H2 O ⋠B(C6 F5 )3 -Catalyzed para-Alkylation of Anilines with Alkenes Applied to Late-Stage Functionalization of Non-Steroidal Anti-Inflammatory Drugs.

Anilines are core motifs in a variety of important molecules including medicines, materials and agrochemicals. We report a straightforward procedure that allows access to new chemical space of anilines via their para-C-H alkylation. The method utilizes commercially available catalytic H2 O ⋅ B(C6 F5 )3 and is highly selective for para-C-alkylation (over N-alkylation and ortho-C-alkylation) of anilines, with a wide scope in both the aniline substrates and alkene coupling partners. Readily available alkenes are used, and include new classes of alkene for the first time. The mild reaction conditions have allowed the procedure to be applied to the late-stage-functionalization of non-steroidal anti-inflammatory drugs (NSAIDs), including fenamic acids and diclofenac. The formed novel NSAID derivatives display improved anti-inflammatory properties over the parent NSAID structure.

Cobalt(III)-Macrocyclic Scaffolds with Anti-Cancer Stem Cell Activity.

Cobalt(III) compounds with tetradentate ligands have been widely employed to deliver cytotoxic and imaging agents into cells. A large body of work has focused on using cobalt(III)-cyclam scaffolds for this purpose. Here, we investigate the cytotoxic properties of cobalt(III) complexes containing 14-membered macrocycles related to cyclam. A breast cancer stem cell (CSC) in vitro model was used to gauge efficacy. Specifically, [Co(1,4,7,11-tetraazacyclotetradecane)Cl2]+ (1) and [Co(1-oxa-4,8,12-triazacyclotetradecane)Cl2]+ (2) were synthesised and characterised, and their breast CSC activity was determined. The cobalt(III) complexes 1 and 2 displayed micromolar potency towards bulk breast cancer cells and breast CSCs grown in monolayers. Notably, 1 and 2 displayed selective potency towards breast CSCs over bulk breast cancer cells (up to 4.5-fold), which was similar to salinomycin (an established breast CSC-selective agent). The cobalt(III) complexes 1 and 2 were also able to inhibit mammosphere formation at low micromolar doses (with respect to size and number). The mammopshere inhibitory effect of 2 was similar to that of salinomycin. Our studies show that cobalt(III) complexes with 1,4,7,11-tetraazacyclotetradecane and 1-oxa-4,8,12-triazacyclotetradecane macrocycles could be useful starting points for the development of new cobalt-based delivery systems that can transport cytotoxic and imaging agents into breast CSCs.

A Breast Cancer Stem Active Cobalt(III)-Cyclam Complex Containing Flufenamic Acid with Immunogenic Potential.

The cytotoxic and immunogenic-activating properties of a cobalt(III)-cyclam complex bearing the non-steroidal anti-inflammatory drug, flufenamic acid is reported within the context of anti-cancer stem cell (CSC) drug discovery. The cobalt(III)-cyclam complex 1 displays sub-micromolar potency towards breast CSCs grown in monolayers, 24-fold and 31-fold greater than salinomycin (an established anti-breast CSC agent) and cisplatin (an anticancer metallopharmaceutical), respectively. Strikingly, the cobalt(III)-cyclam complex 1 is 69-fold and 50-fold more potent than salinomycin and cisplatin towards three-dimensionally cultured breast CSC mammospheres. Mechanistic studies reveal that 1 induces DNA damage, inhibits cyclooxygenase-2 expression, and prompts caspase-dependent apoptosis. Breast CSCs treated with 1 exhibit damage-associated molecular patterns characteristic of immunogenic cell death and are phagocytosed by macrophages. As far as we are aware, 1 is the first cobalt complex of any oxidation state or geometry to display both cytotoxic and immunogenic-activating effects on breast CSCs.

The Bulk Breast Cancer Cell and Breast Cancer Stem Cell Activity of Binuclear Copper(II)-Phenanthroline Complexes.

Mononuclear copper(II)-phenanthroline complexes have been widely investigated as anticancer agents whereas multinuclear copper(II)-phenanthroline complexes are underexplored. Here the synthesis and characterisation of two new binuclear copper(II)-phenanthroline complexes 1 and 2 is reported, comprising of 2,9-dimethyl-1,10-phenanthroline or 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, terminal chloride ligands, and bridging chloride or hydroxide ligands. The binuclear copper(II) complex containing 2,9-dimethyl-1,10-phenanthroline 1 displays nanomolar toxicity towards bulk breast cancer cells and breast cancer stem cells (CSCs) grown in monolayers, >50-fold greater than cisplatin (an anticancer metallodrug) and salinomycin (a gold-standard anti-CSC agent). Spectacularly, 1 exhibits >100-fold greater potency toward three-dimensionally cultured mammospheres than cisplatin and salinomycin. Mechanistic studies show that 1 evokes breast CSC apoptosis by elevating intracellular reactive oxygen species levels and damaging genomic DNA (possibly by an oxidative mechanism). To the best of our knowledge, this is the first study to probe the anti-breast CSC properties of binuclear copper(II)-phenanthroline complexes.

Payload Release Profile and Anti-Cancer Stem Cell Properties of Compositionally Different Polymeric Nanoparticles Containing a Copper(II) Complex.

Cancer stem cells (CSCs) are linked to tumour relapse and metastasis, the main reason for cancer-related deaths. The application of polymeric nanoparticles as drug delivery systems to target CSCs is relatively unexplored. Here, we report the encapsulation of a CSC-potent copper(II) complex 1 by two compositionally different methoxy poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic) acid (PEG-PLGA) copolymers. Specifically, we used PEG-PLGA (5000:10,000 Da, 1:1 LA:GA) and PEG-PLGA (5000:10,000 Da, 4:1 LA:GA) polymers to prepare spherical nanoparticle formulations 1:1 NP15 and 4:1 NP15, respectively, both with a 15% feed of 1. The two formulations show distinct biophysical and in vitro properties. For example, (i) 4:1 NP15 displays a slower payload release profile than 1:1 NP15 in physiologically relevant solutions, (ii) 4:1 NP15 exhibits statistically greater potency towards breast CSCs than bulk breast cancer cells grown in monolayers, whereas 1:1 NP15 is equally potent towards breast CSCs and bulk breast cancer cells, and (iii) 4:1 NP15 shows significantly greater potency towards three-dimensionally cultured mammospheres than 1:1 NP15. This study shows that the release profile and anti-breast CSC properties of PEG-PLGA nanoparticle formulations (containing 1) can be perturbed (and possibly controlled) by modifying the proportion of glycolic acid within the PLGA component.

The Anti-Breast Cancer Stem Cell Potency of Copper(I)-Non-Steroidal Anti-Inflammatory Drug Complexes.

Cancer stem cells (CSCs) are thought to be partly responsible for metastasis and cancer relapse. Currently, there are no effective therapeutic options that can remove CSCs at clinically safe doses. Here, we report the synthesis, characterisation, and anti-breast CSC properties of a series of copper(I) complexes, comprising of non-steroidal anti-inflammatory drugs (NSAIDs) and triphenylphosphine ligands (1-3). The copper(I) complexes are able to reduce the viability of breast CSCs grown in two- and three-dimensional cultures at micromolar concentrations. The potency of the copper(I) complexes towards breast CSCs was similar to salinomycin (an established anti-breast CSC agent) and cisplatin (a clinically used metallopharmaceutical). Cell-based studies showed that the copper(I) complexes are readily, and similarly, internalised by breast CSCs. The copper(I) complexes significantly increase the intracellular reactive oxygen species (ROS) levels in breast CSCs, and their ROS generation profile with respect to time is dependent on the NSAID component present. The generation of intracellular ROS by the copper(I) complexes could be part of the underlying mechanism by which they evoke breast CSC death. As far as we are aware, this is the first study to explore the anti-breast CSC properties of copper(I) complexes.

The Osteosarcoma Stem Cell Activity of a Gallium(III)-Phenanthroline Complex Appended to Salicylate.

We report the synthesis, characterisation, and anti-osteosarcoma properties of a gallium(III) complex (1) comprising of two 1,10-phenanthroline ligands and salicylate, a non-steroidal anti-inflammatory drug. The gallium(III) complex 1 displays micromolar potency towards bulk osteosarcoma cells and osteosarcoma stem cells (OSCs). Notably, the gallium(III) complex 1 exhibits significantly higher toxicity towards OSCs grown in monolayer and three-dimensional cultures than cisplatin, a frontline anti-osteosarcoma drug. Nuclei isolation and immunoblotting studies show that the gallium(III) complex 1 enters osteosarcoma cell nuclei and induces DNA damage. Flow cytometry and cytotoxicity studies (in the presence of prostaglandin E2) indicate that the gallium(III) complex 1 downregulates cyclooxygenase-2 (COX-2) expression and kills osteosarcoma cells in a COX-2-dependent manner. Further, the mode of osteosarcoma cell death evoked by the gallium(III) complex 1 is characterised as caspase-dependent apoptosis.

An Osteosarcoma Stem Cell Potent Nickel(II)-Polypyridyl Complex Containing Flufenamic Acid.

Apoptosis resistance is inherent to stem cell-like populations within tumours and is one of the major reasons for chemotherapy failures in the clinic. Necroptosis is a non-apoptotic mode of programmed cell death that could help bypass apoptosis resistance. Here we report the synthesis, characterisation, biophysical properties, and anti-osteosarcoma stem cell (OSC) properties of a new nickel(II) complex bearing 3,4,7,8-tetramethyl-1,10-phenanthroline and two flufenamic acid moieties, 1. The nickel(II) complex 1 is stable in both DMSO and cell media. The nickel(II) complex 1 kills bulk osteosarcoma cells and OSCs grown in monolayer cultures and osteospheres grown in three-dimensional cultures within the micromolar range. Remarkably, 1 exhibits higher potency towards osteospheres than the metal-based drugs used in current osteosarcoma treatment regimens, cisplatin and carboplatin, and an established anti-cancer stem cell agent, salinomycin (up to 7.7-fold). Cytotoxicity studies in the presence of prostaglandin E2 suggest that 1 kills OSCs in a cyclooxygenase-2 (COX-2) dependent manner. Furthermore, the potency of 1 towards OSCs decreased significantly upon co-treatment with necrostatin-1 or dabrafenib, well-known necroptosis inhibitors, implying that 1 induces necroptosis in OSCs. To the best of our knowledge, 1 is the first compound to implicate both COX-2 and necroptosis in its mechanism of action in OSCs.

Osteosarcoma Stem Cell Potent Gallium(III)-Polypyridyl Complexes Bearing Diflunisal.

We report the anti-osteosarcoma stem cell (OSC) properties of a series of gallium(III)-polypyridyl complexes (5-7) containing diflunisal, a non-steroidal anti-inflammatory drug. The most effective complex within the series, 6 (containing 3,4,7,8-tetramethyl-1,10-phenanthroline), displayed similar potency towards bulk osteosarcoma cells and OSCs, in the nanomolar range. Remarkably, 6 exhibited significantly higher monolayer and sarcosphere OSC potency (up to three orders of magnitude) than clinically approved drugs used in frontline (cisplatin and doxorubicin) and secondary (etoposide, ifosfamide, and carboplatin) osteosarcoma treatments. Mechanistic studies show that 6 downregulates cyclooxygenase-2 (COX-2) and kills osteosarcoma cells in a COX-2 dependent manner. Furthermore, 6 induces genomic DNA damage and caspase-dependent apoptosis. To the best of our knowledge, 6 is the first metal complex to kill osteosarcoma cells by simultaneously inhibiting COX-2 and damaging nuclear DNA.

The Discrete Breast Cancer Stem Cell Mammosphere Activity of Group 10-Bis(azadiphosphine) Metal Complexes.

We report the anti-breast cancer stem cell (CSC) properties of a series of Group 10-bis(azadiphosphine) complexes 1-3 under exclusively three-dimensional cell culture conditions. The breast CSC mammosphere potency of 1-3 is dependent on the Group 10 metal present, increasing in the following order: 1 (nickel complex) <2 (palladium complex) <3 (platinum complex). Notably, 3 reduces the formation and size of mammospheres to a greater extent than salinomycin, an established CSC-active compound, or any reported anti-CSC metal complex tested under similar conditions. Mechanistic studies suggest that the most effective complexes 2 and 3 readily penetrate CSC mammospheres, enter CSC nuclei, induce genomic DNA damage, and trigger caspase-dependent apoptosis. To the best of our knowledge, this is the first study to systematically probe the anti-CSC activity of a series of structurally related Group 10 complexes and to be conducted entirely using three-dimensional CSC culture conditions.

Immunogenic Cell Death of Breast Cancer Stem Cells Induced by an Endoplasmic Reticulum-Targeting Copper(II) Complex.

Immunogenic cell death (ICD) offers a method of stimulating the immune system to attack and remove cancer cells. We report a copper(II) complex containing a Schiff base ligand and a polypyridyl ligand, 4, capable of inducing ICD in breast cancer stem cells (CSCs). Complex 4 kills both bulk breast cancer cells and breast CSCs at sub-micromolar concentrations. Notably, 4 exhibits greater potency (one order of magnitude) towards breast CSCs than salinomycin (an established breast CSC-potent agent) and cisplatin (a clinically approved anticancer drug). Epithelial spheroid studies show that 4 is able to selectively inhibit breast CSC-enriched HMLER-shEcad spheroid formation and viability over non-tumorigenic breast MCF10 A spheroids. Mechanistic studies show that 4 operates as a Type II ICD inducer. Specifically, 4 readily enters the endoplasmic reticulum (ER) of breast CSCs, elevates intracellular reactive oxygen species (ROS) levels, induces ER stress, evokes damage-associated molecular patterns (DAMPs), and promotes breast CSC phagocytosis by macrophages. As far as we are aware, 4 is the first metal complex to induce ICD in breast CSCs and promote their engulfment by immune cells.

The Bulk Osteosarcoma and Osteosarcoma Stem Cell Activity of a Necroptosis-Inducing Nickel(II)-Phenanthroline Complex.

We report the anti-osteosarcoma and anti-osteosarcoma stem cell (OSC) properties of a nickel(II) complex, 1. Complex 1 displays similar potency towards bulk osteosarcoma cells and OSCs, in the micromolar range. Notably, 1 displays similar or better OSC potency than the clinically approved platinum(II) anticancer drugs cisplatin and carboplatin in two- and three-dimensional osteosarcoma cell cultures. Mechanistic studies revealed that 1 induces osteosarcoma cell death by necroptosis, an ordered form of necrosis. The nickel(II) complex, 1 triggers necrosome-dependent mitrochondrial membrane depolarisation and propidium iodide uptake. Interestingly, 1 does not evoke necroptosis by elevating intracellular reactive oxygen species (ROS) or hyperactivation of poly ADP ribose polymerase (PARP-1). ROS elevation and PARP-1 activity are traits that have been observed for established necroptosis inducers such as shikonin, TRAIL and glutamate. Thus the necroptosis pathway evoked by 1 is distinct. To the best of our knowledge, this is the first report into the anti-osteosarcoma and anti-OSC properties of a nickel complex.

Breast Cancer Stem Cell Potency of Nickel(II)-Polypyridyl Complexes Containing Non-steroidal Anti-inflammatory Drugs.

We report the breast cancer stem cell (CSC) potency of two nickel(II)-3,4,7,8-tetramethyl-1,10-phenanthroline complexes, 1 and 3, containing the non-steroidal anti-inflammatory drugs (NSAIDs), naproxen and indomethacin, respectively. The nickel(II) complexes, 1 and 3 kill breast CSCs and bulk breast cancer cells in the micromolar range. Notably, 1 and 3 display comparable or better potency towards breast CSCs than salinomycin, an established CSC-active agent. The complexes, 1 and 3 also display significantly lower toxicity towards non-cancerous epithelial breast cells than breast CSCs or bulk breast cancer cells (up to 4.6-fold). Mechanistic studies suggest that 1 and 3 downregulate cyclooxygenase-2 (COX-2) in breast CSCs and kill breast CSCs in a COX-2 dependent manner. Furthermore, the potency of 1 and 3 towards breast CSCs decreased upon co-treatment with necroptosis inhibitors (necrostatin-1 and dabrafenib), implying that 1 and 3 induce necroptosis, an ordered form of necrosis, in breast CSCs. As apoptosis resistance is a hallmark of CSCs, compounds like 1 and 3, which potentially provide access to alternative (non-apoptotic) cell death pathways could hold the key to overcoming hard-to-kill CSCs. To the best of our knowledge, 1 and 3 are the first compounds to be associated to COX-2 inhibition and necroptosis induction in CSCs.

Modulating the Chemical and Biological Properties of Cancer Stem Cell-Potent Copper(II)-Nonsteroidal Anti-Inflammatory Drug Complexes.

Copper(II) complexes bearing nonsteroidal anti-inflammatory drugs (NSAIDs) are known to potently kill cancer stem cells (CSCs), a subpopulation of tumour cells with high metastatic and relapse fidelity. One of the major disadvantages associated to these copper(II) complexes is their instability in the presence of strong cellular reductants (such as ascorbic acid). Here we present a biologically stable copper(II)-NSAID complex containing a bathocuproinedisulfonic acid disodium ligand and two indomethacin moieties, Cu(bathocuproinedisulfonic acid disodium)(indomethacin)2, 2. The copper(II) complex, 2 kills bulk breast cancer cells and breast CSC equally (in the sub-micromolar range) and displays very low toxicity against non-tumorigenic breast and kidney cells (IC50 value > 100 µM). Three-dimensional cell culture studies show that 2 can significantly reduce the number and size of breast CSC mammospheres formed (from single suspensions) to a similar level as salinomycin (an established anti-breast CSC agent). The copper(II) complex, 2 is taken up reasonably by breast CSCs and localises largely in the cytoplasm (>90%). Cytotoxicity studies in the presence of specific inhibitors suggest that 2 induces CSC death via a reactive oxygen species (ROS) and cyclooxygenase isoenzyme-2 (COX-2) dependent apoptosis pathway.

A Triangular Platinum(II) Multinuclear Complex with Cytotoxicity Towards Breast Cancer Stem Cells.

The preparation of multinuclear metal complexes offers a route to novel anticancer agents and delivery systems. The potency of a novel triangular multinuclear complex containing three platinum atoms, Pt-3, towards breast cancer stem cells (CSCs) is reported. The trinuclear platinum(II) complex, Pt-3 exhibits selective toxicity towards breast CSCs over bulk breast cancer cells and non-tumorigenic breast cells. Remarkably, Pt-3 inhibits the formation, size, and viability of mammospheres to a better extent than salinomycin, an established CSC-potent agent, and cisplatin and carboplatin, clinically used platinum drugs. Mechanism of action studies show that Pt-3 effectively enters breast CSCs, penetrates the nucleus, induces genomic DNA damage, and prompts caspase-dependent apoptosis. To the best of our knowledge, Pt-3 is the first multinuclear platinum complex to selectively kill breast CSCs over other breast cell types.

The Next Generation of Anticancer Metallopharmaceuticals: Cancer Stem Cell-Active Inorganics.

Cancer stem cells (CSCs) are heavily linked to fatal incidences of cancer relapse and metastasis. Conventional cancer therapies such as surgery, chemotherapy and radiation are largely futile against CSCs. Therefore, highly original approaches are needed to overcome CSCs and to provide durable, long-term clinical outcomes. Many academia- and pharmaceutical-led studies aimed at developing chemical or biological anti-CSC agents are ongoing; however, the application of inorganic compounds is rare. In this minireview, we discuss how the chemical diversity and versatility offered by metals has been harnessed to develop an unprecedented, emerging class of metallopharmaceuticals: CSC-active inorganics. A detailed account of their mechanism(s) of action is provided, and possible future directions for exploration are also put forward.

Highly Charged, Cytotoxic, Cyclometalated Iridium(III) Complexes as Cancer Stem Cell Mitochondriotropics.

The cancer stem cell (CSC) toxicity and mechanism of action of a series of iridium(III) complexes bearing polypridyl and charged 1-methyl-2-(2-pyridyl)pyridinium ligands, 1-4 is reported. The most effective complex (containing 1,10-phenanthroline), 3, kills CSCs and bulk cancer cells with equal potency (in the micromolar range), indicating that it could potentially remove heterogenous tumour populations with a single dose. Encouragingly, 3 also inhibits mammopshere formation to a similar extent as salinomycin, a well-established anti-CSC agent. This complex induces CSC apoptosis by mitochondrial membrane depolarization, inhibition of mitochondrial metabolism, and intracellular reactive oxygen species (ROS) generation. To the best of our knowledge, this is the first study to investigate the anti-CSC properties of iridium complexes.

The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs.

The platinum drugs, cisplatin, carboplatin, and oxaliplatin, prevail in the treatment of cancer, but new platinum agents have been very slow to enter the clinic. Recently, however, there has been a surge of activity, based on a great deal of mechanistic information, aimed at developing nonclassical platinum complexes that operate via mechanisms of action distinct from those of the approved drugs. The use of nanodelivery devices has also grown, and many different strategies have been explored to incorporate platinum warheads into nanomedicine constructs. In this Review, we discuss these efforts to create the next generation of platinum anticancer drugs. The introduction provides the reader with a brief overview of the use, development, and mechanism of action of the approved platinum drugs to provide the context in which more recent research has flourished. We then describe approaches that explore nonclassical platinum(II) complexes with trans geometry or with a monofunctional coordination mode, polynuclear platinum(II) compounds, platinum(IV) prodrugs, dual-threat agents, and photoactivatable platinum(IV) complexes. Nanoparticles designed to deliver platinum(IV) complexes will also be discussed, including carbon nanotubes, carbon nanoparticles, gold nanoparticles, quantum dots, upconversion nanoparticles, and polymeric micelles. Additional nanoformulations, including supramolecular self-assembled structures, proteins, peptides, metal-organic frameworks, and coordination polymers, will then be described. Finally, the significant clinical progress made by nanoparticle formulations of platinum(II) agents will be reviewed. We anticipate that such a synthesis of disparate research efforts will not only help to generate new drug development ideas and strategies, but also will reflect our optimism that the next generation of approved platinum cancer drugs is about to arrive.

Polypyridyl Zinc(II)-Indomethacin Complexes with Potent Anti-Breast Cancer Stem Cell Activity.

Cancer stem cells (CSCs) are thought of as a clinically pertinent subpopulation of tumors, partly responsible for cancer relapse and metastasis. Research programs aimed at discovering anti-CSC agents have largely focused on biologics and purely organic molecules. Recently, we showed that a family of redox-active copper(II) complexes with phenanthroline-based ligands and nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, are capable of potently and selectively killing breast CSCs. Herein we present analogous redox-inactive, zinc(II)-phenanthroline-indomethacin complexes with the ability to kill breast CSCs and bulk breast cancer cells with equal potency (in the submicro- or micromolar range). A single dose of the zinc(II) complexes could theoretically be administered to eliminate whole tumor populations. Excitingly, some of the zinc(II) complexes decrease the growth and viability of mammospheres to a comparable or higher degree than salinomycin, a compound known to effectively kill breast CSCs. As far as we are aware this is the first report to examine the anti-breast CSC activity of zinc(II)-containing compounds.

A Copper(II) Phenanthroline Metallopeptide That Targets and Disrupts Mitochondrial Function in Breast Cancer Stem Cells.

The breast cancer stem cell (CSC) and bulk breast cancer cell potency of a series of metallopeptides containing dichloro(1,10-phenanthroline)copper(II) and various organelle-targeting peptide sequences is reported. The mitochondria-targeting metallopeptide 1 exploits the higher mitochondrial load in breast CSCs over the corresponding non-CSCs and the vulnerability of breast CSCs to mitochondrial damage to potently and selectively kill breast CSCs. Strikingly, 1 reduces the formation and size of mammospheres to a greater extent than salinomycin, an established CSC-potent agent. Mechanistic studies show that 1 enters CSC mitochondria, induces mitochondrial dysfunction, generates reactive oxygen species (ROS), activates JNK and p38 pathways, and prompts apoptosis. To the best of our knowledge, 1 is the first metallopeptide to selectivity kill breast CSCs in vitro.