Homo- and Hetero-Multinuclear Iridium(III) Complexes with Cytotoxic Activity

Abstract

Towards the efforts to expand the bioactivity and to reduce toxic and adverse properties of known metal-based drugs, various multinuclear complexes have recently been studied. They have shown enhancement of target specificity and selectivity. Different from small organic compounds and traditional metal-based complexes with anticancer activity, iridium(III) multinuclear or heteronuclear metallodrugs have confirmed potential advantages due to their unique biological and chemical diversities, better activity and different anticancer mechanisms. Ir(III) coordination compounds, similar to most Pt group compounds, are of excessive interest because of their potential cytotoxic activity, effective cellular uptake and tolerance by healthy cells. Although mononuclear Ir(III) complex compounds have been extensively studied as promising candidates for antitumor application, the research on the antineoplastic potential of homo- or hetero-multinuclear iridium(III) complexes is not as abundant; nevertheless, intensive investigations have been conducted in the recent years towards developing complexes that are anticipated to have improved therapeutic potential and biotarget selectivity. Multimetallic iridium(III) frameworks have offered interesting possibilities for designing new antitumor agents by exploiting the action of different metal cations at the same time. This method was very successful in the design of homo- and hetero-multinuclear cyclometalated and half-sandwich organometallic Ir(III) compounds. In the described background, many homonuclear and heteronuclear Ir(III) complexes have been estimated and have exposed promising advantages in cancer therapy. This review intends to summarize newly reported innovative and promising multinuclear Ir(III)-based complexes and to afford a wide-ranging overview of current development and perspectives for the practical impact of these complexes in the tumor therapy field. It is anticipated that this analysis will provide significant direction for the further progress of active homonuclear and heteronuclear iridium-based anticancer agents.

1. Introduction

Metal-based anticancer compounds, particularly the complexes of platinum group metals, are amongst the most extensively applied antineoplastic agents in clinics. The healing potential and possible application of such compounds are at a disadvantage because of resistance problems, toxic and severe dose-limiting side effects, or unsatisfactory antimetastatic and diagnostic properties. All this has provoked the consideration of scientists for the discovery of suitable alternatives. Among the best antitumor metal-based drugs are the complexes of the Pt group, including Ir(III) complexes [1,2,3,4]. They have been proven to be effective antineoplastic agents with good cytotoxicity [5,6], high selectivity [7], minor adverse effects and unique target specificity [8,9], offering a wide spectrum of bioactivities. Their structural and electronic properties have been mostly controlled by the type of involved biologically active ligands since the ligands influence significantly the target site recognition. Compared to classical metallodrugs, numerous Ir(III) cyclometalated compounds have displayed enhanced cytotoxicity, inherent luminescence properties [10,11,12] and specificity in targeting organelles [13,14,15,16,17]. Consequently, the physicochemical characteristics of organoiridium complex compounds gave them substantial bioactivity together with notable properties to be proposed as potential antineoplastic agents. Additionally, they have been exploited for imaging usage as they have exceptional luminescence features such as high quantum yields photostability, long excited-state lifetime, small photobleaching and large Stokes shift. In this context, the progress of Ir(III) compounds, which possessed high cytotoxicity, has directed these complexes to be applied as theranostics [18]. Iridium(III) complex compounds have suitable coordination numbers and geometry, obtainable by their different oxidation states, adjustable chemical interactions, ligand exchange varieties and flexible physical features. As target-specific, iridium(III) complex compounds have been observed to accumulate in various organelles for instance mitochondria [19,20,21], endoplasmic reticulum [22], nucleus [23], lysosomes [24,25], etc., where mitochondria and lysosomes were the key targets [26]. They can generate ROS and decrease mitochondrial membrane potentials (MMPs) by creating mitochondrial dysfunction or triggering mitochondrial apoptotic functions. By acquiring in-depth knowledge about the chemicophysical properties of iridium(III) complexes and their outstanding antitumor activity in various mechanistic pathways, many research groups have designed different types of Ir(III) organometallic complexes, including multinuclear ones [27,28,29], as appropriate chemotherapeutics with diverse from Pt(II) and Pt(IV) agents antineoplastic mechanism, and high cytotoxic potency against resistant to cisplatin tumor cells [30]. In many cases, their cytotoxicity was better than that of the classical platinum drugs [31,32].

Iridium is a rare third-row transition metal belonging to the platinum group. It is often considered to be one of the most inert metals. In this group, the reactivity of Rh, Ir, Pd and Pt are much closer to each other than to the chemical activity of Ru and Os. Rh and Ir readily form complexes with a +3 oxidation state, that, similar to Co analogs, have kinetical inertness [2]. The typical stability of Ir(III) complex compounds makes them potential drug candidates which are capable of reaching the targets without any modifications. The variety of biologically active (N^N, C^N, O^N, etc.) ligands for coordination with iridium is an additional reason for its application, because the bioligands regulate the targets’ recognition. Iridium(III) complex compounds represent two main classes, viz., half-sandwich and cyclometalated Ir(III) complexes. In comparison with half-sandwich iridium(III) complexes, the cyclometalated ones possess improved optical characteristics [33] and could inhibit tumor cells by light exciting, making them appropriate possible agents for phototherapeutic use. Numerous cyclometalated [34,35,36,37] and half-sandwich organoiridium(III) compounds with cyclopentadienyls [38,39,40,41] have been obtained and utilized as antineoplastic agents [42,43].

In recent years, polynuclear metal-based compounds have attracted the consideration of medicinal chemistry researchers because of their exceptional antineoplastic activity compared to the activity of mononuclear corresponding complexes [44,45]. Ma et al.l. have recently reviewed Pt-containing heterometallic complexes with different transition metals, such as Ru, Au, Pd, Rh, Ti, Gd, Eu, Ir, Re, Fe, Cu, Tc, etc. for the treatment of malignant diseases [46]. The synthesis, antitumor activity, mechanisms of action, and interactions between platinum and additional metallic centers have been comprehensively discussed. The multinuclear complex compounds have shown improved cytotoxicity, selectivity and DNA-binding ability. Most of them were able to overcome cisplatin-acquired resistance, which can be explained by the synergistic effects of various metal centers, such as Ru [47], Pt [48], Au [49,50], Re [51] and Ir [52]. Concerning heterometallic anticancer complexes, platinum-based complexes constitute the most extensively studied compounds. Numerous examples of platinum-containing heterometallic complexes have been designated, displaying great potential for overcoming the limitations of traditional clinically used platinum drugs, such as resistance, toxic effects, and diagnostic issues. These multinuclear metal complexes represent combinations of platinum with gold [53], ruthenium [54,55], rhodium [56], palladium [57], and undoubtedly iridium [58], affording theragnostic heterometallic complexes [59]. Multinuclear homometallic or heterometallic complexes have recently been reviewed [60] with an emphasis on multinuclear ruthenium(II), rhodium(III), osmium(II) and iridium(III) derived compounds with various metal-containing species in a framework of homometallic or heterometallic units with cytotoxic and antimicrobial activity. Specific consideration has been paid to the evaluation of structural analogs with diverse numbers or types of metal centers, coordinated with the biologically active ligands [60]. The multinuclear complexes, reported in the literature have shown enhanced permeability and retention, a greater degree of selectivity and lower adverse effects than their mononuclear counterparts [61]. In many cases, malignant cells retained the resulting macromolecules because of their damaged lymphatic drainage, whereas healthy cells readily excreted them [61]. The choice of the metal centers, forming the respective hetero-multinuclear complex, is crucial as it determines the possible therapeutic and synergistic effects [62].

With the recognized features of iridium(III) complexes for anticancer application, their utilization as biomolecular recognition probes for monitoring cellular changes and their potential for bioimaging applications, the majority of active iridium(III) complexes have been combined with other cytotoxic metals during the last decades. The incorporation of a second metallic center with distinct biotargets and advantageous physicochemical characteristics into iridium compounds is a good approach to improve the antineoplastic efficiency of iridium-based agents and to overcome the possible disadvantages. Many diiridium(III) [63] and iridium(III) heterometallic complexes have exposed a great antiproliferative potential and their photo- and bioimaging assets have also been improved. The design of such compounds offers growth in the resulting biological activity through the multiple metal centers or by combining different metal centers with different mechanisms of action and bioeffects. This review provides a wide-ranging analysis and outlook of recently reported representatives of iridium-containing homonuclear and heteronuclear complexes examined for anticancer treatment and might guide investigators in discovering the challenges and prospects in this developing field.

2. Homonuclear Iridium(III) Complexes

2.1. Iridium(III) Cyclopentadienyl Dinuclear Complexes

Half-sandwich Cp*-Ir(III) organometallic complexes with pseudo-octahedral geometry, where Cp* is a derivative of 1,2,3,4,5-pentamethylcyclopenta-1,3-dienyl, have been demonstrated to be potent anticancer metal-based drugs. Dinuclear Ir(III) complexes of bioactive ligands with different structures have shown significant activity and have exhibited different modes of activity as compared to the respective mononuclear corresponding complexes or to similar homonuclear analogous complexes.

A series of homologous dinuclear iridium complexes, presented in Figure 1, [Ir(η⁵-C₅Me₄R)Cl(μ-Cl)]₂ (R = Me, Figure 1a; R = H, Figure 1b; R = Pr, Figure 1c; R = 4-C₆H₄F, Figure 1d; R = 4-C₆H₄OH, Figure 1e) have been prepared, characterized, and assessed for their cytotoxic action on rodent and human tumor cells, such as mouse melanoma B16, rat glioma C6, colorectal carcinoma SW620 and HCT116, breast adenocarcinoma MCF-7, ovarian A2780 carcinoma, and human fetal lung MRC5 fibroblast cells [64]. The evaluation of properties of the adapted Cp* ring by substitution of the Me group with a variety of additional groups and their influence on the cytotoxic activity has also been presented. Predominantly, a substitution of one Me group with H has been found to be satisfactory for improved cytotoxicity.

Half-sandwiched Ir(III) complexes are attractive organometallic antineoplastic agents. The half-sandwich Cp* iridium complex [Ir₂(m-pzpy)(η⁵-Cp*)₂Cl₃]PF₆ (Figure 2), containing the bridging pzpy ligand (Hpzpy = N’-[(1Z)-1-(pyridin-2-yl)ethylidene]pyrazine-2-carbohydrazonamide) as the tridentate one has been synthesized [65]. In the dinuclear iridium complex, the ligands coordinate to one Ir(III) center bidentately and to the other ligand in a monodentate fashion. The cytotoxicity of Ir(III) complex has been estimated against the colorectal carcinoma cell lines HT 29, HCT116 p53þ/þ, HCT116 p53/ and the normal human epithelial resulting from the retina ARPE-19 cells. However, the Ir(III) complex exhibited only moderate cytotoxic activity (IC₅₀ > 100 μM), which was lesser than that of cisplatin and its Rh(III) analogs, Table 1.

Related studies have been extended to other Cp* iridium polypyridyl complexes. The best result has been found for bis-intercalators comprising rigid linkers which favor slow kinetical dissociation and probably further groove reactions. Potential bimetallic bis-intercalators, for instance, the tetracations [{(η⁵-Cp*)Ir(dppz)}₂(μ-pyz)]⁴⁺, and [{(η⁵-C₅Me₅)Ir(pp)}₂(μ-4,4′-bpy)]⁴⁺, where pp is dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[2,3-a:2′,3′-c]phenazine (dppz) or benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (dppn)), which comprise {(η⁵-Cp*)Ir(dppz)}²⁺ groups bridged by rigid bipyridinyl ligands, such as pyrazine (pyz) or 4,4′-bipyridyl (4,4′-bpy), have been designed by Nazif et al. [66]. The DNA-binding characteristics and in vitro cytotoxicity of the binuclear complexes with the formula [{Ir(NˆN)(η⁵-C₅Me₅)}₂L]⁴⁺ (Figure 3a–c) have been studied by the same group [66]. While, the pyz-bridged complex ion appeared to be only a mono-intercalator, NOESY- NMR spectra revealed strong bis-intercalation for the 4,4′-bpy compound, that sandwiched two DNA base pairs through its parallel dppz ligands. As the binuclear complexes displayed inhibitory effects analogous to these of [(η⁵-Cp*)IrCl(dppz)]⁺ where the covalent nucleobase binding was more stable, it could be hypothesized that the character and strength of the possible binding mechanisms were not of principal significance for the resulting potency. The antiproliferative effects of the compounds have been determined against colon HT-29 and breast MCF-7 carcinoma cells. IC₅₀ values of 3.5/1.8 and 3.1/3.8 μM, respectively, were calculated for the pyz and 4,4′-bpy complexes against MCF-7/HT-29 cell line, Table 1. The dipyrido[3,2-a:2′,3′-c]phenazine (dppz) complex, shown in Figure 3b, bonded through a bis-intercalation manner to DNA including the involvement of both the dppz ligands in the base pairs. In contrast, complexes in Figure 3a,c with smaller dipyridoquinoxaline (dpq) and bigger benzodipyridophenazine (dppn) bioligands did not intercalate into DNA base pairs. Therefore, the size of the hypothetically intercalating diimine bioligands is of significance to the design of active bis-intercalators. The outcomes of the lengths of rigid bridging ligands on DNA-binding modes have been examined. For instance, the distance between the effectively parallel dppz bioligands in the complex, shown in Figure 3b, is around 10.2 Å, which is the perfect distance (3 × 3.4 Å) for two base sandwiching pairs.

Improved cytotoxicity has been realized by the corresponding complexes comprising larger dipyridinyl bridging bioligands, precisely 4-[(E)-2-(4-pyridinyl)ethenyl]pyridine (dpee) in Figure 4a, 4-(2-pyridin-4-ylethynyl)pyridine (dpey) in Figure 4b or 1,4-di(2-pyridin-4-ylethynyl)benzene (dpeb) in Figure 4c. Engaging longer rigid bridge ligands (Figure 4) led to improved potency (IC₅₀ = 0.61 and 0.49 μM) for the complexes [{(η⁵-Cp*)Ir(dppz)}₂(μ-B)](CF₃SO₃)₄, where B = dpee, dpey, Figure 4a,b, against the less quickly proliferating MCF-7 cell line, Table 1. Interestingly, complexes dpee (Figure 4a) and dpey (Figure 4b) fix to DNA only in a mono-intercalation manner, although their iridium–iridium distances (13.1 and 13.5 Å, respectively), were close to the perfect distance for 3-base sandwiching pairs, which is 13.6 Å [67]. The reported diiridium complexes have been found to be only mono-intercalators but they strongly cleaved the supercoiled DNA of pBR322 plasmid to produce the nicked forms in dark conditions [67]. The addition of DNA, complex dpeb, Figure 4c, (with iridium–iridium distance of 20.6 Å) quickly formed kinetically-preferred monointercalated adducts, which were gradually transformed to thermodynamically stable intertwined bis-intercalation modes. UV/Vis and CD spectral investigations were in agreement with a stable intertwined bis-intercalation mode for ion pairs in Figure 4c with 1,4-di(2-pyridin-4-ylethynyl)benzene), whose much longer metal–metal distance allowed a stack of five aromatic chromophores to be sandwiched between its effective parallel dppz bioligands. The obtained IC₅₀ values of the nuclease complexes in Figure 4a,b were smaller than those of the bis-intercalator Figure 4c by one order of magnitude. Additionally, the complex in Figure 4c possesses photoinduced nuclease action. The dpeb correspondent (Figure 4c) was much less active (IC₅₀ = 2.2 μM) in the inhibition of MCF-7 proliferation, in spite of its capability to bind to DNA in the more stable bis-intercalative manner, Table 1, [67]. The results suggested that the nuclease characteristics of the dpee and dpey complex compounds could probably be responsible for their better cytotoxic activity. It is also notable that the inhibiting properties of the non-intercalating complexes [{(η⁵-Cp*)Ir(5,6-Me₂phen)}₂(μ-B)](CF₃SO₃)₄ with identical bridging ligands (dpey, dpeb) were similar to those of the corresponding dppz complex compounds against HT-29 and MCF-7 cells [67]. Noticeable antileukemic action (IC₅₀ = 6–7 μM) connected with bigger ROS levels and apoptotic induction has been documented for the complexes towards Jurkat cells. It has been shown that the nature of polypyridyl chelating N,N-ligand appeared to be more significant for the consequential antiproliferative action than the bridging ligand, while the bridging ligands regulated the type of nuclease action, since dpee- and dpey derivatives cleaved DNA in dark conditions, which was detected for the dpeb derivative only when irradiated.

Dinuclear chloridoiridium(III)-Cp* complexes, shown in Figure 5, with higher in vitro antineoplastic activity than cisplatin and analogous Ru derivatives have been discovered by Parveen et al. [68]. Organometallic iridium complexes have been prepared by stirring a methanolic solution of [(Cp*)IrCl₂]₂ with deprotonated 1,n-bis[3-hydroxy-2-methyl-4(1H)-pyridinon-1-yl]alkane (n = 4, 6, 8, 12) and the products have been extracted with diethyl ether and dichloromethane to obtain the respective complexes. The cytotoxicity of the complexes has been assessed against colon HCT116, non-small cell lung NCI-H460, cervix SiHa and colon SW480 cells. The IC₅₀ of the complex, presented in Figure 5d, against the tested cells was less than 1 µM (one order of magnitude more potent than cisplatin in chemo-resistant SW480 colon cancer cell line). The iridium(III) complex, shown in Figure 5d, has induced the formation of ROS in a concentration-dependent manner. To get an idea of the overall toxicity of the compounds, the most active derivative, shown in Figure 5d, was chosen for hemolytic investigations with mouse red blood cells. The complex was non-hemolytic up to 125 µM and less noxious in comparison with Pt-based antitumor agents. All the studied dinuclear iridium(III)-Cp* complexes (Figure 5) have shown in vitro capability superior to cisplatin. The mechanism of action seemed to be associated with DNA damage and ROS-mediated stress pathways. The most cytotoxic complex, shown in Figure 5d, was well tolerated without any mortality or detectable abnormalities observed in the developing zebrafish embryos. This compound has not demonstrated any disturbance in vascular vessel formation, showing the anticipated low toxic effects, despite its marked cytotoxicity in vitro in tumor cell lines.

A series of binuclear half-sandwich Ir(III) complexes [Ir₂(μ-Lⁿ)(η⁵-Cp*)₂Cl₂](PF₆)₂ with 4,4′-biphenyl-based bridging Schiff base bioligands, such as N,N’-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl) methanamine and N,N’-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl)ethanamine, Figure 6, have been reported by Štarha et al. [69]. These complexes have been tested against human ovarian A2780 carcinoma, cisplatin-resistant ovarian A2780R carcinoma, breast MCF-7 carcinoma, osteosarcoma HOS, colon HT-29 carcinoma, pancreatic PANC-1 carcinoma cells and against two healthy human cell lines. Complex, containing N,N’-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl)ethanamine bridging ligand having a six-membered MN₂C₃ chelate ring, exposed higher activity towards A2780 (IC₅₀ = 3.1 μM) and MCF-7 (IC₅₀ = 6.0 μM) cells, as compared with that of N,N’-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl) methanamine bridging ligand with a five-membered MN₂C₂ chelate rings, Table 1. The more active complex exceeded twice the in vitro cytotoxicity of cisplatin and displayed higher selective effects towards A2780 and MCF-7 cells over healthy human cells. Contrary to cisplatin, the complex has not induced cell cycle modifications with high superoxide anion levels at the human ovarian A2780 carcinoma cell line. The investigated iridium(III) dinuclear complexes were the first binuclear compounds with two {Ir(η-ar)Cl} moieties linked by nitrogen-donor bridging ligands chelating two metallic ions.

The above work has been followed by the synthesis of less active corresponding monochloridoiridium(III) complexes with analogous tetradentate nitrogen-donor ligands, produced from 4,4′–methylenedianiline [70] and benzene-1,4-diamine [71]. The binuclear half-sandwich Ir(III) complexes [Ir₂(μ-L1)(η⁵-Cp*)₂Cl₂](PF₆)₂ (Figure 7a), [Ir₂(μ-L2)(η⁵-Cp*)₂Cl₂](PF₆)₂ (Figure 7b) and [Ir₂(μ-L3)(η⁵-Cp*)₂Cl₂](PF₆)₂ (Figure 7c) with tetradentate 4,4-methylenedianiline-based nitrogen-donor ligands (L1–L3) have been synthesized and identified by elemental analysis, mass spectral analysis, NMR and FTIR spectroscopy by Masaryk et al. [70]. The complexes have been tested against human prostate cancer DU-145, melanoma A375, human liver hepatocellular carcinoma HepG2, human lung cancer A549 and breast cancer MCF-7 cells by MTT assay. The most potent complex of the series, shown in Figure 7b, has shown comparable cytotoxic activity to cisplatin in the HepG2 cell line with IC₅₀ of 12.6 μM, and acceptable IC₅₀ for A549 cells (16.5 μM), Table 1. The IC₅₀ of clinically used cisplatin against HepG2 and A549 cell lines were 12.0 μM and 12.3 μM, correspondingly. For the other tumor cells, the same complex showed lower cytotoxicity. The complexes in Figure 7a,c have shown comparatively weak cytotoxicity in the tested cells. Additionally, the studied complexes (Figure 7) partially oxidized NADH to NAD⁺, that could be connected with the redox-mediated mode of cytotoxicity.

Kavukcu et al. have described the preparation of a series of monometallic and dimetallic iridium(III)–arene compounds with various aromatic and aliphatic groups and examined their activity on Vero and HepG2 cells through cell death mechanisms [72]. The electro-neutral [Ir₂(μ-mpa)₂(η⁵-Cp*)₂Cl₂] complex (Figure 8) involved 2-mercapto-N-phenylacetamide (Hmpa), which possessed diverse coordination modes (as a S-donor bridging ligand) whereas its Ru correspondent [Ru₂(μ-mpa)₂(η⁶-pcym)₂] with mpa ligand coordinated as a N,S-chelating ligand bridging the two metallic centers through the S atom of mpa. The complex was synthesized by using [IrCl₂(Cp*)]₂ in dry methyl cyanide at room temperature. The bimetallic Ir(III)–pentamethylcyclopentadienyl complex with S,S-double coordination, shown in Figure 8, has exhibited a reduction of tumor cell viability in mg mL⁻¹, demonstrating a higher cytotoxic activity, comparable with the potency of its ruthenium analog. The complex has been shown to exhibit an increase in RIPK1 immunoreactivity in both cell lines.

The complexes with triphenylamine-modified thiosemicarbazone (TSC) have been reported by Shao et al. [73]. Due to the “enol” configuration of TSC, the studied compounds produced unique dimeric configurations. The most active complex is shown in Figure 9. In comparison with cisplatin, the antineoplastic activity in vivo and in vitro has confirmed that this complex might efficiently inhibit cancer growth, displaying better cytotoxic activity. The complex was active against A549 and HeLa tumor cell lines, with low selectivity against the same cell lines over the normal BEAS-2B cells tested. Meanwhile, it could circumvent adverse effects in the trials of safety estimation. Aided by the suitable fluorescence characteristics, the studied compounds can enter cancer cells in an energy-dependent manner, can be accumulated in lysosomes, and produce lysosome integrity damage. Ir(III) complexes blocked the cell cycle and enhanced the levels of ROS, which led to apoptosis. In particular, half-sandwich Ir(III)-thiosemicarbazone complexes were projected to be hopeful candidates for antitumor therapy.

2.2. Iridium Cyclometalated Dinuclear Complexes

Coordination cyclometalated Ir(III) complexes have exposed promising activity as novel modules of antitumor metal-based drugs. These metal coordination compounds with exceptional luminescent characteristics and the ability to photosensitize O₂-forming ROS have been exploited in imaging diagnostic methods and photodynamic anticancer therapy.

The binuclear organo-Ir(I) complex [IrCl(cod)]₂ with square-planar geometry, where cod = diene 1,5-cyclooctadiene (C₈H₁₂), has displayed strong antimetastatic action in the Lewis lung model nonetheless it has not shown primary tumors’ inhibition (Figure 10) [74,75].

Wang et al. have reported new N-substituted carbazole mono- and binuclear Ir(I) complexes, Figure 11, [76]. To confirm an extended π-conjugated ligand structure, acetylene moiety was incorporated between the bipyridine units and the N-substituted carbazoles. The studied homonuclear complex has demonstrated higher absorbance in the visible spectrum and higher ¹O₂ quantum yields because the complex possessed a conjugated carbazole bridge ligand. Thus, the newly synthesized bimetallic complex could be a potential candidate for numerous applications like triplet photosensitizer for Triplet-Triplet Annihilation Up-conversion (TTA-UC).

A series of highly charged binuclear Ir(III) cationic complexes have been studied for their antineoplastic effects against breast cancer MCF-7 and MDA-MB-231 cells [77]. The alkane linker between the two iridium(III) centers has been varied from 7 to 16 carbon atoms (Figure 12) to investigate its effect on the bioactivity of the compounds. The potency of the complexes improved with increasing the chain lengths. The most active complex, Figure 12, comprised a 16-carbon chain linker. However, while the complex, shown in Figure 12, was cytotoxic against the metastatic MDA-MB-231 cell line (3 μM), it was comparatively non-active against the non-metastatic MCF-7 cells (29 μM). The anticancer activity, lipophilicity (log P) and DNA-binding ability have been studied. Because of their cationic nature, the compounds bonded to ct-DNA with high affinity as demonstrated by circular dichroism spectroscopy. The dinuclear Ir(III) cationic complexes in Figure 12 induced significant variations in the circular dichroism spectral intensity of DNA samples, signifying that they bonded to DNA with high affinity and caused substantial structural distortion. Additional studies might help to understand the mechanisms of cell death for instance apoptotic induction. Further investigations covering wild-type and cisplatinum-resistant cells can afford more insights into the effectiveness of the cytotoxic complexes, as shown in Figure 12.

Unique Ir(III) luminescent coordination compounds also consist of dinuclear bis-tridentate Ir(III) complexes, described by Liu et al., which include analogous cyclometalated ligands that were deliberated above [78]. Dinuclear Ir(III) complexes having terpyridine-capped fluorenyl bridge ligands and a variety of polypyridyl- or cyclometalating terminal tridentate ligands displayed photodynamic beneficial in vitro effects against melanoma SK-MEL-28 cell line upon visible light irradiation, with sub-micromolar photoinduced cytotoxicity and phototherapeutic indices. The reported complexes incorporated 9,9-dioctyl-2,7-di(terpyridyl)-9H-fluorene as a bridging ligand, with variation at the terminal tridentate bioligands, such as 4′-phenyl-2,2′:6′,2″-terpyridine (N^N^N), 1,3-dipyridyl-4,6-dimethylbenzene (N^C^N), 4,6-diphenyl-2,2′-bipyridine (C^N^N), or 2,4,6-triphenyl-pyridine (C^N^C). The complexes phosphoresced brightly in compromised cells and displayed photoactivated cell uptake, confirming the theranostic properties of the studied compounds. Together, both bridging and terminal ligands influenced the photophysical and biological activity significantly. Their properties depended on the bridging linker length. Even though the compounds have exposed very potent photophysical characteristics, their cytotoxic activity has not been correlated with the ROS production quantum yield or their photocleaving ability. Based on photophysical properties, the complex with 1,3-dipyridyl-4,6-dimethylbenzene (N^C^N), shown in Figure 13, was projected to be the most potent in vitro PDT candidate. For an in vitro PDT application, there has been a clear suggestion that terminal tridentate N^C^N ligands performed best in combination with Me substituents on the central cyclometalating rings.

Tang et al. have synthesized and reported binuclear (Figure 14a–c) and mononuclear (Figure 14d–f) Cp*Ir(III) complexes of pyrrole thioamide ligands [79]. Hybrid ligands containing S and N donors are highly adaptable ligands able to coordinate to a very extensive range of metallic centers, because of their soft-hard hybrid donor characteristics. The interactions of pentamethylcyclopentadienyl complex [(η⁵-Cp*)IrCl₂]₂ with pyrrole-2-thioamide ligands (holding several substituents on the pyrrole ring) and triethylamine base have been studied. Two series of iridium(III) complexes incorporating π-hydrocarbon ligands along with dianionic pyrrole thioamide ligands have been obtained—the first series consisted of binuclear complexes with S-bridging pyrrole thioamide bioligands, whereas the second series comprised mononuclear complexes with ancillary PPh₃ ligands and a pyrrole thioamide dianion ligand coordinating through S and the pyrrole N atoms, forming 5-membered chelating ring. Complex compounds have been evaluated by NMR, IR and ESI mass spectrometry. The iridium(III) complexes illustrated the potentially varied coordination properties of the hybrid hard/soft donor pyrrole thioamide ligands. The newly obtained complexes were tested for cytotoxicity towards adenocarcinoma human alveolar basal epithelial A549 cell line with marginal potency. The mononuclear complex, presented in Figure 14d, showed high cytotoxic activity at low concentrations.

3. Heteronuclear Iridium(III) Complexes

In addition to mononuclear complexes, hetero-multinuclear complexes have attracted great consideration and have been studied for cancer treatment during the last decades. Heteronuclear complexes are those complexes which have more than one metallic center by using different metal cations. Such complexes combine the advantages of different metal ions in one molecule. In the group of iridium(III) complexes apart from multinuclear homometallic iridium(III) complexes, the heteronuclear Ir(III) complexes are of great interest. The variation of the central metal cation has a critical role in the resulting properties of these compounds. The coordination of two metal cations would suggestively extend the potential of their action mode. The inclusion of two different metal ions within a single molecule could improve their efficiency as antineoplastic agents through different mechanisms. This improvement could be connected to their interactions with various biotargets or with the enhanced physicochemical characteristics of the obtained heterometallic complexes. In addition, the presence of dissimilar modes of action led to synergistic results, modulation in oxidation-reduction properties and stability changes, which might lead to improved anticancer activities and higher selectivity compared to the respective mononuclear precursors [80]. The improved potency and capability to overcome the possible resistance, projects the obtained multinuclear compounds as prominent antineoplastic candidates.

The synthesis and characterization of a novel photoactive heterometallic platinum-iridium complex, its photochemical and photobiological properties, including phototoxicity against a variety of tumor cells (human ovarian A2780, lung A549, and prostate PC3 cell lines), have been reported by Shi et al. [81]. The photoactivated chemotherapy (PACT) diazido Pt(IV) complex has been conjugated to a photodynamic therapy (PDT) cyclometalated Ir(III) complex, giving the heterometallic platinum-iridium complex. The hetero binuclear complex trans, trans, trans-[Pt(py)₂(N₃)₂-(OH)(OOCCH₂CH₂CONHCH₂-bpyMe)Ir(ppy)₂]Cl, presented in Figure 15, has exhibited charge transfer between the acceptor photochemotherapeutic platinum(IV) and the donor photodynamic iridium(III) parts, displaying synergistic activity.

The platinum(IV)-iridium(III) complex was stable in dark conditions, but underwent photodecomposition more quickly than the parent Pt(IV) complex trans, trans, trans-[Pt(py)₂(N₃)₂(OH)₂] upon irradiation, which generated Pt(II) species, azidyl radicals, highly oxidizing excited-state Ir(III)* species and ROS (¹O₂) during the photodecomposition of the complex. Pt(II) species formed Pt-GMP adducts. Additionally, the Ir(III)* excited state oxidized NADH to NAD⋅ radicals and NAD⁺. The newly obtained complex was highly photocytotoxic against the tested tumor cells. In contrast, the mononuclear platinum and iridium fragments were much less active. The cellular platinum accumulation was higher for the dinuclear complex compared with the parent Pt(IV) complex possibly because of its positive charge and higher lipophilicity. Photoirradiation of the dinuclear complex in tumor cells damaged nuclei and released chromosomes. It has been found that iridium localized strongly in small cellular compartments. The luminescent platinum-iridium complex generated ROS and singlet oxygen ¹O₂ upon irradiation. This heteronuclear bimetallic platinum-iridium complex with photodynamic therapy and photoactivated chemotherapy (PACT) motifs combined in one molecule, provided an original and promising strategy for multimodal phototherapy [81]. Various multinuclear supramolecular assemblies based on platinum(II), ruthenium(II) and iridium(III) vertices for anticancer applications have been lately reviewed by Li et al. [82].

Tripathy et al. have obtained and studied antineoplastic activity of a heterometallic dinuclear Ir–Ru complex [{(ppy)₂Ir}(µ-phpy){Ru(p-cym)Cl}](PF₆)₂ with pyrazino[2,3-f][1,10]phenanthroline as a bridge ligand between the metal ions with distorted octahedral and piano-stool geometry (Figure 16) [83]. The authors have utilized the advantages of η⁶-arene Ru(II) and cyclometalated Ir(III) fragments along with a trinucleating polypyridyl-based ligand 2,3-di(pyridin-2-yl)pyrazino[2,3-f][1,10] phenanthroline (phpy) for the design of effective antitumor heterometallic binuclear complex. The obtained complex appeared to have more promising cytotoxic activity than the homonuclear diruthenium counterpart against various cell lines, such as breast (MCF7), ovarian (SKOV3), prostate (PC3) and endometrial (Ishikawa) cancer cells. The mechanism of cell death induced by the compound, presented in Figure 16, was further explored via cell cycle flow cytometric analysis, which exposed the stalling of the cells in the G₁ phase. Western blots have been performed to check for some fluctuations in expression levels of proteins answerable for cell apoptosis upon treatment with the complex, shown in Figure 16. Particularly, the Bcl-2 and Bax apoptotic regulators and the cleavage of poly(ADP-ribose) polymerase (PARP), included in DNA repair and apoptosis, have been examined in response to treatment with [{(ppy)₂Ir}(µ-phpy){Ru(p-cym)Cl}](PF₆)₂, Figure 16, [84]. No substantial growth in the levels of any of the three proteins has been recorded, demonstrating that the complex, shown in Figure 16, induced nonapoptotic cell death through the flow cytometric and Western blots analyses. Additionally, the examination of the cellular morphology revealed wide cytoplasmic vacuolization, a characteristic of autophagy [85]. The complex [{(ppy)₂Ir}(µ-phpy){Ru(p-cym)Cl}](PF₆)₂, Figure 16, was the first illustration of heteronuclear iridium–ruthenium complex which induced autophagic form of cell death against the resistant MCF7 cell line (IC₅₀ = 0.92 µM, Table 1), however additional studies would be essential to understand the mode of action of the studied complex [83].

Nallas et al. have reported a bipyrimidine-bridged trimetallic complex of the formula {[(bpy)₂Ru(bpm)]₂IrCl₂}⁵⁺, where bpy = 2,2′-bipyridine, and bpm = 2,2′-bipyrimidine, Figure 17, [86]. The complex coupled a catalytically active iridium(III) metal with a light-absorbing Ru(II) metal within the polymetallic basis. The detailed analysis of the spectroscopic, electrochemical, and spectrochemical properties of the polymetal complex was explained. It has been suggested that polymetal systems could be considered to function as photochemical devices, as the trimetallic complex shared the catalytical properties of Ir(III) with the photoactive properties of Ru(II) ions [87]. Within the framework of the progress of heterodimetallic complexes, similar trimetallic complex compounds of the form {[(bpy)₂Ru(BL)]₂MCl₂}ⁿ⁺ integrating the bridging ligands BL = 2,3-bis(2-pyridyl)pyrazine (dpp), 2,3-bis(2-pyridyl)quinoxaline (dpq), and 2,3-bis(2-pyridyl)benzoquinoxaline) (dpb) and the metal Ir(III), Rh(III) or Os(II) ions have also been investigated by the same group [88,89]. These polymetallic systems exhibited unique photophysical and electrochemical properties.

Recently, luminescent 2,2′-bipyrimidine ruthenium(II)-iridium(III)–arene monometallic and homo- and hetero-dimetallic complexes have been obtained and characterized [90]. The monochlorido Ir–Ru complex [Ir(η⁵-Cp*)Cl(μ-bpm)Ru(η⁶-pcym)Cl](PF₆)₂, shown in Figure 18, holding tetradentate 2,2′-bipyrimidine (bpm), has been investigated, along with diruthenium and diiridium analogs, in breast carcinoma MDA-MB-468 and colon carcinoma Caco-2 cells. Although ruthenium(II)-iridium(III) complex was less active (IC₅₀ = 1.9 μM) against MDA-MB-468 cell line (Table 1) than diiridium analog (IC₅₀ = 1.8 μM) and diruthenium complex (IC₅₀ = 0.9 μM), the heterometallic ruthenium(II)-iridium(III) compound (IC₅₀ = 6.2 μM, Table 1) exceeded both homometallic analogs (IC₅₀ = 32.4 and 46.0 μM for diiridium and diruthenium complexes) in Caco-2 cell line. Additionally, the Ir–Ru complex [Ir(η⁵-Cp*)Cl(μ-bpm)Ru(η⁶-pcym)Cl](PF₆)₂, shown in Figure 18, was more active towards the Caco-2 cell line than the respective mononuclear parts [Ir(η⁵-Cp*)(bpm)Cl]PF₆ with IC₅₀ = 50.4 μM and [Ru(η⁶-pcym)(bpm)Cl]PF₆ with IC₅₀ = 49.6 μM.

The same authors designed a novel type of heterobimetallic iridium-rhenium complex [Ir(η⁵-Cp*)Cl(μ-bpm)ReCl(CO)₃]Cl (Figure 19), by means of the conjugation of Ir(III)–Cp* and Re(I)-tricarbonyl motifs through the bridging bpm ligand, which has displayed significant anticancer potency [91]. The potency of the synthesized complex has been evaluated against triple-negative breast cancer (TNBC) MDA-MB-468 cells and normal immortalized human keratinocyte HaCaT cells. The complex displayed almost five-fold higher cytotoxic effect against the MDA-MB-468 cell line (IC₅₀ = 24.1 μM, Table 1) than its dirhenium homometallic and ruthenium–rhenium heterometallic correspondents. The compound was very selective against the MDA-MB-468 cell line compared to the noncancerous HaCaT cells (IC₅₀ = 234.8 μM). The iridium-rhenium complex demonstrated a sparkling binding tendency to DNA and HSA. Complex [Ir(η⁵-Cp*)Cl(μ-bpm)ReCl(CO)₃]Cl (Figure 19) was able to generate apoptosis of the tumor cells by inhibition of G₂/M phase in combination with a plentiful quantity of ROS production and mitochondrial dysfunction through reduction of MMP. The complex (Figure 19) revealed very low toxic effects together with strong binding efficiency with bioactive molecules.

New bimetallic Au(I)-Ir(III) complexes (Figure 20) with a bis(diphenylphosphino) methanide bridging ligand [Ir(C^N)₂(P^P)]⁺, in coparison with the monometallic precursor [Ir(N^C)₂(dppm)]⁺, have been reported [92]. The different Au(I) ancillary ligands, triphenylphosphine, a chloride or a thiocytosine, shown in Figure 20, did not expose any significant effects on the physical characteristics, which was principally owing to metal -ligand charge-transfers based on iridium(III). A study of cytotoxic activity, cell death mechanisms, reactive oxygen species generation, MMP integrity and cell biodistribution in tumor A549 cell line was performed for different Au(I) ancillary ligands. The gold(I) fragment, together with the ancillary ligands, appeared to be critical for the biological activity in the lung carcinoma A549 cell line versus the endothelial cell line. These bimetallic complexes have induced apoptosis with the formation of cytoplasmic vacuolization. The emission characteristics of Ir(III) fraction (green irradiation) have permitted the examination of cellular distribution, which presented the complexes more positioned in the cytoplasm, and the superimposition with red irradiation showed a mitochondrial accumulation. The existence of Au fragment has increased the ROS production and thioredoxin reductase (TrxR) inhibition compared to that demonstrated by the monoiridium complex [Ir(N^C)₂(dppm)]⁺. Iridium peptide bioconjugates and the respective Au(I)-Ir(III) bimetallic complexes have been recently described, in which an ortho-metallated iridium(III) species of the form [Ir(C^N)₂(N^N)]⁺ was functionalized with a peptide coupled to cytotoxic Au(I) fragment [93]. Lysosomal accumulation was detected for the studied compounds, as opposed to the expected accumulation in mitochondria triggered by the Au(I) complexes.

Ir(III)-Cu(II) heterobinuclear complex compounds have been synthesized by the combination of phosphanes (P-coordinated to iridium) and fluoroquinolones (O,O-coordinated to copper) as ligands [94]. Newl heterobinuclear iridium(III)–copper(II) coordination compounds with the formula [Ir(η⁵-Cp*)Cl₂(μ-L)Cu(phen)(H₂O)_n]NO₃, bearing phosphines derived from fluoroquinolones (L), viz., sparfloxacin (Hsfx), ciprofloxacin (Hcfx), lomefloxacin (Hlfx) and norfloxacin (Hnfx), have been obtained and investigated as possible antitumor chemotherapeutic agents. The novel heteronuclear iridium(III)–copper(II) complexes have been obtained by stirring [Cu(phen)(NO₃)₂] with [Ir(η⁵-Cp*)Cl₂(μ-L)] at room temperature. The piano-stool complexes, for instance, the best-acting complex, [Ir(η⁵-Cp*)Cl₂(μ-pcfx)Cu(phen)(H₂O)]NO₃, shown in Figure 21, were expressively more active than cisplatin in different human tumor cells, but they were practically ineffective in non-carcinogenic HEK-293T cell line. The cytotoxic activity of the complexes has been evaluated in vitro against melanoma, breast, lung, and prostate cancer cells and against nontumor human embryonic kidney cells. The heteronuclear iridium(III)–copper(II) complexes displayed higher cytotoxic activity than cisplatin against the tested cell lines (A549, MCF7, DU145) except for the WM2664 cells. IC₅₀ values have been measured by the MTT method in two different methods (after 24 or 24 + 48 h). Particularly the antineoplastic potency of [Ir(η⁵-Cp*)Cl₂(μ-pcfx)Cu(phen)(H₂O)]NO₃ in DU145 cell line was remarkable not only owing to the very low IC₅₀ in picomolar range (IC₅₀ = 1.3 × 10⁻⁶ μM at 24 h exposure), see Table 1, but also due to the unusual recovery of the tested cells (IC₅₀ = 125.7 μM at 24 h exposure and 48 h recovery time in a drug-free setting). Analogous results have been obtained for the same compound and its analogs in the MCF-7 cell line, while the contrary effect has been detected in the most sensitive lung carcinoma A549 cell line (IC₅₀ = 35.5 nM for 24 h exposure, Table 1, and IC₅₀ = 0.4 nM for 24 h exposure and 48 h recovery time in a drug-free setting). The studied complex [Ir(η⁵-Cp*)Cl₂(μ-pcfx)Cu(phen)(H₂O)]NO₃ was further loaded into liposomes. Liposomes loaded with this complex efficiently accumulate inside adenocarcinoma and prostate carcinoma cell lines with colocalization in the nuclei. Significantly for upcoming research in the field of heterometallic antitumor Ir–Cp^x compounds, this complex displayed noticeably higher cytotoxicity in tumor cells than the mononuclear analog, Figure 21 [95]. It could be efficiently accumulated inside prostate carcinoma and lung adenocarcinoma cells with a selective location in the nuclei. Cytometric analyses have revealed the domination of apoptosis over the other cell death types along with a significant increase in ROS generation.

The structure-activity relationships for a series of iridium(III) complexes covering a ferrocenyl moiety have been discovered by Tabrizi et al. [96]. The existence of ferrocene might improve the bioactivity of antineoplastic agents, as reported earlier [97]. Ir(III) arene complex compounds of naphthoquinone derivatives with the general formula [Ir(η⁶-L1)(L2)(3,5-(NO₂)₂pcyd)](PF₆), shown in Figure 22, where L1 = p-methylphenyl)ethynylferrocene; L2 = lawsone (Figure 22a), lapachol (Figure 22b), juglone (Figure 22c), plumbagin (Figure 22d) and 3,5-(NO₂)₂pcyd = 3,5-dinitrophenylcyanamide, have been synthesized and studied for their appropriateness as possible antitumor drugs [96]. The DNA-binding interactions of the compounds with calf thymus DNA have been investigated by absorption, emission, and viscosity measurements. Their cytotoxic activity against the tumor cells including liver hepatocellular carcinoma HepG-2, colon adenocarcinoma (HT-29), colon carcinoma HCT-8, breast MCF-7 and ovary A2780 carcinoma cells have been studied. Remarkably, almost all the tested complexes exhibited substantial cytotoxic activity against the tumor cells and the complex with lapachol, Figure 22b, appeared as the most cytotoxic compound in comparison with the other derivatives. The structure-activity relationships showed that the lipophilicity of tested complexes was the most important factor which determined their cytotoxic activity. Consequently, the most lipophilic complex with lapachol, Figure 22b, gave rise to the best antineoplastic activity based on IC₅₀ values ranging from 4 to 8 µM in cases of different tumor cells. Complexes in Figure 22 induced early- and late-stage apoptosis in the breast cancer cell line. The compounds also induced the formation of intracellular ROS in the case of the MCF-7 cell line. The origin of the ROS production was connected with thioredoxin reductase, that played a key role in preserving the cell redox status. The studied complexes were found to induce high levels of tumor cellular death in an apoptotic way which was in correlation with the effective inhibition of thioredoxin reductase at nanomolar concentrations with additional strengthening of the ROS generation contributing to cytotoxic activity.

Ferrocene-attached half-sandwich Ir(III) phenylpyridine complex compounds have been synthesized and characterized [98]. The complexes have shown improved anticancer activity than the clinically used cisplatin and their rhodium corresponding complexes. Complex [Ir(η⁵-Cp*)Cl(ppyfc)] (Hppyfc = ferrocene-modified 2-phenylpyridine) (Figure 23) was highly potent, but did not display any selectivity with comparable cytotoxic activity at the tumor cells and the normal BEAS-2B cells (IC₅₀ = 3.4 μM). The ferrocene-iridium(III) complexes could efficiently inhibit cell migration and colony formation. Complexes could interact with protein and transport through serum protein, efficiently catalyzing the nicotinamide−adenine dinucleotide oxidation and inducing the reactive oxygen species (ROS, ¹O₂) accumulation, confirming the antitumor mechanism of oxidation. Additionally, laser scanning confocal detection indicated that the compounds could enter cells followed by a non-energy-dependent cell uptake mechanism, efficiently accumulating in the lysosomes, leading to lysosome damage, and reduction of the mitochondrial membrane potential. Therefore, ferrocene-appended Ir(III) compounds possessed the prospect of becoming original multifunctional therapeutics, involving lysosome-targeted imaging agents and antineoplastic drugs [98].

Palao et al. have obtained biscyclometalated Ir(III) complex compounds, Figure 24a–c, including borondipyrromethene (BODIPY)-based ancillary ligands as smarter fluorescent photosentizers [99], where BODIPY unit was grafted to different chelating cores (acetylacetonate and bipyridine) by BODIPY meso-position. The boron–dipyrromethene (BODIPY)–Ir(III) complexes in Figure 24a,b exhibited higher absorption coefficients, higher fluorescence emission and also more effective ¹O₂ generation in visible light, than that shown by the complex in Figure 24c. In vitro photodynamic therapy activity of the complexes in Figure 24a,b in the HeLa cell line showed that these complexes are capably internalized into the tumor cells, demonstrating high photocytotoxic activity, even at low concentrations, making them hypothetically appropriate candidates in theranostic applications [99].

An important approach to improve the bioactivity of iridium organometallic compounds is the inclusion of active bioorganic ligands with recognized functions to design novel metal-based compounds with good therapeutic characteristics. In these cases, the combination of highly fluorescent BODIPY (boron dipyrromethene) ligands with an oil-soluble compound like avobenzone (1-(4-tert-butylphenyl)-3-(4-methoxyphenyl) propane-1,3-dione, AVBH), might be used as sunray blocking agents as important ingredients in lotions or creams have been obtained by Gupta et al., which gives a new platform for developing novel dicationic metal supramolecules as antineoplastic agents [100]. Recent investigations have revealed that AVBH has notable cytotoxicity against various human tumor cells [101] Moreover, the fluorescent dye BODIPY possesses exceptional photo-physicochemical properties. Therefore, BODIPY ligands and their complexes have been chosen because of their crucial application in tumor treatment on account of their emission and absorption effects which facilitated the bio-tagging trials. The structures of BODIPY (BDP = 4-dipyridine boron dipyrromethene and BDPCC = 4-ethynylpyridine boron dipyrromethene) iridium bimetallic complexes [Ir₂(Cp*)₂(AVB)₂BDP][2CF₃SO₃] and [Ir₂(Cp*)₂(AVB)₂BDPCC][2CF₃SO₃] with excellent anticancer activity and bioimaging capability are presented in Figure 25, [100]. The complexes were characterized by various analytical methods, and their structural parameters were further optimized by density functional theory (DFT). These complexes have been proven to be active against human lung A549, cervical HeLa and breast MCF-7 cancer cell lines with IC₅₀ values between 1 and 5 μM. The complexes also were tested against non-malignant mouse embryo fibroblast NIH T3T cells. The BDPCC-based iridium complex was found to have stronger antineoplastic properties than the BDP-containing iridium complex with activity analogous to that of the chemotherapeutic doxorubicin. The studied iridium complexes have also been found to interact strongly with genomic DNA causing the unwinding of the double helix. The BDPCC-based complex [Ir₂(Cp*)₂(AVB)₂BDPCC][2CF₃SO₃] showed a comparatively better activity.

Tetranuclear square BODIPY metallocycles have shown selective toxicity against MCF-7 and U87 cells [102]. The binding study has revealed the capability of the complexes to interact with proteins [103].

4. Metallacages

The development of metal-based macrocycles using bioactive ligands represents a key direction for the design of novel antineoplastic agents. In contrast to platinum(II) and ruthenium(II) assemblies, the progress of iridium-based macrocycles incorporating cyclometalated or half-sandwich Ir(III) complexes is limited. Nevertheless, some Ir(III) assemblies have shown higher effectiveness than cisplatin against the proliferation of numerous tumor types. In recent decades, the investigations involving metalla-macrocycles, tested as antitumor candidates, have increased [82], including the reported hexanuclear complexes [104], and the 5,8-dihydroxy-1,4-naphthoquinonato tetranuclear metallacycles with bipyridyl linkers [105].

Three new pentamethylcyclopentadienyl Ir(III) metalla-rectangles, Figure 26, have been obtained by a self-assembly approach using the embelin-derived metallaclips (η⁵-C₅Me₅)₂Ir₂(μ₄-C₆HRO₄-κO)Cl₂ (R = (CH₂)₁₀CH₃)). Two additional ditopic bridging linear ligands, containing pyrazine (Figure 26a), 4,4′-bipyridine (Figure 26b), or 1,2-bis(4-pyridyl)ethylene (Figure 26c), have been used to complete the metal-based rectangular structures. The compounds have been studied by IR, ¹H- and ¹³C-NMR, and ESI-MS spectral and elemental analyses. Tetranuclear iridium complexes, Figure 26, were assessed for antiproliferative action against lung, prostate, and cervical cancerous and noncancerous (HEK-293) cells [106]. The iridium(III) fragments have been linked through the hydrophobic benzoquinone embelin, purposefully chosen because of its cellular permeability [107]. Higher and biologically potential selectivity against tumor over healthy cells was found for the most cytotoxic tetranuclear iridium complex [Ir₄(μ-emb)₂(μdpee)₂(η⁵-Cp*)₄](CF₃SO₃)₄, depicted in Figure 26c, which displayed markedly lower IC₅₀ values in tumor cells (0.6 μM in HeLa) compared with HEK-293 cell line (70.8 μM), Table 1, [106]. The capability of the tetracationic metallamacrocycles to accumulate in mitochondria has been established by flow cytometry and confocal microscopy investigations. Cell cycle examination displayed accumulation in the sub-G₁ phase, signifying that treatment with such complexes led to DNA fragmentation. The complexes induced apoptosis in the early and late stages. The studied metalla-rectangles appeared to interact with DNA and mitochondria membranes, most probable due to the positive charge of the metallamacrocycles and the existence of lipophilic side chains, therefore giving these compounds the required characteristics for possible in vivo studies.

Ir(III)-Cp* metalla-prismatic structures (Figure 27) [(Cp*Ir)₆(tpt)₂(dhnq)₃]⁶⁺, where tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine and dhnq = 5,8-dihydroxy1,4-naphthoquinonato ligands, have been obtained and identified by different analytical methods. The cytotoxic and photodynamic activities have been assessed on human HT-29 colon cancer cells, displaying IC₅₀ around 1 μM, which decreased to nanomolar concentrations at irradiation, demonstrating a perfect synergistic effect between the cytotoxic and phototoxic activity [108]. Photoactive supramolecular metallamacrocycles integrating Ir(III) complexes have been reported by Martir et al. [109].

Table 1. IC₅₀ values of polynuclear iridium complexes.

Compound	IC50	Cell Lines	Ref.
[Ir2(m-pzpy)(η5-Cp*)2Cl3]PF6	>100 μM	HT-29	[65]
[{(η5-Cp*)Ir(dppz)}2(μ-pyz)]4+	3.5 μM	MCF-7	[66]
	1.8 μM	HT-29
[{(η5-C5Me5)Ir(dppz)}2(μ-4,4′-bpy)]4+	3.1 μM	MCF-7
	3.8 μM	HT-29
[{(η5-Cp*)Ir(dppz)}2(μ-dpee)]4+	0.61 μM	MCF-7	[67]
[{(η5-Cp*)Ir(dppz)}2(μ-dpey)]4+	0.49 μM	MCF-7
[{(η5-Cp*)Ir(dppz)}2(μ-dpeb)]4+	2.2 μM	MCF-7
[Ir2(μ-L*)(η5-Cp*)2Cl2](PF6)2	3.1 μM	A2780	[69]
	6.0 μM	MCF-7
[Ir2(μ-L**)(η5-Cp*)2Cl2](PF6)2	12.6 μM	HepG2	[70]
	16.5 μM	A549
[{(ppy)2Ir}(µ-phpy){Ru(p-cym)Cl}](PF6)2	0.92 µM	MCF7	[83]
[Ir(η5-Cp*)Cl(μ-bpm)Ru(η6-p-cym)Cl](PF6)2	1.9 μM	MDA-MB-468	[90]
	6.2 μM	Caco-2
[Ir(η5-Cp*)Cl(μ-bpm)ReCl(CO)3]Cl	24.1 μM	MDA-MB-468	[91]
[Ir(η5-Cp*)Cl2(μ-pcfx)Cu(phen)(H2O)]NO3	1.3 pM	DU145	[94]
	35.5 nM	A549
[Ir4(μ-emb)2(μ-dpee)2(η5-Cp*)4](CF3SO3)4	0.6 μM	HeLa	[106]
[(Cp*Ir)6(tpt)2(dhnq)3]6+	1 μM	HT-29	[108]

Abbreviations: pzpy = N’-[(1Z)-1-(pyridin-2-yl)ethylidene]pyrazine-2-carbohydrazonamide); dppz = dipyrido[2,3-a:2′,3′-c]phenazine; pyz = pyrazine; 4,4′-bpy = 4,4′-bipyridine; dpee = 4-[(E)-2-(4-pyridinyl)ethenyl]pyridine; dpey = 4-(2-pyridin-4-ylethynyl)pyridine; dpeb = 1,4-di(2-pyridin-4-ylethynyl)benzene; L* = N,N′-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl)ethanamine; L** = (1E)-(6-methylpyridin-2-yl)-N-(4-(4-(((E)-(6-methylpyridin2-yl)methylidene)amino)benzyl)-phenyl)methanimine; bpm = 2,2′-bipyrimidine; pcfx = ciprofloxacin; fcdpm = 5-ferrocenyldipyrromethene; ppy = pyridine; p-cym = p-cymene; phen = phenanthroline; emb = embelin; phpy = 2,3-di(pyridin-2-yl)pyrazino[2,3-f][1,10] phenanthroline; tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine; dhnq = 5,8-dihydroxy1,4-naphthoquinone.

Table 1. IC₅₀ values of polynuclear iridium complexes.

Compound	IC50	Cell Lines	Ref.
[Ir2(m-pzpy)(η5-Cp*)2Cl3]PF6	>100 μM	HT-29	[65]
[{(η5-Cp*)Ir(dppz)}2(μ-pyz)]4+	3.5 μM	MCF-7	[66]
	1.8 μM	HT-29
[{(η5-C5Me5)Ir(dppz)}2(μ-4,4′-bpy)]4+	3.1 μM	MCF-7
	3.8 μM	HT-29
[{(η5-Cp*)Ir(dppz)}2(μ-dpee)]4+	0.61 μM	MCF-7	[67]
[{(η5-Cp*)Ir(dppz)}2(μ-dpey)]4+	0.49 μM	MCF-7
[{(η5-Cp*)Ir(dppz)}2(μ-dpeb)]4+	2.2 μM	MCF-7
[Ir2(μ-L*)(η5-Cp*)2Cl2](PF6)2	3.1 μM	A2780	[69]
	6.0 μM	MCF-7
[Ir2(μ-L**)(η5-Cp*)2Cl2](PF6)2	12.6 μM	HepG2	[70]
	16.5 μM	A549
[{(ppy)2Ir}(µ-phpy){Ru(p-cym)Cl}](PF6)2	0.92 µM	MCF7	[83]
[Ir(η5-Cp*)Cl(μ-bpm)Ru(η6-p-cym)Cl](PF6)2	1.9 μM	MDA-MB-468	[90]
	6.2 μM	Caco-2
[Ir(η5-Cp*)Cl(μ-bpm)ReCl(CO)3]Cl	24.1 μM	MDA-MB-468	[91]
[Ir(η5-Cp*)Cl2(μ-pcfx)Cu(phen)(H2O)]NO3	1.3 pM	DU145	[94]
	35.5 nM	A549
[Ir4(μ-emb)2(μ-dpee)2(η5-Cp*)4](CF3SO3)4	0.6 μM	HeLa	[106]
[(Cp*Ir)6(tpt)2(dhnq)3]6+	1 μM	HT-29	[108]

Abbreviations: pzpy = N’-[(1Z)-1-(pyridin-2-yl)ethylidene]pyrazine-2-carbohydrazonamide); dppz = dipyrido[2,3-a:2′,3′-c]phenazine; pyz = pyrazine; 4,4′-bpy = 4,4′-bipyridine; dpee = 4-[(E)-2-(4-pyridinyl)ethenyl]pyridine; dpey = 4-(2-pyridin-4-ylethynyl)pyridine; dpeb = 1,4-di(2-pyridin-4-ylethynyl)benzene; L* = N,N′-(biphenyl-4,4′-diyldimethylidyne)bis-2-(pyridin-2-yl)ethanamine; L** = (1E)-(6-methylpyridin-2-yl)-N-(4-(4-(((E)-(6-methylpyridin2-yl)methylidene)amino)benzyl)-phenyl)methanimine; bpm = 2,2′-bipyrimidine; pcfx = ciprofloxacin; fcdpm = 5-ferrocenyldipyrromethene; ppy = pyridine; p-cym = p-cymene; phen = phenanthroline; emb = embelin; phpy = 2,3-di(pyridin-2-yl)pyrazino[2,3-f][1,10] phenanthroline; tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine; dhnq = 5,8-dihydroxy1,4-naphthoquinone.

The octanuclear iridium metallocube (Figure 28) has been obtained by incorporating Cp*-Ir building fragments bridged by an alkyl-functionalized ligand, and connected by tetrapyridyl porphyrin fragments [110]. The cubic supramolecular macrocycle has shown strong antitumor activity toward MCF-7, B16, and A549 cancer cells with IC₅₀ under 0.1 μM and good selectivity over the normal NIH-3-T3 cell line. The proposed mechanism of action was apoptotic induction with DNA interaction.

Ir(III) metalla-rectangle IrBAnPy, shown in Figure 29, has been obtained by the coordination of an anthracene-functionalized dipyridyl ligand BAnPy₂, resulting in the formation of a robust rectangular [111]. Its antineoplastic properties have been examined, revealing promising cytotoxic activity against A549 and H460 cancer cells in comparison with the non-malignant MRC5 cells. The detected IC₅₀ were 16.24 µM for H460 and 2.38 µM for A549, respectively. In contrast, the IC₅₀ for non-malignant MRC5 cell line was 29.44 µM. Micronucleus and cell death assay have been performed to determine the damage occurring to the cancer cellular nucleus.

Metal-organic cages (MOCs) have shown well-defined structural shapes and cavities, and possess promising applications in many fields [112,113].

Ir₈Pd₄-heteronuclear metal-organic cage Ir₈Pd₄-MOC (MOC-51) has been constructed from bipodal metal-based ligand [Ir(ppy)₂(qpy)(BF₄)] (qpy = 4,4′:2′,2′′:4′′,4′′′-quaterpyridine; ppy = 2-phenylpridine) with a palladium(II) salt [114]. The cubic barrel-shaped metal-organic cage showed large ¹O₂ quantum yields under visible irradiation, consequently displaying great potential in cell imaging and PDT. In comparison with the Ir(III) metallo-based ligand, the Ir₈Pd₄-MOC has shown less dark toxicity toward HeLa cells and higher mitochondria-targeting efficiency.

Liu et al. have recently described [Pd₄Ir₈]¹⁶⁺ supramolecular cubic cage MOC-53 containing multiple iridium(III) metallo-based ligands, localized in mitochondria with high ¹O₂ production activating efficiently apoptosis [115]. The bimetallic supramolecular cage has shown superior antineoplastic activity, bright phosphorescence and excellent photostability. The reported supramolecular cubic cage has shown higher phototoxicity toward the HeLa cell line.

5. Conclusions and Perspective

This review surveyed iridium(III) multinuclear or heteronuclear complexes and their use in anticancer drug discovery. Although promising mononuclear iridium(III) complexes have been extensively studied and provided a positive hope for developing effective antineoplastic agents, the studies on the antitumor application of homo- and hetero-multinuclear Ir(III) complexes are insufficient and this area needs further examination. This research field is currently extremely active and it has been expanded over the last years. Numerous research teams have developed various multinuclear iridium complexes and have investigated their properties in different conditions (diverse cell lines, concentrations and incubation time, etc.), thus a complete validation of the structure-activity relationship is difficult to achieve. It is greatly appreciated that the common range of IC₅₀ values of the newly reported iridium(III) multinuclear complexes is less than 100 μM. In most of the cases, these complexes have demonstrated selectivity and good tolerance by healthy cells. Their anticancer mechanism of action has been found to be different from that of platinum drugs, offering cytotoxicity against cisplatin-resistant cancer cells. Multinuclear iridium(III) complexes were either derived from their mononuclear analogs or were prepared with similar ligands of diverse denticities, which permitted the coordination of a different number of metallic centers. These strategies have enabled a comparison of the effect of metal cations on the resulting antineoplastic activity and, in most cases, multinuclear complexes displayed better activity than the mononuclear ones. Obviously, the studies, reported in the literature, have confirmed that the design of multinuclear Ir(III) complexes became a viable strategy and a suitable research direction. Nevertheless, as demonstrated in this review, it is difficult to compare complexes with different nuclearity and a systematic study associated with the possible biological applications of these complexes is still lacking. It is expected that this review will offer vital guidance for the further development of these promising anticancer candidates.