The effect of protic electron donor aromatic substituents on ferrocenic and [3]ferrocenophanic anilines and anilides: Some aspects of structureeactivity relationship studies on organometallic compounds with strong antiproliferative effects

Article abstract of DOI:10.1016/j.jorganchem.2013.05.047

A new family of nitrogenous derivatives is synthesized, characterized and evaluated for the investigation of the impact of some structural motifs such as functionalization and conjugation on the antiproliferative activity of ferrocenic complexes against cancer cells. These compounds are 4,40-([3]ferrocenophan-1-ylidenemethylene)dianilides and tetramethylated dianilines derived from Michler's ketone. An alternative McMurry direct heterocoupling method for 4,40-([3]ferrocenophan-1-ylidenemethylene)dianiline synthesis is described and electrochemical studies are also discussed.

Full text of DOI:10.1016/j.jorganchem.2013.05.047

Accepted Manuscript
The Effect of Protic Electron Donor Aromatic Substituents on Ferrocenic and
[3] Ferrocenophanic Anilines and Anilides: Some Aspects of Structure-Activity
Relationship Studies on Organometallic Compounds with Strong Antiproliferative
Effects
José de Jesús Cá zares-Marinero, Eric Labbé , Siden Top, Oliver Buriez, Christian
Amatore, Gé rard Jaouen
PII:
S0022-328X(13)00437-3
10.1016/j.jorganchem.2013.05.047
JOM 18069
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 29 March 2013
Revised Date: 20 May 2013
Accepted Date: 22 May 2013
Please cite this article as: J. de Jesús Cá zares-Marinero, E. Labbé , S. Top , O. Buriez, C. Amatore,
G. Jaouen, The Effect of Protic Electron Donor Aromatic Substituents on Ferrocenic and [3]
Ferrocenophanic Anilines and Anilides: Some Aspects of Structure-Activity Relationship Studies on
Organometallic Compounds with Strong Antiproliferative Effects, Journal of Organometallic Chemistry
(2013), doi: 10.1016/j.jorganchem.2013.05.047.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Highlights:
1. Strongly cytotoxic ([3]ferrocenophan-1-ylidenemethylene)dianiline was prepared.
2. Ferrocenophane compounds derived from Michler’s ketone were synthesized.
3. N-acylation and N-methylation decreases activity of dianilines.
4. Intramolecular electron transfer process is observed in nitrogenous derivatives.
5. Aniline oxidation is influenced by the nature of organometallic moiety

ACCEPTED MANUSCRIPT
The Effect of Protic Electron Donor Aromatic Substituents on Ferrocenic and
[3]Ferrocenophanic Anilines and Anilides: Some Aspects of Structure-Activity
Relationship Studies on Organometallic Compounds with Strong Antiproliferative
Effects
José de Jesús Cázares-Marineroa, Eric Labbéb, Siden Top*a, Oliver Buriezb, Christian Amatoreb
and Gérard Jaouen*a
aChimie ParisTech, Ecole Nationale Supérieure de Chimie de Paris, Laboratoire Charles Friedel, 11 rue Pierre et
Marie Curie, 75231 Paris Cedex 05, France.
bEcole Normale Supérieure, Département de Chimie, UMR CNRS-ENS-UPMC 8640, 24 rue Lhomond, 75231 Paris
Cedex 05, France.
Dedicated to Prof. W. A. Herrmann on the occasion of his 65th birthday
Correspondence :
* Pr Gérard Jaouen
Tél : 33 1 43 26 95 55
e-mail : gerard-jaouen@chimie-paristech.fr
* Dr Siden Top
Tél : 33 1 44 27 66 99
e-mail : siden-top@chimie-paristech.fr
Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223,
11 rue P. et M. Curie, 75231 Paris cedex 05, France.
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Keywords: anilines, anilides, ferrocene, ferrocenophane, bioorganometallic, cancer, cytotoxicity.
A B S T R A C T
A new family of nitrogenous derivatives is synthesized, characterized and evaluated for the
investigation of the impact of some structural motifs such as functionalization and
conjugation on the antiproliferative activity of ferrocenic complexes against cancer cells,.
These
compounds
are
4,4’-([3]ferrocenophan-1-ylidenemethylene)dianilides
and
tetramethylated dianilines derived from Michler’s ketone. An alternative McMurry direct
heterocoupling method for 4,4’-([3]ferrocenophan-1-ylidenemethylene)dianiline synthesis is
described and electrochemical studies are also discussed.
1. Introduction
Bioorganometallic chemistry, a burgeoning discipline involving the study of molecules bearing
at least one carbon-metal covalent bond (C-M) and their biological applications, has grown
exponentially in the last few years [1, 2]. A pertinent model of an organometallic moiety is ferrocene,
which has been shown to be a desirable motif for the design of new products in medicinal chemistry
research [3, 4].
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Generally, the antiproliferative properties of metal-based drugs are attributed to their direct
interaction with deoxyribonucleic acid (DNA), as proposed from the coordination complex cisplatin
[5]. Nevertheless, our group has focused on the synthesis of complexes that appear to be related to
non-genomic mechanisms, and which could represent an alternative for the treatment of drug-
resistant or incurable cancers. We found that organic polyphenols such as 1 (resveratrol) and 2 could
be activated by judicious incorporation of organometallic units on their skeletons. For example, the
ferrocene derivative 3 (Figure 1) showed strong antiproliferative activity, not only against MCF-7 cells,
but also against hormone-independent MDA-MB-231 breast cancer cells (IC50 = 0.6 µM), while 2
stimulated hormone-dependent MCF-7 breast cancer cell growth and had no effect on MDA-MB-231
cells [6,7].
Figure 1. Chemical structures of resveratrol 1, 4,4’-(2-phenylbut-1-ene-1,1-diyl)diphenol
2 and 4,4’-(2-ferrocenylbut-1-ene-1,1-diyl)diphenol 3.
It has been proposed that the antiproliferative activity of ferrocene compounds is related to the
generation of reactive oxygen species (ROS) in cancer cells [8, 9, 10, 11], and quinone methides such as
3-qm (Scheme 1) [12]. Therefore, the conjugation of the ferrocenyl group to the 4-hydroxyphenyl
group is essential for their activity [13].
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H
H
O
O
NH
NH
[O]
[O]
Fe
Fe
Fe
Fe
OH
OH
3
4
3-qm
4-iqm
0.64 ± 0.06 µM
0.86 ± 0.04 µM
H
N
N
O
O
Fe
Fe
4-ac
0.47 ± 0.04 µM
4-ac-iqm
Scheme 1. Formation of 4-[2-ferrocenyl-1-(4-hydroxyphenyl)but-2-en-1-ylidene]cyclohexa-2,5-
dienone 3-qm and 4-(2-ferrocenyl-1-phenylbut-2-en-1-ylidene)cyclohexa-2,5-dienimine 4-iqm
through the oxidation of diphenol 3 and monoaniline 4, respectively. No oxidation of 4-ac to 4-
ac-iqm occurs under the same conditions. IC50 values of 3, 4 and 4-ac against MDA-MB-231.
According to the mechanism shown in scheme 1, other protic and oxidizable functionalities
should also exhibit the same behavior. Indeed, during our investigations, it was observed that
compounds bearing amino (4) and acetamido (4-ac) functions also exhibited strong cytotoxic effects
against breast cancer cell lines [14,15]. It has been reported that 4, similarly to 3, could readily
undergo a base-promoted oxidation sequence [16], and the formation of iminoquinone methide (iqm)
intermediates under biological conditions has been proposed [17,18]. The high reactivity of these
species towards nucleophiles, in particular, towards thiols, polyhydroxylated compounds and cytosine,
as well as electron-deficient dienophiles [19], has also been reported. The cytotoxic properties of
amino analogues may be strongly related to the electron donor capacity of the heteroatom to activate
the π-conjugated system, and therefore, acidic protons in such substituents should be an important
element in the molecular design to assure cytotoxic effects. For 4-ac-like amide derivatives, it was
proposed that same oxidative pathway may be followed after enzymatic hydrolysis of the amide
functionality to afford 4-like compounds [20].
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On the other hand, we have recently shown that the replacement of the 1-
ferrocenylpropylidene (Fcpd) group by [3]ferrocenophan-1-ylidene (Fpd) yielded more effective
species against cancer cells (Figure 2) [21,22,23]. For example, the monoaniline 5 (IC50 = 0.21 ± 0.03
µM)[24] was four times more active than 4 (IC50 = 0.86 ± 0.04 µM) against triple negative MDA-MB-
231 breast cancer cells. Moreover, the additional amine substituent produced the dianiline 6 (0.05 ±
0.01 µM), which, in turn, was four times more active than the monoaniline 5. Nonetheless, it was
suspected that these constricted species do not undergo the same redox activation patterns as the
Fcpd derivatives, and consequently, exert their antiproliferative effects through different mechanisms
of action.
Figure 2. Chemical structures of monoaniline 5, dianiline 6 and diacetanilide 6-ac with
their IC50 values against hormone-independent MDA-MB-231 breast cancer cells.
Herein, we describe the synthesis and characterization of a new series of three organometallic
amides bearing non-functionalized lateral alkyl chains. With these species, we explored the impact of
chain length, the role of functionalization and the importance of organometallic moieties on their
antiproliferative activities. We also describe the synthesis and characterization of three new
tetramethylated dianilines derived from Michler’s ketone; with these, we evaluated the importance of
the hydrogen atoms on primary dianilines such as 6. To explore the impact of the Fpd motif on the
cytotoxicity, we also synthesized the organic dianiline 7. We expected these new compounds to show
very low antiproliferative effects to support our proposal regarding the formation of iminoquinone
methide-type derivatives as active metabolites. An alternative direct method for the synthesis of the
known 4,4’-([3]ferrocenophan-1-ylidenemethylene)dianiline 6 is also described. Finally, a mechanism
based on electrochemical studies explaining the impediments to iminoquinone methide-type
derivative formation from 4-ac-like N-[4-(2-ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]alkanamides is
proposed.
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2. Results and discussion
2.1 Synthesis
2.1.1 Alternative synthesis of 6 and synthesis of derived diamides 6-bu, 6-he and 6-oc.
The protocol reported for the organometallic dianiline 6 involves two steps [24]. First, a 3-day
McMurry cross-coupling reaction took place between [3]ferrocenophan-1-one and N,N’-
[carbonylbis(4,1-phenylene)]diacetamide to afford the intermediate diamide. This was followed by a
4-hour hydrolysis step to give the final product in 34% yield. We now describe its synthesis in only
one step by using a McMurry cross-coupling reaction between [3]ferrocenophan-1-one and 4,4’-
diaminobenzophenone, which gave a 42% yield in just 15 min (Scheme 2).
On
the
other
hand,
N,N’-[([3]ferrocenophan-1-ylidenemethylene)bis(4,1-
phenylene)]dialkanamides derived from dianiline 6 and butyric (bu), hexanoic (he) and octanoic (oc)
acids could be prepared through two pathways (Scheme 2). Pathway A involved a McMurry cross-
coupling reaction between [3]ferrocenophan-1-one and the corresponding N,N’-[carbonylbis(4,1-
phenylene)]dialkanamide, which was prepared previously through the amidation reaction (ii) of 4,4’-
diaminobenzophenone with acyl chlorides, to produce dialkanamides 6-he and 6-oc in 54% and 50%
yields, respectively. In pathway B, 6 reacted with the corresponding acyl chloride to form
dialkanamides 6-he and 6-oc in 83% and 80% yields, respectively.
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Alternative pathway for 6 synthesis
O
O
Cl
H
n
H2N
NH2
Fe
O
ii
i
n
NH2
N
N
O
O
6
he n = 4 (98%)
oc n = 6 (91%)
(42 %)
O
Fe
n
NH2
H
H
n
N
N
ii
O
i
Cl
n
O
O
Fe
Fe
n
H
O
Pathway B
Pathway A
n = 2 (83% via amidation)
n = 4 (54% via McMurry)
6-bu
6-he
6-oc n = 6 (80% via amidation)
(50% via McMurry)
Scheme 2. Alternative synthetic pathway for dianiline 6 and synthesis of N,N’-
[([3]ferrocenophan-1-ylidenemethylene)bis(4,1-phenylene)]dialkanamides 6-bu, 6-he and 6-
oc. Reagents and conditions: (i) THF, TEA, 15 min. (ii) Zn/TiCl4, THF, 90-100°C, 15 min.
2.1.2. Synthesis of organic dianiline 7 and tetramethylated dianilines 6-tm, 7-tm and 8-tm.
For the evaluation of the effect of the [3]ferrocenophan-1-ylidene (Fpd) motif on the
antiproliferative activity, an organic analogue of dianiline 6, bearing the 1-phenylpropilydene (Phpd)
group instead of Fpd, i.e. dianiline 7, was also prepared via the McMurry cross-coupling between 4,4’-
diaminobenzophenone and propiophenone in just 5 min after catalyst preparation (Scheme 3). Finally,
for evaluation of the effect of the removal of protons from the nitrogen atoms in dianilines 6 and 7,
[bis(4-(dimethylamino)phenyl]methanone (Michler’s ketone) was reacted with [3]ferrocenophan-1-
one and propiophenone, respectively, under McMurry cross-coupling conditions to produce the
corresponding methylated analogues 6-tm and 7-tm. Surprisingly, the tetramethylated dianiline 8-tm
7

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was formed in 3 h, while we were unable to isolate its protic analogue, the 4,4’-(2-ferrocenylbut-1-en-
1,1-diyl)dianiline.
Fe
Fe
Fpd
Phpd
Fcpd
O
Y
Zn/TiCl4
Y O
+
R
R
THF, reflux,
R
R
N
R
N
R
N
R
N
R
5 min for 7
20 min for 7-tm
25 min for 6-tm
3 h for 8-tm
Y
R
Y
R
Phpd
Fpd
4,4'-diaminobenzophenone H
Me
7(35%) Phpd
7-tm(74%) Phpd Me
H
propiophenone
[3]ferrocenophan-1-one
Michler'sketone
6-tm(41%)
8-tm(38%)
propionylferrocene Fcpd
Fpd
Fcpd Me
Me
Scheme 3. Synthesis of primary dianiline 7 and tertiary dianilines 6-tm, 7-tm and 8-tm.
It is important to mention that dianilines such as 6 and 7 showed particular behaviors in
acetone-d. For example, 1H-NMR analysis (Spectrum 1) of a sample of 7 stored in acetone-d for 13 days
showed a deformation of the triplet at 0.90 ppm (CH3) and the quartet at 2.50 ppm (CH2). An integral
decrease of the singlets at 4.42 ppm (NH2) and 4.63 ppm (NH2) and partially overlapped signals at 4.44
ppm and 4.67 ppm were also observed. In the aromatic region, new signals appeared, especially in the
ranges 6.74-6.85 ppm and 6.91-7.00 ppm. 1H-NMR analysis in DMSO-d did not show any changes, even
over a longer period (Spectrum 2). It was suspected that this particular behavior with acetone could
be the result of imine formation, highlighting the capability of the dianiline to react with electrophilic
species such as ketones.
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Spectrum 1. 1H-NMR (300 Hz) analysis of dianiline 7 stored in (CD3)2CO for 13 days
Spectrum 2. 1H-NMR (300 Hz) analysis of dianiline 7 stored in (CD3)2SO for 28 days
9

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2.2 Biological activity
2.2.1 Comparison of the antiproliferative activities of diamides 6-bu, 6-he and 6-oc and diamine 6.
The cytotoxic effects of diamides 6-bu, 6-he and 6-oc bearing the [3]ferrocenophan-1-ylidene
(Fpd) motif on hormone-independent MDA-MB-231 breast cancer cells were determined first. Figure 3
shows the percentage of cell growth inhibition at 1 µM. The values for 6-bu, 6-he, and 6-oc were 27%,
23%, and 18%, respectively. These results prove that the efficiencies of such species vary with the
chain length. Thus, the longer the alkyl side-chain of the compound, the lower its cytotoxicity; the
order of cytotoxicity was 6-bu > 6-he > 6-oc. At 10 µM, the inhibition value for 6-he was 51%, while
those of 6-bu and 6-oc were below 50% which indicates that they were less active than the
diacetanilide 6-ac (IC50 = 5.64 µM). It should be noted that these four anilides were far less active than
the diamine 6 (IC50 = 0.05 µM).
Figure 3. MDA-MB-231 breast cancer cell growth inhibition by
dianilides 6-bu, 6-he and 6-oc at 1 µM.
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2.2.1 Comparison of the antiproliferative activity of primary (6-7) and tertiary (6-tm, 7-tm and 8-tm)
dianilines.
For the evaluation of the impact of the [3]ferrocenophan-1-ylidene (Fpd) motif in the skeleton
of the organometallic compound 6, the organic analogue 7 (bearing the 1-phenylpropylidene (Phpd)
motif instead of Fpd) was also tested against hormone-independent MDA-MB-231 breast cancer cells.
The results (Figure 4) show the prominent antiproliferative effect of the organometallic amine 6 (IC50
= 0.05 ± 0.01 µM) against these cancer cell lines (76 times more active than aniline 7, with IC50 = 3.84 ±
0.23 µM). However, the results of the Fpd series seem to indicate that aromatic substituent
modification with electron-withdrawing groups such as acyl moieties has a greater impact on the
antiproliferative activity than Fpd replacement with the Phpd group. Compound 7 was also tested
against hormone-dependent MCF-7 breast cancer cells, and showed an IC50 value of 5.01 ± 0.21 µM.
Figure 4. Chemical structures of primary dianilines 6 and 7 with their IC50
values against hormone-independent MDA-MB-231 breast cancer cells.
Having seen the differences between the primary amines 6 and 7, and having recognized the
importance of the organometallic unit Fpd and the amine function in the production of strong
cytotoxic effects, we now evaluate the impact of the absence of protons on their nitrogenous
substituents. The results on MDA-MB-231 (Figure 5) show that the tetramethylated analogue of the
organic dianiline 7, 7-tm, was unable to inhibit cell growth, even at a concentration of 10 µM. The
tetramethylated dianiline 6-tm inhibited only 20% cell growth at a concentration of 10 µM, while its
protic analogue 6 inhibited 50% cell growth at a concentration of just 0.05 µM. The tetramethylated
aniline 8-tm, inhibited 15% of cell growth at a concentration of 10 µM.
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Figure 5. MDA-MB-231 cell growth inhibition (%) by tetramethylated dianilines 6-tm, 7-
tm and 8-tm at concentrations of 1 µM (gray) and 10 µM (black).
Once again, we highlight the impact of aromatic substituent modification on the cytotoxic
responses. Thus, we can assume that these modifications on the Fpd series, particularly on 6, affect the
compound activity negatively in the order N-dimethylation >> N-diacylation > Phpd replacement. The
ferrocenophanic dianiline 6, combining both structural imperatives, i.e., an Fpd motif and protic amino
substituents, was consequently the most cytotoxic compound investigated in this study, and one of the
most cytotoxic organometallic compounds reported against breast cancer cells.
2.3 Electrochemistry
The redox properties of the ferrocifen family are key issues for understanding their oxidative
metabolism. In this context, these compounds are usually investigated by means of cyclic voltammetry
in the absence and presence of a base model having a pKa value close to those of peptides or DNA
nitrogen intracellular bases [25,26]. Previous electrochemical studies have established that the
monoaniline 4 undergoes a base-promoted oxidation sequence to form iminoquinone methide-type
metabolites, which could play a very important role in the cytotoxic properties of the compound. First,
we explored the electrochemical behavior of the Fpd compound, the dianiline 6. The corresponding
CVs are displayed in Figure 6. Two oxidation waves were observed at 0.43 V (O1) and 0.90 V (O2).
Comparison with the electrochemical behavior reported for 4 [14, 19] prompted us to ascribe wave O1
to the monoelectronic oxidation of the ferrocene group, and wave O2 to the oxidation of both amine
groups of the ferricenium cation electrogenerated at O1 (the intensity of wave O2 is ca. twice that of
O1).
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Figure 6. Cyclic voltammogram for 6 with (dashed) and without (bold) 50 equivalents of
imidazole. Recorded in a solution of 0.1 M Bu4NBF4 in MeOH, with Pt (0.5 mm) working
electrode, stainless steel rod counter electrode, and Ag/AgCl/LiClsat in EtOH reference
electrode, at a scan rate of 0.5 Vs-1.
Interestingly, peculiar behavior was observed when the model compound imidazole was
added. Ferrocene oxidation became irreversible and shifted slightly to a higher potential, and the
prominent second wave assigned to aniline oxidation disappeared. This modification of the cyclic
voltammogram upon the addition of imidazole shows the occurrence of a base-triggered oxidation
sequence, similar to the one reported for the aniline 4, featuring a base-promoted intramolecular
proton-coupled electron transfer between the amine group and ferricenium moiety, ultimately leading
to an aminyl radical, which grafts onto the Pt electrode [19].
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In the absence of a base, the difference in E of the first oxidation wave [O1] for both
compounds 4 and 6 was 0.04 V; the organometallic moiety in 6 was the easiest to oxidize (i.e., at less
positive potential values). However, the difference in the oxidation potential values for the second
oxidation wave [O2] was 0.13 V; this time, the aniline function in 4 was the most easily oxidized. Cyclic
voltammetry was also performed for organic compounds 7 and aniline (C6H5NH2), and the potential
values are reported in Table 1. Compound 7 showed an irreversible oxidation wave at 0.79 V assigned
to the aniline moiety transformation, while aniline itself (recorded in MeCN) oxidized at 1.20 V, which
was consistent with a study showing the aniline potential oxidation to be around 1.00 V [27].
Table 1. Oxidation waves [O1] and [O2] for selected compounds.
Compound
O1 (V)
O2 (V)
C6H5NH2
-
1.20
0.90
6
0.43
7
4
-
0.79
0.77
0.47
The Fc+ antenna in compound 4 makes aniline oxidation “easier”, i.e., oxidation is observed at
less positive potential values (0.77 V vs. 1.20 V for aniline itself). This effect was also observed for the
ferrocenophanic compound 6; however, the effect was attenuated (0.90 V vs. 1.20 V for aniline itself)
when compared with 4. Interestingly, this oxidation potential value for organic 7 was rather similar to
that of 4. This seems to indicate that the overall electronic effects are prone to affect the energetics of
the oxidation of the aniline moiety, and therefore its oxidation potential values were similar for
compounds 4 and 7. Thus, the primary aniline oxidation potential may be influenced by the
substituent, decreasing in the following order: 1.20 V for H > 0.90 V for Fpd in 6 >> 0.79 V for Phpd in
7 > 0.77 V for Fcpd in 4. Rather similar potential values related to the oxidation of the amine moiety in
aniline and compound 6 seem to indicate that the dianiline 6 could only be partially conjugated to the
ferrocenophanic unit (its oxidation being “blind” towards the electronic influence of the ferricenium
species electrogenerated at O1), while in 4 and even 7, the electronic delocalization is possible in this
planar and flexible structures. The voltammograms of the tetramethylated dianilines 6-tm, 7-tm and
8-tm support this observation. Their potential values for the oxidation of the amine group were 0.83 V,
0.70 V and 0.56 V, respectively, thus respecting the established order Fpd > Phpd > Fcpd.
14

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Despite showing intermediate behavior between ferrocenophanic and ferrocenic compounds,
the organic dianiline 7 did not exhibit the same strong cytotoxic effects as the dianiline 6 and
monoanilines 4 and 5. This means that, despite the differences in reactivity observed in both the Fpd
and Fcpd series, which are both electrochemically active and therefore oxidizable, Fpd and Fcpd
compounds can be converted into ferricenium species that promote ROS production, which, in turn,
can activate tumor suppressor genes such as p53, leading to apoptosis or senescence in cancer cells, or
may cause direct DNA damage.
Neither the anilide 4-ac nor dianilides 6-bu, 6-he and 6-oc displayed the second oxidation
wave [O2] under the same conditions. 6-bu was chosen as the representative example (Figure 7). The
reversible wave observed at 0.48 V (a potential value slightly more positive than that of 6) was
assigned to the oxidation of the organometallic moiety displaying the expected energetic effect of
electron-withdrawing substituents on the oxidation of ferrocene.
Figure 7. Cyclic voltammogram for 6-bu with (dashed) and without (bold) 50 equivalents
of imidazole. Recorded in a solution 0.1 M of Bu4NBF4 in MeOH, with a Pt (0.5 mm)
working electrode, stainless steel rod counter electrode, and Ag/AgCl/LiClsat in EtOH
reference electrode, at a scan rate of 0.5 Vs-1.
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This could be explained by the fact that, after ferrocene oxidation, the nitrogen atom in 6-bu
does not undergo intramolecular electron transfer with ferricenium (as was proposed for aniline 4
activation) owing to a resonance effect with the adjacent carbonyl group of the amide function,
considering that the oxygen atom is more electronegative than nitrogen (the Pauling values for O and
N are 3.5 and 3.0, respectively [28]) (Scheme 4). Thus, nitrogen electrons could competitively and
formally displace towards the oxygen atom, resulting in an iminolic form. One can also envision that, if
the electron transfer occurs between nitrogen and ferricenium, the aminyl radical cation would also
experience the resonance effect and result in the formation of iminolic radical species, offering a
competitive parallel pathway.
R
N
OH
H
R
O
H
N+
H
Fe3+
R
N
OH
Fe3+
H
H
N
R
O
H
N
R
O
Fe2+
e-
H
H
e-
Fe2+
Fe3+
H
R
O
N+
H
R
O
R
O
N
N
H
H
Fe2+
Fe2+
Fe2+
N
H
N
H
H
N
N+
N
H
N+
R
O
H
N
R
N
N
H
N
O
H
H
R
N
Fe2+
O
Fe2+
H
e-
Fe3+
Scheme 4. Proposed mechanism for the electrochemical oxidation of N-[4-(2-
ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]alkanamides.
16

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3. Conclusions
We have demonstrated that the blockade of protic substituents by acyl or methyl groups in
nitrogenous aromatic derivatives dramatically decreases the antiproliferative activity of the
compounds. Notably, organic derivatives are far less cytotoxic than organometallic compounds.
However, aromatic substituent modification has a greater impact on the antiproliferative activity of
organometallic compounds than the replacement of the Fpd motif with the organic Phpd group. The
mechanism of activation seems to follow mutatis mutandis the intramolecular electron transfer
process previously observed for phenol derivatives. Despite their differences in terms of redox
activation pathways, ferrocenophanic and ferrocenic compounds are thought to produce ferric
metabolites capable of generating ROS in cancer cells. These latter species may be responsible for
cytotoxic effects via tumor suppressor gene activation, such as p53, or by direct DNA damage.
4. Experimental
4.1 General procedures
All reagents and solvents were obtained from commercial suppliers and used without further
purification. Tetrahydrofuran (THF) was distilled from Na/benzophenone under an argon atmosphere. Thin-
layer chromatography (TLC) was performed on silica gel 60 GF254. Column chromatography was
performed on silica gel Merck 60 (40-63 ꢀm). All of the products were characterized by conventional
techniques. Infrared (IR) spectra were recorded on a Jasco FT/IR-4100 Fourier transform infrared
spectrometer by using the potassium bromide (KBr) pellets technique, and all data are expressed in
1
wavenumbers (cm-1). Melting points (mp) were obtained with a Kofler device and are uncorrected. H and
13C NMR spectra were recorded on a 300 MHz Bruker spectrometer and chemical shifts (δ) are expressed in
ppm. The mass spectra (MS) were obtained on a DSQII and ITQ 1100 Thermo Scientific spectrometer for
both electronic impact (EI) and chemical ionization (CI) methods, and API 3000 PE Sciex Applied
Biosystems for the electrospray ionization (ESI) method. A purity of >99% was confirmed by analytical
reverse phase HPLC with columns Kromasil C18, 10 ꢀm, L = 25 cm, D = 4.6 mm and Macherey-Nagel C18,
5 ꢀm, L = 15 cm, D = 4.6 mm, using different mixtures of MeOH/H2O and MeCN/H2O as eluents (flow rate
= 1 mL/min, λ = 254 nm). Elemental analyses were performed by the Laboratory of Microanalysis at ICSN
of CNRS at Gif sur Yvette, France.
17

ACCEPTED MANUSCRIPT
4.2. Synthesis and characterization of compounds
4.2.1 Alternative pathway for synthesis of 4,4’-([3]ferrocenophan-1-ylidenemethylene)dianiline, 6[24]
In a Schlenk tube, Zn powder (36 mmol, 2.35 g) was suspended in THF (60 mL) at room
temperature and TiCl4 (18 mmol, 2.0 mL) was added slowly with a syringe while stirring. The reaction
mixture was heated to 90-100°C. After 1 h at reflux, a solution of [3]ferrocenophan-1-one (3.9 mmol,
0.94 g) and 4,4’-diaminobenzophenone (3.9 mmol, 0.83 g) in THF (30 mL) and DMSO (1mL) was
added. DMSO was used to improve the solubility of 4,4’-diaminobenzophenone. The reflux was
maintained for 15 min. Then, the mixture was poured into water and extracted with AcOEt. The
organic layer was washed with water, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude product was purified on a silica gel column by using petroleum ether/AcOEt
(75:25) as eluent. The product was obtained in 42% yield (0.70 g).
4.2.2 Amidation reactions
The corresponding primary amine was dissolved in 30 mL of THF (in the case of 4,4’-
diaminobenzophenone, DMSO was used to complete its solubilisation). With stirring, acyl chloride and
triethylamine (TEA) were added successively. After 15 min, water was added and the crude product
was extracted with AcOEt. The organic phase was washed with saturated NaHCO3 solution, dried over
MgSO4 and filtered. The solvents were evaporated under reduced pressure and the product was
purified on a silica gel column.
N,N’-[carbonylbis(4,1-phenylene)]dihexanamide, he
Reagents and quantities: 4,4’-diaminobenzophenone (4.72 mmol, 1.00 g), hexanoyl chloride
(9.44 mmol, 1.3 mL), TEA (9.44 mmol, 1.3 mL). The mixture was heated at 55°C. Eluent for column:
1
petroleum ether/ethyl acetate (1:1). The product was obtained in 98% yield (1.90 g). H-NMR (300
MHz, (CD3)2SO, ppm): δ 0.88 (t, J = 7.0 Hz, 6H, 2CH3), 1.20-1.40 (m, 8H, 4CH2), 1.55-1.70 (quint, J = 7.3
Hz, 4H, 2CH2), 2.35 (t, J = 7.4, 4H, 2CH2CO), 7.69 (d, J = 8.8 Hz, 4H, 4CHAr), 7.76 (d, J = 8.8 Hz, 4H, 4CHAr),
10.23 (s, 2H, 2NH).
N,N’-[carbonylbis(4,1-phenylene)]dioctanamide, oc
Reagents and quantities: 4,4’-diaminobenzophenone (4.72 mmol, 1.00 g), octanoyl chloride
(9.44 mmol, 1.6 mL), TEA (9.44 mmol, 1.3 mL). The mixture was heated at 55°C. Eluent for column:
1
petroleum ether/ethyl acetate (1:1). The product was obtained in 91% yield (2.00 g). H-NMR (300
18

ACCEPTED MANUSCRIPT
MHz, (CD3)2CO, ppm): δ 0.88 (t, J = 7.0 Hz, 6H, 2CH3), 1.20-1.40 (m, 16H, 8CH2), 1.69 (quint, J = 7.3 Hz,
4H, 2CH2), 2.44 (t, J = 7.4, 4H, 2CH2CO), 7.73 (d, J = 8.8 Hz, 4H, 4CHAr), 7.87 (d, J = 8.8 Hz, 4H, 4CHAr),
9.69 (s, 2H, 2NH).
N,N’-[([3]ferrocenophan-1-ylidenemethylene)bis(4,1-phenylene)]dibutyramide, 6-bu
Reagents and quantities: 6 (2.36 mmol, 0.99 g), butyryl chloride (4.72 mmol, 0.5 mL), TEA
(4.72 mmol, 0.7 mL). Eluent for column: chloroform/ethyl acetate/acetic acid (2:1:1). The first fraction
was the desired product 6-bu, the second fraction was identified as the monoamide/monoamine, and
the third was the remaining unreacted 6. 6-bu was obtained in 83% yield (1.10 g). mp: 147-148 °C
(methanol), 1H-NMR (300 MHz, (CD3)2CO, ppm): δ 0.91 (t, J = 7.4 Hz, 3H, CH3), 0.96 (t, J = 7.4 Hz, 3H,
CH3), 1.64 (sext, J = 7.4 Hz, 2H, CH2), 1.70 (sext, J = 7.4 Hz, 2H, CH2), 2.27 (t, J = 7.4 Hz, 2H, CH2CO), 2.35
(t, J = 7.4 Hz, 2H, CH2CO), 2.32-2.42 (m, 2H, CH2Cp’), 2.67-2.76 (m, 2H, CH2C=C), 3.93 (t, J = 1.8 Hz, 2H,
CpHβ’), 3.98 (t, J = 1.8 Hz, 2H, CpHβ), 4.01 (t, J = 1.8 Hz, 2H, CpHα), 4.27 (t, J = 1.8 Hz, 2H, CpHα’), 6.94 (d, J
= 8.7 Hz, 2H, 2CHAr), 7.17 (d, J = 8.7 Hz, 2H, 2CHAr), 7.39 (d, J = 8.7 Hz, 2H, 2CHAr), 7.68 (d, J = 8.7 Hz, 2H,
2CHAr), 8.99 (s, 1H, NHcis), 9.16 (s, 1H, NHtrans). 13C-NMR (75 MHz, (CD3)2CO, ppm): δ 14.0 (CH3), 14.5
(CH3), 19.5 (CH2), 19.6 (CH2), 29.2 (CH2Cp’), 39.6 (2CH2CO), 41.5 (CH2C=C), 68.9 (Cpβ’), 69.4 (Cpβ), 70.9
(Cpα), 71.1 (Cpα’), 84.5 (Cpipso), 87.6 (Cp’ipso), 118.7 (2CAr), 119.6 (2CAr), 130.4 (2CAr), 131.6 (2CAr), 135.1
(C=C), 138.5 (CAr), 139.2 (2CAr), 139.4 (CAr), 141.1 (C=C), 171.6 (COcis), 171.8 (COtrans). IR (KBr,
υmax/cm-1): 3298 (N-H stretch), 3101 (aromatic C-H stretch), 2958, 2873 (alkyl C-H stretch), 1662
(N=CO stretch), 1597 (aromatic C=C stretch) 1523 (N-H bend), 1400 (C-N stretch). MS (CI, m/z): 578
[MNH4]+, 561 [MH]+. Anal. Calc. for C34H36FeN2O2•CH3OH (%): C, 70.94; H, 6.80; N, 4.73. Found, C,
71.01; H, 6.84; N, 4.40. HPLC (RT), 2.09 min (Macherey-Nagel C18, 5 micron, 4.6 x 150 mm, MeOH,
1mL/min).
N,N’-[([3]ferrocenophan-1-ylidenemethylene)bis(4,1-phenylene)]dioctanamide, 6-oc
Pathway B. Reagents and quantities: 6 (0.59 mmol, 0.25 g), THF (15 mL), octanoyl chloride
(1.18 mmol, 0.2 mL), TEA (1.43 mmol, 0.2 mL). Eluent: chloroform/ethyl acetate/acetic acid (2:1:1).
The product was obtained in 80% yield (0.32 g). The first fraction was the desired product, the second
fraction was identified as the monoamide/monoamine, and the third was the remaining starting
material. mp : 119-120°C, 1H-NMR (300 MHz, (CD3)2CO, ppm): δ 0.86 (t, J = 7.0 Hz, 3H, CH3), 0.88 (t, J
= 7.0 Hz, 3H, CH3), 1.21-1.45 (m, 16H, 8CH2), 1.55-1.78 (m, 4H, 2CH2), 2.29 (t, J = 7.4 Hz, 2H, CH2CO),
2.37 (t, J = 7.4 Hz, 2H, CH2CO), 2.34-2.40 (m, 2H, CH2Cp’), 2.68-2.76 (m, 2H, CH2C=C), 3.93 (t, J = 1.8 Hz,
2H, CpHβ’), 3.98 (t, J = 1.8 Hz, 2H, CpHβ), 4.02 (t, J = 1.8 Hz, 2H, CpHα), 4.27 (t, J = 1.8 Hz, 2H, CpHα’), 6.93
(d, J = 8.7 Hz, 2H, 2CHAr), 7.17 (d, J = 8.7 Hz, 2H, 2CHAr), 7.39 (d, J = 8.7 Hz, 2H, 2CHAr), 7.67 (d, J = 8.7
Hz, 2H, 2CHAr), 8.98 (s, 1H, NHcis), 9.15 (s, 1H, NHtrans). 13C-NMR (75 MHz, (CD3)2CO, ppm): δ 14.3
19

ACCEPTED MANUSCRIPT
(2CH3), 23.3 (2CH2), 26.2 (CH2), 26.3 (CH2), 29.2 (CH2Cp’), 29.8 (2CH2), 29.9 (2CH2), 32.4 (2CH2), 37.7
(CH2CO), 37.8 (CH2CO), 41.5 (CH2C=C), 68.9 (Cpβ’), 69.4 (Cpβ), 70.9 (Cpα), 71.1 (Cpα’), 84.6 (Cpipso), 87.6
(Cp’ipso), 118.7 (2CAr), 119.6 (2CAr), 130.4 (2CAr), 131.6 (2CAr), 135.1 (C=C), 138.5 (CAr), 139.1 (CAr),
139.2 (CAr), 139.4 (CAr), 141.1 (C=C), 171.8 (COcis), 172.0 (COtrans). IR (KBr, υmax/cm-1): 3298 (N-H
stretch), 3101 (aromatic C-H stretch), 2924, 2854 (alkyl C-H stretch), 1662 (NC=O stretch), 1597
(aromatic C=C stretch), 1523 (N-H bend), 1400 (C-N stretch). MS (CI, m/z): 690 [MNH4]+, 673 [MH]+.
Anal. Calc. for C42H52FeN2O2 (%): C, 74.99; H, 7.79; N, 4.16. Found: C, 74.54; H, 7.90; N, 4.04. HPLC (RT),
3.14 min (Macherey-Nagel C18, 5 micron, 4.6 x 150 mm, MeOH, 1 mL/min).
4.2.3 McMurry cross-coupling reactions
In a Schlenk tube under argon, zinc powder (Zn) was suspended in THF at room temperature. With
stirring, titanium chloride (TiCl4) was added slowly with a syringe, and the reaction mixture was
heated to 90-100°C. After 1 h of reflux, a THF solution of both ketones was added. The reaction was
monitored by TLC. The mixture was poured into water and extracted with AcOEt. The organic layer
was washed, dried over MgSO4, filtered and concentrated under reduced pressure. The crude product
was purified on a silica gel column.
Pathway A for the synthesis of 6-oc
Reagents and quantities: Zn (20.8 mmol, 1.36 g), TiCl4 (10.4 mmol, 1.1 mL), THF (30 mL),
ketones (in 17 mL of THF): oc (2.08 mmol, 0.97 g), [3]ferrocenophanon-1-one (2.08 mmol, 0.50 g).
Reaction was stopped 15 min after the addition of ketones. After purification, the product was
obtained in 50% yield (0.70 g).
N,N’-[([3]ferrocenophan-1-ylidenemethylene)bis(4,1-phenylene)]dihexanamide, 6-he
Reagents and quantities: Zn (24 mmol, 1.57 g), TiCl4 (12 mmol, 1.3 mL), THF (30 mL), ketones
(in 20 mL of THF): he (2.4 mmol, 0.98 g) and [3]ferrocenophanon-1-one (2.4 mmol, 0.58 g). Reaction
was stopped 15 min after the addition of ketones. Eluent for column: petroleum ether/ethyl acetate
(7:3). The product was obtained in 54% yield (0.80 g). mp: 133-134°C. 1H-NMR (300 MHz, (CD3)2CO,
ppm): δ 0.86 (t, J = 7.0 Hz, 3H, CH3), 0.89 (t, J = 7.0 Hz, 3H, CH3), 1.25-1.40 (m, 8H, 4CH2), 1.57-1.74 (m,
4H, 2CH2), 2.29 (t, J = 7.4 Hz, 2H, CH2CO), 2.37 (t, J = 7.4 Hz, 2H, CH2CO), 2.30-2.40 (m, 2H, CH2Cp’),
2.68-2.75 (m, 2H, CH2C=C), 3.93 (t, J = 1.8 Hz, 2H, CpHβ’), 3.98 (t, J = 1.8 Hz, 2H, CpHβ), 4.01 (t, J = 1.8 Hz,
2H, CpHα), 4.27 (t, J = 1.8 Hz, 2H, CpHα’), 6.93 (d, J = 8.7 Hz, 2H, 2CHAr), 7.16 (d, J = 8.7 Hz, 2H, 2CHAr),
7.39 (d, J = 8.7 Hz, 2H, 2CHAr), 7.68 (d, J = 8.7 Hz, 2H, 2CHAr), 9.05 (s, 1H, NHcis), 9.22 (s, 1H, NHtrans). 13C-
NMR (75 MHz, (CD3)2CO, ppm): δ 14.2 (CH3), 14.3 (CH3), 23.1 (2CH2), 25.9 (CH2), 26.0 (CH2), 29.2
20

ACCEPTED MANUSCRIPT
(CH2Cp’), 32.2 (2CH2), 37.6 (CH2CO), 37.7 (CH2CO), 41.5 (CH2C=C), 68.9 (Cpβ’), 69.3 (Cpβ), 70.9 (Cpα),
71.0 (Cpα’), 84.5 (Cpipso), 87.6 (Cp’ipso), 118.7 (2CAr), 119.7 (2CAr), 130.4 (2CAr), 131.6 (2CAr), 135.0 (C=C),
138.5 (CAr), 139.1 (2CAr), 139.4 (CAr), 141.0 (C=C), 171.9 (COcis), 172.1 (COtrans). IR (KBr, υmax/cm-1):
3298 (N-H stretch), 3097, 3043 (aromatic C-H stretch), 2927, 2858 (alkyl C-H stretch), 1662 (NC=O
stretch), 1597 (aromatic C=C stretch), 1523 (N-H bend), 1400 (C-N stretch). MS (CI, m/z): 634
[MNH4]+, 617 [MH]+. Anal. Calc. for C38H44FeN2O2 (%): C, 74.02; H, 7.19; N, 4.54. Found: C, 73.92; H,
7.47; N, 4.49. HPLC (RT), 2.47 min. (Macherey-Nagel, C18, 5 micron, 4.6 x 250 mm, MeOH, 1 mL/min).
4,4’-(2-phenylbut-1-ene-1,1-diyl)dianiline, 7
Reagents and quantities: Zn (48 mmol, 3.14 g) and TiCl4 (24 mmol, 2.6 mL). THF (80 mL),
ketones (in 40 mL of THF): propiophenone (6.78 mmol, 0.91 g) and 4,4’-diaminobenzophenone (5.09
mmol, 1.08 g), 1 mL of DMSO. The reaction was stopped 5 min after addition of ketones. Eluent for
column : petroleum ether/ethyl acetate (1:1). The product was obtained in 35% yield (0.57 g). mp :
1
150-151°C, H-NMR (300MHz, (CD3)2SO, ppm): δ 0.84 (t, J = 7.3 Hz, 3H, CH3), 2.42 (q, J = 7.3 Hz, 2H,
CH2), 4.84 (s, 2H, NH2cis), 5.05 (s, 2H, NH2trans), 6.19 (d, J = 8.7 Hz, 2H, 2CHAr), 6.46 (d, J = 8.7 Hz, 2H,
2CHAr), 6.54 (d, J = 8.5 Hz, 2H, 2CHAr), 6.83 (d, J = 8.5 Hz, 2H, 2CHAr), 7.02-7.10 (t, J = 7.5 Hz, 1H, CHAr),
7.07 (d, J = 7.5 Hz, 2H, 2CHAr), 7.15 (t, J = 7.5 Hz, 2H, 2CHAr). 13C-NMR (75 MHz, (CD3)2SO, ppm): δ 13.7
(CH3), 28.6 (CH2), 112.9 (2CAr), 113.5 (2CAr), 125.5 (Php), 127.8 (Phm), 129.5 (Pho), 129.8 (2CAr), 131.1
(CAr), 131.3 (2CAr), 131.4 (CAr), 137.4 (PhC=C), 139.3 (C=C), 143.1 (Phipso), 146.3(CArN), 147.2(CArN). IR
(KBr, υmax/cm-1): 3432, 3352, 3213 (N-H stretch), 3024 (aromatic C-H stretch), 2962, 2927, 2870
(alkyl C-H stretch), 1616 (NH2 scissoring), 1508 (aromatic C=C stretch), 1280 (CAr-N stretch). MS (CI,
m/z): 332 [MNH4]+, 315 [MH]+. Anal. Calc. for C22H22N2•⅓H2O(%): C, 82.46; H, 7.13; N, 8.74. Found: C,
82.49; H, 6.96; N, 8.73. HPLC (RT), 2.79 min (Kromasil C18, 10 microns, 4.6 x 250 mm, MeOH, 1
mL/min).
4,4’-(2-phenylbut-1-ene-1,1-diyl)bis(N,N-dimethylaniline), 7-tm
Reagents and quantities: Zn (72 mmol, 4.71 g), TiCl4 (36 mmol, 3.9 mL), THF (100 mL). Ketones
in (60 mL of THF): propiophenone (7.6 mmol, 1.02 g) and 4,4’-bis(dimethylamino)benzophenone (7.6
mmol, 2.04 g). The reaction was stopped 20 min after addition of ketones. Eluent for column :
petroleum ether/ethyl acetate (9:1). The product was obtained in 74.6% yield (2.10 g). mp: 123-
1
125°C, H-NMR (300 MHz, CDCl3, ppm): δ 0.94 (t, J = 7.4 Hz, 3H, CH3), 2.52 (q, J = 7.4 Hz, 2H, CH2),
2.83 (s, 6H, 2NCH3), 2.97 (s, 6H, 2NCH3), 6.39 (d, J = 8.9 Hz, 2H, 2CHAr), 6.72 (d, J = 8.9 Hz, 2H, 2CHAr),
6.73 (d, J = 8.9 Hz, 2H, 2CHAr), 7.12 (d, J = 8.9 Hz, 2H, 2CHAr), 7.05-7.18 (m, 5H, 5CHAr). 13C-NMR (75
MHz, CDCl3, ppm): δ 14.0 (CH3), 29.3 (CH2), 40.6 (2NCH3), 40.8 (2NCH3), 111.4 (2CAr), 112.1 (2CAr),
125.6 (Php), 127.9 (Phm), 130.0 (Pho), 130.6 (2CAr), 132.0 (2CAr), 132.1 (CAr), 132.9 (CAr), 138.8 (PhC=C),
21

ACCEPTED MANUSCRIPT
139.2 (C=C), 143.6 (Phipso), 148.3 (CArN), 149.2 (CArN). IR (KBr, υmax/cm-1): 3074, 3032 (aromatic C-H
stretch), 2962, 2877 (alkyl C-H stretch), 1608, 1515 (aromatic C=C stretch), 1342 (CAr-N stretch). MS
(EI, m/z): 370 (100%) [M]+•, 355 [M-CH3]+. Anal. Calc. for C26H30N2(%): C, 84.28; H, 8.16; N, 7.56.
Found: C, 83.91; H, 8.16; N, 7.48. HPLC (RT), 3.62 min (Kromasil 10 microns, 4.6 x 250 mm, MeCN, 1
mL/min).
4,4’-([3]ferrocenophan-1-ylidenemethylene)bis(N,N-dimethylaniline), 6-tm
Reagents and quantities: Zn (72 mmol, 4.71 g), TiCl4 (36 mmol, 3.9 mL), THF (100 mL), Ketones
(in 60 mL of THF): 4,4’-bis(dimethylamino)benzophenone (7.6 mmol, 2.04 g) and [3]ferrocenophan-1-
one (7.6 mmol, 1.82 g). The reaction was stopped 25 min after addition of ketones. Eluent for column :
petroleum ether/ethyl acetate (85:15). The product was obtained in 41.4% yield (1.50 g). mp : 210-
212°C. 1H-NMR (300 MHz, CDCl3, ppm): δ 2.30-2.40 (m, 2H, CH2Cp’), 2.69-2.74 (m, 2H, CH2C=C), 2.87
(s, 6H, 2NCH3), 2.98 (s, 6H, 2NCH3), 3.97 (t, J = 1.8 Hz, 2H, CpHβ’), 4.00 (t, J = 1.8 Hz, 2H, CpHα), 4.03 (t, J
= 1.8 Hz, 2H, CpHβ), 4.21 (t, J =1.8 Hz, 2H, CpHα’), 6.45 (d, J = 8.8 Hz, 2H, 2CHAr), 6.71 (d, J = 8.8 Hz, 2H,
2CHAr), 6.92 (d, J = 8.8 Hz, 2H, 2CHAr), 7.12 (d, J = 8.8 Hz, 2H, 2CHAr). 13C-NMR (75 MHz, CDCl3, ppm): δ
28.8 (CH2Cp’), 40.5 (2NCH3), 40.7 (2NCH3), 41.5 (CH2C=C), 68.2 (Cpβ’), 68.6 (Cpβ), 70.3 (Cpα), 70.4
(Cpα’), 85.0 (Cpipso), 87.2 (Cp’ipso), 111.1 (2CAr), 112.0 (2CAr), 130.4 (2CAr), 131.0 (CAr), 131.8 (2CAr),
132.2 (CAr), 132.6 (C=C), 141.1 (C=C), 148.5 (CArN), 149.2 (CArN). IR (KBr, υmax/cm-1): 3085, 3039
(aromatic C-H stretch), 2935, 2893 (alkyl C-H stretch), 1604, 1516 (aromatic C=C stretch), 1342 (CAr-N
stretch). MS (EI, m/z): 476 (100%) [M]+•, 397, 238, 199. Anal. Calc. for C30H32FeN2(%): C, 75.63; H,
6.77; N, 5.88. Found: C, 75.45; H, 6.89; N, 5.60. HPLC (RT), 4.24 min (Kromasil 10 microns, 4.6 x 250
mm, MeCN, 1 mL/min).
4,4’-(2-ferrocenylbut-1-ene-1,1-diyl)bis(N,N-dimethylaniline), 8-tm
Reagents and quantities: Zn (72 mmol, 4.71 g), TiCl4 (36 mmol, 3.9 mL), THF (100 mL). Ketones
(in 60 mL of THF): 4,4’-bis(dimethylamino)benzophenone (7.6 mmol, 2.04 g) and propionylferrocene
(7.6 mmol, 1.84 g). The reaction was stopped 3 h after the addition of ketones. Eluent for column:
petroleum ether/ethyl acetate (9:1). The product was obtained in 38.5% yield (1.40 g). mp : 168-
1
170°C. H-NMR (300 MHz, CDCl3, ppm): δ 1.04 (t, J = 7.5 Hz, 3H, CH3), 2.62 (q, J = 7.5 Hz, 2H, CH2),
2.91 (s, 6H, 2NCH3), 2.96 (s, 6H, 2NCH3), 3.96 (t, J = 1.9 Hz, 2H, CpHα), 4.06 (t, J = 1.9 Hz, 2H, CpHβ), 4.11
(s, 5H, CpHunsubst), 6.59 (d, J = 8.9 Hz, 2H, 2CHAr), 6.70 (d, J = 8.9 Hz, 2H, 2CHAr), 6.92 (d, J = 8.9 Hz, 2H,
2CHAr), 7.05 (d, J = 8.9 Hz, 2H, 2CHAr). 13C-NMR (75 MHz, CDCl3, ppm): δ 15.8 (CH3), 28.2 (CH2), 40.7
(4NCH3), 67.8 (Cpβ), 69.2 (Cpunsubst), 69.4 (Cpα), 88.2 (Cipso), 112.2 (2CAr), 112.3 (2CAr), 130.4 (2CAr),
130.9 (2CAr), 133.8 (CAr), 134.0 (CAr), 135.0 (FcC=C), 138.5 (C=C), 148.8 (2CArN). IR (KBr, υmax/cm-1):
3089, 3031 (aromatic C-H stretch), 2951, 2920, 2862 (alkyl C-H stretch), 1604, 1515 (aromatic C=C
22

ACCEPTED MANUSCRIPT
stretch), 1346 (CAr-N stretch). MS (EI, m/z): 478 (100%) [M]+•, 449 [M-Et]+, 413 [M-Cp]+. Anal. Calc.
for C30H34FeN2(%): C, 75.31; H, 7.16; N, 5.86. Found: C, 75.15; H, 7.25; N, 5.86. HPLC (RT), 4.72 min
(Kromasil 10 microns, 4.6 x 250 mm, MeCN, 1 mL/min).
Cytotoxicity assays. The breast adenocarcinoma cell lines MDA-MB-231 and MCF7 were
obtained respectively from ATCC and Dr Matthias Kassack (Bonn, Germany). Cells were grown in RPMI
medium supplemented with 10% fetal calf serum, in the presence of penicilline, streptomycine and
fungizone in a 75 cm3 flask under 5% CO2. Cells were plated in 96-well tissue culture plates in 200 μL
medium and treated 24 h later with a 2 μL stock solution of compounds dissolved in DMSO with a
Biomek 3000 apparatus (Beckman-Coulter). The control samples received the same volume of DMSO
(1% final volume). After 72h exposure, MTS reagent (Promega) was added, and the sample was
incubated for 3 h at 37°C; the absorbance was monitored at 490 nm and the results are expressed as
the inhibition of cell proliferation calculated as the ratio [(1-(OD490 treated/OD490 control)×100] in
triplicate experiments. For IC50 determination [50% inhibition of cell proliferation], cells were
incubated for 72 h following the same protocol with compound concentrations in the range 5 nM to
100 μM in separate duplicate experiments.
Electrochemistry. Cyclic voltammograms (CVs) were obtained using a three-electrode cell
with a 0.5 mm Pt working electrode, stainless steel rod counter electrode and Ag/AgCl/LiClsat in
ethanol reference electrode, with an μ-Autolab 3 potentiostat driven by General Purpose
Electrochemical System (GPES) version 4.8, EcoChemie B.V., Utrecht, the Netherlands. Solutions
consisted of 5 mL MeOH, analyte (approx. 0.5 mM) and Bu4NBF4 (0.1 M) as supporting electrolyte.
After the CV was obtained in MeOH, 50 equivalents of imidazole were added to the cell, and new data
were obtained.
5. Abbreviations
Fcpd
Fpd
1-ferrocenylpropylidene
[3]ferrocenophan-1-ylidene
6. Acknowledgments
We thank the Agence Nationale de la Recherche for financial support (Mecaferrol) and the National Council
of Science and Technology of Mexico (CONACyT) for the PhD scholarship (JJCM).
7. References
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Ferrocenic and [3]Ferrocenophanic Anilines and Anilides have been synthesized and
evaluated against triple negative MBA-MB-231 breast cancer cells. Electrochemical studies
were discussed.