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Multidrug resistance is a major impediment to chemotherapy and limits the efficacies of conventional anticancer drugs. A strategy to bypass multidrug resistance is to develop new drug candidates capable of inducing apoptosis-independent programmed cell death. However, cellular pathways implicated in alternative programmed cell death are not well-elucidated and multifactorial, making a target-based discovery approach a challenge. Here, we show that a coordination-directed three-component assembly and phenotypic screening strategy could be employed as a viable alternative for the identification of apoptosis-independent organoruthenium anticancer agents. Through an on-plate synthesis and screening of 195 organoruthenium complexes against apoptosis-sensitive and -resistant cancers, we identified two apoptosis-independent hits. Subsequent validation of the two hits showed a lack of induction of apoptotic biomarkers, a caspase-independent activity and an equal efficacy in both apoptosis-sensitive and -resistant colorectal cancers. This validated their apoptosis-independent modes-of-action, paving the way as potential candidates for the treatment of highly-refractory cancer phenotypes. Show less

The complexes cis-[Ru(quin)(dppm)₂]PF₆ and cis-[Ru(kynu)(dppm)₂]PF₆ (quin = quinaldate; kynu = kynurenate; dppm = bis(diphenylphosphino)methane) were prepared and characterized by elemental analysis, electronic, FTIR, ¹H, and ³¹P{¹H} NMR spectroscopies. Characterization data were consistent with a cis arrangement for the dppm ligands and a bidentate coordination through carboxylate oxygens of the quin and kynu anions. These complexes were not able to intercalate CT-DNA as shown by circular dichroism spectroscopy. On the other hand, bovine serum albumin (BSA) binding constants and thermodynamic parameters suggest spontaneous interactions with this protein by hydrogen bonds and van der Waals forces. Cytotoxicity assays were carried out on a panel of human cancer cell lines including HepG2, MCF-7, and MO59J and one normal cell line GM07492A. In general, the new ruthenium(II) complexes displayed a moderate to high cytotoxicity in all the assayed cell lines with IC₅₀ ranging from 10.1 to 36 µM and were more cytotoxic than the precursor cis-[RuCl₂(dppm)₂]. The cis-[Ru(quin)(dppm)₂]PF₆ were two to three times more active than the reference metallodrug cisplatin in the MCF-7 and MO59J cell lines. Show less

Coordinatively saturated ruthenium complexes with a variable net charge are currently under intense investigation for their anticancer potential. These complexes, possessing long wavelength metal-to-ligand charge transfer with DNA photonuclease activity, have shown promising cytotoxic profiles. Although most of the ruthenium complexes exhibit significant photochemotherapeutic activity, their poor entry into cells hinder their development as potential drug molecules. Here, we report the synthesis and characterization of four new ruthenium (II) azo-8-hydroxyquinoline complexes, their mode of in vitro DNA binding and antiproliferative properties against cultured human cancer cell lines. The activity of these compounds prior to photoirradiation is minimal. However, they could induce DNA photonuclease activity through the generation of reactive oxygen species upon exposure to light. The activities exhibited by these complexes were found to be more efficient (>5-fold) than cisplatin, emphasizing their therapeutic potential. Collectively, these results support the idea that ruthenium (II) azo-8-hydroxyquinoline complexes can serve as potential agents in photodynamic anticancer therapy. Show less

Seven rhenium(I) complexes of the general formula fac-[Re(CO)₃(NN)(OH₂)]⁺ where NN = 2,2'-bipyridine (8), 4,4'-dimethyl-2,2'-bipyridine (9), 4,4'-dimethoxy-2,2'-bipyridine (10), dimethyl 2,2'-bipyridine-4,4'-dicarboxylate (11), 1,10-phenanthroline (12), 2,9-dimethyl-1,10-phenanthroline (13), or 4,7-diphenyl-1,10-phenanthroline (14), were synthesized and characterized by ¹H NMR spectroscopy, IR spectroscopy, mass spectrometry, and X-ray crystallography. With the exception of 11, all complexes exhibited 50% growth inhibitory concentration (IC₅₀) values that were less than 20 μM in HeLa cells, indicating that these compounds represent a new potential class of anticancer agents. Complexes 9, 10, and 13 were as effective in cisplatin-resistant cells as wild-type cells, signifying that they circumvent cisplatin resistance. The mechanism of action of the most potent complex, 13, was explored further by leveraging its intrinsic luminescence properties to determine its intracellular localization. These studies indicated that 13 induces cytoplasmic vacuolization that is lysosomal in nature. Additional in vitro assays indicated that 13 induces cell death without causing an increase in intracellular reactive oxygen species or depolarization of the mitochondrial membrane potential. Further studies revealed that the mode of cell death does not fall into one of the canonical categories such as apoptosis, necrosis, paraptosis, and autophagy, suggesting that a novel mode of action may be operative for this class of rhenium compounds. The in vivo biodistribution and metabolism of complex 13 and its ^99mTc analogue 13* were also evaluated in naı̈ve mice. Complexes 13 and 13* exhibited comparable biodistribution profiles with both hepatic and renal excretion. High-performance liquid chromatography inductively coupled plasma mass-spectrometry (HPLC-ICP-MS) analysis of mouse blood plasma and urine postadministration showed considerable metabolic stability of 13, rendering this potent complex suitable for in vivo applications. These studies have shown the biological properties of this class of compounds and demonstrated their potential as promising theranostic anticancer agents that can circumvent cisplatin resistance. Show less

Two new cyclometalated Ru(II) complexes of the general formula [Ru(N-N)₂(1-Ph-βC)](PF₆), where N-N = 4,4'-dimethyl-2,2'-bipyridine (dmb, Ru1), 2,2'-bipyridine (bpy, Ru2), and 1-Ph-βC (1-phenyl-9H-pyrido[3,4-b]indole) is a β-carboline alkaloids derivatives, have been synthesized and characterized. The in vitro cytotoxicities, cellular uptake and localization, cell cycle arrest and apoptosis-inducing mechanisms of these complexes have been extensively explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, inductively coupled plasma mass spectrometry (ICP-MS), flow cytometry, comet assay, inverted fluorescence microscope as well as western blotting experimental techniques. Notably, Ru1 and Ru2 exhibit potent antiproliferative activities against selected human cancer cell lines with IC₅₀ values lower than those of cisplatin and other non-cyclometalated Ru(II) β-carboline complexes. The cellular uptake and localization exhibit that these complexes can accumulate in the cell nuclei. Further antitumor mechanism studies show that Ru1 and Ru2 can cause cell cycle arrest in the G0/G1 phase by regulating cell cycle relative proteins and induce apoptosis through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation and ROS-mediated DNA damage. Show less

Our study demonstrates that four novel kinetically inert C,N-cyclometalated Ru^II complexes of the type [Ru(C^N)(N^N)₂ ][PF₆ ] containing a handle for functionalization on the C^N ligand are very potent cytotoxic agents against several different human cancer cell lines and are up to 400-fold more potent than clinically used cisplatin. In addition, the investigated ruthenium complexes are less cytotoxic in noncancerous cells, and exhibit higher selectivity for cancer cells than conventional platinum anticancer drugs. The high potency of the investigated ruthenium compounds can be attributed to several factors, including enhanced internalization and their capability to change mitochondrial transmembrane potential in cells. The new ruthenium complexes also interfere with protein synthesis with a markedly higher potency than conventional inhibitors of DNA translation. Notably, the latter mechanism has not been hitherto described for other cytotoxic Ru compounds and cisplatin. Show less

Light-activated compounds are powerful tools and potential agents for medical applications, as biological effects can be controlled in space and time. Ruthenium polypyridyl complexes can induce cytotoxic effects through multiple mechanisms, including acting as photosensitizers for singlet oxygen (¹O₂) production, generating other reactive oxygen species (ROS), releasing biologically active ligands, and creating reactive intermediates that form covalent bonds to biological molecules. A structure-activity relationship (SAR) study was performed on a series of Ru(II) complexes containing isomeric tetramethyl-substituted bipyridyl-type ligands. Three of the ligand systems studied contained strain-inducing methyl groups and created photolabile metal complexes, which can form covalent bonds to biomolecules upon light activation, while the fourth was unstrained and resulted in photostable complexes, which can generate ¹O₂. The compounds studied included both bis-heteroleptic complexes containing two bipyridine ligands and a third, substituted ligand and tris-homoleptic complexes containing only the substituted ligand. The photophysics, electrochemistry, photochemistry, and photobiology were assessed. Strained heteroleptic complexes were found to be more photoactive and cytotoxic then tris-homoleptic complexes, and bipyridine ligands were superior to bipyrimidine. However, the homoleptic complexes exhibited an enhanced ability to inhibit protein production in live cells. Specific methylation patterns were associated with improved activation with red light, and photolabile complexes were generally more potent cytotoxic agents than the photostable ¹O₂-generating compounds. Show less

New Ru(II) complexes with lawsone (law) characterized as trans-[Ru(law)(PPh₃)₂(N-N)]PF₆, where PPh₃ means triphenylphosphine and N-N is 2,2'-bipyridine (1), 4,4'-dimethyl-2,2'-bipyridine (2), 4,4'-dimethoxy-2,2'-bipyridine (3), 1,10-phenanthroline (4) or 4,7-diphenyl-1,10-phenanthroline (5), induce apoptosis in tumor cells. Cytotoxicity of the complexes against the tumor cell lines DU-145 (prostate cancer cells), MCF-7 (breast cancer cells), A549 (lung cancer cells) and lung non-tumor cell line MRC-5 demonstrated promising IC₅₀ values, lower than those found for the cisplatin, a drug used as a reference. Due to the high cytotoxic activity and selectivity against A549 cells line, complex (5) was selected for detailed assays. The complex (5) inhibits cells migration in concentrations in a nanomolar range, inducing tumor cell death by apoptosis, as confirmed by flow cytometry experiments. Furthermore, the antiproliferative activity of complex (5) on A549 tumor cells is attributed to a cell cycle arrest at the Sub G1 phase, followed by a decrease in the number of cells at the S phase. In addition, the interaction of the complexes (1-5) with CT-DNA was evaluated by circular dichroism, in which no changes in the secondary structure of DNA were observed, suggesting a weak interaction of the complexes with the biomolecule. On the other hand, complexes (1-5) showed a higher interaction with human serum albumin (HSA) by non-covalent van der Waals forces and hydrogen bonding, resulting in static quenching. Show less

Valproic acid (VPA) is a short-chain, fatty acid type histone deacetylase inhibitor (HDACi), which can cause growth arrest and induce differentiation of transformed cells. Phosphorescent cyclometalated Ir^III complexes have emerged as potential anticancer agents. By conjugation of VPA to Ir^III complexes through an ester bond, VPA-functionalized cyclometalated iridium(III) complexes 1 a-3 a were designed and synthesized. These complexes display excellent two-photon properties, which are favorable for live-cell imaging. The ester bonds in 1 a-3 a can be hydrolyzed quickly by esterase and display similar inhibition of HDAC activity to VPA. Notably, 1 a-3 a can overcome cisplatin resistance effectively and are about 54.5-89.7 times more cytotoxic than cisplatin against cisplatin-resistant human lung carcinoma (A549R) cells. Mechanistic studies indicate that 1 a-3 a can penetrate into human cervical carcinoma (HeLa) cells quickly and efficiently, accumulate in mitochondria, and induce a series of cell-death-related events mediated by mitochondria. This study gives insights into the design and anticancer mechanisms of multifunctional anticancer agents. Show less

Metallo prodrugs that take advantage of the inherent acidity surrounding cancer cells have yet to be developed. We report a new class of pH-activated metallo prodrugs (pHAMPs) that are activated by light- and pH-triggered ligand dissociation. These ruthenium complexes take advantage of a key characteristic of cancer cells and hypoxic solid tumors (acidity) that can be exploited to lessen the side effects of chemotherapy. Five ruthenium complexes of the type [(N,N)₂Ru(PL)]²⁺ were synthesized, fully characterized, and tested for cytotoxicity in cell culture (1_A: N,N = 2,2'-bipyridine (bipy) and PL, the photolabile ligand, = 6,6'-dihydroxybipyridine (6,6'-dhbp); 2_A: N,N = 1,10-phenanthroline (phen) and PL = 6,6'-dhbp; 3_A: N,N = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline (dop) and PL = 6,6'-dhbp; 4_A: N,N = bipy and PL = 4,4'-dimethyl-6,6'-dihydroxybipyridine (dmdhbp); 5_A: N,N = 1,10-phenanthroline (phen) and PL = 4,4'-dihydroxybipyridine (4,4'-dhbp). The thermodynamic acidity of these complexes was measured in terms of two pK_a values for conversion from the acidic form (X_A) to the basic form (X_B) by removal of two protons. Single-crystal X-ray diffraction data is discussed for 2_A, 2_B, 3_A, 4_B, and 5_A. All complexes except 5_A showed measurable photodissociation with blue light (λ = 450 nm). For complexes 1_A-4_A and their deprotonated analogues (1_B-4_B), the protonated form (at pH 5) consistently gave faster rates of photodissociation and larger quantum yields for the photoproduct, [(N,N)₂Ru(H₂O)₂]²⁺. This shows that low pH can lead to greater rates of photodissociation. Cytotoxicity studies with 1_A-5_A showed that complex 3_A is the most cytotoxic complex of this series with IC₅₀ values as low as 4 μM (with blue light) versus two breast cancer cell lines. Complex 3_A is also selectively cytotoxic, with sevenfold higher toxicity toward cancerous versus normal breast cells. Phototoxicity indices with 3_A were as high as 120, which shows that dark toxicity is avoided. The key difference between complex 3_A and the other complexes tested appears to be higher uptake of the complex as measured by inductively coupled plasma mass spectrometry, and a more hydrophobic complex as compared to 1_A, which may enhance uptake. These complexes demonstrate proof of concept for dual activation by both low pH and blue light, thus establishing that a pHAMP approach can be used for selective targeting of cancer cells. Show less

The synthesis and characterization of Pt(II) (1 and 2) and Ru(II) arene (3 and 4) or polypyridine (5 and 6) complexes is described. With the aim of having a functional group to form bioconjugates, one uncoordinated carboxyl group has been introduced in all complexes. Some of the complexes were selected for their potential in photodynamic therapy (PDT). The molecular structures of complexes 2 and 5, as well as that of the sodium salt of the 4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine ligand (cptpy), were determined by X-ray diffraction. Different techniques were used to evaluate the binding capacity to model DNA molecules, and MTT cytotoxicity assays were performed against four cell lines. Compounds 3, 4, and 5 showed little tendency to bind to DNA and exhibited poor biological activity. Compound 2 behaves as bonded to DNA probably through a covalent interaction, although its cytotoxicity was very low. Compound 1 and possibly 6, both of which contain a cptpy ligand, were able to intercalate with DNA, but toxicity was not observed for 6. However, compound 1 was active in all cell lines tested. Clonogenic assays and apoptosis induction studies were also performed on the PC-3 line for 1. The photodynamic behavior for complexes 1, 5, and 6 indicated that their nuclease activity was enhanced after irradiation at λ = 447 nm. The cell viability was significantly reduced only in the case of 5. The different behavior in the absence or presence of light makes complex 5 a potential prodrug of interest in PDT. Molecular docking studies followed by molecular dynamics simulations for 1 and the counterpart without the carboxyl group confirmed the experimental data that pointed to an intercalation mechanism. The cytotoxicity of 1 and the potential of 5 in PDT make them good candidates for subsequent conjugation, through the carboxyl group, to "selected peptides" which could facilitate the selective vectorization of the complex toward receptors that are overexpressed in neoplastic cell lines. Show less

A photochemically dissociating ligand in Ru(bpy)₂(dmphen)Cl₂ [bpy = 2,2'-bipyridine; dmphen = 2,9-dimethyl-1,10-phenanthroline] was found to be more cytotoxic on the ML-2 Acute Myeloid Leukemia cell line than Ru(bpy)₂(H₂O)₂²⁺ and prototypical cisplatin. Our findings illustrate the potential potency of diimine ligands in photoactivatable Ru(ii) complexes. Show less

Ruthenium complexes capable of light-triggered cytotoxicity are appealing potential prodrugs for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). Two groups of Ru(II) polypyridyl complexes with 2-(2-pyridyl)-benzazole ligands were synthesized and investigated for their photochemical properties and anticancer activity to compare strained and unstrained systems that are likely to have different biological mechanisms of action. The structure-activity relationship was focused on the benzazole core bioisosterism and replacement of coligands in Ru(II) complexes. Strained compounds rapidly ejected the 2-(2-pyridyl)-benzazole ligand after light irradiation, and possessed strong toxicity in the HL-60 cell line both under dark and light conditions. In contrast, unstrained Ru(II) complexes were non-toxic in the absence of light, induced cytotoxicity at nanomolar concentrations after light irradiation, and are capable of light-induced DNA damage. The 90-220-fold difference in light and dark IC₅₀ values provides a large potential therapeutic window to allow for selective targeting of cells by exposure to light. Show less

Lysicamine is a natural oxoaporphine alkaloid, which isolated from traditional Chinese medicine (TCM) herbs and has been shown to possess cytotoxicity to hepatocarcinoma cell lines. Reports on its antitumor activity are scarce because lysicamine occurs in plants at a low content. In this work, we demonstrate a facile concise total synthesis of lysicamine from simple raw materials under mild reaction conditions, and the preparation of the Ru(II), Rh(III), Mn(II) and Zn(II) complexes 1-4 of lysicamine (LY). All the compounds were fully characterized by elemental analysis, IR, ESI-MS, ¹H and ¹³C NMR, as well as single-crystal X-ray diffraction analysis. Compared with the free ligand LY, complexes 2 and 3 exhibited superior in vitro cytotoxicity against HepG2 and NCI-H460. Mechanistic studies indicated that 2 and 3 blocked the cell cycle in the S phase by decreasing of cyclins A2/B1/D1/E1, CDK 2/6, and PCNA levels and increasing levels of p21, p27, p53 and CDC25A proteins. In addition, 2 and 3 induced cell apoptosis via both the caspase-dependent mitochondrial pathway and the death receptor pathway. in vivo study showed that 2 inhibited HepG2 tumor growth at 1/3 maximum tolerated dose (MTD) and had a better safety profile than cisplatin. Show less

Four phosphorescent cyclometalated iridium(III) complexes containing benzimidazole moiety have been designed and synthesized. These Ir(III) complexes can effectively inhibit several cancerous processes, including cell migration, invasion, colony formation, and angiogenesis. Interestingly, they show a much higher singlet oxygen quantum yield in an acidic solution than in a neutral solution. Upon irradiation at 425 nm with low energy (1.2 J cm^-2), they can induce apoptosis through lysosomal damage, evaluation of reactive oxygen species level, and activation of caspase-3/7. The highest phototoxicity index is >476, with almost no dark cytotoxicity observed for Ir4. Ir4 can also inhibit tumor growth effectively in nude mice in vivo after photodynamic therapy. An in vitro assay against 70 kinases indicates that maternal embryonic leucine zipper kinase (MELK), PIK3CA, and AMPK are the possible molecular targets. The half maximal inhibitory concentration of Ir4 toward MELK is 1.27 μM. Our study demonstrates that these Ir(III) complexes are promising anticancer agents with dual functions, including metastasis inhibition and lysosome-damaged photodynamic therapy. Show less

Phosphorescent Ir(III) complexes are expected to be new multifunctional theranostic platforms that enable the integration of imaging capabilities and anticancer properties. Mitophagy is an important selective autophagic process that degrades dysfunctional mitochondria. Until now, the regulation of mitophagy is still poorly understood. Herein, we present two phosphorescent cyclometalated iridium(III) complexes (Ir1 and Ir2) that can accumulate in mitochondria and induce mitophagy. Because of their intrinsic phosphorescence, they can specially image mitochondria and track mitochondrial morphological alterations. Mechanism studies show that Ir1 and Ir2 induce mitophagy by depolarization of mitochondrial membrane potential, depletion of cellular ATP, perturbation in mitochondrial metabolic status, and induction of oxidative stress. Moreover, no sign of apoptosis is observed in Ir1- and Ir2-treated cells under the same conditions that an obvious mitophagic response is initiated. We demonstrate that Ir1 is a promising theranostic agent that can induce mitophagy and visualize changes in mitochondrial morphology simultaneously. Show less