Experimental and Theoretical Studies of the Factors Affecting the Cycloplatination of the Chiral Ferrocenylaldimine ( SC )-[ ( η 5C 5 H 5 ) Fe { ( η 5C 5 H 4 ) – C ( H ) = N – CH ( Me ) ( C 6 H 5 ) } ]

The study of the reactivity of the enantiopure ferrocenyl Schiff base (SC)-[FcCH=N–CH(Me)(C6H5)] (1) (Fc = (η5-C5H5)Fe(η5-C5H4)) with cis-[PtCl2(dmso)2] under different experimental conditions is reported. Four different types of chiral Pt(II) have been isolated and characterized. One of them is the enantiomerically pure trans-(SC)[Pt{κ1-N[FcCH=N–CH(Me)(C6H5)]}Cl2(dmso)] (2a) in which the imine acts as a neutral N-donor ligand; while the other three are the cycloplatinated complexes: [Pt{κ2-C,N [(C6H4)–N=CHFc]}Cl(dmso)] (7a) and the two diastereomers {(Sp,SC) and (Rp,SC)} of [Pt{κ2-C,N[(η5-C5H3)–CH=N–{CH(Me)(C6H5)}]Fe(η5-C5H5)}Cl(dmso)] (8a and 9a, respectively). Isomers 7a-9a, differ in the nature of the metallated carbon atom [CPh (in 7a) or CFc (in 8a and 9a)] or the planar chirality of the 1,2-disubstituted ferrocenyl unit (8a and 9a). Reactions of 7a–9a with PPh3 gave [Pt{κ2-C,N[(C6H4)–N=CHFc]}Cl(PPh3)] (in 7b) and the diastereomers (Sp,SC) and (Rp,SC) of [Pt{κ2-C,N[(η5-C5H3)–CH=N–{CH(Me)(C6H5)}] OPEN ACCESS Inorganics 2014, 2 621 Fe(η5-C5H5)}Cl(PPh3)] (8b and 9b, respectively). Comparative studies of the electrochemical properties and cytotoxic activities on MCF7 and MDA-MB231 breast cancer cell lines of 2a and cycloplatinated complexes 7b-9b are also reported. Theoretical studies based on DFT calculations have also been carried out in order to rationalize the results obtained from the cycloplatination of 1, the stability of the Pt(II) complexes and their electrochemical properties.


Figure 1.
A selection of cyclometallated Pt(II) complexes with a σ(Pt-C Ph ) bond and potent cytotoxic activity reported recently.For illustrative purposes IC50 values (in µM) for A-C in a specific cancer cell line are given: in HCT-116 colon cell line for A compounds: IC50 = 28 ± 2.4 µM (R 1 = H) and 18 ± 1.9 µM (R 1 = Cl); for type B, IC50 in the range 0.95 µM-7.7 µM (IC50 for cisplatin = 40 ± 4.4 µM).Compound C was tested in the A549 breast cancer cell line, IC50 = 4.60 µM (for cisplatin = 8.0 µM) [29][30][31][32].It is well-known that the properties of cycloplatinated compounds, their biological or catalytical activities and their applications are strongly dependent on a wide variety of factors, such as the nature of the donor atoms of the metallated ligand, its denticity, the size of the metallacycle, the electronic and steric effects induced by the presence of substituents and their location and even the nature of the ancillary ligands and their relative arrangement [1][2][3][4][5][6][7][8][9][10].
On the other hand, N-donor ferrocenyl ligands are valuable reagents to achieve heterodimetallic Pt (II) compounds [34][35][36][37][38][39][40][41][42][43].These ligands may produce different types of products depending on the relative arrangement between the N-donor atom and the presence of: (a) one or more σ(C-H) bonds, with the proper orientation as to undergo metallation, and/or (b) additional heteroatoms with good donor abilities and their location [36,37,40,41].Moreover, for monosubtituted ferrocene derivatives metallation of the C5H4 ring induces planar quirality [34,35,38].Despite the variety of cyclometallated Pt(II) complexes derived from achiral N-donor ligands described so far, enantio-or diastereomerically pure platinacycles are scarce [39][40][41][42][43][44][45][46][47] and those containing a Pt-C Fc bond are even less common [39][40][41][42][43].However, the examples described so far are especially outstanding because of their singular properties, behaviors or activities [39][40][41][42][43].A few representative examples (A-D) are shown in Figure 2. Compound A is an excellent catalyst on the asymmetric aza-Claisen rearrangement of Z configured trifuoroacetaamidates giving high enantioselectivities [39] and diastereomers B and C (Figure 2), form an acid-base dependent chiral molecular switch [40,41].Platinacycles D and E are potent cytotoxic agents in lung (A549), breast (MDA-MB231) and colon (HCT-116) cell lines.Moreover, complex E: (a) is less toxic than cisplatin and active in other cancer cell lines (i.e., NCI-H460); (b) induces nuclear translocation of endogenous FOXO3a and a FOXO3a reporter protein in A549 and U2OS cells, respectively; and (c) has a synergic effect with cisplatin [42,43].All these findings suggest that compounds containing simultaneously ferrocenyl units and cycloplatinated rings are promising candidates for the design of multifunctional products with both biological activity and interesting electrochemical properties (due to the presence of a redox active center).This is still an emerging topic and further work is needed to clarify some key points.Unfortunately, isomeric platinacycles differing exclusively in the nature of the metallated carbon have not been described so far, and thus the effect produced by the characteristics of the carbon atom bound to the Pt(II) (i.e., C Fc versus C Ph ) on their properties and activities still remains unknown.
Since during the preparation of D and E (Figure 2), no evidences of the formation of platinacycles arising from the metallation of the phenyl ring were detected [42], we decided to investigate whether for (SC)-[FcC(H)=N-CH(Me)(C6H5)] (1) (Figure 3) [48], the relative orientation between the phenyl ring and the N atom and their proximity could also allow the isolation of platinacycles with a Pt-C Ph bond.Ligand 1 may exhibit different binding modes and hapticities giving a wide variety of Pt(II) complexes (Figure 3).If 1 binds to the Pt(II) atom through the imine nitrogen exclusively, different isomeric forms of the optically active complexes: [Pt{κ 1 -N[FcC(H)=N-CH(Me)(C6H5)]}X2(L)] could be expected (2-4 in Figure 3).They differ in the anti-(E) (in 2 and 3) or the syn-(Z) (in 4 and 5) form of the imine and a trans-(in 2 and 4) or cis-(in 3 and 4) disposition of the X − ligands.Moreover, 1 has several carbon atoms susceptible to undergo metallation.Four types of platinacycles (Figure 3, 6-9) could arise from monometallation of 1.In two of them (6 and 7) there is a σ(Pt-C Ph ) bond, and the imine adopts the anti-(E) (in 6) or syn-(Z) (in 7) forms; while the other two (8 and 9) are diastereomers (Sp,SC) and (Rp,SC) arising from platination of the C5H3 ring that induces planar chirality [34,35,38].
A few examples of cyclometallated Pt(II) compounds with (C,N,C) 2− pincer ligands have been reported [49][50][51][52], and thus the activation of a σ(C Ph -H] and a σ(C Fc -H] bond to give compounds 10 or 11 (Figure 3) cannot be disregarded.Furthermore, and since several groups have achieved cylometallated Pt(IV) complexes in the reaction of cis-[PtCl2(dmso)2] with N-donor ligands [23,53,54], the formation of the platinum(IV) derivatives cannot be ruled out either.
In this work, we report: (a) the synthesis of chiral Pt(II) complexes in which 1 adopts different binding modes {(N), (C Fc , N) − or (C Ph , N) − }, (b) a comparative study of their electrochemical and cytotoxic activities in two breast cancer cell lines, and (c) a theoretical study based on DFT calculations to explain the selectivity of the metallation process and the properties of the new Pt(II) compounds.
Elemental analyses and mass spectra of the major component (Supporting Information) were consistent with those expected for any of the four isomers of [Pt(1)Cl2(dmso)] (Figure 3, types 2-5 with X = Cl and L = dmso (2a-5a)).In the 1 H-NMR spectrum the signals due to the protons of the dmso ligand appeared as one singlet at δ = 3.40 ppm ( 3 JH,Pt = 19 Hz).This is characteristic of trans-[Pt {κ 1 -N[FcC(R 1 )=N-R 2 ]}Cl2(dmso)] (R 1 = H or Me) complexes [20,36,[40][41][42].Moreover, in its ( 1 H-1 H) NOESY spectrum, the signal of the imine proton showed cross peaks with those of the H 2 and H 5 protons of the Fc unit, and also with the resonances of the −CH(Me)-moiety.Thus indicating that imine 1 adopts the anti-(E) form.All these features suggested that in the major product the arrangement of the ligands corresponded to type 2 shown in Figure 1 (with X = Cl and L = dmso, herein after referred to as 2a).
It should be noted that: (a) the molar ratio 2a:7a did not vary significantly when the reaction period (t) increased from 2 h (1.00:0.13) to 2 days (1.00:0.15),and (b) no evidences of the presence of platinacycles with σ[Pt−C(ferrocenyl)] bonds were detected by 1 H-NMR of the crude of the reactions.In view of these and since the formation of platinacycles with bidentate (C Ph or C Fc , N)] − ligands commonly requires the presence of a base (i.e., NaOAc or the ligand itself), [20,36,37,[40][41][42], we also studied the reactivity of 1 toward Pt(II) in the presence of NaOAc.
Treatment of ligand 1 with cis-[PtCl2(dmso)2] and NaAcO (in a molar ratio 1:Pt(II):OAc − = (1:1:2)) in a mixture of toluene: MeOH (4:1) under reflux for 2 h (Scheme 1, step B) followed by the work-up of a chromatographic column, gave small amounts of FcCHO (18 mg), compounds 2a, 7a and traces (ca. 9 mg) of a purple solid (hereinafter referred to as II).Elemental analyses of II agreed with those expected for the platinacycles with 1 acting as a (C,N) − ligand.Its 1 H-NMR spectrum showed two sets of superimposed signals (of relative intensities 1.00:0.90)with the typical pattern of 1,2-disubstituted ferrocene derivatives.Thus, indicating the presence of two species with a Pt−C Fc bond in solution.Since metallation of the C5H4 ring induces planar chirality [20,38,[40][41][42][58][59][60], two diastereomers ((Sp, SC) and (Rp,SC)) could be expected in principle (Figure 3, types 8 and 9 with X = Cl and L = dmso (8a and 9a, respectively)).Consequently II contains a mixture of both isomers.Longer reaction periods (up to 6 days) produced significant variations of the relative abundance of 2a, 7a and II, (from 1.00:0.21:0.09(for t = 2 h) to 1.00:0.46:0.64(for t = 2 days)); and after 6 days, the typical resonances of 2a were not detected in the 1 H-NMR spectrum of the crude of the reaction and only FcCHO, 7a and II were isolated after the work up of the column.These results suggested that the formation of isomers (8a and 9a) was slow.
Since: (a) the crystal structure of 2a revealed that the C−H bond of the ferrocenyl unit is quite close to the Pt(II) ion (distance Pt•••H(9) = 2.866 Å); and (b) the relative proportion 2a: II decreased with time; we tentatively assumed that probably 2a was the precursor of the mixture II.In order to test this hypothesis, compound 2a was treated with NaOAc (in a 1:2 molar ratio) in toluene:MeOH (4:1) under reflux for 6 days and then filtered and concentrated to dryness.NMR studies revealed the presence of FcCHO and the two diastereomers of II in a relative ratio (1.00:0.86)which is close to that obtained when the reaction was performed using ligand 1, cis-[PtCl2(dmso)2] and NaAcO (1.00:0.90).
All the results presented here reveal that despite it is well-known that ferrocene derivatives are more prone to undergo electrophilic attacks than their phenyl analogues [34,35,58,61], for 1, that has different types of σ(C−H) bonds susceptible to metallate, the formation of the platinacycle 7a with a σ[Pt−C(phenyl)] is achieved under milder experimental conditions than those required to isolate the 8a and 9a with a σ[Pt−C(ferrocenyl)] bond.Unfortunately, attempts to separate the two components of II by fractional crystallisation or SiO2 column chromatography failed and only solids enriched in ca.70%-80% could be isolated by successive chromatographic columns.
Since it is well-known that platinacycles with PPh3 ligands are more cytotoxic than their dmso analogues, we decided to study the reactivity of the platinacycles with PPh3.As expected, treatment of II with the equimolar amount of PPh3 gave the diastereomers (Rp, SC) and (Sp, PPh3)] (8b and 9b, respectively) that could be isolated after a careful work-up of a SiO2 column (Scheme 1, step C).In order to compare the electrochemical properties and the cytotoxic activities of isomeric compounds differing only in the nature of the metallated carbon, we also transformed platinacycle 7a in CH2Cl2 following identical procedure and we isolated the Characterization data of 7b-9b (Supplementary Information) agreed with the proposed formulae. 31P{ 1 H}-NMR data for 7b (δ = 19.1 ( 1 JPt−P = 3801 Hz)), 8b (δ = 15.9 ( 1 JPt−P= 4191 Hz)) and 9b (δ = 15.7 ( 1 JPt−P = 4202 Hz)) are similar to those reported for related platinacycles with "(C Ph or C Fc , N,P,Cl)" cores and a trans-arrangement of the PPh3 ligand and the imine nitrogen in good agreement with the transphobia effect [61]. 195Pt NMR spectra of compounds 7b-9b showed a doublet due to coupling with the 31 P-nucleus since it is well-known that an upfield shift in 195 Pt NMR is related to a strong donor interaction [62,63], Thus, differences detected in the 195 Pt chemical shifts of 7b and those of 8b and 9b can be taken as a measure of the different donor abilities of imine 1 adopting the (C Ph ,N) − or the (C Fc ,N) − binding mode.
At this point and due to the increasing interest in platinacycles with (C,N,C') 2− pincer ligands we turned our attention to 8b and 9b.Molecular models revealed that in both isomers the C−H bond on the ortho site of the phenyl ring has the proper orientation as undergo metallation to give platinacycles with 1 acting as a (C Fc ,N, C Ph ) 2− pincer ligand (Figure 2, types 10 and 11 with X = Cl and L = PPh3 (10b and 11b)) and having central and planar chirality.In view of this, we also studied the reactions of the 8b (or 9b) with equimolar amounts of a NaOAc under reflux for 2 days in the toluene: MeOH (4:1) mixtures.However, no significant change was observed and 1 H-NMR spectra of the raw materials did not reveal the presence of any new Pt(II) complex.

Study of the Cytotoxic Activity
Two human breast cancer cell lines (MCF7 and MDA-MB231) were used to test the cytotoxic activity of complex 2a and the platinacycles (7b-9b).For comparison purposes, cisplatin (as positive control) was also evaluated under the same experimental conditions.The IC50 values corresponding to the inhibition of cancer cell growth at 50% level are listed in Table 1.
Table 1.Summary of: (a) the cytotoxic activities (IC50 (µM)) of compound 2a and the platinacycles 7b-9b in the MCF7 and MDA-MB231 breast cancer cell lines a ; and (b) their electrochemical properties (Electrochemical data (anodic (Epa) and cathodic (Epc) potentials, separation between peaks (ΔE = Epa -Epc) and the ratio between the intensities of the anodic and cathodic peaks Ipa/Ipc)) b .The comparison of in vitro cytotoxic activities of 2a, 7b, 8b and 9b against the MCF7 and MDA-MB231 breast cancer cell lines (Table 1 and Figure 5a) shows that complex 2a, where 1 acts as an N-donor group, did not show any significant effect on the MCF7 cell line and only a moderate activity in MDA-MB231 cell line and platinacycles 7b-9b were more potent than 2a in the two cell lines assayed.In fact, their potencies increase according to sequence (1).Among the tested compounds, complex 2a is a weaker cytotoxic agent than 7b; while platinacycle 9b, (and in a lesser extent, 8b) are the most potent ones (IC50 in the ranges: 9-11 µM for MCF7 and 5.8-6.3µM for the triple negative (ER, PR and no HER2 over expression) MDA-MB231 cell line).In particular, in particular the activity of 9b is ca.1.3 times (in MDA-MB231) or 2.1 times (in the MCF7) bigger than that of cisplatin.These findings indicate that platinacycles with Pt-C Fc bond (8b and 9b) are more active than 7b, where the Pt(II) ion is bound to a C Ph atom.The cytotoxic activity of the two diastereomers (8b and 9b) is a bit different, if significant.This agrees with results obtained for compounds D and E (Figure 2) and suggest that the planar quirality of the metallated ring is not as important as the type of metallated carbon.Some authors have also found that for substituted ferrocene derivatives with stereogenic carbon atoms and their transition metal complexes, the absolute configuration does not affect significantly their cytotoxic activity [64][65][66].
Taking into account these results, trend (1) can be re-written as that is a relationship between binding mode of 1 to the Pt(II) and the cytotoxic activity of the complexes.

Electrochemical Properties
In a first attempt to elucidate the effects produced by the mode of binding of the Schiff base to the Pt(II) atoms, and the nature of the metallated carbon atom on the electronic environment of the iron (II), we also studied the electrochemical properties of 2a,7b, 8b and 9b.It should be noted that 1 H-NMR spectra of these products in CD3CN revealed they were stable in this solvent.The electrochemical studies were carried out by cyclic voltammetry of freshly prepared solutions (10 −3 M) in acetonitrile using (Bu4N)[PF6] as supporting electrolyte.All these experiments were carried out at different scan rates υ (υ = from 0.05 to 1.0 V × s −1 ).A summary of the electrochemical data for all the compounds under study is presented in Table 1.
The cyclic voltammograms (CVs) (Figure 5b) exhibited one anodic peak with a directly associated reduction in the reverse scan, intensities ratio Ipa/Ipc was close to 1.0 and the relationship between the Ipa and υ ½ was lineal.These findings are consistent with those expected for a simple reversible one-electron process [67].The main difference between the CVs of 2a,7b,8b and 9b is the position of the waves, that shift to the anodic region as follows: For the platinacycles 7b-9b the trend is similar to that obtained for cyclopalladated compounds arising from the metallation of the Ph ring or the Fc unit of (1SC,2RC)-[(η 5 -C5H5)Fe{(η 5  It is well-known that the proclivity of ferrocene derivatives to oxidize is strongly dependent on the nature of the substituents [41,59,61,67,68].In general, the presence of electron withdrawing groups produces an increase of the Epa value; while donor groups have the opposite effect.On these basis, sequence (4) provides conclusive evidences of the influence of the binding mode of the ligand 1 to the Pt(II) on their the electrochemical properties of the complexes.
Compounds 8b and 9b with the (C Fc ,N) − ligand are more prone to oxidize than 2a where 1 binds to the Pt(II) through the N atom exclusively; while platinacycle 7b is even more resistant to oxidation.Thus indicating that in 7b the metallacycle has greater electron pulling effect on the ferrocenyl array than in 8b, 9b.Comparison of electrochemical data of platinacycles 7b-9b and their biological activity, reveals that 7b is simultaneously less prone to oxidize and a weaker cytotoxic agent than 8b and 9b.This relation shift has also been found for other ferrocene-containing compounds [69,70].

Theoretical Studies
In a first attempt to rationalise the experimental results and in particular, to explain: (a) why for 1 the cycloplatination of the phenyl ring occurs; and (b) the electrochemical properties of the Pt(II) complexes, we decided to undertake DFT calculations of the imine 1 [in the anti-(E) and syn-(Z) forms, herein after referred to as 1 E and 1 Z , respectively], the isomers of [Pt{FcC(H)=CH(Me) (C6H5)}Cl2(dmso)] (2a and 3a) and platinacycles 6-9 of Figure 2 (X = Cl and L = dmso (6a-9a)).
All the calculations were carried out using the B3LYP hybrid functional [70,71] and the LANL2DZ basis set [72,73] implemented in the Gaussian03 program [74].The geometries of compounds and were optimized without imposing any restriction.The bond lengths and angles of the optimized geometry of 2a were consistent with those obtained from the X-ray studies (the differences did not clearly exceed 3σ).
For each one of the molecules under study in their optimized geometries the total energy (ET) was calculated.The results obtained showed that the free ligand in the syn-(Z) conformation (1 Z ) is 5.29 kcal/mol higher in energy than the anti-(E) isomer (1 E ).Thus, suggesting that the energy required for the process 1 E → 1 Z (Scheme 2, step A) is expected to be small, but slightly greater than for the

anti-(E)→syn-(Z) isomerization of the ligand in trans-[Pt{FcC(H)=CH(Me)−(C6H5)]}Cl2(dmso)] (2a) (Scheme 2, B).
Complex 2a has several sites susceptible to metallate which would produce either 6a (Scheme 2, step C) or the isomers 8a and 9a (Scheme 2, steps D and E); but for 3a only the metallation of the phenyl ring, to give 7a, is possible (Scheme 2, step F).In steps C-F the formation of the platinacycles requires the release of one of the Cl − ligands and the activation of the corresponding σ(C−H) bond.On the other hand, the experimental work shows that despite the Fc unit is more prone to undergo electrophillic attacks than the phenyl ring [22], the formation of 7a is achieved under milder experimental conditions than for 8a and 9a.
As a first approach to rationalize experimental results, first we determined the energy for the optimized geometries of 6a-9a.The minimum value was obtained for 9a; for 8a, it was only 0.71 kcal/mol higher; but for 7a and 6a the calculated energies exceed, (by 4.05 and 16.42 kcal/mol, respectively), that obtained for 9a.
We also calculated the energy variations for reactions C-F, where complex 2a is transformed into the platinacycles 6a, 8a and 9a; or 3a into 7a.These processes involve the activation of the C-H bonds and the formation of the monocycloplatinated products.Data  Using the ET values for isomers 6a-9a and T = 298 K, we also calculated the Boltzmann's distribution to estimate the relative proportion of the four isomers: 51.3 (for 8a), 38.6 (for 9a), 10.0 (for 7a) and 0.1% (for 6a).Except for 6a, that was neither isolated nor detected in any of the reactions studied, the relative amounts of the isomers 7a-9a formed when the reaction was performed using cis-[PtCl2(dmso)2], the ligand 1 and NaOAc (in a 1:1:2 molar ratio) follow the trend 7a << 9a < 8a that is in good agreement with the results reached from the Boltzman's analyses.
Experimental work revealed that 2a could be converted into 8a and 9a, but this process required the presence of a two fold excess of NaOAc and long refluxing periods (6 days).However, attempts to achieve platinacycles with 1 acting as (C Fc ,N,C Ph ) 2− from 8a or 9a failed.ΔET calculated for steps G and H of Scheme 2 is high (more than 2.7 times the ΔET of D or of E).This may explain why no evidences of the formation of compounds 10a or 11a were detected in any of the reactions investigated.
On the other hand, experimental results indicate the nature of the C atom bound to the Pt(II) in platinacycles 7b-9b produces great changes in their electrochemical properties.Since the oxidation requires the removal of the electron from the highest occupied molecular orbital (HOMO), molecular orbital calculations were also performed for platinacycles 7a-9a, for comparison purposes we also included complex 6a in these calculations.
For 6a and 7a, the HOMO (Figure 6), is mainly formed by the contribution of a 5d orbital of the Pt(II), a 3p orbital of the chloride, and a π orbital of the metallated phenyl ring.The HOMOs of 8a and 9a are markedly different since they are mainly centered on the Fe(II) and the Pt(II) atoms.These findings confirm that for 6a-9a the HOMO is not only iron based.For 6a-9a LUMO orbitals (Figure 6) have a similar shape that results from the contribution of a π* orbital of the >C=N− group and to a lesser extent an atomic orbital of the Fe(II).In order to explain the different proclivity of compounds 7b-9b to undergo oxidation, we determined the energy of the HOMO (EHOMO).As shown in Table 2 EHOMO increases according to the sequence 6a ≈ 7a << 8a ≤ 9a, indicating that platinacycles 6a and 7a, with the (C Ph , N) − ligand, are less prone to oxidize than their analogues formed by metallation of the Fc unit.This trend agrees with the results obtained from CV that revealed that 7b less more prone to oxidize than 8b or 9b.

Preparation of the Compounds
A suspension containing 250 mg (5.92 × 10 −4 mol) of cis-[PtCl2(dmso)2] and 40 mL of MeOH (HPLC-grade) was refluxed until complete dissolution of the Pt(II) complex.Then, the hot solution was filtered out and filtrate was treated with 188 mg (5.92 × 10 −4 mol) of ligand 1.The reaction mixture was protected from the light with aluminium foil and refluxed for 2 h.After this period the solution was concentrated to dryness on a rotary evaporator, Afterwards the residue was dissolved in the minimum amont of CH2Cl2 and passed through a SiO2-column chromatography (13.5 cm × 2.3 cm) using CH2Cl2 as eluant.The first band collected gave, after concentration to dryness small amounts of FcCHO (18 mg).Once this band was collected the subsequent elution with CH2Cl2 produced the release of an adidional and wide band, that contained complex 2a.This band was collected and concentrated to ca. 5 mL.Slow evaporation of the solvent at 282 K produced the formation of small crystals of 2a that were collected and air-dried.(yield: 275 mg, 70.2%).

(SC)-[Pt{κ 2 -C,N[(C6H4)(Me)CHN=C(H)Fc]}Cl(dmso)] (7a)
This compound was obtained as a minor side-product during the preparation of 2a. and it was isolated from the column described in the preceding paragraph as follows.After the elution of the band containing 2a, the column was eluted with a CH2Cl2: MeOH (100:0.5)mixture.This produced the release of a tiny and narrow band that was collected and concentrated to dryness on a rotary evaporator.The solid formed was air-dried and then dried in vacuum for 2 days (yield: 34 mg, 9.3%).This mixture was obtained by using two alternative procedures (a and b) using either 1 and cis-[PtCl2(dmso)2] as starting materials (method a) or complex 2a (method b).
Method a. Cis-[PtCl2(dmso)2] (250 mg, 5.92 × 10 −4 mol) and imine 1 (188 mg, 5.92 × 10 −4 mol) were treated with 40 mL of toluene.Afterwards, a solution formed by NaOAc (98 mg, 1.19 × 10 −3 mol) and 10 mL of MeOH was added.The reaction mixture weas protected from the light with aluminum foil and refluxed for 6 days.After this period, the hot solution was filtered out and the filtrate was concentrated to dryness on a rotary evaporator.The residue was kept in a SiO2 dissicator overnight.Then it was dissolved in the minimum amount of CH2Cl2 (ca. 10 mL) and passed through a SiO2-column chromatography (13.5 cm × 2.3 cm) using CH2Cl2 as eluant.Concentration to dryness of the yellowish band eluted gave small amounts of FcCHO (22 mg).Then elution with a CH2Cl2: MeOH (100:0.5)mixture, produced two bands of which the first eluted one gave after evaporation of the solvent complex 7a (42 mg).The second band that was purple and remained on the top of the column was later on collected increasing the polarity of the eluant to 100:1.The solid II, containing the two diastereomers (8a and 9a, in a molar ratio 1.00:0.89),was isolated after concentration and the subsequent drying in vacuum for 2 days (140 mg).Method b.Compound 2a (120 mg, 1.8 × 10 −4 mol) was dissolved in 20 mL of toluene, then a solution formed by 30 mg of NaOAc (3.6 × 10 −4 mol) and 5 mL of MeOH was added.Then the flask containing the reaction mixture was protected from the light with aluminum foil and refluxed for 6 days.After this period, the nearly black solution was filtered through a Celite pad and the filtrate was concentrated to dryness on a rotary evaporator.The purple solid formed, that contained 8a and 9a (1.00:0.86),was washed with (3 × 5 mL portions) of n-hexane, air-dried and then dried in vacuum for 2 days (75 mg, 66%).

(SC)-[Pt{κ 2 -C,N[(C6H4)CH(Me)−N=C(H)Fc]}Cl(PPh3)] (7b)
Complex 7a (21 mg, 3.4 × 10 −5 mol) was dissolved in 15 mL of CH2Cl2, then, PPh3 (9 mg, 3.4 × 10 −4 mol) was added.The reaction mixture was protected from the light with aluminum foil and refluxed for 1 h.After this period, the undisolved materials were removed by filtration and the pale orange filtrate was concentrated on a rotary evaporator to ca. 5 mL.The container was kept overnight on a refrigerator.After this period the solution was passed through a fast and short SiO2 column (1.0 cm × 0.5 cm) using CH2Cl2 as eluant.The orange band released was collected and concentrated to dryness on a rotary evaporator.The solid formed was collected, air-dried and afterwards dried in vacuum for 2 days (yield: 27 mg, 69%).]Fe(η 5 -C5H5)}Cl(dmso)] (8a and 9a) and 50 mL of CH2Cl2, PPh3 (27 mg, 1.02 × 10 −4 mol) was added.The reaction mixture was refluxed for 1 h and then filtered out.The filtrate was concentrated to ca. 5 mL and passed through a short SiO2-column chromatography.The use of a CH2Cl2:MeOH (100: 0.2) mixture as eluant produced the release of a deeporange band which was collected and concentrated to dryness on a rotary evaporator.Finally, the solid was dried in vacuum for 3 days and the two diastereomers in a ca 1.0:0.9molar ratio (58 mg, 70%).

8b and 9b)
A 50 mg amount of the mixture of diastereomers (8b and 9b) was dissolved in the minimum amount of CH2Cl2 and passed through a SiO2 column (2.5 cm × 3.0 cm) using a CH2Cl2:MeOH (100:0.01)mixture as eluant.The purple band released was collected in ca.20 mL portions up to a total volume of 180 mL and then concentrated to dryness on a rotary evaporator giving 9b (23 mg).The solution obtained with the subsequent elution with 160 mL, lead after concentration, a mixture of the two isomers; while the final nine fractions collected produced isomer 8b (19 mg).

Conclusions
The results presented in this work reveal that the enantiopure imine 1 is a valuable ligand to achieve different types of chiral heterodimetallic complexes containing Pt(II) and Fe(II).Among the organic compounds that exhibit a similar rich and versatile chemistry in the presence of Pt(II) atoms, ligand 1 is, to the best of our knowledge, the first that can: (a) generate a variety of chiral complexes with ferrocenyl units and (b) adopt three different binding modes ((N) (in 2a), (C Ph ,N) − (in 7a and 7b) or (C Ph ,N) − (in 8a, 8b, 9a and 9b)), and thus leading to isomeric forms of cyclometallated Pt(II) complexes differing in Pt-C bond (C Ph (in 7a or 7b) or C Fc (in 8a, 8b, 9a and 9b)) or the planar chirality {Sp (in 8a and 8b) or Rp (in 9a and 9b)}.It has been reported that cyclopalladation of (1SC, 2RC) [(η 5 -C5H5)Fe{(η 5 -C5H4)−CH=N−CH(R)−CH(OH)(C6H5)}] (R = Me or i Pr) gives palladacycles in which this imine acts as a (C Fc ,N) − or (C Ph ,N) − ligand [59], but for Pt(II) only the diastereomers (Sp,1SC,2RC) and (Rp,1SC,2RC) of [Pt{κ 2 -C,N[(η 5 -C5H3)−CH=N−CH(Me)−CH(OH)(C6H5)]Fe(η 5 -C5H5)}Cl(dmso)] (D and E in Figure 2) were isolated.Thus, indicating that the intercalation of the "(RC) -CH(OH)-" unit between the stereogenic carbon of 1 and phenyl ring is important as to hinder the formation of platinacycles with a Pt−C Ph bond and a [6.6] bicyclic system (the six-membered metallacycle fused with the Ph ring).
Moreover, we have also demonstrated that an accurate control of the experimental conditions permits to control the preferential formation of a specific type of product as well as the nature of the C−H bond involved in the cycloplatination process.Although the selectivity of the cyclometallation process is low, it provides a simple method for achieving enantio-or diastereomerically pure organometallic Pt(II) complexes with one stereogenic carbon center and with (in 8a, 8b, 9a and 9b) or without (in 2a, 7a and 7b) planar chirality which are extremely scarce, but attracting more and more interest due to their potential utilities and applications.
The new Pt(II) complexes presented herein appear to be excellent candidates for use not only as precursors in asymmetric synthesis or catalysis, but also as electrochemical reagents or as cytotoxic agents.For instance, an accurate selection of the product allows the fine-tuning of the anodic and cathodic potentials in a wide range (i.e., Epa(minimum) = −0.157V (for 9b) and Epa(maximum) = 0.101 V (for 7b)).Thus, these compounds are also attractive in view of their potential utility as selective and specific electrochemical sensors or detectors.
Computational studies have also allowed us to explain the effects produced by the nature of the metallated carbon atom on the relative stability of platinacycles with "Pt(C,N)(X)(L) cores" (Figure 2 6-9), the regioselectivity of the process and also the shift of the waves detected in the cyclic voltammograms.
On the other hand, the biological studies reveal that platinacycle 7b is a weaker cytotoxic agent than cisplatin, but 8b and 9b are even more potent agents.This indicates that the binding mode of 1 in the platinacycles ((C Ph ,N) − or (C Fc ,N) − ) is important as to modify their biological activity.Moreover, the IC50 values of 8b and 9b in the MDA-MBD231 breast cancer cell line fall in the range obtained for compounds D and E (Figure 2), which exhibit a synergic effect with cisplatin [43].These findings open up new research paths mainly focused on the following areas: (a) the evaluation of the cytotoxic effect of 8b and 9b in other cancer cell lines including some cisplatin resistant ones, such as HCT-116 colon cancer cell line; (b) additional studies to bring information about their mechanism of action; (c) the chemical modification of the complexes i.e. by replacement of one or the two ancillary ligands to get neutral of ionic derivatives, to be tested on these two breast cancer cell lines.
polarimeter and the concentration is specified in the characterisation section of the corresponding compound.Mass spectra (FAB + ) were obtained with a VG-Quatro Fisions instrument using 3-nitrobenzylalcohol (NBA) as matrix.

A1.2. Crystallography
A prismatic crystal of trans-[Pt{κ 1 -N[FcC(H)=N-CH(Me)(C6H5)]}Cl2(dmso)] (2a) (Table A1) was selected and mounted on a MAR345 diffractometer with a image plate detector.Unit-cell parameters were determined from 15,901 reflections in the range 3° ≤ Θ ≤ 31° and refined by least-squares method.Intensities were collected with a graphite monochromatised Mo-Kα radiation.The number of reflections measured in the range was 14,219 of which 6,010 were non-equivalent by symmetry (Rint(on I) = 0.0031) and 5106 reflections were assumed as observed applying the condiction I > 2σ(I).Lorentz-polarisation and absorption corrections were made.The structure was solved by Direct methods using SHELXS computer program [78] and refined by full-matrix least-squares equation using The SHELX97 computer program [79] using 6010 reflections (very negative intensities were not assumed).The function minimised was Σ w ││Fo│ 2 − │Fc│ 2 │ where w = [σ 2 (I) + (0.0654P) 2 + 4.8984P] −1 and P = (│Fo│ 2 + 2│Fc│ 2 )/3, were taken from the literature [80].The chirality of the structure was determined from the Flack coefficient [81] which is equal to 0.00(2) for the given results.All hydrogen atoms were computed and refined using a riding model, with an isotropic temperature factor equal to 1.2 times the equivalent temparature factor of the atom linked to it.The final R factors as well as other relevant parameters concerning the resolution and refinement of the crystal structure are presented in Table A1.

A1.3.1. Cell Culture
Breast cancer MCF7 and MDA-MB231 cells (from European Collection of Cell Cultures, ECACC) were grown as a monolayer culture in minimum essential medium (DMEM with L-glutamine, without glucose and without sodium pyruvate) in the presence of 10% heat-inactivated fetal calf serum, 10 mM of D-glucose and 0.1% streptomycin/penicillin in standard culture conditions.

A1.3.2. Cell Viability Assays
For these studies, compounds were dissolved in 100% DMSO at 50 mM as stock solution; then, serial dilutions have been done in DMSO (1:1) (in this way DMSO concentration in cell media was always the same); finally, 1:500 dilutions of the serial dilutions of compounds on cell media were done.The assay was performed as described by Givens et al. [82].In brief, MDA-MB231 and MCF7 cells were plated at 5,000 cells/well or 10,000 cells/well respectively, in 100 µL media in tissue culture 96 well plates (Cultek, Boston, USA).After 24 h, media was replaced by 100 µL/well of serial dilution of drugs.Each point concentration was run in triplicate.Reagent blanks, containing media plus colorimetric reagent without cells were run on each plate.Blank values were subtracted from test values and were routinely 5%-10% of uninhibited control values.Plates were incubated for 72 h.Hexosamidase activity was measured according to the following protocol: the media containing the cells was removed and cells were washed once with PBS 60 µL of substrate solution (p-nitrophenol-Nacetyl-β-D-glucosamide 7.5 mM (Sigma, San Luis,MO, USA N-9376), sodium citrate 0.1 M, pH = 5.0, 0.25% Triton X-100 (Sigma-Aldrich Quimica, Tres Cantos, Madrid, Spain), was added to each well and incubated at 37 °C for 1-2 h; after this incubation time, a bright yellow color appeared; then, plates could be developed by adding 90 µL of developer solution (Glycine 50mM, pH = 10.4;EDTA 5 mM), and absorbance was recorded at 410 nm.

A1.4. Electrochemical Studies
Electrochemical data for compounds under study were obtained by cyclic voltammetry under nitrogen at 298 K using acetonitrile (HPLC-grade) as solvent, tetrabutylamonium hexafluorophosphate {(Bu4N)[PF6]} (0.1 M) as supporting electrolyte and a M263A potentiostat from EG&G instruments.The half-wave potentials were referred to an Ag-AgNO3) 0.1 M in acetonitrile) electrode separated from the solution by a medium porosity fritted disk.A platinum wire auxiliary electrode was used in conjunction with a platinum disk working Tacussel-Edi electrode (3.14 mm 2 ).Cyclic voltammograms of ferrocene were preformed before and after each sample to ensure the repeatability of the results, in particular to test the stability of the Ag-AgNO3 electrode.Cyclic voltammograms of freshly prepared solutions (10 −3 M) of the samples in acetonitrile were run and potentials measured were the referred to the ferrocene that was used as internal internal reference.In these experimental conditions the standard error of the measured potentials is ± 5 mV.In all the experiments, the cyclic voltammograms were run using scan-speeds (v) in the range: 0.05 V s −1 ≤ v ≤ 1.0 V s −1 .

R 1 =
H or Cl R 2 = Me, H, F and R 3 = Cl, H, F

Figure 2 .
Figure 2. Representative examples of diastereomerically pure cyclometallated Pt(II) complexes containing a σ(Pt-C Fc ) bond [39-43].(For types B and C, R is Me or i Pr).

Figure 3 .
Figure 3. Schiff base 1 selected for this study and schematic view of the different types of Pt(II) complexes (2-11) that may be formed.Compounds 2-11 differ in the conformation of the ligand (anti-(E) in 2, 4, 6, 8-11 or syn-(Z) in 3, 5 and 7), the binding mode of the imine ((N) in 2-5, (C,N) − in 6-9 or (C,N,C') 2− in 10 and 11), the type of metallated C atom (C Ph in 6 and 7 or C Fc in 8 and 9), the number of Pt-C bonds contained (one in 6-9 or two in 10 and 11) or the planar chirality of the Fc unit (Sp in 8 and 10 or Rp in 9 and 11).(X, L = monoanionic and neutral ligand, respectively).

Figure 5 .
Figure 5. (a) Comparative plot of the cytotoxic activities (IC50 values (µM)) of complexes 2a, the platinacycles 7b-9b and cisplatin in the MCF7 and MDA-MB231 breast cancer cell lines and (b) cyclic voltammograms (at a scan rate of 0.1 V × s −1 ) of the new Pt(II) complexes containing the aldimine 1 acting as a neutral N-donor (in 2a) or as a monoanionic ((C Ph , N) − (in 7b) or (C Fc , N) − (in 8b and 9b)) ligand.

Scheme 2 . 1 E
Scheme 2. Summary of the different processes studied by DFT calculations that involve: (a) the anti-(E) → syn-(Z) isomerization of the free ligand (1) or of compound 2a (steps A and B, respectively); (b) the transformation of complex 2a into platinacycles 6a, 8a and 9a by activation of the σ(C Ph −H) (step C) or the σ(C Ph −H) bonds (steps D and E); (c) the conversion of complex 3a into the platinacycle 7a (step F) and d) the platination of the phenyl ring of 8a or 9a (steps G and H, respectively) to give the biscycloplatinated derivatives 10a and 11a.(The values below or next to the arrows are the variations of energies (in kcal/mol) for the corresponding step).