👤 García-Fontán S

Two novel phosphine ligands, Ph₂PCH₂N(CH₂CH₃)₃ (1) and Ph₂PCH₂N(CH₂CH₂CH₂CH₃)₂ (2), and six new metal (Cu(I), Ir(III) and Ru(II)) complexes with those ligands: iridium(III) complexes: Ir(η5-Cp*)Cl₂(1) (1a), Ir(η5-Cp*)Cl₂(2) (2a) (Cp*: Pentamethylcyclopentadienyl); ruthenium(II) complexes: Ru(η6-p-cymene)Cl₂(1) (1b), Ru(η6-p-cymene)Cl₂(2) (2b) and copper(I) complexes: [Cu(CH₃CN)₂(1)BF₄] (1c), [Cu(CH₃CN)₂(2)BF₄] (2c) were synthesized and characterized using elemental analysis, NMR spectroscopy, and ESI-MS spectrometry. Copper(I) complexes turned out to be highly unstable in the presence of atmospheric oxygen in contrast to ruthenium(II) and iridium(III) complexes. The studied Ru(II) and Ir(III) complexes exhibited promising cytotoxicity towards cancer cells in vitro with IC₅₀ values significantly lower than that of the reference drug-cisplatin. Confocal microscopy analysis showed that Ru(II) and Ir(III) complexes effectively accumulate inside A549 cells with localization in cytoplasm and nuclei. A precise cytometric analysis provided clear evidence for the predominance of apoptosis in induced cell death. Furthermore, the complexes presumably induce the changes in the cell cycle leading to G2/M phase arrest in a dose-dependent manner. Gel electrophoresis experiments revealed that Ru(II) and Ir(III) inorganic compounds showed their unusual low genotoxicity towards plasmid DNA. Additionally, metal complexes were able to generate reactive oxygen species as a result of redox processes, proved by gel electrophoresis and cyclic voltamperometry. In vitro cytotoxicity assays were also carried out within multicellular tumor spheroids and efficient anticancer action on these 3D assemblies was demonstrated. It was proven that the hydrocarbon chain elongation of the phosphine ligand coordinated to the metal ions does not influence the cytotoxic effect of resulting complexes in contrast to metal ions type. Show less

It is a major challenge to design novel multifunctional metal-based chemotherapeutic agents for anti-tumor and anti-metastasis applications. Two complexes (OA-Ir and OA-Ru) were synthesized via CuAAC (copper-catalyzed azide-alkyne cycloaddition) reaction from nontoxic Ir-N₃ or Ru-N₃ species and low toxic alkynyl precursor OA-Alkyne, and exhibited satisfactory anti-tumor and anti-metastasis pharmacological effects. Conjugation of Oleanolic acid (OA) and metal-arene species significantly enhanced the cytotoxicity in A2780 cells compared to the precursors through mitochondrial-induced autophagy pathway. Moreover, the two complexes could inhibit the cell metastasis and invasion through damage of actin dynamics and down-regulation of MMP2/MMP9 proteins. Combination of two precursors improved the lipophilicity and biocompatibility, simultaneously enhanced the cell uptake and the mitochondrial accumulation of metal-arene complexes, which caused mitochondrial membrane potential damage, oxidative phosphorylation, ATP depletion and autophagy. Besides, OA-Ir and OA-Ru displayed excellent activity to disintegrate the 3D multicellular tumor spheroids, showing potential for the treatment of solid tumors. This work provides a new way for developing novel metal-based complexes via CuAAC reaction for simultaneously inhibiting tumor proliferation and metastasis. Show less

Glycoconjugation is a powerful tool to improve the anticancer activity of metal complexes. Herein, we modified commercial arylphosphanes with carbohydrate-derived fragments for the preparation of novel glycoconjugated ruthenium(II) p-cymene complexes. Specifically, d-galactal and d-allal-derived vinyl epoxides (VEβ and VEα) were coupled with (2-hydroxyphenyl)diphenylphosphane, affording the 2,3-unsaturated glycophosphanes 1β and 1α. Ligand exchange with [Ru(C₂O₄)(η⁶-p-cymene)(H₂O)] gave the glycoconjugated complexes Ru1β and Ru1α which were subsequently dihydroxylated with OsO₄/N-methylmorpholine N-oxide to Ru2β and Ru2α containing O-benzyl d-mannose and d-gulose units respectively. Besides, aminoethyl tetra-O-acetyl-β-d-glucopyranoside was condensed with borane-protected (4-diphenylphosphanyl)benzoic acid by HATU/DIPEA under MW heating, to afford the amide 3∙BH₃. Zemplén deacylation with MeONa/MeOH gave the deprotected d-glucopyranoside derivative 4∙BH₃. The glycoconjugated phosphane complexes Ru3 and Ru4 were obtained by reaction of the phosphane-boranes 3∙BH₃ and 4∙BH₃ with [Ru(C₂O₄)(η⁶-p-cymene)(H₂O)]. The employed synthetic strategies were devised to circumvent unwanted phosphine oxidation. The compounds were purified by silica chromatography, isolated in high yield and purity and characterized by analytical and spectroscopic (IR and multinuclear NMR) techniques. The behaviour of the six glycoconjugated Ru complexes in aqueous solutions was assessed by NMR and MS measurements. All compounds were screened for their in vitro cytotoxicity against A2780/A2780R human ovarian and MCF7 breast cancer cell lines, revealing a significant cytotoxicity for complexes containing the 2,3-unsaturated glycosyl unit (Ru1β, Ru1α). Additional studies on five other human cancer cells, as well as time-dependent toxicity and cell-uptake analyses on ovarian cancer cells, confirmed the prominent activity of these two compounds - higher than cisplatin - and the better performance of the β anomer. However, Ru1β, Ru1α did not show preferential activity against cancer cells with respect to fetal lung fibroblast and human embryonic kidney cells as models of normal cells. The effects of the two ruthenium glycoconjugated compounds in A2780 ovarian cancer cells were further investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential. The latter is a relevant factor in the mechanism of action of the highly cytotoxic Ru1β, inducing cell death by apoptosis. Show less

In this work, we report on the development of fluorescent half-sandwich iridium complexes using a fluorophore attachment strategy. These constructs consist of pentamethylcyclopentadienyl (Cp*) iridium units ligated by picolinamidate donors conjugated to green-emitting boron-dipyrromethene (bodipy) dyes. Reaction studies in H₂O/THF mixtures showed that the fluorescent Ir complexes were active as catalysts for transfer hydrogenation, with activities similar to that of their non-fluorescent counterparts. The iridium complexes were taken up by NIH-3T3 mouse fibroblast cells, with 50% inhibition concentrations ranging from ~20-70 μM after exposure for 3 h. Visualization of the bodipy-functionalized Ir complexes in cells using fluorescence microscopy revealed that they were localized in the mitochondria and lysosome but not the nucleus. These results indicate that our fluorescent iridium complexes could be useful for future biological studies requiring intracellular catalyst tracking. Show less

Four neutral cyclometalated iridium(III) (Ir^III) dithioformic acid complexes ([(ppy)₂Ir(S^S)], Ir1-Ir4) were designed and synthesized. Toxicity assay revealed that these complexes showed favorable anticancer activity, especially for human non-small cell lung cancer cells (A549). Ir1 exhibited the best anticancer activity (11.0 ± 0.4 μM) was about twice that of cisplatin, meanwhile, which could availably restrain A549 cells migration. Complexes could target mitochondria, induce a decrease in mitochondrial membrane potential (MMP), result in an increase of intracellular reactive oxygen species (ROS) and disruption of the cell cycle, and ultimately generate apoptosis. Western blotting experiment indicated that complexes could inhibit the expression of B cell CLL/lymphoma-2 protein (Bcl-2), induce the expression of BCL2-associated X protein (Bax) and lead to a massive release of Cytochrome C (Cyt-c), which amplified apoptosis signals by activating downstream pathway to promote apoptosis. All these confirmed the existence of mitochondrial anticancer channels for these complexes. Above all, cyclometalated iridium(III) dithioformic acid complexes possess the prospect of becoming a multifunctional cancer therapeutic platform, including mitochondria-targeted imaging, anti-migration, and anticancer agents. Show less

Despite their outstanding properties as potential photosensitizers for photodynamic therapy (PDT), Ir(III) biscyclometalated complexes need both further developments to overcome remaining limitations and in-depth investigations into their mechanisms of action to reach clinic application in the treatment of cancer. This work describes the synthesis of a family of Ir(III) complexes of general formula [Ir(C^N)₂(N^N')]Cl (N^N' = thiabendazole-based ligands; C^N = ppy (2-phenylpyridinate) (Series A), or dfppy (2-(2,4-difluorophenyl)pyridinate) (Series B)) and their evaluation as potential PDT agents. These complexes are partially soluble in water and exhibit cytotoxic activity in the absence of light irradiation versus several cancer cell lines. Furthermore, the cytotoxic activity of derivatives of Series A is enhanced upon irradiation, particularly for complexes [1a]Cl and [3a]Cl, which show phototoxicity indexes (PI) above 20. Endocytosis was established as the uptake mechanism for [1a]Cl and [3a]Cl in prostate cancer cells by flow cytometry. These derivatives mainly accumulate in the mitochondria as shown by colocalization confocal microscopy experiments. Presumably, [1a]Cl and [3a]Cl induce death on cancer cells under irradiation through apoptosis triggered by a multimodal mechanism of action, which likely involves damage over mitochondrial DNA and mitochondrial membrane depolarization. Both processes seem to be the result of photocatalytic oxidation processes. Show less

Despite the clinical success of photodynamic therapy (PDT), the application of this medical technique is intrinsically limited by the low oxygen concentrations found in cancer tumors, hampering the production of therapeutically necessary singlet oxygen (¹O₂). To overcome this limitation, we report on a novel mitochondria-localized iridium(III) endoperoxide prodrug (2-O-IrAn), which, upon two-photon irradiation in NIR, synergistically releases a highly cytotoxic iridium(III) complex (2-IrAn), singlet oxygen, and an alkoxy radical. 2-O-IrAn was found to be highly (photo-)toxic in hypoxic tumor cells and multicellular tumor spheroids (MCTS) in the nanomolar range. To provide cancer selectivity and improve the pharmacological properties of 2-O-IrAn, it was encapsulated into a biotin-functionalized polymer. The generated nanoparticles were found to nearly fully eradicate the tumor inside a mouse model within a single treatment. This study presents, to the best of our knowledge, the first example of an iridium(III)-based endoperoxide prodrug for synergistic photodynamic therapy/photoactivated chemotherapy, opening up new avenues for the treatment of hypoxic tumors. Show less

To reduce the side effects of marketed cancer drugs against triple negative breast cancer cells we have reported mitochondria targeting half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes for MDA-MB-468 cell therapy and diagnosis. Out of five Ir(iii) complexes (IrL1-IrL5), [iridium(iii)-Cp*-2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline]PF₆ (IrL1) has exhibited the best cytoselectivity against MDA-MB-468 cells compared to normal HaCaT cells along with excellent binding efficacy with DNA as well as serum albumin. The subcellular localization study of the complex revealed the localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction via MMP alteration and ROS generation. Moreover, the IrL1 complex facilitated a substantial G₁ phase cell-cycle arrest of MDA-MB-468 cells at the highest tested concentration of 5 μM. The study verdicts support the prospective therapeutic potential of the IrL1 complex in the treatment and eradication of triple negative breast cancer cells. These results validate that these types of scaffolds will be fairly able to exert great potential for tumor diagnosis as well as therapy in the near future. Show less

Cancer stem cells (CSCs) represent a difficult to treat cellular niche within tumours due to their unique characteristics, which give them a high propensity for resistance to classical anti-cancer treatments and the ability to repopulate the tumour mass. An attribute that may be implicated in the high rates of recurrence of certain tumours. However, other characteristics specific to these cells, such as their high dependence on mitochondria, may be exploited for the development of new therapeutic agents that are effective against the niche. As such, a previously described phosphorescent N-heterocyclic carbene iridium(III) compound which showed a high level of cytotoxicity against classical tumour cell lines with mitochondria-specific effects was studied for its potential against CSCs. The results showed a significantly higher level of activity against several CSC lines compared to non-CSCs. Mitochondrial localisation and superoxide production were confirmed. Although the cell death involved caspase activation, their role in cell death was not definitive, with a potential implication of other, non-apoptotic pathways shown. A cytostatic effect of the compound was also displayed at low mortality doses. This study thus provides important insights into the mechanisms and the potential for this class of molecule in the domain of anti-CSC therapeutics. Show less

Title: Synthesis, physicochemical characterization and antiproliferative activity of phosphino Ru(II) and Ir(III) complexes. Abstract: Herein, we present the synthesis of new complexes based on ruthenium(II) (Ru(η6-p-cymene)Cl2PPh2CH2OH (RuPOH) and Ru(η6-p-cymene)Cl2P(p-OCH3Ph)2CH2OH (RuMPOH)) and iridium(III) (Ir(η5-Cp*)Cl2P(p-OCH3Ph)2CH2OH (IrMPOH) and Ir(η5-Cp*)Cl2PPh2CH2OH (IrPOH)) containing phosphine ligands with/without methoxy motifs on phenyl rings (P(p-OCH3Ph)2CH2OH (MPOH) and PPh2CH2OH (POH)). The complexes were characterized by mass spectrometry, NMR spectroscopy (1D: 1H, 13C{1H}, and 31P{1H} and 2D: HMQC, HMBC, and COSY NMR) and elemental analysis. All the complexes were structurally identified by single-crystal X-ray diffraction analysis. The Ru(II) and Ir(III) complexes have a typical piano-stool geometry with an η6-coordinated arene (RuII complexes) or η5-coordinated (IrIII compounds) and three additional sites of ligation occupied by two chloride ligands and the phosphine ligand. Oxidation of NADH to NAD+ with high efficiency was catalyzed by complexes containing P(p-OCH3Ph)2CH2OH (IrMPOH and RuMPOH). The catalytic property might have important future applications in biological and medical fields like production of reactive oxygen species (ROS). Furthermore, the redox activity of the complexes was confirmed by cyclic voltamperometry. Biochemical assays demonstrated the ability of Ir(III) and Ru(II) complexes to induce significant cytotoxicity in various cancer cell lines. Furthermore, we found that RuPOH and RuMPOH selectively inhibit the proliferation of skin cancer cells (WM266-4; IC50, after 24 h: av. 48.3 μM; after 72 h: av. 10.2 μM) while Ir(III) complexes were found to be moderate against prostate cancer cells (DU145). Show less

Quantifying the content of metal-based anticancer drugs within single cancer cells remains a challenge. Here, we used single-cell inductively coupled plasma mass spectrometry to study the uptake and retention of mononuclear (Ir1) and dinuclear (Ir2) Ir^III photoredox catalysts. This method allowed rapid and precise quantification of the drug in individual cancer cells. Importantly, Ir2 showed a significant synergism but not an additive effect for NAD(P)H photocatalytic oxidation. The lysosome-targeting Ir2 showed low dark toxicity in vitro and in vivo. Ir2 exhibited high photocatalytic therapeutic efficiency at 525 nm with an excellent photo-index in vitro and in tumor-bearing mice model. Interestingly, the photocatalytic anticancer profile of the dinuclear Ir2 was much better than the mononuclear Ir1, indicating for the first time that dinuclear metal-based photocatalysts can be applied for photocatalytic anticancer treatment. Show less

Oncosis (from Greek ónkos, meaning "swelling") is a non-apoptotic cell death process related to energy depletion. In contrast to apoptosis, which is the main form of cell death induced by anticancer drugs, oncosis has been relatively less explored but holds potential to overcome drug resistance phenomena. In this study, we report a novel rationally designed mitochondria-targeted iridium(III) complex (OncoIr3) with advantageous properties as a bioimaging agent. OncoIr3 exhibited potent anticancer activity in vitro against cancer cells and displayed low toxicity to normal dividing cells. Flow cytometry and fluorescence-based assays confirmed an apoptosis-independent mechanism involving energy depletion, mitochondrial dysfunction and cellular swelling that matched with the oncotic process. Furthermore, a Caenorhabditis elegans tumoral model was developed to test this compound in vivo, which allowed us to prove a strong oncosis-derived antitumor activity in animals (with a 41% reduction of tumor area). Indeed, OncoIr3 was non-toxic to the nematodes and extended their mean lifespan by 18%. Altogether, these findings might shed new light on the development of anticancer metallodrugs with non-conventional modes of action such as oncosis, which could be of particular interest for the treatment of apoptosis-resistant cancers. Show less

Title: Exploring the in vitro anticancer activities of Re(I) picolinic acid and its fluorinated complex derivatives on lung cancer cells: a structural study. Abstract: Fifteen rhenium(I) tricarbonyl complexes of the form fac-[Re(N,O')(CO)3(X)], where N,O'-bidentate ligand = 2-picolinic acid (Pico); 3,5-difluoropyridine-2-carboxylic acid (Dfpc); 3-trifluoromethyl-pyridine-2-carboxylic acid (Tfpc) and X = H2O; pyrazole (Pz); pyridine (Py); imidazole (Im); and methanol (CH3OH) were synthesized using the '2 + 1' mixed ligand approach with an average yield of 84%. The complexes were characterized using the following spectroscopic techniques: IR, 1H and 13C NMR, UV/Vis, and single-crystal X-ray diffraction. The effect of the fluorine atoms on the backbone of the N,O'-bidentate ligand was investigated and a trend was noticed in the carbonyl stretching frequencies: with Pico < Tfpc < Dfpc. The in vitro biological screening on Vero (healthy mammalian), HeLa (cervical carcinoma) and A549 (lung cancer) cells revealed one toxic complex, fac-[Re(Pico)(CO)3(H2O)], with respective LC50 values of 9.0 ± 0.9, 15.8 ± 4.9 (SI = 0.570) and 20.9 ± 0.8 (SI = 0.430) μg/mL. As a result, it can be used as a positive control drug of toxicity. Show less

Pd(II)-compounds are presently regarded as promising anticancer drugs, as an alternative to Pt(II)-based drugs (e.g., cisplatin), which typically trigger severe side-effects and acquired resistance. Dinuclear Pd(II) complexes with biogenic polyamines such as spermine (Pd2Spm) have exhibited particularly beneficial cytotoxic properties, hence unveiling the importance of understanding their impact on organism metabolism. The present study reports the first nuclear magnetic resonance (NMR)-based metabolomics study to assess the in vivo impact of Pd2Spm on the metabolism of healthy mice, to identify metabolic markers with possible relation to biotoxicity/side-effects and their dynamics. The changes in the metabolic profiles of both aqueous and lipophilic extracts of mice kidney, liver, and breast tissues were evaluated, as a function of drug-exposure time, using cisplatin as a reference drug. A putative interpretation was advanced for the metabolic deviations specifically triggered by Pd2Spm, this compound generally inducing faster metabolic response and recovery to control levels for all organs tested, compared to cisplatin (except for kidney lipid metabolism). These results constitute encouraging preliminary metabolic data suggestive of potential lower negative effects of Pd2Spm administration. Show less

Stimulation of plasma membrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), locally increases the abundance of reactive oxygen species (ROS). These ROS then oxidize cysteine residues in proteins to potentiate downstream signaling. Spatial confinement of ROS is an important regulatory mechanism of redox signaling that enables the stimulation of different RTKs to oxidize distinct sets of downstream proteins. To uncover additional mechanisms that specify cysteines that are redox regulated by EGF stimulation, we performed time-resolved quantification of the EGF-dependent oxidation of 4200 cysteine sites in A431 cells. Fifty-one percent of cysteines were statistically significantly oxidized by EGF stimulation. Furthermore, EGF induced three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamics simulations indicated widespread redox regulation of cryptic cysteine residues that are solvent exposed only upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates served as two distinct modes by which EGF specified the cryptic cysteine residues that became solvent exposed and redox regulated. Because proteins that are structurally regulated by different RTKs or cellular perturbations are largely unique, these findings suggest that solvent exposure and redox regulation of cryptic cysteine residues contextually delineate redox signaling networks. Show less

The recent years have witnessed a rapid increase in the number of scientific articles in biomedical domain. These literature are mostly available and readily accessible in electronic format. The domain knowledge hidden in them is critical for biomedical research and applications, which makes biomedical literature mining (BLM) techniques highly demanding. Numerous efforts have been made on this topic from both biomedical informatics (BMI) and computer science (CS) communities. The BMI community focuses more on the concrete application problems and thus prefer more interpretable and descriptive methods, while the CS community chases more on superior performance and generalization ability, thus more sophisticated and universal models are developed. The goal of this paper is to provide a review of the recent advances in BLM from both communities and inspire new research directions. Show less

The ruthenium-based anticancer agent BOLD-100/KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target-profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells. Show less

As a basic structure of most polypyridinal metal complexes, [Ru(bpy)₃]²⁺, has the advantages of simple structure, facile synthesis and high yield, which has great potential for scientific research and application. However, sonodynamic therapy (SDT) performance of [Ru(bpy)₃]²⁺ has not been investigated so far. SDT can overcome the tissue-penetration and phototoxicity problems compared to photodynamic therapy. Here, we report that [Ru(bpy)₃]²⁺ is a highly potent sonosensitizer and sonocatalyst for sonotherapy in vitro and in vivo. [Ru(bpy)₃]²⁺ can produce singlet oxygen (¹O₂) and sono-oxidize endogenous 1,4-dihydronicotinamide adenine dinucleotide (NADH) under ultrasound (US) stimulation in cancer cells. Furthermore, [Ru(bpy)₃]²⁺ enables effective destruction of mice tumors, and the therapeutic effect can reach deep tissues over 10 cm under US irradiation. This work paves a way for polypyridinal metal complexes to be applied to the noninvasive precise sonotherapy of cancer. Show less

A series of multichromophoric ruthenium(II) complexes with the formulation [Ru(tpy-BODIPY)(tpy-R)]Cl₂ (1-4), having a heteroleptic Ru(II)-bis-tpy (tpy = 4'-phenyl-2,2':6',2″-terpyridine) moiety covalently linked to a boron-dipyrromethene (BODIPY) pendant, have been prepared and characterized and their application as a phototherapeutic and photodetection agent in cancer therapy has been explored. Ligand L¹ with a terpyridine-BODIPY moiety and complex 1 as its PF₆ salt (1a) have been structurally characterized by a single-crystal X-ray diffraction study. Complex 1a has a distorted-octahedral RuN₆ core with a Ru(II)-bis-terpyridine unit that is covalently linked to one photoactive BODIPY unit. The complexes exhibit strong absorbance near 502 nm (ε ≈ (3.7-7.8) × 10⁴ M^-1 cm^-1) and high singlet oxygen sensitization ability, giving singlet oxygen quantum yield (Φ_Δ) values ranging from 0.57 to 0.75 in DMSO. An emission-based study using complex 4 and Singlet Oxygen Sensor Green (SOSG) displays the formation of singlet oxygen inside the cells and also in the buffer medium upon light irradiation. DNA (pUC19) photocleavage experiments using ROS scavengers/stabilizers reveal photoinduced generation of singlet oxygen by a type-II process and of the superoxide anion radical by a type-I process. Complex 4 having a pendant biotin moiety as a cancer cell targeting group shows high photocytotoxicity with a remarkable phototherapeutic index (PI) value of >1400 in HeLa cancer cells with a low light dose activation (400-700 nm, 2.2 J cm^-2). The complexes display reduced activity in noncancerous HPL1D cells. The emission property of the complexes is used for cellular imaging, thus making them suitable as next-generation theranostic PDT agents. Show less

Morpholine motif is an important pharmacophore and, depending on the molecular design, may localize in cellular acidic vesicles. To understand the importance of the presence of pendant morpholine in a metal complex, six bidentate N,O-donor ligands with or without a pendant morpholine unit and their corresponding ruthenium(II) p-cymene complexes (1-6) are synthesized, purified, and structurally characterized by various analytical methods including X-ray diffraction. Complexes 2-4 crystallized in the P2₁/c space group, whereas 5 and 6 crystallized in the P1̅ space group. The solution stability studies using ¹H NMR support instantaneous hydrolysis of the native complexes to form monoaquated species in a solution of 3:7 (v/v) dimethyl sulfoxide-d₆ and 20 mM phosphate buffer (pH* 7.4, containing 4 mM NaCl). The monoaquated complexes are stable for at least up to 24 h. The complexes display excellent in vitro antiproliferative activity (IC₅₀ ca. 1-14 μM) in various cancer cell lines, viz., MDA-MB-231, MiaPaCa2, and Hep-G2. The presence of the pendant morpholine does not improve the dose efficacy, but rather, with 2-[[(2,6-dimethylphenyl)imino]methyl]phenol (HL1) and its pendant morpholine analogue (HL3) giving complexes 1 and 3, respectively, the antiproliferative activity was poorer with 3. MDA-MB-231 cells treated with the complexes show that the acidic vesicles remain acidic, but the population of acidic vesicles increases or decreases with time of exposure, as observed from the dispersed red puncta, depending on the complex used. The presence of the 2,6-disubstituted aniline and the naphthyl group seems to improve the antiproliferative dose. The complex treated MDA-MB-231 cells show that cathepsin D, which is otherwise present in the cytosolic lysosomes, translocates to the nucleus as a result of exposure to the complexes. Irrespective of the presence of a morpholine motif, the complexes do not activate caspase-3 to induce apoptosis and seem to favor the necrotic pathway of cell killing. Show less

Four bipyridine-type ligands variably derivatized with two bioactive groups (taken from ethacrynic acid, flurbiprofen, biotin, and benzylpenicillin) were prepared via sequential esterification steps from commercial 2,2'-bipyridine-4,4'-dicarboxylic acid and subsequently coordinated to ruthenium(II) p-cymene and iridium(III) pentamethylcyclopentadienyl scaffolds. The resulting complexes were isolated as nitrate salts in high yields and fully characterized by analytical and spectroscopic methods. NMR and MS studies in aqueous solution and in cell culture medium highlighted a substantial stability of ligand coordination and a slow release of the bioactive fragments in the latter case. The complexes were assessed for their antiproliferative activity on four cancer cell lines, showing cytotoxicity to the low micromolar level (equipotent with cisplatin). Additional biological experiments revealed a multimodal mechanism of action of the investigated compounds, involving DNA metalation and enzyme inhibition. Synergic effects provided by specific combinations of metal and bioactive fragments were identified, pointing toward an optimal ethacrynic acid/flurbiprofen combination for both Ru(II) and Ir(III) complexes. Show less

Several complexes of general formula [Ru(halide)(η⁶-p-cymene)(α-diimine)]⁺, in the form of nitrate, triflate and hexafluorophosphate salts, including a newly synthesized iodide compound, were investigated as potential anticancer drugs and bactericides. NMR and UV-Vis studies evidenced remarkable stability of the complexes in water and cell culture medium. In general, the complexes displayed strong cytotoxicity against A2780 and A549 cancer cell lines with IC₅₀ values in the low micromolar range, and one complex (RUCYN) emerged as the most promising one, with a significant selectivity compared to the non-cancerous HEK293 cell line. A variable affinity of the complexes for BSA and DNA binding was ascertained by spectrophotometry/fluorimetry, circular dichroism, electrophoresis and viscometry. The performance of RUCYN appears associated to enhanced cell internalization, favored by two cyclohexyl substituents, rather than to specific interaction with the evaluated biomolecules. The chloride/iodide replacement, in one case, led to increased cellular uptake and cytotoxicity at the expense of selectivity, and tuned DNA binding towards intercalation. Complexes with iodide or a valproate bioactive fragment exhibited the best antimicrobial profiles. Show less

Title: GSH-resistant and highly cytoselective ruthenium(II)- Abstract: To avoid the side effects of the current popular platinum-based anticancer drugs, researchers have made tireless attempts to design appropriate GSH-resistant Ru(ii)-arene complexes. In this regard, luminescent ruthenium(ii)-p-cymene-imidazophenanthroline complexes were developed as promising highly cytoselective cancer theraputic agents for HeLa and Caco-2 cells. Show less

A family of six Ru(II) polypyridyl complexes (1-6) which contain phenanthroline-based ligands functionalized with alkyl chains of different lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The influence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to significantly affect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen)₃]²⁺ and [Ru(TAP)₂phen]²⁺. However, an effect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1, 2, 4, and 5 all showed changes in their MLCT bands that could be analyzed using conventional fitting methods, such as the Bard equation. In contrast, complexes 3 and 6, possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1-6 were carried out in the presence of stDNA which confirmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a significant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a significant effect on photophysical and biological properties. Show less

A series of nine Ru^II arene complexes bearing tridentate naphthoquinone-based N,O,O-ligands was synthesized and characterized. Aqueous stability and their hydrolysis mechanism were investigated via UV/vis photometry, HPLC-MS, and density functional theory calculations. Substituents with a positive inductive effect improved their stability at physiological pH (7.4) intensely, whereas substituents such as halogens accelerated hydrolysis and formation of dimeric pyrazolate and hydroxido bridged dimers. The observed cytotoxic profile is unusual, as complexes exhibited much higher cytotoxicity in SW480 colon cancer cells than in the broadly chemo- (incl. platinum-) sensitive CH1/PA-1 teratocarcinoma cells. This activity pattern as well as reduced or slightly enhanced ROS generation and the lack of DNA interactions indicate a mode of action different from established or previously investigated classes of metallodrugs. Show less

Title: Luminescent naphthalimide-tagged ruthenium(ii)-arene complexes: cellular imaging, photocytotoxicity and transferrin binding. Abstract: Two water-soluble piano-stool shaped ruthenium(ii)-arene complexes, [RuII(η6-p-cymene)(L)Cl2] [RuLCl] and [RuII(η6-p-cymene)(L)(PTA)Cl] [RuLPTA], were designed as emissive photocytotoxic agents tagged with morpholine as the lysosome targeting moiety. Here, L = N-(2-morpholinoethyl)-4-(2-aminoethyl)amino-naphthalimide, and PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane. The crystal structure of [RuLCl] exhibits the pseudooctahedral 'three-legged piano-stool' geometry, wherein Ru(ii) is bound to the η6-p-cymene moiety as a base and two chlorides and the amine-N of the ligand L occupies the three legs of the stool. The complexes exhibited both the possibility of covalent adduct formation via the hydrolyzed Ru-Cl bond and non-covalent intercalation binding through planar naphthalimide moieties. The complexes showed enhanced photo-cytotoxicity under low-power blue LED light irradiation (λmax = 448 nm) mediated by 1O2, thereby acting as potential PDT agents. Fluorescence microscopy studies revealed that luminescent complexes preferentially localized in both the lysosomes and nucleus for effectively targeting and damaging the nuclear DNA for PDT effects. Due to enhanced lipophilicity of [RuLCl], it showed higher internalization into MCF-7 cell, measured in terms of the ruthenium content using ICP-MS. The interaction of the complexes with human transferrin (hTf) proteins was studied through molecular docking calculations, suggesting favorable binding through histidine residues and possible internalization into cancer cells via TfR-mediated endocytosis. The luminescence properties of the complexes were well-utilized to study their cellular uptake mechanism via endocytosis using fluorescence microscopy. Show less

The synthesis, photoactivation and biological activity of a new piano-stool Ru(II) complex is herein reported. The peculiarity of this complex is that its monodentate ligand which undergoes the photodissociation is an asymmetric bis-thiocarbohydrazone ligand that possesses a pyridine moiety binding to Ru(II) and the other moiety contains a quinoline that endows the ligand with the capacity of chelating other metal ions. In this way, upon dissociation, the ligand can be released in the form of a metal complex. In this article, the double ability of this new Ru(II) complex to photorelease the ligand and to chelate copper and nickel is explored and confirmed. The biological activity of this compound is studied in cell line A549 revealing that, after irradiation, proliferation inhibition is reached at very low half maximal inhibitory concentration (IC₅₀) values. Further, biological assays reveal that the dinuclear complex containing Ni is internalized in cells. Show less

Title: Role of the (pseudo)halido ligand in ruthenium(II) Abstract: The reactions of the dimeric complexes [RuX2(η6-p-cymene)]2 (X = Br, I, SCN) with L-proline (ProH) and trans-4-hydroxy-L-proline (HypH), in methanol in the presence of NaOH, afforded [RuX(κ2N,O-Pro)(η6-p-cymene)] (X = Br, 1b; I, 1c; SCN, 1d) and [RuX(κ2N,O-Hyp)(η6-p-cymene)] (X = Br, 2b; I, 2c; SCN, 2d), respectively. Alternatively, the one-pot, sequential addition of the appropriate α-amino carboxylate and X- salt to [RuCl2(η6-p-cymene)]2 led to [RuX(κ2N,O-Pro)(η6-p-cymene)] (X = N3, 1e; NO2, 1f; CN 1g) and [Ru(N3)(κ2N,O-Hyp)(η6-p-cymene)] (2e). Complexes [Ru(κ3N,O,O'-O2CCH(NH2)(R)O)(η6-p-cymene)] (R = CH2, 3h; R = CHMe, 4h; R = CH2CH2, 5h) were prepared from the reaction of [RuCl2(η6-p-cymene)]2 with the appropriate α-amino acid and NaOH in refluxing isopropanol. Treatment of the L-serine (SerH2) derivative [RuCl(κ2N,O-SerH)(η6-p-cymene)] (3a) with 1,3,5-triaza-7-phosphaadamantane (PTA) in water at reflux produced [Ru(κ2N,O-Ser)(κP-PTA)(η6-p-cymene)]Cl ([3i]Cl). The products were isolated in good to excellent yields, and were characterized by elemental analysis, IR and multinuclear NMR spectroscopy. The structures of 1f and 2b-e were ascertained by X-ray diffraction studies. The behaviour of the complexes in water and cell culture medium was investigated by multinuclear NMR and UV-Vis spectroscopy, revealing a considerable influence of the monodentate ligand on the aqueous chemistry. Complexes 1d-e, 2d-e, 3h, 4h and [3i]Cl, showing substantial inertness in aqueous media, were assessed for their cytotoxicity towards A2780 and A2780cisR cancer cell lines and the noncancerous HEK 293T cell line. A selection of compounds was also investigated for Ru uptake in A2780 cells and interactions with cytochrome c as a model protein. Combined, these studies provide insights into the previously debated role of the 'leaving' ligand on the biological activity of Ru(II) arene α-amino acid complexes. Show less

Title: Impact of aliphatic acyl and aromatic thioamide substituents on the anticancer activity of Ru(II)- Abstract: Six different acylthiourea ligands (L1-L6) and their corresponding Ru(II)-p-cymene complexes (P1-P6) were designed to explore the structure-activity relationship of the complexes upon aliphatic chain and aromatic conjugation on the C- and N-terminals, respectively. The compounds were synthesized and adequately characterized using various analytical and spectroscopic techniques. The structures of P2-P6, solved using single crystal X-ray diffraction (XRD), confirmed the neutral monodentate coordination of the S atoms of the acylthiourea ligands to Ru(II) ions. In silico studies showed an increase of lipophilicity for the ligands with an increase in alkyl chain length or aromatic conjugation at the C- or N-terminal, respectively. Subsequently, mitogen-activated protein kinases (MAPK) were predicted as one of the primary targets for the complexes, which showed good binding affinity towards extracellular signal-regulated kinases (ERK1, ERK2 and ERK5), c-Jun N-terminal kinase (JNK) and p38 of the MAPK pathway. Henceforth, the complexes were tested for their anticancer activity in lung carcinoma (A549) and cisplatin-resistant lung carcinoma (cisA549R) cells and human umbilical vein epithelial normal cells (HUVEC). Interestingly, an increase in chain length or aromatic conjugation led to an increase in the activity of the complexes, with P5 (7.73 and 13.04 μM) and P6 (6.52 and 14.45 μM) showing the highest activity in A549 and cisA549R cells, which is better than the positive control, cisplatin (8.72 and 44.28 μM). Remarkably, we report the highest activity yet observed for complexes of the type [(η6-p-cymene)RuIICl2(S-acylthiourea)] in the tested cell lines. Aqueous solution studies showed that complexes P5 and P6 are rapidly hydrolyzed to produce solely aquated species that remained stable for 24 h. Staining assays and flow cytometric analyses of P5 and P6 in A549 cells revealed that the complexes induced apoptosis and arrested the cell cycle predominantly in the S phase. In vivo studies demonstrated the higher toxicity of cisplatin and a comparatively higher survival rate of mice injected with the most active complex P6. Histological analyses revealed that treatment with P6 at high doses of up to 8 mg kg-1 did not cause any palpable damage to the tested organs. Show less

Cancer is the leading cause of death in the developed world. In the last few decades, photodynamic therapy (PDT) has augmented the number of medical techniques to treat this disease in the clinics. As the pharmacological active species to kill cancer cells are only generated upon light irradiation, PDT is associated with an intrinsic first level of selectivity. However, since PDT agents also accumulate in the surrounding, healthy tissue and since it is practically very challenging to only expose the tumor site to light, some side effects can be observed. Consequently, there is a need for a selective drug delivery system, which would give a second level of selectivity. In this work, a dual tumor targeting approach is presented based on mesoporous silica nanoparticles, which act by the enhanced permeability and retention effect, and the conjugation to folic acid, which acts as a targeting moiety for folate receptor-overexpressed cancer cells. The conjugates were found to be nontoxic in noncancerous human normal lung fibroblast cells while showing a phototoxic effect upon irradiation at 480 or 540 nm in the low nanomolar range in folate receptor overexpressing cancerous human ovarian carcinoma cells, demonstrating their potential for cancer targeted treatment. Show less