Professor Gérard Jaouen

Ian S Butler

M J Mcglinchey

In 1982 she helped to set up the Organometallic Chemistry Laboratory at the ENSCP. Her two

Ferrocifen Type Anti Cancer Drugs

Email : gerard.jaouen@chimie-paristech.fr to the memory of Professor Lord (Jack) Lewis, an inspiring mentor Abstract: Despite current developments in therapeutics focusing on biotechnologicallyoriented species, the unflagging utility of small molecules or peptides in medicine is still producing strong results. In 2014 for example, of the 41 new medicines authorized for sale, 33 belonged to the category of small molecules, while in 2013 they represented 24 of 27, according to the FDA. This can be explained as the result of recent forays into new or longneglected areas of chemistry. Medicinal organometallic chemistry can provide us with an antimalarial against resistant parasitic strains, as attested by the phase II clinical development of ferroquine, with a new framework for conceptual advances based on three-dimensional space-filling, and with redox or indeed catalytic intracellular properties. In this context, bioferrocene species with antiproliferative potential have for several years been the subject of sustained effort, based on some initial successes and on the nature of ferrocene as a stable aromatic, with low toxicity, low cost, and possessing reversible redox properties. We show here the different antitumoral approaches offered by ferrocifen derivatives, originally simple derivatives of tamoxifen, which over the course of their development have proved to possess remarkable structural and mechanistic diversity. These entities act via various targets, some of which have been identified, that are triggered according to the concentration of the products.

They also act according to the nature of the cancer cells and their functionality, by mechanistic pathways that can operate either synergistically or not, in successive, concomitant or sequential ways, depending for example on newly identified signaling pathways inducing senescence or apoptosis. Here we present a first attempt to rationalize the behavior of these entities with various anticancer targets.

Introduction, Contextualizing the Problem

We are now witnessing an explosion of cases of cancer, a disease whose multiform nature makes it very difficult to treat. We have reached the stage where we may be said to have entered "the Age of Cancer" 1 with 6.5 million cancer deaths in 2003, reaching 12 million in 2013, and predictions for 2030 of 22 million deaths, 35 million by 2050. It is vital to give the lie to these dark predictions. The numbers reflect an increase that is statistically greater than the increase in population of the planet.

In parallel with this explosion of the disease, major innovations in the therapeutic approach to cancers, complementing established methods such as surgery and ionizing radiation, have appeared in the last decade. Progress has been made for example in improved targeting of cancerous sites, whether by the use of biodegradable nanoparticles, dendrimers, monoclonal antibodies, peptides or saccharides bound to appropriate ligands or indeed the use of microfluidics. [START_REF] Alison | The Cancer Handbook[END_REF][START_REF] Bower | Lecture Notes: Oncology[END_REF] All this contributes to the progress of chemotherapy and hormone therapy.

But the improvements are also driven by a flood of biological information supporting the understanding of cancers and permitting greater personalization of treatment. Chemistry still often plays a central role, however, in bringing these new approaches to fruition.

It must nevertheless be borne in mind that the cost of these new highly targeted treatments is a pressing problem, placing a heavy burden on the health budgets of the majority of developed and developing countries. Herceptin, Mabthera, Avastin, flagship products of Roche, earned 21 billion dollars in 2013. 1 Within the current progression, Kadcyla looks poised to enter the same territory. These drugs exemplify the biopharmaceutical approach (selective monoclonal antibodies for well-targeted cancers, but at exorbitant public cost).

If we hope to find new active principles while holding down costs, we need to look in the direction of innovative chemotherapies, ideally outside the well-worn paths of traditional chemistry. Interestingly, these days, the traditional disciplines of chemistry appear to be undergoing a profound and rapid evolution, for example in chemical biology, in their drive to explore new directions beyond the well traveled pathways, and to build structures of increasing complexity and diversity. Molecular chemistry is no exception to this fundamental trend.

In particular, we are currently perceiving a scientific interest in a diversity of structures produced by inorganic syntheses. [START_REF] Barry | [END_REF] This approach has already broken through into the domain of healthcare, whether in therapy, thanks for instance to the importance of Pt coordination metallodrugs or in diagnostic imaging, with MRI contrast agents based on Gd, or indeed in radiopharmaceuticals designed around the radionucleide 99m Tc, to give only a few examples.

In this context the value of platinum coordination complexes in oncology is well known with three of these complexes (vide infra) being used, alone or in combination, in 70% of cancer treatments. 5 This is despite the well-documented disadvantages of these DNA alkylating agents; their use is in fact sometimes only the result of a lack of alternatives. The coordination complexes of Pt induce cell death primarily by apoptosis thus they are ineffective in the treatment of tumor cells resistant to this pathway. This is often the case with cancers that are difficult to treat and that have poor outcomes. Considering these gaps in the therapeutic arsenal a coordination chemistry of Ru has been explored and some promising molecules such as NAMI-A or KP1019 have been obtained (Chart 1). 6 medicinal organometallic chemistry may provide a key, thanks to the novel behavior of these molecules. [START_REF] Barry | [END_REF]6 The primary difference between a coordination complex of the cisplatin type and an organometallic compound is in their chemical binding mechanism, resulting in different chemistries, depending on the category to which the complex belongs. The metallodrugs of Pt require aquation to activate them and often have DNA as their primary target which is probably not exclusive. 5 Whereas the organometallics, which tend to be more lipophilic and have a carbon-metal (C-M) covalent bond, function differently on protein targets. This complementarity may prove to be a valuable asset. Of added interest is that the identification of multiple targets within cancerous cells may support the synthesis of multiplerecognition drugs, with the objective of overcoming resistance to apoptosis by inducing cell growth arrest via another pathway, senescence for example. [START_REF] Hearn | Proc. Natl. Acad. Sci. U S A[END_REF] The arrival of organometallic frameworks constructed by design, with a precise biological endpoint in mind, was somewhat delayed, owing to an emphasis on catalysis which for some years had the effect of relegating other aspects of the field to obscurity. This situation changed with the arrival of bioorganometallics. [START_REF] Jaouen | [END_REF][START_REF] Jaouen | [END_REF] It is now clear that an innovative organometallic medicinal chemistry is supported by the distinctive properties of these complexes. Among the most studied series of organometallic anticancer drug candidates are the lead molecules shown below (Chart 2). [START_REF] Barry | [END_REF]6,[START_REF] Jaouen | [END_REF]10 Chart 2 : Selected examples of organometallic anticancer drug candidates These molecules and related species have been conceived for a specific purpose. The social challenge represented by those cancers that still have alarmingly poor outcomes (e.g. glioblastoma, melanoma, ovarian, pancreatic and triple negative breast cancers) [START_REF] Hearn | Proc. Natl. Acad. Sci. U S A[END_REF] can only be met by a capacity for medical innovation, drawing from a judicious alliance of ground breaking chemistry, novel biology and the latest tools of medical intervention. These cancers share the characteristic of intrinsic resistance to pro-apoptotic stimuli, which limits the effectiveness of the available chemotherapies, hence the need to discover new therapeutic approaches able to induce cell death or senescence by other means. In this endeavor new medicinal chemistry based on the unique behavior of organometallic complexes of transition metals may provide a turning point.

Selection of antiproliferative bioferrocene compounds with varied structures

In the search for medically-interesting organometallic molecules with novel mechanistic properties, various metals, many of them rare, have been considered for the central position in the complex. These include, amongst others, Au, Ru, Os, Re, Tc, Rh, Ir, Ti. 4, 6, 7, 11-19 Within this framework a number of complexes have been the subject of in-depth mechanistic studies. [START_REF] Barry | [END_REF]6,18 One series of organometallic complexes with particularly intense activity is that of the N-heterocyclic carbene complexes, particularly those of gold. [START_REF] Cinellu | Bioorganometallic Chemistry[END_REF] This research stems from the discovery of these heterocyclic ligands by Lappert starting in 1973.

One could probably attribute the first biological application of these complexes, using this type of carbene ligand with Ru and Rh as antibacterials, to former collaborators of Lappert, the Cetinkayas, in 1996. 20 This pioneering work was not followed up at the time as it was eclipsed by the second generation of Grubbs metathesis catalysts, which appeared in 1999.

Here we can see a possible link between bioorganometallics and homogeneous catalysis in terms of the nature of the complexes that can be used. This has just been brilliantly confirmed by Sadler et al. who have shown that a Noyori-type hydrogenation catalysis can be carried out in cancer cells to provide a new mechanism of anticancer activity. 21 Of the transition metals that are more abundant than those cited in the previous paragraph, iron, especially in the form of ferrocene, a compact metallocene possessing stability in non-oxidating media, low toxicity, and reversible redox behaviour, has recently played an important role in bioorganometallics, as an antiparasitic or an antibacterial, [START_REF] Dubar | Bioorganometallic Chemistry[END_REF][START_REF] Arbi | [END_REF][24] and indeed as an antitumoral agent. The latter aspect is amply illustrated in recent publications. 25- 27 Some examples of the vast and regularly increasing number of bioferrocene compounds with antitumoral potential are shown in Chart 3 below. They illustrate the richness of the activity in this field, the variety of the structures brought into play and the diversity of possible angles of attack. This bodes well for the variety of mechanisms involved even if only a minority of them has so far been reliably elucidated. The ferrocifens 1a-b (Fc-OH-Tam and Fc-diOH) derived from hydroxytamoxifen (OH-Tam) are among the earliest organometallic SERMs (selective estrogen receptor modulators) known since they were described for the first time in 1996. [START_REF] Top | [END_REF][29][30] They possess the unusual feature of being antiproliferative on both hormone-dependent (MCF-7) and hormoneindependent (MDA-MB-231) breast cancer cells (IC 50 of Fc-OH-Tam : 0.8 and 0.5 µM respectively) breast cancer cells, with good efficacies. Such a dual effect is remarkable since the corresponding purely organic form shows antiestrogenic activity only on hormonedependent MCF-7 breast cancer cells. This reveals the significance of the ferrocenyl (Fc) unit and has led to many further studies (vide infra).

In particular, other ferrocene SERMs have since been brought to light. An example is 2 based on a three-dimensional oxabicyclo[2.2.1]heptene scaffold. 31 These entities, like 1, recognize both ERα and its isoform ERβ. The species in this series are cytotoxic at micromolar concentrations (IC 50 ≈ 7.5 µM) on both ER+ and ER-breast cancer cells.

Molecular modeling studies suggest that these scaffolds bind to the ER receptor in a particular way. Studies of these new structures are encouraging.

Following a parallel approach to that used with the ferrocifens, another SERM, derived from raloxifen, 3, was prepared. 32 This compound reveals cytotoxic activity on a number of cancer cell lines (breast, ovary, cervix, lung, colon) with IC 50 values in the low micromolar range. This compound activates a caspase-3 in ovarian cells.

Another series similar to the type 1, that of the ansa ferrocenophanes such as 4 (ansa-Fc-diOH) has been reported. 33 It is significantly more active than the acyclic series 1 with IC 50 values on MDA-MB-231 cells in the order of 0.09 µM.

Unlike the compounds mentioned above (1 -4) which are SERMs that recognize and modulate estrogen receptors but which additionally show another type of cytotoxic activity against cancer cells, the ferrocenyl derivative of the antiandrogen nilutamide 5 does not show significant recognition of the androgen receptor and thus is not a SARM (Selective Androgen Receptor Modulator). 34 However it shows significant anticancer activity against PC-3 hormone-independent prostate cancer cells (IC 50 = 4 µM).

A number of natural compounds modified by a ferrocenyl unit are grouped together (6 -10). They illustrate what can be expected from this type of modification. Several ferrocenyl taxoids such as 6 were prepared by acylation of paclitaxel and docetaxel with ferrocenyl carboxylic acids. The compounds obtained, such as 6, showed good activity against cell lines of multiresistant adenocarcinomas of the colon. The mechanisms of action of these species are currently under study. 35 Several studies on ferrocenyl flavonoids have recently been carried out. A representative example is the dibrominated ferrocenyl flavone 7. This type of flavone shows fairly low cytotoxicity on B16 murine melanoma cells but acts as an excellent antivascular agent. 36 This antiangiogenic effect is interesting and merits further study. Related series such as ferrocenyl pterocarpene 8 and coumestan give IC 50 values between 20 and 45 µM against various cancer cell lines while some members of the group are genotoxic. 37 Curcumin is employed in the Asian diet for its medicinal properties. An example of its modification by ferrocen can be seen with 9. 38 The spacer chain between the curcumin skeleton and the ferrocenyl group influences the biological results both in terms of the IC 50 values on B16 murine cells (IC 50 = 2.2 -7.1 µM) and in the inhibition of tubulin polymerization and rounding up of endothelial cells. This reflects the role of the organometallic on the modification of the biological activities of the curcuminoids.

The extreme toxicity of illudin, produced by mushrooms, does not permit chemical application in its natural state. A modification using ferrocene reduces its toxicity on fibroblasts and increases its selectivity on cancer cells with species such as 10. 39 Heterocyclic skeletons bearing a ferrocenyl substituent also present some interesting behaviors. For example with the bioorganometallic oxindole, 11, kinase inhibition is obtained at submicromolar concentrations. 40 There are also ferrocenyl-intercalator conjugates targeting DNA, such as acridine, with elevated cytotoxicity. This is the case for example with compound 12 which has IC 50 values in the order of 1 µM on several cancer cell lines (KB, Hep, Hela, Colo-206). 41 Ferrocenyl alkyl nucleobases, as for example 13 with thymine, were easily synthesized. 42 The antitumoral activity of 13 with models of solid tumors, for example with Ca755 (murine mammary carcinoma) and LLC (lung mouse carcinoma) were studied in vivo.

A therapeutic synergism exists between 13 and the anticancer drug cyclophosphamide, shown by a 70% inhibition of tumor growth. Other analogs of ferrocenyl nucleosides showing apoptosis-inducing activity were obtained via a very elegant enantioselective synthesis by Schmalz et al. 43 A promising example of a diastereomer treating ex vivo primary lymphoblasts isolated from children is 14 which appears to make it possible to overcome resistance. Other interesting organometallic nucleoside analogs with ferrocenyl linkers have recently been published. 44 Azolactone ferrocene 15 is a potential inhibitor of the catalytic activity of the topoisomerase IIβ (IC 50 = 100 µM) by cleavable complex formation, while a related thiomorpholide amido methyl ferrocene competes with ATP binding (IC 50 = 50 µM). 45 Balaji et al. recently published a novel study on mitochondrial targeting of photocytotoxic compounds of ferrocenyl conjugates with salts of N-alkylpyridinium such as the one shown in 16. 46 This type of conjugate, 16, reveals photocytotoxicity in HeLa cancer cells (IC 50 = 1.3 µM) that is markedly higher than that found in normal 3T µM for the organic hybrid. The estrogen receptor ERα and histone deacetylase (HDAC) are not the principal targets of this hybrid. 47 The literature also reveals several ferrocenyl conjugates of peptides, as for example the derivative of naphthyl 18. 48 In this series there is a wide variety of peptide entities. For example 18 gives an IC 50 value of 0.14 µM on the H 1 299NSCLC cell line, and IC 50 = 1.06 µM for the sk-MCI-28 malignant melanoma cell line. This type of compound induces apoptosis through oxidative damage via a redox mechanism by ROS.

Sugars are also playing their part, as attested by 19, carbohydrate ferrocene conjugates with amide-triazole linkers. 49 Compounds derived from xylose and ribose show a cytotoxic effect on hormone-dependent (MCF-7, IC 50 = 3.18 µM) and hormone-independent (MDA-MB-231, IC 50 = 7.31 µM) cancer cells lines, while conjugates derived from glucose and galactose do not.

The synthesis of N-benzyl-substituted aminoferrocene-based prodrugs has recently been improved, 20, allowing studies of its antiproliferative effect. Interestingly complex 20 is highly cytotoxic on cell lines with different p53 status including those with mutated p53 which are associated with cancers with poor prognosis. Product 20 is not toxic in vivo and is beneficial on BDF1 mice with L1210 leukemia, with survival increasing from 13.7 to 17.5 days at doses of 26 µg/kg. 50 This overview reveals the number and diversity of initiatives being explored using conjugates of ferrocene. Given that many biological studies both in vitro and in vivo have focused on the ferrocifens, where we also have more mechanistic data, this topic will now be covered in greater detail.

The ferrocifens and other members of the metallocifen family

The ferrocifen family is so-called because the first examples in the series were ferrocenyl analogues of hydroxytamoxifen (series A ; Chart 4). The interest in this particular set of complexes prompted an SAR study aimed, on the one hand, towards finding more active molecules and, on the other hand as a route to understanding their mechanism of action. This family is comprised of about 200 complexes that can be placed in six groups. Series C and D have in common the skeletal motif phenyl-but-1-ene-ferrocene in either the open or ansa series. This SAR study led to the extension to complexes of other metallocenes (series B), with two sidechains (series G), with a ferrocenyl unit attached to the sidechain (series E), and more recently to hybrid SAHA-ferrocifens (series F) and complexes with modified alkyl chain (series H).

OH O(CH 2 ) n N(CH 3 ) 2

Series A ferrocenyl analogs of OH-Tam (n= 2-5, 8)

Series B

OH-Tam derivatives of other metals : Ru, Re,

Os (R = O(CH 2 ) n N(CH 3 ) 2 , OH) Fe R 1 R 2 Fe R 1 R 2 Series D ansa ferrocenyl analogs (R 1 , R 2 = H, OH, NH 2 ) OH R OM Series C R 1 , R 2 = H, OH, NH 2 R 1 O(CH 2 ) n Fc R 2 Series E ferrocenyl side chain (R 1 , R 2 = H, OH) N H O R O Fe Series F Ferrocifen SAHA hybrids (R = NHOH, NH 2 ) R O(CH 2 ) 3 N(CH 3 ) 2 O(CH 2 ) 3 N(CH 3 ) 2
Series G 2 amino side chains, R = Fc, ansa-Fc, Ph

Synthesis of complexes with a hydroxy-phenyl-but-1-ene skeleton

Two synthetic routes have been developed for access to these molecules. The first method (Scheme 1) involves addition of an organolithium reagent (MeOC 6 H 4 Li) to an ester (2-ethyl-2 ferrocenyl acetate) to give the dimethylether derivative, followed by its demethylation using BBr 3 which furnishes the ferrocenyl diphenol (Fc-diOH, 2). [START_REF] Top | [END_REF] Scheme 1 : First pathway of synthesis of Fc-diOH One of the inconveniences of this method is the necessity to protect and then deprotect functional groups that react with lithium reagents. However, this problem can be overcome by taking advantage of the McMurry coupling of two different ketones 51 whereby Fc-mono and diOH can be obtained in good yield (Scheme 2, 53% for Fc-diOH). 52 The benefit of this method is its tolerance to many functional groups, and the majority of the ferrocifen family including the ansa series have been prepared by this method. E/Z mixture, and the disubstituted system is a minor product. [START_REF] Top | [END_REF] In the other cases, (n = 4, 5, 8)

the synthesis (route B) begins by alkylation of the dihydroxybenzophenone using the corresponding dichloroalkane [Cl(CH 2 ) n Cl ; n = 4, 5, 8] that yields both the mono and dialkylated ketones. Now, McMurry coupling with propionylferrocene gives the halogenated complexes which, in the final step, react with dimethylamine hydrochloride in an autoclave to furnish the required products. 29,30 Scheme 3 : Synthetic routes to ferrocifens with amino side chains of various length

In certain cases, it is possible to separate the Z and E isomers ; however, in protic media isomerisation to an approximately 50/50 mixture is rapid, consequently it is the mixture that has been used in the biological studies. These complexes have a higher lipophilicity than OH-Tam itself (cf Table 1) and is enhanced with increased chain length allowing in vitro easier entrance into cells. They are soluble in organic solvents, but much less so in the aqueous media encountered in biology. Thus, to study their effects in vitro on cell cultures, 1 mM solutions are prepared in DMSO then diluted in the culture medium (final maximum concentration of DMSO of 0.5%). Selected ferrocifens are shown in Chart 5.

Chart 5: Selection of ferrocifens of interest

A study of the interactions of the ferrocifens with the alpha and beta forms of the estradiol receptor (ERα and ERβ)

The estrogenic effects of hydroxy-tamoxifen, and more generally of SERMs, are widely described as being mediated by their interactions with the two forms, α and β, of the estrogen receptor. The presence of ERα in breast tumours is a marker leading to their classification as hormone-dependent tumours, and is a good indicator of their positive response to hormone therapy. However, in the case of ERβ, almost 20 years after its discovery by Gustafsson, and even after numerous studies, its exact role has still to be elucidated. 53 From the beginning of the studies on Fc-OH-Tam and Fc-diOH the Relative Binding Affinity (RBA) has shown the possibilities of association with both the α et β isoforms of the estrogen receptor. The RBAs of ferrocenyl complexes have been systematically studied for their effect on the growth of the hormone-dependent cancer cells MCF-7. In general, the complexes maintain an affinity for ERα and ERβ even if the measured values are lower than for OH-Tam (Table 1). For those complexes bearing an amino chain it has been shown that the best antiestrogenic effect is found with a 3 or 4 carbon side-chain rather than the 2-carbon one in OH-Tam. This has been rationalised in terms of the greater volume of a ferrocenyl moiety thus requiring a longer arm than is needed for a phenyl group. This antiestrogenic effect is observed at low concentrations and leads to inhibition of the growth of MCF-7 cells practically equivalent to that seen for OH-Tam, but is suppressed upon addition of a low concentration of estradiol (10 nM), and at higher concentrations the complex becomes cytotoxic. In the case of complexes devoid of an amino chain, such as Fc-diOH or Fc-monoOH, one sees at low concentrations (0.1 µM) an estrogenic effect similar to that observed with the corresponding purely organic phenols. When the concentration of incubation increases we discern cytotoxic effect attributable to the presence of the ferrocene.

We can say that the construction of these complexes based on the structure of OH-Tam raises hopes for the possibility of association with the receptor. Molecular modelling studies have shown that Fc-OH-Tam (n = 3,4) forms an anti-estrogenic type association with ERα, present in MCF-7 (Figure 1). The interaction of Fc-OH-Tam with ERβ is also antiestrogenic, but with Fc-diOH, agonist-type binding is favoured. The steric hindrance generated by the basic chain stabilises the indicated conformation via an association with Asp351 ; this prevents the recruitment of the peptide effectors to trigger an estrogenic effect as occurred with Fc-diOH in a different conformation by association between helices H12 and H4. These studies show that the active site of ERα can accommodate molecules in which the planar aromatic ring in the α position has been replaced by the bulkier ferrocenyl moiety. 29,30 This possibility has been verified for other tamoxifen-like analogues where diphenol derivatives of other metallocenes (ruthenocene, osmocene, cyclopentadienyl rhenium tricarbonyl), also maintain an affinity for the estradiol receptor. These tamoxifen-like complexes have an antiestrogenic antiproliferative effect on MCF-7 cells, identical to that of the ferrocifens where the mono and diphenolic complexes have an estrogenic effect. These complexes do not have an antiproliferative effect on the hormone-independent breast cancer cells (MDA-MB-231) at an incubation concentration of 1 µM and distinguishes them from Fc-OH-Tam; clearly, the presence of iron engenders a particular cytotoxicity.

The possibility of interaction of organometallic complexes with estrogen receptors is not matched by their behaviour with androgens. Thus, the incorporation of a ferrocenyl substituent at the17α position in ethynyl-dihydrotestosterone or dihydrotestosterone (DHT), or on nilutamide, a non-steroidal antiandrogen, results in the loss of recognition of the organometallic compound by the receptor. This may be rationalised in terms of the structure of the active site of the androgen receptor that cannot accommodate such a bulky substituent at the 17α position of DHT ; this contrasts with the flexibility of ERα that has available space because of the flexible methionines present. 34,55 One of the suggested approaches concerning the possible role of ERβ was that of redox activity. In this context, we have studied the antiproliferative effect of two tamoxifenlike ferrocifens (Fc-OH-Tam [3] and Fc-OH-Tam [4]) on a series of cancer cells where the presence or absence of ERα or ERβ has been established by quantification of their level of mRNA. We see in Table 2 that, at a concentration of 1 µM, OH-Tam and two ferrocifens are active on the MCF-7 line containing ERα. In the case of PC-3 where ERα is only at the limit of detection, but ERβ++ is clearly present, there is a significant antiproliferative effect with Fc-OH-Tam (n = 3 and 4). On the cell line NCI-ACHN, which contains ERβ but not ERα, there is a very strong antiproliferative effect. This is also the case on the cell line NCI-786

where ERα and β are absent. Thus, one can clearly state that ERβ does not play an essential role in the antiproliferative effects of the ferrocifens.

An SAR study of the ferrocifen family

The antiantiproliferative effect of these complexes was first of all studied by determining their IC 50 values on cancer cells (MDA-MB-231, MCF-7, PC-3) but also in certain cases on noncancerous cells. The first characteristic of these complexes is that their cytotoxicity is much higher on cancer cells than on normal cells. This has been shown on melanocytes (NHEM, neonatal human epidermal melanocytes) 56 and astrocytes 57 for Fc-diOH and Fc-OH-Tam and on GBM cells (glomerular basement membrane) for Fc-diPh. 58 This allowed us to show that the ferrocenyl -double bond -phenol motif was essential for expression of a strong cytotoxic effect. Ferrocene itself is not toxic for the cells (IC 50 = 150 µM), and shifting the ferrocenyl unit to the end of the side-chain leads to much less toxic complexes (series E; IC 50 around 7.5 µM). This is the case even when the double bond is replaced by an sp 3 carbon (IC 50 = 3 -4 µM). 59 This approach allowed us to identify that, in addition to the tamoxifen-like systems, a series of mono-and di-substituted complexes bearing OH and/or NH 2 substituents have a strong antiproliferative effect on MDA-MB-231 cells (Table 3). 60,61 More recently, we have synthesised a series of ansa complexes in which the two cyclopentadienyl rings are linked via a three-carbon bridge. 33 The IC 50 values obtained for these complexes are very clearly lower than for their open-chain homologues. It has also been noted that the dose-effect curves of the complexes in these two series are different, in particular for the diphenol complexes which have been studied on the NCI panel of 60 cell lines. For Fc-diOH the curves have a single slope, whereas for ansa-Fc-diOH the curves exhibit two slopes connected by a plateau. 61 These two compounds are the most active against those cancers (leukemia, central nervous system, renal cancer) for which there are no satisfactory treatments. Moreover, comparison with the 171 molecules in the NCI database reveals that the behaviour of these molecules is different from those already in the database. 61 This study led to the identification of 3 hits, Fc-diOH, Fc-OH-Tam and ansa-Fc-diOH whose mechanisms of action and effects in vivo have been studied.

Effects of the ferrocifens at the cellular level.

One of the common characteristics of the ferrocifens is that as concentrations approach the IC 50 values they show the arrest of cellular growth by senescence whenever this is possible, and not cell death by apoptosis. This senescence is associated with the observation of a cytostatic effect of the complexes which expresses itself by a flattening and an increase in size of the cells incubated at a concentration of about 1 µM with Fc-OH-Tam, 64 Fc-diOH and even lower with ansa-Fc-diOH. 63 This induction of senescence was ascertained with SA-βgalactosidase staining and senescence associated secreting phenotype (SASP) as measured by the secretion of IL-8 and TNF-α, in cancer cell models characterised by various sensitivities to pro-apoptotic stimuli (MDA-MB-231, MCF-7, U373, Hs683, B16F10). 63,64 The secretion of these cytokines may be related to AP-1 transcription factor activity at 1 µM but not at 10 µM. This observation is in accord with the result observed in vivo after innoculation of nude mice with B16F10 cells that are inactivated in the presence of 1 or 10 µM of ansa-FcdiOH. In effect, none of the animals having received cells treated with 1 µM of complex developed tumours, whereas two out of five animals having received cells treated with 10 µM of complex did so. Despite a different oxidation pathway in terms of generation of quinone methides (vide infra) the two complexes induce similar cellular changes related to senescence induction in those cell models. At elevated concentrations (5 -25 µM) the complex Fc-OH-Tam induces a rapid rounding of the cancerous cells (melanomas, MDA-MB-231, MCF-7)

followed by their detachment. 56,65 This effect can be related to the presence of the amino chain which, when protonated at physiological pH, apparently behaves like a detergent. It is also seen with tamoxifen derivatives of other metallocenes (ruthenocene, osmocene). 66 In these variations of cell cycles we observe different behaviour as a function of the incubation concentration. Fc-diOH at a concentration of 1 µM and ansa-Fc-diOH at a concentration of 10 nM lead after 48 h, to an accumulation of cells in the S phase (on MDA-MB-231 and 9L cells). At a more elevated concentration of ansa-Fc-diOH (0.5 -2 µM) one sees an accumulation of 9L cells in the G0/G1 phase and about 30% of the cells in apoptosis. 67 Incubation of 10 µM of Fc-diOH and of ansa-Fc-diOH with mouse melanoma cells B16F10 led to 80% apoptosis. 63 One should also note the particular behaviour of the complex Fc-diPh which lacks the phenolic group (Chart 5). This complex has an elevated cytotoxicity on the leukemia cells HL-60 (IC 50 = 1.04 µM) but a more modest one on MDA-MB-231 cells (IC 50 = 7.5 µM). At a concentration of 1 µM it induces apoptosis of HL-60 with an accumulation of cells in the G0/G1 phase. 58 This singular behaviour is probably associated with the production of ROS (see below). It appears then that the type of inactivation or cell death pathway (senescence, apoptosis) varies according to the complexes, the cell lines and the dosages used.

We note, however, that the special feature of the ferrocifens is their efficacy, by senescence at low dosages, on cell lines resistant to apoptosis.

Mechanisms of action related to the redox properties of ferrocene

The particular antiproliferative effects of the ferrocifens appear to be associated with the unique redox properties of ferrocene, that is to say a reversible oxidation of Fe(II) to Fe(III) at a potential around 0.40 V. 68 This behaviour differs from that of ruthenocifen whose oxidation rapidly becomes irreversible, and of osmocifen whose oxidation is always irreversible. This turns into a large difference in the acidity of the phenolic protons. 66 Ruthenocifen and osmocifen can also be oxidised in their corresponding quinone methides but at a lower rate than ferrocifen (18 times to 3 times depending on the complexes). This could be associated with the redox potential of the three metallocenes (0.47, 1.03, 0.83V, respectively for ferrocene, ruthenocene and osmocene), which results in an easier oxidation of ferrocifens than ruthenocifens and osmocifens. 66

Production of reactive oxygen species (ROS)

The first studies of the antiproliferative effects of ferrocenyl complexes were carried out by Köpf-Maier and Neuse on ferrocenium salts. 69 Subsequently, this was repeated by Osella who showed by EPR that the generation of reactive oxygen species (ROS) is implicated in this cytotoxicity and more specifically that OH • radicals can be produced starting from Fe(II) by a Fenton-type reaction. 70 Even though the IC 50 values of these complexes are very much larger than those of the ferrocifens (250 µM versus 0.5 µM), the assumption that ROS are involved in the toxicity of the ferrocifens is a factor that has been explored. The quantities of ROS produced after 10 minutes incubation of MDA-MB-231 cells in the presence of 1 µM of several representative complexes and their precursors (ferrocene and OH-Tam) are shown in Figure 2.

It shows that ferrocene (Fc) and hydroxytamoxifen (OH-Tam) do not produce ROS at the 1 µM incubation concentration but all the ferrocenyl complexes do so. The effect of ROS on the toxicity of the ferrocifens has been demonstrated by the fact that addition of antioxidants such as N-acetyl cysteine or vitamin E lead to loss of their antiproliferative effect. 64,71 In contrast, production of ROS is not linked to the presence of a phenol and there is no correlation between the IC 50 values and the quantity of ROS measured for the complex. This is particularly true for the complex Fc-diPh, which is one of the two compounds that produce the most ROS but which shows only medium cytotoxicity towards MDA-MB-231

(IC 50 = 7.5 µM). This production of ROS could be the origin of the toxicity of this complex on HL-60 leukemia cells that are sensitive to apoptosis. In effect, this complex that lacks the phenolic group cannot lead to formation of a quinone methide that could play a decisive role in the toxicity of the most active complexes (see below) and leads to cell death by apoptosis and not to inactivation via senescence as with the other complexes. One must emphasise that the protocol used to measure the ROS is important. Thus, a 30 min pre-incubation of the cells in the presence of a solution of H 2 DCFDA (10 µM), followed by rinsing, then incubation in the presence of the tracer (1 µM) allows observation of the generation of ROS, 64,65 but when one incubates the cells in the presence of the complexes before adding H 2 DCFDA formation of ROS is not observed. 62 This is probably related to the fact that ROS are very reactive species and so H 2 DCFDA, the probe used to detect them, needs to be present in cells at the time of their formation.

Formation of quinone methides

A redox process involving two electrons and two protons leading to formation of a phenoxy radical and then a quinone methide has been suggested as being the origin of the high toxicity of these molecules (Scheme 4). 68 This is based on the electrochemical oxidation of Fc-monoOH which shows that the reversible oxidation of ferrocene becomes irreversible in the presence of pyridine or imidazole; it is accompanied by deprotonation of the phenol and leads to formation of the quinone methide. Each step in this reaction scheme has been confirmed by electrochemical and EPR studies of the particular complex stabilised with two ortho methyl substituents in the phenol (R = CH 3 ). 68,72 Fe

OH Fe OH Fe O Fe O -e - N -pyH + -e -/-H + Fe O R R R R R R R R R R R = H or CH 3
Scheme 4 : Mechanism for the formation of quinone methides 68,72 The considerable strength of the ROS signal suggests that it could play a role in the first step, that is to say the oxidation of Fe(II) to Fe(III). The quinone methides of Fc-OH-Tam, Fc-mono and diOH have been prepared by chemical oxidation using Ag 2 O. An X-ray crystal structure of the quinone methide derived from the phenol stabilised by two methyl substituents (Scheme 4 ; R = CH 3 ) has been obtained. 73 Moreover, a study of the metabolism of Fc-OH-Tam, Fc-mono and diOH using rat liver microsomes shows the formation of three major metabolites (Scheme 5): (1) the quinone methide (oxidation product), (2) the indene obtained by cyclisation of the quinone methide in an acidic medium and (3) the allylic alcohol (hydroxylation product). 74 Scheme 5 : Main metabolites formed upon oxidation of ferrocifens by rat microsomes (CYP 450). R = H, OH, O(CH 2 ) 3 N(CH 3 ) 2 . 74 In the case of Fc-OH-Tam, two additional metabolites are obtained, Fc-diOH and the demethylated complex. Interestingly, it has been shown that the vinyl alcohol, a close relative of the allylic alcohol obtained, exhibits a strong antiproliferative effect (IC 50 = 0.2 µM). Since we know that P450 cyctochromes exist in cancerous cells, such a metabolite could play a role in the toxicity of the ferrocifens.

The case of ansa-Fc-diOH is different since chemical oxidation or incubation in the presence of microsomes does not lead to formation of the corresponding quinone methide, but this does not prevent this complex from having one of the lowest IC 50 values (89 nM on MDA-MB-231). In an electrochemical study it was not possible to detect the QM from this complex, nevertheless the complex does undergo an initial oxidation of Fe(II) to Fe(III). One can rationalise this behaviour in terms of the steric strain imposed by the three-carbon bridge that prevents the two cyclopentadienyl rings from becoming parallel and does not allow the planarity required for formation of a quinone methide. This active species may be the radical precursor to the QM, as shown below (Chart 6).

Chart 6 : Quinone methide radical derived from ansa-Fc-diOH

In all cases the quinone methides and their radical precursors are potentially very reactive intermediates and one could regard the ferrocifens as pro-drugs that allow the generation of these cytotoxic entities in the right place.

The chemical search for targets

Interaction of the ferrocifens with DNA.

DNA has long been considered as the principal target for metallodrugs, in particular cisplatin. This is not the case with the ferrocifens. It has been shown that they do not react with purine bases 61 and that DNA lesions appear clearly only at an elevated concentration of ferrocifens (10 µM) and do not require the presence of phenol. 29 There are two literature examples that describe interactions of the ferrocifens with DNA. In the first case, studied by Osella, one sees a degradation of the DNA based on the Comet assay on mesothelium cells (BBR cells from cancer of the pleura) after 2 h incubation in the presence of Fc-diOH (0.5 µM) that is not seen in the presence of Fc-OH-Tam. 75 The other example involves the effect of Fc-diPh on leukemia HL 60 cells. This complex induces apoptosis with an accumulation of cells in the G0/G1 phase and is followed by fragmentation of DNA, a result based on phosphatidylserine externalisation and increase of caspases 3 et 7. 58

Interaction with thiols and thioredoxin reductases (TrxRs)

Quinone methides are known to undergo nucleophilic additions, for example by thiols.

In the case of quinone methides derived from ferrocifens obtained by chemical oxidation using Ag 2 O, it has been found that they can participate in 1,8 Michael additions with certain thiols such as methyl N-acetyl cysteine or glutathione; this is the preferred reaction in basic media. An in vitro study of the interaction of the ferrocifens with purified thioredoxin reductases (TrRx) confirmed that quinone methides interact with TrxRs when these active sites contain accessible selenols, but do not react with the corresponding thiols. 76 This is a notable difference between the ferrocifens and OH-Tam. Indeed, the quinone methide of the latter is more reactive than those of the ferrocifens with the nucleophiles present in cells. It is also noteworthy that there is a difference in chemical reactivity between the quinone methides of Fc-OH-Tam and Fc-monoOH. The QM derived from Fc-monoOH can cyclise in a protic medium to form an indene, and this molecule can no longer undergo Michael additions. The effect of Fc-OH-Tam and Fc-monoOH on Jurkat cells shows that the strongest inhibition of the thioredoxin reductase is induced after incubation in the presence of Fc-OH-Tam. This indicates two things (1) that Fc-OH-Tam behaves like a pro-drug that is transformed in the cell into its quinone methide and (2) the very strong activity of Fc-OH-Tam is due to the fact that it remains in the cell in the state of a QM whereas the quinone methide of Fc-monoOH is partly transformed into an inactive indene (Scheme 6) Scheme 6 : Reaction of quinone methides with selenols and competitive cyclisation to indene in protic medium. 76

In vivo studies with Fc-diOH, Fc-OH-Tam and ansa-Fc-diOH

In vivo studies have been performed with the 3 hits: Fc-diOH, Fc-OH-Tam and ansa-Fc-diOH. There were several challenges to succeed in the transfer from in vitro to in vivo experiments. The first one was to find a formulation suitable for lipophilic molecules and the second one was to allow phenols to remain in the blood to reach their target (cancer cells) while it is well known that phenols are rapidly metabolized in the liver. The first experiments performed with oral administration of Fc-OH-Tam under its citrate salt and of Fc-diOH protected with palmitate functions on xenografted prostate tumors on nude mice did not induce regression of the tumors. 77 This problem was solved with the development of nanotechnologies and more precisely with that of LNCs (lipid nanocapsules). These LNCs consist of a lipidic heart, in which ferrocifens are soluble, surrounded by modified PEG possessing a lipophilic head and a hydrophilic tail. All these constituents are FDA approved which is a significant advantage for their development. In addition their stealthiness increases their time of circulation in the body. This effect can be increased by addition of a second layer of PEG (mspPEG-2000). The effect of these LNCs loaded with Fc-diOH was first tested on rat with implanted tumors from glioblastoma (rat 9L cells) by direct injection in the tumor or intravenously. Significant to almost total regression of the tumors was observed with the second generation of LNCs. An increase in the number of injections does not seem to improve the effect of the drug (LNCs loaded with ansa-Fc-diOH). 67 Very interesting results have been obtained on orthotopic tumors (9L cells implanted in rat brain) when chemotherapy, LNCs loaded with Fc-diOH and injected in rat brain, was coupled with radiotherapy. However it should be kept in mind that some LNCs exhibit a strong toxicity if administered directly in the brain while they are very efficient if delivered via an injection in the carotid. 78 It remains that this formulation is particularly well suited for ferrocifens delivery.

Hybrid Ferrocifen-SAHA and related species

The idea of this study was to combine two active entities in the same molecule in order to modulate simultaneously several mechanisms of action. The choice was made to focus on the coupling of ferrocifens with SAHA (suberoylanilide hydroxamic acid). SAHA is an inhibitor of histone deacetylases (HDAC) which belong to an important class of epigenetic drugs. The hybrid compounds were obtained by replacing the amine chain of Fc-OH-Tam by a SAHA fragment. Thus, Fc-Tam-SAHA and Fc-Tam-PSA (the corresponding amide, PSA = N 1 -phenylsuberamide) were prepared as well as their organic analogues (Tam-SAHA and Tam-PSA; Scheme 7). 47 The antiproliferative effect of these molecules was studied on breast cancer cells MDA-MB-231 cells (Table 4). Interestingly, a synergic effect between SAHA and Fc-Tam was observed for Fc-Tam-SAHA (IC 50 value of 0.7 µM versus 3.6 µM for SAHA and 2.6 µM for Fc-Tam). This synergic effect was not observed for organic molecules Tam-SAHA or Tam-PSA (IC 50 = 8.6 and 25.2 µM respectively). However, both Fc-Tam-SAHA and Tam-SAHA have been found

to exhibit an HDAC inhibition effect with similar activity. This effect is weaker than that observed for SAHA. This result seems to prove that antiproliferative effect of Fc-Tam-SAHA is not mainly related to its HDAC inhibition effect. Moreover, the antiproliferative effect of compounds against hormone-dependent breast cancer cells does not correlate with the interaction of compounds with estradiol receptors even though they were recognized by estradiol receptors with RBA values in the range 4-22%. Interestingly, it was found that Fc-Tam-SAHA and Fc-Tam-PSA significantly increase the p21mRNA expression in MCF-7 cells. This expression may account for the antiproliferative effect of these compounds as ferrocifens were found to produce ROS. However, the antiproliferative behaviour of Fc-Tam-PSA is still not fully understood.

Complexes with two amino chains

Another family of complexes is made up of species possessing two amino chains (Chart 4, Series G). These are secondary products obtained in the McMurry synthesis, and different complexes have been prepared. 79,80 It has been found that although these complexes do not possess a phenolic group they have an affinity for the estradiol receptor which expresses itself, at low concentration (0.1 µM), by a proliferative effect on MCF-7 cells and then a toxic effect leading to IC 50 values around 0.5 µM. Interestingly, this time the antiproliferative effect is not associated with the presence of a metallocene but is uniquely attributable to the presence of the double amino chains. Furthermore, these complexes exhibit strong antibacterial effects. One hypothesis of the mechanism of action involves complexation of a zinc atom. We note that these are the only complexes that possess a strong antibacterial effect; 23 likewise, other symmetric amines, such as ridaifin B, have also been found to exhibit a strong antibacterial effect. 80

Conclusion

Organometallic chemistry, which has been one of the major strengths of molecular chemistry over the past sixty years, particularly in terms of homogenous catalysis, is now seeing the supremacy of this established topic challenged by emerging fields such as organometallic medicinal chemistry where derivatives of metallocenes, such as ferrocene, provide archetypal examples. 81 In this article we have attempted to bring together mechanistic data, both chemical and biological, relating to the ferrocifens. This series was conceived initially as ferrocenyl derivatives of tamoxifen -the primary prodrug to combat hormone-dependent breast cancers -in an attempt to extend the range of applicability to hormone-independent cancers.

However, these complexes soon broke free from this important but limited tether when their activity against other currently incurable types of cancer became apparent; we have now reached the point at which we can begin to hope that they may be considered as possible complements to the coordination complexes of cisplatin and related systems. Furthermore, it quickly became apparent that the ferrocifens, unlike the platinum complexes, targeted primarily not the DNA bases but rather the proteins.

Among the library of ferrocenyl compounds that have been synthesised, attention has focussed on the following three hits: Fc-OH-Tam (the first example prepared in the acyclic series), its corresponding diphenol Fc-diOH, and its cyclic analogue ansa-Fc-diOH. Our understanding of the behaviour of these three hits has benefitted both the chemical and biological studies, as shown in the sections above. This has allowed us to analyse their similarities and differences and to achieve a deeper understanding on a number of fronts.

The study of the metabolites obtained by oxidation, either chemically with Ag 2 O, or biologically with cytochrome P450 (from liver microsomes or purified) have revealed the importance in the open series (Fc-OH-Tam, Fc-diOH et Fc-monoOH) of a quinone methide metabolite subject to evolution. These QMs are electrophiles which are selective for thiolates and selenols, which is not the case for their more reactive organic analogues. This provides an explanation for the selective inhibitory activity of the organometallics on thioredoxin reductase, an enzyme overexpressed in cancerous cells. Nevertheless, during the incubation of these complexes with Jurkat cells, the effect of Fc-monoOH seems to be diminished because of cyclisation of the quinone methide. Moreover, these ferrocifenyl entities, which are frequently producers of ROS, are active essentially on cancerous cells and are much less so on healthy cells. The ferrocifens take advantage of the micro-environment of the cancerous cell and make possible this selectivity associated with a general lowering of toxicity.

Chemically, the case of ansa-Fc-diOH is different since the QM species expected to be formed upon oxidation has never been detected. This does not prevent the complex from having one of the strongest antiproliferative effects found in this family of molecules.

Although it was not possible to detect the signature of this QM electrochemically, initial oxidation of the iron is evident. One can explain this behaviour in terms of the high level of strain imposed by the three-carbon bridge which prevents the two cyclopentadienyl rings from becoming fully parallel and does not allow the planarity required for the QM. The active species could therefore be the radical precursor of the QM. This may be a more widespread type of behaviour.

Although ansa-Fc-diOH and Fc-diOH have shown differences in their oxidative progress concerning the formation, or not, of a quinone methide, their biological behaviour provides some analogies. For example, selectivity for the same cell lines (kidney, leukemia, central nervous system) in the NCI panel of 60 cell lines. Moreover, as for Fc-OH-Tam, the two species induce senescence at low concentration (0.1 µM) and, depending on the cancer cell, a possibility of apoptosis at higher concentrations (> 1 µM) as a function of the sensitivity of the cell to pro-apoptotic stimuli. The discovery of this senescence is not without therapeutic interest since it could provide a promising alternative to cytotoxic compounds, in particular to avoid the problem of their chemoresistance. Senescence involves an irreversible cessation of proliferation. This effect has also been established in a number of benign adenomas. 82 One can envisage taking advantage of senescence for the stabilisation of patients in advanced stages of cancer who no longer respond to cytotoxic agents on cells resistant to apoptosis but respond to senescence, also in the treatment of precancerous or early stage tumours, and as a potential strategy to combat resistance.

It must be stressed that, when working with these often lipophilic organometallic entities, the formulation is key to the efficacy of the system. In this context, stealth lipid nanocapsules (LNCs) have proved to be useful when their composition can be adapted to their mode of administration and to the type of tumour studied (ectopic or orthotopic).

The ferrocifens mentioned above have shown their ability to interact according to both their structure and the nature of the cancer cell targets (whether or not these respond to proapoptopic stimuli), as well as their formulation; in addition, this ability to interact also depends on their biological concentration which allows the activation of any given target, dependent of course on their presence inside the particular cell. The scheme below (Chart 7)

summarises the effects of concentration:

Chart 7 : In vitro effects of ferrocifens on cancer cells vary according to their concentration in the biological medium

The concentration regions where one or another biological signalling pathway predominates are quite well defined, but their capacity for action, synergic or otherwise, requires further elucidation and other pathways must still be identified. The possibility of activity at concentrations appropriate for Fenton reactions, which engender a genotoxic effect by producing hydroxyl radicals, is encouraging for the potential utility of our molecules. In addition, multitargetting of the ferrocifens provides a broad spectrum of efficacy and the potential to significantly delay or even suppress drug resistance.

It is clear that other targets still remain to be found, as illustrated by the ferrocifen-SAHA hybrids that possess a strong antiproliferative effect even though they cannot lead to active QM species. Their activity could be associated with initiation of the expression of the p21 gene recognised as a powerful inducer of senescence also observed with Fc-diOH and ansa-Fc-diOH.

Although the systems above do not possess the Fc-alkene-paraphenol motif, it is still true that research on molecules containing this structural element remains a useful approach in the quest for new prodrugs ; for example, the case of Feroscan 53 shown below : 83 Oxidation leads to a novel type of heterocyclic quinone and opens new pathways to explore in the area of biodiversity.

One can now see the richness of this family of the ferrocifens and the promise of applications that must be studied in more depth. This exploration will still take time to develop, but in light of the results already obtained there is reason of optimism. One can hope that this review may stimulate other, more biochemically focused groups, to extend these studies to include metabolomic or microarray expression profiling of appropriate cell lines to deconvolute the multiple pathways involved.

  Jaouen, a,b,c * Anne Vessières, a,b Siden Top a,b a Sorbonne Universités, UPMC Univ Paris 06, IPCM, F-75005 Paris, France b CNRS, UMR 8232, IPCM, F-75005 Paris, France c PSL Research University, Chimie ParisTech, 11 rue Pierre et Marie Curie, F-75005 Paris, France Corresponding author : Prof G. Jaouen, Chimie ParisTech, 11 rue Pierre et Marie Curie, F-75005 Paris, France. Tel : 33 (0)1 43 26 95 55

Chart 1 :

 1 Selected examples of inorganic anticancer drugs However it is now crucial to find a new and effective angle of attack, probably by taking advantage of novel mechanisms of action. It is in this context that the development of a new

Chart 3 :

 3 Selected bioferrocene compounds

  3 cells (IC 50 = 27.5 µM) with visible light (400 -700nm). Apoptosis is induced via an intrinsic mitochondrial pathway giving rise to reactive oxygen species (ROS). DNA photocleavage activity occurs by formation of hydroxyl radicals (OH • ). Another recently published conjugate, 17, ferrocenyl tamoxifen-SAHA may open a new therapeutic route through the intervention of other targets. This hybrid 17 shows good cytotoxicity on triple negative MDA-MB-231 breast cancer cells (IC 50 = 0.7 µM), while FcTam and SAHA alone give IC 50 values of 2.6 µM and 3. µM respectively, and IC 50 = 8.6

Scheme 2 :

 2 Scheme 2 : Synthesis of Fc-monoOH and Fc-diOH via a McMurry cross coupling reaction

Figure 1 :

 1 Figure 1 : Image of Fc-OH-Tam (n=4) docked in the ligand binding domain (LBD) of ERα with an antagonist conformation. (Adapted from the LBD X-ray structure)54

Figure 2 :

 2 Figure 2 : ROS production in MDA-MB-231 (1 µM, 10 min, H 2 DCFDA as fluorescent probe, mean of two experiments)

Scheme 7 :

 7 Scheme 7 : Structure of SAHA, PSA and of their ferrocifen and tamoxifen hybrids

Scheme 8 :

 8 Scheme 8 : Ferrocifens with modified alkyl chain

Table 1 :

 1 Lipophilicity and Relative binding affinity (RBA) values for the estrogen receptor (α and β forms) of some ferrocifens29,30

		RBA (%) 0°C, 3h30 a) ERα ERβ	LogPo/w
	OH-Tam	38.5 ± 3	24	3.2 (Z) ; 3.4 (E)
	Fc-OH-Tam[3]	11.5 ± 3	12 ± 1	4.3 (Z) ; 4.5 (E)
	Fc-OH-Tam[4]	12 ± 2	11 ± 1	4.0 (Z) ; 4.2 (E)
	Fc-monoOH	5 ± 0.2	11	6.1 (Z) ; 6.2 (E)
	Fc-diOH	9.6 ± 0.9	16.3 ± 1.5	5.0
	a) mean of 2 experiments (when available) ± standard error

Table 2

 2 Antiproliferative effect of OH-Tam and Fc-OH-Tam with n = 3 or 4 on various cell lines and comparison with their ERα and ERβ content.

	Cell line a	ERα	ERβ	% remaining cells (incubation 1 µM, 5 days) b)
		status	status
				4-OH-Tam	Fc-OH-Tam[3] Fc-OH-Tam[4]
	MCF-7	++	+	75 ± 2	62 ± 2	58 ± 0
	MDA-MB-231	-	+++	94 ± 3	37 ± 6	44 ± 6
	PC-3	+/-	++	100 ± 1	36 ± 14	35 ± 5
	NCI-ACHN	-	+++	100 ± 1	2.5 ± 0.5	2,5 ± 0.5
	NCI-786	-	-	98 ± 5	3 ± 0	3.5 ± 0.5
	a) breast cancer cells : MCF-7, MDA-MB-231 ; prostate : PC-3 ; kidney cancer cells : NCI-
	ACHN, NCI-786 ; b) mean of two experiments ± standard error

Table 3

 3 IC 50 values of a selection of complexes in the ferrocenyl acyclic and ansa series

	R 1	R 2	IC 50 (µM) on MDA-MB-231 cells a
			acyclic series	ansa series
	OH O(CH 2 ) 3 N(CH 3 ) 2	0.5	-
	OH	OH	0.64 ± 0.06	0.089 ± 0.006
	OH	H	1.54 ± 0.13	0.47 ± 0.06
	NH 2	H	0.86 ± 0.04	0.21 ± 0.03
	NH 2	NH 2	n.o. b)	0.05 ± 0.01
	NH 2	OH	0.55 ± 0.05	0. 061 ±
				0.005
	H	H	7.5 ± 0.7 c	-
	a) values from ref 61 after 5 days of incubation, mean of 2 experiments ± standard error; b)
	molecule not obtained ; c) IC 50 value on HL-60 = 1.04 µM 62

Acknowledgements

We wish to thank our co-workers named in the cited papers and Dr Patricia de Cremoux. Barbara McGlinchey and Michael McGlinchey are acknowledged for fruitful discussions and their linguistic talents; the LabEx Michem for scientific support. We also thank the Pierre-Gilles De Gennes Foundation, the University PSL, Feroscan and IRCP for generous support.

reviews, and has filed 16 patents. His achievements in the field have been recognized by several international awards. He was awarded Knight of the "Légion d'Honneur" (2006), and was elected as a fellow of the European Academy of Sciences and a member of the Academia Europaea (2012) Anne Vessières, a Director of Research in the CNRS, earned doctorates in both organometallic chemistry (1974) and biochemistry (1980) from the University of Rennes (France). was awarded the Elsevier Award for Outstanding Achievements in Bioorganometallic Chemistry Siden Top, born in Cambodia, is a Director of Research in the CNRS. In 1972 he was awarded a fellowship to study in France where he obtained a "Docteur Ingénieur" degree in Dijon (1976) and

Table of Content

The mechanisms of action of ferrocifens depend on several features : chemical structures, used concentrations, nature of cancer cells


L Pasquini

P Bonifacio

S Randich

D Galli

R G Gratton

B Wolff

Beryllium abundance in turn-off stars of NGC 6752 ⋆

Keywords: abundances -stars: globular clusters -NGC 6752 -stars: formation, age, late-type

Aims. To measure the beryllium abundance in two TO stars of the Globular Cluster NGC 6752, one oxygen rich and sodium poor, the other presumably oxygen poor and sodium rich. Be abundances in these stars are used to put on firmer grounds the hypothesis of Be as cosmochronometer and to investigate the formation of Globular Clusters. Methods. We present near UV spectra with resolution R∼ 45000 obtained with the UVES spectrograph on the 8.2m VLT Kueyen telescope, analysed with spectrum synthesis based on plane parallel LTE model atmospheres.

Results. Be is detected in the O rich star with log(Be/H)=-12.04 ±0.15, while Be is not detected in the other star for which we obtain the upper limit log(Be/H)<-12.2. A large difference in nitrogen abundance (1.6 dex) is found between the two stars. Conclusions. The Be measurement is compatible with what found in field stars with the same [Fe/H] and [O/H]. The 'Be age' of the cluster is found to be 13.3 Gyrs, in excellent agreement with the results from main sequence fitting and stellar evolution. The presence of Be confirms the results previously obtained for the cluster NGC 6397 and supports the hypothesis that Be can be used as a clock for the early formation of the Galaxy. Since only an upper limit is found for the star with low oxygen abundance, we cannot decide between competing scenarios of Globular Cluster formation, but we can exclude that 'polluted' stars are substantially younger than 'unpolluted' ones. We stress that the Be test might be the only measurement capable of distinguishing between these scenarios.

Introduction

In a recent paper we have successfully tested the use of beryllium (Be) as a cosmochronometer for the early phases of Galactic evolution (Pasquini et al. 2004). Be is produced by the interaction of Galactic cosmic rays (GCRs) with the interstellar medium, through the spallation of heavy element nuclei, most noticeably: carbon, nitrogen, and oxygen. Due to its origin, and in particular to the fact that GCRs are generated and transported globally on a Galactic scale, Be is expected to be characterized by a smaller dispersion than the products of type II supernovae, whose abundances in the early Galaxy are affected Send offprint requests to: lpasquin@eso.org ⋆ Based on observations collected at the ESO VLT, Paranal Observatory, Chile , program 075.D-0807(A) by the dispersed character of star formation and inefficient mixing of gas. This led to the suggestion that Be represents a "cosmic clock" (Suzuki et al. 2001;[START_REF] Beers | The Light Elements and Their Evolution[END_REF]. To test this hypothesis Pasquini et al. (2004) carried out the first measurements of Be abundances in a globular cluster (GC); namely they observed Be in two turn-off (TO) stars of the metal poor ([Fe/H]∼ -2) GC NGC 6397, for which an independent age estimate was available. Be was detected in both stars at a level consistent with that of stars in the field with the same [Fe/H] abundance. By comparing their Be values with models of Galactic evolution of Be as a function of time, they concluded that the cluster formed about 0.2-0.3 Gyr after the onset of star formation in the halo, in very good agreement with the cluster age derived from main sequence fitting. In order to prove that this result is not fortuitous, it is extremely important to carry out the same test on additional GCs.

As discussed by Pasquini et al. (2004), Be and Li measurements in GCs provide as well important constraints on the hotly debated issue of cluster formation. Detailed studies of chemical abundances in GC stars have revealed that anomalies are present in all the clusters studied, showing that they are not a homogeneous populations as far as the chemical composition of the stellar atmospheres is concerned (e.g. Gratton et al. 2004 and references therein). More specifically, whereas GCs appear to be extremely homogeneous in Fe and Fe-peak elements, star-to-star variations are seen for those elements involved in H-burning at high temperature. This behaviour is not present among field stars, which do not exhibit significant star-to-star variations in abundance ratios like [O/Na] or [Mg/Al] at a given overall metallicity. This intriguing abundance pattern, and in particular the fact that abundance anomalies are seen not only among evolved cluster stars but also in unevolved stars at the TO, suggests that part of the gas forming the stars that we observe today has been processed and ejected by a previous generation of stars. When considering that GCs are almost as old as the age of the Universe, this implies that this hypothetical previous generation might have been part of the elusive metal-free Population III, the first stars formed. By studying the current chemical composition of GC stars we may therefore try to determine the composition of the ejecta of the first stars and to understand their basic characteristics (mass, chemical composition).

In a series of papers we have investigated the light elements Li and Be in TO stars of GCs (Bonifacio et al. 2002;Pasquini et al. 2004Pasquini et al. , 2005;;Bonifacio et al. 2007, in preparation), showing that these elements are indeed very useful to investigate the process of GC formation. We recall that 7 Li and 9 Be are both destroyed at relatively low temperatures (∼2.5 and ∼3.5 million K respectively) in stellar interiors. These temperatures are significantly below those at which typical reactions responsible for the chemical anomalies in GCs occur. Thus, in the hypothesis that a fraction of GC stars is born from the ejecta of a previous generation of stars, these stars should in principle show low Be and Li abundances. Note, however, that Li and Be form through different mechanisms. Whereas, as mentioned, 9 Be can only be produced by GCR spallation on the ISM, 7 Li is produced during Big Bang Nucleosynthesis, and the Galaxy is later enriched by several mechanisms, such as GCRs spallation, AGB stars, and, possibly, Novae (e.g., Travaglio et al. 2001).

Our observational results can be summarized as follows: the Li abundance is constant in NGC 6397 (Bonifacio et al. 2002), while Li is found to vary from star-to-star in NGC 6752 and 47 Tuc. In both these GCs, Li abundances are correlated with O and anti-correlated with Na abundances (Pasquini et al. 2005;Bonifacio et al. 2006).

As for Be, the mere existence of Be in in the two TO stars of NGC 6397 suggests that the gas which formed the stars we now observe must have been sitting for at least a few hundred million years in the ISM exposed to the GCR spallation before the stars formed.

NGC 6397 could, on the other hand, be a somewhat 'special' case, because it is one of the GCs where chemical anomalies are present at the lowest level, and, as mentioned above, the Li abundance is strikingly constant. It may therefore be questioned whether this cluster is a fair representative of all GCs. NGC 6752, on the opposite, is a prototype of GC with large chemical anomalies; these anomalies have been found all over the colour-magnitude diagram; the Na-O anticorrelation has been discovered among main sequence stars (Gratton et al. 2001, hereinafter G01), as well as Li -Na anticorrelation (Pasquini et al. 2005). This cluster is therefore a very good prototype for anomalies; it has an intermediate metallicity ([Fe/H]=-1.48, G01), and its age is virtually the same of NGC 6397 according to main sequence fitting (Gratton et al. 2003).

Sample Stars and Observations

Stars at the TO of NGC 6752 have been studied extensively for chemical abundances (Gratton et al. 2001, 2003, Carretta et al. 2005, James et al. 2004, Pasquini et al. 2005). We selected two stars from the G01 sample having different composition; star 4428 is representative of the O-rich, Li rich component, while the star 200613 is representative of the Li poor component and, by inference, should be also oxygen poor . By observing stars at the extremes of the chemical distribution, we aimed at maximizing the chances of observing possible differences in Be abundance.

We recall that the Be lines are located at 313 nm and the Be observations of GC turnoff stars are very challenging, because the spectral region is crowded, the Be lines tiny and the UV flux of these relatively cool stars is low. When adding the relatively high absorption introduced by the earth's atmosphere, it clearly emerges that the TO stars of even the closest GCs are very faint for observations of Be lines. NGC 6752 TO stars are almost one magnitude fainter in the V band than the NGC 6397 objects, but the relatively low reddening of the cluster (E(B-V) = 0.05: Gratton et al. 2003) allows a substantial flux recovery at the UV wavelengths, so that these observations, even if very demanding, are feasible with UVES at the VLT .

Table 1 summarizes the characteristics of the stars, including the abundances of the single elements, as derived from the literature. The stellar parameters listed in Table 1 are those adopted in our spectroscopic analysis. Only abundances of those elements which are known to vary from star to star (O, Na, Li) are listed in Table 1; we also add in the table the results for Be and N as resulting from this paper. The reader can find additional element abundances in G01, James et al. (2004), andCarretta et al. (2005).

The observations were carried out in service mode at the VLT observatory in several runs during summer 2005 with the UVES spectrograph (Dekker et al. 2000). We used a 1 ′′ slit providing a resolving power of 45,000. The blue CCD was binned to minimize the CCD read out noise. In addition, all observations were taken at low airmass, to limit the atmospheric absorption, which is particularly high at these UV wavelengths. A total of 10 exposures of 90 minutes each were obtained per star. The observations were reduced using recipes from the UVES pipeline (Ballester et al. 2000). Given the fairly low S/N ratio of each observation in the Be region, we have been especially careful in reducing the data. The two major difficulties were identified in the correct estimate of the background and in the elimination of the 'cosmic ray' events. We have therefore used two different reductions, either using the standard calibration and pipeline, or using the D lamp for flat fielding, which gives better spectra in the blue. This lamp, however, has some emission lines in its spectrum; it can be used in the spectral region of the Be lines, but not over the whole spectrum.

The reduced spectra were averaged leading to a S/N ratio in the Be region of 20 and 10 per 0.025 Å pixel for stars 4428 and 200613 respectively. The spectra in the Be region, together with our synthetic best fits (cfr. next section) are shown in Figure 1.

Abundance analysis

The method used has been described in Pasquini et al. (2004). A full synthesis of the region around the Be doublet has been performed using the line list given in Pasquini et al.(2004). For each star the best abundance was found by a χ 2 fit to the whole feature. The best fits, shown in Fig. 1, correspond to Be abundances of log(Be/H) = -12.04 for the O-rich, Li-rich star 4428, while only an upper limit of log(Be/H) < -12.2 could be found for star 200613. Fitting errors were estimated with Monte Carlo simulations assuming a Poisson noise. The dispersion around the mean in 1000 Monte Carlo samples was of 0.04 dex for star 4428. These should be viewed as lower limit to the errors, since at these low S/N ratios other sources of non-Poisson noise (e.g. shot-noise) could be important. To these errors associated to the noise in the data one must add the errors which derive from the uncertainty in the analysis procedure (atmospheric parameters, etc.). Since we are using lines of singly ionized Be, surface gravity is the parameter which most affects the Be abundance computation. The values of log g have been estimated by comparing the position of the stars in the color-magnitude diagram with theoretical isochrones. The uncertainty in such determination is dominated by possible errors in the distance moduli; we can then safely assume an error of 0.15 dex in log g that translates into an uncertainty of 0.07 dex in the Be abundance. The error arising from a change of 100 K in the effective temperature is about two times lower: 0.03 dex. If we add these values to the errors due to noise (0.04 dex and 0.11 dex, respectively), we obtain an estimate of the total error on the Be abundance of 0.15 dex. Note that this error does not include possible systematic errors due to shortcomings and inadequacies of our modeling (model atmospheres, atomic data, etc.).

We recall that, while G01 used the same temperature for all NGC 6752 stars, given the strong dependence of Li on T ef f , Pasquini et al. (2005) investigated the effects of adopting an additional temperature scale, and derived for these stars T ef f from the by color as well, finding 6013 K for star 4428 and 5948 K for star 200613. These temperatures, when entered into the ischrones of Straniero et al. (1997) imply slightly higher gravities for both stars, namely log g = 4.43 for star 4428 and 4.46 for star 200613.The combined effect of this change in atmospheric parameters is an increase of the Be abundance by 0.12 dex with respect to the values given in Table 1.

We have also been able to measure the N abundance in these two stars, confirming that a very high N difference is present among them. This can be easily seen in Figure 2, which shows the NH band at 336 nm for the two stars. Clearly their N abundance is different.

We estimate the nitrogen abundance by spectral synthesis of two NH UV bands, around 336 nm and around 340.5 nm. Given the complexity of the bands it is not possible to carry out a formal fitting procedure of the whole spectral region. We have therefore inferred a "best abundance" by fitting the most prominent features. Despite several systematic and random uncertainties, we can compute a difference of about 1.6 dex in N abundance among the two stars. For star 200613 the value of [N/Fe]=1.6 is compatible with what found by Carretta et al. (2005) ([N/Fe]=1.7), who used different spectra and different spectral features (CN bands). As far as star 4428 is concerned, the value found ([N/Fe]=0) is 1 dex below that found by Carretta et al. (2005), which should be considered, however, as an upper limit rather than a detection ([N/Fe]=1.1).

The discrepancy between the two [N/Fe] abundance of the 'unpolluted' star 4428 is critical, since the evolution of nitrogen in GCs is one of the key data to understand the formation process of the clusters. In fact, while a value of [N/Fe]=0 is perfectly compatible with that found in metal-poor field stars, a value of [N/Fe]=1 (as determined by Carretta et al.) would be definitely much higher than that observed in un-evolved stars in the field, and it would point out to the need for a different source of N production in the formation process of GC. However, a high N abundance is clearly incompatible with the present spectra of this star.

Since the carbon isotopic ratio is a very sensitive diagnostic of mixing in stellar material which correlates well with [N/Fe] ratios (Spite et al. 2006), it would therefore be interesting to cross-check the inference derived from N abundances with carbon isotopic ratios. Unfortunately we do not have 12 C/ 13 C ratios for these two stars, nor for any of the dwarf stars in this cluster since all the 13 CH features are too weak to be measured (Carretta et al. 2005).

An unusually high N value was found in one NGC 6397 unpolluted star (Pasquini et al. 2004) and a general N overabundance in GC has been postulated, for instance, by Li and Burstein (2003). [N/Fe] among field stars has been studied in the last years by several groups, which found that [N/Fe] is approximately constant with metallicity in the range -3.0 ≤[Fe/H]≤ 0.4, as first shown by Carbon et al. (1987) and recently confirmed by Israelian et al. (2004) and Ecuvillon et al. (2004). According to our results, in NGC 6752, at odds with NGC 6397, the 'unpolluted' star 4428 does not show evidence for strong N overabundance with respect to field stars; also for this element, abundances in field and 'unpolluted' stars of NGC 6752 are extremely similar.

Discussion

The age of NGC 6752 and comparison of Be in GC with field stars

As mentioned in Sect. 1, the first detection of Be in a GC offered the opportunity to test the theoretical proposal that Be could be used as a cosmochronometer for the early evolution of the Galaxy. Adopting a standard model of GCRs spallation for the production of Be in the Galaxy, Pasquini et al. (2004) found an excellent agreement between the "Beryllium age" of NGC 6397 (13.4-13.5 Gyr, assuming a Galactic age of 13.7 Gyr), and the age of the cluster derived by Gratton et al. (2003) from main sequence fitting and theoretical isochrones (13.5 ± 1.1 Gyr). The use of Be as a cosmochronometer has been further exploited by Pasquini et al. (2005) to study the halo-thick disk formation, finding some evidence for a faster star formation in the halo and a slower one in the disk. The detection of Be in the stars of a second GC, NGC 6752, characterized by a higher metallicity than NGC 6397, and presenting strong chemical inhomogeneities, is expected to provide additional constraints to the evolution of Be in the early Galaxy.

The age of NGC 6752 and comparison of Be in GC with field stars

It is clear that the use of Be as cosmochronometer requires a wider observational support. In this sense observations of additional GCs and new comparison between "Be" and "evolutionary" ages are necessary steps.

To this goal, we will adopt as the representative Be abundance of NGC 6752 the value of log(Be/H) = -12.04 ± 0.15 measured in star 4428, which is the prototype of "unpolluted" stars in this cluster. With its high Li and O, and low Na, the atmosphere of this star is in fact expected to be composed mainly -if not entirely -of "unprocessed" gas (cfr. next section).

In Figure 3 we show the Be abundance vs. time according to the same Galactic chemical evolution model adopted in Pasquini et al. (2005) (from Valle et al. 2002), together with the measured values of Be in NGC 6397 and NGC 6752. The assumed age for both clusters is the "isochrone age" derived by Gratton et al. (2003), 13.5 ± 1.1 Gyr and 13.4 ± 1.1 Gyr for NGC 6397 and NGC 6752, respectively. The age difference of 0.1 Gyr between the two clusters is much smaller than the error on the age determination. As in the case of NGC 6397, also for Fig. 3. Evolution of Be with time in the Galaxy according to a three-zone Galactic chemical evolution model (Valle et al. 2002). The three curves refer to the halo, thick disk, and thin disk. The data points show the Be abundance in the young open cluster IC2391 (age 55 Myr), the Sun (age 4.5 Gyr), the globular clusters NGC 6397 (isochrone age 13.5 ± 1.1 Gyr) and NGC 6752 (isochrone age 13.4 ± 1.1 Gyr). The model result is normalized to the solar meteoritic abundance. The inset shows a detail of the early Galactic evolution. The Be abundance of IC2391 is from Randich et al. (2002); the Be abundances of the two GCs are from Pasquini et al. (2004) for NGC 6397 and from this work for NGC 6752. The isochrone ages are from Gratton et al. (2003).

NGC 6752 the agreement with the model predictions is excellent, reinforcing the validity of Be as an alternative (or additional) age estimator. A direct comparison between the observed and predicted abundance of Be in NGC 6752 would imply a "Be age" of 13.3-13.4 Gyr for this cluster, or about 0.1 Gyr less than the age of NGC 6397.

As we considered somewhat fortuitous the extremely good agreement between the "Be" and "isochrone"ages for NGC 6397, we also consider fortuitous the excellent agreement between the two age determinations for NGC 6752. It is worth noticing however, that, although the determination of the absolute value of the age of each cluster may be affected by large uncertainties with either method, the agreement in the relative age difference can be considered as a more robust result, as the effects of systematic errors should cancel. Pasquini et al. (2004) noticed that the Be abundance of the NGC 6397 stars is fully consistent with the wellknown Be vs. Fe trend (Gilmore et al. 1992;Molaro et al. 1997;Boesgaard et al. 1999 -hereafter B99). In Figure 4 we show the Be vs. Fe relation for the field stars from B99, together with the NGC 6397 points and the NGC 6752 value determined in this work. Figure 5 is similar, but Be is plotted as a function of O rather than Fe. Note that, as discussed by Pasquini et al. (2004), O abundances of field (B99) and cluster (G01) stars are on the same scale.

The agreement between the cluster and the field stars is impressive: they are indeed indistinguishable in the Be vs. Fe plane. In the Be vs. O plane the situation is slightly different, because the GC formation process depletes O; this point will be discussed in the next session. However, insofar as 'unpolluted' cluster stars are considered, they are basically indistinguishable from field stars not only as far as nucleosynthesis elements are concerned, but this similarity extends to primordial nucleosynthesis products (Li) and to elements produced by GCRs spallation (Be).

A very simple conclusion is that Be production (or more precisely, the Be/Fe ratio) seems to ignore the environment where the stars are born. We believe that this is a strong evidence in favour to the fact that the Be production in the early Galaxy is largely independent of the local ISM abundances; rather it reflects some global process, as we proposed in our previous work.

The approximately linear and tight relation between Be an Fe also suggests that their production sites are linked, and that they are also fairly independent of the formation site in the early Galaxy. The simplest explanation is that they are produced by the same type of events: supernovae, and that the medium was quite homogeneous at a given time, as far as Fe and Be are concerned.

It will be very difficult to prove the unicity of this solution, but the data collected so far are compatible with it. One test could be the observations of the Be lines for many stars of different populations and similar [Fe/H]; would the trends found by Pasquini et al. (2005) be confirmed by larger samples of stars, not only the hypothesis of Be as a clock would be re-inforced, but also new insights about the mechanisms of halo and disk formation would be gained.

Beryllium and globular cluster formation

Our Be and N observations must be inserted in the general context of cluster chemical anomalies (see e.g. Gratton et al. 2004). In particular (see e.g. the discussion in Bonifacio et al. 2002, Pasquini et al. 2004, and Pasquini et al. 2005), one of the possibilities of explaining the presence of Li in GC stars is that this element, destroyed in the previous generations of stars, has been also produced by them, for instance during the AGB phase, and brought to the surface by the so called Cameron-Fowler (Cameron and Fowler 1971) mechanism.

Be cannot be produced in stars, so its mere detection in NGC 6752 (as well as in NGC 6397) shows beyond any doubt that the gas which formed the stars being here studied, has been sitting in a cloud and exposed to the GCRs for a few hundred million years.

In principle, at least for the NGC 6752 stars, one could invoke that the chemical composition we see now is the product of a more recent mixing (or mixture) of 'pristine' (Be-rich) and 'processed' (Be-poor) gas (see e.g. Charbonnel and Prantzos 2006 for a similar approach).

The observations of NGC 6397 are difficult to be explained simply by a dilution scenario. The large N abundance observed in most stars of this cluster indicate transformations of a fraction of the original O into N, in agreement with the only moderate excess of O (roughly a factor of about 2 smaller than in field stars having the same excess of α-elements). This would suggest a similar dilution factor for the "pristine" material in that cluster, requiring a significant Li content in the "processed" material in order to recover the Spite plateau value, approximately observed in all NGC 6397 stars. Such a Li production would in turn favour the AGB stars as sources of the "processed" material. A further requirement is that this "processed material" should also be rich in Be, since the Be content of NGC 6397 is the correct one for its [Fe/H]: this requires exposition to GCRs for a few 10 8 yrs. These coincidences seems rather hard to accept, mainly if present in several clusters.

On the other hand, the case of NGC6752 seems easier. In this case, the most direct measure of the dilution of pristine material with processed ones in O-poor stars is provided by a comparison of the abundance of O with that observed in the most O-rich stars in the cluster, that we may assume to be composed of "unprocessed" material. Star 4428 is a good candidate for such group: indeed it has an abundance pattern similar to that of field stars of the same metallicity for all observed elements (Be, Li, CNO, Na, etc., if the N abundance provided by our robust NH observation is adopted). For what concerns stars made of "processed" material, unfortunately, the O abundance is not available for star 200613; however, we may assume for this star the same dilution factor we would obtain for stars having a similar composition, like e.g. star 4907 (G01, Carretta et al. 2005), for which [O/Fe]=-0.25. The difference in O content between stars 4428 and 4907 indicates a dilution factor of ∼ 4. This is slightly more than the difference in the Li content (∼ 3); however, this difference is likely within the observational uncertainties (while not excluding some fresh Li production). We should then expect a similar difference in the Be abundances. Our spectrum of star 200613 shows only an upper limit, that is fully compatible with a similar abundance of Be. Hence current observations of NGC 6752 may well be explained by a variable mixing of "pristine" and "processed" material, and the processed material could originate both in AGB stars and massive rotating ones. Again, the presence of Be indicates that the 'pristine' material has been exposed to GCRs for a few 10 8 yrs. This strongly suggest that the material from which this cluster formed followed a chemical evolution similar to those of field stars of similar metallicity.

Be abundance variations in the cluster ?

Due to measurement errors and low S/N of star 200613, our upper limit value does not allow us to exclude that its Be abundance is lower than that of star 4428, nor that, instead, the two stars have a similar Be content. We can however firmly exclude that it has a Be abundance which is a factor of 2 larger. The determination of a Be abundance difference between polluted and unpolluted stars is possibly the only way to understand how many generations of stars have preceeded the one we actually observe.

While it is well established that GCs have suffered by pollution from a previous generation of stars in the CNO elements and perhaps He (see e.g. Carretta et al. 2005, Piotto et al. 2005), it is not clear which progenitor has been responsible for the cluster chemical pollution. Several authors (e.g. Ventura et al. 2002, D'Antona et al. 2005) Both scenarios have in common that the polluters are a second generation of stars having the same metallicity of the cluster and composition of the field, which pollute the ISM in light elements but not in Fe. The "polluted" stars were a subsequent (third) generation, born from (or contaminated by ) the ejecta of the second generation.

The unpolluted stars should therefore be "second generation"1 stars, similar in everything to the field stars, while the "polluted" objects are heavily contaminated, third generation. If this idea, common to both scenarios, were true, then we may expect a difference in Be abundance between the two generations of objects. The third generation of objects should have a younger age, and therefore a larger Be abundance. On the other hand the polluting material should be Be-depleted due to the high temperatures necessary to form the excess nitrogen. Therefore there are a priori three possibilities:

1. the third generation stars have higher Be abundance; in this case the age difference is large enough that a significant amount of Be has built up and/or the fraction of Be-depleted polluting material is small enough not to cancel this signature. 2. the third generation stars have lower Be abundance; in this case the age difference between the second and third generation is small and the pollution by Bedepleted material dominates. 3. the third generation stars have the same Be abundance as the second generation stars; this situation may arise in a number of ways by fine tuning the age difference between the two generations and the fraction of Bedepleted polluted material incorporated into the new stars; ranging from very small age difference and little pollution to large age difference and large pollution.

Our observations clearly rule out the first of the three cases, but the other two remain viable. The possibility of fine tuning age and pollution in the third case is however limited by the observations of O and Li. From these two elements the dilution factor is in the range three to four and should be the same also for Be. Therefore, in principle, if the two stars had the same Be abundance we could estimate the age difference using this dilution factor and Fig. 3, assuming that the total amount of CNO, necessary for the Be production, has not changed.

If the Be content of star 200613 were similar to that of the unpolluted star 4428, for sake of completeness, we should mention that there is another scenario which could be envisaged: polluted and unpolluted stars belong to the same generation, and the metals and the CNO anomalies are produced by the same preceding generation, but perhaps by stars of different stellar masses. The previous generation of GC stars was extremely metal poor (perhaps even zero metal), dominated by stars of large masses; these exploded and ejected mass very quickly and their total mass was a substantial fraction of the GC total masses. The Supernovae contributed to the global enrichment of metals in the Galaxy, without leaving any local inhomogeneity, while CNO processing and ejection through the slower stellar winds, prior to SNe explosions, produced the differential enrichment in the cluster of Na, N, Mg, and the depletion of Li in the cluster gas. It should be however noted, that this scenario requires that the SN explosions do not lead to a total mixing of the gas, which would cancel the inhomogeneities created by the winds. This may be difficult to reconcile with the cluster homogeneity in Fe, which is better than 10%. Additionally, it might be difficult to reconcile the required nucleosynthesis with current understanding of the yields from core collapse SNe.

An accurate determination of the Be abundance in the polluted star 200613 should enable us to discriminate among competing scenarios.

Fig. 1 .

 1 Fig. 1. UVES spectra of the two turn-off stars 200613 and 4428 of the GC NGC 6752. The dotted lines correspond to the Be abundances log(Be/H)= -12.2 (upper limit) for star 200613 and log(Be/H)= -12.04 for star 4428.

Fig. 2 .

 2 Fig. 2. UVES spectra of stars 200613 and 4428 around the NH band at 336 nm. The band is much stronger in star 200613, indicating a strong N overabundance. Solid lines are the observed spectra; thin lines are synthetic spectra computed with parameters appropriate to the program stars, and N abundances of [N/Fe]=0, 0.4, 0.8, 1.2, 1.6 and 2.0.

Fig. 4 .

 4 Fig. 4. Be abundance vs. Fe for the NGC 6397 (filled circles) and the NGC 6752 TO star (filled square) and the star from B99, (open circles). The stars in the clusters are indistinguishable from the field stars.

Fig. 5 .

 5 Fig. 5. Be abundance vs. O for the NGC 6397 (filled circles) and the NGC 6752 TO stars (filled square) and the star from B99, (open circles). The 'unpolluted', O-rich stars in the clusters closely follow the field stars.

Table 1 .

 1 NGC 6752 sample stars, their atmospheric parameters and abundances. The atmospheric parameters, [Fe/H], [O/H] and [Na/Fe] are from G01; Log(Li/H) from Pasquini et al. (2005), [N/Fe] and log(Be/H) from this work.

	Star	T eff	log g [Fe/H] [O/H] [Na/Fe] log(Li/H) [N/Fe] log(Be/H)
	4428	6226 4.28	-1.52	-1.28 -0.35	2.50	0	-12.04 ± 0.15
	200613 6226 4.28	-1.56	/	-0.09	2.13	1.6	< -12.2

  favour intermediate mass AGB stars, while others (Meynet and Maeder 2006, Prantzos and Charbonnel 2006) consider massive, rotating donors.

We adopt here the term second generation in a rather loose sense, simply to indicate that GC stars where preceded by several generations of stars.

Acknowledgements. We are grateful to the referee, R. Cayrel, for pointing out the consequences of excluding a large Be abundance in star 200613. PB acknowledges support from the MIUR/PRIN 2004025729 002 and from EU contract MEXT-CT-2004-014265 (CIFIST). We would like to emphasize as these observations are absolutely challenging and we thank the UVES team for building such a wonderful instrument and the VLT operations for carrying them on.