Dichloro Ru(II)-p-cymene-1,3,5-triaza-7-phosphaadamantane (RAPTA-C): A Case Study

Abstract

The use of organometallic compounds to treat various phenotypes of cancer has attracted increased interest in recent decades. Organometallic compounds, which are transitional between conventional inorganic and organic materials, have outstanding and one-of-a-kind features that offer fresh insight into the development of inorganic medicinal chemistry. The therapeutic potential of ruthenium(II)-arene RAPTA-type compounds is being thoroughly investigated, specifically owing to the excellent antimetastatic property of the initial candidate RAPTA-C. This review gives a thorough analysis of this complex and its evolution as a potential anticancer drug candidate. The numerous mechanistic investigations of RAPTA-C are discussed, and they are connected to the macroscopic biological characteristics that have been found. The “multitargeted” complex described here target enzymes, peptides, and intracellular proteins in addition to DNA that allow it to specifically target cancer cells. Understanding these may allow researchers to find specific targets and tune a new-generation organometallic complex accordingly.

Cisplatin (cis-diamminedichloroplatinum(II)) is the most popular metal-based coordination complex in cancer-therapeutic settings.¹ After receiving U.S. Food and Drug Administration (FDA) approval in 1978 to treat ovarian and testicular cancer, cisplatin was widely commercialized to treat a variety of cancers, despite having a toxicity profile toward normal cells that caused adverse side effects even at modest dosages. The gradual emergence of acquired resistance with cisplatin’s continued use was another disadvantage. Patients often responded well to cisplatin-based chemotherapy at first but subsequently relapsed because of the emergence of cisplatin resistance (either acquired or intrinsic), which significantly lowered its therapeutic efficacy.²⁻⁵

With the goal of overcoming resistance, cisplatin derivatives such as oxaliplatin,⁶ carboplatin,⁷ etc., were synthesized and have now become renowned (Table 1). However, the efficacy of these derivatives is continuously constrained by side effects. Other metals that possess pertinent pharmacological characteristics have since been investigated to overcome these side effects.⁸⁻¹⁰ Conspicuously, Ru-based complexes have drawn interest since some have been discovered to circumvent the established Pt resistance pathways¹¹⁻¹³ and function via different mechanisms.¹³⁻¹⁵ In comparison to Pt-based drugs, Ru-based ones have some exceptional qualities that make them desirable chemotherapeutic agents.^12,15−18 These qualities include improved tumor cell selectivity and reduced toxicity toward normal tissues through interaction with protein-based receptors. The known mechanisms of action (MoA) for these against tumors include mitochondrial, DNA damage, and death receptor pathways.¹⁹⁻²¹ The therapeutic benefits against cancer were seen to be enhanced when Ru-based complexes were administered in combination with various other known treatments, including photodynamic therapy (PDT),²² photothermal therapy (PTT),²³ targeted therapy,²⁴ nanotechnology,²⁵ etc. However, the challenge in creating new anticancer drugs remains how to enhance absorption and therapeutic selectivity of the drug for cancer cells.^{13,15,18,19,21,26−32}

Table 1. Some of the Well-Known Pt-Based Chemotherapeutic Drugs.

Only three Ru-based compounds, imidazolium-trans-tetrachloro(dimethylsulfoxide) imidazoleruthenium(III) (NAMI-A),^14,33trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019),³⁴ and its sodium salt (N)KP1339, have been studied and investigated in clinical trials for cancer.^14,35 Although the clinical trials for NAMI-A were terminated, NKP1339 in the form of BOLD-100 is being comprehensively researched (Figure 1). BOLD-100 is the most advanced Ru-based treatment currently under investigation as an anticancer therapeutic. For the treatment of advanced gastric, pancreatic, colon, and bile duct cancers, BOLD-100 is being studied in combination with FOLFOX [a chemotherapy regimen composed of the medicines folinic acid, fluorouracil (5-FU), and oxaliplatin] (Figure 2) in a global phase 2 trial. BOLD-100 successfully completed a phase 1 monotherapy dose escalation trial in patients with advanced cancer, demonstrating its general safety and tolerance. The U.S. FDA has recently given BOLD-100 the orphan drug designation (ODD) for pancreatic and stomach cancers.

Figure 1.

Some well-researched Ru-based complexes for anticancer therapy.

Figure 2.

FOLFOX: A combination-chemotherapy regime used to treat colorectal cancer.

Ru(II)-arene 1,3,5-triaza-7-phosphaadamantane [Ru(arene)Cl₂(PTA)], also known as the RAPTA complex, is another Ru-based compound that is advancing well. The Dyson group developed the famous piano-stool framework; a Ru(II) complex that contains an η⁶-coordinated arene to assist in stabilizing the +2 oxidation state of Ru in addition to two labile chlorido ligands and an amphiphilic PTA ligand.³⁶ PTA is comparatively sterically undemanding compared to other phosphine ligands (cone angle 103°)³⁷ and is expected to help the RAPTA framework to become more water soluble than other Ru(II)-arene complexes.³⁸ It is worthwhile to mention that, in parallel, the research on Ru(II)-arene ethylene diammine (RAED)-type complexes developed by Sadler’s group is also progressing rapidly (Figure 1). However, fundamental differentiation can be made between the two—while the former is noncytotoxic and antimetastatic, the latter has shown good cytotoxicity across various cancer cell lines.

Among many RAPTA-type candidates, having revealed its efficacy in anticancer action through both in vitro and in vivo assessments, RAPTA-C (Figure 3) appears to be a fitting applicant for clinical investigation.³⁶⁻³⁹ RAPTA-C is ineffective in cytotoxicity assays (IC₅₀ > 300 μM in most cancer cell lines tested, Table 2) but has strong antitumor effects in vivo with significantly less toxicity.^18,40−47 RAPTA-C alters proteins and histone DNA to demonstrate antimetastatic, antiangiogenic, and antitumor actions.^48,49 RAPTA-C seems to be quite pragmatic compared to other Ru-based medications that have recently been studied in clinical trials, particularly when used in conjunction with other targeted medications. Numerous frameworks related to its structure have been popping up in the literature owing to the excellent activity exhibited by RAPTA-C, which makes understanding the chemistry and MoA related to the complex a prerequisite. This review is an exclusive case study on RAPTA-C and its investigations as an anticancer therapeutic drug.

Figure 3.

Structural skeleton of RAPTA-C.

Table 2. Poor Cytotoxic Profile of RAPTA-C in Various Cell Lines.

	IC50(μM)
Cell line	48 h	72 h	96 h
CH1	–	–	6566
SW480	–	–	17166
A549	–	–	>51566
TS/A	–	>30039	–
HBL-100	–	>30039	–
A2780	–	353;67 24748	–
CisRA2780	–	50748	–
MCF-7	>100068	–	–
HCC1937	>100068	–	–

Discovery, Structural Features, and Activation Mechanism

Dyson’s group put forward the earliest report on RAPTA-C.⁵⁰ The synthesis involved refluxing a mixture of Ru(II)-p-cymene precursor and PTA (1:2 ratio) in methanol for about 24 h (Scheme 1).

Scheme 1. Synthesis of RAPTA-C⁵⁰.

As reported, the single-crystal X-ray diffraction (XRD) structure of RAPTA-C displayed the characteristic piano-stool structure (Figure 4). The Ru–C bond length average was about 2.20 and 2.21 Å in each asymmetric molecule reported; the C₆-ring of the p-cymene was coordinated to the Ru(II) ion with a hapticity (η) of 6. The remaining components of the coordination sphere were the PTA group (mean Ru–P bond length, 2.296(2)–2.298(3) Å) and two chlorido ligands.⁵⁰ The p-cymene ligand is said to stabilize the +2 oxidation state by binding as a π-acceptor and a η⁶ electron (e^–) donor. The Ru-p-cymene binding energy was discovered to be 21.2 kcal/mol. The potential energy surface (PES) diagram, which explains how the p-cymene rotates, was discovered to be very flat. The highest occupied molecular orbital (HOMO) was mostly concentrated on the Ru ion and chlorido centers (bonded d-p-orbital interaction), with little involvement of π-orbitals of p-cymene and Ru d-orbitals and none from PTA. Similarly, the lowest unoccupied molecular orbital (LUMO) (antibonding) was localized on the Ru ion (d-orbitals perpendicular to the p-cymene plane), chlorido ligands, and p-cymene but not on the PTA.⁵¹

Figure 4.

Classic piano-stool structure of Ru(II)-arene complexes (in figure: RAPTA-C).

The MoA of metal-based anticancer candidates has been the subject of various investigations.^{12,46,52−55} For these coordination complexes, hydrolysis is thought to initiate the activation mechanism. For cisplatin, the usually accepted MoA is the hydrolysis of the Pt–Cl bond intracellularly (initial step) before it reaches the DNA target; the complex releases the chlorido ligand in the intracellular medium until equilibrium, and it is thought that the hydrolysis product(s) are the actual antitumor vehicles (Figure 5). Numerous techniques have been used to determine the rate of hydrolysis of the M–X bond in such coordination complexes.⁵⁶⁻⁵⁹ In Ru(II)-arene complexes, it is now well known that the presence of a π-bonded arene can significantly alter their properties. There is an elegant balance between e^– donation from the arene ligand into the vacant 4d orbitals of Ru and back-donation from the filled 4d⁶ orbitals into unoccupied arene orbitals. The remaining ligands on the Ru^II-arene system can affect the accessibility of the metal’s 4d⁶ e^–(s), which in turn can control the kinetic substitution parameters of the Ru–X bond.

Figure 5.

A simple depiction of the MoA of cisplatin. [Redrawn from ref (60).]

Taking a look at the solution behavior, RAPTA-C was primarily found as its monohydrolyzed [RuCl(H₂O)(η⁶-p-cymene)(PTA)]⁺ form in aqueous solutions, along with minor quantities of both the hydroxo-aqua [Ru(OH)(η⁶-p-cymene)(H₂O)(PTA)]⁺ species and the original complex. It was seen that the hydrolysis of RAPTA-C was significantly lower at extracellular concentrations of Cl^– ions (∼100 mM) compared to intracellular concentrations (∼4 mM). These findings suggested that, similar to cisplatin, RAPTA-C, unless metabolized differently, as via interaction with proteins, would withstand the surroundings in the blood circulation and that its activation may only transpire upon hydrolysis that may happen intracellularly, where the [Cl^–] falls to ∼4 mM. It was also seen that the second chlorido substitution reaction happened more quickly than the first, most likely because of the initial deprotonation of the aqua ligand (Scheme 2).

Scheme 2. Most Probable Activation Mechanism of RAPTA-C⁵⁷.

The rate of hydrolysis of RAPTA-C was approximately 3 times quicker than that of RAED and 2 times faster than those of imidazolium[trans-[RuCl₄(1H-imidazole)₂] (KP418) and cisplatin.^57,61 Substituting a bidentate cyclobutane-1,1-dicarboxylate (a more slowly hydrating ligand) in place of the labile chlorido ligands in the cisplatin gave rise to a more stable carboplatin. Owing to this, carboplatin yields the same reaction products in vitro at equal dosages as cisplatin but has a lower reactivity and slower DNA binding kinetics. The decreased reactivity restricts protein–carboplatin adducts (which is how the complex is excreted), and hence carboplatin has a lower excretion rate than other drugs: the more of it is kept in the body, the longer its effects last.¹ With a similar aim, the two labile chlorido ligands in the RAPTA-C were swapped out for a dicarboxylate ligand. A parallel change as observed for its Pt analogues was witnessed.⁶²

One of the earliest findings depicted that RAPTA-C did not cause DNA damage at physiological pH (7.5 or higher) (hydroxo form predominated, not active). The extent of DNA impairment increased when the pH was decreased (aqua species dominated, active). Given that DNA is negatively charged and that the pH value above which RAPTA-C inhibited DNA movement nearly matched the pK_a of PTA, it could be anticipated that, if these two species were present together, their contact would be encouraged. This factor was particularly beneficial to improve the selectivity of the coordination complex since it is well known that cancer cells, having a reductive environment, show pH less than 7.0.⁵⁰ The pK_a of the coordinated H₂O ligand of the activated aqua adduct is also crucial for drug activation, just as significant as the rate of hydrolysis.^50,55 Interestingly, the PTA ligand in RAPTA-C had a pK_a value of 3.13 in 0.1 M NaCl solution (used to maintain the original complex form), indicating that it was unlikely that the PTA ligand would be protonated in vivo under physiological conditions.³⁹ However, a study measured the impact of the PTA ligand on the physicochemical characteristics of RAPTA-C in the gas phase using ion mobility mass spectrometry (IM-MS) and collision-induced dissociation (CID) methods. Electrospray ionization (ESI) of RAPTA-C produced two molecular ions: in-source oxidation ([Ru^IIICl₂(p-cymene)(PTA)]⁺) or protonation ([RuCl₂(p-cymene)(PTA+H)]⁺) products. Control investigations demonstrated that the infusion solvent composition significantly impacted the balance between these two ionization pathways.

The binding of RAPTA-C in physiological conditions has often led to the cleavage of the ligands in its coordinative sphere in order to accommodate the biomolecular target(s). Of course, the labile chlorido ligands are the first to go; however, in some cases, the PTA ligand was seen to be cleaved, and with longer incubation times, the p-cymene is also lost, although the latter was seldom observed.^20,63,64

Cytotoxicity, Metastatic Action, and ADME Properties

Fascinatingly, in the initial report pertaining to the biological activity of RAPTA-C, only the antimicrobial qualities of the complex had been assessed, i.e., antiviral against the Herpes simplex and polio viruses, antibacterial against Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, as well as antifungal against Candida albicans, Cladosporium resinae, and Trichrophyton mentagrophytes.⁶⁵ It was deduced in the initial report itself that the biological activity of RAPTA-C did not seem to be connected to DNA binding; instead, it appeared to be due to specific interactions with proteins,⁶⁵ which was well-proven later on.

Exploring the cytotoxicity of a possible anticancer candidate in cancer cell line(s) is probably the most fundamental step in cancer-drug research. However, the results for RAPTA-C in the initial test against the epithelial cell line derived from human breast milk (HBL-100) and mouse mammary adenocarcinoma (TS/A) cell lines were rather disheartening (Table 2). Fortunately, seeing the low toxicity of the drug, the researchers proceeded to measure the in vivo activity of the complex. When administered on a regimen of 2 × 200 and 4 × 100 mg/kg/day, RAPTA-C was observed to lessen the number of lung metastases in CBA mice. The most effective treatment for preventing the development of lung metastases seemed to be a 200 mg/kg dosage given twice. It is significant to note that 2/5 mice in this treatment group did not have any metastases at the conclusion of the experiment.³⁹ These results seemed worthy enough for the research into the activity of RAPTA-C to continue; nontoxicity toward normal cells while showing metastatic activity was a very appealing concept. The reduced toxicity of RAPTA-C was seen as a contributing factor in the mice’s enhanced survival.

Another early study showed that, in an in vivo experimental model (mice), the growth of Ehrlich ascites carcinoma (EAC) cells was significantly inhibited following RAPTA-C treatment (40 mg/kg was the optimum dosage). Following exposure to RAPTA-C, the cell-cycle was arrested in the G2/M phase, which was induced by lowering the production of cyclin E (a crucial controller of the G1-S/G2-M transition)⁶⁹⁻⁷¹ and inflating the expression of p21 (cyclin-dependent kinase inhibitor 1).^72,73 Further, the cytochrome c (cyt c) level was raised as well as that of the proapoptotic proteins, which activated procaspase-9 (following caspase cascade),⁷⁴ encouraging apoptosis. Inhibition of cell growth caused by RAPTA-C in EAC cells was by activation of c-Jun N-terminal kinases (JNK) (the primary target of many DNA-damaging agents).⁷⁵ This work drew attention to modifications in the expression and function of vital proteins known to control the cell-cycle and apoptosis (Figure 6).³⁶

Figure 6.

Probable mechanism of cell death of RAPTA-C.

The metabolism and proliferation of isolated endothelial cells (HUVEC) and an immortalized endothelial cell line (ECRF24) following treatment with RAPTA-C were found to be inhibited; apoptosis was induced in order to limit the proliferation. Interestingly, it was shown that tumor cells were marginally less responsive than endothelial cells, indicating that the complex had inherent angiostatic capability, which was further seen to be dose dependent. Large avascular zones were evident due to a marked inhibition of vessel development in the chicken chorioallantoic membrane (CAM), which indicated that RAPTA-C may have an anticancer effect via angiogenesis suppression. Besides, the suppression of proliferation was thought to primarily consist of antiangiogenic action, as no cytostatic impact was seen. This effect was surprisingly similar to that of the antimetastatic Ru(III)-agent NAMI-A,⁷⁶ which was gaining immense popularity around the same time. This led to the belief that RAPTA-C could be the next in line for metastasis treatment. The study put forward the idea that RAPTA-C may be a selective inhibitor of the vascular endothelial growth factor (VEGFR) and fibroblast growth factor receptor 1 (FGF-R1) receptors, which are implicated in tumor angiogenesis.⁷⁷

RAPTA-C therapy effectively decreased tumor development: at a dosage of 0.2 mg kg^–1 per day, tumor growth was reduced by roughly 75% in the human A2780 ovarian cancer (CAM model). It is notable that RAED-C, when tested on tumors produced from A2780 in athymic mice, inhibited tumor development by around 50% at a dosage that was 10 times greater than the amount employed for RAPTA-C in this study.⁷⁸ Also, 50% inhibition was seen on Swiss athymic mice with human colorectal adenocarcinoma (LS174T) (100 mg kg^–1 per day) upon RAPTA-C treatment.³⁷ The RAPTA-C-treated tissues had significant sections of nonproliferating tumor cells, while the control samples contained live tumor tissues with many blood vessels. Microvessel density analysis of treated tumors (100 mg kg^–1) showed a substantial reduction in the number of blood vessels, indicating a potent antiangiogenic effect. Smooth muscle actin (SMA) staining showed that RAPTA-C treatment produced pericyte-covered, normalized, more mature blood vessels.³⁷ Notably, again, during the course of therapy, the average body weight of animals in the treatment groups did not drastically alter (Figure 7).

Figure 7.

Antitumor and antiangiogenic action observed in various in vivo models upon RAPTA-C treatment.

Following delivery, a drug’s pharmacokinetics are typically described by its absorption, distribution, metabolism, and elimination (ADME). The half-life period (T_1/2) of RAPTA-C ranged between 10.39 and 12.21 h in healthy Swiss CD-1 mice. The distribution volumes (V_d) ranged from 100 to 163 mL, depending on the dosage. The amount of blood cleared of the complex by all elimination processes (metabolism and excretion) in one unit of time, or total clearance (Cl_tot), ranged from 6.5 to 9.2 mL/h (Table 3). The study also suggested that RAPTA-C had more blood clearance and better tissue penetration than NAMI-A. Depending on the dosage of the therapy, the amount of Ru retained by the body 1 h after the final injection was around 8–14% of the administered dose of RAPTA-C. For NAMI, the amount of recovered Ru, 1 h after the final treatment, was around 20% of the administered dosage and reduced to 10% after 24 h when administered in a single 200 mg/kg IV therapy. Atomic absorption spectroscopy measured the amount of Ru in the blood, plasma, and a few organs (liver, kidney, spleen, and lung) in healthy Swiss CD-1 mice. Compared to the liver, kidney, and spleen, RAPTA-C was removed from the lungs more quickly, in contrast to NAMI-A. The findings also show that RAPTA-C was excreted from the spleen at a modest pace.³⁹ However, another study in healthy Balb/c mice reported that 2 h after injection, the quantity of Ru in the urine (kidney) was approximately 28 times greater than that in the liver, spleen, lungs, and heart, indicating quick and effective renal elimination when studied as radioactively labeled 103 Ru-RAPTA-C.³⁷

Table 3. Pharmacokinetic Data of Ruthenium Complexes.

Ru complex	Dose	Cltot(mL h–1)	Vd(L)	T1/2(h)
In Phase I Clinical Trials(46)
NAMI-A	300 mg m–2	7.8	7.9	49
KP1019	600 mg	56.4	5.43	68.1
In Swiss CD-1 Mice(39)
RAPTA-C	4 × 100 mg/kg/day	6.5	0.1	10.39
	2 × 200 mg/kg/day	9.2	0.163	12.21
	1 × 200 mg/kg/day	9.0	0.153	11.47

Biological Interaction Partners

Targeted therapy, a type of cancer treatment, targets the proteins that control how cancer cells multiply and spread. It forms the cornerstone of precision medicine. Researchers are at a greater benefit in developing cancer therapies targeting these proteins as they learn about proteins and their functions that fuel cancer.^63,79−89 Restricting these proteins/enzymes subsequently hinders the functions they perform in cancerous cells, thereby causing an adverse effect on those cells.

Cyt c, a small hemeprotein associated with the mitochondrion’s inner membrane, is connected to cell death and respiration. The intrinsic mechanism of apoptosis is triggered when cyt c is released into the cytoplasm as a result of the permeabilization of the mitochondrial outer membrane.⁸⁶ Lysozyme levels in the blood can reach hazardous levels in some malignancies, most notably myelomonocytic leukemia, due to excessive lysozyme synthesis by cancer cells. Low blood potassium and renal failure are two symptoms that can be brought on by high lysozyme levels in the blood, although they may get better or go away with therapy for underlying cancer.⁸⁷

Noting the significance of cyt c and lysozyme expression in cancer cells, Casini et al. studied the binding behavior of RAPTA-C with horse heart cyt c and hen egg white lysozyme using ESI-MS and found that the reactivity of the complex toward the latter was significantly lower. They observed four different adducts of RAPTA-C with cyt c; two corresponded to monoruthenated species, while the other two were attributed to biruthenated species.⁹⁰ In contrast, cisplatin formed mono-, bis-, and tris-adducts under almost identical circumstances. Histidine (His) 33 was discovered to be a primary Ru interaction site in cyt c.⁹⁰

Further, it was discovered that cisplatin and transplatin were far less reactive than RAPTA-C, which exhibited a high affinity for ubiquitin (Ub) and cyt c but not superoxide dismutase (SOD) (Table 4).⁹² Ub is a small 76-amino-acid regulatory protein that plays a role in synthesizing new and degrading damaged proteins.⁸⁸ Proteins are tagged for removal by Ub, which binds to them (ubiquitination), forming a Ub-proteasome (UPS) complex, which controls elements in both tumor-promoting and -suppressing pathways.⁸⁸ Deregulation of the cell-cycle and checkpoint control, carried out by the UPS, is one of the fundamental aspects of cancer.⁹¹ It is reasonable to assume that the binding property toward Ub and cyt c may be due to higher steric hindrance of RAPTA-C and potential hydrophobic interactions caused by the arene ring, which could increase the selectivity of the complex compared to simple coordination complexes such as cisplatin. Cisplatin and RAPTA-C revealed similar affinity for amino acid residues in protein binding.⁹²

Table 4. Main Peaks Seen in the Deconvoluted Mass Spectra of Protein Mixtures Treated with Cisplatin, Transplatin, and RAPTA-C Following a 24 h Incubation at 37 °C^a.

	Ub	Cyt c	SOD
Cisplatin	Ub–Pt(NH3),	Cyt c–Pt(NH3),	SOD–Pt(NH3)2
	Ub–Pt(NH3)2	Cyt c–Pt(NH3)2
Transplatin	Ub–Pt(NH3)2,	Cyt c–Pt(NH3)2,	SOD–Pt(NH3)2,
	Ub–[Pt(NH3)2Cl]	Cyt c–[Pt(NH3)2Cl]	SOD–[Pt(NH3)2Cl]
RAPTA-C	Ub–[Ru(η6-p-cym)],	Cyt c–[Ru(η6-p-cym)],	SOD–[Ru(η6-p-cym)],
	Ub + 2[Ru(η6-p-cym)],	Cyt c–[Ru(η6-p-cym)(PTA)],	SOD–[Ru(η6-p-cym)(PTA)Cl]
	Ub–[Ru(η6-p-cym)(PTA)]	Cyt c–[Ru(η6-p-cym)(PTA)]
	+ Ub–[Ru(η6-p-cym)],	+ Cyt c–[Ru(η6-p-cym)]
	Ub–[Ru(η6-p-cym)(PTA)Cl]
	+ Ub–[Ru(η6-p-cym)]

^aData from ref (92). p-cym = p-cymene.

Dyson’s group also studied how RAPTA-C reacted with protein targets thioredoxin reductase (TxrR) and cathepsin B (cat B) and found that the complex acted as rather a strong cat B inhibitor (IC₅₀ = 2.5 μM), but its TxrR inhibition [IC₅₀ = 37.1 μM (TrxR1) and > 200 μM (TxrR2)] was much less prominent. TxrR is a suitable target for antitumor treatment since it is vital for cell growth and survival. Additionally, the enzyme levels are increased in a number of cancer forms, including malignant mesothelioma.⁸¹ Cancers that are invasive and metastatic are associated with overexpression of cat B.⁹³ Hence, the higher affinity of RAPTA-C toward cat B inhibition was consistent with in vivo research (RAPTA-C lowers the number and bulk of metastases).^36,37,39,94 The docking results obtained in the study showed numerous adduct-forming bonds that extended beyond the coordinative bond of Ru(II) to the sulfur of the active-site cysteine, stabilizing the overall protein–complex adduct.⁹⁵ In a computational study, the optimized geometry of the N-acetyl-l-cysteine-N′-methylamide (which replicates the side chain of cysteine in the cat B active site and nearby peptide groups) and RAPTA-C exhibited pseudo-octahedral coordination around the Ru with a piano stool geometry, wherein the plane of the receptor lay almost perpendicular to the equatorial plane of p-cymene. For Ru, the M–S bond length was observed to be 2.39 Å. RAPTA-C bound to cat B with a considerably negative binding free energy of −32.7 kcal/mol, indicating a thermodynamically advantageous binding.⁹⁶

Later in 2009, the group reported the binding of RAPTA-C with rabbit metallothionein (MT-2). Shortcomings in MT function may cause cells to undergo continuous replication, which eventually results in cancer since MTs play a crucial role in controlling transcription factors. Studies have discovered higher levels of MT expression in various cancers. Greater MT expression levels may also cause chemotherapeutic treatment resistance.⁶³ Prior to the binding, the chlorido ligands and the PTA ligand (to a lower extent) of RAPTA-C were lost while the p-cymene invariably bound to the Ru(II) core. The competitive binding study done between Ub and MT-2 divulged the increased capacity of MT-2 to bind to free RAPTA-C in solution. The ability of MT-2 to strip RAPTA-C from Ub over cisplatin may be significant regarding its pharmacological features, viz., RAPTA-C might get easily detached and detoxified by MT-2. The increased overall toxicity of cisplatin observed during the study was explained by the fact that the disintegration of Pt adducts from Ub following the addition of MT-2 was significantly lower. This implied that RAPTA-C was more likely to be active in cancers overexpressing MTs.⁹⁷

Speaking of sulfur-containing compounds, RAPTA-C was seen to form adducts pretty quickly with glutathione (GSH). A variety of cellular functions, such as cell differentiation, proliferation, and apoptosis, are dependent on GSH levels in the cell, and any change is linked to the development of many human disorders, including cancer. Elevated GSH levels increase the antioxidant competence of the cell under consideration, which become resistant to oxidative stress, as seen in many cancer cells. A GSH deficiency, or a lower GSH/glutathione disulfide (GSSG) ratio, leads to a higher probability of oxidative stress that has been linked to cancer growth.⁸⁰ Herein also, it was established that adding GSH allowed the release of adducts generated between RAPTA-C and the model protein Ub. It is presumed that, when these species (Ru–protein adducts) interact with GSH, they may cleave intracellularly, making it potentially accessible to display its MoA.⁹⁸

RAPTA-C was tested for its partiality toward protein Ub or DNA in a competitive binding experiment by Artner et al., employing a combination of Ub and 5′-dATTGGCAC-3′ oligonucleotide. Specifically, following hydrolysis, RAPTA-C generated Ub + (p-cymene)Ru and DNA + n(p-cymene)Ru(PTA) adducts, where n = 1–3. The PTA ligand was seen to be cleaved during the Ub contact, as already witnessed in prior studies; however, with a longer incubation time (24 h), the p-cymene was also seen to be released from the oligonucleotide’s bis-adduct, resulting in a DNA + (p-cymene)Ru(PTA) + Ru(PTA) adduct, illustrating the variations in the reactivity of the complex with proteins and DNA. Direct and specific coordination of the Ru(II) ion to both adenine and cytosine were found on the adducts formed.²⁰ Holtkamp et al. reported that RAPTA-C, supplied at a dosage that was 40 times higher than that of cisplatin, had an accumulation over time that was just 10 times more than the latter, which foretold that RAPTA-C was more resistant to extracellular reactions than cisplatin.³⁸

Ru-based complexes are known to bind plasma proteins with a high affinity,^83,99 making them likely RAPTA-C’s initial intravenous binding partners. In fast-growing tumors, essential transport proteins such as albumin and transferrin are known to accumulate. While albumin and transferrin were determined to be the primary binding partners for cisplatin, RAPTA-C was seen to react almost exclusively with albumin, which is very surprising as most of the literature based on these complexes presumes that Ru will interact with transferrin in a better manner due to its similarity with Fe.³⁸

A variety of cancer forms have also been identified to have somatic loss-of-function mutations of BRCA1 and BRCA2, most notably high-grade serous ovarian adenocarcinoma. Methylation of the BRCA1 gene promoter, which is linked to a decrease in BRCA1 expression, has also been discovered in these cancer types. Proteins that are essential for the precise repair of DNA double-strand breaks are encoded by BRCA1 and BRCA2.¹⁰⁰ The BRCA1 protein reacted with RAPTA-C and caused intermolecular cross-links, resulting in dimers or larger aggregates. The complex disrupted the BRCA1 proteins’ secondary structure, increasing the amount of α-helices and decreasing the amount of β-sheets.¹⁰¹ The Zn(II) ion was released from the BRCA1 proteins in a dose-dependent manner upon the interaction of RAPTA-C. A functional model of RAPTA’s effects on the BRCA1 protein is presumed in which binding of the complex to the Zn finger of the protein’s RING domain causes Zn to be displaced, thereby disturbing its secondary structure.¹⁰¹

For effective cellular function and proliferation, DNA integrity is essential. Cell-cycle checkpoint proteins can recognize high amounts of DNA damage, and their activation causes cell-cycle arrest to stop the transfer of damaged DNA during mitosis. Cell death might occur if the damaged DNA cannot be effectively repaired. Cancer cells frequently have relaxed DNA damage-sensing/repair mechanisms. More significantly, they can bypass cell-cycle checkpoints, allowing the cells to proliferate rapidly. This also makes cancer cells more vulnerable to DNA damage, as replicating damaged DNA increases the risk of cell death. Numerous anticancer drugs, including cisplatin, doxorubicin, 5-FU, etoposide, and gemcitabine, were created as a result of the idea of targeting DNA that bestows them with excellent cell-killing properties (Figure 8).

Figure 8.

MoA of alkylating agents.¹⁰²

Groessl et al. examined how the DNA model molecule, dGMP, interacted with the Ru-based anticancer therapeutic candidates KP418, KP1019, and RAPTA-C and assessed the hydrolytic stability of the complexes under artificially realistic physiological settings. RAPTA-C’s nucleotide binding characteristics were pH dependent, with up to 10-fold more Ru being present in dGMP bound form at pH 6.0 over 7.4. RAPTA-C was quite reactive toward dGMP and showed substantially better affinity toward the biomolecule at more acidic pH values than the Ru(III) complexes KP1019 and KP418. These findings put forward the idea of anticancer complexes made selective for cancer cells that have lower pH values than healthy cells.^102,103

Compared to the untreated DNA control, RAPTA-C effectively blocked DNA replication and halted the amplification entirely at a concentration of 600 μM, as observed by Ratanaphan et al. Contrary to the behavior of cisplatin, this work noted that RAPTA-C produced unique Ru–DNA adducts (mostly monofunctional) at adenine and cytosine residues and to a lesser extent at guanine.^62,68,104 When the binding process of RAPTA-C to double-stranded DNA was studied, it was found that the N7 atom of the guanine bases readily bound to the complex through its Ru core; this contradicted what was observed by Ratanaphan et al.⁶² RAPTA-C could quickly fit into the main groove because of the flexibility of DNA and mimicked the distinctive DNA distortion brought on by cisplatin by bending the DNA. RAPTA-C, following 1,2-GG intrastrand cross-linking on the DNA, caused a local kink. Potentially, RAPTA-C could attach in two different ways depending on how the arene and PTA ligands are positioned in relation to the DNA. With the arene near the Watson–Crick hydrogen bonds of the base-pairs, the PTA ligand is either oriented toward the backbone of the ruthenated strand in configuration I or the opposite (configuration II). Extended classical molecular dynamics simulations showed that binding mode I was around 6 kcal/mol more preferred than II.¹⁰⁵ The reactions of RAPTA-C with building blocks of DNA such as 9-methylguanine, 9-methyladenine, guanosine, inosine, hypoxanthine, adenosine, cytidine, thymidine, and uridine were investigated by Dorcier et al. using NMR and ESI-MS spectroscopy.⁶⁴ The complex showed moderate binding behavior toward all, with the loss of chlorido and PTA ligands upon adduct formation; however, the arene ligand seemed to be intact in all cases, contradicting the results observed by Artner et al.²⁰

Ru anticancer medicines often exhibit lesser reactivity toward double-strand DNA than Pt compounds, although their biological mechanisms of action are unknown. The Ru(III) compounds KP1019 and KP1339 appear to primarily target cytosolic proteins in cells. The quantity of histone proteins and DNA sites in the nucleus, as well as their fundamental regulatory role, make chromatin potentially a superior therapeutic target for metallodrugs and other substances. Wu et al. studied in detail about the structural, kinetic, and thermodynamic behavior of RAPTA-C on binding to nucleosome core particle (NCP), which is the fundamental repeating constituent of chromatin’s histone-packaged DNA. NCP comprises of about 146 base pairs of DNA wrapped in 1.67 left-handed superhelical turns around a histone octamer.¹⁰⁶ Three distinct histone binding sites were produced after RAPTA-C’s treatment to the NCP, while no significant adduct formation was observed at any DNA locations. All three RAPTA-C sites were located on a single face of the NCP’s two symmetrical sections. The sites have different coordinating ligands, such as side chains of glutamate and lysine (Site 1), two glutamates (Site 2), or a single histidine (Site 3). Complete coordinative replacement of the initial chlorido ligands was required at sites 1 and 2. However, in the instance of site 3, a carboxylate oxygen atom of a proximal glutamate side chain in close proximity to the Ru²⁺ ion indicated that there might be weak or partial coordination. The arene and PTA ligands seemed to function to promote protein site selection via shape and hydrophobic complementarity, in addition to their steric requirements, which appeared to account for the moderate reactivity with double-helical DNA. However, each of the three RAPTA-C sites had many hydrophobic interactions with the histone components. It was even possible to recognize cooperativity in the creation of double adducts over this area. Ru²⁺ ion in RAPTA-C exhibited higher site discrimination than other soft-acid ions like Ni²⁺ and Co²⁺,¹⁰⁷ which was likely a result of its overall steric hindrance from the bulky arene and PTA ligands.⁴²

The nucleosome was thought to be a probable therapeutic target for RAPTA-C since about 5% of the intracellular Ru concentration was linked to chromatin. It is noteworthy in this context because, in a comparable cellular investigation on cisplatin, less than 1% of Pt accumulation was seen on the same. In comparison to the binding constants for cisplatin [K = 8.52 × 10² M^–1 (HSA)]¹⁰⁸ or KP1019 [K = 10.6 × 10⁴ M^–1 (albumin) and 5.6 × 10⁴ M^–1 (transferrin)]¹⁰⁹ association with serum proteins, it was discovered that the total binding constant for the RAPTA-C–NCP (K = 2.3 × 10⁵ M^–1) adducts was significantly larger. Additionally, at saturating drug stoichiometry, there were eight RAPTA-C adducts per NCP in the solution. It is interesting to note that the RAPTA-C adducts’ positions on the broad face of the nucleosome in the crystal structure correspond to regions that may have an influence on cellular processes such as chromatin compaction, interactions with nuclear factors, and histone post-translational modifications.⁴²

The MoA of RAED-C was generally more comparable to that of cisplatin in terms of DNA-targeting activity and apoptotic profile compared to RAPTA-C, which preferentially produced protein adducts in the cell. RAPTA-C-treated cells considerably recovered (both regular and cisplatin-resistant cancer cells) when compared to the ones treated with cisplatin. Due to the larger covalent character of the adduct bonds, Pt (cisplatin) had a higher bonding strength than Ru (RAPTA-C and RAED-C) compounds, which suggests that Ru adducts’ greater lability may permit these lesions to clear out more quickly after drug exposure. This may be a beneficial factor in the clearance and reduced toxicity qualities of Ru-based anticancer drugs.

Metastatic cells are among the least likely to be affected by (nontargeted) cytotoxic substances since they are particularly resistant to apoptosis. RAPTA-C has a minimal cytotoxic potential, which was in line with the fact that extremely large doses of the compound were needed to generate detectable quantities of DNA adducts. The replacement of the PTA group in RAPTA-C by the ethylenediamine ligand in RAED-C switched the adduct formation profile from primarily targeting the proteins associated with chromatin (RAPTA) to the DNA (RAED). It is probable that the mono- vs bifunctional difference in the capacity of RAPTA-C and RAED-C to coordinate also plays a role in the differences in their activity profiles. RAPTA-C has the capacity to create cross-links inside and between proteins and nucleic acids in this manner.⁴⁸ It was reported that RAPTA-C showed a somewhat stronger affinity for HeLa cells’ chromatin (6% of Ru found associated with chromatin) than it did for A2780 cells (4% of Ru found associated with chromatin), which could confer a better activity of the complex in the former cell line. The activity of RAPTA-C was demonstrated to trigger death by interfering with the mitotic process, which could be explained by significant chromatin adduct levels. Additionally, this is in line with the significant degree of G2/M phase arrest brought on by the complex. It was also witnessed that RAPTA-C adducts can prevent the regulator of chromatin condensation 1 (RCC1) and other nuclear proteins that appear to bind to acidic patches from binding, which is likely to contribute to their biological effects (Figure 9).⁴⁹

Figure 9.

Biological interaction partners of RAPTA-C and its preferred site of attack.

Combination Therapy

The most effective treatment for various cancers involves a mix of surgery, radiation therapy, chemotherapy, and/or other cancer medications. Localized cancer is treated by surgery and radiation treatment, whereas cancer medicines also eradicate cancer cells that have traveled to distant places. Whether a single therapy or combination therapy is required frequently depends on the stage and kind of cancer. Combination therapy is the most efficient way to treat cancer. Combination therapy is motivated by the notion that utilizing drugs with different MoAs may lessen the likelihood that cancer cells would develop treatment resistance.

In order to find the best low-dose combinations, Weiss et al. used a population-based stochastic search method and the feedback system control (FSC) technique to explore a vast parametric space including nine angiostatic medicines at four doses. The ideal medication cocktail was found, which included erlotinib, RAPTA-C, and BEZ-235. Compared to the similar single-drug dosage efficiencies in vitro, it permitted dose reductions of 5, 11, and 6 times, respectively. Interestingly, as compared to the individual drugs, the mixtures showed increased endothelial cell selectivity. In two preclinical in vivo tumor models, this combination medication was successfully translated. When compared to the optimum single-drug dosages, tumor growth was slowed synergistically and substantially with lower drug doses.¹¹⁰

It was hypothesized that a rise in pO₂ is associated with a fall in interstitial fluid pressure and normalization of temporal vascular modulation. Such normalization decreases the pore size of the vasculature in solid tumors, which limits vascular permeability of molecules bigger than 12 nm in diameter while allowing smaller-sized molecules to enter tumor space more readily and quickly. Giving small-molecule chemotherapeutics, such as doxorubicin (8 nm in diameter) and RAPTA-C (1 nm), during this normalizing phase significantly increased their ability to extravasate and decrease tumor development in general. Surprisingly, RAPTA-C was a more effective tumor growth inhibitor when given at a lower dose (0.4 mg/kg) than doxorubicin (3 mg/kg). This observation is extremely amazing considering that RAPTA-C has little toxicity and had to be administered daily over several days at levels of 100 mg/kg to have a comparable tumor-inhibiting effect when used alone.³⁷ RAPTA-C therapy at 400 mg/kg did not alter tumor development when given alone during this normalization window. This ineffective dosage of RAPTA-C dramatically reduced tumor development by 83.5% when combined with axitinib. It would appear that the combination of axitinib and RAPTA-C reduced tumor development via both vascular modulation and antiangiogenic pathways, based on histology and in vitro tests in ECRF24 endothelial cells (Table 5).¹¹¹

Table 5. Combination Therapies Tried and Tested Involving RAPTA-C.

Combination	RAPTA-C alone	How the combination improved the activity
RAPTA-C + axitinib (A2780 transplanted on CAM)	Treatment at 400 mg/kg did not affect tumor growth	Inhibited tumor growth by 83.5%
RAPTA-C + erlotinib (nude mice xenografted with A2780 tumors)	Treatment at 100 mg/kg inhibited tumor growth by 37%	Inhibited tumor growth by 48–53%
RAPTA-C + BEZ-235 + erlotinib (A2780 transplanted on CAM)	Treatment at 100 mg/kg	An 11-fold increase in activity was observed
RAPTA-C + radiation (plasmid DNA)	–	Enhanced resistance of RAPTA-C-modified DNA against the damage induced by radicals

Human ovarian cancer (A2780 and A2780cisR) and endothelial (ECRF24 and HUVEC) cell survival were significantly inhibited by combination of RAPTA-C and the endothelial growth factor receptor (EGFR) inhibitor erlotinib. Additionally, in A2780 cells, but not in endothelial cells, erlotinib dramatically increased the cellular absorption of RAPTA-C in comparison to treatment with RAPTA-C alone. Drug combinations caused chromosomal bridges to develop in A2780 and A2780cisR, which delayed abscission and persists beyond mitotic departure, indicating the beginning of cellular senescence. In vivo A2780 tumors established on the CAM model and a preclinical model in nude mice are used to further assess the therapeutic potential of these substances and their combination. Effective antiangiogenic and antiproliferative efficacy in vivo was confirmed by immunohistochemical analysis, based on a considerable drop in microvascular density and a reduction in proliferating cells.¹¹¹

The side effects due to any oncology treatment can be reduced by possible chemoradiation therapies that offer lower doses or by effectively targeting malignancies with radiation utilizing innovative ion beam methods. The so-called synergistic impact, which is the augmentation of the damage caused to the tumor by concurrent therapy over the effect of the individual chemotherapy and radiation treatments, is the major benefit of such therapies. A mature CAM was exposed to angio-occlusive PDT along with the subsequent topical application of RAPTA-C. A dosage of 70 (μg/embryo)/day in this experiment prevented nearly 100% of vascular regeneration in the PDT region. This result is intriguing because the developmental CAM was easily apparent but only moderately inhibited by the same dosage.⁷⁷ In an experimental investigation, the combined effects of radiation and cisplatin/RAPTA-C on DNA damage were investigated. Free RAPTA-C had no discernible combination action with radiation. The quantity of Ru adducts was comparatively less for the same starting ratio of cisplatin and RAPTA-C during incubation. DNA modification by cisplatin caused the radiation damage to be increased, but DNA modification by RAPTA-C causes the radiation damage to be decreased. Such an outcome may result from RAPTA-C’s DNA unfolding property. The DNA shape is quite crucial: the double-strand-break (DSB) damage is definitely less severe than single-strand-break (SSB) damage. The greater stability of RAPTA-C-modified DNA against radiation-induced secondary reactive species might potentially contribute to the radioprotectivity of RAPTA-C.¹¹²

RAPTA-C showed an intriguing activity against metastatic tumors along with antiangiogenic effects, even though it had a rather poor in vitro cytotoxicity. Such results demonstrate the possibility of creating metallotherapeutics that do not necessarily have great cytotoxic action but may be used in conjunction with surgical intervention and complementary chemotherapy regimens. With a lower profile of side effects, such a method may be able to improve the clinical success of the treatment plan. The growth of such RAPTA complexes has advanced quickly in the hunt for more effective anticancer agents since the breakthrough of the archetypal RAPTA-C and the early identification of its antimetastatic capabilities. This synthetic effort has resulted in a wide variety of molecules with a variety of biological profiles, ranging from the development of more focused structures and complicated macromolecular conjugates to the small change of individual ligands of the RAPTA structure.

RAPTA-C has been tested in a number of preclinical models as a result of its special characteristics, and its efficacy in these models is quite encouraging, especially given how quickly it leaves the circulation and organs and how little overall toxicity it exhibits. Surprisingly, even at lower doses, RAPTA-C is more effective in preventing tumor development when given during tumor normalization than the drug doxorubicin. It is evident that there is minimal association between the action of RAPTA-C in vivo and in normal cell-proliferation tests. Correlations between in vitro and in vivo activity could only be made with more specialized cell-based tests in the relevant cell lines. Given this paradigm, it is conceivable that certain newly generated RAPTA complexes may merit additional investigation in more suitable assays, leading to the discovery of a larger spectrum of pertinent pharmacological action. The findings with respect to RAPTA-C may highlight the key targets, and from there, the potential outcomes of chemotherapeutic treatment may be anticipated. However, the advancements gained with the RAPTA compounds so far are very encouraging, and further rational design and enhanced proteomics combinations could result in the discovery of more effective chemotherapeutics with potential anticancer capabilities.

Conclusions

RAPTA-C, a Ru(II)-arene complex fostered by Dyson’s group, is an up-coming organometallic complex that has been proven to be very much worthy of clinical trials. This Review provides an overview of the physicochemical mechanisms underlying RAPTA-C’s preference for cancer cells over normal cells, which is mainly through a pH-dependent action. The activation mechanism of the complex wherein it gets delivered as a prodrug and subsequent release of active species in an acidic environment of the cancer cell via hydrolysis has received particular attention. There are several MoAs by which the complex prevents the development of tumors, including binding to proteins/DNA, inhibition of transferases and kinases, and enzymatic methods. The complex is seen to be very preferential toward proteins over DNA, although interaction with the latter is also quite significant. For protein macromolecules, RAPTA-C binds mostly through sulfur of a cysteine residue, on par with the HSAB principle. Competitive binding studies have shown that this property might be very useful in the clearance of such protein–Ru adducts. RAPTA-C alters proteins and histone-DNAs to demonstrate antimetastatic, antiangiogenic, and antitumor actions. The success of the antimetastatic/antiangiogenic property of RAPTA-C demonstrates the possibility for creating complementary metal-based complexes that do not necessarily display significant cytotoxicity but may be used in combination with complementing chemotherapy regimens. A method like this may help the therapeutic regime’s clinical outcome be more effective while also reducing adverse effects.

The authors declare no competing financial interest.