Chemistry and catalysis advances in organometallic chemistry and catalysis

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Figure 47.15

Cyclic voltammograms of 4 (1 mM) in H

O with Na

(0.1 M) as the supporting electrolyte, recorded at a glassy

carbon electrode (3 mm diameter) at a scan rate of 50 mV/s and in the presence (a) of

β-CD or (b) Me-β-CD (20 equiv).

host–guest inclusion complex with a CD, the resulting complex generally will not undergo any direct electrochemical

reaction [50–52]. Indeed, the complex must dissociate and release its electroactive moiety, which depicts a classical CE

process. The inclusion constant of the 4

•Me-β-CD complex, evaluated in MeOH and MeOH/H

O (1 : 1), was found equal

to 80 and 825 M

−1

, respectively, in agreement with the polarity medium [49].

Importantly, CDs allowed the solubilization of ferrocifens in pure water, that is, in the absence of any organic solvent.

Typical cyclic voltammograms obtained after complete solubilization of ferrocifen 4 in the presence of either

β-CD or

Me-

β-CD are shown in Fig. 47.15.

As expected, the oxidation of 4 occurred at a more positive potential, with respect to the studies performed in less polar

media such as MeOH and MeOH/H

O evidencing a stronger complexation of 4 by the CD. The oxidation wave showed a

more pronounced plateau shape, thus featuring a fully developed CE process proceeding under pure kinetic control. On the

contrary, the current on the reverse scan exhibited a peak shape rather than a plateau, conﬁrming that the electrogenerated,

electrically charged, polar ferricenium species was not complexed by the CD. Interestingly, when the same experiment

was performed with

β-CD instead of Me-β-CD the oxidation peak of O

appeared at a less positive potential value and

its intensity was smaller, suggesting that 4 was more strongly complexed by the former randomly methylated CD (Fig.

47.15). Accordingly, the peak current of O

was smaller in the presence of

β-CD, a lower amount of 4 being solubilized in

that case. Interestingly, the cyclic voltammogram obtained for an unsubstituted ferrocene (Cp

Fe) in the presence of

β-CD,

under conditions identical to those mentioned in Fig. 47.15, gave no signiﬁcant electrochemical response (the Cp

Fe/CD

complex was not soluble in water). This suggested that, in the case of ferrocifens, the presence of the phenol groups,

which presumably extrude from the cavity, provides a higher solubility of the inclusion complex. In line with this view,

the replacement of

β-CD by Me-β-CD made the complex Cp

Fe/Me-

β-CD sufﬁciently soluble in water to be observed by

cyclic voltammetry, which appears related to the higher solubility of Me-

β-CD compared to β-CD [53]. As expected, the

intrinsic solubility of the CD determines the solubility of the corresponding host–guest complexes.

Other ferrocifen compounds were investigated under the conditions favoring the best complexation, that is, in the presence

of Me-

β-CD (Fig. 47.16) [49].

Qualitatively, the time required to partially or totally dissolve the four complexes decreased in the order 5

> 14 > 4 > 6.

Here again, comparison of the cyclic voltammograms of compounds 5, 14, and 4 showed that the presence of phenol groups

was of great importance for solubilizing the ferrocene-CD adducts. Indeed, compounds 4 and 14 were totally solubilized

after 1 day, whereas 5 was only partially dissolved after 2 days. This also explains the much lower oxidation current of

5 compared to those of 14 and 4. The smaller peak current at O

observed with 4 compared to 14 was likely due to a

stronger complexation for the former compound in agreement with a more pronounced trend to adopt a plateau shape for

4 [thus indicating a kinetically disfavored decomplexation before the electron transfer (CE mechanism) than for 14]. As

expected, the absence of the apolar ethyl–vinyl fragment increased the solubility of the complex in water (compare 4 and

6). However, the cyclic voltammogram shape indicated that the complexation dynamics were faster for 6 than for 4.

646

FERROCIFENS: AN ELECTROCHEMICAL OVERVIEW

−6

−4

−2

(a)

(b)

(c)

(d)

−0.1

0.1

0.2

0.3

0.4

0.5

0.6

−0.1

0.1

0.2

0.3

0.4

0.5

0.6

OH

I (

−6

−4

−2

−0.1

0.1

0.2

0.3

0.4

0.5

0.6

I (

−6

−4

−2

8

I (

A)

−6

−4

−2

8

I (

1

E (V vs SCE)

E (V vs SCE)

E (V vs SCE)

E (V vs SCE)

−0.1

0.2

0.5

0.8

1

6

OH

O1

R1

14

4

Figure 47.16

Cyclic voltammograms of ferrocifen-type molecules 4 (a), 14 (b), 6 (c), and 5 (d) (1 mM) in H

O with Na

(0.1 M) as

the supporting electrolyte, recorded at a glassy carbon electrode (3 mm diameter) at a scan rate of 50 mV/s in the presence of Me-

β-CD

(20 equiv).

Cyclic voltammetry was used above to rationalize the relationship between the oxidizability and the biological activity of

the ferrocifen compounds in a model environment. Since we just showed that using CD/ferrocifens complexes constituted a

valuable alternative to the poor water-solubility of ferrocifens, it was then important to ensure if their oxidation mechanism

was retained or not after complexation by CDs. As shown in Fig. 47.17, the addition of an excess of pyridine to a 4/Me-

β-CD

solution led to a circa threefold increase of the peak current of O

together with a pronounced change in the wave shape,

with a loss of reversibility suggesting an EC sequence.

Considering the initial sluggish one-electron CE process current, it was inferred that the maximum current value obtained

in the presence of pyridine corresponded roughly to a three-electron process. This evolution of the CVs showed that the

presence of pyridine kinetically favored the dissociation of the complex 4/CD and opens a subsequent two-electron pathway

after the initial formation of the ferricinium derivative. This appeared fully consistent with the former investigation of

pyridine-induced effects on the voltammetry of 4 in apolar medium [20]. This strongly suggests that the overall mechanism

previously depicted for oxidation of 4 in the presence of a base (see Fig. 47.3) still prevails when 4 is initially complexed

by a CD; note that this implies that oxidation of [4, CD] produces an uncomplexed 4

•

+

(EC) or that 4 is oxidized

after decomplexation (CEC). So, a three-electron oxidation sequence is most likely triggered in the presence of pyridine,

irrespective of the fact that 4 is borne initially by a CD. The corresponding mechanism is summarized in Fig. 47.18.

Note that the third electron transfer most likely corresponds to a further oxidation of the ferrocene moiety of the QM

structure. In addition, the passage from a one-electron plateau shape to a three-electron peak shape for wave O

in the

presence of both CD and pyridine (Fig. 47.17) suggests that pyridine interacts with the phenolic group of the adduct [4,

CD], allowing a faster decomplexation of 4 along the EC-initiated sequence in Fig. 47.18 (if pyridine reacted only with

uncomplexed compound 4 (namely, as in the CEC sequence of Fig. 47.18), a plateau shape would still be observed with a

circa threefold current intensity). Similarly, although not indicated in Fig. 47.18 for simplicity, the neutral species (phenoxy

SUPRAMOLECULAR INTERACTIONS OF FERROCIFENS WITH CYCLODEXTRINS AND LIPID BILAYERS

647

O

1

R

1

I (

μ

(a)

(b)

(c)

Me-

β-CD

−4

−2

−0.1

0.1

0.2

0.3

0.4

0.5

0.6

0.7

+

E (V vs SCE)

Figure 47.17

Cyclic voltammograms of 4 (1 mM) in H

O with Na

(0.1 M) as the supporting electrolyte, recorded at a glassy

carbon electrode (3 mm diameter) at a scan rate of 50 mV/s, in the presence of Me-

β-CD (20 equiv) and (a) in the absence of pyridine

or in the presence of (b) 0.1 and (c) 0.2 M, respectively, of pyridine.

Fe +

− e

+ e

K

−e, −pyH

•

Fe +

−e, −H

+

CD

−e

4

[4, CD]

+ Py/

−PyH

+

Figure 47.18

Electrooxidation of 4 in the presence of CD in the absence and the presence of pyridine.

radical, and QM) formed along the oxidation sequence may also exist as inclusion complexes. One may also consider a

competitive complexation between pyridine and the ferrocene moiety with the CD host, leading thus to a decrease in the

value of the association constant.

The in vitro effect of the organometallic complexes alone or encapsulated in Me-

β-CD was investigated using the

hormone-independent breast cancer cell lines MDA-MB-231 after 4 days of culture to assess whether or not the inclusion

of 4 or 6 into the CD affected their bioavailability. The corresponding results are reported in Fig. 47.19 [49].

648

FERROCIFENS: AN ELECTROCHEMICAL OVERVIEW

50

100

% Proteins/control

OH

4

6

control

(4)

− [0.5

μm]

(6)

− [0.5

μm]

(4)

− [0.5

μm] + CD

(6)

− [0.5

μm] + CD

(4)

− [1

μm]

(6)

− [1

μm]

(4)

− [1

μm] + CD

(6)

− [1

μm] + CD

Figure 47.19

Comparison of the antiproliferative effect of ferrocenyl complexes 4 and 6 (0.5 or 1

μM) alone or encapsulated in Me-β-

CD (20

μM) on hormono-independent breast cancer cell lines (MDA-MB231) after culturing for 96 h. Results are the average value of

two independent experiments.

Clearly, no statistically signiﬁcant difference could be observed between the free and CD-complexed ferocifens, their

antiproliferative activities being identical within the precision of their determinations [19, 54]. A similar result was also

observed with tamoxifen citrate

β-CD nanoparticles [55]. With respect to the association constant determined in MeOH/H

O,

one may also consider that complexes are most likely dissociated at such low concentrations although the polarity (and

therefore the association constant) is much higher in pure water. Nevertheless, as 4 and 6 may be delivered intravenously

at much more signiﬁcant doses thanks to the higher solubilities of their inclusion complexes compared to the uncomplexed

compounds, this opens encouraging routes for more effective formulations of ferrocifens for anticarcinogenic therapies.

47.3.2

Interaction with Lipid Bilayers

We have shown above that ferrocifens delivery to cells could be improved by CD complexation. However, it is clear that

whether ferrocifens are delivered free or encapsulated, they must cross the bilipidic cellular membranes. The purpose of this

section is to investigate their interactions with such bilayers.

Electrochemistry may also be used to probe the interactions between ferrocifens and nonpolar molecular architectures,

with respect to the hydrophobic architectures/barriers that compose cell membranes as well as lipidic cargoes/vectors (e.g.,

liposomes) using model systems consisting of glassy carbon electrodes modiﬁed with a planar bilayer of 1,2-dimyristoyl-

sn-glycero-3-phosphocholine (DMPC) (Fig. 47.20) [56].

Such model systems established that the afﬁnity of neutral ferrocifen derivatives toward the lipid bilayer depended on

both their size and their polarity. Conversely, the electrogenerated ferricenium derivatives were expulsed reversibly from

the bilayer owing to their positive charge. This led to unsymmetrical redox processes reﬂecting a different partition of the

species between the lipid ﬁlm and the solution. Fig. 47.21a illustrates this typical behavior for compound 7 and shows the

accumulation of the parent ferrocifen in the DMPC-modiﬁed glassy carbon electrode (GCE) as a function of time. The

corresponding apparent coverage of the electrode was investigated by monitoring the oxidation peak growth. As depicted

in Fig. 47.21b, distinct kinetic features were obtained for compounds 7 and 5. Presumably, favorable interactions between

the phenolic hydroxy group in compound 7 and the polar extremities of the lipid molecules sustain the observed faster

incorporation of 7 with respect to 5.

On the other hand, considering the 5

× 10

−10

mol/cm

coverage of a surface as a reference for a close-packed layer

of comparable ferrocene molecules in SAMs [57], the saturation observed at circa 3

× 10

−10

mol/cm

for compound 7

shows that the loading of 7 in the bilipid ﬁlm is considerable, being enough to drastically disorganize the original bilayer

SUPRAMOLECULAR INTERACTIONS OF FERROCIFENS WITH CYCLODEXTRINS AND LIPID BILAYERS

649

Figure 47.20

Schematic representation of the glassy carbon electrode (GCE) modiﬁed with DMPC ﬁlm (1,2-dimyristoyl-sn-glycero-3-

phosphocholine) used to investigate the interactions between ferrocifens with a model membrane.

−2.0

−1.0

0.0

1.0

2.0

3.0

(a)

(b)

−0.2

0.0

0.2

0.4

0.6

E (V vs SCE)

0.8

O

7

I*

R

7

Time

7

5

100

150

200

250

300

Time (min)

0.5

1.5

2.5

3.5

Time (min)

(x 10

−

mol/cm

)

Figure 47.21

(a) Cyclic voltammograms of 7 (0.2 mM) recorded at a glassy carbon electrode (3 mm diameter) modiﬁed with a DMPC

ﬁlm as a function of the equilibration time (0, 45, 90, 150, and 210 min) in H

O

+ EtOH (20%) + KCl (0.1 M) and at v = 100 mV/s.

(b) Evolution of the apparent coverage of the DMPC-modiﬁed GCE electrode for 5 and 7 (0.2 mM each in EtOH (8/2)

+ KCl (0.1 M))

as a function of the time of incubation in the corresponding ferrocifen solution. I* is the normalized current, I*

= I (μA)/C (mM).

structure. The presence of small molecules such as anesthetics (chloroform for instance) is well known to induce a strong

reorganization, typically, swelling of membranes [58]. This swelling is, in fact, at the origin of the anesthetic effect through

the increased pressure created onto ion-channels crossing the outer membrane of the nerves; so, one cannot exclude similar

behavior for ferrocifens.

Interestingly, these results established that within the limit that a DMPC is a sufﬁciently correct model of a cell membrane,

large delivery ﬂuxes of ferrocifens are expected to disrupt cell membranes. This may be an important factor to consider in view

of side toxicity of these species at sufﬁciently high concentration. In other words, if their delivery to cell membranes is highly

desirable it should not be excessive unless the delivery is properly targeted to tumor cells. Then ferrocifens may act both as

biochemical agents (i.e., via the QM generated by their oxidation) and as a kind of detergent disrupting the cell membranes.

This should lead to noncommon cell death processes in agreement with experimental observations still under the way.

650

FERROCIFENS: AN ELECTROCHEMICAL OVERVIEW

47.4

CONCLUSION

Cyclic voltammetry has proved to be very useful in investigating not only the reactivity, but also the solubilization and

interaction with lipidic bilayers of organometallic ferrocene-based anticancer drugs. It was notably established that ferrocifen

cytotoxicity relies on a proton transfer coupled sequentially or not with an intramolecular electron transfer between a protic

and oxidizable functionality (phenol, aniline, catechol, etc.) and the ferrocenyl moiety. This process in which the ferricenium

group acts as an intramolecular “antenna” leads ultimately to the formation of extremely cytotoxic species (QM, quinone

imines, ortho-quinone) even under moderate oxidizing conditions. On the other hand, we established that CDs may be an

excellent alternative to increase their solubility in water and their bioavailability as the ferrocifens complexation by CDs

did not affect the ultimate production of the cytotoxic species following their oxidation. Cyclic voltammetry also provided

valuable information on the interactions and on the reactivity of ferrocifens in the presence of lipid bilayers used as simple

models of cell membranes.

In conclusion, this chapter illustrates how electrochemical techniques allow the elucidation of the mechanisms involved

in the metabolism of a drug, as well as characterizing the interactions of these molecules with supramolecular structures,

such topics being essential for the development of more active drugs and the establishment of coherent reaction frames

related to their vectorization and administration.

ACKNOWLEDGMENTS

The authors thank Prof. G´erard Jaouen, Prof. Anne Vessi`eres, and Dr. Elizabeth Hillard (UMR CNRS 7223, Paris) for

supplying the ferrocifen complexes synthesized in their laboratory and for this fruitful collaboration. This work was supported

in part by the Centre National de la Recherche Scientiﬁque (UMRs 8640 and 7223), the Minist`ere de la Recherche (MESR),

the Ecole Normale Sup´erieure, Universit´e P. et M. Curie, and the Agence Nationale de la Recherche (No. ANR-06-BLAN-

0384-01, “FerVect”).

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REFERENCES

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