A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome.

BACKGROUND: G-quadruplex DNA (G4) is a non-canonical structure forming in guanine-rich regions, which play a vital role in cancer biology and are now being acknowledged in both nuclear and mitochondrial (mt) genome. However, the impact of G4-based targeted therapy on both nuclear and mt genome, affecting mt function and its underlying mechanisms remain largely unexplored. METHODS: The mechanisms of action and therapeutic effects of a G4-binding platinum(II) complex, Pt-ttpy, on mitochondria were conducted through a comprehensive approaches with in vitro and in vivo models, including ICP-MS for platinum measurement, PCR-based genetic analysis, western blotting (WB), confocal microscope for mt morphology study, extracellular flux analyzer, JC1 and Annexin V apoptosis assay, flow cytometry and high content microscope screening with single-cell quantification of both ROS and mt specific ROS, as well as click-chemistry for IF study of mt translation. Decipher Pt-ttpy effects on nuclear-encoded mt related genes expression were undertaken via RNA-seq, Chip-seq and CUT-RUN assays. RESULTS: Pt-ttpy, shows a highest accumulation in the mitochondria of A2780 cancer cells as compared with two other platinum(II) complexes with no/weak G4-binding properties, Pt-tpy and cisplatin. Pt-ttpy induces mtDNA deletion, copy reduction and transcription inhibition, hindering mt protein translation. Functional analysis reveals potent mt dysfunction without reactive oxygen species (ROS) induction. Mechanistic study provided first evidence that most of mt ribosome genes are highly enriched in G4 structures in their promoter regions, notably, Pt-ttpy impairs most nuclear-encoded mt ribosome genes' transcription through dampening the recruiting of transcription initiation and elongation factors of NELFB and TAF1 to their promoter with G4-enriched sequences. In vivo studies show Pt-ttpy's efficient anti-tumor effects, disrupting mt genome function with fewer side effects than cisplatin. CONCLUSION: This study underscores Pt-ttpy as a G4-binding platinum(II) complex, effectively targeting cancer mitochondria through dual action on mt and nuclear G4-enriched genomes without inducing ROS, offering promise for safer and effective platinum-based G4-targeted cancer therapy.

Antifungal and Antiparasitic Activities of Metallocene-Containing Fluconazole Derivatives.

The search for new anti-infectives based on metal complexes is gaining momentum. Among the different options taken by researchers, the one involving the use of organometallic complexes is probably the most successful one with a compound, namely, ferroquine, already in clinical trials against malaria. In this study, we describe the preparation and in-depth characterization of 10 new (organometallic) derivatives of the approved antifungal drug fluconazole. Our rationale is that the sterol 14α-demethylase is an enzyme part of the ergosterol biosynthesis route in Trypanosoma and is similar to the one in pathogenic fungi. To demonstrate our postulate, docking experiments to assess the binding of our compounds with the enzyme were also performed. Our compounds were then tested on a range of fungal strains and parasitic organisms, including the protozoan parasite Trypanosoma cruzi (T. cruzi) responsible for Chagas disease, an endemic disease in Latin America that ranks among some of the most prevalent parasitic diseases worldwide. Of high interest, the two most potent compounds of the study on T. cruzi that contain a ferrocene or cobaltocenium were found to be harmless for an invertebrate animal model, namely, Caenorhabditis elegans (C. elegans), without affecting motility, viability, or development.

Strategies for the Nuclear Delivery of Metal Complexes to Cancer Cells.

The nucleus is an essential organelle for the function of cells. It holds most of the genetic material and plays a crucial role in the regulation of cell growth and proliferation. Since many antitumoral therapies target nucleic acids to induce cell death, tumor-specific nuclear drug delivery could potentiate therapeutic effects and prevent potential off-target side effects on healthy tissue. Due to their great structural variety, good biocompatibility, and unique physico-chemical properties, organometallic complexes and other metal-based compounds have sparked great interest as promising anticancer agents. In this review, strategies for specific nuclear delivery of metal complexes are summarized and discussed to highlight crucial parameters to consider for the design of new metal complexes as anticancer drug candidates. Moreover, the existing opportunities and challenges of tumor-specific, nucleus-targeting metal complexes are emphasized to outline some new perspectives and help in the design of new cancer treatments.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

BASHY Dyes Are Highly Efficient Lipid Droplet-Targeting Photosensitizers that Induce Ferroptosis through Lipid Peroxidation.

Ferroptosis is an iron-dependent lipid-peroxidation-driven mechanism of cell death and a promising therapeutic target to eradicate cancer cells. In this study, we discovered that boronic acid-derived salicylidenehydrazone (BASHY) dyes are highly efficient singlet-oxygen photosensitizers (PSs; ΦΔ up to 0.8) that induce ferroptosis triggered by photodynamic therapy. The best-performing BASHY dye displayed a high phototoxicity against the human glioblastoma multiform U87 cell line, with an IC50 value in the low nanomolar range (4.40 nM) and a remarkable phototoxicity index (PI > 22,700). Importantly, BASHY dyes were shown to accumulate in lipid droplets (LDs) and this intracellular partition was found to be essential for the enhanced phototoxicity and the induction of ferroptosis through lipid peroxidation. The safety and phototoxicity of this platform were validated using an in vivo zebrafish model (Danio rerio).

In Situ Bioconjugation of a Maleimide-Functionalized Ruthenium-Based Photosensitizer to Albumin for Photodynamic Therapy.

Maleimide-containing prodrugs can quickly and selectively react with circulating serum albumin following their injection in the bloodstream. The drug-albumin complex then benefits from longer blood circulation times and better tumor accumulation. Herein, we have applied this strategy to a previously reported highly phototoxic Ru polypyridyl complex-based photosensitizer to increase its accumulation at the tumor, reduce off-target cytotoxicity, and therefore improve its pharmacological profile. Specifically, two complexes were synthesized bearing a maleimide group: one complex with the maleimide directly incorporated into the bipyridyl ligand, and the other has a hydrophilic linker between the ligand and the maleimide group. Their interaction with albumin was studied in-depth, revealing their ability to efficiently bind both covalently and noncovalently to the plasma protein. A crucial finding is that the maleimide-functionalized complexes exhibited significantly lower cytotoxicity in noncancerous cells under dark conditions compared to the nonfunctionalized complex, which is a highly desirable property for a photosensitizer. The binding to albumin also led to a decrease in the phototoxicity of the Ru bioconjugates in comparison to the nonfunctionalized complex, probably due to a decreased cellular uptake. Unfortunately, this decrease in phototoxicity was not compensated by a dramatic increase in tumor accumulation, as was demonstrated in a tumor-bearing mouse model using inductively coupled plasma mass spectrometry (ICP-MS) studies. Consequently, this study provides valuable insight into the future design of in situ albumin-binding complexes for photodynamic therapy in order to maximize their effectiveness and realize their full potential.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Since these diseases target the most vulnerable communities who are disadvantaged by health and socio-economic factors, new agents should be, if possible, easy-to-prepare to allow for commercialization based on their low cost. In this study, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030. Overall, the discovery of these new compounds with broad-spectrum activity opens new avenues for the development of treatments for several current human infections, either caused by fungi or by parasites, including other NTDs, as well as newly emerging diseases. ONE-SENTENCE SUMMARY: Simple derivatives of the well-known antifungal drug fluconazole were found to be highly effective in vivo against fungal infections, and also potent against the parasitic nematode Brugia, which causes lymphatic filariasis and against Trichuris, one of the soil-transmitted helminths that infects millions of people globally.

Structurally Simple Osmium(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy in the Near Infrared.

Five osmium(II) polypyridyl complexes of the general formula [Os(4,7-diphenyl-1,10-phenanthroline)2 L]2+ were synthesized as photosensitizers for photodynamic therapy by varying the nature of the ligand L. Thanks to the pronounced π-extended structure of the ligands and the heavy atom effect provided by the osmium center, these complexes exhibit a high absorption in the near-infrared (NIR) region (up to 740 nm), unlike related ruthenium complexes. This led to a promising phototoxicity in vitro against cancer cells cultured as 2D cell layers but also in multicellular tumor spheroids upon irradiation at 740 nm. The complex [Os(4,7-diphenyl-1,10-phenanthroline)2 (2,2'-bipyridine)]2+ was found to be the most efficient against various cancer cell lines, with high phototoxicity indexes. Experiments on CT26 tumor-bearing BALB/c mice also indicate that the OsII complexes could significantly reduce tumor growth following 740 nm laser irradiation. The high phototoxicity in the biological window of this structurally simple complex makes it a promising photosensitizer for cancer treatment.

Polymeric encapsulation of a ruthenium(ii) polypyridyl complex: from synthesis to in vivo studies against high-grade epithelial ovarian cancer.

The in vitro to in vivo translation of metal-based cytotoxic drugs has proven to be a significant hurdle in their establishment as effective anti-cancer alternatives. Various nano-delivery systems, such as polymeric nanoparticles, have been explored to address the pharmacokinetic limitations associated with the use of these complexes. However, these systems often suffer from poor stability or involve complex synthetic procedures. To circumvent these problems, we report here a simple, one-pot procedure for the preparation of covalently-attached Ru-polylactide nanoparticles. This methodology relies on the ring-opening polymerization of lactide initiated by a calcium alkoxide derivative formed from calcium bis(trimethylsilyl amide) and a hydroxyl-bearing ruthenium complex. This procedure proceeds with high efficiency (near-quantitative incorporation of Ru in the polymer) and enables the preparation of polymers with varying molecular weights (2000-11000 Da) and high drug loadings (up to 68% w/w). These polymers were formulated as narrowly dispersed nanoparticles (110 nm) that exhibited a slow and predictable release of the ruthenium payload. Unlike standard encapsulation methods routinely used, the release kinetics of these nanoparticles is controlled and may be adjusted on demand, by tuning the size of the polymer chain. In terms of cytotoxicity, the nanoparticles were assessed in the ovarian cancer cell line A2780 and displayed potency comparable to cisplatin and the free drug, in the low micromolar range. Interestingly, the activity was maintained when tested in a cisplatin-resistant cell line, suggesting a possible orthogonal mechanism of action. Additionally, the internalization in tumour cells was found to be significantly higher than the free ruthenium complex (>200 times in some cases), clearly showcasing the added benefit in the drug's cellular permeation and accumulation of the drug. Finally, the in vivo performance was evaluated for the first time in mice. The experiments showed that the intravenously injected nanoparticles were well tolerated and were able to significantly improve the pharmacokinetics and biodistribution of the parent drug. Not only was the nanosystem able to promote an 18-fold increase in tumour accumulation, but it also allowed a considerable reduction of drug accumulation in vital organs, achieving, for example, reduction levels of 90% and 97% in the brain and lungs respectively. In summary, this simple and efficient one-pot procedure enables the generation of stable and predictable nanoparticles capable of improving the cellular penetration and systemic accumulation of the Ru drug in the tumour. Altogether, these results showcase the potential of covalently-loaded ruthenium polylactide nanoparticles and pave the way for its exploitation and application as a viable tool in the treatment of ovarian cancer.

Towards Selective Delivery of a Ruthenium(II) Polypyridyl Complex-Containing Bombesin Conjugate into Cancer Cells.

An increasing number of novel Ru(II) polypyridyl complexes have been successfully applied as photosensitizers (PSs) for photodynamic therapy (PDT). Despite recent advances in optimized PSs with refined photophysical properties, the lack of tumoral selectivity is often a major hurdle for their clinical development. Here, classical maleimide and versatile NHS-activated acrylamide strategies were employed to site-selectively conjugate a promising Ru(II) polypyridyl complex to the N-terminally Cys-modified Bombesin (BBN) targeting unit. Surprisingly, the decreased cell uptake of these novel Ru-BBN conjugates in cancer cells did not hamper the high phototoxic activity of the Ru-containing bioconjugates and even decreased the toxicity of the constructs in the absence of light irradiation. Overall, although deceiving in terms of selectivity, our new bioconjugates could still be useful for advanced cancer treatment due to their nontoxicity in the dark.

A New Derivative of Retro-2 Displays Antiviral Activity against Respiratory Syncytial Virus.

Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in newborns, with all children being infected before the age of two. Reinfections are very common throughout life and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.

In Vivo Sustained Release of the Retrograde Transport Inhibitor Retro-2.1 Formulated in a Thermosensitive Hydrogel.

A recently developed inhibitor of retrograde transport, namely Retro-2.1, proved to be a potent and broad-spectrum lead in vitro against intracellular pathogens, such as toxins, parasites, intracellular bacteria and viruses. To circumvent its low aqueous solubility, a formulation in poly(ethylene glycol)-block-poly(D,L)lactide micelle nanoparticles was developed. This formulation enabled the study of the pharmacokinetic parameters of Retro-2.1 in mice following intravenous and intraperitoneal injections, revealing a short blood circulation time, with an elimination half-life of 5 and 6.7 h, respectively. To explain the poor pharmacokinetic parameters, the metabolic stability of Retro-2.1 was studied in vitro and in vivo, revealing fast cytochrome-P-450-mediated metabolism into a less potent hydroxylated analogue. Subcutaneous injection of Retro-2.1 formulated in a biocompatible and bioresorbable polymer-based thermosensitive hydrogel allowed for sustained release of the drug, with an elimination half-life of 19 h, and better control of its metabolism. This study provides a guideline on how to administer this promising lead in vivo in order to study its efficacy.

Red-Absorbing Ru(II) Polypyridyl Complexes with Biotin Targeting Spontaneously Assemble into Nanoparticles in Biological Media.

Four new ruthenium(II) polypyridyl complexes were synthesized to study the effect of poly(ethylene glycol) and/or biotin conjugation on their physical and biological properties, including their hydrophilicity, their cellular uptake, and their phototoxicity. Unexpectedly, these complexes self-assembled into nanoparticles upon dilution in biological media. This behavior leads to their accumulation in lysosomes following their internalization by cells. While a significant increase in cellular uptake was observed for the biotin-conjugated complexes, it did not result in an increase in their phototoxicity. However, their high phototoxicity upon irradiation at long wavelengths (645-670 nm) and their self-assembling behavior make them a promising backbone for the development of new lysosome-targeted photosensitizers for photodynamic therapy.

Is antitumor Pt(IV) complex containing two axial lonidamine ligands a true dual- or multi-action prodrug?

This work studied the mechanism of action of a Pt(IV) complex 2 bearing two axial lonidamine ligands, which are selective inhibitors of aerobic glycolysis. The presence of two lonidamine ligands in 2 compared to the parent Pt(II) complex increased its antiproliferative activity, cellular accumulation, and changed its cell cycle profile and mechanism of cell death. In 3D cell culture, 2 showed exceptional antiproliferative activity with IC50 values as low as 1.6 µM in MCF7 cells. The study on the influence of the lonidamine ligands in the Pt complex on glycolysis showed only low potency of ligands to affect metabolic processes in cancer cells, making the investigated complex, not a dual- or multi-action prodrug. However, the Pt(IV) prodrug effectively delivers the cytotoxic Pt(II) complex into cancer cells.

Metallodrug Profiling against SARS-CoV-2 Target Proteins Identifies Highly Potent Inhibitors of the S/ACE2 interaction and the Papain-like Protease PLpro.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100 structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PLpro . In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PLpro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.

Physical, spectroscopic, and biological properties of ruthenium and osmium photosensitizers bearing diversely substituted 4,4'-di(styryl)-2,2'-bipyridine ligands.

Capitalising on the previous identification of a distyryl coordinated Ru(II) polypyridine complex as a promising photosensitizer for photodynamic therapy, eight new complexes were synthesized by modifications of the ligands or by changing the metal coordinated. We report in this work the effects of these modifications on the physical, spectroscopic, and biological properties of the synthesized complexes. Subtle structural modifications of the distyryl ligand only had a moderate effect on the corresponding complexes' visible light absorption and singlet oxygen quantum yield. These modifications however had a significant effect on the lipophilicity, the cellular uptake and the phototoxicity of the complexes. Although the lipophilicity of the complexes had a somewhat expected effect on their cellular uptake, this last parameter could not be directly correlated to their phototoxicity, revealing other underlying phenomena. Overall, this work allowed identification of two promising ruthenium complexes as photosensitisers for photodynamic therapy and provides some guidance on how to design better photosensitizers.

Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity.

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.

Increased Lipophilicity of Halogenated Ruthenium(II) Polypyridyl Complexes Leads to Decreased Phototoxicity in†vitro when Used as Photosensitizers for Photodynamic Therapy.

In the fight against cancer, photodynamic therapy is generating great interest thanks to its ability to selectively kill cancer cells without harming healthy tissues. In this field, ruthenium(II) polypyridyl complexes, and more specifically, complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) as a ligand are of particular interest due to their DNA-binding and photocleaving properties. However, ruthenium(II) polypyridyl complexes can sometimes suffer from low lipophilicity, which hampers cellular internalisation through passive diffusion. In this study, four new [Ru(dppz-X2 )3 ]2+ complexes (X=H, F, Cl, Br, I) were synthesized and their lipophilicity (logP), cytotoxicity and phototoxicity on cancerous and noncancerous cell lines were assessed. This study shows that, counterintuitively, the phototoxicity of these complexes decreases as their lipophilicity increases; this could be due solely to the atomic radius of the halogen substituents.

Rationally Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy.

The utilization of photodynamic therapy (PDT) for the treatment of various types of cancer has gained increasing attention over the last decades. Despite the clinical success of approved photosensitizers (PSs), their application is sometimes limited due to poor water solubility, aggregation, photodegradation, and slow clearance from the body. To overcome these drawbacks, research efforts are devoted toward the development of metal complexes and especially Ru(II) polypyridine complexes based on their attractive photophysical and biological properties. Despite the recent research developments, the vast majority of complexes utilize blue or UV-A light to obtain a PDT effect, limiting the penetration depth inside tissues and, therefore, the possibility to treat deep-seated or large tumors. To circumvent these drawbacks, we present the first example of a DFT guided search for efficient PDT PSs with a substantial spectral red shift toward the biological spectral window. Thanks to this design, we have unveiled a Ru(II) polypyridine complex that causes phototoxicity in the very low micromolar to nanomolar range at clinically relevant 595 nm, in monolayer cells as well as in 3D multicellular tumor spheroids.

Increasing the Cytotoxicity of Ru(II) Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo Ligands.

Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)2(sq)](PF6) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure-activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP)2 core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes 1-4 in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes 5 and 6, on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes 1-6 led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes 1-4 expressed much higher cytotoxicities than complexes 5 and 6. Complex 1 constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC50 = 0.07-0.7 µM in different cancerous cell lines), complex 1 was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex 1 with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex 1 a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex 1 for further in vivo studies.