# Synthesis, Structure, Electrochemistry, and Cytotoxic Properties of Ferrocenyl Ester Derivatives

Authors: Li Ming Gao; Ramón Hernández; Jaime Matta; Enrique Meléndez
Journal: Metal-Based Drugs
 (2009)

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## Abstract

A series of ferrocenyl ester complexes, varying the lipophilic character of the pendant groups, was prepared and characterized by spectroscopic and analytical methods.  The syntheses ofFe(C5H4CO2CH3)2, Fe(CpCOOCH3)(CpCOO
CH2CH3), and
Fe(CpCOOCH2CH3)2 are reported. The solid-state structure of Fe(C5H4CO2CH3)2 has been determined by X-ray crystallography. Fe(C5H4CO2CH3)2 has the cyclopentadienyl rings virtually in an eclipsed conformation with the pendant groups not completely opposite to each other. Cyclic voltammetry characterization showed that the functionalized ferrocenes oxidize at potentials, Epa, higher than ferrocene as a result of the electro withdrawing effect of the pendant groups on the cyclopentadienyl ligand. The cytotoxicities of Fe(C5H4CO2CH2CH2OH)2, Fe(C5H4CO2CH2CH=CH2)2, Fe(C5H4CO2CH3)2, Fe(CpCOOCH3)(CpCOOCH2CH3), and Fe(CpCOOCH2CH3)2 in colon cancer HT-29 and breast cancer MCF-7 cell lines were measured by the MTT biological viability assay and compared to ferrocene and ferrocenium. Fe(C5H4CO2CH2CH=CH2)2 showed the best IC50 values, 180(10)
μM for HT-29 and 190(30)
μM for MCF-7 cell lines, with cytotoxicities similar to ferrocenium.
The cytotoxic data suggest that as we increase the lipophilic character of the functionalized ferrocene,
the cytotoxicity improves approaching to the cytotoxic activity of
ferrocenium.


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## Body

## 1. Introduction

Modern
organometallic chemistry has been greatly influenced by the accidental
discovery of bis(cyclopentadienyl)iron(II) (ferrocene) in 1951 [1, 2]. In fact,
ferrocene (Cp2Fe) has been the most extensively studied
organometallic species. Its applications in catalysis, organic synthesis, and
industrial processes are numerous [3–6]. The thermal
stability, inert character in concentrated acid and base solutions as well as
the redox properties of ferrocene makes this species a versatile compound in
many research areas.Until
1979, the biological properties of metallocenes were unexplored. The report of
titanocene dichloride (Cp2TiCl2) as the first metallocene
to possess antitumor activity opened a new area of research,
bioorganometallics, which has been developing rapidly in the last twenty eight
years [7, 8].In 1984, Köpf-Maier et al. reported
the anticancer activity of ferrocenium complex in Ehrlich ascites tumor [9]. Since
then, ferrocene has been studied for potential biological and medicinal
applications [8, 10, 11]. Originally, the oxidized species of ferrocene,
ferrocenium (Cp2Fe+) was reported to be responsible for
the cytotoxic properties on DNA mediated through its capacity to generate
oxygen-free radical species [12, 13]. Ferrocene has been recently reported to
have antitumor properties due to the metabolic formation of ferrocenium ions
which induces oxidative damage to DNA [14, 15]. As a result, many
functionalized ferrocenes have been prepared and tested in cancer cells [11].We have initiated a project on functionalized ferrocene chemistry, with
pendant groups (functional groups, R) on the Cp rings varying their polar and
lipophilic characters,
see Scheme1.  The objective of this
study is to understand how the polar and lipophilic characters of the pendant
groups change the anticancer activity of the corresponding ferrocenyl
derivatives. We have investigated the synthesis, structure, electrochemistry,
and cytotoxic properties of five ferrocenyl esters in HT-29 colon cancer and
MCF-7 breast cancer cell lines. The objective of this study is to report these
novel findings.Scheme 1
Structure of ferrocenyl ester complexes.R=CH3,CH2CH3,CH2CH2OH, and CH2CH=CH2.


## 2. Results and Discussion

### 2.1. Synthesis and Structure

The syntheses of
Fe(C5H4CO2CH3)2, Fe(C5H4CO2CH2CH=CH2)2,
and Fe(C5H4CO2CH2CH2OH)2 have been reported previously [16, 17]. We applied
the methodology developed by Busetto et al. to synthesize Fe(C5H4CO2CH2CH3)2 and Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
(mixed ferrocene) [17]. Interestingly, using this
synthetic methodology, the reaction of FeCl2 and one equivalent of NaC5H4CO2CH2CH3 and NaC5H4CO2CH3, even at room
temperature, allowed the selective incorporation of two distinct functionalized
Cp ligands, affording the mixed ferrocene complex in good yield without the
formation of Fe(C5H4CO2CH3)2 and Fe(C5H4CO2CH2CH3)2.The NMR, IR, and
elemental analysis corroborate the identities of the ferrocenyl ester complexes.
The IR spectral data showedν(C=O) broadbands at 1708 cm−1 (Fe(C5H4CO2CH2CH3)2 and 1712 cm−1 (Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
corresponding to the carbonyl groups of the esters.  In the H1 NMR spectrum, Fe(C5H4CO2CH2CH3)2 exhibits two set of
resonances at 4.42
and 4.84 ppm. These Cp protons exhibit
coupling belonging to an AA′BB′ spin system. For the Fe(C5H4CO2CH2CH3)(C5H4CO2CH3),
the H1 NMR spectrum shows two sets of resonances at 4.42 and 4.85 ppm corresponding
to the Cp protons of C5H4CO2CH2CH3 and C5H4CO2CH3 rings. While we
might expect four sets
of resonances in the vinyl region corresponding to two different substituted Cp
rings, the fact that we observed only two sets suggests overlapping between these Cp proton
signals. Particularly interesting in the C13 NMR spectrum, at room
temperature, two sets of resonances for each carbon atom are observed in a
ratio of 1:1. This suggests that two possible conformational diastereoisomers, syn and anti, coexist in solution as
discussed below (Scheme 2) [18].Scheme 2
Conformational diastereoisomers
of Fe(C5H4CO2CH2CH3)(C5H4CO2CH3).In
solution, rapid ring oscillation about Cp–Fe–Cp axis allowsH2 and H5 (H2′ and H5′) and H3 and H4 (H3′ and H4′) to become
equivalents. This situation applies for both substituted Cp rings. Since both
Cp rings have ester groups, overlapping of the H2/H5 and
H2′/H5′, H3/H4 and H3′/H4′ signals in the H1 NMR
spectrum is expected. However, the position of the ester groups yields to
conformational diastereoisomers, syn and anti, which differ in the orientation of the carbonyl groups [18]. This condition applies when the C(Cp)–C(CO) bond is slow
in the NMR scale time. When the rotation is fast, having enough energy to
overcome the rotational barrier of the C(Cp)–C(CO) bond, such stereolabile
chirality disappears. In the C13 NMR spectrum, the carbonyl
orientation (syn and anti) makes significantly different
magnetic environment on the Cp carbons as well as on the carbonyl groups such that
two sets of
resonances corresponding to syn and anti conformers can be observed. Variable
temperature NMR studies, in toluene-d8, showed that at 80°C in the H1
spectrum, the multiplet signal at 4.85 ppm became a single line and the
multiplet at 4.43 ppm broadened. These signals belong to the Cp rings. In the C13
NMR spectrum at 80°C, the carbon signals of one conformer begin to increase in
intensity at expense of the other conformer, reaching a ratio of 1:2. This suggests that
conformational diastereoisomers, syn and anti, at room temperature, have
very similar stability but at high temperature, one diastereoisomer
(presumably) anti becomes more
populated.In general, the present synthetic route
developed by Busetto et al. [17] for the ferrocenyl ester complexes (Cp functionalization and formation of the
corresponding functionalized ferrocene) represents a convenient alternative procedure
for the functionalization of metallocenes and has been used by our group to
synthesize functionalized titanocenes [19].The solid-state structure of Fe(C5H4CO2CH3)2 was investigated by single-crystal X-ray diffraction, see Figure 1. Crystal
data and structure refinement are summarized in the Supplementary Material available online at
doi:10.1155/2009/420784
(Cambridge Crystallographic Data Centre and the deposition number is CCDC
705387). Bonding parameters are included in Supplementary Table 1.Figure 1
Solid-state structure of Fe(C5H4CO2CH3)2 drawn 50% thermal ellipsoids. Average Fe-Cp bonds, Fe(C1–C5)
2.049(28) Å and Fe(C6–C10) bond 2.048 (11) Å. Torsion
angles for C(8)–C(7)–C(11)–O(2) 6.9(4)° and O(4)–C(13)–C(5)–C(1) 15.2(4)°.As shown by X-ray, this ferrocene
is a sandwich complex with the cyclopentadienyl ligands adopting an eclipsed
conformation and with the pendant groups not completely opposite to each other.
The average Fe–C(Cp) distances for Fe(C5H4CO2CH3)2 are 2.049 and 2.048 Å′  which is very similar to those reported for
ferrocene, 2.045 Å′  [20].  The
shortest Fe–C(Cp) bond distances are on C(5) and C(7). The shorter bond
distances could be attributed to the inductive (electro-withdrawing) effect of
the ester groups on these carbon atoms. The
ester groups are not coplanar with the Cp ring, one of the OCH3 is bent toward the iron atom (below the Cp plane) with a
torsion angle of 15.2° while the other is bent away from the iron atom by 6.9°.
The C(Cp)–C(CO) bonds (C(5)–C(13) and C(7)–C(11)) are
somewhat shorter than a typical C-C single bond, 1.477(4) versus 1.54 Å′  (for a single bond), suggesting these bonds
have partial double bond character as a result of conjugation with the Cp π
system [21]. The later might explain the high C(Cp)–C(CO) rotational barrier
encountered in the Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
complex.  In
addition, the two CO2Me groups are anti to each other.While the solid structure of Fe(C5H4CO2CH3)2 has been reported previously, our solid-state structure has a fundamental
difference to that published by Cetina et al. [22]. That
is, in the lattice, they determined a hydrogen bonding network between the
carbonyl group and the CH3 of the adjacent molecule. We could not
determine such hydrogen bonding network between the carbonyl group and the CH3 of the adjacent molecule even though our data was collected at lower
temperature (173 K versus 293 K) and we obtained better refinement parameter (R
(I>2 sigma (I)) 0.0397
versus 0.046).


### 2.2. Cyclic Voltammetry

Electrochemical characterization of the
subject complexes was performed by mean of cyclic voltammetry (CV). It is well known that ferrocene
easily undergoes one electron oxidation to form ferrocenium (Cp2Fe+)
in a reversible manner. Thus, we investigated the functionalized ferrocenes electrochemical
behaviors and compared to ferrocene in organic solvent. Table 1 presents the CV results. As it can be
seen in the cyclic voltammograms (Figure 2), ferrocene
and Fe(C5H4CO2CH3)2, as
well as all the remaining functionalized ferrocenes undergo a reversible redox
process with an ipa/ipc ratio
close to one. All the functionalized ferrocene
demonstrated oxidation potentials, Epa,
higher than
ferrocene. This is the result of the electro-withdrawing (inductive) effect of the ester groups on the
Cp rings. Since ferrocene is known to undergo one-electron redox process, it
can be deduced that the subject complexes also undergo one-electron redox
change. Thus, this major redox process on the functionalized ferrocenes is
associated to the ferrocene/ferrocenium couple.Table 1
Redox potential of
ferrocenyl esters inCH3CN 1M CH3CN 1M [NBun4]PF6 at a scan rate of 100 mV/s, referenced to ferrocene/ferrocenium redox couple.
Ferrocene concentration of 2×10−3 M.  E1/2 is an average of the anodic and
cathodic peaks potentials.
ComplexE1/2 (mV)ΔE (mV)Fe(C5H4CO2CH3)245475Fe(CpCOOCH2CH3)244874Fe(CpCOOCH3)(CpCOOCH2CH3)45278Fe(C5H4CO2CH2CH=CH2)245385Fe(C5H4CO2CH2CH2OH)245689Figure 2
Cyclic voltammograms
of complexes 2: Fe(CpCOOMe)2, 3: Fe(CpCOOEt)2, 4: Fe(CpCOOMe)(CpCOOEt),
5: Fe(CpCOOCH2CH=CH2)2, and 6: Fe(CpCOOCH2CH2OH)2 (2×10−2 M) in CH3CN (1×10−3 M Bu4NPF6)
at room temperature. The working electrode was a platinum disk, reference
electrode was Ag/AgCl, and the scan rate was 100 mv/s.


### 2.3. Cytotoxic Studies—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT): Colorimetric Assay for Cytotoxicity Analysis on the Colon Cancer HT-29 and Breast MCF-7 Cell Lines.

The cytotoxicities
of the ferrocenyl ester complexes on the HT-29 colon cancer and MCF-7 breast
cancer cell lines were measured using a slightly modified MTT assay [23, 24].
Ferrocene was initially evaluated at time intervals of 72, 96, and 120 hours at
concentrations that ranged from 10–1200 μM, in
HT-29, to determine its optimal activity
(Supplementary Figure 1S). Ferrocene
displayed comparable activity at all three time intervals, with an IC50 value of 3.6×10−4 M. Since exposing the cells to ferrocene at
longer periods of time did not increase its cytotoxic activity, all subsequent
experiments were performed at a drug exposure time of 72 hours. Ferrocene,
ferrocenium, and the functionalized complexes (–CO2R, R=Me, Et)
were tested in concentrations which ranged from 10–1200 μM. In
addition, two control experiments were run 100% Medium and 5% DMSO/95% Medium. Both
control experiments behaved identical demonstrating that 5% DMSO in the Medium
does not have any cytotoxic effect on these cells. Table 2 summarizes the results
of the cytotoxicity experiments and Figure 3 depicts the cytotoxic curves from
MTT assays showing the effect of the ferrocene complexes on the viability of
HT-29 colon cancer cell line. The IC50 value represents the
concentration of the ferrocene at which the cell growth is inhibited by 50%. It
can be noted that bis(carboethoxycyclopentadienyl)ferrocene showed activity
comparable to ferrocene, with values of 370 and 360 μM,
respectively but it has less cytotoxicity than ferrocenium (180 μM).
In contrast,
(carboethoxycyclopentadienyl)-(carbomethoxycyclopentadienyl)-ferrocene and the
bis(carbomethoxycyclopentadienyl)-ferrocene exhibited less cytotoxic activity
than ferrocene and ferrocenium. The carbomethoxy functionalization has been
shown previously to inactivate or lower the cytotoxic activity of resulting
titanocene complexes [19]. The present results clearly show how the presence of
the carbomethoxy substituent in the cyclopentadienyl ring lowers the activity
of ferrocene in a stepwise manner, being the bis(carbomethoxy)ferrocene less
active than (carbomethoxy)(carboethoxy)ferrocene. The carboethoxy
functionalization does not improve the activity of the ferrocene complexes as
compared to ferrocene.Table 2
Cytotoxicities of ferrocenes studied on HT-29 colon cancer and MCF-7 breast
cancer cell lines at 72 hours, as determined by MTT assay. IC values are the
average of four independent measurements with their standard deviations ().
HT-29MCF-7ComplexIC50 (μM)IC50 (μM)FeCp2360(30)1500(100)Fe(Cp–COOEt)2370(10)250(20)Fe(Cp–COOMe)(Cp–COOEt)500(20)320(30)Fe(Cp–COOMe)2720(50)520(20)Fe(CpCOOCH2CH2OH)2370(20)340(30)Fe(CpCOCH2CHCH2)2180(10)190(30)Fe[Cp2]BF4180(10)150(05)Figure 3
Dose-response curves
for functionalized cyclopentadienyl ferrocenes against HT-29 cells at 72 hours
drug exposure. Legend: ferrocene-diamonds, bis(carbomethoxy)-squares,
bis(carboethoxy)-triangles, and (carbomethoxy)(carboethoxy)-circles.Other ferrocenyl
complexes, varying the lipophilic character of the carboalkoxy groups, were
also evaluated for cytotoxic activity against HT-29 cells. Ferrocene complexes
with either terminal alcohol or allyl groups were tested in concentrations that
ranged from 13 to 1300μM at a 72-hour time interval. IC50 values
for these complexes are also summarized in Table 2, and Figure 4 shows the
cytotoxic curves for these complexes along with ferrocene and ferrocenium for comparison.
The ferrocene alcohol complex shows an IC50 value of 370 μM
which is comparable to ferrocene,
while (allyl) complex shows higher cytotoxic activity than ferrocene and equal
to the ferrocenium at the time interval studied, with an IC50 value
of 180 μM.Figure 4
Dose-response curves
for functionalized ferrocene complexes on HT-29 colon adenocarcinoma cells at
72 hours. Complex with terminal alcohol (diamonds), complex with terminal allyl
(squares), ferrocene (triangles), and ferrocenium ion (circles).The initial work
performed by Kopf-Maier et al. demonstrated that ferrocenium possesses in vivo anticancer activity in breast
cancer [9]. Based on this precedent, we investigated our functionalized
ferrocenes in MCF-7 breast cancer cell line. Surprisingly, ferrocene showed to
be less cytotoxic in breast cancer than in colon cancer. With regard to the
functionalized ferrocenes and similar to the results on HT-29 cell line, a
pattern in the cytotoxicity as we change the functional group (pendant group)
is evident. First, in the ferrocenyl with carboalkoxy groups, the incorporation
of methyl ester (–CO2CH3) groups on the Cp rings decreases the cytotoxic
activity of the resulting complexes. Second, see Figure 5, the increase in the
lipophilic character on the carboalkoxy substituents such as in Fe(C5H4CO2CH2CH=CH2)2,
where the ethyl group is substituted by an olefin, increases the cytotoxic
activity. Such behavior has been reported previously by two-independent research
groups, as described below [12, 25].Figure 5
Dose-response curves for functionalized
ferrocene complexes on MCF-7 breast cancer cells at 72 hours.
Ferrocene(diamonds), ferrocenium(squares), bis(carboethoxy)(triangles), and
terminal allyl (circles).A wide variety of ferrocenyl
derivatives have been synthesized by several research groups aimed to improve
their anticancer activity as well as to understand the structure-activity
relationship. Very active ferrocenyl derivatives have been synthesized
containing estrogen receptor modulators as pendant groups [26]. Among them,
1,1′-bis-(4′-hydroxyphenyl)-2-ferrocenyl-but-1-ene showed strong antiproliferative
activity in both hormone-dependent (MCF-7) and hormone-independent (MDA-MDA231)
breast cancer cells with IC50=0.7 and 0.6 μM, respectively [26]. On
the other hand, ferrocenyl carbohydrate conjugates prepared by Orvig et al. showed
IC50 values between 87–468 μM on HTB-129
human breast cancer cell line [27]. Our ferrocenyl esters showed IC50 values similar to those of the ferrocenyl carbohydrates [27] and ferrocenium
derivatives tested on MCF-7 cell line: ferroceniumcarboxylic acid
tetrafluoroborate (IC50=340 μM), decamethylferrocene
tetrafluoroborate (IC50=37 μM), 1,1′-dimethylferrocenium tetrafluoroborate (IC50=320 μM), and ferrocenium boronic
acid tetrafluoroborateIC50=317 μM) [12].  It should be clearly noticed
that, as reported previously, the increase in the lipophilic character on the
ferrocene or ferrocenium complex increases the cytotoxic activity [12, 25].For colon cancer, the cytotoxic data is more
limited for a more meaningful comparison and assessment. On a recent review on
the medicinal properties of ferrocenes, it was described a series of ferrocene
conjugates (functionalized) anchored to polyaspartimides (water soluble carrier
polymers) and evaluated on Colo and Hela colon cancer cell lines [11, 28]. The
IC50 values determined are in the 10−4–10−5 M
range, analogous to some of our most active species. From these studies, Neuse et
al. found that increasing the hydrophilicity of the polyaspartimide side chains
does not necessarily improve the cytotoxicity of the resulting ferrocenes,
similar to our findings [28]. On the other hand, opposite to our findings,
Kenny et al. studied the antiproliferative effect of a series of
N-(ferrocenyl)benzoyl dipeptide esters on H1229 lung cancer cells and found
that increasing the alkyl chain length of the amino acid lower the cytotoxic
activity [29]. However, since the data come from different cancer cell lines,
the comparison of the IC50 values becomes less accurate.Finally, an important conclusion
derived from this study is that minor changes in the pendant group on the Cp
ring may have notable impact in the cytotoxic activity of the resulting
ferrocenes. This is in good agreement with previous reports [12, 25]. Increasing
the lipophilic character of the ferrocenyl esters apparently improves their
biological activity, as a result of better transport of these species into the
target place inside the cell [12, 13, 25]. This increased lipophilic character
on the ferrocenes increases the cell uptake (improving cell membrane
permeability of the ferrocene), and more ferrocene molecules could become available
inside the cell to express their activity. It has been proposed by Lander et
al. that not only ferrocenium cation but also ferrocene is able to express
oxidative stress [15]. Ferrocene is able to generate H2O2 by autooxidation, forming ferrocenium ions. The immune-stimulatory properties
of ferrocene are postulated to be mediated by redox-sensitive signaling
proteins [15].


## 2.1. Synthesis and Structure

The syntheses of
Fe(C5H4CO2CH3)2, Fe(C5H4CO2CH2CH=CH2)2,
and Fe(C5H4CO2CH2CH2OH)2 have been reported previously [16, 17]. We applied
the methodology developed by Busetto et al. to synthesize Fe(C5H4CO2CH2CH3)2 and Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
(mixed ferrocene) [17]. Interestingly, using this
synthetic methodology, the reaction of FeCl2 and one equivalent of NaC5H4CO2CH2CH3 and NaC5H4CO2CH3, even at room
temperature, allowed the selective incorporation of two distinct functionalized
Cp ligands, affording the mixed ferrocene complex in good yield without the
formation of Fe(C5H4CO2CH3)2 and Fe(C5H4CO2CH2CH3)2.The NMR, IR, and
elemental analysis corroborate the identities of the ferrocenyl ester complexes.
The IR spectral data showedν(C=O) broadbands at 1708 cm−1 (Fe(C5H4CO2CH2CH3)2 and 1712 cm−1 (Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
corresponding to the carbonyl groups of the esters.  In the H1 NMR spectrum, Fe(C5H4CO2CH2CH3)2 exhibits two set of
resonances at 4.42
and 4.84 ppm. These Cp protons exhibit
coupling belonging to an AA′BB′ spin system. For the Fe(C5H4CO2CH2CH3)(C5H4CO2CH3),
the H1 NMR spectrum shows two sets of resonances at 4.42 and 4.85 ppm corresponding
to the Cp protons of C5H4CO2CH2CH3 and C5H4CO2CH3 rings. While we
might expect four sets
of resonances in the vinyl region corresponding to two different substituted Cp
rings, the fact that we observed only two sets suggests overlapping between these Cp proton
signals. Particularly interesting in the C13 NMR spectrum, at room
temperature, two sets of resonances for each carbon atom are observed in a
ratio of 1:1. This suggests that two possible conformational diastereoisomers, syn and anti, coexist in solution as
discussed below (Scheme 2) [18].Scheme 2
Conformational diastereoisomers
of Fe(C5H4CO2CH2CH3)(C5H4CO2CH3).In
solution, rapid ring oscillation about Cp–Fe–Cp axis allowsH2 and H5 (H2′ and H5′) and H3 and H4 (H3′ and H4′) to become
equivalents. This situation applies for both substituted Cp rings. Since both
Cp rings have ester groups, overlapping of the H2/H5 and
H2′/H5′, H3/H4 and H3′/H4′ signals in the H1 NMR
spectrum is expected. However, the position of the ester groups yields to
conformational diastereoisomers, syn and anti, which differ in the orientation of the carbonyl groups [18]. This condition applies when the C(Cp)–C(CO) bond is slow
in the NMR scale time. When the rotation is fast, having enough energy to
overcome the rotational barrier of the C(Cp)–C(CO) bond, such stereolabile
chirality disappears. In the C13 NMR spectrum, the carbonyl
orientation (syn and anti) makes significantly different
magnetic environment on the Cp carbons as well as on the carbonyl groups such that
two sets of
resonances corresponding to syn and anti conformers can be observed. Variable
temperature NMR studies, in toluene-d8, showed that at 80°C in the H1
spectrum, the multiplet signal at 4.85 ppm became a single line and the
multiplet at 4.43 ppm broadened. These signals belong to the Cp rings. In the C13
NMR spectrum at 80°C, the carbon signals of one conformer begin to increase in
intensity at expense of the other conformer, reaching a ratio of 1:2. This suggests that
conformational diastereoisomers, syn and anti, at room temperature, have
very similar stability but at high temperature, one diastereoisomer
(presumably) anti becomes more
populated.In general, the present synthetic route
developed by Busetto et al. [17] for the ferrocenyl ester complexes (Cp functionalization and formation of the
corresponding functionalized ferrocene) represents a convenient alternative procedure
for the functionalization of metallocenes and has been used by our group to
synthesize functionalized titanocenes [19].The solid-state structure of Fe(C5H4CO2CH3)2 was investigated by single-crystal X-ray diffraction, see Figure 1. Crystal
data and structure refinement are summarized in the Supplementary Material available online at
doi:10.1155/2009/420784
(Cambridge Crystallographic Data Centre and the deposition number is CCDC
705387). Bonding parameters are included in Supplementary Table 1.Figure 1
Solid-state structure of Fe(C5H4CO2CH3)2 drawn 50% thermal ellipsoids. Average Fe-Cp bonds, Fe(C1–C5)
2.049(28) Å and Fe(C6–C10) bond 2.048 (11) Å. Torsion
angles for C(8)–C(7)–C(11)–O(2) 6.9(4)° and O(4)–C(13)–C(5)–C(1) 15.2(4)°.As shown by X-ray, this ferrocene
is a sandwich complex with the cyclopentadienyl ligands adopting an eclipsed
conformation and with the pendant groups not completely opposite to each other.
The average Fe–C(Cp) distances for Fe(C5H4CO2CH3)2 are 2.049 and 2.048 Å′  which is very similar to those reported for
ferrocene, 2.045 Å′  [20].  The
shortest Fe–C(Cp) bond distances are on C(5) and C(7). The shorter bond
distances could be attributed to the inductive (electro-withdrawing) effect of
the ester groups on these carbon atoms. The
ester groups are not coplanar with the Cp ring, one of the OCH3 is bent toward the iron atom (below the Cp plane) with a
torsion angle of 15.2° while the other is bent away from the iron atom by 6.9°.
The C(Cp)–C(CO) bonds (C(5)–C(13) and C(7)–C(11)) are
somewhat shorter than a typical C-C single bond, 1.477(4) versus 1.54 Å′  (for a single bond), suggesting these bonds
have partial double bond character as a result of conjugation with the Cp π
system [21]. The later might explain the high C(Cp)–C(CO) rotational barrier
encountered in the Fe(C5H4CO2CH2CH3)(C5H4CO2CH3)
complex.  In
addition, the two CO2Me groups are anti to each other.While the solid structure of Fe(C5H4CO2CH3)2 has been reported previously, our solid-state structure has a fundamental
difference to that published by Cetina et al. [22]. That
is, in the lattice, they determined a hydrogen bonding network between the
carbonyl group and the CH3 of the adjacent molecule. We could not
determine such hydrogen bonding network between the carbonyl group and the CH3 of the adjacent molecule even though our data was collected at lower
temperature (173 K versus 293 K) and we obtained better refinement parameter (R
(I>2 sigma (I)) 0.0397
versus 0.046).


## 2.2. Cyclic Voltammetry

Electrochemical characterization of the
subject complexes was performed by mean of cyclic voltammetry (CV). It is well known that ferrocene
easily undergoes one electron oxidation to form ferrocenium (Cp2Fe+)
in a reversible manner. Thus, we investigated the functionalized ferrocenes electrochemical
behaviors and compared to ferrocene in organic solvent. Table 1 presents the CV results. As it can be
seen in the cyclic voltammograms (Figure 2), ferrocene
and Fe(C5H4CO2CH3)2, as
well as all the remaining functionalized ferrocenes undergo a reversible redox
process with an ipa/ipc ratio
close to one. All the functionalized ferrocene
demonstrated oxidation potentials, Epa,
higher than
ferrocene. This is the result of the electro-withdrawing (inductive) effect of the ester groups on the
Cp rings. Since ferrocene is known to undergo one-electron redox process, it
can be deduced that the subject complexes also undergo one-electron redox
change. Thus, this major redox process on the functionalized ferrocenes is
associated to the ferrocene/ferrocenium couple.Table 1
Redox potential of
ferrocenyl esters inCH3CN 1M CH3CN 1M [NBun4]PF6 at a scan rate of 100 mV/s, referenced to ferrocene/ferrocenium redox couple.
Ferrocene concentration of 2×10−3 M.  E1/2 is an average of the anodic and
cathodic peaks potentials.
ComplexE1/2 (mV)ΔE (mV)Fe(C5H4CO2CH3)245475Fe(CpCOOCH2CH3)244874Fe(CpCOOCH3)(CpCOOCH2CH3)45278Fe(C5H4CO2CH2CH=CH2)245385Fe(C5H4CO2CH2CH2OH)245689Figure 2
Cyclic voltammograms
of complexes 2: Fe(CpCOOMe)2, 3: Fe(CpCOOEt)2, 4: Fe(CpCOOMe)(CpCOOEt),
5: Fe(CpCOOCH2CH=CH2)2, and 6: Fe(CpCOOCH2CH2OH)2 (2×10−2 M) in CH3CN (1×10−3 M Bu4NPF6)
at room temperature. The working electrode was a platinum disk, reference
electrode was Ag/AgCl, and the scan rate was 100 mv/s.


## 2.3. Cytotoxic Studies—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT): Colorimetric Assay for Cytotoxicity Analysis on the Colon Cancer HT-29 and Breast MCF-7 Cell Lines.

The cytotoxicities
of the ferrocenyl ester complexes on the HT-29 colon cancer and MCF-7 breast
cancer cell lines were measured using a slightly modified MTT assay [23, 24].
Ferrocene was initially evaluated at time intervals of 72, 96, and 120 hours at
concentrations that ranged from 10–1200 μM, in
HT-29, to determine its optimal activity
(Supplementary Figure 1S). Ferrocene
displayed comparable activity at all three time intervals, with an IC50 value of 3.6×10−4 M. Since exposing the cells to ferrocene at
longer periods of time did not increase its cytotoxic activity, all subsequent
experiments were performed at a drug exposure time of 72 hours. Ferrocene,
ferrocenium, and the functionalized complexes (–CO2R, R=Me, Et)
were tested in concentrations which ranged from 10–1200 μM. In
addition, two control experiments were run 100% Medium and 5% DMSO/95% Medium. Both
control experiments behaved identical demonstrating that 5% DMSO in the Medium
does not have any cytotoxic effect on these cells. Table 2 summarizes the results
of the cytotoxicity experiments and Figure 3 depicts the cytotoxic curves from
MTT assays showing the effect of the ferrocene complexes on the viability of
HT-29 colon cancer cell line. The IC50 value represents the
concentration of the ferrocene at which the cell growth is inhibited by 50%. It
can be noted that bis(carboethoxycyclopentadienyl)ferrocene showed activity
comparable to ferrocene, with values of 370 and 360 μM,
respectively but it has less cytotoxicity than ferrocenium (180 μM).
In contrast,
(carboethoxycyclopentadienyl)-(carbomethoxycyclopentadienyl)-ferrocene and the
bis(carbomethoxycyclopentadienyl)-ferrocene exhibited less cytotoxic activity
than ferrocene and ferrocenium. The carbomethoxy functionalization has been
shown previously to inactivate or lower the cytotoxic activity of resulting
titanocene complexes [19]. The present results clearly show how the presence of
the carbomethoxy substituent in the cyclopentadienyl ring lowers the activity
of ferrocene in a stepwise manner, being the bis(carbomethoxy)ferrocene less
active than (carbomethoxy)(carboethoxy)ferrocene. The carboethoxy
functionalization does not improve the activity of the ferrocene complexes as
compared to ferrocene.Table 2
Cytotoxicities of ferrocenes studied on HT-29 colon cancer and MCF-7 breast
cancer cell lines at 72 hours, as determined by MTT assay. IC values are the
average of four independent measurements with their standard deviations ().
HT-29MCF-7ComplexIC50 (μM)IC50 (μM)FeCp2360(30)1500(100)Fe(Cp–COOEt)2370(10)250(20)Fe(Cp–COOMe)(Cp–COOEt)500(20)320(30)Fe(Cp–COOMe)2720(50)520(20)Fe(CpCOOCH2CH2OH)2370(20)340(30)Fe(CpCOCH2CHCH2)2180(10)190(30)Fe[Cp2]BF4180(10)150(05)Figure 3
Dose-response curves
for functionalized cyclopentadienyl ferrocenes against HT-29 cells at 72 hours
drug exposure. Legend: ferrocene-diamonds, bis(carbomethoxy)-squares,
bis(carboethoxy)-triangles, and (carbomethoxy)(carboethoxy)-circles.Other ferrocenyl
complexes, varying the lipophilic character of the carboalkoxy groups, were
also evaluated for cytotoxic activity against HT-29 cells. Ferrocene complexes
with either terminal alcohol or allyl groups were tested in concentrations that
ranged from 13 to 1300μM at a 72-hour time interval. IC50 values
for these complexes are also summarized in Table 2, and Figure 4 shows the
cytotoxic curves for these complexes along with ferrocene and ferrocenium for comparison.
The ferrocene alcohol complex shows an IC50 value of 370 μM
which is comparable to ferrocene,
while (allyl) complex shows higher cytotoxic activity than ferrocene and equal
to the ferrocenium at the time interval studied, with an IC50 value
of 180 μM.Figure 4
Dose-response curves
for functionalized ferrocene complexes on HT-29 colon adenocarcinoma cells at
72 hours. Complex with terminal alcohol (diamonds), complex with terminal allyl
(squares), ferrocene (triangles), and ferrocenium ion (circles).The initial work
performed by Kopf-Maier et al. demonstrated that ferrocenium possesses in vivo anticancer activity in breast
cancer [9]. Based on this precedent, we investigated our functionalized
ferrocenes in MCF-7 breast cancer cell line. Surprisingly, ferrocene showed to
be less cytotoxic in breast cancer than in colon cancer. With regard to the
functionalized ferrocenes and similar to the results on HT-29 cell line, a
pattern in the cytotoxicity as we change the functional group (pendant group)
is evident. First, in the ferrocenyl with carboalkoxy groups, the incorporation
of methyl ester (–CO2CH3) groups on the Cp rings decreases the cytotoxic
activity of the resulting complexes. Second, see Figure 5, the increase in the
lipophilic character on the carboalkoxy substituents such as in Fe(C5H4CO2CH2CH=CH2)2,
where the ethyl group is substituted by an olefin, increases the cytotoxic
activity. Such behavior has been reported previously by two-independent research
groups, as described below [12, 25].Figure 5
Dose-response curves for functionalized
ferrocene complexes on MCF-7 breast cancer cells at 72 hours.
Ferrocene(diamonds), ferrocenium(squares), bis(carboethoxy)(triangles), and
terminal allyl (circles).A wide variety of ferrocenyl
derivatives have been synthesized by several research groups aimed to improve
their anticancer activity as well as to understand the structure-activity
relationship. Very active ferrocenyl derivatives have been synthesized
containing estrogen receptor modulators as pendant groups [26]. Among them,
1,1′-bis-(4′-hydroxyphenyl)-2-ferrocenyl-but-1-ene showed strong antiproliferative
activity in both hormone-dependent (MCF-7) and hormone-independent (MDA-MDA231)
breast cancer cells with IC50=0.7 and 0.6 μM, respectively [26]. On
the other hand, ferrocenyl carbohydrate conjugates prepared by Orvig et al. showed
IC50 values between 87–468 μM on HTB-129
human breast cancer cell line [27]. Our ferrocenyl esters showed IC50 values similar to those of the ferrocenyl carbohydrates [27] and ferrocenium
derivatives tested on MCF-7 cell line: ferroceniumcarboxylic acid
tetrafluoroborate (IC50=340 μM), decamethylferrocene
tetrafluoroborate (IC50=37 μM), 1,1′-dimethylferrocenium tetrafluoroborate (IC50=320 μM), and ferrocenium boronic
acid tetrafluoroborateIC50=317 μM) [12].  It should be clearly noticed
that, as reported previously, the increase in the lipophilic character on the
ferrocene or ferrocenium complex increases the cytotoxic activity [12, 25].For colon cancer, the cytotoxic data is more
limited for a more meaningful comparison and assessment. On a recent review on
the medicinal properties of ferrocenes, it was described a series of ferrocene
conjugates (functionalized) anchored to polyaspartimides (water soluble carrier
polymers) and evaluated on Colo and Hela colon cancer cell lines [11, 28]. The
IC50 values determined are in the 10−4–10−5 M
range, analogous to some of our most active species. From these studies, Neuse et
al. found that increasing the hydrophilicity of the polyaspartimide side chains
does not necessarily improve the cytotoxicity of the resulting ferrocenes,
similar to our findings [28]. On the other hand, opposite to our findings,
Kenny et al. studied the antiproliferative effect of a series of
N-(ferrocenyl)benzoyl dipeptide esters on H1229 lung cancer cells and found
that increasing the alkyl chain length of the amino acid lower the cytotoxic
activity [29]. However, since the data come from different cancer cell lines,
the comparison of the IC50 values becomes less accurate.Finally, an important conclusion
derived from this study is that minor changes in the pendant group on the Cp
ring may have notable impact in the cytotoxic activity of the resulting
ferrocenes. This is in good agreement with previous reports [12, 25]. Increasing
the lipophilic character of the ferrocenyl esters apparently improves their
biological activity, as a result of better transport of these species into the
target place inside the cell [12, 13, 25]. This increased lipophilic character
on the ferrocenes increases the cell uptake (improving cell membrane
permeability of the ferrocene), and more ferrocene molecules could become available
inside the cell to express their activity. It has been proposed by Lander et
al. that not only ferrocenium cation but also ferrocene is able to express
oxidative stress [15]. Ferrocene is able to generate H2O2 by autooxidation, forming ferrocenium ions. The immune-stimulatory properties
of ferrocene are postulated to be mediated by redox-sensitive signaling
proteins [15].


## 3. Experimental

### 3.1. General Procedure

All reactions
were performed under an atmosphere of dry nitrogen using Schlenk glassware or a
glovebox, unless otherwise stated. Reaction vessels were flame dried under a
stream of nitrogen, and anhydrous solvents were transferred by oven-dried
syringes or cannula.  Tetrahydrofuran was
dried and deoxygenated by distillation over K-benzophenone under nitrogen. Infrared
spectra were recorded on a Brucker Vector-22 spectrometer with the samples as
compressed KBr discs. The NMR spectra were obtained on a 500 MHz Bruker
spectrometer.  Elemental analyses were obtained
from Atlantic Microlab, Inc., Ga, USA. Electrochemical characterization was
carried out on a BAS CV050W voltammetric analyzer of Bioanalytical Systems, Inc. with a three-stand electrode cell. Cyclic
voltammetric experiments were performed in deoxygenatedCH3CN
solution of ferrocene complexes with 1M of [NBun4PF6 as supporting electrolyte and ferrocene complex concentration of 2×10−3M. The three electrodes used were platinum
disk as the working electrode, Ag/AgCl as a reference electrode, and Pt
wire as an auxiliary electrode. The working electrode was polished with 0.05 μm
alumina slurry for 1–2 minutes, and
then rinsed with double-distilled and deionized water. This cleaning process is
done before each CV experiment and a sweep between 0 and 2000 mV is performed
on the electrolyte solution to detect any possible deposition of ferrocene on
the electrode surface.Dimethyl carbonate, diethyl carbonate, diallyl carbonate, ethylene carbonate, diethyl ether (anhydrous≥99.7%), anhydrous FeCl2, [Cp2Fe]BF4, and CDCl3 were purchased from Sigma-Aldrich.
NaCp was prepared in situ by reacting freshly distilled cyclopentadiene and NaH
in THF. Silica gel was heated at
about 200°C while a slow stream of dry nitrogen was passed
through it.The syntheses of sodium
1-carbomethoxycyclopentadienide
(NaCpCOOCH3) and sodium
1-carboethoxycyclopentadienide
(NaCpCOOCH2CH3) have been reported previously by our
group [18].The colon cancer cell line HT-29 and
the breast adenocarcinoma cell line MCF7 were purchased from American Type
Culture Collection, Va, USA, and were at 37°C
and 95% Air/5% CO2. Growth medium for HT-29 was McCoy’s 5A complete
medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotic/antimycotic. Growth medium for MCF7 was Eagle’s Minimum Essential
Media supplemented with 10% (v/v) fetal bovine serum, 1% (v/v)
antibiotic/antimycotic, nonessential amino acids, and 0.01 mg/mL bovine insulin.
MTT and Triton X-100 used for the cytotoxic assay were obtained from Sigma. All MTT
manipulations were performed in a dark room.


### 3.2. Synthesis

#### 3.2.1. Synthesis of 1,1′-bis(carbomethoxy)-ferrocene (Fe(CpCOOCH3)2)

To a solution of
NaCpCOOCH3 (0.47 g, 3.1 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g, 1.5 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.45 g (95%) of orange
viscous oil. The product was redissolved in chloroform/hexane (1:9) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 3.85(s, 6H; –OCH3), 4.43[t, 4 H, J3(H,
H) = 1.5 Hz, AA′BB′; Cp], 4.85[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 51.71(–OCH3), 71.61(CH; Cp),
72.62(CH; Cp), 72.89(ipso-C; Cp), 170.82(C=O). IR
(KBr, cm−1): 2957, 1703, 1470, 1288, 1197, 1147, 965, 780. Anal. Calcd for C14H14O4Fe: C, 55.67; H, 4.64. Found:
C, 56.01; H, 4.74.


#### 3.2.2. Synthesis of 1,1′-bis(carboethoxy)-ferrocene (Fe(CpCOOCH2CH3)2

To a solution of
NaCpCOOCH2CH3 (0.50 g, 3.16 mmol) in THF 20 mL, was added
solid FeCl2 (0.2 g, 1.58 mmol). The solution was stirred for 24 hours
at room temperature. The solvent was removed under vacuum, and CH2Cl2 was added; the red suspension was first filtered on a Celite pad and then
chromatographed on silica gel eluting with Et2O to give 0.48 g (92%)
of orange viscous oil. The product was resolved in chloroform/hexane (1:12) at −20°C and orange solid could be obtained. H1NMR
(500 MHz, CDCl3): δ(ppm) 1.38(t, 6H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 4.31(q, 4H, J3 (H,
H) = 7.0 Hz; –OCH2CH3), 4.42[t, 4H, J3 (H,
H) = 1.5 Hz, AA′BB′; Cp], 4.84[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 14.53(–OCH2CH3),
60.43(–OCH2CH3), 71.53(CH; Cp), 72.71(CH; Cp),
73.16(ipso-C; Cp), 170.43(C=O). IR
(KBr, cm−1): 2985, 2936, 1708, 1479, 1457, 1379, 1282, 1144, 1030,
1017, 774. Anal. Calcd for C16H18O4Fe: C,
58.22; H, 5.46.  Found: C, 58.19; H,
5.48.


#### 3.2.3. Synthesis of 1-(carbomethoxy)-1′-(carboethoxy)-ferrocene (Fe(CpCOOCH3) (CpCOOCH2CH3)

To a solution of
NaCpCOOCH3 (0.23 g, 1.58 mmol) and NaCpCOOCH2CH3 (0.25 g, 1.58 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g,
1.58 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2 was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.46 g (92%) of orange
viscous oil. The product was dissolved in chloroform/hexane (1:10) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 1.39(t, 3H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 3.85, 3.86*(s, 3H; –OCH3),
4.32(q, 2H, J3 (H, H) = 7.0 Hz; –OCH2CH3),
4.43[m, 4H; Cp], 4.85[m, 4H; Cp].C13NMR(125 MHz, CDCl3): δ(ppm) 14.52, 14.55*(–OCH2CH3),
51.76(–OCH3), 60.46, 60.48*(–OCH2CH3),
71.57, 71.61*(CH; Cp), 72.73, 72.77*(CH; Cp), 72.86, 73.21*(ipso-C; Cp),
170.43, 170.49*(C=O), 170.87, 170.95*(C=O). IR
(KBr, cm−1): 2995, 2950, 1712, 1472, 1284,1143, 1030, 774, 514. Anal. Calcd for C15H16O4Fe: C,
57.00; H, 5.07.  Found: C, 57.19; H, 5.08.Complexes [Fe(C5H4CO2CH2CH=CH2)2]
and [Fe{C5H4CO2(CH2)2OH}2]
were prepared as described by Busetto et al. from sodium cyclopentadenide and
diallyl carbonate or solid ethylene carbonate [17].


### 3.3. Cytotoxic Assay

Biological activity was determined
using the MTT assay originally described by Mossman [19a]but using 10% Triton
in isopropanol as a solvent for the MTT formazan crystals [19b]. HT-29 and MCF7
cells were maintained at 37°C
and 95% Air/5% CO2 in McCoy’s 5A (ATCC) complete medium, which had
been supplemented with 10% (v/v) fetal bovine serum (ATCC) and 1% (v/v)
antibiotic/antimycotic (Sigma).
Asynchronously, growing cells were seeded at 1.5×104 cells per well
in 96-well plates containing 100 μL of complete growth medium, and allowed to
recover overnight. Various concentrations of the complexes (10–1300 μM)
dissolved in 5% DMSO/95% Medium were added to the wells (eight wells per concentration,
experiments performed in quadruplicate plates). The complexes solutions were
prepared first by dissolving the corresponding ferrocene in DMSO and then
Medium was added to a final composition of 5% DMSO/95% Medium. In addition to
the cells treated with the ferrocenes, two controls experiments were run one
without any addition of solvent mixture (5% DMSO/95% Medium) and one adding 5%
DMSO/95% Medium to the cells. Both control experiments behaved identical,
showing that 5% of DMSO in the Medium did not render toxic to these types of
cells. The cells were incubated for an additional 70 hours. After this time,
MTT dissolved in complete growth medium was added to each well to a final
concentration of 1.0 mg/mL and incubated for two additional hours. After this
period of time, all MTTs
containing medium were removed, cells were washed with cold PBS and
dissolved with 200 μL of a 10% (v/v) Triton X-100 solution in isopropanol.
After complete dissolution of the formazan crystals, well absorbances were
recorded in triplicates on a 340 ATTC Microplate Reader (SLT Lab Instruments) at
570 nm with background subtraction at 630 nm. Concentrations of compounds
required to inhibit cell proliferation by 50% (IC50) were calculated
by fitting data to a four-parameter logistic plot by means of SigmaPlot
software from SPSS, Ill,
USA.


### 3.4. X-Ray Crystallographic Analysis

A light orange needle crystal with0.15×0.04×0.01 mm in size was mounted on a cryoloop with Paratone oil. Data was collected in a nitrogen gas
stream at −173°C on a Bruker Smart system. Data collection was 99.6%
complete to 25° in θ. The data was integrated
using the Bruker SAINT software program. The structure was solved by direct
methods and all nonhydrogen atoms were refined anisotropically by full-matrix
least-squares (SHELXL-97).  The crystal structure has been
deposited at the Cambridge Crystallographic Data Centre and the deposition
number is CCDC 705387.


## 3.1. General Procedure

All reactions
were performed under an atmosphere of dry nitrogen using Schlenk glassware or a
glovebox, unless otherwise stated. Reaction vessels were flame dried under a
stream of nitrogen, and anhydrous solvents were transferred by oven-dried
syringes or cannula.  Tetrahydrofuran was
dried and deoxygenated by distillation over K-benzophenone under nitrogen. Infrared
spectra were recorded on a Brucker Vector-22 spectrometer with the samples as
compressed KBr discs. The NMR spectra were obtained on a 500 MHz Bruker
spectrometer.  Elemental analyses were obtained
from Atlantic Microlab, Inc., Ga, USA. Electrochemical characterization was
carried out on a BAS CV050W voltammetric analyzer of Bioanalytical Systems, Inc. with a three-stand electrode cell. Cyclic
voltammetric experiments were performed in deoxygenatedCH3CN
solution of ferrocene complexes with 1M of [NBun4PF6 as supporting electrolyte and ferrocene complex concentration of 2×10−3M. The three electrodes used were platinum
disk as the working electrode, Ag/AgCl as a reference electrode, and Pt
wire as an auxiliary electrode. The working electrode was polished with 0.05 μm
alumina slurry for 1–2 minutes, and
then rinsed with double-distilled and deionized water. This cleaning process is
done before each CV experiment and a sweep between 0 and 2000 mV is performed
on the electrolyte solution to detect any possible deposition of ferrocene on
the electrode surface.Dimethyl carbonate, diethyl carbonate, diallyl carbonate, ethylene carbonate, diethyl ether (anhydrous≥99.7%), anhydrous FeCl2, [Cp2Fe]BF4, and CDCl3 were purchased from Sigma-Aldrich.
NaCp was prepared in situ by reacting freshly distilled cyclopentadiene and NaH
in THF. Silica gel was heated at
about 200°C while a slow stream of dry nitrogen was passed
through it.The syntheses of sodium
1-carbomethoxycyclopentadienide
(NaCpCOOCH3) and sodium
1-carboethoxycyclopentadienide
(NaCpCOOCH2CH3) have been reported previously by our
group [18].The colon cancer cell line HT-29 and
the breast adenocarcinoma cell line MCF7 were purchased from American Type
Culture Collection, Va, USA, and were at 37°C
and 95% Air/5% CO2. Growth medium for HT-29 was McCoy’s 5A complete
medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotic/antimycotic. Growth medium for MCF7 was Eagle’s Minimum Essential
Media supplemented with 10% (v/v) fetal bovine serum, 1% (v/v)
antibiotic/antimycotic, nonessential amino acids, and 0.01 mg/mL bovine insulin.
MTT and Triton X-100 used for the cytotoxic assay were obtained from Sigma. All MTT
manipulations were performed in a dark room.


## 3.2. Synthesis

### 3.2.1. Synthesis of 1,1′-bis(carbomethoxy)-ferrocene (Fe(CpCOOCH3)2)

To a solution of
NaCpCOOCH3 (0.47 g, 3.1 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g, 1.5 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.45 g (95%) of orange
viscous oil. The product was redissolved in chloroform/hexane (1:9) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 3.85(s, 6H; –OCH3), 4.43[t, 4 H, J3(H,
H) = 1.5 Hz, AA′BB′; Cp], 4.85[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 51.71(–OCH3), 71.61(CH; Cp),
72.62(CH; Cp), 72.89(ipso-C; Cp), 170.82(C=O). IR
(KBr, cm−1): 2957, 1703, 1470, 1288, 1197, 1147, 965, 780. Anal. Calcd for C14H14O4Fe: C, 55.67; H, 4.64. Found:
C, 56.01; H, 4.74.


### 3.2.2. Synthesis of 1,1′-bis(carboethoxy)-ferrocene (Fe(CpCOOCH2CH3)2

To a solution of
NaCpCOOCH2CH3 (0.50 g, 3.16 mmol) in THF 20 mL, was added
solid FeCl2 (0.2 g, 1.58 mmol). The solution was stirred for 24 hours
at room temperature. The solvent was removed under vacuum, and CH2Cl2 was added; the red suspension was first filtered on a Celite pad and then
chromatographed on silica gel eluting with Et2O to give 0.48 g (92%)
of orange viscous oil. The product was resolved in chloroform/hexane (1:12) at −20°C and orange solid could be obtained. H1NMR
(500 MHz, CDCl3): δ(ppm) 1.38(t, 6H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 4.31(q, 4H, J3 (H,
H) = 7.0 Hz; –OCH2CH3), 4.42[t, 4H, J3 (H,
H) = 1.5 Hz, AA′BB′; Cp], 4.84[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 14.53(–OCH2CH3),
60.43(–OCH2CH3), 71.53(CH; Cp), 72.71(CH; Cp),
73.16(ipso-C; Cp), 170.43(C=O). IR
(KBr, cm−1): 2985, 2936, 1708, 1479, 1457, 1379, 1282, 1144, 1030,
1017, 774. Anal. Calcd for C16H18O4Fe: C,
58.22; H, 5.46.  Found: C, 58.19; H,
5.48.


### 3.2.3. Synthesis of 1-(carbomethoxy)-1′-(carboethoxy)-ferrocene (Fe(CpCOOCH3) (CpCOOCH2CH3)

To a solution of
NaCpCOOCH3 (0.23 g, 1.58 mmol) and NaCpCOOCH2CH3 (0.25 g, 1.58 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g,
1.58 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2 was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.46 g (92%) of orange
viscous oil. The product was dissolved in chloroform/hexane (1:10) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 1.39(t, 3H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 3.85, 3.86*(s, 3H; –OCH3),
4.32(q, 2H, J3 (H, H) = 7.0 Hz; –OCH2CH3),
4.43[m, 4H; Cp], 4.85[m, 4H; Cp].C13NMR(125 MHz, CDCl3): δ(ppm) 14.52, 14.55*(–OCH2CH3),
51.76(–OCH3), 60.46, 60.48*(–OCH2CH3),
71.57, 71.61*(CH; Cp), 72.73, 72.77*(CH; Cp), 72.86, 73.21*(ipso-C; Cp),
170.43, 170.49*(C=O), 170.87, 170.95*(C=O). IR
(KBr, cm−1): 2995, 2950, 1712, 1472, 1284,1143, 1030, 774, 514. Anal. Calcd for C15H16O4Fe: C,
57.00; H, 5.07.  Found: C, 57.19; H, 5.08.Complexes [Fe(C5H4CO2CH2CH=CH2)2]
and [Fe{C5H4CO2(CH2)2OH}2]
were prepared as described by Busetto et al. from sodium cyclopentadenide and
diallyl carbonate or solid ethylene carbonate [17].


## 3.2.1. Synthesis of 1,1′-bis(carbomethoxy)-ferrocene (Fe(CpCOOCH3)2)

To a solution of
NaCpCOOCH3 (0.47 g, 3.1 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g, 1.5 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.45 g (95%) of orange
viscous oil. The product was redissolved in chloroform/hexane (1:9) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 3.85(s, 6H; –OCH3), 4.43[t, 4 H, J3(H,
H) = 1.5 Hz, AA′BB′; Cp], 4.85[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 51.71(–OCH3), 71.61(CH; Cp),
72.62(CH; Cp), 72.89(ipso-C; Cp), 170.82(C=O). IR
(KBr, cm−1): 2957, 1703, 1470, 1288, 1197, 1147, 965, 780. Anal. Calcd for C14H14O4Fe: C, 55.67; H, 4.64. Found:
C, 56.01; H, 4.74.


## 3.2.2. Synthesis of 1,1′-bis(carboethoxy)-ferrocene (Fe(CpCOOCH2CH3)2

To a solution of
NaCpCOOCH2CH3 (0.50 g, 3.16 mmol) in THF 20 mL, was added
solid FeCl2 (0.2 g, 1.58 mmol). The solution was stirred for 24 hours
at room temperature. The solvent was removed under vacuum, and CH2Cl2 was added; the red suspension was first filtered on a Celite pad and then
chromatographed on silica gel eluting with Et2O to give 0.48 g (92%)
of orange viscous oil. The product was resolved in chloroform/hexane (1:12) at −20°C and orange solid could be obtained. H1NMR
(500 MHz, CDCl3): δ(ppm) 1.38(t, 6H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 4.31(q, 4H, J3 (H,
H) = 7.0 Hz; –OCH2CH3), 4.42[t, 4H, J3 (H,
H) = 1.5 Hz, AA′BB′; Cp], 4.84[t, 4H, J3 (H,
H) = 1.5 Hz; AA′BB′; Cp].C13NMR(125 MHz,
CDCl3): δ(ppm) 14.53(–OCH2CH3),
60.43(–OCH2CH3), 71.53(CH; Cp), 72.71(CH; Cp),
73.16(ipso-C; Cp), 170.43(C=O). IR
(KBr, cm−1): 2985, 2936, 1708, 1479, 1457, 1379, 1282, 1144, 1030,
1017, 774. Anal. Calcd for C16H18O4Fe: C,
58.22; H, 5.46.  Found: C, 58.19; H,
5.48.


## 3.2.3. Synthesis of 1-(carbomethoxy)-1′-(carboethoxy)-ferrocene (Fe(CpCOOCH3) (CpCOOCH2CH3)

To a solution of
NaCpCOOCH3 (0.23 g, 1.58 mmol) and NaCpCOOCH2CH3 (0.25 g, 1.58 mmol) in THF 20 mL, was added solid FeCl2 (0.2 g,
1.58 mmol). The solution was stirred for 24 hours at room temperature. The
solvent was removed under vacuum, and CH2Cl2 was added;
the red suspension was first filtered on a Celite pad and then chromatographed
on silica gel eluting with Et2O to give 0.46 g (92%) of orange
viscous oil. The product was dissolved in chloroform/hexane (1:10) at −20°C and orange solid could be obtained.H1NMR
(500 MHz, CDCl3): δ(ppm) 1.39(t, 3H, J3 (H, H) =
7.0 Hz; –OCH2CH3), 3.85, 3.86*(s, 3H; –OCH3),
4.32(q, 2H, J3 (H, H) = 7.0 Hz; –OCH2CH3),
4.43[m, 4H; Cp], 4.85[m, 4H; Cp].C13NMR(125 MHz, CDCl3): δ(ppm) 14.52, 14.55*(–OCH2CH3),
51.76(–OCH3), 60.46, 60.48*(–OCH2CH3),
71.57, 71.61*(CH; Cp), 72.73, 72.77*(CH; Cp), 72.86, 73.21*(ipso-C; Cp),
170.43, 170.49*(C=O), 170.87, 170.95*(C=O). IR
(KBr, cm−1): 2995, 2950, 1712, 1472, 1284,1143, 1030, 774, 514. Anal. Calcd for C15H16O4Fe: C,
57.00; H, 5.07.  Found: C, 57.19; H, 5.08.Complexes [Fe(C5H4CO2CH2CH=CH2)2]
and [Fe{C5H4CO2(CH2)2OH}2]
were prepared as described by Busetto et al. from sodium cyclopentadenide and
diallyl carbonate or solid ethylene carbonate [17].


## 3.3. Cytotoxic Assay

Biological activity was determined
using the MTT assay originally described by Mossman [19a]but using 10% Triton
in isopropanol as a solvent for the MTT formazan crystals [19b]. HT-29 and MCF7
cells were maintained at 37°C
and 95% Air/5% CO2 in McCoy’s 5A (ATCC) complete medium, which had
been supplemented with 10% (v/v) fetal bovine serum (ATCC) and 1% (v/v)
antibiotic/antimycotic (Sigma).
Asynchronously, growing cells were seeded at 1.5×104 cells per well
in 96-well plates containing 100 μL of complete growth medium, and allowed to
recover overnight. Various concentrations of the complexes (10–1300 μM)
dissolved in 5% DMSO/95% Medium were added to the wells (eight wells per concentration,
experiments performed in quadruplicate plates). The complexes solutions were
prepared first by dissolving the corresponding ferrocene in DMSO and then
Medium was added to a final composition of 5% DMSO/95% Medium. In addition to
the cells treated with the ferrocenes, two controls experiments were run one
without any addition of solvent mixture (5% DMSO/95% Medium) and one adding 5%
DMSO/95% Medium to the cells. Both control experiments behaved identical,
showing that 5% of DMSO in the Medium did not render toxic to these types of
cells. The cells were incubated for an additional 70 hours. After this time,
MTT dissolved in complete growth medium was added to each well to a final
concentration of 1.0 mg/mL and incubated for two additional hours. After this
period of time, all MTTs
containing medium were removed, cells were washed with cold PBS and
dissolved with 200 μL of a 10% (v/v) Triton X-100 solution in isopropanol.
After complete dissolution of the formazan crystals, well absorbances were
recorded in triplicates on a 340 ATTC Microplate Reader (SLT Lab Instruments) at
570 nm with background subtraction at 630 nm. Concentrations of compounds
required to inhibit cell proliferation by 50% (IC50) were calculated
by fitting data to a four-parameter logistic plot by means of SigmaPlot
software from SPSS, Ill,
USA.


## 3.4. X-Ray Crystallographic Analysis

A light orange needle crystal with0.15×0.04×0.01 mm in size was mounted on a cryoloop with Paratone oil. Data was collected in a nitrogen gas
stream at −173°C on a Bruker Smart system. Data collection was 99.6%
complete to 25° in θ. The data was integrated
using the Bruker SAINT software program. The structure was solved by direct
methods and all nonhydrogen atoms were refined anisotropically by full-matrix
least-squares (SHELXL-97).  The crystal structure has been
deposited at the Cambridge Crystallographic Data Centre and the deposition
number is CCDC 705387.



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*Source: 420784-2009-03-24.xml*