Ruthenium–Cyclopentadienyl–Cycloparaphenylene Complexes: Sizable Multicharged Cations Exhibiting High DNA-Binding Affinity and Remarkable Cytotoxicity

Two novel sizable multicharged cationic complexes, of the formulae [(η6–-[12]CPP)[Ru(η5–-Cp)]12]Χ12 and [(η6–-[11]CPP)[Ru(η5–-Cp)]11]Χ11, CPP = cycloparaphenylene, Cp = cyclopentadienyl, X = [PF6]−, (1), (3) and [Cl]−, (2), (4), were synthesized and characterized using NMR techniques, high-resolution mass spectrometry, and elemental analyses. Complexes (1) and (3) were stable in acetone and acetonitrile solutions over 48 h. In contrast, the water-soluble (2) and (4) begin to decompose in aqueous media after 1 h, due to the [Cl]− tendency for nucleophilic attack on ruthenium of the {Ru(η5–-Cp)} units. Fluorescence quenching experiments conducted during the stability window of (2) with the d(5′-CGCGAATTCGCG-3′)2-EtBr adducts revealed remarkably high values for Ksv = 1.185 × 104 ± 0.025 M−1 and Kb = 3.162 × 105 ± 0.001 M−1. Furthermore, the cytotoxic activity of (2) against A2780, A2780res, and MCF-7 cancer cell lines shows that it is highly cytotoxic with IC50 values in the range of 4.76 ± 1.85 to 16 ± 0.81 μΜ.


Introduction
In recent years, cycloparaphenylenes have attracted significant attention from researchers due to their unique photophysical and photochemical properties [1,2].The synthesis of both classic [3][4][5][6] and modified [7][8][9] cycloparaphenylenes, along with their potential applications, has opened a new frontier in chemistry.This field is not extensively explored yet, but it holds great promise for biomedical applications.In one instance, 10 [CPP] was observed to form vesicles, under specific conditions, that could cause some cytotoxicity after internalization by the cells.This example illustrates the potential use of these supramolecular structures as functional carbon biomaterials [10].Jasti and colleagues synthesized a modified m-cycloparaphenylene serving as a fluorophore agent for biological imaging.This subcellular-targeted cycloparaphenylene, designed for both one-and two-photon live-cell imaging, exhibits no cytotoxicity up to 50 µM.Investigations into cellular uptake indicate internalization through endocytic pathways [11].In a separate study, a water-soluble cycloparaphenylene was synthesized and functionalized with sulfonate groups.This water-soluble compound demonstrates minimal cytotoxicity and possesses the capability to enter the cellular environment, functioning as a bioimaging substance.Additionally, this compound exhibits insensitivity to pH changes, a crucial property given the varying pH values in different cellular parts [12].
Extensive evaluations of numerous transition metal complexes have revealed promising potential as anticancer agents [13][14][15][16].Among them, ruthenium compounds have demonstrated remarkable cytotoxicity, showing significant promise for clinical applications.More particularly, ruthenium-arene organometallic compounds have attracted considerable research attention, primarily for two reasons: (a) their in vitro significant cytotoxicity in various cancer cell lines, and (b) their low cytotoxicity in healthy cells.The latter presents a critical advantage compared to platinum anticancer complexes.The exploration of the cytotoxic activity of ruthenium-arene compounds started with the study of the half-sandwich complex [(η 6 -C 6 H 6 )Ru(metronidazole)Cl 2 ] (where metronidazole is 1-β-hydroxyethyl-2-methyl-5-nitroimidazole) [17].A few years later, Saddler's group reported that the complex [(η 6 -cym)Ru(en)Cl]PF 6 exhibits antitumor activity similar to carboplatin across a wide range of cancer cell lines [18].This finding triggered a series of investigations into half-sandwich ruthenium-arene complexes with various chelate ligands.Among these complexes, those containing aromatic diamines instead of ethylenediamine have exhibited remarkable cytotoxicity and demonstrated a capacity for binding to DNA, as has been reported by our group [19][20][21][22].
However, the lack of selectivity towards cancer cells and the irreversible coordinative binding of ruthenium-arene half-sandwich complexes to targeted biomolecules prompted the design of complexes with kinetically inert ligands.These complexes are able to bind non-coordinatively and reversibly to biomolecules, potentially demonstrating anticancer activity.A category of compounds displaying these characteristics is the class of fullsandwich ruthenium-arene complexes.More specifically, cationic full-sandwich complexes containing η 5 -cyclopentadienyl units have shown anticancer activity, albeit through a different mechanism than that of the half-sandwich arene complexes.The absence of binding due to the lack of leaving groups results in a different mechanism of action [23,24].It has been reported that {Ru(η 5 -Cp*)} complexes with various η 6 -arene ligands show remarkable cytotoxicity, depending on the size and the lipophilicity of the arene ligand [25].Bulkier and more lipophilic ligands tend to increase the complexes' cytotoxicity.Kudinov et al. discovered that complexes [(η 5 -L1)Ru(L2)]BF 4 comprising an amino acid of the type [(C 5 Me 4 CH 2 OOCCH(CH 2 R)NHBoc)Ru(C 10 H 8 )]BF 4 (R = tryptophan, phenylalanine) showed notable cytotoxicity in various cancer cell lines with IC 50 = 21-96 µM, which is similar to that of cisplatin [26].The same group explored the selective labeling of the tryptophan residue in melittin, an existing peptide in bee venom with potential anticancer activity, using the organometallic fragment {RuCp} [27].The substantial stability of the [(η 5 -C 5 H 5 )Ru(η 6 -Trp-melittin)] allowed for establishing the biodistribution of the modified protein in mice through inductively coupled plasma MS analysis of ruthenium [28].It also has been reported that the bimetallic complex [(η 5 -Cp*)Ru(η 6 -benz-η 5 -Cp)Fe(η 5 -Cp)]Cl and the trinuclear bimetallic one [(η 5 -Cp*)Ru(η 6 -benz-η 5 -Cp)Fe(η 5 -Cp-η 6 -benz)Ru(η 5 -Cp*)]Cl exhibited significant cytotoxic activity against various cancer cell lines (A2780, SK-OV-3, MDA-MB-231) [29].Given this significant cytotoxicity observed in polynuclear fullsandwich ruthenium-arene complexes, we considered that cycloparaphenylenes may be ideal ligands.

Results and Discussion
The 1 H NMR spectrum of (1) showed two singlet peaks.The signal at 7.08 ppm was assigned to η 6 -coordinated phenyl rings with the {RuCp} units, shifted upfield by −0.54 ppm compared to the free [12]CPP.This shift reflects the contribution of ruthenium to the arene electron density [34,36].On the other hand, the cyclopentadienyl protons appeared as a single singlet peak at 5.63 ppm, indicating a symmetrical distribution of the Cp rings around the [12]CPP.Similarly, a 1 H NMR spectrum was observed in the case of (3) (Figure S1).The corresponding [Cl] − salts, (2) and (4), showed similar spectra, but the observed signals were significantly upfield, likely due to hydrophobic intramolecular interactions in D 2 O (Figure 1).Scheme 1. Synthetic procedure for the synthesis of (3), and structure numbering of (3p).
The 1 H NMR spectrum of (1) showed two singlet peaks.The signal at 7.08 ppm was assigned to η 6 -coordinated phenyl rings with the {RuCp} units, shifted upfield by −0.54 ppm compared to the free [12]CPP.This shift reflects the contribution of ruthenium to the arene electron density [34,36].On the other hand, the cyclopentadienyl protons appeared as a single singlet peak at 5.63 ppm, indicating a symmetrical distribution of the Cp rings around the [12]CPP.Similarly, a 1 H NMR spectrum was observed in the case of (3) (Figure S1).The corresponding [Cl] − salts, (2) and (4), showed similar spectra, but the observed signals were significantly upfield, likely due to hydrophobic intramolecular interactions in D2O (Figure 1).Scheme 1. Synthetic procedure for the synthesis of (3), and structure numbering of (3p).
The synthesis and stability of compounds ( 1) and ( 3) suggest that the critical factor for the η 6 -coordination of the {Ru(η 5 --Cp)} units lies in the convexity of the CPP acquired by the phenyl ring.In smaller CPPs, the nonplanar phenyl rings adopt a shallow boat conformation due to the ring strain, resulting in a dihedral angle of the out-of-plane carbon atom calculated as 15.6° for [5]CPP [37].This conformation seems to hinder the η 6coordination of the {Ru(η 5 --Cp)} unit.However, when ruthenium coordinates, it reduces this angle, causing an increase in the corresponding angle of the neighboring phenyl rings to maintain the ring shape.Consequently, only three {Ru(η 5 --Cp)} units can coordinate alternately to [6]CPP [34].In larger CPPs, this angle is smaller [37], reducing the impact on the neighboring phenyl rings upon {Ru(η 5 -Cp)} coordination, facilitating the continuous coordination of the units.Although it is assumed that the {Ru(η 5 --Cp)} moiety tends to favor the concave side of a curved aromatic organic molecule [38,39] due to its higher negative electrostatic potential compared to the convex surface [40], its ability to coordinate concavely with smaller CPPs is impeded due to steric reasons.However, in larger CPPs where steric hindrance is insignificant due to the size of CPP, the deviation from the planarity in the phenyl rings becomes marginal.Consequently, the {Ru(η 5 --Cp)} moiety coordinates from the exterior side of the nanoring.

Stability Studies of the Complex in Aqueous Media
Although initially isolated as [PF6] − salts, complexes (1) and (3) were converted into their [Cl] − salts, which are soluble in aqueous media, in order to study their cytotoxic properties.Both complexes (1) and ( 3) are stable in acetone and acetonitrile for over 48 h.In contrast, their [Cl] − salts, ( 2) and ( 4), are stable in aqueous media only for a short period, releasing solvolyzed {Ru(η 5 --Cp)} species.Monitoring the 1 H NMR spectrum of a freshly prepared solution of (2), we observed a new peak at about 7.75 ppm, attributed to the proton signals of an unbound phenyl ring.Essentially, this indicates that a {Ru(η 5 --Cp)} unit has dissociated from (2), leaving a phenyl ring unbound.In parallel, a signal at 6.5 ppm appeared, attributed to cyclopentadienyl protons of various {RuCp} solvolyzed species.
In fact, the integrals ratio of these new signals is 4:5, precisely reflecting the ratio between the protons of an unbound phenyl group in the [12]CPP and the ligand cyclopentadienyl, indicating their association originated from the same release reaction.Over Although it is assumed that the {Ru(η 5 -Cp)} moiety tends to favor the concave side of a curved aromatic organic molecule [38,39] due to its higher negative electrostatic potential compared to the convex surface [40], its ability to coordinate concavely with smaller CPPs is impeded due to steric reasons.However, in larger CPPs where steric hindrance is insignificant due to the size of CPP, the deviation from the planarity in the phenyl rings becomes marginal.Consequently, the {Ru(η 5 -Cp)} moiety coordinates from the exterior side of the nanoring.

Stability Studies of the Complex in Media
Although initially isolated as [PF 6 ] − salts, complexes (1) and ( 3) were converted into their [Cl] − salts, which are soluble in aqueous media, in order to study their cytotoxic properties.Both complexes (1) and ( 3) are stable in acetone and acetonitrile for over 48 h.In contrast, their [Cl] − salts, ( 2) and ( 4), are stable in aqueous media only for a short period, releasing solvolyzed {Ru(η 5 -Cp)} species.Monitoring the 1 H NMR spectrum of a freshly prepared solution of (2), we observed a new peak at about 7.75 ppm, attributed to the proton signals of an unbound phenyl ring.Essentially, this indicates that a {Ru(η 5 -Cp)} unit has dissociated from (2), leaving a phenyl ring unbound.In parallel, a signal at 6.5 ppm appeared, attributed to cyclopentadienyl protons of various {RuCp} solvolyzed species.
In fact, the integrals ratio of these new signals is 4:5, precisely reflecting the ratio between the protons of an unbound phenyl group in the [12]CPP and the ligand cyclopentadienyl, indicating their association originated from the same release reaction.Over time, these signals broadened, and additional peaks appeared due to the formation of various RuCp species on one side, and phenyl ring protons located in random sites on (2) on the other side.Both the solvent and the [Cl] − seem to contribute to the decomposition of (2), participating in the formation of various {Ru(η 5 -Cp)} species.
Integrating the four-proton signals of the unbound phenyl rings of (2), appearing around 7.9 ppm (Figure 2, yellow ring) and comparing them with those of the remaining [(η 6 -[12]CPP)[Ru(η 5 -Cp)] n ](PF 6 ) n , n < 12, (Figure 2, green ring), enables the determination of the decomposition percentage.Also, we can confirm the obtained results by integrating the five-proton signals of all the cyclopentadienyls, nanoringcoordinated, and released, appearing between 5.1-6.1 ppm (Figure 2 red and orange rings).However, the decomposition rate of (2) and ( 4) differs.Complex (4) exhibits a faster release of {Ru(η 5 -Cp)} species, indicating a higher decomposition rate.This observation further supports the hypothesis that the curvature of a CPP, which is related to its size, significantly influences the coordination and the release of the {Ru(η 5 -Cp)} units.After 24 h, both complexes have lost a similar percentage of {Ru(η 5 -Cp)} units: 21% for (2) and 23% for (4), suggesting a similar equilibrium.Figure 4 presents the decomposition (2) and (4) over time.
Molecules 2024, 29, x FOR PEER REVIEW 8 of 16 decomposition rate of (2) and ( 4) differs.Complex (4) exhibits a faster release of {Ru(η 5 --Cp)} species, indicating a higher decomposition rate.This observation further supports the hypothesis that the curvature of a CPP, which is related to its size, significantly influences the coordination and the release of the {Ru(η 5 --Cp)} units.After 24 h, both complexes have lost a similar percentage of {Ru(η 5 -Cp)} units: 21% for (2) and 23% for (4), suggesting a similar equilibrium.Figure 4 presents the decomposition (2) and (4) over time.Fluorescence spectroscopy stands out as a highly sensitive and effective method for investigating the binding affinity of small molecules with DNA [41].Ethidium bromide (EtBr) exhibits three different binding modes with DNA: (i) primarily through intercalation, which significantly enhances DNA fluorescence [42,43]; (ii) within the helix minor  Fluorescence spectroscopy stands out as a highly sensitive and effective method for investigating the binding affinity of small molecules with DNA [41].Ethidium bromide (EtBr) exhibits three different binding modes with DNA: (i) primarily through intercalation, which significantly enhances DNA fluorescence [42,43]; (ii) within the helix minor groove; and (iii) via electrostatic interactions due to its cationic nature [44].The displacement of EtBr from DNA-EtBr adducts via a DNA binder results in a notable quenching in emission intensity.The degree of the quenching directly correlates with the competitive binding constant (K sv ).Molecular simulation studies investigating the interaction between the dodecamer d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 and EtBr indicate its predominant stacking between the terminal base pairs CG of the sequence, with minimal interaction with the inner AT base pairs [45].Investigating the affinity of (2) with the d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 -EtBr adducts, we prepared a solution d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 in buffer phosphates (100 mM, pH = 7.0) to which we gradually added an amount of EtBr until the emission intensity reached the saturation point where any further addition has a negligible impact.Subsequently, a sample of the above stock solution was titrated with complex (2).It was observed that the emission intensity of the d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 -EtBr decreased rapidly as the concentration of (2) increased, while the λ of the emission maximum remained almost unchanged (Figure 5).This observation strongly suggests the displacement of the EtBr from the DNA-EtBr adduct due to its the interaction with (2).This outcome was unexpected, considering that ( 2) is capable of participating only in electrostatic interactions with the negatively charged phosphate groups of the d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 -EtBr.
On the other hand, it is known that cations with high charge density exhibit a considerable impact on the persistence length of the DNA [46].Several methods have demonstrated significant bending of B-DNA induced by multivalent cations [47][48][49], even though some random sequence DNA maintains in B conformation [50,51].Rouzina and Bloomfield proposed a mechanism by which multivalent cations induce DNA bending [46].According to their model, a multivalent cation binds to the B-DNA major groove electrostatically, repelling the sodium counterions from neighboring phosphates.This results in a strong binding of the cation to the groove, accompanied by groove closure and DNA bending toward the captured cation.In the context of the electrostatic interaction between the dodecavalent cation [(η 6 -- [12]CPP)[Ru(η 5 --Cp)]12] 12+ and the d(5′-CGCGAATTCGCG-3′)2-EtBr, it is possible that significant bending of the helix occurs, causing the release of EtBr and subsequent quenching of the emission intensity of the sample.This release occurs not through the replacement of EtBr by another molecule, but simply due to structural alterations of the B-DNA conformation induced by the strong electrostatic interaction.The above results reflected in the competition Stern-Volmer quenching constant, the value of which was determined from the slope of the F/Fo = f([Q]) plot (Figure S3) as Ksv = 1.185 × 10 4 ± 0.025 M −1 , indicating a great displacement of the EtBr from the duplex d(5′-CGCGAATTCGCG-3′)2.The calculated binding constant Kb, obtained through the double logarithmic plot log[(F0--F)/F] versus log[Q] (Figure S4), is 3.162 × 10 5 ± 0.001 M −1 .This value indicates a strong affinity of ( 2) with the d(5′-CGCGAATTCGCG-3′)2, similar to those of the DNA intercalators [52], signifying more than a simple external electrostatic interaction.

Cytotoxic Activity
To study the effects of (2) and [12]CPP on cell growth in vitro, three human cancer cell lines were treated with different concentrations of these compounds for 72 h and their IC50 values were calculated.The cytotoxic effects of the two compounds were monitored by employing real-time imaging using the Incucyte ZOOM system and the results are shown in Figure 6.Complex (2) exhibited the most potent effect on the proliferation of the ovarian cancer cell line A2780 and its cisplatin-resistant counterpart (A2780 Cis-res) (Figure 6a) depicting similar IC50 values (4.85 and 4.76 µΜ, respectively) (Table 1).It is interesting that both cell lines were equally sensitive to complex (2), suggesting that this On the other hand, it is known that cations with high charge density exhibit a considerable impact on the persistence length of the DNA [46].Several methods have demonstrated significant bending of B-DNA induced by multivalent cations [47][48][49], even though some random sequence DNA maintains in B conformation [50,51].Rouzina and Bloomfield proposed a mechanism by which multivalent cations induce DNA bending [46].According to their model, a multivalent cation binds to the B-DNA major groove electrostatically, repelling the sodium counterions from neighboring phosphates.This results in a strong binding of the cation to the groove, accompanied by groove closure and DNA bending toward the captured cation.In the context of the electrostatic interaction between the dodecavalent cation [(η 6 - [12]CPP)[Ru(η 5 -Cp)] 12 ] 12+ and the d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 -EtBr, it is possible that significant bending of the helix causing the release of EtBr and subsequent quenching of the emission intensity of the sample.This release occurs not through the replacement of EtBr by another molecule, but simply due to structural alterations of the B-DNA conformation induced by the strong electrostatic interaction.The above results reflected in the competition Stern-Volmer quenching constant, the value of which was determined from the slope of the F/F o = f ([Q]) plot (Figure S3) as K sv = 1.185 × 10 4 ± 0.025 M −1 , indicating a great displacement of the EtBr from the duplex d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 .The calculated binding constant K b , obtained through the double logarithmic plot log[(F 0 -F)/F] versus log[Q] (Figure S4), is 3.162 × 10 5 ± 0.001 M −1 .This value indicates a strong affinity of (2) with the d(5 ′ -CGCGAATTCGCG-3 ′ ) 2 , similar to those of the DNA intercalators [52], signifying more than a simple external electrostatic interaction.

Cytotoxic Activity
To study the effects of ( 2) and [12]CPP on cell growth in vitro, three human cancer cell lines were treated with different concentrations of these compounds for 72 h and their IC 50 values were calculated.The cytotoxic effects of the two compounds were monitored by employing real-time imaging using the Incucyte ZOOM system and the results are shown in Figure 6.Complex (2) exhibited the most potent effect on the proliferation of the ovarian cancer cell line A2780 and its cisplatin-resistant counterpart (A2780 Cis-res) (Figure 6a) depicting similar IC 50 values (4.85 and 4.76 µM, respectively) (Table 1).It is interesting that both cell lines were equally sensitive to complex (2), suggesting that this compound has the potential to be used as a second-line treatment after the development of cisplatin resistance in ovarian cancer.Complex (2) showed a weaker, but still potent, cytotoxic effect on the breast adenocarcinoma cell line MCF-7 (Figure 6a) with the IC 50 value being 16 µM (Table 1).The A2780 Cis-res cells were quite susceptible to the cytotoxic effect of the [12]CPP compared to the other two cell lines (Figure 6b).Specifically, the IC 50 value of [12]CPP in the resistant cells was ~10 µM, while it was ~17.5 µM and ~19.5 µM in the A2780 parental and in the MCF-7 cells, respectively (Table 1).Overall, complex (2) was more efficient than the [12]CPP one, especially in the ovarian cancer cells.
Molecules 2024, 29, x FOR PEER REVIEW 10 compound has the potential to be used as a second-line treatment after the develop of cisplatin resistance in ovarian cancer.Complex (2) showed a weaker, but still po cytotoxic effect on the breast adenocarcinoma cell line MCF-7 (Figure 6a) with the value being 16 µM (Table 1).The A2780 Cis-res cells were quite susceptible to the cyto effect of the [12]CPP compared to the other two cell lines (Figure 6b).Specifically, th value of [12]CPP in the resistant cells was ~10 µM, while it was ~17.5 µΜ and ~19.5 µ the A2780 parental and in the MCF-7 cells, respectively (Table 1).Overall, complex (2 more efficient than the [12]CPP one, especially in the ovarian cancer cells.
To a THF (5 mL) solution containing 89.8 mg (0.40 mmol) SnCl 2 •2H 2 O, we added 0.1 mL HCl 12 M (1.20 mmol) at room temperature and stirred for 0.5 h.The resulting solution was then added to another 5 mL solution of THF containing 0.05 mL conc.HCl and 110 mg of (4p).After stirring for 12 h at 50 • C we added 5 mL of an aqueous solution NaOH (10% w/v) and performed extraction with DCM.The residue obtained was purified via column chromatography (SiO 2 , 1/2, CH 3 Cl/hexane) to give [11]CPP as a pale yellow solid.Yield 3,2%.The 1 H NMR spectrum consistent with the literature data in CDCl 3 [3] and it was recorded in acetone-d 6 (Figure S7).

Stability Studies of the Complex in Aqueous Media
The stability of the [Cl] − and [PF 6 ] − salts of the complexes in organic solvents and aqueous media was monitored by 1 H NMR (Figures S8 and S9) and HR-ESI-MS (Figures S10 and S11) spectroscopy.In a typical experiment, 1-2 mg of each complex was dissolved in 0.5 mL of the appropriate solvent in order to obtain a 2 mM solution and the 1 H NMR spectrum of the sample was recorded at intervals of 15 min for the initial 5-h period.

Fluorescence Measurements
Fluorescence emission study was carried out using a Jasco FP-8300 fluorimeter equipped with a xenon lamp source.All the experiments were performed using a 10 mm path length cuvette in a 100 mM phosphate buffer at pH 7.0.Successive amounts of complex (2) from a stock solution of 1 mM were added to a 20 µM of d(5'-CGCGAATTCGCG-3') 2 saturated with ethidium bromide EtBr (5.07 µM) [55].The DNA-EtBr sample was titrated with (2) and the emission spectra were recorded at wavelengths of 500-800 nm with excitation at 480 nm in a 1 cm quartz cell.The excitation and emission slit widths were kept at 5 nm each.All measurements were recorded following a 5 min incubation at 298 K to minimize the exposure of (2) to the aqueous environment and ensure that it remains intact (see stability studies).Details on the calculations of K sv and K b are presented in the Supplementary Materials.

Cell Growth Assay
To monitor the cell growth and evaluate the cytoxicity effects of (2) and [12]CPP, the IncuCyte Zoom system (Essen BioScience, Hertfordshire, UK) and software were used, as has been previously described [20].The IC 50 values of the compounds were calculated from a log(concentration) versus normalized response curve fit using Graphpad Prism version 8.01.Two to three independent, biological experiments were conducted in triplicate for each compound in each cell line.

Figure 2 .
Figure 2. The positive mode of the high-resolution ESI mass spectrum of (1) showing a main cluster-peak at m/z = 1405.1395.Inset the calculated cluster-peak for the triple-charged cation [C 132 H 108 P 9 F 54 102 Ru 12 ] 3+ .

Figure 6 .
Figure 6.Effects of (2) and [12]CPP complexes on cell growth.The human breast cancer ce MCF-7 and the human ovarian cancer cell lines A2780 and A2780 Cis-res were treated for 72 h 1-80 µМ and 1-35 µΜ of the (2) (a) and the [12]CPP (b) complexes, respectively.Cell growt was determined through confluency measurements using the Incucyte Zoom live cell analysi tem.Data from two to three independent biological experiments performed in triplicate are sh Error bars represent the S.E. of the mean.

Figure 6 .
Figure 6.Effects of (2) and [12]CPP complexes on cell growth.The human breast cancer cell line MCF-7 and the human ovarian cancer cell lines A2780 and A2780 Cis-res were treated for 72 h with 1-80 µM and 1-35 µM of the (2) (a) and the [12]CPP (b) complexes, respectively.Cell growth rate was determined through confluency measurements using the Incucyte Zoom live cell analysis system.Data from two to three independent biological experiments performed in triplicate are shown.Error bars represent the S.E. of the mean.

Figure S9. 1 H
NMR spectrum of (4) in D 2 O after 48 h and 72 h.
• C oil bath and stirred vigorously for 16 h.After cooling the reaction mixture at room temperature, the crude product was extracted with DCM/H 2 O, concentrated to about 1 mL, and purified by column chromatography (SiO 2 , 10/90, ethyl acetate/hexane) affording (3p).