Mathilde Bouché

Cécilia Hognon

Stéphanie Grandemange

Antonio Monari

Philippe C Gros

Recent advances in iron-complexes as drug candidates for cancer therapy: reactivity, mechanism of action and metabolites

Introduction

The high therapeutic potential of platinum-based compounds against solid tumors has shed light on the inherent benefits of metal-based drug candidates in the fight against cancer. This is well exemplified by the prevalence of cisplatin in treatment regimens, where the latter is administered alone or in cotreatment with other chemotherapeutics or radiotherapy. 1,2 Yet, the significant side effects of cisplatin, and the low survival improvement with platinum-based drugs impeded their advance to clinics, stimulating intense interest in finding alternative metals. 3,4 In this field, the cooperation of specialists at the interface between chemistry, biology, molecular imaging, bioinformatics and biophysics has enabled significant progress in the comprehension of the mechanism of action of metallodrugs. 5,6,7,8,9,10 This contributes to accelerate the translation of lead candidates to clinical trials and emphasizes the bright future of metal complexes in cancer therapy. Among transition metals, iron is a key player that is widely abundant in the biosphere and omnipresent in living systems. Moreover, this metal benefits a rich redox chemistry that grants access to a large scope of reactivities, making iron-based coordination complexes particularly attractive for biomedicine and especially for cancer treatment. However, uncovering their cellular targets and intricate modes of action is a complex task, further complicated by the presence of high levels of endogenous iron, and requires ingenious strategies to discriminate drug metabolites from the so called 'labile iron pool'. As a most illustrative example, significant progress in deciphering the cellular targets, modes of action and metabolites enabled ferroquine's advance to phase II clinical trial as a lead compound. This successful translation paved the way for in-depth studies of other molecular iron complexes, and for the rationalization of their modes of action and cellular trafficking. Moreover, the rise of sub-cellular chemical imaging, high-throughput analytical techniques and omics allowed the in-depth investigation of iron complexes' reactivity and metallomics at the cellular level. 11,12,13 Furthermore, the progress in the comprehension of iron complexes' cellular reactivity has foreseen a rise in pre-clinical studies in view of advancing more candidates to clinical trial. This also confirms that investigating the intricate relationships between the structural modifications of iron complexes, and their reactivity, cellular trafficking and mechanisms of action is of paramount to allow proper translational approaches. For this reason, the present perspective is not intended to comprehensively detail the synthetic strategies to access ironcontaining complexes as this has been thoroughly covered in recent years, 14,15,16 and we will rather focus on the structureactivity relationship of iron complexes as anticancer drug candidates and their effects at the cellular and body levels. While significant work on iron-containing polymetallic and heterobimetallic species, supramolecular coordination assemblies, and nanoformulations have advanced the field, those topics are not detailed herein and the reader is referred to recent thematic reviews for further detail. 17,18 Differently from those approaches, we will focus on molecular iron complexes, the relationship between their structure, metabolization and This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins antiproliferative activities. Efforts to characterize their molecular targets and mode of action using innovative techniques will be further discussed (Figure 1). Hence, we aim to provide a useful guidance for researchers willing to explore the mechanism of action of metallodrugs at the molecular level and further their pre-clinical evaluation. Meanwhile, we will also discuss the most important perspectives in the development of anticancer iron-complexes, providing a comprehensive vision that combines assessment of both their inherent chemical and biological effects, that is fundamental to develop novel and less toxic chemotherapeutic agents.

Ferrocenyl Derivatives

Ferrocene finds broad application in chemistry, and is a gold standard in electrochemistry due to its reversible one electron oxidation from +II to + III oxidation state, and high stability toward air and water. 19 Its redox activity is also of interest for cancer treatment, due to its oxidation potential (0.4 V versus standard hydrogen electrode) that enables its redox cycling in living systems. This can affect cancer cells by induction of oxidative stress, and motivated intense screening as anticancer candidates.

Ferrocene-containing drug candidates

Ferrocerone was firstly administered as treatment for irondeficiency anemia in the 1970s, although significant side effects prompted its interruption. 20 Contrastingly, ferroquine is one of the most advanced metal-based therapeutic, undergoing phase II clinical trial against infectious diseases. This raised significant interest for analogues of ferroquine that display antimalarial properties 21 and to repurpose them for anticancer research. 22 Overall, ferrocenyl-based drug candidates display antiproliferative activity at sub-millimolar concentrations against numerous cancer cell lines, including hormone dependent and independent breast cancers (MCF7 and MDA-MB-231 respectively), cervical cancer (HeLa), lung cancer (A549), leukemia (CCRF-CEM, Jurkat) and so forth. Remarkably, most ferrocenyl complexes showed negligible effects on non-cancerous lines, hence displaying an elevated safety index that is beneficial in view of clinical evaluation. 23,24 Ferrocenyl complexes showed redox activity that produces high levels of reactive oxygen species (ROS), notably by a Fentontype reaction. 25 At low concentration ROS are key players in cell signaling and involved in the maintenance of cellular redox homeostasis. 26 However, at elevated levels ROS provoke oxidative damages leading to apoptosis or senescence, thus offering alternative therapeutic mechanisms that can be effective in overcoming the acquired resistance of cancer cells towards DNA-binding drugs. 27,28 Ferrocenes altered the cellular redox balance, 29,30 triggered DNA oxidative damages and overall displayed a pro-apoptotic activity that induced cell cycle stop at the G2/M checkpoint following DNA replication. 31,32 Ferrocene appended with hydroxyl-Schiff bases induced a G2/M arrest in HeLA cells while the Schiff base alone arrested cells in G1, emphasizing the effect of the ferrocene moiety in altering cancer cell cycle. 33 A ferrocenyl-chromone complex induced about 60% arrest in G2/M phase in MCF7, HepG2 and CCRF-CEM, while the normal cell cycle could be recovered after exposure to a drug-free medium, possibly due to partial DNA repair in the absence of ferrocene. 34 Contrastingly, 1ferrocenyl-2-(5-phenyl-1H-1,2,4-triazol-3-ylthio)ethanone stopped A549 cells at the G1-checkpoint, and the authors suggested a ROS mediated senescence induction through the p38 MAPK pathway. 35 Ferrocenylseleno-dopamine derivatives induced G1 cell arrest on HepG2, stimulated the activation of caspases 3 and 9 while down regulating the anti-apoptotic Bcl-2 protein, and triggered cell apoptosis with partial necrosis. 36 Of Please do not adjust margins Please do not adjust margins note, no significant differences in the cellular effects of two chiral isomers of ferrocene were observed on A549 and H322. 37 Drug metabolites can be more active than their parent complexes, as illustrated by cisplatin, and their screening is of paramount importance. Ferroquine is metabolized by the hepatic microsomal systems via an oxidative route, leading mostly to the formation of mono-and N,N-demethyl ferroquine byproducts. 38,39 Noteworthy, the kinetics were highly dependent on the microsomes' species, ranging from 80% oxidative metabolization in mice to less than 20% in human microsomes. Toxicology investigations highlighted the high in vivo tolerability of ferrocenium ions, 40 with median lethal doses up to LD 50 = 440 mg.kg -1 in C57BL/6 mice, while a single I.P. injection of 0.2 mg.kg -1 was sufficient to inhibit tumor growth, showing an elevated safety index. 41

Ferrocifens

Taking advantage of the inherent redox activity of ferrocene in biological media and aiming at developing tailored and selective anticancer drug candidates, ferrocenes were conjugated to the estrogen receptor modulator, hydroxytamoxifen, that is currently used in clinical regimens. 42 Such anticancer drug candidates, termed ferrocifens (Figure 1), efficiently inhibited the proliferation of a large range of cancers while showing negligible effects on non-cancerous cells, thus offering a beneficial safety index. Ferrocifens are especially promising for the treatment of hormone dependent breast cancer cells, such as MCF7 that does not respond to hydroxytamoxifen alone, via a mechanism of action involving the binding to estrogen receptors ERα and ERβ. 42 Such feature attracted considerable attention in the field and numerous ferrocifen derivatives were developed to fine-tune their pharmacological profiles and antiproliferative activities. Remarkably, ferrocifens were overall found to have a cytostatic effect via senescence induction, 42 as evidenced by characteristic morphological changes, overexpression of senescence markers, e.g.

β-galactosidase (SA-β-gal), 30 pro-inflammatory interleukins and tumor necrosis factor TNF-α. 43 Hydroxyferrocifen induced cell accumulation in S phase and arrest in G0/G1 phase at longer time in MCF7 or ZR-75-1, while it permanently blocked MDA-MB-231 cells in S or G2/M phases. 30 Tonolo et al. showed the relationship between structure, metabolization pathway and antiproliferative activity, exemplified by two isomeric ferrocifens that show dramatic changes in their metabolization and hence anticancer efficacy (Figure 2). 44 Mimicking either enzymatic oxidation with H 2 O 2 /horseradish peroxidase (HRP) or a chemical oxidation with Ag 2 O, 1,1-bis(4'-hydroxyphenyl)-2-ferrocenyl-but-1-ene evolved via a metal-centered 2 e-oxidation to its quinone methide (QM) metabolite that can alkylate endogenous nucleophiles. 44,45,46 On the other hand, the chemical oxidation of 1,2-bis(4'-hydroxyphenyl)-1-ferrocenyl-but-1-ene (Z) involved a metal-independent 1e-oxidation to diquinone methide that readily tautomerized to the phenol-quinone metabolite. 44,47,48 The authors concluded that ferrocifens acted as prodrugs that could be redox activated to their QM active metabolites. 46 Thioredoxin reductase (TrxR) is a regulator of redox homeostasis overexpressed in numerous cancers, and a wellestablished target for some metallodrug candidates that bind to its selenocysteine residue and inhibit its biological activity. 49,50 Both dimethylaminopropyloxyphenyl-ferrocifen and its QM metabolite significantly inhibited the cellular activity of TrxR, while hydroxyferrocifen showed a negligible effect. 51 Remarkably, Tonolo et al. showed that a ferrocifen, whose mechanism of action was believed to occur via inhibition of TrxR and redox homeostasis disturbance, significantly accumulated in the mitochondria (35 % in MCF7) despite inducing only moderate decrease of the mitochondrial membrane potential. 44 Hence, the authors suggested that the high drug accumulation in specific organelles did not necessarily correlate with the primary target responsible for their antiproliferative activity. Remarkably, direct DNA binding was rarely observed with ferrocenyl and ferrocifens, 52,42 except when conjugated to DNA targeting moieties. 53 This contrasts with the paradigm of metal based drug candidates that covalently bind DNA, and emphasizes the potential of ironbased drug candidates to display novel mechanisms of anticancer action that may provide opportunities for the treatment of cancers that are resistant to standard treatment. Investigation of the cellular trafficking of ferrocifens by optical imaging has been hampered by their low fluorescence quantum yield, 54 and instead infra-red (IR) or Raman vibrational spectroscopies have been used tracking metal-carbonyl vibrations that are active in the 1850-2100 cm -1 range, where endogenous molecules are IR-inactive. 55,56,57 A ferrocifen appended with rhenium-tricarbonyl tag enabled its quantification in MDA-MB-231 cells by IR and showed a dose dependent cellular accumulation that was supported by Fe and Re quantification using ICP-OES. 58 Atomic force microscopy (AFM)-IR mapping at 1950 cm -1 showed the complex colocalization with both the nucleic acids phosphates stretching (1080 cm -1 ) and protein amid band (1640 cm -1 ) suggesting a predominant accumulation in the nucleus, although This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins instrumental limitations precluded the mitochondria-nucleus discrimination. The high hydrophobicity of ferrocifens lowers their anticancer activity and limits their direct pre-clinical evaluation on animal models. Several formulations were investigated to overcome this issue, using micelles or lipidic nanocapsules, achieving mixed results depending on the formulation used, the disease model and the administration strategy. 59,60,61,62 However, ferrocenyl-hydroxytamoxifen and ansa-ferrocifen loaded in lipidic nanocarrier (65 nm diameter, -4 mV surface charge) administered by intravenous injection (45 mg.kg -1 ) to melanoma engrafted nude mice (SK-Mel28) successfully decreased the rate of tumor growth, and the ansa-ferrocifen showed a greater antiproliferative effect. 63 This was attributed to its metabolization to QM and induction of dual pro-senescent and pro-apoptotic effects compared to ferrocenylhydroxytamoxifen that failed to generate the active metabolite. Surprisingly, ferrocifens incorporating 18 F atoms that are active in PET imaging, 74,64 are yet to be administered to living systems to track their in vivo biodistribution. However, such fundamental investigation, also dependent on medical imaging, should be expected to take place in the near future. Noteworthy, the start-up company Feroscan is pushing forward the preclinical investigation of nanoparticulate ferrocifens, 20 and additional reports of in vivo testing of the most promising candidates is foreseen.

JAHA

Jay Amin hydroxamic acid, termed JAHA (Figure 1), are ferrocenes appended to the class I and II histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), an FDA approved agent for T-cell lymphomas treatment. 65 HDAC inhibitors stimulated the expression of estrogen receptors that are deficient in triple negative breast cancer, for a dual therapeutic effect. JAHA exerted significant inhibition of HDAC of class I compared to its parent drug SAHA, although no activity was observed on class II HDAC. 66 JAHA induced antiproliferative effects on both hormone-dependent and independent breast cancer cell lines, (MCF7 and MDA-MB-231 respectively) and induced MCF7 accumulation in the G2/M phase. 67 Molecular docking on selenocyanide-JAHA suggested the binding of the ferrocene in the active site pocket of HDAC, favored by hydrogen bonding between the selenocyanide moiety and Y312 residue that does not occur with its parent drug. 68 Its administration by oral gavage to BALB/c mice showed a median lethal dose of 605.6 mg.kg -1 while both tumor inhibition and low systemic toxicity were observed at a 20 mg.kg -1 dose in MDA-MB-231 xenografted mice. However, further pre-clinical evaluation of JAHA derivatives is necessary to highlight their therapeutic potential, notably as prospective anti-lymphomas drug.

Aminoferrocene-based activatable metallodrug candidate

Aminoferrocenes (Figure 1) are relatively unstable species in aqueous media that readily degrade to free cyclopentadienyl and Fe 2+ ions and can further catalyze ROS production. 69,70,71 Their functionalization with a stimuli-responsive linker formed N-alkylaminoferrocene prodrugs (Figure 1) that could be selectively activated to the unstable aminoferrocene. A ROSactivatable phenylboronic acid pinacol ester linker was used for tumor microenvironment targeted drug release, 69 while photolabile derivatives of o-nitrophenyl were used to achieve light-triggered activation. 72 Of note, some aminoferrocenes are highly lipophilic (log p above 4), and aggregate in aqueous media, which limited both their degradation and ROS production. 73 Pharmacokinetic profiling by radio-HPLC of Nalkylaminoferrocene incubated with human plasma or rat blood evidenced its rapid metabolization to ferrocenium. 74 Reshetnikov et al. showed that a N-ferrocenyl-N-(1-alkyl-1H1,2,3-triazol-4-yl)aminocarbonyloxymethylboronic acid pinacol ester bearing a fluorescent coumarin moiety was metabolized to a fluorescent N-alkylaminoferrocene intermediate. 75 Its trafficking in A2780 and NHDF cells (Normal Human Dermal Fibroblasts) highlighted its privileged accumulation in mitochondria and ROS mediated metabolization. On the other hand, conjugation of a fluorescent alkylated piperidine moiety to aminoferrocene showed the offon release of a fluorescent metabolite in lysosomes (Figure 3). 76

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Ex-vivo exposure of liver slices to N-alkylated aminoferrocene suggested its negligible effect on liver viability, which was supported by the absence of visible toxicity to BDF1 mice treated via intraperitoneal injection of up to 6 mg.kg -1 of drug. 77 In L1210 inoculated BDF1 mice, its administration extended their survival by 17.5 days compared to 13.7 days for vehicle treated mice, and blood drawn L1210 cells showed both a disturbance in their mitochondrial function and enhanced levels of ROS. Further investigation of tailored Nalkylaminoferrocenes and activation by a broader range of stimuli is anticipated considering their potential for selective anticancer treatment in personalized medicine.

Cyclopentadienyl-iron complexes

Half-sandwich cyclopentadienyl-iron complexes (Figure 1) bear only one cyclopentadienyl ligand that can stabilize the metal by both electron-donor or acceptor effects, hence influencing the three additional coordination sites allowing a further fine-tuning of iron's reactivity. 78 These compounds induce either necrosis or apoptosis depending on the ligands that complete the iron coordination sphere, although reports remain scarce and more biological testing are needed to conclude on their cellular effects. 79,80 Of note, Cingolani et al. reported on the preliminary stability in aqueous medium and cellular effects of a cyclopentadienone containing iron complex at the oxidation degree 0, 81 which contrasts with the predominant iron(II/III)-based drug candidates. Undoubtedly, more progress in biological evaluation of such compounds should be achieved in the coming years.

Polypyridyl-iron derivatives

Photoactive iron(III)-polypyridyl complexes

Iron-polypyridyl complexes were investigated as an alternative to photo-active ruthenium-polypyridyl complexes that showed significant tumor growth control in vivo. 82,83 The challenge to access photoactive iron-complexes for photochemotherapy (PC) and photodynamic therapy (PDT) also motivated significant interest in the field to overcome the early photobleaching or slow body clearance of FDA approved photosensitizers (PS). 84,85 Chakravarty et al. showed the potential of iron(III) complexes to display photoactivity under red-light irradiation when bearing tripodal polypyridyl ligands such dipyridoquinoxalines (dpq), 86 dipicolylamines (dpa), 87,88 and Schiff base derivatives. 89 Appending an anthracenyl to Fe(III)dipicolylamine complex had a dual effect by shifting the ligand-to-metal charge-transfer (LMCT) band to the near IR window, while allowing its intercalation in DNA. 87,88 This favored its cellular accumulation in the nucleus which resulted in the significant production of hydroxyl radicals ( • OH) and DNA cleavage after light irradiation. • OH was predominantly produced while only negligible levels of other ROS such as singlet oxygen ( 1 O 2 ) were observed among Fe(II)-dpq analogs. 86 Functionalization of Fe(III)-dpa complex with pyrene showed DNA intercalation, preferred accumulation in the nucleus of HeLa cells, and a light-triggered pro-apoptotic activity. 90 On the other hand, triphenylphosphonium appended Fe(III)-dpa complex targeted mitochondria, disrupted their activity and induced cell death. 91 Remarkably, functionalization of a polypyridyl-Fe(III) complex with a naturally occurring coumarin enabled the access to an LMCT based photoactivatable complex, hence enabling its intracellular tracking in the mitochondria under red-light. 92 Conjugating a BODIPY photosensitizer to Fe(III)-dpa significantly enhanced its antiproliferative activity on HeLa cells from IC 50 = 75 µM to IC 50 = 2 µM under visible or red light irradiation (600-720 nm), and localized in mitochondria due to the BODIPY moiety. 93 Its in vivo effects were evaluated on BT474 (human breast adenocarcinoma) xenografted nude mice that received 5 mg.kg - 1 dose of iron complex, followed by photo-irradiation. Although tumor growth measurements indicated enhanced inhibitory effect of the iron-containing drug candidate compared to vehicle, negligible effects of the infra-red photo-activation were observed, an event attributed to the low photoconversion under infra-red light. 94 Remarkably, a Fe(III)-dpa-BODIPY analog bearing a vitamin B6 ligand displayed enhanced antiproliferative activity toward MCF7 and HeLa cells, preferential accumulation in lysosome rather than mitochondria, and enhanced uptake by the vitamin B6 channels. 95 Biotin-functionalization of a Fe(III)-phenanthroline (phen) complex did not affect its in vitro antiproliferative activity although it hindered its intercalation in DNA, suggesting a mechanism of action independent of DNAbinding. 96 Cellular trafficking of the biotin-Fe(III) conjugate was comparable to the free biotin and diverged from the nontargeted Fe(III)-phen complex, showing an uptake mechanism mainly by receptor mediated endocytosis and partial diffusion pathway, cytosolic localization at 3h incubation and nucleus translocation after 12h incubation.

Bipyridyl-and phenanthroline-iron(II) complexes

Contrastingly to Fe(III) complexes, Fe(II)-polypyridyl are poorly emissive with a short-lived excited states, due to the ultrafast deactivation of the photophysically active metal-toligand charge-transfer ( 3 MLCT) state to the dark metal-centered 3 MC and 5 MC states. Hence, their use as photosensitizers and tracking their localization by optical techniques is challenging. However, significant success has been achieved recently in increasing the fluorescence lifetime by producing Fe(II) complexes displaying low energy MLCT band and increasing the 3 MLCT-3 MC gap. 97,98 Gasser et al. reported on a Fe(II)phen complex and its hydrophilic analog bearing SO 3 -moieties, both showing comparable photophysical properties despite a poor singlet oxygen production ( 1 O 2 yields < 1 % in PBS) and limited improvement in light-triggered cell cytotoxicity, which were associated to the short life-time of the excited state. 99 However, both complexes were stable in human plasma, exhibited low photobleaching and the hydrophilic Fe(II)-phen complex showed reduced dark cytotoxicity compared to its lipophilic analog. Extension of the π-system of phen ligands with -OMe or -NMe 2 terminating moieties red-shifted the optical absorption of their corresponding Fe(II)-phen Of note, a Fe(II)-(py 5 -OH) analog displayed SOD-like activity which could contribute to its antiproliferative effect. 103 A range of Fe(II)-bipyridine (bpy) and Fe(II)-phenanthroline (phen) complexes (Figure 1) with increasing planarity were synthesized and showed increased lipophilicity and uptake by HeLa, MCF7 and A375. 104 After accumulation in the cellular membrane Fe(II)-bpy was internalized by a transferrinmediated cellular uptake, and translocated to the nucleus at 24h after accumulation in the lysosomes and diffusion the cytoplasm. Its cellular localization may also be correlated to its biological effects as Fe(II)-bpy scavenged intracellular ROS, induced cell cycle arrest in S phase via a down regulation of CDK2 and CyclinA, and triggered significant DNA damages and p53-mediated apoptosis. Contrastingly, Fe(II)-phen neither accumulated in the nucleus nor caused DNA damage despite its comparable mechanism of uptake. It scavenged ROS, induced G0/G1 cell arrest, and inhibited the AKT pathway. Those examples also highlight the fine interplay between iron complexes and the cellular machinery. Indeed, while high concentration of iron usually triggers oxidative stress production, some complexes may reduce it by ROS scavenging.

The different mechanism of action between Fe(II)-bpy and Fe(II)-phen was suggested to relate to a threshold in lipophilicity that could direct their cellular trafficking (Figure 4). 104 The hydrophilic Fe(II) complex of 2-selenoimidazole[4,5-f]1,10-phenanthroline showed both enhanced pro-apoptotic activity and anti-angiogenic activity on HUVEC cells by inhibiting VEGF and the AKT pathway, decreasing the neovascularization of Chick embryo chorioallantoic membranes (CAM). 105 Fe(II)-phen complexes also sensitized U87 cells (glioblastome) to TNF-related apoptosis-inducing ligand (TRAIL) and synergized to induce a pro-apoptotic activity involving p38 and p53 activation. 106 In addition, Fe(II)-phen complexes were stable for 48h toward both glutathione (GSH) and bovine serum albumin (BSA), and penetrated cells primarily by a transferrin receptor (TfR) mediated endocytic pathway. 107 They scavenged ROS in Caski cells in a dose dependent manner, which contrasts with most iron complexes. Both the morphological changes of the cell and accumulation in subG1 phase suggested a pro-apoptotic activity. Negligible changes in the hematological analysis of blood lipid, liver and kidney function or pathological investigation of major organs were observed in vivo. Intravenous injection of Fe(II)-phen to U87 xenografted nude mice significantly inhibited tumor growth by a pro-apoptotic mechanism through ROS overproduction, while showing negligible systemic toxicity. 106 Fe(II)-phen synergized with cisplatin in the treatment of esophageal squamous cell carcinoma (ESCC) due to its cellular transport via TfR that is overexpressed in ESCC. 108 Its greater in vitro safety index compared to cisplatin (2.7 vs. 1.4) is reflected by its low side effects on kidney, lungs, neurons and hematology of BALB/C mice. 109 Therefore, in view of their elevated translational potential, more pre-clinical investigation are anticipated in the near future to accelerate their progression to clinical trial evaluation.

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Terpyridyl-iron complexes

Fe(II)-terpyridine (tpy) complexes (Figure 1) are also characterized by poor photoactivity. 110 Their functionalization with anthracene or pyrene showed a moderate ligand centered fluorescence, although no MLCT luminescence was observed due to the unfavorable competition with lower energy MC states and rapid non-radiative decay. For the same reasons the phenyl-functionalized complex showed no photoactivity. 111 Fe(II)-tpy complexes partially intercalated into DNA, however, because of their non-radiative deactivation, their 1 O 2 production was negligible, and they rather cleaved DNA by • OH production under green light, hence inhibiting HeLa proliferation. The complexes localized primarily in the nucleus of HeLa cells which contrasts with the cytoplasmic diffusion of the free ligand, and emphasized the iron-related effect on both the cellular trafficking and light induced activity. Remarkably, Gattuso et al. combined insights from molecular modeling and simulation, spectroscopy, and cellular biology to investigate the effects of phenyl-or pyridyl-functionalization of Fe(II)-tpy complexes on their interaction modes with DNA and their subsequent cellular effects (Figure 5). 112 A 14 bp model B-DNA composed of poly(dA)-poly(dT) and poly(dC)poly(dG) was used to investigate by classical all-atoms molecular dynamic simulations three interaction modes, e.g. major groove binding, minor groove binding and intercalation. While persistent minor groove bindings were obtained for all the Fe(II)-tpy complexes, the presence of a pendant phenyl-and pyridyl-moiety induced the formation of a stable intercalation mode. The presence of the pendant moieties also significantly limited the diffusion of the complex in the DNA minor groove as compared to their unfunctionalized analog, while no persistent interaction was observed in the major groove. Of note, the analysis of the non-covalent interaction pathways revealed the formation of a hydrogen bond between the pyridylsubstituent of the Fe(II)-tpy complex with the electron rich amine moiety of guanine. Minor groove binding and intercalation of both Fe(II)-tpy complexes significantly bended DNA, and showed a partial eviction of the guanine from the ideal B-DNA stacking. While contribution from molecular modeling to elucidating the molecular mechanism of action of iron-based drug candidates is scarce, this example illustrates the significant progress of simulation techniques in providing an atomistic resolution of the interactions taking place at the molecular level between the drug candidates and their biomolecular targets. Such comprehension will certainly prove fundamental in view of designing tailored drugs, and hence more important breakthroughs are expected in the near This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins future 113,114 to rationalize the interaction with DNA or protein targets, oxidative stress induction and drug internalization pathways either via passive lipid membrane permeation or by interaction with cellular transporters.

Salen-and salophen-iron derivatives

Iron complexes bearing N,N-bis(salicylidene)ethylenediamine (salen) or N,N-bis(salicylidene)phenylenediamine (salophen) ligands (Figure 1), mimic the catalytic activity of the iron active sites of enzymes such as the cytochrome P450, dioxygenase and so forth. 115,116 Although they showed low interaction with DNA, 117 their [Fe(salen)] + metabolite interacted via electrostatic interaction with phosphates of the DNA backbone, 118 which is beneficial for iron-mediated production of ROS, oxidative DNA cleavage and elevated antiproliferative activity. 119 Moreover, the Fe-salophen complex remained stable over 3.5 h in physiological conditions while rapid conversion was observed in acidic conditions. 120 Those complexes displayed high antiproliferative activity, IC 50 as low as 58 nM were observed on A2780 cells for the phenyltetrazole analog of Fe-salophen complex. 121 Negligible effect on healthy cells, notably skin fibroblast, human hepatocytes and primary lung were evidenced. 122 On platinumresistant ovarian cancer cells, Fe-salophen complexes induced chromatin fragmentation, activation of caspases 3, 8, 9 and PARP cleavage (Figure 6). 122 SMS-KCNR NB cells treated with Fe-salophen complex featured a more pronounced ROS production than the ones exposed to the free ligand, DNA degradation and cell cycle arrest in G1 phase. 123 In depth investigation also showed disruption of mitochondria's membrane, stimulation of caspases 3,7 and regulation of proliferation signaling pathways (MAPK and JNK) due to treatment with Fe-salophen. Surprisingly, while a dose of 3 µM significantly decreased the viability of HUVEC cells, doses in the range of 0.3-1 µM stimulated their proliferation, showing dose-dependent effects. Noteworthy, Gust et al. suggested that Fe-salophen complexes inhibit cell proliferation via interconnected necroptosis and ferroptosis pathways, which should receive more attention in the future. 124,125 Repeated IP administration of Fe-salophen to albino rats showed overall negligible effect in vivo, although cases of hepatocyte degeneration and pulmonary mononuclear cell infiltrates that are characteristic of inflammation, were observed at elevated drug doses. 122 Remarkably, a Fe-salophen was magnetically active, hence enabling its use for hyperthemia on VX2 xenografted rabbits after intra-arterial administration, and paving the way for alternative combination therapies. 126

Thiosemicarbazone-iron derivatives

Iron overload has been identified as a pro-tumoral factor, in part due to the enhanced need for nutrients and oxygen of highly proliferative cancer cells. 127 Hence thiosemicarbazones (TSC) were developed to target excess iron in cancer cells and chelate the "labile iron pool" (LIP) to, on one hand prevent its association to hemoglobin, ferritin and so forth to limit cancer cell proliferation, and on the other hand induce local antiproliferative activity due to the formation of toxic iron-TSC complexes. The reader is referred to compelling reviews for further details concerning their structure-activity relationship. 128,129,130 TSC, e.g. triapine, di-2-pyridylketone 4,4dimethyl-3-thiosemicarbazone and so forth, formed stable complexes with iron (Figure 1) that could regulate iron homeostasis by both direct chelation of the LIP and inhibition of iron transporters. Moreover, those iron complexes displayed additional antiproliferative activity by producing high levels of ROS, damage DNA and induced G1/S cell arrest. 131,132,133,134 Remarkably, triapine and its iron complex behave differently in CT-26 (colon cancer) xenografted mice, the iron complex bound proteins and was excreted via the urines to a larger extent than the free ligand. 135 This fact was also correlated with the different pharmacological properties of the ligand and its complex that direct their cellular trafficking (Figure 7). 135 Although iron-TSC complexes are stable toward bioreducing agents, e.g. GSH and metalothioneins, 136 they engage numerous Please do not adjust margins Please do not adjust margins targets, notably by inhibition of ribonucleotide reductase that is involved in DNA synthesis. 137 The complexes inhibited the p21 gene and cyclins D, which contributed to cell cycle regulation, while they upregulated anti-oncogenes such as N-myc. 138,139,140 Tracking the chelation of the radionuclide 59 Fe by TSC suggested the TSC-mediated inhibition of Fe-transferrin adducts, while showing negligible effect toward Fe-S cluster proteins that are involved in balancing the redox homeostasis and gene regulation. 128 Triapine advanced to phase II clinical trials and showed encouraging anti-leukemia effects despite limited efficacy on solid tumors, and significant side effects including vomiting and anemia, 141,142 foreseeing more upcoming pre-clinical evaluation of TSC analogs.

Bio-inspired iron complexes

A variety of bio-mimic ligands and natural extracts are efficient iron chelators, i.e. flavonoids, rosins, salinomycin and so forth, 143,144,145 and offer alternative therapeutic strategies displaying overall elevated biocompatibility. Among them, bleomycin (BLM), a glycoprotein that possesses both a DNA interaction domain and metal binding domain, showed high therapeutic potential against a variety of cancers. Indeed, its coordination to Fe 2+ ion and activation of O 2 (Figure 1) led to DNA cleavage, preferentially at GC and GT sequences. 146,147 A squared pyramidal low spin BLM-Fe(III)-OOH intermediate was suggested to trigger H4' atom transfer from the C4' cytosine of DNA to the metal, 148,149 which contrasts from hemelike chemistry that occurs in enzymes such as cytochrome P450 and deoxygenases, 150 although the exact mechanism of action remains debated (Figure 8). 151 BLM displayed high antiproliferative activity against a range of cancer cells, and stopped cell cycle in both G2 and mitosis. BLM successfully completed phase IV clinical trial in combination to electroporation techniques, i.e. lipid membrane's permeabilization by application of an electric field, to overcome its limited spontaneous uptake. It is currently commercialized under the brand name of Blenoxane® for the treatment of various cancers, despite limitations inherent to its systemic toxicity. 152,153 Its metabolization was linked to bleomycin hydrolases, and BLM showed predominant excretion through the kidneys with up to 65 % of the injected drug detected in the urines after 24h. Consequently, screening for BLM derivatives and overall bio-mimetic iron chelates is required for increasing its safety index and pharmacokinetics.

Iron-nitrosyl complexes for NO delivery

Iron is a key player in the biosynthesis and transport of nitric oxide (NO), a signaling molecule that contributes to programed cell death at high concentrations. 154 Hence, biomimetic iron complexes were developed to grant intracellular NO delivery and inhibit cancer cell proliferation by both enhancing the activity of p53 gene, and suppressing the activity of key reparation protein of mammals, the O6-methyl-guanine-DNAmethyl-transferase 2 and mitochondrial respiration. 155,156 Iron complexes delivering NO were recently reviewed, 157,158 which highlighted the therapeutic potential of nitrosylation of Fe/S clusters to afford tetrahedral dinitrosyl iron complexes [Fe(NO) 2 ] (DNIC) (Figure 1) that release NO under various light or redox stimuli and ligand substitution. 159,160,161 DNIC showed promising anti-proliferative and pro-apoptotic activities in the micromolar range, 162,163,164 notably acting via inhibition of survival associated proteins and activation of JNK proliferation signaling pathway. 165 Finetuning the coordination sphere to stabilize the Fe-NO bond 166 enabled NO delivery with half-lives in the order of minutes and achieved high PC3 tumor inhibition at 0.2 mg.kg -1 doses. 165,167 Advance of both nitroprusside [Fe(NO)(CN) 5 ] 2-to phase I clinical trial against several cancers and leiomyoma as an anti-angiogenic drug, and of Oxacom as an hypotensive drug against endometriosis, highlighted the therapeutic promise of iron complexes for NO This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins delivery. 168,169 Furthermore, DNIC showed high potential for sensitizing hypoxic cells to γ-radiation damage 170 or to chemotherapeutics, 171 thus offering opportunities in combination with adjuvant therapies.

Conclusions and Perspectives

Overall, significant developments in the field of metallodrugs, and especially anticancer iron complexes, were achieved recently with growing emphasis on studying their behavior and mechanism of action at the cellular level. As a matter of fact, the examples discussed in this perspective illustrate the variety in structures of molecular iron complexes that were screened for anticancer applications, ranging from non-heme iron complexes, photoactive and bio-inspired iron complexes. Those showed elevated anticancer activities with IC 50 down to the nanomolar range on a broad range of cancer cell lines, hence offering opportunities for the treatment of both solid and soft tumors, which contrasts with the reference platinum-based chemotherapies that are limited to the treatment of solid tumors. Another beneficial aspect of iron complexes for biomedical application lies in their negligible effects on non-cancerous cells and overall elevated safety index that foresees a safer use and significant progress in cancer management for patients. The wide applicability of iron complexes can probably be explained by the diversity in cellular effects that were observed both in vitro and in vivo, since such molecular complexes can interfere with the maintenance of intracellular redox balance, disturb iron homeostasis, intercalate in DNA and so forth. Considering the redox activity of iron complexes in biological environment and their affinity for thiols and selenols that are abundant in proteins and enzymes, it is of paramount importance to screen for metabolization pathways. Hence, metabolites can be more active than their parent compounds, and overlooking this aspect can lead to misconception in the design of libraries of drug candidates that ultimately leads to a dead end. In this context, we emphasize the importance of rationalizing the structure-activity relationship and uncovering the active species. While carrying such study on cells and living systems can be both lengthy and pricey, and often results in a bottleneck, a recent work by Gasser et al. described a good alternative for in vitro screening of the behavior of metal complexes in plasma or microsomes to mimic blood and liver conditions. 172 Moreover, the wide array of mechanisms of action and targets observed for iron complexes drug candidates highlights the difficulty of a compelling investigation due to the complex biological environment and their intertwined pathways. The case of iron complexes is especially complicated due to the presence of elevated levels of endogenous iron that, on one hand make it difficult to quantify the uptake of the iron complex, and on the other hand can lead to cross-talk with endogenous iron and disturbance of the iron homeostasis in the case of labile complexes. Such lock out will be a key point to leverage in order to facilitate the investigation of iron complexes and other metal-based therapeutics in living systems, and to this end progress in the neighboring fields of speciation by metallomic study, 173,174 sub-cellular imaging 175,176 and bioinformatics 177 will be of essential. While increasing knowledge in the cellular effects and behavior of iron complexes is welcome, in the near future it will be important for the field to investigate their mechanism of action via an interdisciplinary approach, bringing together bioinorganic and theoretical chemists, cellular biologists, bioinformaticians, and specialists in medical imaging. The synergy between those neighbor fields appears to us as a key element to acquire in-depth knowledge in their cellular effects, as exemplified by the groundbreaking progress in the comprehension of cellular effects of ferrocifens that fastened their pre-clinical investigation in view of evaluation in clinical trials. Hence, we anticipate growing collaborative work in the near future to contribute in unveiling the richness in therapeutic opportunities offered by iron complexes as anticancer drug candidates, and we expect this will stimulate further translational studies to establish a new paradigm in cancer treatment.

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Figure 1 .

 1 Figure 1. Iron complexes discussed in this review.

Figure 2 .

 2 Figure 2. Pathways proposed for the metabolization of A) 1,1-bis(4'-hydroxyphenyl)-2ferrocenyl-but-1-ene into active QM, and B) 1,2-bis(4'-hydroxyphenyl)-1-ferrocenylbut-1-ene (Z) into inactive phenol-QM. Adapted with permission from reference 44.

Figure 3 .

 3 Figure 3. A) Structure of the aminoferrocene prodrugs 1, 2, 5 and their activation pathways A1 and A2 respectively to their photoinactive and fluorescent lysosome specific metabolites. Representative photographs of acridine orange stained DU-145 cells B) in drug-free medium, C) incubated with prodrug 1 or D) prodrug 2. DU-145 cells treated with prodrug 5 and Lysotracker Red under E) Lysotracker Red specific channel, F) channel specific of drug activation, G) overlap of (E) and (F). Adapted with permission from 76.

Figure 4 .

 4 Figure 4. A) Structure of the selected Fe(II)-bpy/-phen complexes. B) Relationship between structure, lipophilicity and cellular uptake in MCF7 cells incubated with the complexes for 3h or C) followed at several times points over 3 h. D) Representative photographs showing the localization of the complexes by fluorescence in MCF7 (top), A375 (middle) and HeLa (bottom) cells, and E) Evolution of the fluorescence intensity across the cells. Adapted with permission from reference 104.

Figure 5 .

 5 Figure 5. A) Synthesis of the three Fe(II)-tpy complexes of interest, B) representative examples of the interaction of FeC-3 with DNA obtained by molecular dynamic, i.e. major groove binding (left), minor groove binding (middle) and intercalation (right). C) Table summarizing the DNA interaction mode of the three Fe(II)-tpy complexes. Adapted with permission from 112.

Figure 6 .

 6 Figure 6. A) Synthesis of Fe-salophen complex, and its effect in SKOV-3 on B) the cell viability, C) DNA adducts by TUNNEL assay and D) expression of caspase 9, 8, 3 and PARP-1. Adapted with permission from 122.

Figure 7 .

 7 Figure 7. A) Structure of triapine and its iron complex, comparison of their biodistribution in B) serum and C) urin. Evolution of the cytosolic levels of D) triapine and E) Fe-triapine complex. Adapted with permission from 135.

Figure 8 .

 8 Figure 8. Reactivity and intermediates hypothesized for BLM-iron interaction with the C4' cytosine. Adapted with permission from 151.

L1210

  photophysical and photochemical phenomena taking place in complex (biological) environments. He is also interested in describing the structural and dynamical behavior of complex biological architectures submitted to external stress sources via enhanced sampling molecular dynamics and free energy techniques. He particularly tackles DNA photolesions induction and repair, the photosensitization of other biological systems, such as vision related proteins, and the photophysics of organometallic compounds. He is author of more than 150 publications and is member of the executive committee of the French Society of Photobiology, and of the CNRS "Comité National" for the physical chemistry and numerical methods in biology sections.

  up to 708 nm, although negligible cytotoxicities and low luminescence quantum yields (< 0.1 %) were correlated with poor 1 O 2 production.100 Of note, their counter ion exchange from PF 6 -to Cl -efficiently enhanced their water solubility but showed no effect on photophysical properties and cytotoxicity, hence emphasizing the access to long-lived photoactive state in Fe(II) complexes as a key issue in determining their phototoxicity. Che et al. highlighted the impact of the coordination sphere onto the mechanism of action of two Fe(II) complexes bearing rigid quinquepyridine (qqy) or flexible 2,6-bis[hydroxybis(2pyridyl)methyl]pyridine (py 5 -OH) pentadentate ligands, showing high antiproliferative activity on a range of cancer cell lines (IC 50 values in the range of 0.8-3.1 µM or 0.6-3.4 µM respectively), although the Fe(II)-(py 5 -OH) complex showed a reduced cytotoxicity toward CCD-19Lu cells (normal lung fibroblast). 101,102 While both complexes induced DNA damage, pro-apoptotic activity and regulated the MAPK proliferation signaling pathway, in depth study of their DNA cleavage ability, ROS production, genes and pathways regulation highlighted profound mechanistic differences. Fe(II)-qqy complex produced elevated levels of ROS, cleaved DNA and arrested HeLa cells in S phase independently from p53, while Fe(II)-(py 5 -OH) complex produced ROS by a Fenton reaction that arrested HeLa cells in G0/G1 phase by activation of p38 and p53 pathways. Both complexes regulated different genes expression, although similarities with a known drug, ciclopirox, suggested a mechanism involving the regulation of iron levels.

6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Acknowledgements

Support from the Université de Lorraine and CNRS is gratefully acknowledged. The authors wish to thank the Université de Lorraine LUE i-site program for funding under the Mirabelle+ program.

Notes and references

Conflicts of interest

The authors declare no competing interest.


Callum Yeardley

Alan R Kennedy

Philippe C Gros

Sabrina Touchet

Michael Fairley

Ross Mclellan

Antonio J Martínez-Martínez

Charles T O'hara

Structural and metal-halogen exchange reactivity studies of sodium magnesiate biphenolate complexes

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Metal-halogen exchange involving aromatic halides is a powerful transformation in synthetic chemistry. It allows the facile conversion of aromatic halides to useful functionalised arenes via metal aryl intermediates. Traditionally this exchange has been carried out utilising monometallic lithium-and magnesium-based reagents. Organolithium reagents typically require the use of cryogenic conditions and exhibit a poor functional group tolerance, whilst organomagnesiums often display a greater functional group tolerance but are generally less reactive. However, the reactivity of organomagnesium reagents can be increased by combining them with an alkali metal one to generate an alkali metal magnesiate. 1 As structure is inextricably linked to reactivity in organomagnesium chemistry, and much like monometallic Grignard reagents whose solution-state structure is dictated by the Schlenk equilibrium, alkali metal magnesiates also show a structural complexity which might not be immediately evident or expected. 2,3 The synergic behaviour arising from the combination of two metal centres within a complex gives these reagents unique and often enhanced reactivity, allowing them to perform transformations that would otherwise be unachievable using their monometallic parent complexes. 1,4,5 Not only do these bimetallic reagents allow for reactions to be carried out under milder conditions 6 (i.e., carried out at temperatures at or close to ambient temperature), they also exhibit a much greater functional group tolerance compared to monometallic bases. 7 Consequently, bimetallic magnesiates have attracted much attention for use in metal-halogen exchange reactions. Trialkylmagnesiates are also used extensively. 8 The enhanced reactivity of these species can, in some cases, enable exchange to occur using sub-stoichiometric quantities of magnesiate. 9 Despite their reactivity, it has been shown that exchange does not always occur with full consumption of all the alkyl ligands leading to undesired side reactions. 10 Replacement of unreactive alkyl ligands with alkoxides is therefore an attractive principle for tuning the chemoselectivity of magnesiate complexes. Knochel and co-workers have recently utilised 2-ethylhexanol to synthesise alkylmagnesium alkoxides s BuMgOR•LiOR and s Bu 2 Mg•2LiOR (where R = 2ethylhexyl). 11 These reagents undergo magnesium-halogen exchange with a range of aryl and hetero-aryl substrates at ambient temperatures in non-polar solvents. Furthermore, the inclusion of alkoxide ligands also provides facile access to the realm of asymmetric synthesis. [12][13][14][15][16] Noyori has reported the efficient enantioselective alkylation of aldehydes using a lithium magnesiate containing the chiral BINOL alkoxide ligand. 17 Please do not adjust margins Please do not adjust margins Furthermore, the groups of Gros and Mongin have utilised lithium magnesiates containing the chiral diols (R,R)-TADDOL and (R)-BIPHEN [(R,R)-TADDOL is (4R,5R)-2,2-dimethyl-1,3dioxolane-4,5-diyl)bis(bis(4-(tert-butyl)phenyl)methanol and (R)-BIPHEN is 5,5´,6,6´-tetramethyl-3,3´-di-tert-butyl-1,1´biphenyl-2,2´-diol] as alkoxide ligands in metal-halogen exchange reactions to access a range of chiral alcohols. 18,19 A well-defined and robust organometallic complex with complete saturation of the metal coordination sites is essential in order to limit excess aggregation and subsequent loss of enantioselectivity. Our group has successfully characterised a series of dilithium dialkylmagnesiates containing the (rac)-BIPHEN ligand and demonstrated their reactivity in metalhalogen exchange. 20 Using the enantiomerically pure form of the ligand, (S)-BIPHEN, Gros and co-workers have synthesised a range of chiral 3-substituted isobenzofuranones with good enantiomeric ratios and yields from reaction of the monoalkyl lithium magnesiate (S)-BIPHEN n BuMgLi with ethyl-2iodobenzoate and subsequent electrophilic quenching with a range of aldehydes. 21 Despite the wealth of knowledge relating to the structure of lithium magnesiates and their reactivity in metal-halogen exchange, the same cannot be said for sodium magnesiates, particularly those bearing alkoxide ligands. Sodium magnesiates have primarily been employed as powerful bases in deprotonation chemistry 22,23 and as efficient catalysts for a range of transformations. [24][25][26][27][28] To the best of our knowledge there are no examples of metal-halogen exchange involving sodium magnesiates. Herein, we report the synthesis of two new disodium dialkylmagnesiates containing the (rac)-BIPHEN ligand (Scheme 1) and discuss their preliminary reactivity in metal-halogen exchange.

Results and Discussion

Syntheses of Sodium Magnesiates from (rac)-BIPHEN

Prior to preparing the desired sodium magnesiates, synthesis of the homometallic sodium precursor was initiated starting from the (rac)-BIPHEN ligand. The parent (rac)-BIPHEN-H 2 ligand was doubly deprotonated using n BuNa (two molar equivalents) in hexane solution. To aid crystallisation, the majority of the hexane was removed in vacuo and replaced with THF. Cooling the resultant solution to -18 °C generated the desired disodium phenoxide [BIPHEN] 2 Na 4 (THF) 4 1 (unoptimised 26% crystalline yield). In an attempt to prepare the desired magnesiate, a hexane solution of the sodium reagent was generated in-situ as described herein, followed by co-complexation with n Bu 2 Mg (one molar equivalent). THF was added and the mixture heated gently to ensure complete dissolution. After cooling to -18°C, X-ray quality colourless crystals were isolated and were determined to be the n Bu-free [BIPHEN rich] complex [BIPHEN] 2 MgNa 2 (THF) 4 2 (37% crystalline yield. Maximum yield of 50% based on metals). In an attempt to prepare heteroleptic (mixed alkyl/phenoxide) magnesiates, N,N,Nʹ,Nʹtetramethylethylenediamine (TMEDA) and N,N,Nʹ,Nʹʹ,Nʹʹpentamethylethylenetriamine (PMDETA) were utilised as chelating donor ligands. To emphasise, instead of using THF in the aforementioned reaction, TMEDA and PMDETA (two molar equivalents) were utilised. Using this slightly modified synthetic methodology, the higher order magnesiates [(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2 3 (56 % yield) and [(rac)-BIPHEN]Na 2 Mg n Bu 2 (PMDETA) 2 4 (55 % yield) were synthesised.

X-ray crystallographic studies

The structure of the disodium phenoxide, which crystallised as a dimeric THF solvate, was revealed to be the tetranuclear complex [BIPHEN] 2 Na 4 (THF) 4 1. Each molecule has crystallographically imposed 2-fold symmetry, and as two of the Na centres sit on the twofold axis, there are three chemically distinct sodium environments (Figure 1). Despite using the racemic form of the BIPHEN ligand during synthesis, the crystal Please do not adjust margins Please do not adjust margins of 1 measured was enantiopure, chiral space group P4 1 2 1 2, with one crystallographically unique BIPHEN ligand per asymmetric unit [Flack parameter, 0.018 (14)]. Spontaneous resolution upon crystallisation is thought to occur for approximately 5 to 10% of all racemic compounds. This forms conglomerates, or physical mixtures, of separate Rand S-crystals. [START_REF] Jacques | Enantiomers, Racemates and Resolutions[END_REF][START_REF] Srisanga | [END_REF] In the absence of any driver to enantiopurity, this is presumably the case here.

Analysis of the organometallic framework of 1 shows the central sodium atom Na1 is four coordinate with a distorted tetrahedral geometry (sum of bond angles 664. Comparison of the key bond lengths and bond angles of 1 with its related tetranuclear lithium complex (BIPHEN) 2 Li 4 (THF) 4 reported in our previous work, 20 revealed an enhanced puckering of the four membered ring at the top of the structure (Na3-O2-Na3ʹ-O2ʹ ring in Figure 1). For a comparison see the Supporting Information. Turning to the BIPHEN-rich 2 which also crystallised with crystallographically imposed symmetry. The central magnesium centre Mg1 sits on three twofold axes and is four coordinate with a distorted tetrahedral geometry (sum of bond angles 665.6°). The Mg-O bond length is 1.962(3) Å and this is in good agreement with its homometallic analogue. 31 The related sodium atom sites Na1/Na1 are situated on a twofold axis and are also four coordinate, again adopting a distorted tetrahedral geometry (sum of bond angles 662.1°). They are bound to a single oxygen atom of each of the BIPHEN ligands [Na1-O1 bond distance, 2.229( 4) Å] and their coordination spheres are completed by two molecules of THF [Na1-O2 bond distance, 2.273( 5) Å]. Similar to the situation observed with 1, the single crystal of 2 examined was determined to be chiral, crystallising in the chiral space group I222 [Flack parameter, -0.16 (17)].

Solution studies

The isolation of the BIPHEN-rich complex 2 is perhaps surprising considering the stoichiometry of starting reagents used in the reaction. It can be attributed to the disproportionation (ligand reorganisation) of the expected disodium dialkyl magnesiate product, [(rac)-BIPHEN]MgNa 2 Bu 2 (THF) 4 (Scheme 2). This type of reorganisation is a common pathway to generally undesired compounds in related bimetallic chemistry. 32,33 It is possible that the formation of 2 may be due to thermal decomposition; however, no other products from this Schlenk-like equilibrium were isolated from the reaction (Scheme 2). The reaction was subsequently repeated using the bulkier dialkylmagnesium reagent Mg(CH 2 SiMe 3 ) 2 . Co-complexation of Mg(CH 2 SiMe 3 ) 2 with 1 again produced the BIPHEN-rich 2 as the only isolable reaction product and not the desired magnesiate [(rac)-BIPHEN]Na 2 MgR 2 (THF) 4 . Despite multiple attempts at preparing both dialkylmagnesiates, their solid-state structure could not be determined -the only isolable product on each occasion was 2.

In an attempt to preclude the aforementioned Schlenk-like equilibrium, the bidentate donor TMEDA was employed. It was expected that the strong chelation of the diamine with the Na centre may inhibit further reaction to form undesired 2. This was indeed the case and the formation and isolation of [(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2 3 was facile. As noted previously, by employing PMDETA, the related complex 4 was prepared. The excellent solubilities of 3 and 4 in arene solvents has allowed them to be characterised by 1 H and 13 C NMR spectroscopy (Tables 2 and3). The CH 2 -Mg methylene hydrogen atoms for 3 appear at δ -0.31 and -0.78 ppm as two broad multiplets rather than a single resonance due to the diastereotopic nature of these hydrogen atoms. The corresponding CH 2 -Mg was located at 8.84 ppm in the 13 C NMR spectrum. Comparison of the 1 H and 13 C NMR spectra of 3 with those of 4 showed a good level of agreement with the exception of the resonance 20 To further investigate the role in which TMEDA and PMDETA play in suppressing disproportionation, C 6 D 6 and d 8 -THF solutions of complexes 3 and 4 were prepared. Immediate displacement of TMEDA and PMDETA by THF was observed for both 3 and 4 in d 8 -THF. Monitoring the solutions by 1 H NMR spectroscopy showed no disproportion occurred in either solution of 3 or 4 at 300 K. Heating these solutions to 50°C for a further 12 hours showed no change in 1 H NMR spectra of 3 or 4 in C 6 D 6 . In d 8 -THF however, complex 3 showed multiple new resonances in the aliphatic and aromatic regions of the 1 H NMR spectrum which was accompanied by a colour change in the solution from colourless to yellow. Surprisingly, the 1 H NMR spectrum of 4 in d 8 -THF showed comparatively little decomposition compared to that of complex 3 under the same conditions and suggests the parent magnesiate 4 remains intact i.e., no ligand rearrangement occurs after heating in d 8 -THF (see Supporting Information).

Metal-halogen exchange studies

The reactivity of 3 and 4 were investigated to assess their potential in metal-halogen exchange reactions ultimately generating of aryl and heteroaryl magnesium reagents (5-10, Table 3). Investigations into Mg-I exchange were performed using a 2:1 ratio of organic substrate to exchange reagent (3 or 4) in order to achieve high efficiency double Mg-I exchange. Initially examining the reactivity of substrates bearing electron donating functionalities, the reaction of 3 and 4 with 2iodoanisole produced 5 in a 60 and 30% yield respectively after 15 minutes. Concomitant formation of 4-iodobutane was observed as evidenced by resonances at  2.76, 1.43, 1.13 and 0.69 ppm in the associated 1 H NMR spectra in both cases. Formation of an additional product [29% yield for 3 and (41%) for 4,  3.33 ppm] was tentatively assigned to be the intermediate monoalkyl-monoaryl-magnesium species resulting from exchange of a single alkyl ligand. Continued monitoring of the reactions over a further 12 hours showed an increase in the yield of 5 to 90% with full consumption of 3, 13 Please do not adjust margins Please do not adjust margins while a maximum yield of 77% was achievable using 4. Furthermore, no changes were observed in the 1 H NMR spectra after full consumption of 3 and 4 which indicates no subsequent reaction between the newly formed diaryl magnesiate and the iodobutane generated in situ.

Table 3: Metal-halogen exchange of aryl iodides using 3 and 4.

Reactions were performed in a Young's cap NMR tube using 0.0377 mmol of magnesiate and 0.0754 mmol of Iodoarene. Unless otherwise stated yields were calculated from 1 H NMR spectroscopic data by integration against an internal standard of 1,2,3,4-tetraphentlnapthalene.

[a] Full consumption of magnesiate observed within 18 hours. [b] Incomplete conversion of magnesiate after 18 hours.

[c] Calculated from 1 H NMR spectroscopic data by integration against an internal standard of hexamethylcyclotrisiloxane. [d] Full consumption of magnesiate observed within 15 minutes.

Having demonstrated the ability of 3 and 4 to perform magnesium-halogen exchange reactions, the scope was expanded to investigate the tolerance of both magnesiates towards metaand para-substituted electron rich substrates. Reaction with 3-and 4-iodoanisole produced 6 and 7 in considerably diminished yields of 14% and 6% respectively after 15 minutes using 3 while no appreciable reactivity was observed using 4. This significantly decreased reactivity is likely due to the more remote nature of the Lewis basic MeO-group with respect to the site of metal-halogen exchange c.f., Directed ortho-Metallation (DoM) chemistry. 34 After 1 hour an increase in the yields of 6 and 7 to 41% and 20% respectively were observed using 3 whilst with 4, 6 was obtained in only a 6% yield and no formation of 7 was observed. In all cases there were significant quantities of the starting material 4-iodoanisole remaining. Continued monitoring of the reaction gave a maximum yield of 91% for 6 accompanied by full consumption of 3 whilst a maximum conversion of 79% was observed for 7 without complete consumption of the parent magnesiate after an additional 12 hours. In the case of 4, full consumption of the magnesiate was not observed in either case and gave 6 and 7 in yields of 65% and 20% respectively. Complexes 3 and 4 were next tested with iodobenzene and furnished 8 in a low yield of 10% after 15 minutes using 3 whilst 4 again showed no reaction.

After 1 hour, 8 was obtained in a 28% yield using 3; however, no increase in the yield was observed in the case of 4. Full conversion of 3 to 8 was observed after prolonged monitoring to give a maximum yield of 90% whilst 4 remained only partially consumed giving 8 in a moderate yield of 57%.

Turning to substrates containing electron withdrawing functionalities, the reactivity of 3 and 4 were investigated using 4-iodobenzotrifluoride. Complex 3 produced 9 in an 87% yield after less than 15 minutes with no observable increase in the yield after 1 hour. Complex 4 reacted slower producing 9 in yields of 24% and 56% after 15 minutes and 1 hour respectively. Conversion of 4 to 9 reached a maximum yield of 84% after a further 12 hours of reaction time. The tolerance of heteroaromatic substrates was next examined. Thus, complexes 3 and 4 were reacted with 2-iodopyridine to give 10 in yields of 78% and 80% respectively within 15 minutes. Despite the sluggish reactivity of 4 compared to 3 for the majority of substrates, reaction with the electron deficient 2iodopyridine gave a comparable yield. No increase in the yield of 5f was observed over time using 3 or 4. Perhaps counterintuitively, 3/4 appear to react slower than their THFsolvated lithium analogues in metal-halogen exchange reaction. 20 This comparison has to be taken tentatively due to the difference in Lewis bases solvating the different metal centres. Intriguingly, a lower reactivity could potentially benefit selectivity/enantioselectivity which will be studied in due course.

Conclusions

Four novel organometallic complexes starting from the (rac)-BIPHEN ligand have been characterised in both solid (1, 2 and 4) and solution-state (1-4). The heterobimetallic sodium magnesiates 3 and 4 been prepared and utilised in metalhalogen exchange reactions using a range of electron rich and electron poor substrates. Magnesiate 3 generally possesses high levels of reactivity in non-polar solvents particularly with electron deficient substrates; whereas, 4 is largely less reactive. It is possible that this difference in reactivity may be exploited in the future as it is likely that 4 will display higher levels of selectivity due to its lower reactivity. To the best of our knowledge these reactions represent the first examples of metal-halogen exchange utilising sodium magnesiates. Building on these results, the next stage in our research will utilise the enantiomerically pure ligands and apply the resulting magnesiates in asymmetric synthesis.

Sodium magnesiates incorporating the (rac)-BIPHEN ligand have been prepared and characterised and subsequently used in metal-halogen exchange reactions.

General Consideration

All reactions were carried out under a protective atmosphere of argon using stabdard Schlenk techniques. Non-deuterated solvents were dried by refluxing over sodium in the presence of benzophenone under a nitrogen enviroment. Deuterated solvents were degassed using the freeze, pump, thaw method and stored over 4 Å molecular sieves. (rac)-BIPHEN-H 2 , n BuLi and Mg n Bu 2 were purchased from Sigma-Aldrich and used without any further purification.

TMEDA and PMDETA were purchased from Alfa Aesar, dried by refluxing over CaH 2 and stored over 4 Å molecular sieves under a protective atmosphere of nitrogen. Iodobenzene, 4-Iodobenzotrifluoride, 2, 3 and 4-Iodoanisole were obtained from Sigma-Aldrich; and 2-Iodopyridine from Fluorchem. All substrtes were stored over 4 Å molecular sieves and used without any further purification. NMR Spectra were recordeded on a Bruker AVIII 400 MHz spectrometer operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. Single-crystal X-ray diffraction data were collected at 123 K using an Oxford Diffraction Gemini and Xcalibur diffractometers with monochromated Cu (λ= 1.5418 Å) and Mo (λ= 0.71 Å) radition. The structures were solved by direct methods and refined to convegence on F 2 and against all independent reflections by full-matrix least squares and ShelXl programs.

Gerenal Synthesis of n BuNa

NaO t Bu (3.84g, 40 mmol) was suspended in hexane in an argon filled schlenk and cooled to 0 °C. Commercial n BuLi (25 ml, 40 mmol, 1.6 M solution) was added dropwise to give a white suspension. The suspension was allowed to warm to ambient temperature and was stirred for 12 hours. The reaction was then filtered under argon and the white solid washed with hexane (3 x 20 ml). The solid was then dried in vacuo and stored in an argon filled glovebox (typical yield 2.7 g, 84 %).

General Synthesis of MgR 2 (where R = CH 2 SiMe 3 )

Mg turnings (4g) were added to a round bottom flask along with 100 ml of ether. A solution of Me 3 SiCH 2 Cl (19 ml) in ether (50ml) was then added dropwise to the Mg turnings. The solution was then heated to reflux and and stirred for 1 hour. A solution of dioxane (10 ml) in ether (80 ml) was then added dropwise to the Grignard solution to give a vicous grey suspension. The suspension was stirred for 12 hours at ambient temperature. The suspension was then filtered through Celite and glasswool and washed with ether (20 ml). The solvent was removed in vacuo and the resulting solid was purified by sublimation under reduced pressure at 160 °C to give the final product as a white microcrystaline powder. The powder was stored in an argon filled glovebox. All magnesiates were prepared and isolated prior to use in metal-halogen exchange reactions.

Synthesis of [BIPHEN] 2 Na 4 (THF) 4

[BIPHEN] 2 Na 4 (THF) 4 was prepared by the addition of a solution of (rac)-BIPHEN-H2 (0.177g, 0.5 mmol) in hexane (2 ml) to a suspension of freshly prepared n BuNa (0.08 g, 1 mmol) in hexane (5 ml) at -10 °C. The suspension was stirred for 1 hour with slow warming to room temperature. The solvent was removed in vacuo and THF (3 ml) was added to give a colourless solution. The solution was subsequently stored at -33 °C to yield colourless crystals of [BIPHEN] 2 Na 4 (THF) 4 (yield 0.145 g, 26 %). 1 [BIPHEN] 2 Na 2 Mg(THF) 4 was prepared by the addition of a solution of (rac)-BIPHEN-H2 (0.177g, 0.5 mmol) in hexane (2 ml) to a suspension of freshly prepared n BuNa (0.08 g, 1 mmol) in hexane (5 ml) at -10 °C. The suspension was stirred for 30 minutes after which commercial Mg n Bu 2 (0.5 ml, 0.5 mmol, 1 M solution) was added to create a white suspension. The suspension was allowed to warm to ambient temperature and THF was added dropwise to give a pale yellow solution. The solution was concentrated in vacuo and the solution stored at -33 °C to give [BIPHEN] 2 Na 2 Mg(THF) 4 as colourless crystals (yield 0.197 g, 37%).

[BIPHEN] 2 Na 2 Mg(THF) 4 can also be prepared by using Mg(CH 2 SiMe 3 ) as a replacement for [(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2 was prepared by the addition of a solution of (rac)-BIPHEN-H2 (0.177g, 0.5 mmol) in hexane (2 ml) to a suspension of freshly prepared n BuNa (0.08 g, 1 mmol) in hexane (5 ml) at -10 °C. The suspension was stirred until a colourless solution was formed after which commercial Mg n Bu 2 (0.5 ml, 0.5 mmol, 1 M solution) was added to create a white suspension. The suspension was stirred at -10 °C for 5 minutes after which time TMEDA (0.15 ml, 1 mmol) was added to give a pale yellow solution. The solution was concentrated in vacuo and the solution stored at -33 °C. Affter storage at sub-ambient temperature the soluton was filtered to yield [(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2 as a microcrystaline powder (yield 0.216 g, 56 %). 1

Synthesis of [(rac)-BIPHEN]Na 2 Mg n Bu 2 (PMDETA) 2

[(rac)-BIPHEN]Na 2 Mg n Bu 2 (PMDETA) 2 was prepared by the addition of a solution of (rac)-BIPHEN-H2 (0.177g, 0.5 mmol) in hexane (2 ml) to a suspension of freshly prepared n BuNa (0.08 g, 1 mmol) in hexane (5 ml) at -10 °C. The suspension was stirred until a colourless solution was formed after which commercial Mg n Bu 2 (0.5 ml, 0.5 mmol, 1 M solution) was added to create a white suspension. The suspension was stirred at -10 °C for 5 minutes after which time PMDETA (0.21 ml, 1 mmol) was added to give a white suspension. The suspension was concentrated in vacuo then heated gently to give a colourless solution. The solution was then allowed to cool slowly to ambient temperature to give colouless crystals of [(rac)-BIPHEN]Na 2 Mg n Bu 2 (PMDETA) 2 (yield 0.245 g, 56 %). 1

General Procedure for NMR Scale Metal-Halogen Exchange Reactions using 3

All reactions were carried out on an NMR scale using a 2:1 ratio of substrate to magnesiate.

[(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2 (0.029 g, 0.0377 mmol) was combined with tetraphenylnapthalene (0.010 g, 0.025 mmol) as an internal standard and 0.5 ml of d 8 -toluene in a J Young's NMR tube. The desired substrate (0.0754 mmol) was then added and the reation monitored at regular intervals by 1 H NMR spectroscopy and yields obtained were calculated from NMR spectroscopic integrals relative to the internal standard.

General Procedure for NMR Scale Metal-Halogen Exchange Reactions using 4

All reactions were carried out on an NMR scale using a 2:1 ratio of substrate to magnesiate.

[(rac)-BIPHEN]Na 2 Mg n Bu 2 (PMDETA) 2 (0.033 g, 0.0377 mmol) was combined with tetraphenylnapthalene (0.010 g, 0.025 mmol) (5a-c, e and f) or hexamethylcyclotrisiloxane (0.011 g, 0.050 mmol) (5d) as internal standards and 0.5 ml of d 8 -toluene in a J Young's NMR tube. The desired substrate (0.0754 mmol) was then added and the reation monitored at regular intervals by 1 H NMR spectroscopy and yields obtained were calculated from NMR spectroscopic integrals relative to the internal standard.

Crystalographic and Refinement data for Compounds 1, 2 and 4

Data for all compounds were measured at low temperature using Oxford Diffraction instruments. Refinement was to convergence and against F 2 using all unique reflections. 1 Structure 2 contained a region of disordered solvent thatcould not be adequately modelled.

The contribution of this feature to the structure was removed using the SQUEEZE routine within the PLATON program suite. 2 A total of 79 electron equivalents was removed form approximately 248 Å 3 of space. This corresponds to approximately 2 THF molecules per unit cell. All three structures include moieties that have been modelled as disordered over two sites. For 1 and 2 some THF ligands have been treated in this way and for

  a

Scheme 1 :

 1 Scheme 1: Preparation of sodium dialkylmagnesiates.

Figure 1 :

 1 Figure 1: Solid state structure of [BIPHEN] 2 Na 4 (THF) 4 .0.4(THF) 1. Hydrogen atoms and disorder in THF molecules are omitted for clarity. Thermal ellipsoids are set to 50 % probability level. Key bond distances (Å) and bond angles (°): Na(1)-O(1), 2.217(2); Na(1)-O(1´), 2.217(2); Na(1)-O(2), 2.313(2); Na(1)-O(2´), 2.313(2); Na(2)-O(1), 2.199(2); Na(2)-O(1´), 2.199(2); Na(2)-O(3), 2.308(3); Na(2)-O(3´), 2.308(3); Na(3)-O(2), 2.265(2); Na(3)-O(2´), 2.298(2); Na(3)-O(4), 2.298(3); Na(3)-C(4), 2.878(3); Na(3)-C(5), 2.808(3); Na(3´)-O(2), 2.298(2); Na(3´)-O(2´), 2.265(2); Na(3´)-O(4´), 2.298(3); O(1)-Na(1)-O(1´), 92.49(11); O(1)-Na(1)-O(2), 110.87(7); O(2)-Na(1)-O(2´), 78.01(10); O(1)-Na(2)-O(1´), 93.45(12), O(3)-Na(2)-O(3´), 103.62(14); O(2)-Na(3)-O(2´), 79.27(8); O(2)-Na(3´)-O(2´), 79.27(8).

  1°) and is bound to all four oxygen atoms of both BIPHEN ligands [range of Na-O bond distances, 2.215(3) -2.315(3) Å]. Na2 is also four coordinate with a distorted tetrahedral geometry (sum of bond angles, 661.1°) and is bound to one oxygen atom from each BIPHEN ligand [Na2-O1 2.200(3) Å]. The coordination sphere of Na2 is complete by coordination to two molecules of THF [Na2-O3 2.310(4) Å]. The other sodium sites Na3/Na3´ each make short contacts with 5 atoms. Each such Na centre coordinates to three O atoms [one from each BIPHEN ligand and one from a THF molecule]. The remaining contacts are η 2 -π-interactions to an adjacent phenyl group [Na3-C4 and Na3-C5 bond distances, 2.878(3) and 2.808(3) Å respectively].

Figure 2 :

 2 Figure 2: Solid-state structure of [BIPHEN] 2 MgNa 2 (THF) 4 2. Hydrogen atoms and disorder in THF molecules are omitted for clarity. Thermal ellipsoids are set to 50 % probability level. Key bond distances (Å) and bond angles (°): Mg(1)-O(1), 1.960(16); Mg(1)-Na(1), 3.0914(14); Mg(1)-Na(1´), 3.0913(14); Na(1)-O(1), 2.2372(19); Na(1)-O(2), 2.270(7); O(1)-Mg(1)-O(1´), 102.72(9); O(1)-Mg(1)-O(1´´), 92.32(9); O(1)-Mg(1)-O(1´´´), 137.67(10); Na(1)-Mg(1)-Na(1´), 180; O(1)-Na(1)-O(1´´), 78.38(38); O(2)-Na(1)-O(2´´), 101.5(2).

Figure 3 :

 3 Figure 3: Solid state structure of [(rac)-BIPHEN]Na 2 MgBu 2 (PMDETA) 2 4. Hydrogen atoms and disorder in PMDETA molecules are omitted for clarity. Thermal ellipsoids are set to 50 % probability level. Key bond distances (Å) and bond angles (°): Mg(1)-O(1), 2.011(3); Mg(1)-O(2), 2.009(3); Mg(1)-C(43), 2.203(6); Mg(1)-C(44), 2.204(5); Na(1)-O(1), 2.279(3); Na(1)-N(1), 2.614(4); Na(1)-N(2), 2.558(4); Na(1)-N(3), 2.520(4); Na(2)-O(2), 2.267(3); Na(2)-N(4), 2.492(5); Na(2)-N(6), 2.618(4); Na(1)-C(44), 2.690(5); Na(2)-C(43), 2.688(6); O(1)-Mg(1)-O(2), 99.35(13), C(43)-Mg(1)-C(44), 107.7(2); Na(1)-C(44)-Mg(1), 79.3(2); Na(2)-C(43)-Mg(1), 79.1(2); O(1)-Na(1)-C(44), 82.0(1); O(2)-Na(2)-C(43), 91.9(3).

Scheme 2 :

 2 Scheme 2: Simplified Schlenk equilibrium demonstrating the formation of 2.

MgBu 2 . 1 H

 21 NMR (400.13 MHz, 298 K, d 8 -THF): δ 1.15 (36H, bs, C(CH 3 ) 3 ), 1.45 (12H, s, CH 3 ), 1.77 (14H, m, OCH 2 CH 2 , THF), 2.04 (12H, s, CH 3 ), 3.61 (10H, m, OCH 2 CH 2 , THF), 6.72 (4H, s, Ph). 13 C NMR (100.62 MHz, 298 K, d 8 -THF): δ 16.90 (CH 3 ), 20.43 (CH 3 ), 26.18 (THF), 31.05 (C(CH 3 ) 3 ), 34.62 (C(CH 3 ) 3 ), 68.02 (THF), 120.70, 125.82, 132.69, 132.89, 134.21, 161.31 (Ph) Synthesis of [(rac)-BIPHEN]Na 2 Mg n Bu 2 (TMEDA) 2

Variable Temperature Stability Studies of 3 and 4 S10: 1 HTable 3 , product 9 using 3 S18:

 4133 NMR Spectrum of [(rac)-BIPHEN]Na 2 MgBu 2 (TMEDA) 2 in C 6 D 6 after heating to 50 °C for 12 hours S11: 1 H NMR Spectrum of [(rac)-BIPHEN]Na 2 MgBu 2 (TMEDA) 2 in d 8 -THF after heating to 50 °C for 12 hours Metal-Halogen Exchange reactions Table 3, product 5 using 3 S14: Product 5 in d 8 -Tol with 1,2,3,4-Tetraphenylnapthalene as internal standard, yield 90% (25 °C, 18 h) Product 9 in d 8 -Tol with 1,2,3,4-Tetraphenylnapthalene and ferrocene as internal standard, yield 87% (25 °C, 15 min) Table 3, product 10 using 4 S25: Product 10 in d 8 -Tol with 1,2,3,4-Tetraphenylnapthalene as internal standard, yield 80% (25 °C, 15 min)

  . WestCHEM, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, UK. b. Université de Lorraine, CNRS, L2CM, F-54000 Nancy, France.

c. Supramolecular Organometallic Chemistry Laboratory CIQSO-Center for Research in Sustainable Chemistry & Department of Chemistry University of Huelva Campus El Camen, 21007 Huelva, Spain † Dedicated to Professor Robert E. Mulvey, a fantastic mentor and colleague, on the occasion of his 60 th birthday. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x ARTICLE Journal Name 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Table 1 :

 1 Key 1 H NMR chemical shifts for 3 and 4 in C 6 D 6 at 300K. Mg-CH 2 H atoms which appear as one broad multiplet at -0.75 ppm, instead of two distinct multiplets. A low temperature NMR spectroscopic study failed to separate this multiplet (see Supporting Information). The data for 3 and 4 are consistent with values reported in literature for the related lithium magnesiates.

		1 H Chemical Shift (ppm)
		3	4
	Ar	7.18	7.14
	t Bu	1.70	1.77
	Me	1.80, 2.23	1.90, 2.31
	Mg-CH 2	-0.78, -0.31	-0.75
	TMEDA	1.79, 1.86	-
	PMDETA	-	1.82, 1.86, 1.93

Table 2 :

 2 Key 13 C NMR chemical shifts for 3 and 4 in C 6 D 6 at 300K.

Synthesis of [BIPHEN] 2 Na 2 Mg(THF) 4

  H NMR (400.13 MHz, 298 K, d 8 -THF): δ 1.30 (36H, bs, C(CH 3 ) 3 ), 1.56 (12H, bs, CH 3 ), 1.73 (10H, m, OCH 2 CH 2 , THF), 2.07 (12H, bs, CH 3 ), 3.58 (10H, m, OCH 2 CH 2 , THF), 6.69 (4H, s, Ph). 13 C NMR (100.62 MHz, 298 K, d 8 -THF): δ 16.71 (CH 3 ), 20.34 (CH 3 ), 26.15 (THF), 30.27 (C(CH 3 ) 3 ), 34.75 (C(CH 3 ) 3 ), 68.00 (THF), 116.19, 125.91, 132.40,

	133.12, 133.70, 165.12 (Ph)

  H NMR (400.13 MHz, 298 K, C 6 D 6 ): δ -0.79 (2H, m, MgCH 2 ), -0.314 (2H, m, MgCH 2 ), 1.25 (6H, t, CH 3 ), 1.69 (9H, bs, C(CH 3 ) 3 ), 1.79 (8H, bs, CH 2 , TMEDA), 1.80 (6H, s, CH 3 ), 1.85 (33H, bs, CH 3 TMEDA and C(CH 3 ) 3 ), 2.22 (6H, s, CH 3 ), 7.18 (2H, s, Ph). 13 C NMR (100.62 MHz, 298 K, C 6 D 6 ): δ 8.84 (2 x MgCH 2 ), 14.90 (2 x CH 3 , Butyl), 17.48 (2 x CH 3 , (rac)-BIPHEN), 20.77 (2 x CH 3 , (rac)-BIPHEN), 31.70 (C(CH 3 ) 3 , (rac)-BIPHEN), 32.81-33.74 (CH 2 , Butyl), 35.34 (C(CH 3 ) 3 , (rac)-BIPHEN), 45.53 (TMEDA), 56.99 (TMEDA), 119.61, 127.26, 132.10, 132.74, 135.90, 162.71 (Ph).

  Ph).13 C NMR (100.62 MHz, 298 K, C 6 D 6 ): δ 8.78 (2 x MgCH 2 ), 14.87 (2 x CH 3 , Butyl), 17.95 (2 x CH 3 , (rac)-BIPHEN), 20.81 (2 x CH 3 , (rac)-BIPHEN), 32.25 (C(CH 3 ) 3 , (rac)-BIPHEN), 33.53 (CH 2 , Butyl), 34.00 (CH 2 , Butyl), 35.45 (C(CH 3 ) 3 , (rac)-BIPHEN), 44.16 (PMDETA), 45.59 (PMDETA), 54.09 (PMDETA), 57.12 (PMDETA), 119.85, 126.58, 132.62, 134.11, 134.80, 163.06 (Ph).

	H NMR (400.13 MHz, 298 K, C 6 D 6 ): δ -
	0.75 (4H, m, MgCH 2 ), 1.26 (6H, t, CH 3 ), 1.77 (18H, bs, C(CH 3 ) 3 ), 1.82 (16H, bs, CH 2 , PMDETA),
	1.86 (6H, s, CH 3 , PMDETA), 1.90 (6H, s, CH 3 ), 1.93 (24H, bs, CH 3 , PMDETA), 2.31 (6H, s, CH 3 ),
	7.14 (2H, s,

Table S1 :

 S1 3 one butyl group is disordered. Selected structural and refinement data are tabulated below and full details are available in cif format via the CCDC, deposition numbers 1988061 to 1988063. Formula C 65.6 H 99. Na 4 O 8.4 C 64 H 96 MgNa 2 O 8 C 50 H 96 MgN 6 Na 2 O 2 Comparison of key bond lengths and bond angles between (1) and (BIPHEN) 2 Li 4 (THF) 4 [where Am = Na for 1 and Am = Li for (BIPHEN) 2 Li 4 (THF) 4 ]. (BIPHEN) 2 Na 4 (THF) 4 (BIPHEN) 2 Li 4 (THF) 4

	No.
				404	192	583
	Parameters
	2θmax			146.36	54.00	146.41
	Rint			0.0202	0.0234	0.0506
	Goodness of Fit Bond Lengths (Å)		1.074	1.043	1.038
	R[on F, obs refs AM(1)-O(1)		0.0453	0.0426 2.217(2)	0.0713 1.879(3)
	only]
	wR (on F 2 , all AM(1)-O(2)			2.313(2)	2.070(3)
	data)	AM(2)-O(1)		0.1365	0.1128 2.199(2)	0.1896 1.875(3)
	Largest diff. AM(3)-O(2)			2.265(2)	1.901(3)
	peak/hole / e Å -3 Bond Angles (°)		0.52/-0.27	0.21/-0.19	1.09/-0.28
	Compound O(1)-AM(1)-O(2) Flack Parameter O(1)-AM(1)-O(1´)	1 0.018(14)	2 -0.16(17) 110.87(7) 92.49(11)	4 133.04(5) 0.07(4) 92.27(17)
	FW			1114.20	1063.69	883.61
	Crystal System O(2)-AM(1)-O(2´)	Tetragonal	Orthorhombic 78.01(10)	Monoclinic 76.46(13)
	Space Group O(1)-AM(2)-O(1´)	P4 1 2 1 2	I222 93.45(12)	P n 92.50(18)
	Wavelengh/Å O(2)-AM(3)-O(2´)	1.54184	0.71073 79.27(8)	1.54184 84.53(12)
	a/Å	13.33310(10)	11.3684(11)	12.9924(3)
	b/Å	13.33310(10)	15.6863(16)	14.0108(3)
	c/Å			37.0015(5)	18.5667(12)	15.4464(5)
	β/°90	90	100.040(3)
	Volume/Å3	6577.81(13)	3311.0(5)	2768.71(13)
	Z			4	2	2
	Temperature/K		123(2)	156(2)	123(2)
	Reflections
				25584	7323	27167
	Collected
	Reflections
				6448	3269	8631
	Unique
	Reflections
				6182	2829	7768
	Observed

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

Acknowledgements

We gratefully acknowledge the Leverhulme Trust (research grant RPG-2016-281 to CTOH) and the Royal Society (International Exchange grant IEC\R2\170207 to CTOH and ST) for the generous sponsorship of this research. The data used within this publication can be accessed at xxxxx. The X-ray data have been deposited with the CCDC and are available from the data centre on quoting reference numbers 1988061-1988063.

ARTICLE

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Conflicts of interest

There are no conflicts to declare.

Structural and metal-halogen exchange reactivity studies of sodium magnesiate biphenolate complexes