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The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC

Five titanium(iv) phenolato complexes were synthesized and fully characterized, and they showed high cytotoxicity, high hydrolytic stability, and enhanced solubility. The halogenated derivatives are identified as the most promising drug candidates.

A new family of titanium(IV) complexes based on [ONON] diaminobis(phenolato) ligands with Me, Br, Cl, and F ortho substitutions was synthesized and characterized. X-ray structures of three derivatives revealed homoleptic L

Asymmetrical 2,2′-((ethane-1,2-diylbis((2-hydroxyethyl)azanediyl))bis(methylene))diphenol substituted compounds and their C1-symmetrical diaminobis(phenolato)-bis(alkoxo) titanium(iv) complexes were synthesized, with one symmetrical analogue. X-ray crystallography corroborated tight ligand binding. Different substitutions on the two aromatic rings enabled fine-tuning of the complex properties, giving enhanced solubility, high anticancer activity (IC50 < 4 μM), and significant hydrolytic stability., Substituting differently the two aromatic rings of bis(phenolato)-bis(alkoxo) ligands yields C1-symmetrical titanium(iv) complexes that are highly soluble and stable in biological media and display marked cytotoxicity toward various cancer cells.

Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein-protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53-iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.

The controlled release of drugs by an external stimulus is of pivotal interest and importance as a means of increasing administration efficacy. Accordingly, many responsive systems have been developed based on primarily pH, temperature, and light changes. Here, a novel electrochemical triggered release of a doxorubicin (Dox)-loaded hydroxyapatite (HAp) nanoparticle (NP) system is presented. Dox is loaded onto HAp NPs by producing a stable dispersion in DMSO. The Dox-HAp NPs are electrophoretically deposited on a stainless steel (S.S) surface. The adsorbed Dox-HAp NPs are released either by applying a moderate electrochemical potential pulse or upon scanning the potential. Two mechanisms were proposed. The first is that the positive potential induces the desorption of the Dox-HAp NPs. Alternatively, the positive potential could drive the oxidation of water and generation of protons, causing the dissolution of the Dox-HAp NPs. In situ characterization techniques, such as atomic force microscopy (AFM) and confocal microscopy, were used to gain insight on the release mechanism. All measurements allude to the electrochemically driven dissolution of the Dox-HAp NPs and release of the embedded drug. In vitro antitumor activity against both HT-29 and A2780 cancer cells revealed that the efficacy of the released Dox was not significantly affected by the electrochemical process. We believe that the electrochemically triggered release of NPs could be applied to many other responsive systems.

Titanium(IV) complexes of diaminobis(phenolato)-bis(alkoxo) ligands are promising anticancer drugs, showing marked in-vivo efficacy with no toxic side-effects in mice, hence, it is of interest to elucidate their mechanism of action. Herein, we employed a fluoro-substituted derivative, FenolaTi, for mechanistic analysis of the active species and its cellular target by quantitative

Orally administered anticancer drugs facilitate treatment, but the acidic conditions in the stomach often challenge their availability. PhenolaTi is a TiIV -based nontoxic anticancer drug with marked in-vivo efficacy. We report that nanoformulation protects phenolaTi from decomposition in stomach-like conditions. This is evidenced by similar NMR characteristics and similar in-vitro cytotoxicity toward murine (CT-26) and human (HT-29) colon cancer cells before and after incubation of nanoformulated phenolaTi (phenolaTi-F) at pH 2, unlike results with the unformulated form of the complex. Furthermore, administration of phenolaTi-F in animal drinking water revealed a notable inhibition of tumor growth in Balb/c and immune-deficient (Nude) mice inoculated with CT-26 and HT-29 cells, respectively. In-vivo efficacy was at least similar to that of the corresponding intraperitoneal treatment with phenolaTi-F and the clinically employed oral drug, capecitabine. No body weight loss or clinical signs of toxicity were evident in the phenolaTi-F-treated animals. These findings demonstrate a new convenient mode of cancer treatment through oral administration by safe titanium-based drugs.

Titanium(IV) anticancer complexes are promising candidates for treatment of various cancers, and previous studies have pointed to possible interactions between Ti(IV) anticancer complexes and the serum proteins albumin and transferrin. Herein, we explored the binding of phenolaTi, a leading diaminobis(phenolato)bis(alkoxo) Ti(IV) anticancer complex, to serum proteins, and derived the binding constants and thermodynamic parameters. The results were compared with those obtained for a salan Ti(IV) bis(isopropoxo) complex and titanocene dichloride, studied under similar conditions. Human serum albumin (HSA) binds phenolaTi in a spontaneous, exothermic process, with a dissociation constant (Kd) of 47 ± 7 μM at room temperature. In the presence of transferrin, the Kd of phenolaTi increases by 2-fold, reflecting the competition between the two proteins over the complex, which was more dominant for the other, less hydrolytically stable complexes tested. Examining the kinetics of the binding, it reaches a maximum after ca. 6 h, and the bond partially dissociates after 24-36 h, presumably due to partial ligand hydrolysis in the absence of cells; nevertheless, the proteins HSA and transferrin have a negligible effect on cytotoxicity after 72 h of incubation, with a possible negative impact on cell entry at short incubation periods. Overall, HSA serves as a carrier for phenolaTi through both its known drug binding sites, presumably in its intact form, which is the species that actively penetrates the cells and inflects cytotoxicity.

Titanium(IV) complexes of diaminobis(phenolato)-bis(alkoxo) ligands are promising anticancer drugs, showing marked in-vivo efficacy with no toxic side-effects in mice, hence, it is of interest to elucidate their mechanism of action. Herein, we employed a fluoro-substituted derivative, FenolaTi, for mechanistic analysis of the active species and its cellular target by quantitative 19F NMR detection to reveal its biodistribution and reactivity in extracellular and intracellular matrices. Upon administration to the serum-containing medium, FenolaTi interacted with bovine serum albumin. 20 h post administration, the cellular accumulation of FenolaTi derivatives was estimated as 37% of the administered compound, in a concentration three orders-of-magnitude higher than the administered dose, implying that active membrane transportation facilitates cellular penetration. An additional 19% of the administered dose that was detected in the extracellular environment had originated from post-apoptotic cells. In the cell, interaction with cellular proteins was detected. Although some intact Ti(IV) complex localized in the nucleus, no signals for isolated DNA fractions were detected and no reactivity with nuclear proteins was observed. Interestingly, higher accumulation of FenolaTi-derived compounds in the endoplasmic reticulum (ER) and interaction with proteins therein were detected, supporting the role of the ER as a possible target for cytotoxic bis(phenolato)-bis(alkoxo) Ti(IV) complexes.

Summary PhenolaTi is an advanced non-toxic anticancer chemotherapy; this inert bis(phenolato)bis(alkoxo) Ti(IV) complex demonstrates the intriguing combination of high and wide efficacy with no detected toxicity in animals. Here we unravel the cellular pathways involved in its mechanism of action by a first genome study on Ti(IV)-treated cells, using an attuned RNA sequencing-based available technology. First, phenolaTi induced apoptosis and cell-cycle arrest at the G2/M phase in MCF7 cells. Second, the transcriptome of the treated cells was analyzed, identifying alterations in pathways relating to protein translation, DNA damage, and mitochondrial eruption. Unlike for common metallodrugs, electrophoresis assay showed no inhibition of DNA polymerase activity. Reduced in vitro cytotoxicity with added endoplasmic reticulum (ER) stress inhibitor supported the ER as a putative cellular target. Altogether, this paper reveals a distinct ER-related mechanism by the Ti(IV) anticancer coordination complex, paving the way for wider applicability of related techniques in mechanistic analyses of metallodrugs., Graphical Abstract, Highlights • First in-depth mechanistic analysis of a non-toxic Ti-based anticancer metallodrug • A comprehensive RNA-seq analysis to map the transcriptomic netowrk initiated by phenolaTi. • Unraveling a distinct mechanism through ER stress, not through direct DNA binding • Toward understanding and applicability of diverse better-tolerable chemotherapies, Organometallic Chemistry; Biochemistry; Genomics; Cancer

Due to the toxicity of platinum compounds used in the clinic as anticancer chemotherapies, titanium serves as a safe and attractive alternative. Lately, we introduced a new family of Ti complexes based on readily available phenolato ligands, demonstrating incredibly high hydrolytic stability, with the lead compound phenolaTi demonstrating wide cytotoxic activity toward the NCI‐60 panel of human cancer cell lines, with an average GI50value of 4.7±2 μm. Herein, we evaluated in vivo: a) the safety, and b) the growth inhibitory capacity (efficacy) of this compound. PhenolaTi was found to be effective in vivo against colon (CT‐26) and lung (LLC‐1) murine cell lines in syngeneic hosts and toward a human colon cancer (HT‐29) cell line in immune‐deficient (Nude) mice, with an efficacy similar to that of known chemotherapy. Notably, no clinical signs of toxicity were observed in the treated mice, namely, no effect on body weight, spleen weight or kidney function, unlike the effects observed with the positive control Pt drugs. Studies of combinations of phenolaTi and Pt drugs provided evidence that similar efficacy with decreased toxicity may be achieved, which is highly valuable for medicinal applications.

Advanced anticancer phenolato titanium(IV) complexes were combined with known chemotherapeutic anticancer drugs applied in the clinic and were analyzed in vitro on cell lines most sensitive to the Ti(IV) complex and relevant to the clinical application of the known drugs. Combination of the Ti(IV) complex with cisplatin on ovarian cells showed mostly an additive behavior, also on a line resistant to cisplatin. Combination of the Ti(IV) complex with fluorouracil on colon cells gave near additive behavior, and that with oxaliplatin gave a synergistic behavior at a wide range of Ti : Pt ratios, but only when the drugs were administered together. Increasing the time intervals between the administration of Ti and of Pt turned the behavior to antagonistic, suggesting some deactivation of Pt by the Ti agent. For combinations where the drugs were applied together, the behavior depended on the effect level, and higher effects gave greater synergism, implying that technical aspects such as solubility are influential. Nevertheless, more complex patterns recorded for combinations where the drugs had been applied separately suggested multiple mechanisms with different concentration dependence. Overall the results point to high medicinal potential for the tested compounds for anticancer combination treatments.

International audience; Vanadium complexes are attractive potential alternatives to platinum-based anticancer drugs. Two vanadium(V) complexes, based on a common chelating tetradentate diaminobis(phenolato) ligand featuring a branched connectivity but differing in their labile ligands, were synthesized and characterized. Whereas the isopropoxido complex was obtained as a mixture of cis and trans isomers with regard to the orientation of the labile group vs. the amine sidearm, the salicylato-containing complex was obtained as a single trans isomer. X-ray structures of the complexes featured octahedral geometry for both. The two complexes exhibited high cytotoxic activity toward different cancer cells, often higher than that of cisplatin, including toward cisplatin-resistant ovarian cancer cells. These complexes demonstrated rapid hydrolysis, releasing the labile ligand within several minutes, with no indications of release of free chelating ligand after water exposure, suggesting that polynuclear hydrolysis products are involved in the cellular activity.

Six homoleptic Ti(IV) compounds of dianionic tridentate Schiff base ligands were synthesized from chiral amino acids, 2-hydroxybenzaldehyde and Ti(OiPr)4. The compounds were spectroscopically characterized and the molecular geometries were established by X-ray crystallography. The ligands coordinated the titanium via carboxylate-O-, imine-N-, and phenoxide-O atoms. Two isomers were identified; each based on a trans-N2O4 donor set, but one with trans carboxylate-O atoms and another with each carboxylate-O atom trans to a phenoxide-O atom. Photophysical profiles exhibited faster excited-state relaxation in the solid phase than in solution. Marked cytotoxicities were recorded toward human ovarian A2780 and colon HT-29 cancer cells with IC50 values ranging between 23±2 and 103±3 µM. Comparative hydrolytic stability studies by NMR in 10% D2O solutions provided t1/2 values of up to 15±2 h, with little correlation to cytotoxicity implying a role of hydrolysis products in the reactivity and identifying steric bulk as a contributor to stability and solubility.

Hydrolytically stable and water soluble Ti(IV) complexes can be used for various applications, including industrial, medicinal and more. Herein, a novel tetra-nuclear Ti(IV) complex with citrate ligands was obtained by reacting Ti(IV) isopropoxide and citric acid in an aqueous solution. The three dimensional structure of the complex [Ti4O6(Hcit)3(cit)]9− 1, as it appears in the compound [Co(NH3)6]3[Ti4O6(Hcit)3(cit)]⋅20H2O was determined by single-crystal X-ray crystallography. Complex 1 possesses an adamantane-like {Ti4O6} core where each metal atom is coordinated to a tri-dentate citrate ligand with one dangling CH2COOH group. An additional already known Ti(IV) complex [Ti(Hcit3−)3]5− 2 was synthesized for comparison. The high hydrolytic stability of complexes 1 and 2 has been substantiated based on NMR techniques. The cytotoxicity of the complexes was tested on three cancer cell lines: human ovarian A2780 and drug-resistant variants A2780cis and A2780ADR. Complex 1 showed a slight cytotoxicity towards the ovarian cells, while complex 2 was inactive.

Racemic and enantiomerically pure titanium(IV) complexes with ortho-brominated or para-nitrated chiral diaminobis(phenolato) ligands were prepared with NH and NMe cyclohexyldiamino bridges through ligand to metal chiral induction. The hydrolytic behavior of the complexes was evaluated, identifying the N-methylated complex as the most stable. A representative NH complex hydrolyzed to first give a dimeric structure in solution as deduced by NMR diffusion measurements, followed by formation of clusters with higher nuclearity, as was supported by X-ray characterization of a tetranuclear cluster obtained in trace amounts following 30 days in water solutions. The cytotoxicity of the enantiomerically pure and racemic complexes was measured on HT-29 human colon cancer cell line based on the MTT assay; all stereochemical configurations of the N-methylated complex were inactive, whereas for the NH complexes, the racemic mixtures were mostly inactive but the pure enantiomers exhibited similarly high cytotoxicity, supporting a polynuclear active species. Analysis of the two enantiomers of the most active brominated complex for their cytotoxicity on human ovarian A2780, cisplatin resistant A2780cp and multi-drug-resistant A2780adr cell lines as well as for their apoptosis induction on the A2780 line revealed similar reactivity, supporting a similar mechanism for the two enantiomers.

Two differently substituted fluorescent salen Ti(iv) complexes were developed. One was inactive on human cancer cells, whereas the other showed high cytotoxicity. Based on live cell imaging, both complexes penetrated the cell, but were not detected in the nuclei. Moreover, the inactive complex was trapped in endocytic vesicles, whereas the active complex accumulated in the perinuclear region and inflected phototoxicity upon continuous irradiation.

Titanium(IV) coordination complexes represent attractive alternatives to platinumbased anticancer drugs. The advantage of the titanium metal lies in its low toxicity, and the hydrolysis of titanium(IV) coordination complexes in biological water-based environment to the safe and inert titanium dioxide is an enormous benefit. On the other hand, the rapid hydrolysis of titanium(IV) complexes in biological environment and their rich aquatic chemistry hampered the exploration and the development of effective compounds. Titanium(IV) complexes were the first to enter clinical trials for cancer treatment following the success of platinum-based chemotherapy, with the pioneering compounds titanocene dichloride and budotitane. Despite the high efficacy and low toxicity observed in vivo, the compounds failed the trials due to insufficient efficacy to toxicity ratio and formulation complications. The rapid hydrolysis of the complexes led to formation of multiple undefined aggregates and difficulties in isolating and identifying the particular active species and its precise cellular target. Numerous derivatives with different labile ligands or substitutions on the inert ones contributed to improve the complex anticancer features, and the best ones were comparable with, and occasionally better than cisplatin. Hydrolytic stability was improved in some cases but remained challenging. The following generation of phenolato-based complexes that came three decades later exhibited high activity and markedly improved stability, where no dissociation was observed for weeks in biological solutions. Complexes of no labile ligands whatsoever that remain intact in solution demonstrated in vitro and in vivo efficacy, with no signs of toxicity to the treated animals. Mechanistic insights gained for the different complexes analyzed include, among others, possible interaction with DNA and induction of apoptosis. Such complexes are highly promising for future exploration and clinical development.

Titanium(IV) coordination complexes represent attractive alternatives to platinumbased anticancer drugs. The advantage of the titanium metal lies in its low toxicity, and the hydrolysis of titanium(IV) coordination complexes in biological water-based environment to the safe and inert titanium dioxide is an enormous benefit. On the other hand, the rapid hydrolysis of titanium(IV) complexes in biological environment and their rich aquatic chemistry hampered the exploration and the development of effective compounds. Titanium(IV) complexes were the first to enter clinical trials for cancer treatment following the success of platinum-based chemotherapy, with the pioneering compounds titanocene dichloride and budotitane. Despite the high efficacy and low toxicity observed in vivo, the compounds failed the trials due to insufficient efficacy to toxicity ratio and formulation complications. The rapid hydrolysis of the complexes led to formation of multiple undefined aggregates and difficulties in isolating and identifying the particular active species and its precise cellular target. Numerous derivatives with different labile ligands or substitutions on the inert ones contributed to improve the complex anticancer features, and the best ones were comparable with, and occasionally better than cisplatin. Hydrolytic stability was improved in some cases but remained challenging. The following generation of phenolato-based complexes that came three decades later exhibited high activity and markedly improved stability, where no dissociation was observed for weeks in biological solutions. Complexes of no labile ligands whatsoever that remain intact in solution demonstrated in vitro and in vivo efficacy, with no signs of toxicity to the treated animals. Mechanistic insights gained for the different complexes analyzed include, among others, possible interaction with DNA and induction of apoptosis. Such complexes are highly promising for future exploration and clinical development.

Vanadium complexes are attractive candidates for anticancer chemotherapy, although often suffering from rich aqueous chemistry and hydrolytic instability. We have introduced an LVO family of vanadium oxo complexes, L being a diaminotris(phenolato) chlelating ligand, demonstrating high hydrolytic stability in water along with promising in vitro and in vivo efficacy. Herein we analyzed mechanistic aspects of the reactivity of such complexes in cellular environment. A representative complex exhibited high activity toward all lines in the NIH NCI-60 panel, with an average GI50 value of 0.7 ± 0.5 μM, and with a unique reactivity pattern implying a distinct mechanism. Free ligands demonstrated cytotoxicity similar to that of their vanadium complexes, were identified in cells treated with the complex, and induced apoptosis as did the parent complex, all implying their participation as active species. Cell cycle studies pointed to possible arrest mostly at the S phase, with some variations for the complex and ligand on the two lines analyzed. Nevertheless, the vanadium ion apparently accelerated cellular entry, as the activity was evident following markedly shorter periods of incubation with the extracellular complex when compared with the free ligand. The results displayed herein overall highlight the role of the vanadium complex as a pro-drug.

Titanium(IV) complexes exhibit high potential as anti-tumor agents, particularly due to their low intrinsic toxicity and cytotoxicity toward cisplatin resistant cells. Nevertheless, Ti(IV) complexes generally undergo rapid hydrolysis that previously hampered their utilization as anticancer drugs. We recently overcame this difficulty by developing a highly stable Ti(IV) complex that is based on tetra-phenolato, hexadentate ligand, formulated into organic nanoparticles. Herein we investigated the activity of this complex in vitro and in vivo. Although inactive when tested directly due to poor solubility, when formulated, this complex displayed (a) high cytotoxicity toward cisplatin resistant human ovarian cells, A2780-cp, with resistance factor of 1.1, (b) additive behavior in combination with cisplatin toward ovarian and colon cancer cells, (c) selectivity toward cancer cells as implied by its mild activity toward non-cancerous, fibroblast lung cells, MRC-5, (d) high stability and durability as manifested by the ability to maintain cytotoxicity, even following one week of incubation in 100% aquatic medium solution, and (e) in vivo efficacy toward solid tumors of human colon cancer cells, HT-29, in nude mice without any clinical signs of toxicity. These features support the formulated phenolato Ti(IV) complex being an effective and selective anti-tumoral agent.

Two diaminobis(phenolato) Ti(IV) complexes were separately combined with azatoxin, camptothecin or cisplatin and underwent in vitro cytotoxicity analysis toward HT-29 and NCI-H1229 cancer cell lines. The methylated salan derivative exhibited synergistic effects with cisplatin at varying ratios toward both lines, when the compounds were administered fresh and simultaneously, implying great medicinal potential.

Titanium compounds are promising anticancer metallodrugs; nevertheless, they generally suffer from hydrolytic instability. Phenolato complexes were developed to address this issue; the first LTiX 2 -type complexes that included two labile groups demonstrated high activity in vitro and in vivo, cancer selectivity and no clinical signs of toxicity to treated animals, and improved hydrolytic stability, giving defined clusters within hours. The following LTi-type complexes gave both similarly high activity and selectivity in vitro and in vivo, independently of formulations, as also established on the NCI-60 panel, and also hydrolytic stability for weeks in water. The complexes operate through apoptotic pathway.

Nanoparticles of titanium(IV) complexes of phenolato ligands were formed and evaluated for cytotoxicity toward human HT-29 colon cancer, murine T-25 lymphoma, and murine HU-2 multidrug-resistant (MDR) cells. The nano-formulation, besides increasing the complexes' shelf lives, is particularly efficient in overcoming limitations in solubility and cell-penetration, thus enhancing biological accessibility; large complexes that were inactive when measured in a non-formulated form showed marked activity when nano-formulated. For active and accessible small complexes, the effect of the formulation was negligible. Most complexes showed similar activity toward MDR cells and their drug-sensitive analogues, further increasing their therapeutic potential. An exception is a particularly hydrophobic complex, which is presumably more accessible to interaction with the membrane ABCB1 (MDR1) transporter active in the multidrug resistance of HU-2 cells. The most efficient compound is a mononuclear complex of a single hexadentate ligand, combining particularly high activity and hydrolytic stability with accessibility aided by the nano-formulation.

Diaminobis(phenolato) ("salan") titanium(IV) complexes of differently substituted aromatic rings were synthesized, and their hydrolytic stability and cytotoxicity were analyzed and compared to those of the C2-symmertrical analogues and their equimolar mixtures. The hydrolytic stability of the asymmetrical complexes was in between those of the symmetrical analogues, implying an additive influence of the ligand structural parameters. Most mixed halogenated/nitrated complexes showed a marked improvement of cytotoxic activity relative to the symmetrical analogues and their mixtures, with IC50 values as low as1 μM corresponding to activity exceeding that of cisplatin by up to 30-fold. In contrast, asymmetrical complexes with substitutions of similar properties revealed an added influence of both, with cytotoxicity in between those of the symmetrical analogues. With the presumption that the active species is generally a polynuclear hydrolysis product kept in mind, it is overall evident that particular ligand design and fine-tuning of the parameters of influence including hydrophilicity and hydrophobicity are essential for maximizing biological efficiency.

Racemic and enantiomerically pure titanium(IV) complexes with ortho-bromo-para-methyl-substituted diaminobis(phenolato) ligands were prepared with NH-, NMe-, and bipyrrolidine-based diamino bridges through ligand-to-metal chiral induction. The hydrolytic stability of the complexes was evaluated, and their cytotoxicity was measured using HT-29 human colon cancer cells based on the MTT assay. All stereochemical forms of the NMe-based complexes, although demonstrating the highest hydrolytic stability, were biologically inactive. For the NH and bipyrrolidine-based active complexes, the pure enantiomers exhibited high cytotoxicity whereas the racemic mixtures were inactive, supporting the involvement of a polynuclear active species. The bipyrrolidine complexes appear to provide the best combination of hydrolytic stability and biological activity, presumably by minimizing steric bulk and consequently enabling biological accessibility.

High-resolution continuum source atomic absorption spectroscopy (HR-CS AAS) is a valuable analytical technique for metal quantification because of its high sensitivity and selectivity for metal atoms as well as its improved background correction mode. However, the quantification of metals in biological materials, e.g. cell lysates, is still challenging because of matrix effects and other experimental complications. A method to quantify the titanium content of tumor cells exposed to titanium-based drugs was developed using HR-CS AAS. This method allows the quantification of titanium in cell suspensions in the low µg L

Cationic titanium(IV) phenolato complexes with a pyridinium ligand were synthesized from their isopropoxo precursors, and featured improved solubility in water/DMSO solutions. High cytotoxicity was recorded toward HT-29 cells, often higher than that of cisplatin. Marked activity enhancement was observed upon substitution with pyridinium ligand, which improved the complexes biological accessibility.

Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(I), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1 −/−) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2′,7′-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels.

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

Eleven bis(dimethylphenolato) Ti(IV) complexes of salen ligands with different steric and electronic properties due to different aromatic substituents at the ortho and para positions are reported, and their cytotoxicity toward HT-29 and OVCAR-1 cells and its dependence on hydrolytic behavior are discussed. Eight complexes of this series were analyzed by X-ray crystallography, confirming the trans geometry of the labile ligands with otherwise relatively similar coordination features to those of cis-salan analogues. Relatively high and similar hydrolytic stability is observed for all complexes, with t(1/2) values for labile ligand hydrolysis of 2-11 h in 10% D(2)O solutions. In contrast, varying cytotoxicities were achieved, identifying selected members as the first trans-Ti(IV) complexes reported as anticancer agents. Steric bulk all around the complex diminished the activity, where a complex with no aromatic substitutions is especially active and complexes substituted particularly at the ortho positions are mostly inactive, including ortho-halogenated and ortho-tert-butylated, with one exception of the ortho-methoxylated complex demonstrating appreciable activity. In contrast, para-halogenation provided the complexes of highest cytotoxic activity in this series (IC(50) as low as 1.0 ± 0.3 μM), with activity exceeding that of cisplatin by up to 15-fold. Reaction of a representative complex with ortho-catechol yielded a "cis"-Ti(IV) complex following rearrangement of the salen ligand on the metal center, with highly similar coordination features and geometry to those of the catecholato salan analogues, suggesting that the complexes operate by similar mechanisms and rearrangement of the salen ligand may occur upon introduction of a suitable chelating target. In additional cytotoxicity measurements, a salen complex was preincubated in the biological medium for varying periods prior to cell addition, revealing that marked cytotoxicity of the salen complex is retained for longer preincubation periods relative to known Ti(IV) complexes, suggesting that the hydrolysis products may also induce cytotoxic effects, thus reducing stability concerns.

The effect of stereochemistry on the cytotoxicity of highly active and hydrolytically stable N-methylated Ti(IV) salan complexes is reported. Four bis(isopropoxo) complexes incorporating N-methylated salan ligands with different aromatic substitution patterns have been prepared in racemic and optically active forms for the first time by ligand-to-metal chiral induction from trans-diaminocyclohexyl-based chiral ligands. The configuration of the metal center that derives from that of the ligand has an enormous influence on cytotoxicity, with the racemic mixture mostly being more active than the single enantiomers that are of either similar or different activity. This implies that the active species is a salan-bound heterochiral polynuclear compound, interacting with a chiral target. Four additional complexes of achiral salan and chiral labile sec-butoxo ligands, analyzed as racemic and as homochiral, revealed no influence of stereochemistry, supporting early dissociation of the labile ligands to give the polynuclear products.

In this paper, we describe our recent results with three families of Ti(IV) complexes, elaborating on their cytotoxic activity, hydrolytic behavior, and the potential connection between the two. Complexes of diamine bis(phenolato) ligands, particularly those of ligands of sequential arrangement of donor atoms, demonstrate appreciable activity towards colon HT-29 and ovarian OVCAR-1 cell, which is higher than that of titanocene dichloride, budotitane and cisplatin. Strong dependence of activity on the particular ligand is observed, where small modifications relating to steric bulk at various locations, electronic effects, ligand lability, general symmetry of the complex and more, have a major effect on the cytotoxic activity. An interesting correlation between cytotoxic activity and hydrolytic behavior is observed. Complexes of cytotoxic activity lead upon addition of water to defined O-bridged clusters within several hours, which is a particularly slow period for such processes, where the bulk of the substituents located near the metal site affects the rate of hydrolysis. In contrast, complexes that are inactive biologically either decompose rapidly, or possess steric bulk too large to form the O-bridged cluster. Additional mechanistic investigations revealed that the activity is independent of transferrin, and that the cell penetration of the active species by an alternative mode occurs relatively rapidly prior to its decomposition to give inactive complexes, among which, the O-bridged cluster. Thus, a relatively inert ligand is required to allow this process and stabilize the active species, while hydrolysis of the more labile groups seems to be essential for its formation. These conclusions are also supported by additional studies conducted with hydroxylamino-triazine compounds, which proved them to be excellent ligands for Ti(IV) forming especially short Ti–N coordinative bonds, thus leading to exceptionally high hydrolytic stability. Some cytotoxic activity was observed for highly inert homoleptic Ti[ONO] 2 complexes of this family, while lack of activity was obtained with analogous substantially more rigid [ONNO]Ti(OiPr) 2 complexes featuring weaker ligand binding and thus hydrolytic instability. Well-defined Ti(IV) complexes of bis(carboxylato) ligands are more difficult to obtain due to the ability of the carboxylato groups to bind in a bidentate mode and bridge two metal centers, nevertheless, such complexes are in general more hydrolytically instable and lack cytotoxic activity.

In this paper we overview our studies on amine–phenolato TiIV complexes as cytotoxic agents, in the context of the results reported thus far with titanocene dichloride, budotitane and derivatives. In particular, we emphasize the studies about the structure–activity relationship performed and important insights gained with the known compounds and our complexes, in regards to their hydrolytic behavior and cytotoxic activity, while pointing to potential mechanistic aspects. Titanocene dichloride and budotitane show cytotoxic activity towards cells that are resistant to cisplatin with reduced side effects. Their main drawback is their hydrolytic instability that has impeded mechanistic investigations and pharmaceutical use. Our new family of cytotoxic complexes was designed to include a single highly electron-donating chelating ligand to afford octahedral TiIV complexes of relatively high hydrolytic stability, with the aim to retain ligand binding throughout the biological activity for achieving controlled processes and allowing mechanistic evaluation. The effect of several parameters on hydrolysis and cytotoxicity were investigated, including those relating to the tetradentate ligand and those relating to the labile groups. Overall we observed high cytotoxic activity that is strongly dependent on the ligand, and which is strongly correlated to the complex hydrolytic behavior. Additional mechanistic studies provide insights regarding the time frame of activity and cell penetration. Some comparisons to titanocene dichloride, budotitane and analogues are highlighted.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

A small difference in diamine bis(phenolato) ligands, namely an additional single methylene unit, directs formation of dinuclear Ti(IV) complexes rather than mononuclear ones as characterized by X-ray crystallography. Varying steric bulk of the ligand affects the coordination number in the dinuclear complexes and the ligand to metal ratio. A ligand with reduced steric bulk leads to a L2Ti2(OiPr)4 type complex featuring two octahedral metal centers bridged only by the two phenolato ligands, whereas a bulky ligand leads to a Ti2(μ-L1)(μ-OiPr)2(OiPr)4 type complex with a single chelating ligand, two bridging isopropoxo ligands, and two terminal isopropoxo groups on each of the two metal centers, which are of trigonal bi-pyramidal geometry.

Ethylene-bridged bis(hydroxylamino-1,3,5-triazine) compounds, that may serve as tetradentate ligands, were synthesized in three steps from 2,4,6-trichloro-1,3,5-triazine. These compounds demonstrate different rotation restrictions around the C(Ar)-N bonds due to their distinctive electronic structure as apparent from their resonative contributors. A dinuclear complex Ti 2(mu-L)2(OiPr)4 (L = bis(triazine)) was synthesized where each octahedral Ti(IV) center is also bound to two isopropoxo groups. The complex rigidity is manifested in a significant deviation from planarity of the aromatic systems, and relatively long Ti-N bonds compared to mononuclear analogous complexes. Increased ligand lability in this complex brings about diminished cytotoxicity toward colon and ovarian cells.

Treating the amine bis(carboxylato) ligand precursor pyridine-2,6-dicarboxylic acid (H 2 L 1 ) with titanium tetra(isopropoxide) gave the mononuclear octacoordinate complex L 1 2 Ti IV (dmf) 2 (1), in which each carboxylato ligand binds in an η 1 fashion and two solvent molecules are also coordinated to the metal. In contrast, employing the diamine bis(carboxylic acid) ligand precursor H 2 L 2 gave a unique hexanuclear Ti IV complex [Ti IV 6 (OiPr) 16 (μ 2 -OiPr) 4 (μ-L 2 ) 2 ] (2), where L 2 = (pyridin-2-ylmethyl)amino diacetate. The crystal structure reveals that all Ti IV atoms exhibit octahedral geometry, where both carboxylate and isopropoxide groups serve as bridging ligands. Surprisingly, no oxido ligands appear in the structure, which indicates that the complex, obtained at room temp., was not exposed to any traces of water. The diamine bis(carboxylato) ligand coordinates in η 2 modes of both carboxyl groups to give a total of six coordination sites. Employing the same ligand with a slight change in reaction conditions, which included heating to 35 °C, led to the formation of two O-bridged complexes: dinuclear [Ti IV 2 (μ 2 -O)(OiPr) 2 -(L 2 ) 2 ] (3) and tetranuclear [Ti IV 4 (μ 2 -O) 4 (L 2 ) 4 ] (4), where each bis(carboxylato) ligand binds to a single Ti center in an η 1 fashion, and the only bridging ligands are oxide groups, with Ti-O-Ti angles of 180.0° and 166.6° for 3 and 4, respectively, indicating π interactions in the dinuclear species. Employing a different diamine bis(carboxylato) ligand featuring a sequential connectivity of donor atoms allowed the identification of a mononuclear C 2 -symmetrical bis(isopropoxido) complex, L 3 Ti IV (OiPr) 2 , (5) where L 3 = ethylenediamine diacetate. In 5, as opposed to 2 and similarly to 1, 4, and 5, the carboxylato ligand binds in an η 1 fashion. Complexes 3, 4, and 5 are all inactive against ovarian OVCAR-1 and colon HT-29 cells.

We have synthesized a number of CuI complexes of thiophenolato ligands with different alkyl substituents in the ortho positions in a single-step procedure starting from a readily available copper(I) precursor to evaluate the coordination preferences of the metal atom. In most cases similar reaction conditions led to the formation of two complexes: a mononuclear complex and a polynuclear cluster, as evident from X-ray crystallography. One dinuclear complex formed has the unique geometry of an L2Cu2(μ-L)2 core that has not previously been observed for related systems. The steric bulk around the metal atom has a clear effect on the coordination sphere of the complex obtained. Ligands with no ortho substituents and those with one substituent with a size varying from methyl to tert-butyl all lead to the formation of three-coordinate complexes for the mononuclear species, and clusters of different sizes where larger substituents lead to larger cages and all copper centers are three-coordinate as well. The introduction of two methyl substituents (one in each ortho position) leads to either a mononuclear two-coordinate copper complex or a polynuclear cluster where two copper centers are three-coordinate and three are two-coordinate.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

In our attempt to define the parameters affecting anticancer activity of titanium complexes and to assess the role of hydrolytic stability, titanium compounds of oxygen-based ligands were studied. A homoleptic complex of hydroxyamino-1,3,5-triazine ligands was prepared and its hydrolysis was investigated by UV-vis spectroscopy at biologically relevant pH and temperature conditions based on its ligand to metal charge transfer absorption band. This complex exhibits very high hydrolytic stability under the conditions measured with negligible ligand dissociation. Its anticancer reactivity was investigated on ovarian OVCAR-1 and colon HT-29 cells, in comparison with the reference highly labile Ti(OiPr)(4) and TiCl(4)(THF)(2) (where THF is tetrahydrofuran), the inert thermodynamically stable TiO2, and the free aromatic hydroxyamino-1,3,5-triazine ligand. Whereas all reference titanium complexes were found to be completely unreactive against both tumor cell types, suggesting some moderate inertness is required, the homoleptic complex of the triazine ligands clearly possess some mild reactivity despite having no labile groups, and despite its incomplete solubility in the concentrations applied. As the free aromatic ligand is highly active under similar conditions, detailed time-dependence measurements were conducted and indicated that the cytotoxicity of the ligand is more affected by reducing incubation time, and that introducing the titanium complex to the medium prior to cell administration does not increase reactivity at a certain incubation time. These findings suggest that the reactivity of the complex does not result from that of the free ligand following dissociation, but rather involves the titanium center.

Vanadium(V) oxo complexes with no labile ligands, including six octahedral complexes with pentadentate diaminotris(phenolato) ligands and one pentacoordinate complex with a tetradentate aminotris(phenolato) ligand, were synthesized in high yields. All octahedral complexes demonstrated high hydrolytic stability with no signs of decomposition after days in the presence of water, whereas the pentacoordinate complex decomposed within minutes to release the free ligand, demonstrating the marked impact of coordination number and geometry on the complex electrophilicity. All complexes showed marked cytotoxicity toward human colon HT-29 and ovarian OVCAR-3 cells. In particular, the octahedral complexes exhibited especially high activity, higher than that of cisplatin by up to 200-fold. Selected complexes demonstrated similarly high activity also toward the A2780 and the A2780cis cisplatin-resistant line. High cytotoxicity was also recorded after prolonged incubation in a DMSO solution at 4 and 37 °C temperatures and in biological medium. In vivo studies pointed to high efficacy in reducing tumor size, where no clinical signs of toxicity were detected in the treated mice. These results overall indicate high potential of the tested compounds as antitumor agents.

Titanium compounds, in particular, Ti(IV) based diaminobis(phenolato) "salan" complexes demonstrate high cytotoxicity towards a wide range of cancer cell lines in vitro, and still, very little is known on their mode of action. A representative salan Ti(IV) complex was tested both in vitro and in vivo on human HT-29 colorectal adenocarcinoma and A2780 ovarian carcinoma cells. Both cell lines were sensitive in vitro with A2780 demonstrating an enhanced rate of uptake and intracellular accumulation and thus an earlier response to the drug. HT-29 cells responded in vivo by impaired tumor development in nude mice. Both cell lines responded in vitro (but to a different extent) by upregulation of p53 with no apparent effect on p21 followed by cell cycle arrest, apoptosis and necrosis as demonstrated by sub-G1 cell accumulation and staining by Annexin-V and propidium iodide. Furthermore, time dependent activation of cysteine-aspartic proteases9 (caspase9) as well as some minor activation of cysteine-aspartic proteases3 (caspase3) support a direct effect on the apoptotic pathway. The differential response of the two cell lines to the salan titanium(IV) complex suggests that more than one pathway is involved in their growth regulation and thus could inhibit development of drug resistant variants.

The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural analysis and affinity and oxidation kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidation of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidation rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.

The activity of amine bis(phenolate) Ti dibenzyl complexes in 1-hexene polymerization catalysis as a function of the phenolate substituents was investigated. Two series of Ti complexes carrying either a dimethylamino or a methoxy sidearm donor were prepared and characterized. In each series, the substituents on the phenolate rings were either alkyl groups of varying bulk or electron-withdrawing chloro groups. When activated with a suitable Lewis acid, all precatalysts have shown reactivity toward 1-hexene. Different structure-activity trends were observed in the two series: The activity of the catalysts of the “OMe” series was low to moderate and was almost independent of the phenolate substituents. In contrast, the activity of the “NMe2” catalysts had a strong dependence on both the steric and electronic character of the phenolate substituents, ranging from mild to highly active. The difference in polymerization activity trends between the two series may be traced back to their different tendency toward misinsertions. A “NMe2”-Ti complex having Cl groups in the ortho, para positions of the phenolate rings led to the most active catalyst and to a remarkably high-Mw polymer (>4 000 000) obtained withi n1ho fpolymerization at RT. The characterization of this ultrahigh-Mw atactic poly(1-hexene) by means of oscillatory rheometry and stress relaxation experiments indicated typical elastomeric behavior at RT.