EDCI as a coupling agent and DMAP (4-(dimethylamino)pyridine) as a basic catalyst, compounds 12 and 13 being isolated in medium yields of 40 and 51%.
The 'click' metronidazole conjugate 15 was synthesized by the 1,3-dipolar cycloaddition reaction of the alkyne diruthenium intermediate 7 with the metronidazole azide derivative 14 performed in the presence of CuSO4 as a catalyst and sodium ascorbate as a reducing agent (Scheme 4), using an adapted literature procedure [81,82]; conjugate 15 was isolated in 33% yield. The metronidazole azide 14 was synthesized in two steps (activation of the hydroxy group as mesylate followed by the nucleophilic substitution with azide). Of note, this is the first time that the 1,3-dipolar cycloaddition reaction is used for synthesizing conjugates based on the trithiolato diruthenium scaffold.

Conjugates with Ciprofloxacin
For the derivatization of ciprofloxacin, two positions can be considered: the carboxy group in position 3 or the piperazine fragment in position 7 of the fluoroquinolone core [83]. To avoid possible side reactions, the protection of one of these groups was considered prior to attempt connecting this moiety to the trithiolato diruthenium unit. The piperazine fragment of ciprofloxacin was protected using Boc2O (di-tert-butyl dicarbonate) in basic conditions (TEA, triethylamine) following a reported protocol [84] (Scheme 5), and intermediate 16 was isolated in quantitative yield. The 'mixed' hydroxy and amino diruthenium complexes 3 and 4 were reacted with piperazine N-Boc protected ciprofloxacin 16 (Scheme 5). The esterification (conjugate 17) was realized in the presence of EDCI and DMAP, while the amide coupling (conjugate 18) was performed in the presence of HOBt, EDCI, and DIPEA. While the amide conjugate 18 was easily isolated in high yield (87%), the ester conjugate 17 could not be obtained in pure form, as it is prone to hydrolysis/solvolysis during purification. The N-Boc deprotection of 18 was realized in classical acidic conditions [85,86] (TFA, trifluoroacetic acid, Scheme 5), allowing the isolation of compound 19 in 64% yield.

Conjugates with Menadione
Since atovaquone ( Figure 1) and buparvaquone are quinone-based antimicrobial medications for the prevention and treatment of T. gondii [69][70]72] and other parasites, we have considered the development of a small library of compounds in which the 1,4naphtoquinone motif is associated with the trithiolato diruthenium scaffold. To validate the concept, a simpler structure based on the menadione moiety (2-methylnaphthalene-1,4-dione, Figure 1) was approached. First, menadione carboxy derivatives 20, 21 and 22 that can be further anchored on the diruthenium unit were synthesized. This type of modification was previously used to prepare carboxy analogues of lawsone (2-hydroxynaphthalene-1,4-dione) [87][88][89], menadione or plumbagin (5-hydroxy-2-methylnaphthalene-1,4-dione) [90][91][92][93]. Compounds 20, 21 and 22, bearing linkers of different lengths between the 1,4-naphtoquinone moiety and the carboxylic group, were obtained from menadione and succinic, suberic and adipic acid, respectively, in the presence of AgNO3 and (NH4)2S2O3 following literature procedures [90], and were isolated in medium yields of 70, 61 and 52%, respectively (Scheme 6). 20 was further reacted with the hydroxy and amino diruthenium complexes 3 and 4 (Scheme 7). The esterification reaction was realized in the presence of EDCI and DMAP and important issues were encountered in the purification of conjugate 23 due to degradation (59%). The amide coupling was performed in the presence of HOBt, EDCI and DI-PEA, and led to the isolation of 24 in medium yield (54%). Varying the relative proportion between the metal units and the drug fragments in this type of conjugates might lead to improved bio-efficacy [94,95]. To increase the number of 1,4-naphtoquinone molecules anchored on the trithiolato diruthenium core, symmetric intermediates 5 and 6 bearing either three hydroxy or three amino groups were used for the ester and amide couplings with 20, 21 and 22 (Scheme 8). The reaction of 5 with the carboxy 1,4-naphtoquinone derivative 20 in the presence of HOBt, EDCI and DIPEA, allowed only the isolation of the monosubstituted conjugate 25 in a low yield of 27%. At the same time, important degradation of the diruthenium substrate was observed. For the assessment of the biological activity, the compounds were prepared as stock solutions in dimethylsulfoxide (DMSO), in which the compounds are well soluble. 1 H-NMR spectra of similar conjugates (with polypeptides, coumarin units or derivatives with two or three diruthenium units) dissolved in DMSO-d6 or deuterated water, recorded at 25 °C 5 min and 28 days after sample preparation showed no visible changes, demonstrating very good stability of the compounds in this highly complexing solvent and in water. [42,43,80,96] 2.1.6. X-ray Crystallography The crystal structure of the trithiolato diruthenium sulfamethoxazole conjugate 9 was established in the solid state by single-crystal X-ray diffraction (an ORTEP representation is shown in Figure 3), confirming the expected structure. To the best of our knowledge, this is the first example of a structure containing the trithiolato-bridged diruthenium unit and an organic moiety. Data collection and refinement parameters are given in Table 1. Selected bond lengths and angles are presented in Table 2.   Table 2. Comparison of key bond lengths (Å) and angles (°) of the diruthenium moiety in 9 and previously reported mixed complex J (Figure 4, data from ref. [97]).

Complex 9
Complex J

Ru-S
Ru(1)-cent(S1-S3)-Ru (2) 177.30  In the network, an organization in dimers due to the presence of intermolecular Hbonding interactions between the sulfamethoxazole fragments was observed ( Figure 5). These interactions involve sulfonamide NH from one molecule and the carboxyamide oxygen atom of another molecule. Additional H-bonding interactions are observed between the carboxyamide NH and the Cl⁻ counterion. Representative bond lengths and angles for these interactions are given in Table 3.  Table 3).  The activity against T. gondii tachyzoites and HFF (human foreskin fibroblasts) host cells of the new conjugates (13 compounds), of the antimicrobial drugs (8 compounds) and the representative intermediates (9 compounds) has been investigated. The trithiolato intermediates 2-6 have been evaluated previously against T. gondii β-gal under similar conditions [30,43,80], and the corresponding values were introduced in Table 4 and Figure  6 for comparison. Of note, complex 5 exhibited no activity against the parasite [30] and was therefore not included in the discussion of the results. The purity of isolated ciprofloxacin and menadione ester conjugates 17, 23 and 25 was not satisfactory and, therefore, these compounds were not evaluated.

Distance (Å) Angle (°) D-H H•••A D•••A D-H•••A
In a primary screening, transgenic T. gondii tachyzoites constitutively expressing βgalactosidase (T. gondii β-gal) were cultured in HFF monolayers and exposed to concentrations of 1 and 0.1 μM of each compound of interest. In parallel, the cytotoxicity of these compounds was evaluated at the same concentrations in noninfected HFF. As a measure of the parasite proliferation, β-galactosidase activity was determined, while the impact on noninfected HFF was assessed using the alamarBlue assay; the results are summarized in Table 4 and Figure 6.

Diruthenium Intermediates
From the diruthenium intermediates, carboxy and tri-amino derivatives 2 and 6 had no effect on parasite proliferation, but slightly affected HFF viability at 1 μM. In contrast, hydroxy, amino and alkyne functionalized compounds 3, 4 and 7 drastically reduced T. gondii β-gal proliferation when administered at 1 μM but were also toxic to HFF already at 0.1 μM.

Antimicrobial Drugs and Conjugates
Except for sulfadiazine and sulfamethoxazole conjugate 9, all antimicrobial drugs, intermediates based on the antimicrobials, and conjugates did not affect the viability of the HFF even at the highest tested concentration (1 μM) (Table 4, Figure 6).
The poor in vitro anti-Toxoplasma activity of the selected antimicrobial drugs agrees with some previously reported data [44].
If dapsone impaired parasite proliferation even at 0.1 μM, its conjugate 8 inhibited T. gondii β-gal proliferation to 36% only when applied at 1 μM. Both sulfamethoxazole and its conjugate 9 exhibited only reduced effect on the parasite. However, while sulfamethoxazole had little influence on HFF viability, 9 displayed considerable cytotoxicity to host cells at 1 μM.
Remarkably, the sulfadiazine conjugate 10 was not toxic to HFF but inhibited the T. gondii β-gal proliferation to 72% when administrated at 0.1 μM, and completely abolished it at 1 μM. Sulfadiazine was toxic to HFF when administrated at 1 μM, but at 0.1 μM, it did not affect the HFF viability but reduced the T. gondii β-gal proliferation to 57%. When applied at 1 μM, sulfadoxine-conjugate 11 exhibited a stronger effect on the T. gondii βgal proliferation compared to sulfadoxine (11 vs. 83%).
All sulfa-drugs were connected to the trithiolato diruthenium unit via similar strong carbamide bonds. With the exception of the sulfamethoxazole conjugate 9, the nature of the anchored organic drug had little effect on the viability of the host cells, but in some cases, impacted the efficacy against the parasite (e.g., sulfadiazine vs. 10 and sulfadoxine vs. 11). Compared to the trithiolato diruthenium amino analogue 4, conjugates with sulfadrugs were all less cytotoxic to HFF.
Compared to the diruthenium carboxy intermediate 2, ester conjugates with triclosan 12 and metronidazole 13 reduced parasite proliferation more efficiently at 1 μM.
Both triclosan and its ester conjugate 12 presented a similar reduced antiparasitic effect (at 1 μM parasite proliferation was reduced to 71 and 66%, respectively).
Although metronidazole and azide intermediate 14 displayed only low activity against T. gondii β-gal, conjugates 13 (ester) and 15 (triazole) were highly active at 1 μM and almost abolished proliferation. In this case, conjugation to the trithiolato diruthenium unit improved the antiparasitic activity without increasing host toxicity. Alkyne intermediate 7 was more active on the parasite than the carboxy precursor 2, but also more toxic to HFF, while 'click' metronidazole conjugate 15 impacted less the HFF viability than 7.
Compared to amino diruthenium compound 4, amide conjugates with ciprofloxacin 18 and 19, and with menadione 24 were less detrimental to host cell viability.
N-Boc protected ciprofloxacin conjugate 18 exhibited no antiparasitic activity, while the deprotected conjugate 19 had a stronger impact on T. gondii β-gal proliferation in comparison to ciprofloxacin (21 vs . 84% at 1 μM).
When administered at 1 μM, menadione reduced T. gondii β-gal proliferation to 50%, while its derivatives 20, 21 and 22, presenting different chain lengths between the 1,4naphtoquinone unit and the carboxy group, exhibited little antiparasitic effect. Compared to menadione intermediate 20, the amide conjugate 24 presented increased anti-T. gondii β-gal activity, almost abolishing parasite proliferation at 1 μM. Conjugates 26 and 27 presenting three menadione units connected via amide bonds to the trithiolato diruthenium core did not affect parasite proliferation, regardless of the linker size. A similar lack of antiparasitic effect has been previously observed in the case of a coumarin trisubstituted ester derivative [43] and might be associated with the important size of this type of analogue. This observation is corroborated by the results obtained for the monosubstituted conjugate 28, which presented increased efficacy on the parasite compared to intermediate 22. The difference in antiparasitic activity between the monosubstituted derivatives 24 and 28 might be due to the nature of the other two bridging thiols and the length of the linker between the diruthenium scaffold and the menadione moiety.
The first screening allowed the identification of five conjugates, 10 (sulfadiazine), 11 (sulfadoxine), 13 and 15 (metronidazole), 19 (ciprofloxacin) and 24 (menadione) that were more active against T. gondii at 1 μM compared to the respective antimicrobial drug. Concomitantly, these derivatives exhibited low or intermediate impairment of HFF viability at the highest tested concentration (1 μM). The highest antiparasitic activity increase was observed in the case of the metronidazole ester and triazole conjugates 13 and 15, which almost abolished parasite proliferation when administered at 1 μM while the antimicrobial drug was inactive at the same concentration. The most modest amelioration was observed in the case of menadione and its respective conjugate 24 which, at 1 μM, reduced parasite proliferation with 50 and 3%, respectively.

Secondary Screening
Based on the primary screening, conjugates 10, 11, 13, 15 and 24 were selected for the determination of the IC50 values against T. gondii and the assessment of HFF viability after exposure to 2.5 μM. For the selection of the compounds for IC50 determination, two criteria had to be simultaneously satisfied: (i) T. gondii β-gal growth inhibition of 90% or more compared to an untreated control when the compound was applied at 1 μM, and (ii) HFF host cell viability not impaired by more than 50% for a compound applied at 1 μM. The results are summarized in Table 5, and dose-response curves are shown in Figure S1 (S upple me nta r y Supplementary Materials).
For comparison, the results obtained for the carboxy and amino-functionalized diruthenium intermediates 2 and 4, as well as those for pyrimethamine used as the standard, are also shown.  a Data for pyrimethamine, 2 and 4 were previously reported [30,43,80]. b Values at 95% confidence interval (CI); LS is the superior limit of CI and LI is the inferior limit of CI. c The standard error of the regression (SE), represents the average distance that the observed values fall from the regression line. d Control HFF cells treated only with 0.25% DMSO exhibited 100% viability. e The standard deviation of the mean (six replicate experiments).
The most interesting compound of the series is the sulfadoxine conjugate 11, exhibiting a low IC50 (0.063 μM), and only slightly affecting the HFF viability at 2.5 μM (83%). Of note, the IC50 value of 11 is significantly lower than that of the corresponding carboxy diruthenium intermediate (0.181 μM) or that of pyrimethamine (0.326 μM). Interestingly, the sulfadiazine derivative 10 presented poor antiparasitic activity (IC50 0.524 μM, more than 8 times higher compared to 11), and increased toxicity to HFF (62%). These significant differences between these two conjugates underline the importance of the drug fragment for the biological activity, as both conjugates share the same diruthenium moiety and similar bonding between the two units. Sulfadoxine is interesting as this sulfonamide is used in combination with pyrimethamine in the treatment or prevention of malaria [98][99][100][101].
A significant difference was observed between the two metronidazole conjugates 13 and 15. In comparison to 2, the ester conjugate 13 presents a lower IC50 value (0.152 vs. 0.181 μM), but increased HFF toxicity when administered at 2.5 μM (64 vs. 99%). The triazole conjugate 15 exhibited no HFF cytotoxicity but also reduced antiparasitic activity. As both compounds were obtained from the same carboxy diruthenium analogue 2, the differences are likely due to the linking units between the diruthenium moiety and metronidazole.
The amide menadione hybrid 24 was not only more toxic to HFF (viability at 2.5 μM, 32 vs. 51%) but also less active in parasite proliferation inhibition compared to the corresponding amino intermediate 4 (0.481 vs. 0.153 μM).
Overall, the results obtained for conjugates 10, 11, 13, 15 and 24 indicate that this type of hybrid molecules, antimicrobial drug-thiolato-bridged dinuclear ruthenium(II)-arene complex, seems promising and that a fine-tuning of the biological activity can be achieved by a judicious choice of the drugs and connecting units.
The mechanism of action of these trithiolato-bridged dinuclear ruthenium(II)-arene complexes and conjugates has not been yet elucidated. In contrast to almost all other ruthenium(II)-arene complexes presenting labile chlorine or carboxylate ligands, these dinuclear ruthenium(II)-arene compounds do not hydrolyze and are stable in the presence of DNA and amino acids [26]. Oxidation of cysteine (Cys) and glutathione (GSH) to form cystine and glutathione-disulfide (GSSG), respectively, was observed in the presence of some complexes. Still, no correlation between in vitro cytotoxicity and the catalytic activity on the oxidation reaction of glutathione could be established [24,102].
For some compounds, transmission electron microscopy (TEM) detected ultrastructural alterations in the matrix of the T. gondii mitochondria within few hours of treatment, followed by a more pronounced destruction of tachyzoites at later time points [30,43]. Gaining more insight into the mechanisms of action of these dinuclear complexes, responsible for the observed effects on various parasites, will allow a more rational selection of drugs that could be anchored to the diruthenium scaffold (e.g., organic molecules sharing the same molecular target or that can direct the diruthenium fragment to reach a specific biomolecule or organelle).

Chemistry
The experimental chemistry portion, with a full description of experimental procedures and characterization data for all compounds, is presented in the Supplementary Materials.

Crystal-Structure Determination
A crystal of 9 (C60H74ClN3O4Ru2S4•3CH3CH2OH•CHCl3) was mounted in the air at ambient conditions. All measurements were made on a RIGAKU Synergy S area-detector diffractometer [103] using mirror optics monochromated Cu Kα radiation (λ = 1.54184 Å) [104]. The unit cell constants and an orientation matrix for data collection were obtained from a least-squares refinement of the setting angles of reflections in the range 2.82° < θ < 77.133°. A total of 2404 frames were collected using ω scans, with 0.25 s exposure time, a rotation angle of 0.5° per frame, a crystal-detector distance of 65.0 mm, at T = 110(2) K.
Data reduction was performed using the CrysAlisPro [103] program. The intensities were corrected for Lorentz and polarization effects, and an absorption correction based on the multiscan method using SCALE3 ABSPACK in CrysAlisPro [103] was applied. Data collection and refinement parameters are given in Table 1.
The structure was solved by direct methods using SHELXT [105], which revealed the positions of all non-hydrogen atoms of the title compound. All non-hydrogen atoms were refined anisotropically. H-atoms were assigned in geometrically calculated positions and refined using a riding model where each H-atom was assigned a fixed isotropic displacement parameter with a value equal to 1.2 Ueq of its parent atom (1.5 Ueq for methyl groups).
The refinement of the structure was carried out on F 2 using full-matrix least-squares procedures, which minimized the function Σw(Fo 2 − Fc 2 ) 2 . The weighting scheme was based on counting statistics and included a factor to down-weight the intense reflections. All calculations were performed using the SHELXL-2014/7 [106] program in OLEX2 [107].

In Vitro Activity Assessment against T. gondii Tachyzoites and HFF
All tissue culture media were purchased from Gibco-BRL, and biochemical agents from Sigma-Aldrich. Human foreskin fibroblasts (HFF) were purchased from ATCC, maintained in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Waltham, MA, USA) and antibiotics as previously described [108]. Transgenic T. gondii β-gal samples (expressing the β-galactosidase gene from Escherichia coli) were kindly provided by Prof. David Sibley (Washington University, St. Louis, MO, USA) and were maintained, isolated, and prepared for new infections as shown before [108,109].
All the compounds were prepared as 1 mM stock solutions from powder in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). For in vitro activity and cytotoxicity assays, HFF were seeded at 5 × 10 3 /well and allowed to grow to confluence in phenol-red free culture medium at 37 °C and 5% CO2. Transgenic T. gondii β-gal tachyzoites were isolated and prepared for infection as described [108]. T. gondii tachyzoites were released from host cells, and HFF monolayers were infected with freshly isolated parasites (1 × 10 3 /well), and compounds were added concomitantly with infection. In the primary screening, HFF monolayers infected with T. gondii β-gal received 0.1 and 1 μM of each compound, or the corresponding concentration of DMSO (0.01 or 0.1% respectively) as controls and incubated for 72 h at 37˚C/5% CO2 as previously described [96].
For the primary screening at 0.1 and 1 μM, activity was measured as the release of chlorophenol red over time, was calculated as a percentage from the respective DMSO control, which represented 100% of T. gondii β-gal growth. For the IC50 assays, the activity measured as the release of chlorophenol red over time was proportional to the number of live parasites down to 50 per well as determined in pilot assays. IC50 values were calculated after the logit-log-transformation of relative growth and subsequent regression analysis.
All calculations were performed using the corresponding software tool contained in the Excel software package (Microsoft, Redmond, WA, USA). Cytotoxicity assays using uninfected confluent HFF host cells were performed by the alamarBlue assay as previously reported [110]. Confluent HFF monolayers in 96 well-plates were exposed to 0.1, 1 and 2.5 μM of each compound. Non-treated HFF as well as DMSO controls (0.01%, 0.1% and 0.25%) were included. After 72 h of incubation at 37 °C/5% CO2, the medium was removed, and plates were washed once with PBS. 200 μL of Resazurin (1:200 dilution in PBS) were added to each well. Plates were measured at excitation wavelength 530 nm and emission wavelength 590 nM at the EnSpire ® multimode plate reader (PerkinElmer, Inc.). Fluorescence was measured at different time points. Relative fluorescence units were calculated from time points with linear increases.

Conclusions
This study has focused on the synthesis and in vitro evaluation of 13 new conjugates based on trithiolato-bridged ruthenium(II)-arene scaffold tethered with various antimicrobial drugs, aiming at improving the antiparasitic properties and the selectivity.
The type of chemical bond between the two units and their relative proportion was varied. In total, 30 compounds (conjugates, representative intermediates, drugs) were submitted to a first activity screening against T. gondii β-gal tachyzoites cultured in HFF and cytotoxicity determination against HFF host cells, which allowed the identification of five interesting conjugates. The IC50 values against T. gondii and the evaluation of HFF viability after exposure to 2.5 μM led to the selection of the sulfadoxine conjugate 11 as the most promising of this series of 13 conjugates.
Our study suggests that the nature of the drug and of the linker between the drug and the diruthenium(II) moiety greatly impacts biological activity. Overall, anchoring antimicrobial drugs to trithiolato diruthenium(II)-arene moieties is a promising approach for obtaining new compounds presenting different toxicity profiles than the parent organometallic complexes. The conjugates obtained in this study deserve further attention and can be evaluated for other pharmacological applications (e.g., antiproliferative activity on cancer cells or as antibacterials).
Supplementary Materials: The Supplementary Materials are available online at www.mdpi.com/article/10.3390/inorganics9080059/s1. The dose response curves for compounds 10, 11, 13, 15 and 24 as inhibitors of T. gondii β-gal tachyzoites proliferation and the experimental chemistry portion with the full description of experimental procedures. Accession code CCDC 2084579 (compound 9) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_re-quest@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: +44 1223 336033.