What happened to BBR3464 and where to from here for multinuclear platinum-based anticancer drugs?

Abstract

The development of the trinuclear platinum(II) complex BBR3464 (also known as triplatin) in the late 1990s was meant to be a revolution in the field of platinum chemotherapy. What made it remarkable was that it defied many of the known structure–activity rules for platinums; it is cationic, has a single labile trans leaving group on each terminal platinum, and it binds DNA in ways different to mononuclear platinum drugs, like cisplatin and oxaliplatin. The flexible, long-range adducts the drug forms with DNA means that it showed activity in cancers not typically sensitive to platinums, and more importantly, BBR3464 demonstrated an ability to overcome acquired resistance to platinum drugs. But while preclinical and phase I testing showed promise, its more severe side effects which greatly limited the deliverable dose when compared with standard platinums, combined with its lack of biostability, led to a lack of activity in phase II trials and its development was halted. But, from its ashes have risen 4^th generation complexes which target the phosphate backbone of DNA. These, and the original BBR3464 drug, could potentially be further developed and gain regulatory approval through formulation with macrocycle-based drug delivery vehicles.

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Mia graduated from Freeman Catholic College and was dux of the class of 2021. She is currently completing a Bachelor of Pharmacy degree at The University of Sydney and works as an undergraduate research assistant in her spare time. Upon graduation, she intends to undertake postgraduate research and an internship as a hospital pharmacist.

Professor Wheate is a graduate of the Australian Defence Force Academy and served for 10 years in the Royal Australian Navy prior to becoming a university academic. He has a BSc and PhD from the University of New South Wales (UNSW) and an MBA and DSc from the University of Sydney (USYD). He has held academic positions at UNSW, USYD, Western Sydney University, and the University of Strathclyde, and is currently a professor and Director – Academic Excellence at Macquarie University in Sydney, Australia.

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Introduction

The discovery,¹ development, and eventual approval of cisplatin was a significant change in the treatment of cancer (Fig. 1). Nearly 60 years on, platinums are still used to treat around 24 different types of cancer, are included in a quarter of all chemotherapy protocols,² and nearly half of all patients who receive chemotherapy will get a platinum drug at some point during their treatment.³

Fig. 1 The chemical structures of the rule following drugs (a) cisplatin, (b) carboplatin, and (c) oxaliplatin and the rule breaking (d) original 1,1/t,t shown with the four methylene linker chain, (e) BBR3464/triplatin, and (f) triplatinNC. Anions are omitted but are typically chlorides or nitrates.

Cisplatin is a pro-drug. In blood serum, where the chloride concentration is high, its chloride ligands remain attached, although it is known to extensively bind to human serum albumin.⁴ When taken into the cell, the chlorides of the drug are replaced by water molecules, and once activated in this way, cisplatin is able to bind to its cellular target, nuclear DNA.⁵ The drug binds predominantly in the major groove of B-type DNA at the N7 position of guanine, and to a lesser extent adenosine residues, to form short-range and rigid adducts. The most abundant adduct formed is the 1,2-GpG intrastrand adduct that bends and unwinds the DNA which inhibits DNA transcription and replication, and thus induces cellular apoptosis.⁵

In developing next generation drugs from cisplatin, early rules were proposed for structural features that would make a platinum complex anticancer active.⁶ Potential drugs that broke these rules were thought to be likely inactive due to their inability to block DNA transcription and replication. To be active a platinum drug had to (1) be neutral and square planar, but could become positively charged upon aquation; (2) have two semi-labile ligands, typically chlorides or a single dicarboxylate ligand, that could be displaced by water; (3) have ammine and/or amine carrier ligands that remain coordinated to the platinum after DNA binding; and (4) the labile and am(m)ine ligands should be in a cis, rather than trans, configuration on the platinum atom.

These rules persisted and resulted in the development and approval of the second generation drugs carboplatin and oxaliplatin (Fig. 1). But in the late 1980s and early 1990s we started to question these rules. It started with the development of the mononuclear platinum(II) complexes of Farrell where the ligands were in a trans-configuration,⁷ which evolved into his series of multinuclear (or polynuclear) platinum complexes, of which, the trinuclear complex BBR3464 was the most promising, but ultimately, unsuccessful drug.

Dinuclear platinums

These drugs, starting with an uncharged dinuclear complex [cis-{PtCl₂(NH₃)}₂μ-H₂N(CH₂)_nNH₂]Cl₂ (referred to as 2,2/c,c) which resembles two linked cisplatin molecules,⁸ soon developed into the charged dinuclear complexes, best exemplified by 1,1/t,t or [trans-{PtCl(NH₃)₂}₂μ-H₂N(CH₂)_nNH₂]Cl₂ (Fig. 1, where n was typically 2–6; for n = 6 the complex is also called BBR3005), that consist of two platinum(II) atoms that are coordinated to two trans-ammine groups and a single chloride ligand and where each platinum centre is connected by a flexible diaminoalkane chain.⁹

These dinuclear complexes were highly active in cancer cell lines including those with acquired resistance. In vitro assays showed that 1,1/t,t could overcome both cisplatin and oxaliplatin resistance in a mouse leukaemia cell line,^9,10 and overcome cisplatin resistance in a human ovarian carcinoma cell line. This cytotoxicity was thought be due to the unique adducts that the drug formed with DNA. While the cisplatin 1,2-GpG intrastrand adduct results in a rigid, 30–35° bend into the minor groove, 1,1/t,t is able to form both intra- and interstrand adducts, up to four base-pairs apart, which are both flexible and non-directional.^11–13 The DNA adducts of 1,1/t,t are also characterised by their ability to induce B-type to Z- and A-type DNA conformation changes,^14–16 by the higher number of adducts they form with DNA when compared with cisplatin,¹⁰ and1,1/t,t's lower sensitivity to degradation and deactivation by the intracellular antioxidant, glutathione, due to its faster binding to DNA when compared with cisplatin.¹⁷ The family of dinuclear complexes were further improved by the addition of hydrogen-bonding functionality into the bridging ligand. This created dinuclear complexes with spermine (designated BBR3535), spermidine (BBR3571), and spermine-like (BBR3610) bridging linkers.^18–20

Other groups have built on the work of Farrell to develop their own dinuclear platinum complexes. These include a triangular trinuclear platinum(II) complex,²¹ pyrazole-based complexes,²² triazolato-bridged complexes,²³ and platinum(II) complexes that contain flexible chains incorporating carborane (N-dicarba-closo-dodecaborane) for the synergistic application of chemotherapy with boron neutron capture therapy.²⁴

BBR3464 (triplatin)

BBR3464 is a trinuclear platinum drug (Fig. 1) developed by Farrell and his research team, which evolved from their work with the dinuclear platinum complexes and can be considered a 3^rd generation drug in the platinum family. In its structure are two terminal platinum(II) atoms, where each has two trans-ammine ligands and a single chloride leaving group in the trans position from a 1,6-diaminohexane bridging linker. The drug also contains a central platinum(II) atom, which has two trans-ammine ligands and which connects to the terminal platinums through the diaminohexane linkers. The drug is cationic with a charge of 4+, which can increase to 6+ upon aquation of both chloride ligands.

Like other platinums, BBR3464 is thought to derive its cytotoxicity through DNA binding. But while the DNA adducts formed by cisplatin, carboplatin, and oxaliplatin are short-range and rigid, like its dinuclear parents, BBR3464 is distinctive for the long-range, flexible adducts it forms.

The drug is able to form both intra- and interstrand adducts, which includes short range 1,2 adducts, but more commonly long range adducts that span 4, 5, or 6 base pairs.^13,25–27 These adducts induce a local unwinding of the DNA duplex by around 9 to 10° and a bending of the DNA of 15 to 21°.^25,28 The drug is also a very efficient compactor of DNA; it is able to induce compaction at molar ratios (0.01) much lower than cisplatin (0.5–5.0).²⁹ BBR3464 also generates more micro and macroloops in DNA when compared with cisplatin.³⁰

The DNA adducts formed by BBR3464 can be conformational, which are not interchangeable, and can be formed in both the 5′–5′ or 3′–3′ directions. Importantly, each conformer appears to affect DNA in a different way and all are thought to contribute to the drug's cytotoxicity.^26,31 The different adducts and conformers that are formed, are to a large part, dictated and influenced by the preassociation of the drug with the DNA, which occurs before N7 guanosine binding.³² In most cases, the drug will coordinatively bind with the terminal platinum groups located in the DNA major groove, but with the central platinum of the drug in the minor groove.³¹

The minor groove binding of the drug's central platinum is thought to play an important role in how BBR3464 is able to inhibit DNA polymerases; a family of proteins involved in reading DNA strands and building new strands by assembling the nucleotides. When a model A-family polymerase, κF-, was reacted with DNA bound by BBR3464, the central platinum was found to be able to obstruct productive contact of the protein with the minor groove.²⁷ Importantly, BBR3464 adducts of DNA are not easily recognised by high mobility group proteins.³³ These proteins (like HMGB1) play a crucial role in recognising DNA damage caused by platinum-based drugs. While the adducts are not recognised by HMG proteins, the adducts can be removed by nucleotide excision repair.³⁴ The effect of BBR3464 adducts has also been examined on NF-κB binding sites in DNA. The protein NF-κB is a major transcription factor that regulates genes and is involved in the immune response. BBR3464 has a much stronger inhibitory effect on the DNA binding of NF-κB, when compared with cisplatin, and both are much higher than transplatin, which correlates to their cytotoxicity.³⁵

As well as the unique adducts BBR3464 forms with DNA, its high activity is also thought to be a function of its uptake into cells when compared with cisplatin. It is not known definitively how platinum drugs are taken into cells. Initially it was thought that cisplatin entered cells through passive diffusion, but we know now that active mechanisms, like copper transporters and organic cation transporters, also play a role.³⁶ Studies have shown that BBR3464 is also taken up by cells via copper transporters, particularly hCTR1.³⁷

Given the cationic charge on the drug (4+ to 6+) it would be expected that the drug would be attracted to, and interact, with the highly anionic phospholipid membrane of cells. Studies have shown that BBR3464 does interact with the head groups of phospholipids, but interestingly, it is also capable of interacting and binding with the fatty acid tails.³⁸ Such binding was found to affect the resultant liposome membrane fluidity. BBR3464 interacts with phospholipids both covalently and electrostatically, which has implications for its passive uptake into cells.

BBR3464 has similar, or slightly lower, uptake to cisplatin in sensitive murine leukemia cells (when the three-fold higher concentration of platinum in BBR3464 is taken into account) but more than two-fold higher uptake into matched cisplatin resistant cells.³⁹ Similar uptake levels of BBR3464 and cisplatin are also observed in human squamous cell carcinoma cells,⁴⁰ but BBR3464 had at least a two-fold higher uptake in human ovarian cells.³⁷ For the squamous cells, prior polyamine depletion was found to increase the uptake of BBR3464 by 2.4-fold.⁴⁰

One proposed mechanism for the high uptake of BBR3464 is due to its potential binding of heparan sulfate which is a linear polysaccharide involved in enzymatic catalysis, growth factor signalling, and cell adhesion. Binding of the drug to mono- and disaccharides,^41,42 relevant to heparan, has shown that BBR3464 will form both covalent bonds through its platinum atoms and hydrogen bonds through its platinum ammine groups to the saccharides. It is thought that binding to heparan polysaccharides may facilitate cancer cell uptake via heparan sulfate proteoglycan-mediated pathways.

Pre-clinical testing of BBR3464 showed remarkable anticancer activity both in vitro and in vivo. BBR3464 was up to 900-fold more active in vitro than cisplatin in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), ovarian, and cervical cancers, and osteosarcoma⁴³ and melanoma⁴⁴ cell lines. For example, in the cell line Me 665/2/60 the IC₅₀ for cisplatin was 35.3 μM, but was just 0.04 μM for BBR3464.⁴⁴ Likewise, BBR3464 was 20-fold more active in glioma cells than cisplatin,⁴⁵ and nearly 4400-fold more active than cisplatin in neuro-blastoma cells.⁴⁶ BBR3464 also displayed activity in ovarian,^47,48 osteosarcoma,⁴⁹ and neuroblastoma⁵⁰ cell lines that had acquired resistance to cisplatin. Importantly, BBR3464 also demonstrated better activity in cancer cell lines with p53 mutations when compared with wild-type p53 cells.⁴³

More recent research has examined the effect of the co-administration of non-chemotherapy drugs with BBR3464 on cancer cell lines. The tricyclic antidepressant drug desipramine resulted in around a 50% increase in cell apoptosis by the 100 hours time point in colorectal carcinoma cells from a fixed 1 : 1 of BBR3464 and desipramine. But the mechanism by which it enhanced the cytotoxicity of BBR3464 is unclear given that there was no statistical difference in human serum albumin binding, cellular uptake, or p53 stabilisation with, or without, the antidepressant.⁵¹ Likewise, pre-treatment to induce polyamine depletion in head and neck squamous carcinoma cells with α-difluoromethylornithine (DENSPM) and N¹,N¹¹-diethylnorspermine (DFMO) were found to increase the in vitro cytotoxicity of BBR3464.⁴⁰

The in vitro results of BBR3464 were confirmed in various in vivo studies. In three lung tumour xenografts the drug inhibited tumour growth ∼93% which was better than cisplatin (56–70%).⁴³ In a glioma subcutaneous xenograft, BBR3464 (dose of 0.35 mg kg⁻¹) took, on average, around 10 days longer to reach a large tumour size when compared with cisplatin.⁴⁵

The results of first-in-human testing of BBR3464 were published in 2000 from a phase I clinical trial. From a starting dose of 0.03 mg m⁻² day⁻¹ when given every day for five consecutive days in a 500 mL saline intravenous infusion with 1000 mL of pre- and post-hyperhydration, the maximum tolerated dose (MTD) was determined to be 0.12 mg m⁻².⁵² This is significantly lower than the standard doses used clinically for cisplatin (10–100 mg m⁻²), carboplatin (1.5–8 area under the curve) and oxaliplatin (85–130 mg m⁻²).² The dose limiting toxicity at this MTD, while short-lasting, was found to be late onset diarrhoea and neutropenia. In a separate phase I trial, BBR3464 was administered as a single IV dose every 21 days, rather than five consecutive days, at exploratory doses of 0.6 or 0.75 mg m⁻² with protracted intravenous infusion (PVI) of 5-fluorourcil (200 mg m⁻²). It was found that the two drugs were not compatible and the investigators concluded that BBR3464 could not be combined safely with PVI 5-FU at a dose sufficient to be associated with antitumour activity.⁵³

Two full reports of BBR3464 in phase II clinical trials as a single agent have been reported. In the first trial that spanned the United Kingdom and Italy, 45 patients with metastatic or locally advanced gastric or gastro-oesophageal adenocarcinoma were treated with BBR3464. Initially patients were given the drug at a dose of 1.1 mg m⁻² once every 28 days, but due to the side effects, the dose was reduced to 0.9 mg m⁻² every 21 days. The patients were not given pre- or post-IV hydration. Even at the lower dose, patients still experienced grade 4 toxicity. The results showed that BBR3464 was not successful as no patient had a complete response and only one patient had a partial response. Excluding the seven people who could not be evaluated, the remaining patients had no change or progressed with their cancer. The study authors concluded that BBR3464 was not recommended in that disease setting.⁵⁴

In the second phase II trial, BBR3464 was trialled for sensitive and refractory SCLC. While the starting dose was 0.9 mg m⁻², patients who experienced grade 3 or higher diarrhea or grade 4 neutropenia for five days or more and with an associated complication (grade 2 fever) were given a dose reduction to 0.75 mg m⁻². In total, 14 of the 37 patients were given either a dose reduction or had treatment stopped. No patient demonstrated a response to treatment, while 11 patients showed stable disease, 23 showed disease progression, and three were not eligible for evaluation. Overall, the study authors concluded that the lack of significant responses did not support further evaluation of BBR3464 as a single agent in that cancer type.⁵⁵

While an abstract of the drug trialled with advanced ovarian cancer patients is available,⁵⁶ no peer-reviewed follow up has been published. What is known is that the phase II trial involved patients with ovarian cancer who had previously been treated with a platinum- and taxane-based chemotherapy protocol. It involved 18 refactory and 28 relapsed patients, who in total received 164 infusions of BBR3464. There were five partial responses to treatment. Side effects from the treatment were also reported. Consistent with earlier clinical trials, the dose limiting toxicity was neutropenia (grades 3 and 4) for 12 patients which resulted in dose reductions to 0.9 mg m⁻² for six of the patients. Diarrhoea side effects were managed with the drug loperamide. The authors concluded that BBR3464 showed provisional evidence of activity in relapsed ovarian cancer, but as stated, no full follow-up report was published.

Metals and metal-based drugs are known for the increased severity of their side-effects when compared with purely organic-based drugs.⁵⁷ Platinum-based drugs are especially notorious for their dose limiting side effects. Patients receiving platinum-based chemotherapy can expect to experience any combination of 40 specific side-effects. Often these side-effects can be so severe that the patient will require a dose reduction of their platinum, or may even have to stop treatment with their platinum.⁵⁸ One likely cause of BBR3464's failure is the severity of its side effects, even when compared with other platinums, as evidenced by its MTD. Given the high in vitro potency of the drug to cancer cells, the poor clinical results most likely lie in an insufficient concentration of the drug at tumour sites to induce apoptosis; there was simply not enough drug getting to tumours, because of the low administrable dose, to be able to induce apoptosis.

Another issue is the significant blood plasma degradation of the drug before it reached tumour sites, giving BBR3464 a very narrow therapeutic index. It is known that BBR3464 degradation in the bloodstream leads to inactive mono- and dinuclear products and that this degradation is rapid in human blood.⁵⁹ Like all clinically used platinums, BBR3464 is highly reactive with glutathione, and the reaction products of the drug with the peptide have been studied. Glutathione is able to remove both the bridging linker and chloride ligands from BBR3464, resulting in a dinuclear species where a glutathione molecule bridges two platinum centres.⁶⁰

Overall, no clear reasons have been published for why BBR3464 was discontinued from clinical trials, although changes in the development company, and company takeovers, likely resulted in a decision based on financial considerations and the poor phase II results.

Phosphate clamping multinuclear platinums

As discussed earlier, by 2000 it was known that the unique DNA adducts formed by BBR3464, and its dinuclear parents, were influenced by the drug's initial non-covalent pre-association with DNA. Being highly cationic, multinuclear platinum complexes are attracted to the highly anionic phosphate backbone of the DNA helix. Studies have shown that the pre-association of multinuclear platinums with DNA results in non-covalent binding in the DNA minor groove with a preference for adenosine and tyrosine rich regions. Binding is stabilised through van de waals interactions with the floor of the DNA groove and through electrostatic interactions.^61–65

BBR3464, and its related dinuclear complexes like BBR3005 and BBR3610, first associate with the minor groove where they cover a length of around five base-pairs, with a preference for adenosine residues.⁶⁶ As well as electrostatic interactions, there is evidence for the formation of hydrogen bonds between the am(m)ine groups on the platinum and the phosphates of the DNA backbone.^66,67 Binding in this way lowers the rate of aquation of BBR3464 as it reduces access by water to the drug and sterically inhibits the formation of the five membered transition state needed for aquation to occur.²⁶

During and after pre-association, the aliphatic arms of BBR3464 can wrap around the DNA strands and move into the major groove, while the central platinum remains in the minor groove.²⁶ Mono-aquation is then thought to occur, followed by monoadduct formation, aquation of the second chloride ligand before inter- and intrastrand adduct formation.⁶⁷

The understanding that came from the analysis of how BBR3464 interacts with DNA before its covalent binding led to Farrell's development of 4^th generation platinum complexes that can non-covalently bind to DNA. He has classed these platinum complexes as phosphate clamps.

While anticancer active platinum complexes that were co-ordinately saturated with am(m)ine ligands were already know, like the phenanthroline complexes of Aldrich-Wright,^68,69 these were the first rationally developed from knowledge of drug–DNA interactions.

The lead complex in the phosphate clamping family of platinum complexes by Farrell; triplatinNC is a derivative of BBR3464 where the chloride ligands on the terminal platinum atoms have been replaced with additional 1,6-diaminohexane ligands (see Fig. 1). A BBR3464 derivative has also been synthesised where the chloride ligands have been replaced with ammine ligands. Both trinuclear complexes are highly cationic with charges of 8+ and 6+, respectively.

The phosphate clamping family of platinums are distinct in their mode of binding in that they are neither DNA intercalators or groove binders.⁷⁰ TriplatinNC forms hydrogen bonds to the oxygen atoms of DNA phosphate groups, with a preference for adenosine and tyrosine bases, via a distinct bidentate ammine–oxygen–ammine adduct (Fig. 2).⁷¹ The resultant effects of triplatinNC on cells is the direct inhibition of DNA transcription and nuclear condensation.⁵⁹

Fig. 2 The crystal structure of triplatinNC bound to the Dickerson-Drew dodecamer showing minor-groove spanning and backbone tracking (top), and representation of an arginine fork (bottom, left) and phosphate clamp molecular recognition motifs (bottom, right). This figure has been adapted/reproduced from ref. 72 with permission from Royal Society of Chemistry, copyright 2015.

While not able to covalently bind to DNA, triplatinNC does have inherent cytotoxicity. In a panel of five ovarian cancer cell lines it displayed cytotoxicity (IC₅₀: 1.6 to 7.4 μM) that was not as good as cisplatin (IC₅₀: 0.15 to 4.2 μM) and BBR3464 (IC₅₀: 0.016 to 0.355 μM) but it was able to overcome cisplatin resistance.⁷³ Likewise, in a study that compared triplatinNC with cisplatin in a panel of 12 cell lines of lung and ovarian cancers, and osteosarcomas, that were sensitive or resistant to cisplatin, triplatinNC had IC₅₀ values of 2.7 to 7.4 μM and resistance indices of 0.5 to 1.4; compared with cisplatin resistance indices of 2.6 to 12.2.⁷⁴

But while it is less effective at killing cancer cells than BBR3464, it may also be safer. While it displayed near three-fold higher uptake into mouse bone marrow mast cells (which play a role in the response to inflammation and infection), it was up to 447-fold less cytotoxic to these cells than BBR3464.⁷⁵

Interestingly, triplatinNC also appears to have antiviral activity. By binding to cell surface glycosaminoglycans (which is not seen for cisplatin or oxaliplatin), it can prevent the entry of enterovirus 71 (EV71) and human metapneumovirus.⁷⁶ The concentration of triplatinNC required to block entry of EV71 into rhabdomyosarcoma cells was nearly four-fold lower than the concentration needed of suramin, an antiparasitic drug used to treat African sleeping sickness (African trypanosomiasis) and river blindness (onchocerciasis), to achieve the same result.

But despite the interesting in vitro results, triplatinNC is likely to suffer from a similar side effect profile and level of toxicity that was observed for BBR3464, that will likely severely limit the dose that could be administered to patients. Likewise, triplatinNC will likely be subject to the same rapid chemical degradation that was observed for BBR3464, which means little of the drug would reach cancer cells intact. But it could potentially be developed as a useful drug through delivery and formulation strategies.

Future development and rescuing of multinuclear platinums

The high level of side effects and chemical degradation of BBR3464 within the human body does not need to stop the development of multinuclear platinum complexes. Through ligand design or the use of drug delivery vehicles, this class of platinum drugs can be made safer and better targeted to cancer cells. They are excellent at killing cancer cells; instead, the trick is to get them to their target (cancer cells) intact and without them harming or affecting normal cells.

Conversion of the platinum centres in a drug from an oxidation state of 2+ to 4+ has been shown to reduce their reactivity in the blood stream.⁷⁷ An example is the development of satraplatin, a platinum(IV) complex that was designed to be reduced to platinum(II) within cells. But while the drug showed positive Phase III clinical trial activity,⁷⁸ it was ultimately not approved for sale. The potential of platinum(IV) derivatives of dinuclear drugs has been demonstrated,^79,80 but this has not yet been extended to trinuclear drugs.

But ligand design and changing the chemical structures of BBR3464 and triplatinNC and their derivatives is not the only way to rescue these agents. Another solution is formulation and drug delivery.⁸¹ The last few decades have seen the development of many types of drug delivery vehicles, including macrocycles,⁸² nanoparticles, liposomes, polymers,⁸³ and even DNA and RNA in the form of aptamers.⁸⁴ The approved chemotherapy drug Abraxane is a protein-bound nanoparticle formulation of the drug paclitaxel,⁸⁵ Doxil is a liposomal formulation of doxorubicin,⁸⁶ and the development of mRNA COVID vaccines was made possible because of lipid nanoparticles.⁸⁷ But while the active component of cisplatin has been attached to gold⁸⁸ and iron oxide nanoparticles,⁸⁹ and has been encapsulated within liposomes,⁹⁰ neither BBR3464 or other multinuclear platinum complexes are have been examined in detail with nanoparticles and this remains an area of potentially enormous potential.

Steric protection of BBR3464 and triplatinNC is a strategy that can be used to prevent or slow the degradation of multinuclear platinums within the body. Steric protection was the basis for the design of the platinum drug picoplatin; a sterically hindered mononuclear platinum drug which entered phase III clinical trials but never gained regulatory approval.⁹¹ The drug was a derivative of cisplatin where one ammine ligand was replaced by a 2-methyl-pyridine ligand. The methyl group sat above the platinum atom, helping to protect it from binding by thiols.

The chemical structure of BBR3464 and triplatinNC doesn't need to be changed in order to give them steric protection, although this was attempted with a dinuclear platinum(II) complex. The sterically protected dinuclear complex, which utilised a flexible linker that coordinated to the platinum atoms through similar 2-methyl-pyridine-like groups, was shown to react more slowly with guanosine monophosphate, glutathione, and human serum albumin when compared with an unprotected version of the complex. Unfortunately, the sterically hindered complex was poorly active in vitro and unable to overcome cisplatin resistance.⁹²

Instead, steric protection of multinuclear platinums could better be achieved through the use of macrocycles,⁸² which are ring shaped molecules that have one or more portals that lead to a hydrophobic cavity. The four major classes of macrocycles that are used in drug delivery are cyclodextrins, calixarenes, pillararenes, and cucurbiturils.⁸² Cyclodextrins are already used as excipients (non-physiologically active ingredients used in pharmaceutical formulations) in several medicines, including products for erectile dysfunction, pain, gastric reflux and heartburn, allergies, medical imaging, inflammation, seizures, skin infections, and cancer chemotherapy.⁸²

Cucurbiturils (CB[n]; where n is the number of subunits in the macrocycle), named for their pumpkin-like appearance,⁹³ are a family of macrocycles that have been studied extensively with platinum complexes. They are made from the acid-catalysed condensation of glycoluril and formaldehyde and are known for their barrel shape that gives a hydrophobic cavity accessible through two identical carbonyl-lined portals.⁹⁴ Cucurbiturils have been studied as drug delivery vehicles with cisplatin,^95–97 other mononuclear platinums,^98,99 but more importantly, also with multinuclear platinum complexes.^100–102

For multinuclear platinums, the largely hydrophobic chains of the complexes are incorporated into the macrocycle's cavity (Fig. 3). This binding then places the platinum centres at the portals of the macrocycle, where steric protection is provided to nucleophilic attack, especially from thiol-containing molecules.^99,103,104 When two dinuclear complexes, designed to mimic BBR3005 and BBR3571, were encapsulated by cucurbiturils, it significantly slowed their rate of binding to the thiol-containing amino acid cysteine. For one of the dinuclear complexes, CT233, it's half-life with cysteine was five minutes, but when encapsulated by either CB[7] or CB[8], the half-life increased to 40–45 minutes.¹⁰⁵ Encapsulation of platinums in cucurbiturils has also been shown to slow their binding to guanosine.¹⁰⁰

Fig. 3 Molecular models showing the encapsulation of a trinuclear platinum complex by two (A) cucurbit[7]uril and (B) cucurbit[8]uril molecules, over the linker chains of the molecule. Binding is driven by hydrophobic effects between the linkers and the macrocycle cavity, and hydrogen bonding and ion-dipole bonding at the macrocycle portals. This figure has been reproduced from ref. 101 with permission from Royal Society of Chemistry, copyright 2006.

By using different sized cucurbiturils, with 6, 7, or 8 subunits, the strength of the binding of the platinum complexes to the macrocycles can be modulated, and through that, adjustment of the on–off rate and its effect on the drug's cytotoxicity.¹⁰³ Broadly, the lower the binding constant, the less effect it has on the drug. In the mouse leukaemia cell line L1210, BBR3464 has an IC₅₀ of 0.57 μM and an IC₅₀ 0.0245 μM in the cisplatin-resistant daughter cell line L1210/DDP. When encapsulated by CB[7], BBR3464 lost all cytotoxicity (IC₅₀ higher than 37.5 μM). But when encapsulated in the larger CB[8], the IC₅₀ values dropped to 6.6 and 1.4 μM, respectively, and with the even larger CB[10] the IC₅₀ values were 0.7 and 0.2 μM, respectively.

Clinically, cucurbituril encapsulation of platinums has shown some benefit to the drugs. Using an in vivo model the encapsulation of cisplatin within cucurbit[7]uril (cisplatin@CB[7]) was shown to increase in the total concentration of circulating platinum over 24 hours when compared with an equivalent dose of free cisplatin.⁹⁵ The end effect was the ability of cisplatin to overcome its own resistance in ovarian carcinoma xenografts. When cisplatin@CB[7] was administered at a dose of 34 mg kg⁻¹ (equivalent to a dose of 6 mg kg⁻¹ of just cisplatin) the tumour had a doubling time 1.6-fold longer than with treatment of an equivalent dose of free cisplatin.

While some of the side effects of BBR3464 may be potentially attributable to the degradation products of the drug, it is more likely that it is simply a function of its off-target attack of healthy cells in the body. All platinums are known for their wide ranging, and dose limiting side effects, and BBR3464 is no different. These effects arise because the drugs are rapidly taken up into all fast growing cells, like the lining of the gastrointestinal tract, bone marrow, and hair. The side effects also cause damage to the liver, as the body attempts to metabolise the drugs, and to the kidneys, as the body attempts to excrete the drug in urine.

The key to developing a future multinuclear drug, or saving BBR3464, will be their co-development with delivery technologies that better target the drugs to tumours, and result in less of the drug going to healthy tissue. This can potentially be undertaken using active targeting strategies,¹⁰⁶ such as nutrient (like folate or glucose), antibodies, peptides (including cell penetratin peptides), and aptamers which can be chemically attached to delivery vehicles, like macrocycles. Thus, the development of functionalised cucurbiturils,¹⁰⁷ which facilitate the potential attachment of those active cancer targeting groups, could further increase the viability of cucurbiturils in rescuing multinuclear platinums as clinical agents.

Conclusions

While highly active in vitro and in animal models, the severe dose-limiting side effects and rapid degradation of BBR3464 meant that it did not progress beyond phase II clinical trials. Developed from the learnings of BBR3464 pre-association with DNA led to the development of a 4^th generation family of platinum complexes that act as phosphate clamps to DNA. While these complexes are also expected to suffer from the same side effects and degradation problems of BBR3464, the future for platinum chemotherapy, and multinuclear platinum complexes as anticancer drugs, lies not in the redesign of the drug molecules, but in developing effective drug delivery technologies that can better target the drugs to cancerous cells and prevent their attack and degradation from extracellular thiol-containing nucleophiles.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts to declare.

References

1. B. Rosenberg, L. Van Camp and T. Krigas, Nature, 1965, 205, 698–699.
2. I. S. Um, E. Armstrong-Gordon, Y. E. Moussa, D. Gnjidic and N. J. Wheate, Inorg. Chim. Acta, 2019, 492, 177–181.
3. E. Armstrong-Gordon, D. Gnjidic, A. J. McLachlan, B. Hosseini, A. Grant, P. J. Beale and N. J. Wheate, J. Cancer Res. Clin. Oncol., 2018, 144, 1561–1568.
4. G. Ferraro, L. Massai, L. Messorib and A. Merlino, Chem. Commun., 2015, 51, 9436–9439.
5. S. Dhar and S. J. Lippard, in Bioinorg. Med. Chem, ed. E. Alessio, Wiley-VCH Verlag GmbH & Co KGaA, Weinheim, Germany, 2011, pp. 79–95.
6. M. J. Cleare and J. D. Hoeschele, Bioinorg. Chem., 1973, 2, 187–210.
7. N. Farrell, T. T. B. Ha, J.-P. Souchard, F. L. Wimmer, S. Cros and N. P. Johnson, J. Med. Chem., 1989, 32, 2240–2241.
8. N. Farrell, Y. Qu and M. P. Hacker, J. Med. Chem., 1990, 33, 2179–2184.
9. N. Farrell, Y. Qu, L. Feng and B. Van Houten, Biochemistry, 1990, 29, 9522–9531.
10. B. A. J. Jansen, J. van der Zwan, H. den Dulk, J. Brouwer and J. Reedijk, J. Med. Chem., 2001, 44, 245–249.
11. J. Kasparkova, K. J. Mellish, Y. Qu, V. Brabec and N. Farrell, Biochemistry, 1996, 35, 16705–16713.
12. C. Hofr, N. Farrell and V. Brabec, Nucleic Acids Res., 2001, 29, 2034–2040.
13. Y. Qu, N. J. Scarsdale, M. C. Tran and N. Farrell, J. Inorg. Biochem., 2004, 98, 1585–1590.
14. T. D. McGregor, Z. Balcarova, Y. Qu, M.-C. Tran, R. Zaludova, V. Brabec and N. Farrell, J. Inorg. Biochem., 1999, 77, 43–46.
15. A. Johnson, Y. Qu, B. V. Houten and N. Farrell, Nucleic Acids Res., 1992, 20, 1697–1703.
16. N. Farrell, T. G. Appleton, Y. Qu, J. D. Roberts, A. P. S. Fontes, K. A. Skov, P. Wu and Y. Zou, Biochemistry, 1995, 34, 15480–15486.
17. B. A. J. Jansen, J. Brouwer and J. Reedijk, J. Inorg. Biochem., 2002, 89, 197–202.
18. T. D. McGregor, W. Bousfield, Y. Qu and N. Farrell, J. Inorg. Biochem., 2002, 91, 212–219.
19. T. D. McGregor, A. Hegmans, J. Kasparkova, K. Neplechova, O. Novakova, H. Penazova, O. Vrana, V. Brabec and N. Farrell, J. Biol. Inorg. Chem., 2002, 7, 397–404.
20. T. Shingu, V. C. Chumbalker, H.-S. Gwak, K. Fujiwara, S. Kondo, N. P. Farrell and O. Bogler, Neuro-Oncology, 2010, 12, 1269–1277.
21. A. Eskandari, A. Kundu, S. Ghosh and K. Suntharalingam, Angew. Chem., Int. Ed., 2019, 58, 12059–12064.
22. J. A. Broomhead, L. M. Rendina and L. K. Webster, J. Inorg. Biochem., 1993, 49, 221–234.
23. S. Komeda, M. Lutz, A. L. Spek, M. Chikuma and J. Reedijk, Inorg. Chem., 2000, 39, 4230–4236.
24. S. L. Woodhouse, E. J. Ziolkowski and L. M. Rendina, Dalton Trans., 2005, 2827–2829.
25. J. B. Mangrum and N. P. Farrell, Chem. Commun., 2010, 46, 6640–6650.
26. J. J. Moniodis, D. S. Thomas, M. S. Davies, S. J. Berners-Price and N. P. Farrell, Dalton Trans., 2015, 44, 3583–3593.
27. O. Novakova, N. P. Farrell and V. Brabec, Metallomics, 2018, 10, 132–144.
28. J. Kasparkova, J. Zehnulova, N. Farrell and V. Brabec, J. Biol. Chem., 2002, 277, 48076–48086.
29. T. Banerjee, P. Dubey and R. Mukhopadhyay, Biochimie, 2010, 92, 846–851.
30. T. Banerjee, P. Dubey and R. Mukhopadhyay, Biochimie, 2012, 94, 494–502.
31. J. Malina, N. P. Farrell and V. Brabec, Chem. – Asian J., 2011, 6, 1566–1574.
32. R. A. Ruhayel, S. J. Berners-Price and N. P. Farrell, Dalton Trans., 2012, 42, 3181–3187.
33. S. Mondal, S. Banerjee, D. Dey, H. Lahiri, T. Mana, S. S. Jana and R. Mukhopadhyay, Biochimie, 2023, 213, 168–175.
34. J. Zehnulova, J. Kasparkova, N. Farrell and V. Brabec, J. Biol. Chem., 2001, 276, 22191–22199.
35. V. Brabec, J. Kasparkova, H. Kostrhunova and N. P. Farrell, Sci. Rep., 2016, 6, 28474.
36. M. D. Hall, M. Okabe, D.-W. Shen, X.-J. Liang and M. M. Gottesman, Annu. Rev. Pharmacol. Toxicol., 2008, 48, 495–535.
37. P. Kabolizadeh, J. Ryan and N. Farrell, Biochem. Pharmacol., 2007, 73, 1270–1279.
38. Q. Liu, Y. Qu, R. V. Antwerpen and N. Farrell, Biochemistry, 2006, 45, 4248–4256.
39. J. D. Roberts, G. Beggiolin, C. Manzotti, L. Piazzoni and N. Farrell, J. Inorg. Biochem., 1999, 77, 47–50.
40. J. Kjellstrom, S. M. Oredsson and J. Wennerberg, Cancer Cell Int., 2012, 12, 20.
41. A. K. Gorle, A. K. Malde, C.-W. Chang, P. Rajaratnam, M. von Itzstein, S. J. Berners-Price and N. P. Farrell, Inorg. Chem., 2023, 62, 13212–13220.
42. A. K. Gorle, P. Rajaratnam, C.-W. Chang, M. von Itzstein, S. J. Berners-Price and N. P. Farrell, Inorg. Chem., 2019, 58, 7146–7155.
43. G. Pratesi, P. Perego, D. Polizzi, S. C. Righetti, R. Supino, C. Caserini, C. Manzotti, F. C. Giuliani, G. Pezzoni, S. Tognella, S. Spinelli, N. Farrell and F. Zunino, Br. J. Cancer, 1999, 80, 1912–1919.
44. C. Manzotti, G. Pratesi, E. Menta, R. Di Domenico, E. Cavalletti, H. H. Fiebig, L. R. Kelland, N. Farrell, D. Polizzi, R. Supino, G. Pezzoni and F. Zunino, Clin. Cancer Res., 2000, 6, 2626–2634.
45. C. Billecke, S. Finniss, L. Tahash, C. Miller, T. Mikkelsen, N. P. Farrell and O. Bogler, Neuro-Oncology, 2006, 8, 215–226.
46. A. Riccardi, D. Meco, C. Ferlini, T. Servidei, G. Carelli, G. Segni, C. Manzotti and R. Riccardi, Cancer Chemother. Pharmacol., 2001, 47, 498–504.
47. G. Colella, M. Pennati, A. Bearzatto, R. Leone, D. Colangelo, C. Manzotti, M. G. Daidone and N. Zaffaroni, Br. J. Cancer, 2001, 84, 1387–1390.
48. L. Orlandi, G. Colella, A. Bearzatto, G. Abolafio, C. Manzotti, M. G. Daidone and N. Zaffaroni, Eur. J. Cancer, 2001, 37, 649–659.
49. P. Perego, C. Caserini, L. Gatti, N. Carenini, S. Romanelli, R. Supino, D. Colangelo, I. Viano, R. Leone, S. Spinelli, G. Pezzoni, C. Manzotti, N. Farrell and F. Zunino, Mol. Pharmacol., 1999, 55, 528–534.
50. T. Servidei, C. Ferlini, A. Riccardi, D. Meco, G. Scambia, G. Segni, C. Manzotti and R. Riccardi, Eur. J. Cancer, 2001, 37, 930–938.
51. P. Kabolizadeh, B. Engelmann, N. Pullen, J. K. Stewart, J. J. Ryan and N. P. Farrell, J. Biol. Inorg. Chem., 2012, 17, 123–132.
52. C. Sessa, G. Capri, L. Gianni, F. Peccatori, G. Grasselli, J. Bauer, M. Zucchetti, L. Vigano, A. Gatti, C. Minoia, P. Liati, S. Van den Bosch, A. Bernareggi, G. Camboni and S. Marsoni, Ann. Oncol., 2000, 11, 977–983.
53. C. Gourley, J. Cassidy, C. Edwards, L. Samuel, D. Bissett, G. Camboni, A. Young, D. Boyle and D. Jodrell, Cancer Chemother. Pharmacol., 2004, 53, 95–101.
54. D. I. Jodrell, T. R. J. Evans, W. Steward, D. Cameron, J. Prendiville, C. Aschele, C. Noberasco, M. Lind, J. Carmichael, N. Dobbs, G. Camboni, B. Gatti and F. De Braud, Eur. J. Cancer, 2004, 40, 1872–1877.
55. T. A. Hensing, N. H. Hanna, H. H. Gillenwater, M. G. Camboni, C. Allievi and M. A. Socinski, Anti-Cancer Drugs, 2006, 17, 697–704.
56. A. H. Calvert, H. Thomas, N. Colombo, M. Gore, H. Earl, L. Sena, G. Camboni, P. Liati and C. Sessa, Eur. J. Cancer, 2001, 37, S260.
57. T. T. Monisha Jaishankar, N. Anbalagan, B. B. Mathew and K. N. Beeregowda, Interdiscip. Toxicol., 2014, 7, 60–72.
58. R. Oun, Y. E. Moussa and N. J. Wheate, Dalton Trans., 2018, 47, 6645–6653.
59. N. P. Farrell, Chem. Soc. Rev., 2015, 44, 8773–8785.
60. M. E. Oehlsen, Y. Qu and N. P. Farrell, Inorg. Chem., 2003, 42, 5498–5506.
61. N. J. Wheate and J. G. Collins, J. Inorg. Biochem., 2000, 78, 313–320.
62. N. J. Wheate, L. K. Webster, C. R. Brodie and J. G. Collins, Anti-Cancer Drug Des., 2000, 15, 313–322.
63. N. J. Wheate, S. M. Cutts, D. R. Phillips, J. R. Aldrich-Wright and J. G. Collins, J. Inorg. Biochem., 2001, 84, 119–127.
64. N. J. Wheate and J. G. Collins, J. Inorg. Biochem., 2001, 86, 478–478.
65. J. G. Collins and N. J. Wheate, J. Inorg. Biochem., 2004, 98, 1578–1584.
66. R. A. Ruhayel, J. S. Langer, M.-J. Oke, S. J. Berners-Price, I. Zgani and N. P. Farrell, J. Am. Chem. Soc., 2012, 134, 7135–7146.
67. A. Hegmans, S. J. Berners-Price, M. S. Davies, D. S. Thomas, A. S. Humphreys and N. Farrell, J. Am. Chem. Soc., 2004, 126, 2166–2180.
68. S. Kemp, N. J. Wheate, D. P. Buck, M. Nikac, J. G. Collins and J. R. Aldrich-Wright, J. Inorg. Biochem., 2007, 101, 1049–1058.
69. A. M. Krause-Heuer, R. Grunert, S. Kuhne, M. Buczkowska, N. J. Wheate, D. D. Le Pevelen, L. R. Boag, D. M. Fisher, J. Kasparkova, J. Malina, P. J. Bednarski, V. Brabec and J. R. Aldrich-Wright, J. Med. Chem., 2009, 52, 5474–5484.
70. S. Komeda, T. Moulaei, M. Chikuma, A. Odani, R. Kipping, N. P. Farrell and L. D. Williams, Nucleic Acids Res., 2011, 39, 325–336.
71. S. Komeda, T. Moulaei, K. K. Woods, M. Chikuma, N. P. Farrell and L. D. Williams, J. Am. Chem. Soc., 2006, 128, 16092–16103.
72. Y. Qu, R. G. Kipping and N. P. Farrell, Dalton Trans., 2015, 44, 3563–3572.
73. A. L. Harris, X. Yang, A. Hegmans, L. Povirk, J. J. Ryan, L. Kelland and N. P. Farrell, Inorg. Chem., 2005, 44, 9598–9600.
74. E. J. Peterson, V. R. Menon, L. Gatti, R. Kipping, D. Dewashinge, P. Perego, L. F. Povirk and N. P. Farrell, Mol. Pharmaceutics, 2015, 12, 287–297.
75. A. L. Harris, J. J. Ryan and N. Farrell, Mol. Pharmacol., 2006, 69, 666–672.
76. B. Bailly, A. K. Gorle, L. Dirr, A. K. Malde, N. P. Farrell, S. J. Berners-Price and M. von Itzstein, Chem. Commun., 2021, 57, 4666–4669.
77. A. Najjar, N. Rajabi and R. Karaman, Curr. Pharm. Des., 2017, 23, 2366–2376.
78. C. N. Sternberg, D. P. Petrylak, O. Sartor, J. A. Witjes, T. Demkow, J.-M. Ferrero, J.-C. Eymard, S. Falcon, F. Calabrò, N. James, I. Bodrogi, P. Harper, M. Wirth, W. Berry, M. E. Petrone, T. J. McKearn, M. Noursalehi, M. George and M. Rozencweig, J. Clin. Oncol., 2009, 10, 5431–5438.
79. H. Shi, I. Romero-Canelón, M. Hreusova, O. Novakova, V. Venkatesh, A. Habtemariam, G. J. Clarkson, J.-I. Song, V. Brabec and P. J. Sadler, Inorg. Chem., 2018, 57, 14409–14420.
80. V. Pichler, P. Heffeter, S. M. Valiahdi, C. R. Kowol, A. Egger, W. Berger, M. A. Jakupec, M. S. Galanski and B. K. Keppler, J. Med. Chem., 2012, 55, 11052–11061.
81. S. Alassadi and N. J. Wheate, Dalton Trans., 2022, 51, 10835–10846.
82. A. Gu and N. J. Wheate, J. Inclusion Phenom. Macrocyclic Chem., 2021, 100, 55–69.
83. K. Ulbrich, K. Hola, V. Subr, A. Bakandritsos, J. Tucek and R. Zboril, Chem. Rev., 2016, 116, 5338–5431.
84. N. L. McGinely, J. A. Plumb and N. J. Wheate, J. Inorg. Biochem., 2013, 128, 124–130.
85. E. N. Hoogenboezem and C. L. Duvall, Adv. Drug Delivery Rev., 2018, 130, 73–89.
86. P. G. Rose, J. A. Blessing, S. Lele and O. Abulafia, Gynecol. Oncol., 2006, 102, 210–213.
87. J. Bader and B. Finn, Adv. Drug Delivery Rev., 2024, 215, 115461.
88. G. E. Craig, S. D. Brown, D. Lamprou, D. Graham and N. J. Wheate, Inorg. Chem., 2012, 51, 3490–3497.
89. A. J. Wagstaff, S. D. Brown, M. R. Holden, G. E. Craig, J. A. Plumb, R. E. Brown, N. Schreiter, W. Chrzanowski and N. J. Wheate, Inorg. Chim. Acta, 2012, 393, 328–333.
90. J. H. Schellens, G. Groenewegen, T. J. M. Meeru, M. Maliepaard, M. M. Tinbben, D. Pluim, H. W. W. ten Bokke, M. Schot, H. Welbank, G. H. Blijham and J. H. Bleijnen, Proc. Am. Soc. Clin. Oncol., 1999, 1999 ASCO Annual Meeting.
91. G. Hamilton and U. Olszewski, Expert Opin. Drug Metab. Toxicol., 2013, 9, 1381–1390.
92. S. D. Brown, K. D. Trotter, O. B. Sutcliffe, J. A. Plumb, B. Waddell, N. E. B. Briggs and N. J. Wheate, Dalton Trans., 2012, 41, 11330–11339.
93. J. T. Wheate and N. J. Wheate, Supramol. Chem., 2024 DOI:10.1080/10610278.10612024.12380054.
94. J. Lagona, P. Mukhopadhyay, S. Chakrabarti and L. Isaacs, Angew. Chem., Int. Ed., 2005, 44, 4844–4870.
95. J. A. Plumb, B. Venugopal, R. Oun, N. Gomez-Roman, Y. Kawazoe, N. S. Venkataramanan and N. J. Wheate, Metallomics, 2012, 4, 561–567.
96. R. Oun, R. S. Floriano, L. Isaacs, E. G. Rowan and N. J. Wheate, Toxicol. Res., 2014, 3, 447–455.
97. N. Gomez-Roman, F. McGregor, N. J. Wheate and J. A. Plumb, J. Ovarian Res., 2015, 8, 62.
98. S. Kemp, N. J. Wheate, S. Wang, J. G. Collins, S. F. Ralph, A. I. Day, V. J. Higgins and J. R. Aldrich-Wright, J. Biol. Inorg. Chem., 2007, 12, 969–979.
99. S. Kemp, N. J. Wheate, M. P. Pisani and J. R. Aldrich-Wright, J. Med. Chem., 2008, 51, 2787–2794.
100. N. J. Wheate, A. I. Day, R. J. Blanch, A. P. Arnold, C. Cullinane and J. G. Collins, Chem. Commun., 2004, 1424–1425.
101. N. J. Wheate, D. P. Buck, A. I. Day and J. G. Collins, Dalton Trans., 2006, 451–458.
102. A. R. Kennedy, A. F. Florence, F. J. McInnes and N. J. Wheate, Dalton Trans., 2009, 7695–7700.
103. N. J. Wheate, J. Inorg. Biochem., 2008, 102, 2060–2066.
104. M. S. Bali, D. P. Buck, A. J. Coe, A. I. Day and J. G. Collins, Dalton Trans., 2006, 5337–5344.
105. Y. Zhao, M. S. Bali, C. Cullinane, A. I. Day and J. G. Collins, Dalton Trans., 2009, 5190–5198,  10.1039/b905112k.
106. D. Vyas, M. Patel and S. Wairkar, Eur. J. Pharmacol., 2022, 915, 174512.
107. H. Yin, Q. Cheng, D. Bardelang and R. Wang, JACS Au, 2023, 3, 2356–2377.

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